Plant Cell Wall Polymers. Biogenesis and Biodegradation 9780841216587, 9780841212503, 0-8412-1658-4

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ACS

SYMPOSIUM

S E R I E S 399

Plant Cell Wall Polymers Biogenesis and Biodegradation

Norman G . Lewis,

EDITOR

Virginia Polytechnic

Michael G . Paice,

EDITOR

Pulp and Paper Research Institute of Canada

Developed from a symposium sponsored by the Cellulose, Paper, and Textile Division of the American Chemical Society at the Third Chemical Congress of North America (195th National Meeting of the American Chemical Society), Toronto, Ontario, Canada, June 5-11, 1988

American Chemical Society, Washington, DC 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Library of Congress Cataloging-in-Publication Data Plant cell wall polymers. (ACS symposium series, ISSN 0097-6156; 399) "Developed from a symposiu d b th Cellulose, Paper, and Textile Divisio Chemical Congress of North Americ Meeting of the American Chemica Society), Ontario, Canada, June 5-11, 1988."

,

Includes bibliographical references. 1. Plant polymers—Synthesis—Congresses. 2. Plant polymers—Biodegradation—Congresses. 3. Plant Cell Walls—Congresses. I. Lewis, Norman G., 1949- . II. Paice, Michael G., 1949. III. Chemical Congress of North America (3rd: 1988: Toronto, Ont.) IV. American Chemical Society. Meeting (195th: 1988: Toronto, Ont.) V. American Chemical Society. Cellulose, Paper, and Textile Division. VI. Series. QK898.P76P53 1989 581.87'5 ISBN 0-8412-1658-4

89-17541

Copyright ©1989 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per-copy fee through the Copyright Clearance Center, Inc., 27 Congress Street, Salem, M A 01970, for copying beyond that permitted by Sections 107 or 108ofthe U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN T H E U N I T E D STATES O F A M E R I C A

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

ACS Symposium Series M. Joan Comstock, Series Editor 1989 ACS Books Advisory Board Paul S. Anderson Merck Sharp & Dohme Research Laboratories

Mary A. Kaiser Ε I du Pont de Nemours and

Alexis T. Bell University of California—Berkeley

Michael R. Ladisch Purdue University

Harvey W. Blanch University of California—Berkeley Malcolm H . Chisholm Indiana University Alan Elzerman Clemson University John W. Finley Nabisco Brands, Inc. Natalie Foster Lehigh University Marye Anne Fox The University of Texas—Austin G. Wayne Ivie U.S. Department of Agriculture, Agricultural Research Service

John L . Massingill Dow Chemical Company Daniel M . Quinn University of Iowa James C . Randall Exxon Chemical Company Elsa Reichmanis AT&T Bell Laboratories C . M . Roland U.S. Naval Research Laboratory Stephen A . Szabo Conoco Inc. Wendy A . Warr Imperial Chemical Industries Robert A . Weiss University of Connecticut

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Foreword T h e A C S S Y M P O S I U M S E R I E S was founded i n 1974 to p r o v i d e a

m e d i u m for p u b l i s h i n g s y m p o s i a q u i c k l y i n b o o k f o r m . T h e format o f the Series parallels that o f the c o n t i n u i n g A D V A N C E S IN C H E M I S T R Y SERIES except that, i n order to save t i m e , the papers are not typeset but are reproduced as they are submitted by the authors i n c a m e r a - r e a d y form. Papers are reviewed under the supervision o f th A d v i s o r y B o a r d and are selected to m a i n t a i n the integrity o f the s y m p o s i a ; h o w e v e r , v e r b a t i m reproductions o f p r e v i o u s l y p u b lished papers are not accepted. B o t h reviews a n d reports o f research are acceptable, because s y m p o s i a m a y e m b r a c e both types o f presentation.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Preface

XLANT

CELL

WALLS

A R E COMPLEX,

HETEROGENEOUS

STRUCTURES

composed mainly of polymers, such as cellulose, hemicelluloses, and lignins. In spite of several decades of research, cell wall assembly and the biosynthesis and ultimate biodegradative pathways of individual polymers are still far from being fully understood. One simple example will suffice: Even today, no enzyme capabl has been obtained. The objective of the symposium on which this book is based was to bring together scientists from different plant-related disciplines, who do not often interact with each other, to discuss the subjects of cell wall polymer biogenesis and biodégradation. These individuals, linked by a common interest in plant polymers, exchanged, promoted, and at times discarded ideas, often in spirited discussions. This book attempts to place current and emerging concepts that were discussed into a common perspective. This approach is timely because within the past five years several subject areas have advanced rapidly. F o r example, an increased understanding of lignin biogenesis and structure has been achieved by in situ labeling, and new techniques for structural analysis of other plant polymers (e.g., cutin, suberin, and hemicelluloses) have been developed. Additionally, scientists have made substantial progress in localizing the enzymes associated with cell wall polymer formation and have an improved understanding of the temporal and spatial distribution of polymer constituents within the plant cell wall. This progress leads to the conclusion that we can make an optimistic prognosis for the eventual understanding of the mechanism of cell wall assembly. The same facts apply to biodegradative processes: Five years ago, little was known about the enzymology of lignin biodégradation, and the molecular biology of cellulases was in its infancy. Advances have since been made in leaps and bounds; these advances are fully discussed within this volume. This book is divided into eight sections, spanning cell wall development, biogenesis, plant-microbe interactions, and biodegradation. Each section contains informative, up-to-date reviews and original reports by some of the leading researchers in their fields.

xi In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

We are indebted to a number of individuals and organizations for their contributions to this book. The symposium itself was sponsored by the Cellulose, Paper, and Textile Division of the American Chemical Society, Phillip Morris Tobacco Company, the U . S . Department of Energy (the Energy Conversion and Utilization Technology Biobased Materials project through the Solar Energy Research Institute), Weyerhaeuser, Abitibi-Price, and the Chemical Institute of Canada. Virginia Polytechnic Institute and State University and the Pulp and Paper Research Institute of Canada provided valuable assistance with correspondence, and Kathryn Hollandsworth and Janice Baker-Briand were particularly helpful in preparing many of the manuscripts. Cheryl Shanks of the A C S Books Department provided much-needed support during the editing and publication process. W e would be remiss not to mention Helena L i Chum, who tirelessly and selflessly contributed much to the overall success o However, most of all, to the authors and referees who invested their time and effort in writing and refining the manuscripts, we extend our sincerest thanks.

NORMAN G . LEWIS

Virginia Polytechnic Institute and State University Blacksburg, VA 24061-0323 MICHAEL G . PAICE

Pulp and Paper Research Institute of Canada Pointe Claire, Quebec H9R 3J9, Canada March 30, 1989

xii In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 1

Control of Plant Cell Wall Biogenesis An Overview D . H . Northcote Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, England

All the polysaccharides of the cell wall are synthesized in association with phospholipid membranes. The hemicelluloses and pectin polysaccharides are formed at the membranes of the Golgi apparatus, cellulose at the plasma membrane. Control of the rate of polysaccharide synthesized and the type of polymer formed is exerted by the transport of donor nucleoside diphosphate sugar molecules across the membranes, the amount, type, and activity of the synthases (glycosyltransferases) and fusion and targetting of vesicles containing the polysaccharides at specific sites at the plasma membrane. The formation of a polysaccharide typically depends on an enzyme complex organized on a membrane. The complex consists of transporters, glycosyltransferases, epimerases and binding proteins to hold the acceptor molecules. In addition to these, subsidiary proteins may also be present which may act to bring about and control the assembly of the complex and its location on the membrane. They may also act as modulators of the polysaccharide synthesis in conjunction with smaller molecules or ions. During xylem formation, lignin is deposited as well as polysaccharides. Part of the control mechanism for the formation of lignin is the level of phenylalanine ammonia lyase activity. Proteins and lipids are also deposited in the wall and although these constituents are not present in large amounts, they are very important for the function of the wall and the cell during growth.

0097-6156/89/0399-0001$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2

PLANT C E L L W A L L P O L Y M E R S

T h e cell w a l l is f o r m e d f r o m m a t e r i a l s w i t h i n the c y t o p l a s m w h i c h are subsequently t r a n s p o r t e d either as m o n o m e r s or p o l y m e r s to the outside of the cell. D u r i n g g r o w t h a n d differentiation of the cell its c o m p o s i t i o n a n d s t r u c t u r e changes, a n d i t can also alter i n response to e n v i r o n m e n t a l factors. T h e r e is therefore a dialogue between the outside of the cell a n d the s y n t h e t i c a n d t r a n s p o r t systems at the inside of the cell so t h a t the changes i n the w a l l are b r o u g h t about i n a n ordered m a n n e r at p a r t i c u l a r stages of its development (1). T h e m a j o r p o l y m e r s t h a t m a k e u p the w a l l are p o l y s a c c h a r i d e s a n d l i g n i n . These o c c u r together w i t h more m i n o r b u t very i m p o r t a n t c o n s t i t u e n t s such as p r o t e i n a n d l i p i d . W a t e r constitutes a m a j o r a n d very i m p o r t a n t m a t e r i a l of y o u n g , p r i m a r y walls (2). T h e l i g n i n is t r a n s p o r t e d i n the f o r m of its b u i l d i n g u n i t s (these m a y be present as glucosides) a n d is p o l y m e r i z e d w i t h i n the w a l l . T h o s e polysaccharides w h i c h make u p the m a t r i x of the w a l l (hemicelluloses a n d p e c t i n m a t e r i a l ) are p o l y m e r i z e d i n the e n d o m e m b r a n e s y s t e m outside of the cell. F u r t h e r m o d i f i c a t i o n s of the polysaccharides (such as a c e t y l a t i o n ) m a y o c c u r w i t h i n the w a l l after d e p o s i t i o n . C e l l u l o s e is p o l y m e r i z e d at the cell surface b y a c o m p l e x e n z y m e s y s t e m t r a n s p o r t e d to the p l a s m a m e m b r a n e (3). T h e c o n t r o l of the development of the cell w a l l m u s t be r e g u l a t e d at the various processes w h i c h m a k e the constituents a n d w h i c h deposits t h e m to the outside of the cell. These m a y be s u m m a r i z e d as follows: (1) r e g u l a t i o n of synthesis b y the a m o u n t s of the synthase; this is d i r e c t l y c o n t r o l l e d by gene r e g u l a t i o n ; (2) b i o c h e m i c a l feed-back c o n t r o l m e c h a n i s m s w h i c h regulate the level of the precursors of the p o l y m e r s or the a c t i v i t i e s of the synthases w h i c h f o r m t h e m ; (3) r e g u l a t i o n of the segregation a n d t a r g e t t i n g of m a t e r i a l f o r m e d w i t h i n the e n d o p l a s m i c r e t i c u l u m a n d G o l g i a p p a r a t u s ; (4) c o n t r o l of t r a n s p o r t of m o n o m e r s to the synthases of the p o l y m e r s ; (5) c o n t r o l of vesicle fusion a n d t a r g e t t i n g of the m e m b r a n e b o u n d m a t e r i a l to specific sites at the cell surface; (6) receptors for p l a n t g r o w t h substances a n d mechanisms for cell s i g n a l l i n g at the c y t o p l a s m i c surface a n d other cell m e m b r a n e s . T h i s chapter reviews some of these topics i n more d e t a i l for the p o l y s a c c h a r i d e s , l i g n i n , p r o t e i n a n d l i p i d of the w a l l . Polysaccharides T h i s section describes a d e t a i l e d hypothesis for the c o n t r o l of p o l y s a c c h a r i d e synthesis a n d d e p o s i t i o n i n the w a l l d u r i n g g r o w t h . M o s t of the b i o c h e m i c a l studies o n p o l y s a c c h a r i d e synthesis to date have been concerned w i t h the f o r m a t i o n of h o m o p o l y m e r s even w h e n it is k n o w n t h a t the synthesis o f the h o m o p o l y m e r c h a i n occurs in vivo as p a r t of a h e t e r o p o l y s a c c h a r i d e (4-6). C y t o c h e m i c a l investigations have m a d e no s u c h d i s t i n c t i o n s a n d the p o l y m e r s located b y these studies have n e a r l y a l ways been sites at w h i c h h e t e r o p o l y m e r s were present a n d where d e p o s i t i o n i n the w a l l o c c u r r e d . T h e b u l k of the polysaccharides t h a t o c c u r i n the w a l l , w i t h the e x c e p t i o n of cellulose a n d callose, are h e t e r o p o l y m e r s . G e n e r a l l y the polysaccharides of the hemicelluloses a n d pectins are c o m p o s e d o f p o l y -

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1.

NORTHCOTE

Control of Plant Cell Wall Biogenesis

3

mers c o n t a i n i n g different monosaccharides c o m b i n e d b y different linkages ( 2 , 7 - 1 0 ) . U s u a l l y there is a b a c k b o n e m a d e u p of a single c h a i n , b u i l t o f the same m o n o s a c c h a r i d e (two i n the case o f g l u c o m a n n a n s , see b e l o w ) u s u a l l y c o m b i n e d b y a specific l i n k a g e , o n t o w h i c h short branches are a t t a c h e d w h i c h m a y be j u s t a single monosaccharide different f r o m t h a t of the u n i t s of the m a i n c h a i n , e.g., g l u c u r o n o x y l a n , a r a b i n o g l u c u r o n o x y l a n s , x y l o g l u cans. I n a d d i t i o n more c o m p l i c a t e d p o l y m e r s such as the a r a b i n o g a l a c t a n s o c c u r b u t even i n these p o l y m e r s there is a c e n t r a l core o n t o w h i c h the branches are c o n s t r u c t e d (2). T h e influence of the i n c o r p o r a t i o n of the side branches o n the synthesis of the m a i n c h a i n a n d vice v e r s a is of some significance for the c o n t r o l o f the p o l y s a c c h a r i d e synthesis a n d i n t r o d u c e s the i d e a of a n e n z y m e c o m p l e x at the site for the p o l y m e r s y n t h e s i s . T h e b a c k b o n e c h a i n c a n i n m o s t cases be s y n t h e s i z e d separately w h e n a n in vitro s y s t e m is used. T h e s e investigations have s h o w n t h a t the s y n thase a c t i v i t i e s w h i c h transfer the sugar f r o m a nucleoside d i p h o s p h a t e sugar d o n o r to the g r o w i n of p o l y s a c c h a r i d e w h i c h is f o r m e d at a n y stage of the g r o w t h a n d developm e n t of the w a l l (5, 6 , 1 1 , 1 2 ) . T h e r e is also s t r o n g c i r c u m s t a n t i a l evidence w h i c h i n d i c a t e s t h a t these v a r i a t i o n s i n a c t i v i t y are due t o changes i n the a m o u n t s of the synthases w h i c h are available at the p a r t i c u l a r sites at a p a r t i c u l a r t i m e (13). T h u s the development o f the w a l l has some c o n t r o l m e c h a n i s m s d i r e c t l y related to the r e g u l a t i o n of the genome d u r i n g diff e r e n t i a t i o n ; t h i s controls the a m o u n t s of the synthases w h i c h are f o r m e d , p r o b a b l y at the level o f t r a n s c r i p t i o n r a t h e r t h a n t r a n s l a t i o n . Synthesis. T h e synthases are present at the e n d o m e m b r a n e s y s t e m o f the cell a n d have been isolated on m e m b r a n e f r a c t i o n s p r e p a r e d f r o m the cells ( 5 , 6 ) . T h e nucleoside d i p h o s p h a t e sugars w h i c h are used b y the synthases are f o r m e d i n the c y t o p l a s m , a n d u s u a l l y the epimerases a n d the other e n zymes (e.g., dehydrogenases a n d decarboxylases) w h i c h i n t e r c o n v e r t t h e m are also soluble a n d p r o b a b l y o c c u r i n the c y t o p l a s m (14). Nevertheless some epimerases are m e m b r a n e b o u n d a n d t h i s m a y be i m p o r t a n t for the r e g u l a t i o n of the synthases w h i c h use the different epimers i n a hete r o p o l y s a c c h a r i d e . T h i s is e s p e c i a l l y significant because the a v a i l a b i l i t y of the d o n o r c o m p o u n d s at the site of the transglycosylases (the synthases) is of obvious i m p o r t a n c e for c o n t r o l of the synthesis. T h e synthases are l o c a t e d at the l u m e n side o f the m e m b r a n e a n d the nucleoside d i p h o s p h a t e sugars m u s t therefore cross the m e m b r a n e i n order to t a k e p a r t i n the rea c t i o n . M o d u l a t i o n o f t h i s t r a n s p o r t m e c h a n i s m is a n o b v i o u s p o i n t for the c o n t r o l not o n l y for the rate of synthesis b u t for the t y p e of synthesis w h i c h occurs i n the p a r t i c u l a r l u m e n of the m e m b r a n e s y s t e m . O b v i o u s l y the s y n t h a s e c a n n o t f u n c t i o n unless the d o n o r m o l e c u l e is t r a n s p o r t e d to its active site a n d the t r a n s p o r t e r s m a y o n l y be present at c e r t a i n regions w i t h i n the e n d o m e m b r a n e s y s t e m . It has been observed t h a t w h e n i n t a c t cells are fed r a d i o a c t i v e monosaccharides w h i c h w i l l f o r m a n d l a b e l p o l y s a c charides, these cannot a l w a y s be f o u n d at a l l the m e m b r a n e sites w i t h i n the cell where the synthase a c t i v i t i e s are k n o w n to o c c u r (15). A p o s s i b l e r e a son for t h i s difference m a y be the selection of precursors b y the t r a n s p o r t mechanism.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4

PLANT C E L L W A L L POLYMERS

T h e preformed polysaccharides before they are deposited i n the w a l l can be detected i n the G o l g i a p p a r a t u s either b y r a d i o a c t i v e l a b e l l i n g tech­ niques a n d direct analysis of the isolated organelle, or b y c y t o c h e m i c a l s t a i n s , the m o s t specific of w h i c h are i d e n t i f i c a t i o n s u s i n g a n t i b o d i e s . F i g ­ ure 1 shows a section of a d e v e l o p i n g secondary w a l l f r o m the h y p o c o t y l cells of b e a n . T h e a n t i b o d y was raised i n r a b b i t s against oligosaccharides prepared f r o m w a l n u t β 1 —• 4 x y l a n (5) a n d conjugated to b o v i n e s e r u m a l b u m i n . T h e a n t i b o d y to the p r o t e i n was removed b y affinity c h r o m a t o g ­ r a p h y a n d the r e s u l t a n t p u r i f i e d a n t i b o d y was specific to antigens c a r r y i n g β 1 —• 4 xylose u n i t s . It d i d not cross-react w i t h x y l o g l u c a n , β 1 —• 4 g l u c a n , a r a b i n a n or m a n n a n . T h e section s h o w n i n F i g u r e 1 was treated w i t h the a n t i b o d y a n d s t a i n e d w i t h g o l d - l a b e l l e d g o a t - a n t i r a b b i t s e r u m . T h e x y l a n is present at the secondary w a l l (st) a n d i n the vesicles of the G o l g i a p p a r a t u s (v). F i g u r e 2 shows a m e r i s t e m a t i c cell f r o m the root of b e a n , treated w i t h an a n t i b o d y specific for L - a r a b i n o f u r a n o s e a n d s t a i n e d w i t h gold-labelled goat-antirabbi (cw) a n d the developing cell-plate (cp) where p e c t i n was b e i n g l a i d d o w n . Glucomannan Synthesis. A l t h o u g h the heteropolymers are u s u a l l y s i m i l a r to the g l u c u r o n o a r a b i n o x y l a n s , there are heteropolymers i n w h i c h two dif­ ferent monosaccharides occur i n the m a i n c h a i n , e.g., g l u c o m a n n a n s a n d g a l a c t o g l u c o m a n n a n s . F o r a discussion of the c o n s t r u c t i o n a n d c o n t r o l of the synthesis of a h e t e r o p o l y m e r the synthesis of g l u c o m a n n a n i n the h e m i cellulose of g y m n o s p e r m s serves as a g o o d e x a m p l e ( 1 6 , 1 7 ) . A m e m b r a n e p r e p a r a t i o n isolated f r o m pine s t e m tissues i n c o r p o r a t e d glucose i n t o glucans f r o m b o t h U D P G l c a n d G D P G l c . T h e s e were m i x e d p o l y m e r s c o n t a i n i n g β 1 —• 3 a n d β 1 —> 4 l i n k e d glucose. It also c a r r i e d an epimerase w h i c h interconverted G D P G l c a n d G D P M a n (18). T h e m e m ­ brane p r e p a r a t i o n formed a g l u c o m a n n a n i n the presence of added G D P ­ M a n a n d i n the presence of G D P M a n the f o r m a t i o n of g l u c a n c o n t a i n i n g β 1 —• 3 l i n k s f r o m G D P G l c was repressed a n d the β 1 —• 4 g l u c o m a n n a n was f o r m e d (whether a separate β 1 —*· 4 g l u c a n was synthesized i n a d d i ­ t i o n was difficult to determine) ( T a b l e I) (18). T h e a c t i v i t y of the g l u c a n synthase w h i c h used U D P G l c was unaffected b y the presence of G D P M a n . T h e s e observations c a n best be e x p l a i n e d i n t e r m s of a n e n z y m e c o m p l e x carried o n the m e m b r a n e . T h i s c o m p l e x has a m i n i m u m of three a c t i v i t i e s : (1) a n epimerase for the interconversion of G D P M a n a n d G D P G l c ; (2) a synthase w h i c h used U D P G l c ; (3) a synthase w h i c h used b o t h G D P G l c a n d G D P M a n . T h i s l a t t e r synthase h a d a greater affinity for G D P G l c t h a n G D P M a n a n d , in vitro, i n the presence of G D P G l c b u t i n the a b ­ sence of added G D P M a n , i t formed a g l u c a n i n spite of the presence of the epimerase. Since the influence of the presence-of the G D P M a n o n the i n ­ c o r p o r a t i o n f r o m G D P G l c into a different type of p o l y m e r is so direct, the two a c t i v i t i e s , one for the transfer of glucose a n d the other for the transfer of mannose f r o m the G D P sugars, must either be c a r r i e d out b y the same transglycosylase or the two transglycosylases must be very close together so t h a t they can influence one another.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

1.

NORTHCOTE

Control of Plant Cell Wall Biogenesis

S

F i g u r e 1. D e v e l o p i n g secondary thickened w a l l of the h y p o c o t y l of Phaseolus vulgaris. T h e s e c t i o n was treated w i t h a n a n t i b o d y specific for β 1 —• 4 linked D-xylose units and stained w i t h gold-labelled goat-antirabbit serum. T h e l a b e l is seen at the secondary t h i c k e n i n g (st) a n d the vesicles of the G o l g i a p p a r a t u s (v).

F i g u r e 2. M e r i s t e m a t i c cell of the r o o t - t i p of Phaseolus vulgaris. T h e s e c t i o n was treated w i t h a n a n t i b o d y specific for L - a r a b i n o f u r a n o s e a n d s t a i n e d w i t h g o l d - l a b e l l e d g o a t - a n t i r a b b i t s e r u m . T h e l a b e l is seen at the developing cell p l a t e (cp) a n d the y o u n g w a l l (cw) of the m o t h e r c e l l .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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T a b l e I. T h e influence of the presence of exogenous G D P M a n o n the s y n ­ thesis of a m i x e d βΐ —• 3, 1 —• 4 g l u c a n f r o m G D P G l c b y a m e m b r a n e p r e p a r a t i o n f r o m pine s t e m tissue. M e a s u r e m e n t s were m a d e f r o m the i n c o r p o r a t i o n of r a d i o a c t i v e glucose f r o m G D P [ U C ] G l c (18). (- indicates t h a t no g l u c a n c a r r y i n g a βΐ —• 3 linkage was formed.) 1 4

G D P [ U - C ] G l c as the P r i m a r y S u b s t r a t e (1.0 n m o l ) 1 4

GDPMan Additions nmol

βΐ —• 3,1 —» 4 g l u c a n formed nmol. m i n " (mg p r o t e i n ) " 1

1

0.0 0.5 1.0 2.5 5.0 10.0

0.28 -

β! —» 4 g l u c o m a n n a n formed nmol. m i n " ( m g protein) 0.0 0.56 1.0 0.70 0.39

It is likely t h a t i n a d d i t i o n to the synthases a n d epimerases there is also present at the m e m b r a n e i n close p r o x i m i t y to these, t r a n s p o r t e r systems for the transfer of the nucleoside d i p h o s p h a t e donor c o m p o u n d s t o the transglycosylases s i t u a t e d o n the l u m e n side of the m e m b r a n e . T h e p o l y m e r w h i c h formed was either a m i x e d /? 1 —• 3, β I —• 4 g l u c a n or a β 1 —* 4 g l u c o m a n n a n a n d runs of β 1 —• 4 l i n k e d glucose o c c u r r e d i n the g l u c o m a n n a n (18). T h e g l u c o s y l transferase t h a t used G D P G l c added glucose either at the 3 p o s i t i o n of the r e c e i v i n g sugar, or at the 4 p o s i t i o n . It was also s h o w n t h a t the transglucosylase added the g l u c o s y l r a d i c a l to water to f o r m free glucose (18). In t h i s s y s t e m , therefore, the transglycosylase u s i n g G D P G l c was not specific for the receptor molecule since t h i s m a y be w a t e r , glucose at the 3 p o s i t i o n , glucose at the 4 p o s i t i o n , or m a n n o s e at the 4 p o s i t i o n . W h e n a g l u c o m a n n a n was f o r m e d , the linkage was a l w a y s m a d e at the 4 p o s i t i o n . T h e acceptor molecule, a l t h o u g h i t was not specific i n the t r a n s g l y c o s y l r e a c t i o n , influenced h o w the transfer o c c u r r e d . T h e s e diverse actions of the transglycosylase c a n be most easily ex­ p l a i n e d b y p o s t u l a t i n g the existence of a b i n d i n g p r o t e i n w h i c h holds the acceptor molecules. T h e n d u r i n g the transglycosylase r e a c t i o n t h a t forms the g l u c o m a n n a n c h a i n , a g l u c o s y l r a d i c a l c o u l d first be transferred to w a ­ ter w i t h i n an associated b i n d i n g p r o t e i n a n d the r e s u l t a n t sugar c o u l d t h e n be e x t e n d e d b y subsequent transfers to the n o n - r e d u c i n g end of the g r o w i n g c h a i n , either f r o m G D P M a n or G D P G l c . It is possible to have the acceptor sugar precisely o r i e n t a t e d b y such a b i n d i n g p r o t e i n , i n order to receive the new g l y c o s y l residue b y the transglycosylase at the p a r t i c u l a r h y d r o x y l w h i c h is presented to the d o n o r molecule. T h e transglycosylase i t s e l f c o u l d have a d o m a i n at w h i c h the acceptor oligosaccharide was h e l d b u t since the

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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o r i e n t a t i o n of the acceptor m o l e c u l e is altered i n d e p e n d e n t l y of the t r a n s glycosylase there are p r o b a b l y at least t w o p r o t e i n s i n v o l v e d : one used for transfer a n d h o l d i n g the donor a n d another necessary for h o l d i n g the accept o r . T h e transglycosylase p r o t e i n is not therefore specific for the acceptor m o l e c u l e . T h i s is i n contrast to the way i n w h i c h the g l y c o s y l a t i o n of cert a i n g l y c o p r o t e i n s is t h o u g h t to occur (19). D e f i n i t e sequences of sugars are b u i l t o n the p r o t e i n because the various transglycosylases i n v o l v e d are specific for the acceptor oligosaccharide w h i c h changes i n a stepwise m a n ner. A t each stage a definite glycosyltransferase c a n act t o f o r m a definite sequence of sugars. Binding Protein and Enzyme Complex. T h a t sugars a n d oligosaccharides c a n s p e c i f i c a l l y b i n d t o p r o t e i n is w e l l k n o w n . H e x o k i n a s e is k n o w n to h o l d glucose w i t h i n a cleft of the e n z y m e b y h y d r o g e n b o n d s ; i n d e e d , the presence of glucose causes a m o v e m e n t i n the c o n f o r m a t i o n a l s t r u c t u r e o f the p r o t e i n so t h a t i t folds a r o u n p o s i t i o n is presented to the kinase for the transfer of the p h o s p h a t e g r o u p f r o m the A T P (20). Specific associations between proteins a n d o l i g o s a c c h a rides a n d polysaccharides are also w e l l d o c u m e n t e d . L y s o z y m e a n d t a k a amylase are k n o w n to h o l d oligosaccharides i n a r i b b o n - l i k e c o n f i g u r a t i o n b y h y d r o g e n b o n d i n g a n d v a n der W a a l s forces w i t h i n a b i n d i n g - s i t e groove (21). Precise a n d stereospecific i n t e r a c t i o n s are f o r m e d a n d m a i n t a i n e d b y the o r i e n t a t i o n o f h y d r o g e n - b o n d i n g residues w h i c h are i n t u r n fixed b y c o m p l e x h y d r o g e n b o n d networks to other residues w i t h i n the b i n d i n g sites. D u r i n g b i n d i n g , c o n f o r m a t i o n a l changes m a y o c c u r w h i c h allow the c a r b o h y d r a t e s to be o r i e n t e d for the b i n d i n g to progress a n d specific i n t e r a c t i o n between p r o t e i n a n d c a r b o h y d r a t e results (22). D u r i n g the synthesis of a m i x e d p o l y s a c c h a r i d e s u c h as a g l u c u r o n o x y l a n or a x y l o g l u c a n , at least two transglycosylases are i n v o l v e d w h i c h w o r k i n c o n j u n c t i o n w i t h one another (23-27). It is possible to envisage the m a i n c h a i n of either glucose or x y l o s e b e i n g h e l d b y a b i n d i n g p r o t e i n a n d the side chains being g u i d e d o n t o the b a c k b o n e w h i c h is h e l d i n such a w a y as t o present the a p p r o p r i a t e h y d r o x y l to the s u b s t i t u e n t sugar a n d the appropriate transglycosylase. W h a t e v e r the m e c h a n i s m , the c o o r d i n a t e d synthesis of a p o l y s a c c h a r i d e , s u c h as g l u c o m a n n a n or g l u c u r o n o x y l a n or the a r a b i n o g a l a c t a n s of the p e c t i n s or even a h o m o p o l y m e r s u c h as single cellulose c h a i n s (3), needs the c o o p e r a t i o n of a set of p r o t e i n s . These m u s t be o r g a n i z e d close to one a n other i n correct o r i e n t a t i o n for the synthesis t o o c c u r i n a n e c o n o m i c a l a n d r a p i d m a n n e r . T h e r e is t h u s a m u l t i e n z y m e s y s t e m o r g a n i z e d o n the m e m brane a n d h e l d i n a c o o r d i n a t e d way. T h e m e m b r a n e o n a n d i n w h i c h the p r o t e i n s are h e l d becomes a n i m p o r t a n t p a r t of the s y n t h e t i c process. D i s r u p t i o n o f the m e m b r a n e w i l l b r i n g a b o u t loss o f o r g a n i z a t i o n a n d the in vitro p r e p a r a t i o n s m a d e f r o m i n t a c t cells m a y f o r m p o l y s a c c h a r i d e s different f r o m those f o r m e d in vivo (e.g., callose i n s t e a d of cellulose) (28) or for a h e t e r o p o l y s a c c h a r i d e the dependence of one t r a n s g l y c o s y l a s e o n the a c t i o n o f a n o t h e r w i l l b e c o m e less precise ( 2 4 , 2 9 ) i n the in vitro p r e p a r a t i o n t h a n i n the i n t a c t cell.

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It seems likely t h a t the e n z y m e complexes for hemicelluloses, pectins a n d cellulose are c o n s t r u c t e d , at least i n p a r t , o n the e n d o p l a s m i c r e t i c u l u m a n d then transferred to the G o l g i a p p a r a t u s , where they are m o d i f i e d a n d sorted so t h a t they can be segregated w i t h i n the c o m p a r t m e n t s of the G o l g i cisternae ( 3 0 , 3 1 ) . T h e c o m p l e x for cellulose synthesis is not n o r m a l l y a c t i v e w i t h i n the G o l g i a p p a r a t u s a n d i t is t r a n s p o r t e d to active sites at the p l a s m a m e m b r a n e (1). T h e hemicelluloses a n d pectins are f o r m e d w i t h i n vesicles a n d cisternae of the G o l g i a p p a r a t u s a n d the vesicles are t r a n s p o r t e d to the p l a s m a m e m b r a n e , where fusion occurs a n d the polysaccharides are packed i n t o the w a l l (1). It is not k n o w n whether p a r t i c u l a r polysaccharides such as the x y l a n s of the hemicellulose a n d the a r a b i n o g a l a c t a n s of the p e c t i n s are t r a n s p o r t e d i n separate vesicles or together i n one vesicle. N o r is i t k n o w n i f the c o m p l e x for cellulose synthesis is t r a n s p o r t e d by vesicles w h i c h c a r r y hemicellulose a n d p e c t i n polysaccharides. Deposition of Wall Polysaccharides. polysaccharides a n d synthas vesicles to p a r t i c u l a r sites a n d the rate of fusion w i t h the p l a s m a m e m b r a n e c o n s t i t u t e i m p o r t a n t c o n t r o l p o i n t s for the d e p o s i t i o n of the m a t e r i a l i n t o the w a l l . It is k n o w n t h a t at sites of active w a l l d e p o s i t i o n vesicles are d i rected to the p l a s m a m e m b r a n e b y m i c r o t u b u l e s (32). However, i t is possible t h a t other signals a n d receptors at the m e m b r a n e surface m a y be i n v o l v e d i n r e c o g n i t i o n of the sites for i n c o r p o r a t i o n . P a r t of the c o n t r o l for vesicle fusion at the surface is m e d i a t e d by the i o n i c atmosphere at the m e m b r a n e , and C a is necessary for the fusion to o c c u r . T h e rate of vesicle fusion can be a l i m i t i n g process for the rate of cell w a l l f o r m a t i o n , since at a n y one t i m e the n u m b e r of vesicles ready for fusion exceeds the n u m b e r t h a t are f u s i n g a n d d e p o s i t i n g m a t e r i a l into the w a l l . In t h i s way the c o m p o s i t i o n a n d a m o u n t of w a l l m a t e r i a l deposited m a y respond very q u i c k l y to a s t i m u l u s at the cell surface w h i c h allows the rate of vesicle fusion to vary. For instance, the m a t e r i a l deposited i n the cell w a l l , especially the p e c t i n , changes very q u i c k l y at the i n i t i a l stages of p l a s m o l y s i s w h e n the w a l l is j u s t separated f r o m the p l a s m a m e m b r a n e . A new steady state w o u l d t h e n be achieved t h a t p r o d u c e d the requisite n u m b e r of vesicles f r o m the G o l g i a p p a r a t u s to m a i n t a i n the altered rate of fusion (32-34). 2 +

Lignin Precursors and Lignin Formation Phenylalanine Ammonia-Lyase. T h e b u i l d i n g u n i t s of l i g n i n are f o r m e d f r o m c a r b o h y d r a t e v i a the s h i k i m i c a c i d p a t h w a y to give a r o m a t i c a m i n o acids. Once the a r o m a t i c a m i n o acids are f o r m e d , a key e n z y m e for the c o n t r o l of l i g n i n precursor synthesis is p h e n y l a l a n i n e a m m o n i a - l y a s e ( P A L ) (1). T h i s e n z y m e catalyzes the p r o d u c t i o n of c i n n a m i c a c i d f r o m p h e n y l a l a n i n e . It is very active i n those tissues of the plant t h a t become lignified a n d it is also a c e n t r a l enzyme for the p r o d u c t i o n of other p h e n y l p r o p a n o i d d e r i v e d c o m p o u n d s such as flavonoids a n d c o u m a r i n s , w h i c h can o c c u r i n m a n y p a r t s of the plant a n d i n m a n y different organs (35). R a d i o a c t i v e p h e n y l a l a n i n e a n d c i n n a m i c a c i d are d i r e c t l y i n c o r p o r a t e d i n t o l i g n i n i n vascular tissue (36).

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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T h e i n d u c t i o n o f P A L a c t i v i t y at the onset o f vascular d i f f e r e n t i a t i o n c a n be s h o w n b y the use o f p l a n t tissue c u l t u r e s (37-39). X y l e m cells w i t h secondary a n d l i g n i f i e d w a l l s are differentiated over a t i m e course o f 3-14 d a y s b y the a p p l i c a t i o n of the p l a n t g r o w t h factors n a p h t h y l e n e acetic a c i d ( N A A ) a n d k i n e t i n i n the r a t i o 5:1 (1.0 m g / l i t e r N A A , 0.2 m g / l i t e r k i n e t i n ) to tissue cultures of b e a n cells (Phaseolus vulgaris) ( 3 7 , 4 0 ) . T h e t i m e for d i f f e r e n t i a t i o n varies w i t h the t y p e of c u l t u r e , s o l i d or s u s p e n s i o n , a n d w i t h the frequency a n d d u r a t i o n of s u b c u l t u r e , b u t for a n y one c u l t u r e i t is r e l a t i v e l y constant ( 3 7 , 4 1 , 4 2 ) . A t the t i m e of d i f f e r e n t i a t i o n w h e n the x y l e m vessels f o r m , the a c t i v i t y of P A L rises to a m a x i m u m . T h e r i s i n g phase of the e n z y m e a c t i v i t y was i n h i b i t e d b y a c t i n o m y c i n D a n d b y D - 2 , 4 - ( 4 methyl-2,6-dinitroanilino)-N-methylpropionamide ( M D M P ) applied under c a r e f u l l y c o n t r o l l e d c o n d i t i o n s (42). T h i s i n d i c a t e d t h a t b o t h t r a n s c r i p t i o n a n d t r a n s l a t i o n were necessary for the response to the h o r m o n e s . E x p e r i m e n t s u s i n g a n a n t i b o d y for P A L a n d a c D N A p r o b e for the P A L - m R N A have also s h o w n t h a t ther P A L d u r i n g the f o r m a t i o n of l i g n i n w h e n Z i n n i a m e s o p h y l l cells are i n d u c e d to f o r m x y l e m elements i n c u l t u r e ( L i n a n d N o r t h c o t e , u n p u b l i s h e d w o r k ) . T h e i n d u c t i o n of P A L a c t i v i t y b y the two g r o w t h factors can be sepa r a t e d i n t i m e so t h a t they m a y act at different sites w i t h i n the cell to b r i n g a b o u t the response (40). A u x i n a d d e d at the t i m e of s u b c u l t u r e of the tissue changes the p a t t e r n of p r o t e i n synthesis of the cells b y c h a n g i n g the t r a n s c r i p t i o n p a t t e r n of the m R N A after two hours (43). K i n e t i n does not have t h i s effect (44). Hydroxylation and Methylation. T h e p a t h w a y for the p r o d u c t i o n of the l i g n i n b u i l d i n g u n i t s involves h y d r o x y l a t i o n of the a r o m a t i c r i n g a n d m e t h y l a t i o n of the h y d r o x y l groups. It has been d e m o n s t r a t e d t h a t the m e t h y l a t i o n s are b r o u g h t a b o u t by S-adenosylmethionine:caffeic a c i d , 3 - 0 - m e t h y l transferase. T h i s transferase is m e t a specific a n d can also m e t h y l a t e 5h y d r o x y f e r u l i c a c i d a n d 3 , 4 , 5 - t r i h y d r o x y c i n n a m i c a c i d t o s i n a p i c a c i d (45). T h e i n d u c t i o n o f t h i s e n z y m e i n b e a n cultures d u r i n g d i f f e r e n t i a t i o n is c o i n cident w i t h the rise i n P A L a c t i v i t y (46). T h e h y d r o x y l a t i o n o f the a r o m a t i c r i n g o f c i n n a m i c a c i d is b r o u g h t a b o u t b y c i n n a m i c a c i d 4 - h y d r o x y l a s e , a n d a f u r t h e r h y d r o x y l a s e , p - c o u m a r i c a c i d 3 - h y d r o x y l a s e , also o c c u r s t o give caffeic a c i d (47). T h e 4 - h y d r o x y l a s e a c t i v i t y , i n some tissue, is i n d u c e d at the same t i m e as the P A L a c t i v i t y ( 4 8 , 4 9 ) . T h e r e f o r e , some c o o r d i n a t e d i n d u c t i o n of gene expression for the p r o d u c t i o n of l i g n i n precursors d u r i n g d i f f e r e n t i a t i o n is possible. T h e P A L a c t i v i t y t h a t is necessary for l i g n i n f o r m a t i o n o c c u r s i n the c y t o p l a s m or b o u n d to the c y t o p l a s m i c surface of the e n d o p l a s m i c r e t i c u l u m m e m b r a n e s . T h e c i n n a m i c a c i d p r o d u c e d is p r o b a b l y c a r r i e d o n the l i p i d surface of the m e m b r a n e s , since i t is l i p o p h i l i c , a n d i t is s e q u e n t i a l l y h y d r o x y l a t e d b y the m e m b r a n e - b o u n d h y d r o x y l a s e s ( 4 7 , 5 0 ) . I n t h i s w a y there is the p o s s i b i l i t y of at least a two-step c h a n n e l i n g route f r o m p h e n y l a l a n i n e to p - c o u m a r i c a c i d . T h e t r a n s m e t h y l a s e s t h e n direct the m e t h y l groups to the m e t a p o s i t i o n s . T h e r e is a difference between the t r a n s m e t h y lases f r o m a n g i o s p e r m s a n d those f r o m g y m n o s p e r m s , since w i t h the l a t t e r

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p r e p a r a t i o n s the e n z y m e is relative i n a c t i v e o n 5 - h y d r o x y f e r u l a t e . T h u s i t is possible t h a t these t r a n s m e t h y l a s e s m a y have some p a r t i n the c o n t r o l of the t y p e of l i g n i n f o r m e d . T h e g u a i a c y l t y p e ( 3 - m e t h o x y - 4 - h y d r o x y ) is f o u n d i n g y m n o s p e r m s a n d the s y r i n g y l - g u a i a c y l t y p e ( 3 , 5 - d i m e t h o x y - 4 h y d r o x y ) i n dicotyledons (51). Formation and Polymerization of the Building Units of Lignin. T h e acid b u i l d i n g u n i t s are reduced t o the c o r r e s p o n d i n g alcohols before p o l y m e r i z a t i o n t o l i g n i n . T h i s r e d u c t i o n occurs as a two-step process i n v o l v i n g the C o A ester o f the a c i d a n d u s i n g N A D P H as cofactor. T h e enzymes C o A ligase, c i n n a m o y l - C o A : N A D P H oxidoreductase, a n d c i n n a m y l a l c o h o l dehydrogenase are i n v o l v e d , a n d they give f i n a l l y the three b u i l d i n g u n i t s of l i g n i n : p - c o u m a r y l a l c o h o l , c o n i f e r y l a l c o h o l , a n d s i n a p y l a l c o h o l (51). V a r i o u s isoenzymes o f the C o A ligases m a y also c o n t r o l the t y p e o f l i g n i n t h a t is f o r m e d , since the isoenzymes have different affinities a n d a c t i v i t i e s for p - c o u m a r i c , ferulic a n ferent p r o p o r t i o n s i n different p l a n t s a n d i n different tissues o f the same p l a n t (51). T h e a c t i v a t i o n o f the p h e n y l p r o p i o n i c acids a n d t h e i r subsequent red u c t i o n m a y o c c u r i n vesicles t h a t fuse w i t h the p l a s m a m e m b r a n e a n d e m p t y the precursors of l i g n i n i n t o the w a l l (52). T h e N A D P H for the r e d u c t i o n is p r o v i d e d b y the pentose p h o s p h a t e p a t h w a y ( 5 3 , 5 4 ) . T h e c o n t r o l for the f i n a l steps t h a t p r o d u c e the c i n n a m y l alcohols, w h i c h are the i m m e d i a t e b u i l d i n g u n i t s of the l i g n i n , is therefore dependent o n the e n ergy s t a t u s o f the cell, since A T P is necessary for the ligase a c t i v i t y , a n d i t is also dependent o n the d i s t r i b u t i o n of c a r b o h y d r a t e m e t a b o l i s m between the pentose phosphate p a t h w a y a n d g l y c o l y s i s . It is w i t h i n the w a l l t h a t the p o l y m e r i z a t i o n process to f o r m a c o m p l e x l i g n i n cage occurs. T h e c i n n a m y l alcohols m a y reach the w a l l as the free alcohols or as /?-glucosides f o r m e d by glucosyltransferases w i t h U D P G l c (55). F o r p o l y m e r i z a t i o n , the free a l c o h o l is necessary, a n d /?-glucosidases o c c u r i n the walls of tissues t h a t are lignified (56). G l u c o s i d e s m a y be i m p o r t a n t for the t r a n s p o r t of the alcohols t o the w a l l s , b u t they are not o b l i g a t o r y for l i g n i n s y n t h e s i s . T h e y m a y act as reservoirs o f the l i g n i n precursors. T h e p r e c u r sors arise i n cells t h a t are u n d e r g o i n g l i g n i f i c a t i o n , or t h e y m a y arise f r o m n e i g h b o r i n g cells of y o u n g d i f f e r e n t i a t i n g x y l e m , w h i c h themselves are not at the stage o f m a s s i v e l i g n i f i c a t i o n (36). L i g n i f i c a t i o n is b r o u g h t a b o u t b y the o x i d a t i o n of the alcohols to y i e l d mesomeric p h e n o x y r a d i c a l s w i t h h a l f lives of a b o u t 45 sec, so t h a t r a p i d p o l y m e r i z a t i o n occurs. A t the same t i m e , linkages of these r a d i c a l s , a n d hence the l i g n i n , to c a r b o h y d r a t e c a n t a k e place ( 5 6 , 5 7 ) . It is p r o b a b l e t h a t the final o x i d a t i o n of the p h e n o l i c h y d r o x y g r o u p to give the free r a d i c a l s is b r o u g h t a b o u t b y a specific i s o e n z y m e of peroxidase t h a t occurs i n the walls of p l a n t cells (58-60). T h i s i s o e n z y m e , o n electrophoresis, is a n a n o d i c - m i g r a t i n g c o m p o n e n t . I n Zinnia elegans, the a c t i v i t y of w a l l - b o u n d peroxidase increases d u r i n g the onset o f l i g n i f i c a t i o n , a l t h o u g h the t o t a l soluble peroxidase a c t i v i t y of the cell m a y decrease ( 3 9 , 4 6 ) . A n o t h e r i s o e n z y m e of peroxidase m i g h t be r e q u i r e d t o p r o d u c e the h y d r o g e n peroxide o n w h i c h the o x i d a t i v e p o l y m e r i z a t i o n process de-

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p e n d s (61). T h e synthesis a n d a c t i v i t y of some isoenzymes of peroxidase are therefore possible c o n t r o l sites for l i g n i f i c a t i o n . Establishment of Cross Linkages in the W a l l I n the r i g i d s e c o n d a r y w a l l o f w o o d y tissue, the l i g n i n replaces the water of the g r o w i n g cell w a l l a n d f o r m s a h y d r o p h o b i c m a t r i x a r o u n d the m i crofibrils. S t r o n g h y d r o g e n b o n d s o c c u r between the p o l y s a c c h a r i d e s at the m i c r o f i b r i l l a r - m a t r i x interface a n d between c o m p o n e n t s of the m a t r i x . T h e s e , together w i t h the covalent b o n d s f o r m e d between c a r b o h y d r a t e a n d l i g n i n , m a k e the w a l l a composite i n w h i c h the l i n e a r p o l y s a c c h a r i d e p o l y mers are enclosed i n a c r o s s - l i n k e d p o l y m e r cage. T h e w a l l has great tensile s t r e n g t h because of the m i c r o f i b r i l s a n d a r i g i d s t r u c t u r e because o f the l i g nified m a t r i x (2). T h e peroxidase of the w a l l m a y also e s t a b l i s h other covalent linkages between w a l l p o l y m e r s . T h of the p r o t e i n s m a y be o x i d i z e d b y peroxidase to give cross-linkages of i s o d i t y r o s i n e between the p o l y p e p t i d e s ( 6 2 , 6 3 ) . F e r u l i c a c i d occurs i n the cell w a l l s of some herbaceous p l a n t s a n d grasses a n d t h i s m a y be o x i d i z e d t o give d i f e r u l i c a c i d ester linkages j o i n i n g p o l y s a c c h a r i d e c h a i n s ( 6 4 , 6 5 ) . T h e possible f o r m a t i o n of these covalent cross linkages between the p o l y m e r s of the p r i m a r y w a l l is believed b y some workers to l i m i t p l a n t cell w a l l e x t e n s i b i l i t y a n d have some p a r t i n the m e c h a n i s m s for the c o n t r o l of cell g r o w t h a n d e x t e n s i o n b y p l a n t g r o w t h factors (66). P r o t e i n i n the W a l l M o s t of the p r o t e i n s f o u n d i n the cell w a l l are g l y c o p r o t e i n s . T h e s e c a n be enzymes s u c h as isoenzymes of peroxidase, phosphatase a n d a m y l a s e or the h y d r o x y p r o l i n e - r i c h g l y c o p r o t e i n s (67) a n d g l y c i n e - r i c h p r o t e i n s (68). T h e h y d r o x y p r o l i n e - r i c h g l y c o p r o t e i n s m a y be classified o n the basis o f the size of t h e i r sugar p r o s t h e t i c groups. T h e soluble lectins a n d a g g l u t i n i n s a n d the i n s o l u b l e w a l l g l y c o p r o t e i n s have s m a l l oligosaccharides of arabinose ( a L - A r a f ( l -+ 3 ) - 0 - / ? - L - A r a f (1 -+ 2 ) - 0 - / ? - L - A r a f ( l - » 2 ) - 0 - / ? - L - A r a f - l H y p ) l i n k e d to the h y d r o x y p r o l i n e ( H y p ) a n d also single galactose u n i t s a t t a c h e d to serine (69-71), w h i l e the a r a b i n o g a l a c t a n p r o t e i n s are m a i n l y large m o l e c u l a r weight p o l y s a c c h a r i d e s a t t a c h e d to p r o t e i n , the r e s u l t a n t m o l e c u l e b e i n g a b o u t 8 0 - 9 0 % c a r b o h y d r a t e ( 7 2 , 7 3 ) . H y d r o x y l a t i o n of p e p t i d y l p r o l i n e o c c u r s as a p o s t - t r a n s l a t i o n a l process i n the e n d o p l a s m i c r e t i c u l u m (74-76), a n d the a d d i t i o n of the s m a l l a r a b i n o s y l oligosaccharides p r o b a b l y occurs w i t h i n the G o l g i a p p a r a t u s w i t h o u t the necessity for a s s e m b l y o n a l i p i d i n t e r m e d i a t e (77). H o w e v e r , w i t h the large m o l e c u l a r weight a r a b i n o g a l a c t a n p r o t e i n a l i p i d c a r r i e r m i g h t be i n v o l v e d , e s p e c i a l l y as r e p e a t i n g s u b u n i t s w i t h i n the a r a b i n o g a l a c t a n p o r t i o n of the m o l e c u l e have been d e t e c t e d . O l i g o s a c c h a r i d e s l i n k e d to p o l y i s o p r e n y l - p y r o p h o s p h a t e have been f o u n d to c o n t a i n arabinose (78) a n d galactose (79); the galactose was l i n k e d b y 1 —• 6, 1 —+ 4 a n d 1 —• 3 bonds a n d the arabinose was 1 —• 5 l i n k e d . T h e s e i s o p r e n y l d i p h o s p h a t e oligosaccharides were f o r m e d b y m e m b r a n e s of p e a

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cells w h e n they were i n c u b a t e d w i t h the a p p r o p r i a t e r a d i o a c t i v e U D P sugar c o m p o u n d s , a n d they c o u l d serve as precursors of the a r a b i n o g a l a c t a n glycoproteins. T h e possible i n c l u s i o n of l i p i d - l i n k e d intermediates i n the t r a n s g l y c o s y l a t i o n s i n v o l v e d i n g l y c o p r o t e i n a n d p o l y s a c c h a r i d e f o r m a t i o n provides a f u r t h e r step at w h i c h c o n t r o l of the synthase s y s t e m c a n be exercised. H o w e v e r , a l t h o u g h l i p i d intermediates are w e l l established for the f o r m a t i o n of N - l i n k e d glycoproteins a n d , i n some instances, for polysaccharides where g l y c o p r o t e i n s can f u n c t i o n as i n t e r m e d i a t e s d u r i n g the f o r m a t i o n of these polysaccharides (80-82), there is, at present, no evidence for the direct transfer f r o m l i p i d - o l i g o s a c c h a r i d e onto p o l y s a c c h a r i d e . O n e of the i m p o r t a n t consequences of the p a r t i c i p a t i o n of l i p i d - o l i g o s a c c h a r i d e i n t e r m e d i a t e s is t h a t the sequence of sugars f o r m e d on the l i p i d can be successively t r a n s ferred so t h a t a r e p e a t i n g ordered sequence of the m i x e d sugars i n the oligosaccharide c o u l d o c c u r i n the synthesized p o l y m e r (83-85). F o r m a t i o n of L i p i d a n d T r a n s p o r t to the W a l l M a n y cell walls have layers i n the outer regions of the w a l l t h a t c a r r y l i p i d m a t e r i a l . These are c u t i n , s u b e r i n , a n d waxes (67). H o w these are t r a n s p o r t e d to the outside of the cell w a l l is not k n o w n . Pores have not been f o u n d , nor has a v o l a t i l e l i p i d solvent been detected t h a t w o u l d c a r r y the l i p i d t h r o u g h the h y d r o p h i l i c w a l l . T h e f a t t y acids are synthesized i n chloroplasts or p r o p l a s t i d s a n d m o v e d into the c y t o p l a s m a n d the e n d o m e m b r a n e s y s t e m for further m o d i f i c a t i o n a n d synthesis of n e u t r a l fats, p h o s p h o l i p i d s , a n d other c o m p o u n d s (86). T h e f a t t y acids c o u l d be carried b y proteins b y a process s i m i l a r to the way i n w h i c h s e r u m a l b u m i n binds f a t t y a c i d i n the b l o o d s t r e a m of m a m m a l s . O t h e r types of l i p i d m i g h t be f o r m e d i n t o complexes analogous to l o w - d e n s i t y l i p o p r o t e i n s of the type f o u n d i n a n i m a l tissues, where the l i p i d core of the l i p o p r o t e i n is s u r r o u n d e d b y a h y d r o p h i l i c cortex m a d e up of p r o t e i n , p h o s p h o l i p i d , a n d cholesterol (87). T h i s allows the l i p i d to be m o v e d i n an aqueous e n v i r o n m e n t . T h e p r o t e i n of the l i p o p r o t e i n shell c o u l d also act as possible ligands for p a r t i c u l a r receptors at the m e m brane of the cell at w h i c h the e x p o r t occurs. T h e l i p o p r o t e i n s , i f they are present, w o u l d p r o b a b l y be formed w i t h i n the e n d o m e m b r a n e l u m e n a n d w o u l d receive the proteins at the e n d o p l a s m i c r e t i c u l u m . Summary T h i s chapter has a t t e m p t e d to suggest how the synthesis of the m a i n c o n s t i t u e n t s of the w a l l (polysaccharides, l i g n i n , p r o t e i n a n d l i p i d ) are c o n t r o l l e d d u r i n g their d e p o s i t i o n i n t o the w a l l . A hypothesis is developed for the synthesis of polysaccharides. It arises f r o m the evidence for g l u c o m a n n a n synthesis (18), a n d also for cellulose a n d callose synthesis (28). D i f ferent g l y c o s i d i c linkages are f o r m e d f r o m the same m e m b r a n e p r e p a r a t i o n under different c o n d i t i o n s . T h i s is most easily e x p l a i n e d by p o s t u l a t i n g the existence of a b i n d i n g p r o t e i n to h o l d the acceptor molecules of the

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t r a n s g l y c o s y l a s e r e a c t i o n . T h e hypothesis suggests t h a t there is a c o m ­ p l e x o f p r o t e i n s , w i t h different f u n c t i o n s , o r g a n i z e d close together a t t h e m e m b r a n e o f t h e cell. A t r a n s g l y c o s y l a s e , nucleoside d i p h o s p h a t e sugar t r a n s p o r t e r s , a n d t h e b i n d i n g p r o t e i n are a l l necessary. T h e l a t t e r p r o t e i n , since i t h o l d s the g r o w i n g p o l y s a c c h a r i d e c h a i n , c a n m o d u l a t e t h e t r a n s g l y ­ cosylase r e a c t i o n b y o r i e n t a t i n g the receptor m o l e c u l e . I n t h i s w a y t h e same transglycosylase m a y transfer t h e g l y c o s y l g r o u p t o different h y d r o x y l s o f the acceptor, e.g., t o give 1 —• 3 o r 1 —• 4 linkages. Literature Cited

1. Northcote, D. H. In Biosynthesis and Biodegradation of Wood Compo­ nents; Academic Press: London, 1985; Ch. 5. 2. Northcote, D. H. Ann. Rev. Plant Physiol. 1972, 23, 113-132. 3. Northcote, D. H. In Biosynthesis and Biodegradation of Cellulose and Cellulose Materials; 4. Bailey, R. W.; Hassid, W. Z. Proc. Natl. Acad. Sci. 1966, 56, 1586-93. 5. Dalessandro, G.; Northcote, D. H. Planta 1981, 151, 53-60. 6. Dalessandro, G.; Northcote, D. H. Planta 1981, 151, 61-67. 7. Timell, T. E. Adv. Carbohydr. Chem. 1964, 19, 247-302. 8. Timell, T. E. Adv. Carbohydr. Chem. 1965, 20, 409-483. 9. O'Neill, Μ. Α.; Selvendran, R. R. Carb. Res. 1983, 111, 239-255. 10. Aspinall, G. O.; Molloy, J. Α.; Craig, J. W. T. Can. J. Biochem. Phys­ iol. 1969, 47, 1063-70. 11. Bolwell, G. P.; Northcote, D. H. Planta 1981, 152, 225-33. 12. Bolwell, G. P.; Dalessandro, G.; Northcote, D. H. Phytochem. 1985, 24, 699-702. 13. Bolwell, G. P.; Northcote, D. H. Planta 1984, 162, 139-46. 14. Dalessandro, G.; Northcote, D. H. Biochem. J. 1977, 162, 139-46. 15. Bolwell, G. P.; Northcote, D. H. Biochem. J. 1983, 210, 497-507. 16. Ramsden, L.; Northcote, D. H. Phytochem. 1987, 26, 2679-83. 17. Dalessandro, G.; Piro, G.; Northcote, D. H. Planta 1986, 169, 564-74. 18. Dalessandro, G.; Piro, G.; Northcote, D. H. Planta 1988, 175, 60-70. 19. Watkins, W. M. Carbohydr. Res. 1986, 149, 1-12. 20. Steitz, Τ. Α.; Shoham, M.; Bennett, W. S. Phil. Trans. R. Soc. London 1981, B293, 43-52. 21. Matsura, Y.; Kunsunoki, M.; Harada, W.; Kakudo, M. J. Biochem. 1984, 95, 697-702. 22. Quiocho, F. A. Ann. Rev. Biochem. 1986, 55, 287-315. 23. Waldron, K. W.; Brett, C. T. Biochem. J. 1983, 213, 115-22. 24. Waldron, K. W.; Brett C. T. In Biochemistry of Plant Cell Walls; SEB Seminar Series 1985; 28, 79-97. 25. Ray, P. M. Biochim. Biophys. Acta 1980, 629, 431-44. 26. Hayashi, T.; Matsuda, K. J. Biol. Chem. 1981, 256, 11117-22. 27. Hayashi, T.; Matsuda, K. Plant and Cell Physiol. 1981, 22, 1571-84. 28. Jacob, S. R.; Northcote, D. H. J. Cell Sci. 1985 Suppl.2, 1-11. 29. Campbell, R. E.; Brett, C. T.; Hillman, J. R. Biochem. J. 1988, in press.

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66. Fry, S. C. Ann. Rev. Pl. Physiol. 1986, 37, 165-86. 67. Gould, J.; Northcote, D. H. In Bacterial Adhesion: Mechanisms and Physiological Significance; Plenum: New York, 1984; 89-110. 68. Cassab, G. I.; Varner, J. E. Nature 1986, 323, 110. 69. Lamport, D. T. A. In The Biochemistry of Plants: Carbohydrates; Aca­ demic Press: London, 1980; 3, 501-41. 70. Allen, A. K.; Dsai, Ν. N.; Neuberger, Α.; Creeth, J. M. Biochem. J. 1978, 171, 665-74. 71. Muray, R. Η. Α.; Northcote, D. H. Phytochem. 1978, 17, 623-9. 72. Fincher, G. B.; Stone, Β. Α.; Clarke, A. E. Ann. Rev. Pl. Physiol. 1983, 34, 47-70. 73. Cassab, G. I. Planta 1986, 168, 441-46. 74. Wienecke, K.; Glas, R.; Robinson, D. G. Planta 1982, 155, 58-63. 75. Blankenstein, P.; Lang, W. C.; Robinson, D. G. Planta 1986, 169, 238-44. 76. Sauer, Α.; Robinson 77. Owens, R. J.; Northcote, D. H. Biochem. J. 1981, 195, 661-7. 78. Hayashi, T.; Maclachlan, G. Biochem. J. 1984, 217, 791-803. 79. Hayashi, T.; Maclachlan, G. Phytochem. 1984, 23, 487-92. 80. Green, J. R.; Northcote, D. H. Biochem. J. 1978, 170, 599-608. 81. Green, J. R.; Northcote, D. H. Biochem. J. 1979, 178, 661-71. 82. Green, J. R.; Northcote, D. H. J. Cell Sci. 1979, 40, 235-44. 83. Ielpi, L.; Couso, R.; Dankert, R. FEBS Lett. 1981, 130, 253-56. 84. Ielpi, L.; Couso, R.; Dankert, R. Biochem. Biophys. Res. Comm. 1981, 102, 1400-8. 85. Couso, R. O.; Ielpi, L.; Garcia, R. C.; Dankert, R. Eur. J. Biochem. 1982, 123, 617-27. 86. Stumpf, P. K.; Shimakata, T. In Biosynthesis and Function of Plant Lipids Proc 6 University of California; Amer. Soc. Pl. Physiologists: Maryland, 1983; 1-27. 87. Nilsson-Ehle, P.; Garfinkel, A. S.; Schotz, M. C. Ann. Rev. Biochem. 1980, 49, 667-92. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 2

Formation and Functions of Xyloglucan and Derivatives David A . Brummell and Gordon A . Maclachlan Biology Department, M c G i l l University, 1205 Avenue D r Penfield, Montreal, Quebec H3A 1B1, Canada

Xyloglucan is a matrix polysaccharide which in primary cell walls associates with cellulose microfibrils by hydro­ gen bonding to form a relatively rigid structure. Its syn­ thesis by Golgi membranes requires the activity of at least four enzymes: 1,4-β-glucosyl-, 1,6-α-xylosyl-, 1,2β-galactosyl-, and 1,2-α-fucosyl-transferases. Continued chain elongation by the β-glucosyltransferase is depen­ dent upon concurrent α-xylosyl transfer. The xyloglu­ can backbone is substituted with galactosyl and fuco­ syl residues at very specific xylosyl side groups to com­ plete a polysaccharide with regular alternating subunits of Glc ·Xyl and Glc ·Xyl ·Gal·Fuc. Auxin treatment of young dicot tissues induces both growth and the produc­ tion of an endo-1,4-β-glucanase, the main substrate of which appears to be cell wall xyloglucan. Auxin evokes a net increase in xyloglucan levels, but depolymerization and turnover also take place during growth. Although oligosaccharide products of xyloglucan hydrolysis inhibit auxin-stimulated growth, at the same time they stimu­ late endo-1,4-β-glucanase activity versus xyloglucan in vitro. 4

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T h e p r i m a r y cell walls o f g r o w i n g tissues i n higher p l a n t s are c o m p o s e d o f h i g h l y h y d r a t e d p o l y m e r i c m a t e r i a l s consisting m a i n l y o f c o m p l e x p o l y s a c ­ charides, w i t h smaller a m o u n t s o f g l y c o p r o t e i n . G e n e r a l l y 2 0 - 3 0 % o f t h e d r y weight o f the w a l l is cellulose, w i t h the balance o f the p o l y s a c c h a r i d e s b e i n g m a d e u p o f pectins a n d hemicelluloses. I n dicots, t h e p r e d o m i n a n t hemicellulose is x y l o g l u c a n , w h i c h forms about 2 0 % o f the w a l l (1). I n t h e t y p i c a l g r o w i n g region o f p e a stems, for e x a m p l e , t h e r a t i o o f x y l o g l u c a n to cellulose is about 0 . 7 : 1 o n a weight basis (2). I n m o n o c o t s , t h e m a i n

0097-6156/89/0399-0018$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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BRUMMELL & MACLACHLAN

Xyloglucan and Derivatives

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hemicelluloses are glucans a n d a r a b i n o x y l a n s , w i t h x y l o g l u c a n a p p a r e n t l y f o r m i n g o n l y a m i n o r f r a c t i o n of the w a l l . However, i n m o n o c o t s the g l u c a n b a c k b o n e of x y l o g l u c a n is m u c h less s u b s t i t u t e d t h a n i n d i c o t s (3), w h i c h m a y result i n t h e i r u n d e r - e s t i m a t i o n . A r a b i n o x y l a n s i n m o n o c o t s appear t o e x h i b i t m a n y of the same p r o p e r t i e s as do x y l o g l u c a n s i n d i c o t s ( 4 , 5 ) . T h e e x t r a p r o t o p l a s m i c w a l l defines the cell s h a p e , a n d m e t a b o l i c r e g u l a t i o n of cell w a l l s t r u c t u r e is believed t o c o n t r o l the d i r e c t i o n a n d rate of cell e x p a n s i o n . X y l o g l u c a n , u n l i k e cellulose, is c l e a r l y subject to t u r n o v e r d u r i n g g r o w t h . Decreases i n x y l o g l u c a n average m o l e c u l a r weight d u r i n g development have been n o t e d , p a r t i c u l a r l y after a u x i n t r e a t m e n t , as w e l l as the release o f s m a l l q u a n t i t i e s of s o l u b i l i z e d m a t e r i a l i n c l u d i n g x y l o g l u c a n oligosaccharide s u b u n i t s of c h a r a c t e r i s t i c s t r u c t u r e (6). T h i s review examines the synthesis of x y l o g l u c a n a n d i t s f u n c t i o n s i n the cell w a l l s of y o u n g g r o w i n g regions of d i c o t stems. T h e roles i n g r o w t h o f x y l o g l u c a n t u r n o v e r a n d of oligosaccharide fragments d e r i v e d b y p a r t i a l h y d r o l y s i s of x y l o g l u c a n are also considered

Structure of X y l o g l u c a n M a t u r e x y l o g l u c a n s o f dicot p l a n t s possess a v e r y regular s t r u c t u r e c o m posed of a l , 4 - / ? - D - g l u c a n b a c k b o n e w i t h D - x y l o s y l residues 1 , 6 - a - l i n k e d to three ( i n g r o w i n g tissue) or a n average of t w o ( i n seeds) o u t of four s e q u e n t i a l g l u c o s y l residues ( 1 , 7 ) . T h e a d d i t i o n either of L - f u c o s y l - 1 , 2 - a D-galactose ( i n g r o w i n g tissue) or D-galactose alone ( i n seeds) l i n k e d 1,2-/? to specific x y l o s y l residues further extends the side chains at selected i n tervals ( F i g . 1). I n some species t e r m i n a l L - a r a b i n o s y l residues are f o u n d i n s t e a d of fucose (1). X y l o g l u c a n f r o m g r o w i n g m o n o c o t tissue is less s u b s t i t u t e d w i t h x y l o s e a n d galactose t h a n i n dicots (3) a n d a p p e a r s to lack fucose (8), whereas t h a t f r o m g y m n o s p e r m s resembles d i c o t x y l o g l u c a n (9). T h e s t r u c t u r e of x y l o g l u c a n has been d e t e r m i n e d t h r o u g h e x a m i n a t i o n of d i g e s t i o n p r o d u c t s after h y d r o l y s i s w i t h specific e n d o - l , 4 - / ? - g l u c a n a s e s a n d a n a l y s i s of the linkages present i n the r e s u l t i n g fragments after m e t h y l a t i o n . P a r t i a l h y d r o l y s i s of p e a x y l o g l u c a n ( F i g . 1) results i n a n o n a s a c c h a ride ( G l c X y l G a l F u c ) a n d a heptasaccharide (GIC4XVI3) (2). I d e n t i c a l s t r u c t u r e s have been d e t e r m i n e d for sycamore (10), soybean (11), a n d m u n g b e a n (12) x y l o g l u c a n . I n m u n g b e a n s m a l l a m o u n t s of a decasaccharide ( G l c 4 - X y l - G a l 2 - F u c ) were also f o u n d . 4

3

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W h e n x y l o g l u c a n f r o m p e a e p i c o t y l cell w a l l s was h y d r o l y z e d in vitro w i t h p u r i f i e d e n d o - l , 4 - / ? - g l u c a n a s e f r o m Streptomyces, r e d u c i n g sugar was p r o d u c e d at a l i n e a r rate for at least 48 h (2). F r a c t i o n a t i o n o f the h y d r o l y s a t e o n B i o - G e l P 6 c o l u m n s showed t h a t endo-1,4-/?-glucanase cleaved the l , 4 - / ? - g l u c a n b a c k b o n e at u n s u b s t i t u t e d g l u c o s y l residues t o generate e q u a l a m o u n t s of heptasaccharide a n d nonasaccharide ( F i g . 1). T h e s e u n i t s a c c u m u l a t e d w i t h t i m e of h y d r o l y s i s u n t i l they were essentially the o n l y end p r o d u c t s . T r a n s i e n t a m o u n t s of higher m o l e c u l a r weight p r o d u c t s were also f o u n d , i d e n t i f i e d as d i m e r s of the r e s u l t i n g oligosaccharides. These were m a i n l y c o m p o s e d of the 16 saccharide u n i t ( a n o n a s a c c h a r i d e p l u s a

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L POLYMERS

h e p t a s a c c h a r i d e , F i g . 1), w i t h smaller a m o u n t s of 18 (two n o n a - ) a n d 14 (two hepta-) saccharides. Since the nonasaccharide a n d the heptasaccharide were present i n a p p r o x i m a t e l y a 1:1 m o l a r r a t i o a n d the m a i n d i m e r c o n t a i n e d the two oligosaccharides, i t appears t h a t p e a x y l o g l u c a n is c o m p o s e d of these two s u b u n i t s a r r a n g e d p r i m a r i l y i n a l t e r n a t i n g sequence. However, the presence of the 14 a n d 18 saccharide dimers showed t h a t t h i s d i s t r i b u t i o n is not perfect. T h e m o l e c u l a r weight of x y l o g l u c a n varies w i t h species a n d stage of g r o w t h . I n y o u n g cell w a l l s , x y l o g l u c a n has a m o l e c u l a r weight of 250 k D to 350 k D , w h i c h represents a degree of p o l y m e r i z a t i o n ( D P ) of the g l u c a n b a c k b o n e of 800 to 1200, suggesting t h a t 100 or m o r e of each oligosaccharide s u b u n i t are present (2). T r e a t m e n t of x y l o g l u c a n w i t h f u n g a l m i x e d endo- a n d exo-glycosidase p r e p a r a t i o n s , such as f r o m Aspergillus, degraded the molecule to m o n o s a c charides a n d isoprimeverose ( 6 - O - a - D - x y l o p y r a n o s y l - D - g l u c o p y r a n o s e ) (13, 14). T h e presence of t h i teristic of x y l o g l u c a n , a n d the i n c o r p o r a t i o n of U D P - [ C ] x y l o s e i n t o isoprimeverose has been used as a n u n a m b i g u o u s assay for x y l o g l u c a n b i o s y n thesis (14). 1 4

Biosynthesis of the X y l o g l u c a n

Backbone

X y l o g l u c a n , like the other non-cellulosic w a l l polysaccharides, is s y n t h e s i z e d a n d secreted b y m e m b r a n e s of the G o l g i a p p a r a t u s . T h e transferases res p o n s i b l e for x y l o g l u c a n biosynthesis co-sedimented w i t h m a r k e r s for G o l g i membranes i n density gradients ( 1 3 , 1 5 ) , a n d i m m u n o g o l d l o c a l i z a t i o n s t u d ies u s i n g a p o l y c l o n a l a n t i b o d y to x y l o g l u c a n f o u n d the p o l y s a c c h a r i d e to be present i n G o l g i cisternae a n d vesicles (16). N o l a b e l was seen over the e n d o p l a s m i c r e t i c u l u m , i n d i c a t i n g t h a t synthesis of x y l o g l u c a n occurs i n the G o l g i . T h e very regular s t r u c t u r e of the final p o l y m e r i m p l i e s a precise c o o r d i n a t i o n of the enzymes responsible for x y l o g l u c a n biosynthesis. T h e glucose-xylose backbone of x y l o g l u c a n is synthesized b y c o o r d i n a t e d a c t i v i t y of a specific glucosyltransferase a n d a x y l o s y l t r a n s f e r a s e w h i c h a p p e a r to f u n c t i o n i n d e p e n d e n t l y to o n l y a very l i m i t e d e x t e n t . E a r l y studies o n pea membranes suggested t h a t some g l u c o s y l transfer c o u l d o c c u r i n the absence of x y l o s y l transfer, a n d i t was concluded t h a t x y l o s y l transfer t o o k place o n t o a preformed g l u c a n c h a i n (13). However, w i t h m e m b r a n e s f r o m soybean c u l t u r e d cells, i n c o r p o r a t i o n of glucose f r o m U D P - g l u c o s e i n t o x y l o g l u c a n was almost completely dependent o n the c o n c e n t r a t i o n of U D P - x y l o s e i n the i n c u b a t i o n m i x t u r e (14). Conversely, m e m b r a n e s i n c o r p o r a t e d o n l y a s m a l l a m o u n t of l a b e l l e d m a t e r i a l w h e n i n c u b a t e d w i t h U D P - [ C ] x y l o s e alone, b u t s u b s t a n t i a l i n c o r p o r a t i o n ensued i n the presence of U D P - g l u c o s e a n d U D P - [ C ] x y l o s e together (14). T h e p r o d u c t s f o r m e d in vitro were composed m a i n l y of two s u b u n i t s , a pentasaccharide ( G l c 3 - X y l ) a n d a heptasaccharide ( G l c 4 - X y l ) . Pulse-chase e x p e r i m e n t s i n d i c a t e d t h a t the pentasaccharide was converted to the heptasaccharide, a n d t h a t t h i s process was dependent u p o n a n d regulated b y the c o n c e n t r a t i o n of U D P - x y l o s e (17). 1 4

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BRUMMELL & MACLACHLAN

R e c e n t l y , the m o l e c u l a r size of w a t e r - i n s o l u b l e 1,4-/?-linked p o l y s a c charides n e w l y s y n t h e s i z e d b y p e a m e m b r a n e s f r o m U D P - [ C ] g l u c o s e has been d e t e r m i n e d (18). R e a c t i o n p r o d u c t s were first e x h a u s t i v e l y h y d r o l y z e d w i t h p u r i f i e d 1,3-/?-glucanase t o remove 1,3-/?-linked p r o d u c t s . T h e 1,4/?-linked p o l y m e r s so o b t a i n e d were dissolved i n a n h y d r o u s p a r a f o r m a l d e h y d e : d i m e t h y l s u l f o x i d e ( 4 0 - 4 5 % , w / v ) a n d f r a c t i o n a t e d by e x c l u s i o n c h r o m a t o g r a p h y o n c o l u m n s of c o n t r o l l e d pore glass beads. T h e 1,4-/?-linked p r o d u c t s f o r m e d f r o m 1 m M U D P - [ C ] g l u c o s e alone d i s p l a y e d a peak of m o l e c u l a r size equivalent to the e l u t i o n v o l u m e of c o m m e r c i a l a - d e x t r a n w i t h a m o l e c u l a r weight of 70 k D ( F i g . 2). T h i s p r o d u c t c o n t a i n e d o n l y a few [ C ] g l u c o s e u n i t s added w i t h 1,4-/?-linkages to the n o n - r e d u c i n g end of an u n k n o w n endogenous acceptor. It was c l e a r l y u n a b l e to elongate f u r t h e r i n t h i s r e a c t i o n s y s t e m since its size r e m a i n e d the same even i n the presence of h i g h e r s u b s t r a t e c o n c e n t r a t i o n s a n d after longer i n c u b a t i o n p e r i o d s (19). 1 4

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H o w e v e r , w h e n U D P - x y l o s e was present i n the r e a c t i o n m e d i u m i n a d d i t i o n to U D P - [ C ] g l u c o s e p e r i o d a n d the e l u t i o n peak f r o m glass b e a d c o l u m n s increased i n a p p a r e n t size w i t h t i m e to a p p r o a c h t h a t of a u t h e n t i c x y l o g l u c a n ( F i g . 2, cf. 20). E n z y m i c h y d r o l y s i s confirmed t h a t the p r o d u c t c o n t a i n e d isoprimeverose, thereby e s t a b l i s h i n g u n a m b i g u o u s l y t h a t a x y l o g l u c a n b a c k b o n e h a d been f o r m e d . Pulse-chase e x p e r i m e n t s p r o v i d e d u n e q u i v o c a l evidence t h a t the t r u n c a t e d p r o d u c t f o r m e d f r o m U D P - g l u c o s e alone was a precursor for the x y l o g l u c a n f o r m e d w h e n U D P - x y l o s e was also present (18). T h e s e s t u d ies suggested t h a t f o r m a t i o n of the g l u c a n b a c k b o n e of x y l o g l u c a n was res t r i c t e d i n the absence of x y l o s y l transfer, a n d t h a t b o t h glucose a n d x y l o s e must be i n c o r p o r a t e d i n a concerted m a n n e r for the c o m p l e t e x y l o g l u c a n b a c k b o n e to f o r m . S y n t h e s i s of the glucose-xylose b a c k b o n e in vitro d i d n o t require the presence of U D P - g a l a c t o s e or G D P - f u c o s e , a n d the g l u c o s y l - a n d xylosyltransferases appeared to act together i n d e p e n d e n t l y of the a d d i t i o n of t e r m i n a l sugars. 1 4

A d d i t i o n s to t h e X y l o g l u c a n

Backbone

Pea membrane preparations incorporated [ C]fucose from G D P - [ C ] f u cose i n t o large pre-made p r i m e r chains w h i c h d i d not u n d e r g o detectable e l o n g a t i o n w i t h t i m e (20). I n c o r p o r a t i o n of fucose o c c u r r e d o n l y o n t o h i g h m o l e c u l a r weight (up to 300 k D ) acceptors, even w i t h very short i n c u b a t i o n t i m e s ( F i g . 3), a n d these p r o d u c t s h y d r o l y z e d to the n o n a s a c c h a r i d e s u b u n i t of x y l o g l u c a n . T h e transfer of fucose f r o m G D P - [ C ] f u c o s e to these p r o d ucts was p r o m o t e d by, b u t not dependent o n , the presence of U D P - g l u c o s e , U D P - x y l o s e or U D P - g a l a c t o s e i n the r e a c t i o n m i x t u r e . T h i s suggests t h a t fucosyl transfer is i n d e p e n d e n t of glucose a n d x y l o s e transfer ( a n d t h u s c h a i n e l o n g a t i o n ) , a n d t h a t the pre-made p r i m e r s were a l r e a d y p a r t i a l l y g a l a c t o s y l a t e d . Nevertheless, w h e n d o u b l e - l a b e l l i n g e x p e r i m e n t s were p e r f o r m e d i n the presence of a l l four nucleotide sugars needed for x y l o g l u c a n biosynthesis, i n c l u d i n g U D P - [ H ] x y l o s e and G D P - [ C ] f u c o s e , nonasaccharide c o n t a i n i n g b o t h labels c o u l d be i s o l a t e d f r o m the r e a c t i o n p r o d u c t s (20). T h i s i n d i c a t e s t h a t the x y l o g l u c a n b a c k b o n e can be decorated w i t h galactose a n d fucose even w h i l e e l o n g a t i o n occurs. 14

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PLANT C E L L W A L L

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POLYMERS

Go

I

X

X

I

X

I

X

I

I

-G-G-G-G-G-G-G-G-I

I

I

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X

heptasacch.

-Ίοο

nonasacch.

endo. 1,4-ye-glucanase

F i g u r e 1. R e p e a t i n g s u b u n i t s o f p e a e p i c o t y l x y l o g l u c a n . T h e m o l e c u l e is composed of a nonasaccharide (Glc4-Xyl3-Gal-Fuc) a n d a heptasaccharide (Glc4-Xyl3) arranged p r i m a r i l d i m e r is repeated about 100 t i m e s i n t h e average molecule. A r r o w s s h o w where h y d r o l y s i s b y p u r i f i e d p e a o r f u n g a l endo-1,4-/?-glucanase o c c u r s (2). I d e n t i c a l s t r u c t u r e s have been d e t e r m i n e d for sycamore (10), soybean (11) a n d m u n g b e a n (12) x y l o g l u c a n .

264

I

2000 2 &

1500

>

1000 -

1

500

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70

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-β

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40

50

60

70

FRACTION NUMBER F i g u r e 2. E l u t i o n profile f r o m c o l u m n s (100 x 1.0 c m ) o f c o n t r o l l e d p o r e glass beads o f 1,4-/?-linked p r o d u c t s f o r m e d in vitro b y p e a m e m b r a n e s i n 30 m i n . P r o d u c t s were dissolved i n h o t p a r a f o r m a l d e h y d e : D M S O a n d e l u t e d w i t h D M S O i n 1 m l f r a c t i o n s . O p e n circles, 1 m M U D P - [ C ] g l u c o s e alone; closed circles, 1 m M U D P - [ C ] g l u c o s e p l u s 50 μ Μ U D P - x y l o s e . Size m a r k e r s show the m o l e c u l a r weight o f peak e l u t i o n volumes o f s t a n d a r d d e x t r a n s , 264 = 264000 D ; 70 = 70000 D . ( T a k e n w i t h p e r m i s s i o n f r o m Ref. 18. © 1 9 8 8 J . W i l e y k Sons.) 1 4

1 4

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Xyloglucan and Derivatives

Fucosyltransferase i n p e a m i c r o s o m a l p r e p a r a t i o n s also f u c o s y l a t e d e x o g e n o u s l y - a d d e d x y l o g l u c a n acceptors i n a d d i t i o n to endogenous p r i m e r s (21). E x t r a c t s of p e a cell w a l l x y l o g l u c a n were f o u n d to be a p o o r f u c o s y l acceptor, p r e s u m a b l y because they were a l m o s t c o m p l e t e l y f u c o s y l a t e d a l ready. T a m a r i n d seed x y l o g l u c a n , however, w h i c h is g a l a c t o s y l a t e d b u t c o n t a i n s no fucose, s t r o n g l y p r o m o t e d fucosyl transfer f r o m G D P - [ C ] f u c o s e , g i v i n g rise to t y p i c a l x y l o g l u c a n nonasaccharides u p o n endoglucanase digest i o n . X y l o g l u c a n oligosaccharides u p to the o c t a s a c c h a r i d e d i d n o t act as f u c o s y l acceptors b u t i n s t e a d i n h i b i t e d f u c o s y l transfer to b o t h endogenous a n d exogenous acceptors, s h o w i n g t h a t the a c t i v e site of the f u c o s y l t r a n s ferase recognized a fragment longer t h a n the g a l a c t o s y l a t e d o c t a s a c c h a r i d e . T h i s w o r k confirmed t h a t f u c o s y l a t i o n of x y l o g l u c a n proceeds i n d e p e n d e n t l y of the e l a b o r a t i o n of the glucose-xylose b a c k b o n e . N o t e , however, t h a t complete in vitro synthesis of the x y l o g l u c a n m o l e c u l e f r o m the four n u c l e o t i d e sugars w i t h o u t the i n v o l v e m e n t of p r e - m a d e p r i m e r s has so far not been achieved. 1 4

Localization of X y l o g l u c a n in Cell Walls X y l o g l u c a n is believed to p l a y a key role i n the a r c h i t e c t u r e of d i c o t cell walls b y l i n k i n g together cellulose m i c r o f i b r i l s a n d p o s s i b l y other c o m p o nents of the w a l l i n order to achieve s t r u c t u r a l r i g i d i t y (1). M o s t cell w a l l c o m p o n e n t s can be s o l u b i l i z e d b y hot w a t e r , c h e l a t i n g agents or d i l u t e a l k a l i ( 4 % K O H ) . H o w e v e r , the a s s o c i a t i o n between x y l o g l u c a n a n d cellulose is so s t r o n g t h a t it requires concentrated a l k a l i ( 2 4 % K O H ) to dissolve x y l o g l u can f r o m the x y l o g l u c a n - c e l l u l o s e m a c r o m o l e c u l a r c o m p l e x (2). X y l o g l u c a n b i n d s to cellulose in vitro i n a p H - d e p e n d e n t m a n n e r (22), suggesting t h a t the p o l y m e r s are associated by h y d r o g e n bonds. T h e 1,4-/?-linkage of the g l u c a n b a c k b o n e of x y l o g l u c a n creates a l i n e a r m o l e c u l e , a n d the g a l a c t o s y l fucose side c h a i n m a y c u r l a r o u n d one side of the b a c k b o n e , t h u s a l l o w i n g u n i m p e d e d h y d r o g e n b o n d i n g between the other side of the b a c k b o n e a n d cellulose m i c r o f i b r i l s (10). T h e presence of the galactosyl-fucose side c h a i n m a y also prevent f u r t h e r h y d r o g e n b o n d i n g of x y l o g l u c a n w i t h other x y l o g l u c a n molecules, c r e a t i n g cellulose m i c r o f i b r i l s coated w i t h a single layer of x y l o g l u c a n (10). E n z y m i c a n d c h e m i c a l f r a c t i o n a t i o n of cell w a l l s f r o m s y c a m o r e (Acer) a n d Rosa i n d i c a t e d h y d r o g e n b o n d i n g between x y l o g l u c a n a n d cellulose in vivo, a n d i m p l i e d the presence of g l y c o s i d i c linkages b e tween x y l o g l u c a n a n d pectic p o l y m e r s ( 1 0 , 2 3 ) . Subsequent w o r k , however, suggested t h a t the b o n d i n g between x y l o g l u c a n a n d p e c t i n s was p r i n c i p a l l y noncovalent (24). A u t o r a d i o g r a p h y of cell walls f r o m l i v i n g tissue p r e v i o u s l y i n c u b a t e d w i t h [ H]fucose (2) a n d i m m u n o g o l d l o c a l i z a t i o n u s i n g p o l y c l o n a l a n t i b o d ies to x y l o g l u c a n (16) suggested t h a t x y l o g l u c a n was d i s t r i b u t e d t h r o u g h o u t the c e l l u l o s e - c o n t a i n i n g p a r t of the w a l l , a n d was absent o n l y i n the m i d d l e l a m e l l a (16). W h e n cell w a l l "ghosts" (the cell w a l l x y l o g l u c a n - c e l l u l o s e c o m p l e x , w h i c h r e t a i n e d the shape of the cell) were p r e p a r e d b y e x t r a c t i o n of p e a s t e m tissue w i t h hot 7 0 % e t h a n o l , 0.1 M E D T A a n d 4% K O H , the 3

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PLANT C E L L W A L L POLYMERS

residues c o n t a i n e d o n l y x y l o g l u c a n a n d cellulose (2). T r e a t m e n t o f these "ghosts" w i t h a fluorescent fucose-binding l e c t i n clearly showed x y l o g l u c a n d i s t r i b u t e d over the entire surface ( F i g . 4). E l e c t r o n m i c r o s c o p i c a l observations of shadowed "ghost" p r e p a r a t i o n s showed x y l o g l u c a n to be d i s t r i b u t e d b o t h o n a n d between cellulose m i crofibrils (2). E v e n i n the cell walls of m a t u r i n g tissues, as the content of x y l o g l u c a n i n the w a l l s u b s t a n t i a l l y declined, cellulose was s t i l l almost c o m p l e t e l y coated w i t h x y l o g l u c a n (22). T h u s , x y l o g l u c a n exists in situ i n i n t i m a t e a s s o c i a t i o n w i t h cellulose t h r o u g h o u t the w a l l at a l l stages of growth. M e t a b o l i s m of X y l o g l u c a n Structural Function of Xyloglucan. T h e cell w a l l has sufficient r i g i d i t y to counteract the forces of t u r g o r generated b y the p r o t o p l a s m , a n d b r e a k d o w n of key components m a cellulose m i c r o f i b r i l s to move relative to one another a n d the cell to exp a n d . T h a t cell w a l l " g h o s t s " , essentially consisting o n l y of x y l o g l u c a n a n d cellulose, r e t a i n e d the shape of the cell suggests t h a t x y l o g l u c a n not o n l y coats i n d i v i d u a l cellulose m i c r o f i b r i l s b u t also b o n d s the m i c r o f i b r i l s together (2). E n d o h y d r o l y s i s of x y l o g l u c a n m a y be essential i n order to p e r m i t the movement of cellulose m i c r o f i b r i l s relative to one another, a n d consequently cell w a l l extension. A requirement for exposed x y l o g l u c a n i n g r o w t h has been d e m o n s t r a t e d i n studies u s i n g f u c o s e - b i n d i n g lectins (25). T h e lectins b o u n d to the cell w a l l of a z u k i bean e p i c o t y l segments, a n d i n h i b i t e d b o t h a u x i n - i n d u c e d cell w a l l loosening a n d g r o w t h , p r e s u m a b l y by protecting xyloglucan from turnover. Deposition of Xyloglucan. I n dicot s t e m segments, a u x i n r a p i d l y (after a b o u t 1 h) p r o m o t e d a n increase i n the rate of d e p o s i t i o n of cellulosic a n d non-cellulosic cell w a l l m a t e r i a l s , even i f g r o w t h were i n h i b i t e d ( 2 6 , 2 7 ) . I n p e a e p i c o t y l apices, a u x i n t r e a t m e n t for 48 h r e s u l t e d i n a s m a l l increase i n cellulose content b u t more t h a n a d o u b l i n g of x y l o g l u c a n as the cells e x p a n d e d ( T a b l e I). T h e synthesis a n d i n c o r p o r a t i o n of x y l o g l u c a n i n t o the w a l l m a y t h u s be h o r m o n a l l y c o n t r o l l e d . T h e a c t i v i t y of " x y l o g l u c a n s y n t h a s e " ( x y l o g l u c a n xylosyltransferase was a c t u a l l y measured) declined i n the g r o w i n g region of the s t e m of whole p e a p l a n t s as t h i s zone m a t u r e d , b u t t r e a t m e n t of the p l a n t s w i t h a u x i n prevented the decline (28). A u x i n t r e a t m e n t of d e c a p i t a t e d whole plants a n d o f isolated s t e m segments also m a i n t a i n e d the a c t i v i t y of ^ - g l u c a n synthase ( 2 9 , 3 0 ) , a n e n z y m e w h i c h m a y be responsible for the synthesis of the 1,4-/?-glucan b a c k b o n e of x y l o g l u c a n (18). Degradation of Xyloglucan. I n a d d i t i o n to p r o m o t i n g x y l o g l u c a n d e p o s i t i o n , a u x i n t r e a t m e n t brings about a massive i n d u c t i o n (up to 30-fold after 72 h) of e n d o - l , 4 - / ? - g l u c a n a s e a c t i v i t y (31). C o n c o m i t a n t w i t h t h i s i n d u c t i o n , a m a r k e d decline i n the average m o l e c u l a r weight of x y l o g l u c a n , b u t not cellulose, was observed (Table I). These changes o c c u r r e d despite the increases i n net deposits of x y l o g l u c a n a n d cellulose. In vitro i n c u b a t i o n

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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BRUMMELL & MACLACHLAN

V

I

0

I 23

4

25

Glc

1—ι—ι—ι

I

F i g u r e 3. G e l filtration of a l k a l i - s o l u b l e x y l o g l u c a n o n c o l u m n s (95 χ 1.5 c m ) of Sepharose C L - 6 B . P e a m i c r o s o m a l m e m b r a n e s were i n c u b a t e d for v a r i o u s p e r i o d s w i t h G D P - [ C ] f u c o s e a n d u n l a b e l l e d sugar nucleotides. P r o d u c t s were e l u t e d w i t h 0.1 M N a O H i n 1 m l f r a c t i o n s . M o l e c u l a r weights of d e x t r a n m a r k e r s , 1 = 264000 D ; 2 = 70000 D ; 3 = 40000 D ; 4 = 10600 D ; G l c = g l u c o s e . R e d r a w n f r o m C a m i r a n d a n d M a c l a c h l a n (20). 1 4

F i g u r e 4. P e a s t e m m a t e r i a l was s e q u e n t i a l l y e x t r a c t e d w i t h h o t 7 0 % e t h a n o l , 0.1 M E D T A a n d 4 % K O H - 0 . 1 % N a B H to leave x y l o g l u c a n cellulose cell w a l l " g h o s t s " . B i n d i n g of fluorescent f u c o s e - b i n d i n g l e c t i n f r o m Ulex europeus as v i s u a l i z e d b y fluorescence m i c r o s c o p y shows x y l o g l u ­ c a n d i s t r i b u t e d over the w h o l e w a l l surface. P h o t o g r a p h courtesy of D r . T . Hayashi. 4

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of p e a cell w a l l "ghosts" w i t h purified endo-1,4-/?-glucanase d r a m a t i c a l l y increased the n u m b e r of r e s i d u a l r e d u c i n g ends a n d b r o u g h t a b o u t s o l u b i l i z a t i o n of x y l o g l u c a n before any h y d r o l y s i s of cellulose took place (32). T h e l o c a l i z a t i o n of x y l o g l u c a n i n the w a l l , where cellulose m i c r o f i b r i l s are sheathed b y a layer of x y l o g l u c a n , m a y m a k e x y l o g l u c a n m o r e accessible to the e n z y m e t h a n is cellulose. T h i s s t u d y confirmed t h a t i n cell walls a u x i n i n d u c e d endo-1,4-/?-glucanase ( c o m m o n l y called " c e l l u l a s e " ) p r e f e r e n t i a l l y h y d r o l y z e s x y l o g l u c a n over cellulose. T a b l e I. Effects of a u x i n t r e a t m e n t o n p e a e p i c o t y l apices. A p i c a l 5 m m regions of e t i o l a t e d epicotyls were delineated, seedlings sprayed once at zero t i m e w i t h or w i t h o u t 4.5 m M 2 , 4 - D a n d m a r k e d regions e x a m i n e d after 48 h . D a t a c o m p i l e d f r o m refs. 28 a n d 32 Parameter

Zero T i m e

48 h Untreated

Length (mm/seg) Fresh weight ( m g / s e g ) Weight/Length (mg/mm)

5 12 2.4

Xyloglucan ^g/seg) Cellulose (pg/seg)

28 40

Xyloglucan ( D P x l O ) Cellulose ( D P x l 0 ~ ) - 3

3

E n do-1,4-/?-glucanase activity (units/seg)

1.1 8.4 13

Auxin 7.5 44 5.9

25 56 2.2

137 615

59 515

0.2 9.2

0.8 11.0

234

40

In soybean h y p o c o t y l , the a c t i v i t y of cell w a l l endo-1,4-/?-glucanase was higher i n regions of the s t e m a c t i v e l y u n d e r g o i n g cell e x p a n s i o n , a n d t h i s increased a c t i v i t y was correlated w i t h a reduced average m o l e c u l a r weight of cell w a l l x y l o g l u c a n (33). In a z u k i b e a n e p i c o t y l cell w a l l s , a u x i n t r e a t m e n t reduced the average m o l e c u l a r weight of x y l o g l u c a n w i t h i n 2 h , a l t h o u g h a decrease i n the average m o l e c u l a r weight of the t o t a l h e m i c e l lulose f r a c t i o n was observed after o n l y 30 m i n (34). A u x i n also r a p i d l y evoked a r e d u c t i o n i n average m o l e c u l a r weight of the x y l o g l u c a n c o m p o nent of Avena coleoptile cell w a l l s (35). T h e s e changes were not a result of cell e x p a n s i o n , since they o c c u r r e d even w h e n g r o w t h was o s m o t i c a l l y suppressed b y m a n n i t o l . W h e n pea e p i c o t y l segments were pulse-chased w i t h [ C ] g l u c o s e , s u b sequent i n c u b a t i o n w i t h a u x i n brought about a loss of cell w a l l glucose a n d xylose a n d the appearance i n the i n c u b a t i o n m e d i u m of soluble p o l y m e r i c m a t e r i a l c o n t a i n i n g xylose a n d glucose (36). T h e a p p a r e n t s o l u b i l i z a t i o n of cell w a l l x y l o g l u c a n was detectable after o n l y 15 m i n of a u x i n t r e a t m e n t a n d thus o c c u r r e d as q u i c k l y as the increased g r o w t h rate (37). M o r e o v e r , s o l u b i l i z a t i o n increased as g r o w t h rate increased, a n d was not p r e v e n t e d 14

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27

w h e n g r o w t h was i n h i b i t e d b y m a n n i t o l (37), suggesting t h a t x y l o g l u c a n h y d r o l y s i s reflected the w a l l - l o o s e n i n g process. Subsequent w o r k c o n f i r m e d a decrease i n cell w a l l x y l o g l u c a n u p o n a u x i n t r e a t m e n t (38). C o l l e c t i o n of cell w a l l free space s o l u t i o n s u s i n g a low-speed c e n t r i f u g a t i o n t e c h n i q u e also f o u n d a n a u x i n - i n d u c e d p r o d u c t i o n of water-soluble m a t e r i a l s possessing the p r o p e r t i e s of x y l o g l u c a n (39). X y l o g l u c a n s u b u n i t s have been detected i n the b a t h i n g m e d i u m of c u l t u r e d cells d u r i n g g r o w t h (6). E n z y m e s capable of h y d r o l y z i n g x y l o g l u c a n to monosaccharides have been detected i n e x t r a c t s f r o m soybean cell walls ( 3 3 , 4 0 ) . H i g h levels of endo-1,4-/?-glucanase a c t i v i t y were f o u n d i n e l o n g a t i n g regions of the s t e m , where x y l o g l u c a n a p p e a r e d to be cleaved i n t o c o m p a r a t i v e l y large f r a g ­ m e n t s . O t h e r enzymes, however, p r e s u m a b l y i n c l u d i n g a n α-xylosidase, a /?-glucosidase, a /?-galactosidase a n d a n α-fucosidase, showed higher a c t i v i t y i n n o n - e l o n g a t i n g regions. T h e s e findings suggest t h a t p a r t l y degraded x y ­ l o g l u c a n e v e n t u a l l y w i l l become c o m p l e t e l y b r o k e n d o w n a n d t h a t oligosac­ charides w i l l not a c c u m u l a t t r e a t m e n t e n h a n c e d the a c t i v i t y of enzymes c l e a v i n g g l u c o s i d i c , x y l o s i d i c a n d g a l a c t o s i d i c b o n d s (41), a l t h o u g h it was not s h o w n t h a t these enzymes were l o c a l i z e d i n the cell w a l l . T h e f u l l i n d u c t i o n of endo-1,4-/?-glucanase b y a u x i n t r e a t m e n t r e q u i r e d a p e r i o d of m a n y hours or even days (31) r a t h e r t h a n m i n u t e s , p r e c l u d ­ i n g the appearance of new endo-1,4-/?-glucanase as m e d i a t i n g the " r a p i d a c t i o n " of a u x i n o n g r o w t h . C h a n g e s i n the a c t i v i t y of p r e - e x i s t i n g e n d o l , 4 - / ? - g l u c a n a s e seem more likely, p e r h a p s r e l a t e d to the suggestion t h a t a u x i n p r o m o t e s g r o w t h b y c a u s i n g the a c i d i f i c a t i o n of the cell w a l l space b y the p r o t o p l a s m (42). C o n s i s t e n t w i t h t h i s are the findings t h a t buffer of p H 7 prevented b o t h g r o w t h a n d the release of soluble x y l o g l u c a n f r o m the w a l l i n d u c e d b y a u x i n , a n d t h a t buffer of p H 4 caused a s i m i l a r release of x y l o g l u c a n as d i d a u x i n , a n d i n the s h o r t - t e r m also caused a s i m i l a r p r o ­ m o t i o n of g r o w t h (43). A c i d t r e a t m e n t at 0 ° C d i d not result i n x y l o g l u c a n release, suggesting t h a t x y l o g l u c a n d e g r a d a t i o n was e n z y m e m e d i a t e d (39). T h e a c t i v i t i e s of a /?-glucosidase a n d a /?-galactosidase i n Avena coleoptile cell w a l l s were p r o m o t e d b y c h a n g i n g the p H f r o m 7 to a r o u n d 5 (44), b u t i t is n o t k n o w n i f these enzymes act on x y l o g l u c a n i n the w a l l . E n d o - 1 , 4 /?-glucanase, w h i c h does act o n x y l o g l u c a n i n the w a l l , has a b r o a d p H o p t i m u m of 6.0-6.5 (45). F u r t h e r m o r e , the occurrence i n the w h o l e p l a n t of changes i n cell w a l l p H of the m a g n i t u d e of those described above has been questioned (46). T h u s , the m e c h a n i s m b y w h i c h a u x i n regulates the very r a p i d changes observed i n x y l o g l u c a n D P is not yet f u l l y c l a r i f i e d . T h e c o n t i n u e d i n c o r p o r a t i o n of m a t r i x w a l l p o l y m e r s i n t o the w a l l also seems to be essential for l o n g - t e r m g r o w t h (27), i m p l y i n g t h a t i n c o r p o r a t i o n as w e l l as b r e a k d o w n o f x y l o g l u c a n m a y be i m p o r t a n t i n the w a l l - e x p a n s i o n process. F u r t h e r s t u d y of how synthesis a n d b r e a k d o w n of w a l l c o m p o n e n t s are c o o r d i n a t e d to regulate cell w a l l loosening is r e q u i r e d . X y l o g l u c a n S u b u n i t s as

Oligosaccharins

F r a g m e n t s of cell w a l l polysaccharides of specific s t r u c t u r e c a n b r i n g a b o u t

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r e g u l a t o r y effects o n p l a n t cells. O l i g o g l u c o s i d e s f r o m f u n g a l cell walls a n d fragments o f h o m o g a l a c t u r o n a n , released f r o m the p l a n t ' s o w n cell w a l l b y enzymes i n pathogens, can induce the de novo p r o d u c t i o n of m R N A ' s a n d enzymes responsible for the synthesis of p h y t o a l e x i n s , n a t u r a l p l a n t a n t i b i o t i c s (47). F r a g m e n t s of pectic polysaccharides can also i n d u c e the f o r m a t i o n o f protease i n h i b i t o r s w h i c h protect p l a n t s f r o m digestive a t ­ tack b y insects a n d m i c r o b e s . C e l l w a l l p o l y s a c c h a r i d e fragments have several other m o r p h o g e n e t i c effects o n p l a n t s , for they m a y m o d i f y such processes as flowering a n d vegetative g r o w t h (48). B i o l o g i c a l l y a c t i v e cell w a l l p o l y s a c c h a r i d e fragments have been t e r m e d oligosaccharins (48). T h e endo-1,4-/?-glucanase whose f o r m a t i o n was i n d u c e d b y a u x i n u l ­ t i m a t e l y h y d r o l y z e d x y l o g l u c a n to c h a r a c t e r i s t i c oligosaccharides. The n o n a s a c c h a r i d e derived f r o m c u l t u r e d sycamore x y l o g l u c a n , w h e n a d d e d i n v e r y low concentrations to pea s t e m segments, i n h i b i t e d a u x i n - s t i m u l a t e d g r o w t h (49). T h i s report was recently confirmed u s i n g nonasaccharides de­ r i v e d f r o m Rosa cell wall an o p t i m u m for g r o w t h - i n h i b i t o r y a c t i o n , b e i n g less effective or t o t a l l y i n ­ effective at higher concentrations (Table II). T h e o p t i m u m was a r o u n d 10 n M (49) or 1 n M (50) i n the two studies, a n d c o n c e n t r a t i o n s above a b o u t 100 n M were ineffective. C o n s i d e r a b l e v a r i a b i l i t y i n the degree of i n h i b i t i o n by the nonasaccharide was observed, r a n g i n g f r o m 70 to almost 1 0 0 % i n the e x p e r i m e n t s r e p o r t e d by Y o r k et ai (49), a n d f r o m a b o u t 30 to 7 0 % i n those s h o w n b y M c D o u g a l l a n d F r y (50). I n b o t h r e p o r t s the heptasaccharide was f o u n d to be w i t h o u t g r o w t h - i n h i b i t o r y effect.

T a b l e II. Effects of x y l o g l u c a n oligosaccharide s u b u n i t s o n a u x i n - s t i m u l a t e d g r o w t h of p e a e p i c o t y l segments in vivo a n d p e a endo-1,4-/?glucanase a c t i v i t y in vitro. G r o w t h of segments i n 1 μ Μ 2 , 4 - D was measured after 18 h . E n d o - 1 , 4 - / ? - g l u c a n a s e a c t i v i t y was de­ t e r m i n e d v i s c o m e t r i c a l l y after 30 m i n u s i n g t a m a r i n d x y l o g l u c a n as s u b s t r a t e . D a t a c a l c u l a t e d f r o m refs. 50 a n d 51 Xyloglucan Oligosaccharide concentration

Heptasaccharide

Nonasaccharide

I n h i b i t i o n of a u x i n - s t i m u l a t e d g r o w t h O.lnM 1.0 10.0 100.0

3 4 0 0

(%)

0 65 50 20

S t i m u l a t i o n of /?-glucanase a c t i v i t y (%) 50μΜ 100 150 200

210 280 350 410

430 550 640 700

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Effects of x y l o g l u c a n oligosaccharides o n endo-1,4-/?-glucanase a c t i v ­ i t y in viiro have also been observed (51). P e a n o n a - a n d h e p t a s a c c h a r i d e b o t h d r a m a t i c a l l y s t i m u l a t e d p e a e n d o - 1 , 4 - / ? - g l u c a n ase a c t i v i t y as deter­ m i n e d v i s c o m e t r i c a l l y u s i n g t a m a r i n d x y l o g l u c a n as s u b s t r a t e ( T a b l e II). S t i m u l a t i o n was c o n c e n t r a t i o n - d e p e n d e n t , a n d a p p r o a c h e d s a t u r a t i o n at 200 μ Μ . N o n a s a c c h a r i d e was the more effective, c a u s i n g over 7 0 0 % s t i m ­ u l a t i o n at 200 μ Μ , a c o n c e n t r a t i o n at w h i c h the h e p t a s a c c h a r i d e caused 4 0 0 % a c t i v a t i o n . E v e n x y l o g l u c a n p e n t a s a c c h a r i d e caused a d o u b l i n g of a c t i v i t y . T h e s t i m u l a t i o n was b o t h s u b s t r a t e - a n d enzyme-specific. A c t i v a ­ t i o n b y oligosaccharides was not detected u s i n g c a r b o x y m e t h y l c e l l u l o s e as s u b s t r a t e , nor w i t h endo-1,4-/?-glucanase f r o m Trichoderma using xyloglu­ can as s u b s t r a t e . T h u s , at n M c o n c e n t r a t i o n s in vivo, n o n a s a c c h a r i d e p r o d u c e d b y the m e t a b o l i s m of cell w a l l x y l o g l u c a n appears to act as a negative feedback m o d u l a t o r of a u x i n - s t i m u l a t e late endo-1,4-/?-glucanase a c t i o to m a g n i f y the decay rate of cell w a l l x y l o g l u c a n f u r t h e r , a n d l e a d to the p r o d u c t i o n of more oligosaccharide. T h i s c o u l d p e r h a p s raise the c o n c e n t r a ­ t i o n of n o n a s a c c h a r i d e in vivo to above t h a t w h i c h was i n h i b i t o r y t o w a r d s g r o w t h . I n c u l t u r e d s p i n a c h cells, a f u c o s e - c o n t a i n i n g n o n a s a c c h a r i d e ac­ c u m u l a t e d i n the c u l t u r e m e d i u m to a s t e a d y - s t a t e c o n c e n t r a t i o n of a b o u t 430 n M (6). However, the levels of n o n a s a c c h a r i d e e x i s t i n g in vivo at the l o a d - b e a r i n g region of the i n n e r m o s t w a l l layers of cells i n the e x p a n s i o n zone of the s t e m , where r e g u l a t i o n of g r o w t h p r e s u m a b l y o c c u r s , are n o t known. O f course, i n a given tissue n o n a s a c c h a r i d e m a y i n h i b i t g r o w t h a n d s t i m u l a t e endo-1,4-/?-glucanase a c t i v i t y i n d e p e n d e n t l y of one a n o t h e r . T h e o b s e r v a t i o n t h a t heptasaccharide was i n a c t i v e i n one s y s t e m b u t a c t i v e i n the other suggested t h a t the effects were m e d i a t e d b y different m e c h a n i s m s . T h e h e p t a s a c c h a r i d e was not itself i n h i b i t o r y to g r o w t h , n o r d i d i t i n t e r ­ fere w i t h the n o n a s a c c h a r i d e effect (50), suggesting great s p e c i f i c i t y i n the p e r c e p t i o n of n o n a s a c c h a r i d e for g r o w t h i n h i b i t i o n . T h e existence of t w o i n d e p e n d e n t m e c h a n i s m s m a y e x p l a i n the loss of i n h i b i t o r y effect of x y ­ l o g l u c a n nonasaccharides o n a u x i n - s t i m u l a t e d g r o w t h w h e n c o n c e n t r a t i o n s exceeded 10 n M . A t higher c o n c e n t r a t i o n s oligosaccharides m a y act d i r e c t l y o n cell w a l l endo-1,4-/?-glucanase, i n c r e a s i n g i t s a c t i v i t y a n d p r o m o t i n g x y ­ l o g l u c a n h y d r o l y s i s a n d cell w a l l loosening. T h i s m a y t h e n override the m e c h a n i s m p r o d u c i n g the g r o w t h - i n h i b i t o r y effect at lower c o n c e n t r a t i o n s of n o n a s a c c h a r i d e . A n o t h e r a l t e r n a t i v e is t h a t the t w o m e c h a n i s m s are s p a t i a l l y s e p a r a t e d a n d o c c u r i n different regions of the s t e m . P e r h a p s low c o n c e n t r a t i o n s of n o n a s a c c h a r i d e are f o u n d o n l y near cells a p p r o a c h ­ i n g f u l l size, where they act to i n h i b i t a u x i n - s t i m u l a t e d g r o w t h , whereas higher c o n c e n t r a t i o n s are f o u n d i n regions where cells are a l r e a d y f u l l y e x p a n d e d . Here the oligosaccharides m a y s t i m u l a t e the a c t i v i t i e s of v a r ­ ious glycosidases i n the w a l l , r e s u l t i n g i n the c o m p l e t e h y d r o l y s i s of the c o m p a r a t i v e l y large x y l o g l u c a n pieces a n d oligosaccharides t o m o n o s a c c h a ­ rides. T h i s w o u l d remove a n y b i o l o g i c a l a c t i v i t y o f the fragments, a n d the m o n o s a c c h a r i d e s c o u l d be absorbed b y the cell a n d reused.

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Conclusions T h e p i c t u r e t h a t emerges is t h a t x y l o g l u c a n functions i n the walls o f g r o w i n g tissues as a b i n d i n g p o l y s a c c h a r i d e w h i c h c o n t r i b u t e s t o t h e r i g i d i t y o f the cellulose f r a m e w o r k . D u r i n g t h e s t i m u l a t i o n o f g r o w t h evoked b y a u x i n , endo-1,4-/?-glucanase is i n d u c e d w h i c h hydrolyzes x y l o g l u c a n preferentially. S u c h a h y d r o l y s i s is correlated w i t h the w a l l - l o o s e n i n g process. P r e s u m a b l y new synthesis is required i n order t o preserve t h e s t r e n g t h o f the w a l l , a n d to m a i n t a i n i t i n a state capable o f further loosening d u r i n g l o n g - t e r m g r o w t h . T h e r e l a t i o n s h i p between new w a l l synthesis a n d t h e d e g r a d a t i o n of e x i s t i n g w a l l p o l y m e r s i n w a l l loosening is p o o r l y u n d e r s t o o d a t present. T h e role p l a y e d i n g r o w t h b y x y l o g l u c a n oligosaccharide fragments i s j u s t b e g i n n i n g t o be investigated. X y l o g l u c a n nonasaccharide specifically i n h i b i t e d a u x i n - e v o k e d g r o w t h at c e r t a i n concentrations i n the n M range, b u t n o n a - , h e p t a - a n d pentasaccharide a l l s t i m u l a t e d endo-1,4-/?-glucanase a c t i v i t y in vitro i n t h e μΜ range. T h i s l a t t e r effect, i f i t occurs in vivo at concentrations o f oligosaccharide w o u l d be expected t o enhance x y l o g l u c a n d e p o l y m e r i z a t i o n , w a l l loosening a n d g r o w t h . T h e s e a p p a r e n t l y opposite effects o f n o n a s a c c h a r i d e o n g r o w t h a n d the p u t a t i v e w a l l - l o o s e n i n g process c o n s t i t u t e a c h a l l e n g i n g p r o b l e m for future w o r k t o resolve. Acknowledgments T h i s review was prepared w i t h s u p p o r t b y grants f r o m t h e N a t u r a l S c i ­ ences a n d E n g i n e e r i n g Research C o u n c i l o f C a n a d a . W e t h a n k D r s . A n n e C a m i r a n d , R u t h G o r d o n , a n d V l a d i m i r Farkas for useful discussions. Literature Cited

1. McNeil, M.; Darvill, A. G.; Fry, S. C.; Albersheim, P. Ann. Rev. Biochem. 1984, 53, 625-63. 2. Hayashi, T.; Maclachlan, G. A. Plant Physiol. 1984, 75, 596-604. 3. Kato, Y.; Iki, K.; Matsuda, K. Agric. Biol. Chem. 1981, 45, 2745-53. 4. Carpita, N. C. Plant Physiol. 1983, 72, 515-21. 5. Carpita, N. C. Plant Physiol. 1984, 76, 205-12. 6. Fry, S. C. Planta 1986, 169, 443-53. 7. Reid, J. S. G. Adv. Bot. Res. 1985, 11, 125-55. 8. Kato, Y.; Matsuda, K. Plant Cell Physiol. 1985, 26, 437-45. 9. Thomas, J. R.; McNeil, M.; Darvill, A. G.; Albersheim, P. Plant Phys­ iol. 1987, 83, 659-71. 10. Bauer, W. D.; Talmadge, K. W.; Keegstra, K.; Albersheim, P. Plant Physiol. 1973, 51, 174-87. 11. Hayashi, T.; Kato, Y.; Matsuda, K. Plant Cell Physiol. 1980, 21, 140518. 12. Kato, Y.; Matsuda, K. Agric. Biol. Chem. 1980, 44, 1759-66. 13. Ray, P. M. Biochim. Biophys. Acta 1980, 629, 431-4.

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14. Hayashi, T.; Matsuda, K. J. Biol. Chem. 1981, 256, 11117-22. 15. Camirand, Α.; Brummell, D. Α.; Maclachlan, G. A. Plant Physiol. 1987, 84, 753-6. 16. Moore, P. J.; Staehelin, L. A. Planta 1988, 174, 433-45. 17. Hayashi, T.; Nakajima, T.; Matsuda, K. Agric. Biol. Chem. 1984, 48, 1023-7. 18. Gordon, R.; Maclachlan, G. A. J. Appl. Polym. Sci. Symp. 1988, 43, in press. 19. Gordon, R. Ph.D. Thesis, McGill Univ., Montreal, 1988. 20. Camirand, Α.; Maclachlan, G. A. Plant Physiol. 1986, 82, 379-83. 21. Farkas, V.; Maclachlan, G. A. Arch. Biochem. Biophys. 1988, 264, 48-53. 22. Hayashi, T.; Marsden, M. P. F.; Delmer, D. P. Plant Physiol. 1987, 83, 384-9. 23. Chambat, G.; Barnoud 687-93. 24. Fry, S. C. Ann. Rev. Plant Physiol. 1986, 37, 165-86. 25. Hoson, T.; Masuda, Y. Physiol. Plant. 1987, 71, 1-8. 26. Abdul-Baki, Α. Α.; Ray, P. M. Plant Physiol. 1971, 47, 537-44. 27. Brummell, D. Α.; Hall, J. L. Physiol. Plant. 1985, 63, 406-12. 28. Hayashi, T.; Maclachlan, G. A. Plant Physiol. 1984, 76, 739-42. 29. Spencer, F. S.; Ziola, B.; Maclachlan, G. A. Can. J. Biochem. 1971, 49, 1326-32. 30. Ray, P. M. Plant Physiol. 1973, 51, 601-8. 31. Datko, A. H.; Maclachlan, G. A. Plant Physiol. 1968, 43, 735-42. 32. Hayashi, T.; Wong, Y.-S.; Maclachlan, G. A. Plant Physiol. 1984, 75, 605-10. 33. Koyama, T.; Hayashi, T.; Kato, Y.; Matsuda, K. Plant Cell Physiol. 1981, 22, 1191-8. 34. Nishitani, K.; Masuda, Y. Physiol. Plant. 1981, 52, 482-94. 35. Inouhe, M.; Yamamoto, R.; Masuda, Y. Plant Cell Physiol. 1984, 25, 1341-51. 36. Labavitch, J. M.; Ray, P. M. Plant Physiol. 1974, 53, 669-73. 37. Labavitch, J. M.; Ray, Ρ. M. Plant Physiol. 1974, 54, 499-502. 38. Gilkes, N. R.; Hall, M. A. New Phytol. 1977, 78, 1-15. 39. Terry, M. E.; Jones, R. L.; Bonner, B. A. Plant Physiol. 1981, 68, 531-7. 40. Koyama, T.; Hayashi, T.; Kato, Y.; Matsuda, K. Plant Cell Physiol. 1983, 24, 155-62. 41. O'Neill, R. Α.; White, A. R.; York, W. S.; Darvill, A. G.; Albersheim, P. Phytochemistry 1988, 27, 329-33. 42. Rayle, D. L.; Cleland, R. E. Curr. Top. Dev. Biol. 1977, 11, 187-214. 43. Jacobs, M.; Ray, P. M. Plant Physiol. 1975, 56, 373-6. 44. Johnson, K. D.; Daniels, D.; Dowler, M. J.; Rayle, D. L. Plant Physiol. 1974, 53, 224-8. 45. Maclachlan, G. A. Methods Enzymol. 1988, 160, 382-91. 46. Brummell, D. Α.; Hall, J. L. Plant Cell Environ. 1987, 10, 523-43.

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47. Ryan, C. A. Ann. Rev. Cell Biol. 1987, 3, 295-317. 48. Tran Thanh Van, K.; Toubart, P.; Cousson, Α.; Darvill, A. G.; Gollin, D. J.; Albersheim, P. Nature 1985, 314, 615-7. 49. York, W. S.; Darvill, A. G.; Albersheim, P. Plant Physiol. 1984, 75, 295-7. 50. McDougall, G. J.; Fry, S. C. Planta 1988, 175, 412-6. 51. Farkas, V.; Maclachlan, G. A. Carbohydr. Res. 1988, in press. RECEIVED

March 10, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 3

Toward a Working Model of the Growing Plant Cell Wall Phenolic Cross-Linking Reactions in the Primary Cell Walls of Dicotyledons Stephen C . Fry and Janice G . Miller Department of Botany, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh E H 9 3 J H , Scotland

The efficient formation of inter-polymeric cross-links by oxidative coupling of the small number of polymer-bound phenolic side-chains present in the non-lignified, growing plant cell wall requires considerable specificity in the reactions concerned. In this paper we summarize some of the evidence that such specificity exists. We suggest that the cell wall is initially assembled by non-covalent interactions, especially involving the hydrogen-bonding of neutral xyloglucan chains to several microfibrils, thereby tethering these microfibrils. Oxidative coupling of other matrix polymers [acidic polysaccharides and/or basic glycoproteins] via their phenolic side-chains is seen as a subsequent wall-modification reaction whereby xyloglucan chains may be strapped to their current microfibrils so that the existing architecture is rendered more nearly permanent. Efficient strapping (i.e., fastening the maximum amount of material with fewest "buckles") requires chemical specificity—the formation of cross-links at appropriate sites. There is great specificity both in the biosynthetic reactions by which phenolic side-chains become attached to the wall polymers and also in the choice of phenolic partners and orientation during coupling. T h e g r o w i n g plant cell w a l l contains polymers w h i c h bear a s m a l l p r o p o r t i o n o f phenolic side-chains. These side-chains appear t o be subject in vivo to o x i d a t i v e phenolic c o u p l i n g a n d thus t o p a r t i c i p a t e i n c r o s s - l i n k i n g r e actions t h a t m a y be h i g h l y significant i n the c o n t r o l o f w a l l e x t e n s i b i l i t y (and therefore i n cell growth) a n d o f e n z y m i c d i g e s t i b i l i t y (1,2). For phenolic c r o s s - l i n k i n g t o b e b i o l o g i c a l l y effective, despite the presence o f o n l y low levels of phenolic c o m p o u n d s i n the p r i m a r y cell w a l l , the reactions 0097-6156/89/0399-0033$06.00/0 © 1989 American Chemical Society

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must be a c c u r a t e l y a n d efficiently steered: they m u s t be specific. Here we s u m m a r i z e evidence t h a t the necessary specificity exists. Hypothesis In order to t h i n k about the n a t u r e a n d consequences of cell w a l l p o l y ­ mer phenolic c r o s s - l i n k i n g , we need a w o r k i n g m o d e l of the m o d e of as­ s e m b l y a n d the final s t r u c t u r e of the p r i m a r y cell w a l l . U n f o r t u n a t e l y , there is no u n i v e r s a l l y acceptable m o d e l : t h a t proposed b y A l b e r s h e i m a n d co-workers (3) is not now w i d e l y accepted because the p o s t u l a t e d i n t e r p o l y s a c c h a r i d e glycosidic bonds have not been d e m o n s t r a t e d (4); a n d the ' w a r p - w e f t ' m o d e l of L a m p o r t (5) rests o n the a s s u m p t i o n s t h a t extensin (i) forms a defined-porosity network (not proven); (ii) is o r i e n t a t e d a n t i c l i n a l l y to the cell surface [some evidence against (6)]; a n d (iii) is a m a j o r component of a l l p r i m a r y cell walls (not true). Major Structural Polymers of the Cell Wall. A w a l l m o d e l requires a de­ s c r i p t i o n of the m a j o r p o l y m e r s i n v o l v e d . T h e m a j o r p o l y m e r s of the grow­ i n g D i c o t y l e d o n cell w a l l are s h o w n , a p p r o x i m a t e l y to scale, i n F i g u r e 1. T h e y are described i n more d e t a i l elsewhere (2); i n brief, they are: 1. The microfibrillar cellulose. T h i s forms the skeletal scaffolding of the cell w a l l . M i c r o f i b r i l s are about 4 n m i n diameter (6) a n d are of i n d e ­ terminate length. 2. The neutral hemicelluloses. These are x y l o g l u c a n i n D i c o t y l e d o n o u s p r i m a r y walls a n d p r i n c i p a l l y m i x e d - l i n k e d β-(1—»3), (1—*4)-D-glucans i n members of the G r a m i n a c e a e (grasses, i n c l u d i n g cereals). In i s o l a t i o n , x y l o g l u c a n is water-soluble, a l t h o u g h the molecule is a r e l a ­ tively stiff rod a p p r o x i m a t e l y 150 to 1500 n m i n t o t a l contour l e n g t h (8). O n e x y l o g l u c a n h a d a measured a x i a l r a t i o of about 100 (9). In the i n t a c t cell w a l l , the x y l o g l u c a n is firmly a t t a c h e d to the surface of the cellulosic m i c r o f i b r i l s , p r i n c i p a l l y by h y d r o g e n - b o n d i n g (2,8,10-13), a l t h o u g h some more secure bonds m a y also be present (2,10). 3. The acidic polysaccharides. In D i c o t y l e d o n s , the m a j o r acidic p o l y s a c ­ charides are the pectins ( p a r t i a l l y methyl-esterifled h o m o g a l a c t u r o nans a n d r h a m n o g a l a c t u r o n a n s ) a n d s m a l l e r a m o u n t s of a r a b i n o g l u c u r o n o x y l a n s (2). [In g r o w i n g grass cell walls, the a r a b i n o g l u c u r o n o x y lans u s u a l l y p r e d o m i n a t e over pectins.] 4. The basic extensins. These are h y d r o x y p r o l i n e - , l y s i n e - a n d t y r o s i n e r i c h glycoproteins consisting of r i g i d m o l e c u l a r rods about 80 n m l o n g (14,15), b e a r i n g short m o n o - to tetrasaccharide side-chains (2,14). W h e n n e w l y secreted they b i n d i o n i c a l l y to the a c i d i c polysaccharides of the cell w a l l a n d can be e x t r a c t e d w i t h cold salt solutions; later they become m u c h more resistant to s a l t - e x t r a c t i o n a n d are s a i d to be covalently b o u n d , p r o b a b l y v i a d i m e r i z a t i o n of their tyrosine residues to f o r m isodityrosine (15). Some of the acidic a n d basic polymers of the cell w a l l bear pheno­ lic side-chains. T h e acidic polysaccharides carry ferulic a n d p - c o u m a r i c acid a n d related c i n n a m a t e - d e r i v a t i v e s , esterified to specific h y d r o x y groups

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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35

Cellulose m i c r o f i b r i l

Xyloglucan molecule

Extensin molecule

scale 100 nm

F i g u r e 1. T h e p r i n c i p a l s t r u c t u r a l c o m p o n e n t s o f the g r o w i n g cell walls o f a D i c o t y l e d o n . T h e d r a w i n g s are a p p r o x i m a t e l y t o scale.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L P O L Y M E R S

(1,2,17-22). F e r u l o y l a t e d pectins have been found i n the p a r e n c h y m a tous cell walls of m a n y D i c o t y l e d o n s ( m a i n l y i n the C e n t r o s p e r m a e a n d Solanaceae), b u t UV-fluorescence microscopy suggests t h a t at least the epidermal cell walls of a l l D i c o t y l e d o n s c o n t a i n phenolic residues; i t r e m a i n s to be seen whether these phenolic residues are a t t a c h e d to polysaccharides or to c u t i n , b u t l o c a t i o n of even a s m a l l q u a n t i t y of, say, f e r u l o y l - p e c t i n i n the e p i d e r m a l w a l l w o u l d be p a r t i c u l a r l y significant i n the c o n t r o l of g r o w t h because the e x t e n s i b i l i t y of the epidermis controls the e x p a n s i o n of whole stems (23) a n d leaves ( F r y , u n p u b l i s h e d observations). T h e extensins, as a l r e a d y m e n t i o n e d , are r i c h i n the phenolic a m i n o a c i d t y r o s i n e (2). Assembly of a Xyloglucan-Microfibril Framework to the Wall. T h e i n i t i a l assembly of the cell w a l l is l i k e l y t o be v i a non-covalent b o n d i n g , especially h y d r o g e n - b o n d i n g . These i n i t i a l steps are believed to be n o n - e n z y m i c . T h e m i c r o f i b r i l s are synthesized b y enzyme complexes w h i c h are m o b i l e i n the plasma membrane. T h e come to lie i n a p l a n e — t h e i n n e r m o s t , accreting face of the cell w a l l . A t the same t i m e , G o l g i - d e r i v e d vesicles deposit the essentially soluble m a t r i x p o l y m e r s ( F i g . 2a). E v i d e n c e suggesting t h a t the m a t r i x p o l y m e r s are indeed water-soluble when n e w l y secreted was p r o v i d e d by observation of the polysaccharides secreted b y n a k e d protoplasts d i r e c t l y into the c u l t u r e m e d i u m (24). O f the various m a t r i x p o l y m e r s i n t h i s m i x t u r e ( h e m i c e l luloses, pectins a n d e x t e n s i n ) , it is p a r t i c u l a r l y the x y l o g l u c a n t h a t w i l l s t r o n g l y h y d r o g e n - b o n d to the m i c r o f i b r i l s , c l o t h i n g t h e m w i t h a m o l e c u l a r monolayer (10-12). Since the t o t a l contour length of a x y l o g l u c a n molecule is 40 t o 400 times greater t h a n the diameter of a m i c r o f i b r i l (8), it w o u l d seem i n e v i t a b l e t h a t most of the newly deposited x y l o g l u c a n chains w i l l have the o p p o r t u n i t y to b i n d to the several m i c r o f i b r i l s across w h i c h they are r a n d o m l y l a i d d o w n ( F i g . 2b) (25). A n y tendency for a x y l o g l u c a n molecule to re-orientate a n d come to lie w i t h its entire length a l o n g a single m i c r o f i b r i l w i l l be m i n i m i z e d by the presence i n the space between the adjacent m i c r o f i b r i l s of other m a t r i x p o l y m e r s w h i c h are not s t r o n g l y h y d r o g e n - b o n d i n g (pectins a n d extensins) a n d w h i c h w i l l (a) r e t a r d the m o l e c u l a r m o t i o n of the x y l o g l u c a n a n d (b) [by some of t h e m l y i n g w i t h their m a i n - c h a i n s p e r p e n d i c u l a r to the m i crofibrils] p h y s i c a l l y prevent the x y l o g l u c a n chain f r o m l i n i n g up a l o n g a n y favored m i c r o f i b r i l . T h e consequence of t h i s is t h a t the i n d i v i d u a l x y l o g l u can molecules w i l l come t o be hydrogen-bonded a l o n g discrete segments of several m i c r o f i b r i l s , t e t h e r i n g t h e m , and the i n t e r v e n i n g lengths of x y l o g l u can w i l l be suspended between the m i c r o f i b r i l s ( F i g . 2b). It m a y be noted p a r e n t h e t i c a l l y t h a t evidence t h a t extensins are not s t r o n g l y hydrogen-bonded i n the cell w a l l is the ease w i t h w h i c h they can be leached out of the w a l l w i t h 25-50 m M L a C l 3 or AICI3 ( 2 U ) — a t r e a t m e n t t h a t effectively breaks ionic bonds but not hydrogen b o n d s — s o l o n g as the t r e a t m e n t is a p p l i e d before the extensin has become covalently b o n d e d i n the cell w a l l . E v i d e n c e t h a t at least one pectic p o l y s a c c h a ride ( r h a m n o g a l a c t u r o n a n - I ) is not s t r o n g l y h y d r o g e n - b o n d e d i n the cell

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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F i g u r e 2. A s p e c u l a t i v e m o d e l for the assembly a n d g r o w t h o f the D i c o t y l e donous cell w a l l . F o r c l a r i t y , the m o d e l shows t w o m i c r o f i b r i l s = = = , = = = ) a n d o n l y one of the m a n y x y l o g l u c a n molecules ( ) t h a t are p r o p o s e d to interconnect t h e m . T h e loops i n the lower d i a g r a m represent other m a t r i x p o l y m e r s ( e x t e n s i n a n d / o r pectins) whose p h e n o l i c side-chains have b e c o m e o x i d a t i v e l y c o u p l e d . Steps (d) a n d (g) are p r o p o s e d t o be c a t a l y z e d b y cellulase. F o r f u r t h e r d e t a i l s , see ref. (25).

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L POLYMERS

w a l l is the fact t h a t , despite its large size [ D P 2,000 (27); s i m i l a r to a n average x y l o g l u c a n ] , i t is released i n t a c t f r o m the cell w a l l i n t o c o l d n e u t r a l water following t r e a t m e n t w i t h pure e n d o - p o l y g a l a c t u r o n a s e , a n e n z y m e t h a t hydrolyzes h o m o g a l a c t u r o n a n ( w i t h w h i c h the backbone o f the r h a m n o g a l a c t u r o n a n h a d p r e s u m a b l y been contiguous) b u t does not affect the r h a m n o g a l a c t u r o n a n itself. An Important Requirement for Cell Growth. W i t h continued cell e x p a n s i o n , the i n t e r m i c r o f i b r i l l a r segments of x y l o g l u c a n w i l l become stretched t a u t ( F i g . 2c) a n d w i l l e v e n t u a l l y come to bear the b u r d e n of t u r g o r . F u r t h e r g r o w t h w i l l then be impossible unless one or b o t h of two t h i n g s h a p p e n s : 1. the i n t e r - m i c r o f i b r i l l a r segments of x y l o g l u c a n are h y d r o l y z e d ( F i g . 2d) b y a n endo-/?-(l—>4)-D-glucanase (cellulase), a well-established p l a n t cell w a l l enzyme (28); 2. the xyloglucan-cellulose hydrogen-bonds are p h y s i c a l l y p u l l e d o p e n , u n z i p p i n g the x y l o g l u c a hydrogen-bonds are considerabl w h i c h the i n d i v i d u a l polysaccharide chains are c o n s t r u c t e d (13). Consequences and Requirements of Oxidative Phenolic Coupling in the Cell Wall. If the phenolic side-chains present o n pectins a n d / o r extensins c a n cross-link i n a n a p p r o p r i a t e m a n n e r a n d place, they c o u l d f o r m loops t h a t w o u l d encircle m i c r o f i b r i l s a n d thereby strap p a r t i c u l a r x y l o g l u c a n molecules on to p a r t i c u l a r m i c r o f i b r i l s ( F i g . 2f). T h i s w o u l d prevent the " u n z i p p i n g " m o d e of g r o w t h , b u t w o u l d not affect the " h y d r o l y t i c " m o d e . T h u s , w h i l e c r o s s - l i n k i n g c o u l d p o t e n t i a l l y decelerate g r o w t h very r a p i d l y ( F i g . 2 h ) , such a n effect need not be irreversible ( F i g . 2g). T h e i m p o r t a n c e of t h i s conclusion can be considered by reference to a p h y s i o l o g i c a l e x a m ple: T h e r a p i d l y - i m p o s e d i n h i b i t o r y effect of blue light o n the g r o w t h of stems has been hypothesized to be m e d i a t e d b y peroxidase-catalyzed crossl i n k i n g of w a l l p o l y m e r - b o u n d phenolics (29); since the i n i t i a l g r o w t h rate is r a p i d l y restored when the blue light is s w i t c h e d off, the hypothesis o n l y stands i f the m e c h a n i s m of g r o w t h i n h i b i t i o n is reversible. Efficient cross-link f o r m a t i o n by a s m a l l n u m b e r of w a l l p o l y m e r - b o u n d phenolics requires great precision i n the m e t a b o l i c reactions i n v o l v e d . It is not sufficient to f o r m cross-links: the cross-links need to be formed i n the p r o p e r place w i t h i n the p o l y m e r molecule a n d w i t h i n the cell w a l l . E v i d e n c e t h a t cross-links f o r m at a l l [albeit sometimes as a low percentage of the t o t a l w a l l phenolics] is presented elsewhere (1,2,13,16,30-32). Here we present evidence t h a t sufficient m o l e c u l a r specificity exists to be c o m p a t i b l e w i t h useful cross-link f o r m a t i o n . E v i d e n c e for Specificity i n t h e R e a c t i o n s G r o u p s are I n t r o d u c e d i n t o W a l l P o l y m e r s

by

which

Phenolic

Background. In order to m a x i m i z e the efficiency of c r o s s - l i n k i n g based o n a s m a l l number of phenolic groups, i t is i m p o r t a n t t h a t these groups s h o u l d be sited o n the w a l l p o l y m e r s at appropriate loci rather t h a n r a n d o m l y . In the case of the phenolic side-chains of extensins this c r i t e r i o n is met since the s i t i n g of the tyrosines is genetically encoded (27).

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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39

Siting of Ferulic Acid in the Wall Polysaccharides. T h e o r i g i n of the feru l o y l residues of the a c i d i c w a l l polysaccharides is not so s t r a i g h t f o r w a r d . H o w e v e r , there is g o o d evidence t h a t these phenolic side-chains are very specifically s i t e d . T h e p o s i t i o n of the f e r u l o y l groups can most easily be e x p l o r e d b y e n z y m i c " d i s s e c t i o n " of the polysaccharides w i t h c o m m e r c i a l m i x t u r e s of enzymes such as t h a t k n o w n as Driselase (available f r o m S i g m a C h e m i c a l C o . ) . Driselase, w h i c h is w i d e l y used for the i s o l a t i o n o f p l a n t p r o t o p l a s t s because i t possesses enzymes t h a t h y d r o l y z e most of the g l y cosidic linkages of the p r i m a r y cell w a l l , lacks feruloyl-esterase a c t i v i t y and therefore leaves the f e r u l o y l groups a t t a c h e d to the a p p r o p r i a t e sugar u n i t of the p o l y s a c c h a r i d e . I n a d d i t i o n , the endo- a n d exo-glycanases of Driselase cannot h y d r o l y z e those glycose residues t h a t bear f e r u l o y l g r o u p s . T h e expected p r o d u c t s of h y d r o l y s i s of a f e r u l o y l - p o l y s a c c h a r i d e are t h u s m a i n l y monosaccharides p l u s feruloyl-disaccharides (2). [Driselase l a c k s α-xylosidase a c t i v i t y a n d therefore also y i e l d s large a m o u n t s of a s i m p l e disaccharide, D - x y l o p y r a n o s y l - a - ( T h e cell walls of D i c o t y l e d o n s , especially i n the C a r y o p h y H a l e s , y i e l d u p o n Driselase digestion two m a j o r f e r u l o y l - d i s a c c h a r i d e s , n a m e l y 3-0-(3-0-feruloyl-a-L-arabinopyranosyl)-L-arabinose (Fer-Ara2) and 4 - 0 (6-0-feruloyl-/?-D-galactopyranosyl)-D-galactose ( F e r - G a l ) (17). These two c o m p o u n d s together account for 6 0 - 7 0 % of the f e r u l o y l residues of the p r i m a r y walls of c u l t u r e d s p i n a c h cells. In the grasses, the m a j o r f e r u l o y l - d i s a c c h a r i d e o b t a i n e d w i t h Driselase is 3 - 0 - ( 5 - 0 - f e r u l o y l a - L - a r a b i n o f u r a n o s y l ) - D - x y l o s e ( F e r - A r a - X y l ) , b u t larger a m o u n t s of a feruloyl-trisaccharide, 4-0-[3-0-(5-0-feruloyl-a-L-arabinofuranosyl) -/?-Dx y l o p y r a n o s y l ] - D - x y l o s e ( F e r - A r a - X y l ) , u s u a l l y p r e d o m i n a t e because D r i ­ selase is inefficient at h y d r o l y z i n g x y l o b i o s e (19,20). 2

2

T h e i m p o r t a n t conclusion is t h a t m u c h of the w a l l ' s ferulic a c i d is l i n k e d to specific h y d r o x y groups o n specific sugars of specific p o l y s a c c h a r i d e s . T h e specificity is p a r t i c u l a r l y n o t a b l e i n the case of F e r - A r a , since the f e r u l o y l a t e d arabinose residues are i n the rare pyranose r i n g - f o r m (17). It is clear t h a t the f e r u l o y l a t i o n reactions are not r a n d o m , b u t are carefully steered b i o s y n t h e t i c steps. 2

Evidence for Intracellular Feruloylation Reactions. T h e reactions by w h i c h f e r u l o y l residues are added to polysaccharides o c c u r i n t r a c e l l u l a r l y . T h i s was established for the F e r - A r a u n i t s of the pectic polysaccharides of c u l ­ ture s p i n a c h cells b y a d m i n i s t r a t i o n of [ H]arabinose so t h a t the careers of p o l y m e r - b o u n d pentose residues c o u l d be followed f r o m t h e i r i n t r a c e l l u l a r i n c o r p o r a t i o n i n t o nascent polysaccharides a n d g l y c o p r o t e i n s , v i a t h e i r se­ c r e t i o n t h r o u g h the p l a s m a m e m b r a n e , to t h e i r u l t i m a t e fate w i t h i n the w a l l (28). T h e cells were fed [ H]arabinose for various defined periods of t i m e , after w h i c h two d i s t i n c t analyses were p e r f o r m e d ( F i g . 3): a. A s a m p l e of the cells was k i l l e d i n e t h a n o l a n d the a l c o h o l - i n s o l u b l e residue ( c o n t a i n i n g the polysaccharides a n d g l y c o p r o t e i n s ) was d i ­ gested e x h a u s t i v e l y w i t h Driselase. T h i s converted the various pentosec o n t a i n i n g u n i t s of the p o l y m e r s to the f o l l o w i n g m a j o r b r e a k d o w n products: 2

3

3

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

PLANT C E L L W A L L

40

[3H]Arabinose

POLYMERS

Ethanol

Driselase

Washings rejected

Digestion products

time t

Spinach cells

Culture filtrate

- - Polymers

Origin—!

O -

-Xyl-a-(l-6)Glc

o -

-Ara

o -

-xyi

o —

--Fer-Ara2

Ι Λ Λ Λ Λ Λ / V V V V V V N F i g u r e 3. Scheme o f a n e x p e r i m e n t to d e m o n s t r a t e the i n t r a c e l l u l a r n a t u r e of polysaccharide-feruloylation.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3.

Working Model of Growing Plant Cell Wall

FRY & MILLER

arabinan feruloyl-arabinan arabinoxylan xyloglucan

—• [ —• [ —• [ —• [

3

3

3

3

41

H]arabinose H]arabinose + Fer-[ H]Ara2 H]arabinose + [ H]xylose + [ H]xylobiose H ] x y l o s y l - a - ( l —• 6) glucose 3

3

3

— w h i c h were t h e n separated b y paper c h r o m a t o g r a p h y a n d assayed i n d i v i d u a l l y for H b y s c i n t i l l a t i o n - c o u n t i n g , b . A s a m p l e of the c u l t u r e filtrate was also a n a l y z e d b y p a p e r c h r o m a t o g r a p h y to separate the u n c h a n g e d [ H ] a r a b i n o s e f r o m a n y secreted p o l y s a c c h a r i d e present. T h e H i n the l a t t e r ( R p = 0.00) was m e a s u r e d by scintillation-counting. B y step (a), a p i c t u r e c o u l d be formed o f the k i n e t i c s o f the i n t r a c e l l u lar i n c o r p o r a t i o n of H f r o m [ H ] a r a b i n o s e i n t o the m a j o r polysaccharides o f the cell w a l l . R a d i o a c t i v i t y was i n c o r p o r a t e d i n t o p o l y m e r - b o u n d a r a binose a n d xylosyl-glucose u n i t s after l a g periods of a b o u t 3.5 m i n a n d 6.5 m i n , respectively. Thes [ H ] a r a b i n o s e t o be take p o l y s a c c h a r i d e biosynthesis 3

3

3

3

3

3

[ H]Ara — [ H]Ara-l-P ~ 3

3

UDP-[ H]Ara (~ 3

UDP-[ H]Xyl) 3

(1,35) a n d p o s s i b l y for the U D P - s u g a r s to be t r a n s p o r t e d i n t o the e n d o m e m b r a n e s y s t e m where the polysaccharides are s y n t h e s i z e d . A f t e r the l a g , the rate of i n c o r p o r a t i o n of H i n t o p o l y m e r s r e m a i n e d f a i r l y constant for several h o u r s . T h e i n c o r p o r a t i o n of H f r o m [ H ] a r a b i n o s e i n t o F e r - A r a 2 u n i t s showed a l a g o f a b o u t 4.2 m i n , after w h i c h i t too b e c a m e l i n e a r . T h i s means t h a t as l i t t l e as 0.7 m i n after their i n c o r p o r a t i o n i n t o a ( p o s s i b l y s t i l l nascent) p o l y s a c c h a r i d e , [ H ] a r a b i n o s e residues were susceptible t o f e r u l o y l a t i o n . T h e f e r u l o y l a t i o n r e a c t i o n is t h u s l i k e l y to have been o c c u r r i n g i n the e n d o m e m b r a n e s y s t e m , c o - s y n t h e t i c a l l y , as one o f the h i g h l y r e g u l a t e d p a r t s o f the s o p h i s t i c a t e d p o l y s a c c h a r i d e - b i o s y n t h e t i c m a c h i n e r y . T h i s c a n be c o m p a r e d to the c o - t r a n s l a t i o n a l m o d i f i c a t i o n k n o w n t o o c c u r i n m a n y proteins. B y step (b), further evidence was o b t a i n e d t h a t the f e r u l o y l a t i o n was intracellular. T h e e x t r a c e l l u l a r polysaccharides a n d g l y c o p r o t e i n s o n l y s t a r t e d to acquire L after a l a g of a b o u t 25 m i n (34). T h i s is i n t e r preted t o m e a n t h a t before 25 m i n essentially a l l the [ H ] p o l y s a c c h a r i d e was s t i l l i n t r a c e l l u l a r , either i n the G o l g i bodies or packaged i n t o G o l g i d e r i v e d vesicles, b u t not yet passed t h r o u g h the p l a s m a m e m b r a n e . T h e fact t h a t these sugar residues were b e i n g f e r u l o y l a t e d , at the m a x i m a l r a t e , w e l l before 25 m i n s u p p o r t s the conclusion t h a t f e r u l o y l a t i o n was l a r g e l y intracellular. 3

3

3

3

3

3

Possible Extracellular Feruloylation. It has been suggested (36) t h a t feru l o y l a t i o n o c c u r s e x t r a c e l l u l a r l y . E v i d e n c e i n s u p p o r t of t h i s c o n t e n t i o n was the observation t h a t i n m a t u r i n g coleoptiles of b a r l e y the a m o u n t of p o l y s a c c h a r i d e - b o u n d ferulate continued t o increase for at least one d a y after t o t a l p o l y s a c c h a r i d e a c c u m u l a t i o n h a d ceased. However, t h i s fact is o p e n t o the a l t e r n a t i v e e x p l a n a t i o n t h a t r e l a t i v e l y s m a l l a m o u n t s o f a h i g h l y

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f e r u l o y l a t e d polysaccharide continued to be synthesized ( i n t r a c e l l u l a r l y ) i n m a t u r e coleoptiles even after t o t a l w a l l polysaccharide synthesis h a d decelerated a n d been a p p r o x i m a t e l y equalled b y p o l y s a c c h a r i d e b r e a k d o w n so t h a t the net rate of polysaccharide a c c u m u l a t i o n was zero. F u r t h e r evidence a p p a r e n t l y i n favor of some f e r u l o y l a t i o n b e i n g ext r a c e l l u l a r was the observation t h a t [ C ] f e r u l o y l - C o A w i l l b i n d covalently to coleoptile cell walls i n vitro (37). It was suggested t h a t t h i s was due to the o p e r a t i o n of a n e x t r a c e l l u l a r feruloyltransferase c a t a l y z i n g a t r a n s esterification r e a c t i o n whereby the feruloyl residue is transferred f r o m the C o A donor to a polysaccharide acceptor. However, i t r e m a i n s t o be seen (i) whether f e r u l o y l - C o A , the p r o p o s e d donor, occurs e x t r a c e l l u l a r l y ; a n d (ii) whether, i n the in-vitro s y s t e m , the C r e m a i n s as [ C ] f e r u l o y l residues or whether the [ C ] f e r u l o y l residues become b o u n d t o other w a l l - b o u n d (non-radioactive) phenolic groups, perhaps b y o x i d a t i v e c o u p l i n g . [ O x i d a tive c o u p l i n g i n isolated cell walls i n the absence of a d d e d has been observed (38).] S u c h a reactio p o s s i b l y catalase (31). If [ C ] f e r u l o y l - C o A does indeed b i n d to cell w a l l s in vitro v i a the f o r m a t i o n of ester b o n d s , it w i l l be of great interest t o see whether the C can be recovered b y Driselase digestion i n the f o r m of [ C ] F e r - A r a - X y l a n d [ C ] F e r - A r a - X y l i n d i c a t i n g t h a t the h i g h l y specific f e r u l o y l - s u g a r b o n d characteristic of grass cell walls h a d been synthesized b y a n e x t r a c e l l u l a r enzyme s y s t e m . B i o s y n t h e s i s of ester b o n d s i n the cell w a l l has a precedent i n the proposed biosynthesis of c u t i n f r o m f a t t y a c y l C o A thioesters i n the e p i d e r m a l cell w a l l (for a review, see 39). It is possible t h a t Y a m a m o t o et ai (37) have detected the biosynthetic s y s t e m b y w h i c h feruloyl residues are a t t a c h e d to the a l i p h a t i c core of c u t i n rather t h a n to the w a l l polysaccharides. T h e c u r r e n t evidence seems to favor i n t r a c e l l u l a r f e r u l o y l a t i o n of polysaccharides. 14

1 4

14

14

14

1 4

1 4

1 4

2

E v i d e n c e for Specificity i n t h e O x i d a t i v e C o u p l i n g o f P h e n o l i c S i d e - C h a i n s i n the C e l l W a l l T h e previous section has presented evidence t h a t phenolic u n i t s are caref u l l y positioned w i t h i n the w a l l p o l y m e r s . W h e n these u n i t s undergo oxi d a t i v e phenolic c o u p l i n g reactions i n the cell w a l l , the c o u p l i n g reactions themselves are also r e m a r k a b l y specific. T h i s can be i l l u s t r a t e d b y reference to the tyrosine residues of e x t e n s i n . Orientation of Coupling in Protein-Bound Tyrosine Residues. T y r o s i n e can couple to f o r m either of two isomeric d i m e r s , dityrosine a n d i s o d i t y r o s i n e (1,16) ( F i g . 4). T h e choice between these two dimers is governed b y the c o n d i t i o n s under w h i c h the c o u p l i n g is carried o u t ; some examples are given i n T a b l e I. In a n i m a l s t r u c t u r a l proteins in vivo, the o n l y k n o w n d i m e r of tyrosine is d i t y r o s i n e (40,41); i n the extensin of plant cell walls, i n contrast, the o n l y d i m e r formed in vivo is isodityrosine (16). H o w is the c o u p l i n g of t y r o s i n e i n plants confined to the f o r m a t i o n of isodityrosine? T h e r e is n o t h i n g u n i q u e about the l o c a l e n v i r o n m e n t of the tyrosine residues i n (pure) e x t e n s i n , since

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T a b l e I. D i m e r i c P r o d u c t s of the O x i d a t i v e C o u p l i n g of P r o t e i n - B o u n d T y rosine Residues under V a r i o u s C o n d i t i o n s Substrate

System

PH

Products

Reference

R e s i l i n in vivo C o l l a g e n in vivo E x t e n s i n in vivo E x t e n s i n in vitro Bovine serum a l b u m i n in vitro E x t e n s i n in vitro

insect cuticle nematode cyst D i c o t p r i m a r y cell w a l l peroxidase + H 2 O 2

> 7 >7 < 7 9

DÎT DiT Idt DiT

(40) (41) (16) (38)

9 6

DiT Idt

(42) (38)

OH

peroxidase + H 2 O 2 isolated cell w a l l

O

1

kJ

Κ

I

ÇH CHNH 2

2

COOH Tyrosine

ÇH ÇHNH COOH 2

2

•tyrosine

Isodityrosine

F i g u r e 4. S t r u c t u r e s of t y r o s i n e , d i t y r o s i n e a n d i s o d i t y r o s i n e . [ R e p r o d u c e d w i t h p e r m i s s i o n f r o m Journal of Expenmental Botany 38, 853-62; © O x f o r d U n i v e r s i t y P r e s s , 1987.

these residues are q u i t e capable of f o r m i n g d i t y r o s i n e (38) under the same o p t i m a l in vitro c o n d i t i o n s as w o r k for v i r t u a l l y a n y other p r o t e i n (e.g., b o v i n e s e r u m a l b u m i n ) (42). T h e o n l y in vitro s y s t e m i n w h i c h i s o d i t y r o s i n e p r o d u c t i o n p r e d o m i n a t e s is i n e x t e n s i n treated w i t h i s o l a t e d p l a n t cell w a l l s (38) at p H 6. T w o factors associated w i t h the p l a n t cell w a l l were considered w h i c h m i g h t direct exclusive i s o d i t y r o s i n e f o r m a t i o n : a. The pH of the plant cell wall T h i s p H is always l i k e l y t o be b e l o w 7.0 in vivo, a n d therefore w e l l below the o p t i m u m ( p H ca. 9.0) for t o t a l d i m e r i z a t i o n of tyrosine; p H 9.0 was used i n most in vitro assays other t h a n those where cell walls were the source of peroxidase. b . Specific isoperoxidases. T h e cell w a l l c o n t a i n s a n u m b e r o f peroxidase isozymes, q u i t e d i s t i n c t f r o m the m a j o r basic i s o z y m e o b t a i n e d f r o m horseradish a n d used i n most in vitro assays; i t is possible t h a t some of these other isozymes have a p r o p e n s i t y to c a t a l y z e i s o d i t y r o s i n e formation.

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Evidence Against Cell Wall pH and Isoperoxidase Specificity as Determi­ nants of Isodityrosine Formation. T o test these possible e x p l a n a t i o n s for the exclusive f o r m a t i o n of isodityrosine i n the p l a n t cell w a l l , samples of [ C ] t y r o s i n e were o x i d i z e d b y H 2 O 2 i n the presence of three s h a r p l y c o n ­ t r a s t i n g horseradish-isoperoxidases [two l o w - p i (acidic) a n d one h i g h - p i ( b a ­ sic)] at a wide range of p H values (37). T h e two l o w - p i isozymes were considerably poorer t h a n the h i g h - p l isozyme at c a t a l y z i n g t o t a l t y r o s i n e - o x i d a t i o n [the c o m p a r i s o n was based o n the use of a constant 1.2 / / k a t / m l of each isozyme, 1 / i k a t b e i n g the a m o u n t t h a t w i l l catalyze the o x i d a t i o n of p y r o g a l l o l to p u r p u r o g a l l i n at 1 μ η ι ο ΐ / s at p H 6.0 a n d 2 5 ° C ] . T h e l o w e r - p i isozyme o x i d i z e d tyrosine o p t i m a l l y at p H 6, a n d the h i g h - p i isozyme h a d an o p t i m u m of p H 9. T h e rate of o x i d a t i o n by the h i g h - p i isozyme at p H 9 was a b o u t seven t i m e s greater t h a n t h a t of the l o w e r - p l isozyme at p H 6. T h i s confirms t h a t the isozymes e x h i b i t e d considerable differences i n c a t a l y t i c properties, as well as differing i n p i . 14

W h e n the d i m e r s p r o d u c e d b y these isozymes, at a w i d e range of ρ H values, were a n a l y z e d i n d i v i d u a l l y , no great difference was f o u n d (37). A t h i g h p H values ( p H 8-10), b o t h the l o w - p l a n d h i g h - p i isozymes generated ca. 20 times more dityrosine t h a n i s o d i t y r o s i n e . A s the p H was lowered, the y i e l d of i s o d i t y r o s i n e increased a n d t h a t of d i t y r o s i n e decreased u n t i l at p H 3 there was o n l y about 2-3 times more d i t y r o s i n e t h a n i s o d i t y r o s i n e ; however, under no c o n d i t i o n s d i d the y i e l d of i s o d i t y r o s i n e ever exceed t h a t of d i t y r o s i n e . It m a y be concluded t h a t neither p H nor isozyme-specificity is l i k e l y t o direct the exclusive f o r m a t i o n of i s o d i t y r o s i n e i n the p l a n t cell w a l l i n vivo. Indeed, i t m i g h t be argued t h a t isozyme-specificity is i n t r i n s i c a l l y u n l i k e l y t o direct the o r i e n t a t i o n of c o u p l i n g ( d i t y r o s i n e vs. isodityrosine) since the role of the enzyme is thought to be merely the p r o d u c t i o n of t y r o s i n e free r a d i c a l s (44) w h i c h t h e n non-enzymically p a i r off. Role of Neighboring Polysaccharide Molecules in Determining the Orienta­ tion of Tyrosine Residues During Coupling. These considerations suggest a t h i r d possible e x p l a n a t i o n for the exclusive f o r m a t i o n o f i s o d i t y r o s i n e i n the p l a n t cell w a l l i n vivo: t h a t the n e i g h b o r i n g s t r u c t u r a l molecules of the w a l l c o n s t r a i n extensin to prevent dityrosine f o r m a t i o n . T h i s w o u l d m e a n t h a t the b i o l o g i c a l l y relevant s u b s t r a t e for peroxidase i n the p l a n t cell w a l l is not n a k e d e x t e n s i n b u t extensin complexed w i t h another w a l l c o m p o ­ nent, p o s s i b l y a n acidic polysaccharide to w h i c h the e x t e n s i n w o u l d b i n d ionically. Conclusions In c o n c l u s i o n , i t seems fair to say t h a t specificity exists i n b o t h the b i o s y n ­ thesis a n d i n the o x i d a t i v e c o u p l i n g of p o l y m e r - b o u n d phenols i n the grow­ i n g cell w a l l , (a) T y r o s i n e residues are placed at specific sites a l o n g the extensin molecule b y genetically-encoded i n f o r m a t i o n , (b) T y r o s i n e crossl i n k i n g i n vivo is a very specific, carefully steered process i n t h a t i t occurs

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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o n l y w h e n t h e extensin is i n a precise molecular e n v i r o n m e n t , p o s s i b l y as a n i o n i c c o m p l e x w i t h a n a c i d i c p o l y s a c c h a r i d e . T h i s i s evidenced b y t h e fact t h a t cell walls couple their t y r o s i n e residues t o m a k e i s o d i t y r o s i n e r a t h e r t h a n d i t y r o s i n e , whereas t h e same residues i n t h e absence o f a cell w a l l generate m a i n l y d i t y r o s i n e . (c) T h e f e r u l o y l a t i o n a n d p - c o u m a r o y l a t i o n of a c i d i c polysaccharides occurs o n h i g h l y specific h y d r o x y g r o u p s , (d) I t r e m a i n s t o be seen h o w precise o r r a n d o m t h e c o u p l i n g o f p o l y s a c c h a r i d e b o u n d p h e n o l i c side-chains i s . T h e significance o f t h i s precision is t h a t i t suggests t h a t adequate s p e c i ­ ficity exists for t h e c o u p l i n g reactions t o take p a r t efficiently i n t h e " s t r a p ­ p i n g " o f hemicellulose molecules t o m i c r o f i b r i l s m e n t i o n e d earlier. It w i l l b e of great interest i n future research t o explore w h e t h e r a n d t o w h a t extent such " s t r a p p i n g " occurs. A cknowledgment s W e are grateful t o t h e A g r i c u l t u r a l a n d F o o d Research C o u n c i l for t h e a w a r d o f a research g r a n t i n s u p p o r t o f o u r w o r k . Literature Cited

1. Fry, S. C. Ann. Rev. Plant Physiol. 1986, 37, 165-86. 2. Fry, S. C. The Growing Plant Cell Wall: Chemical and Metabolic Anal­ ysis; Longman: London; and Wiley: New York, 1988. 3. Keegstra, K.; Talmadge, K. W.; Bauer, W. D.; Albersheim, P. Plant Physiol. 1973, 51, 188-96. 4. Darvill, A. G.; McNeil, M.; Albersheim, P.; Delmer, D. P. In The Bio­ chemistry of Plants: A Comprehensive Treatise; Vol. 1; Preiss, J., Ed.; Academic Press: New York, 1980; pp. 91-162. 5. Lamport, D. T. A. In Cellulose: Structure, Modification and Hydrol­ ysis; Young, R. Α.; Rowell, R. M., Eds.; Wiley: New York, 1986; pp. 77-90. 6. Stafstrom, J. P.; Staehelin, L. A. Planta 1988, 174, 321-32. 7. Rubin, G. C. This volume. 8. Fry, S. C. J. Exp. Bot. 1989, 40, 1-11. 9. O'Neill, R. Α.; Selvendran, R. R. Carbohydr. Res. 1983, 111, 239-55. 10. Hayashi, T.; Marsden, M. P. F.; Delmer, D. P. Plant Physiol. 1987, 83, 384-9. 11. MacKay, A. L.; Wallace, J. C.; Sasaki, K.; Taylor, I. E. P. Biochemistry 1988, 27, 1467-73. 12. MacKay, A. L.; Wallace, J. C.; Sasaki, K.; Taylor, I. E. P. This volume. 13. Fry, S. C. Mod. Meth. Plant Analysis, New Series, 10, in press. 14. Heckman, J. W.; Terhune, B. T.; Lamport, D. T. A. Plant Physiol. 1988, 86, 848-56. 15. Stafstrom, J. P.; Staehelin, L. A. Plant Physiol. 1986, 81, 234-41. 16. Fry, S. C. Biochem. J. 1982, 204, 449-55. 17. Fry, S. C. Biochem. J. 1982, 203, 493-504. 18. Fry, S. C. Planta 1983, 157, 111-23.

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Kato, Y.; Nevins, D. J. Carbohydr. Res. 1985, 137, 139-50. Ahluwalia, B.; Fry, S. C. J. Cer. Sci. 1986, 4, 287-95. Smith, M. M.; Hartley, R. D. Carbohydr. Res. 1983, 118, 65-80. Gubler, F.; Ashford, A. E.; Bacic, Α.; Blakeney, A. B.; Stone, B. A. Ausi. J. Plant Physiol. 1985, 12, 307-17. Sachs, J. Handbuch der Experimentalphysiologie der Pflanzen; Engelmann: Leipzig, 1865. Hanke, D. E.; Northcote, D. H. J. Cell Sci. 1974, 14, 29-50. Fry, S. C. Physiol. Plant. 1989, 75, in press. Smith, J. J.; Muldoon, E. P.; Lamport, D. T. A. Phytochemistry 1984, 23, 1233-40. McNeil, M.; Darvill, A. G.; Fry, S. C.; Albersheim, P. Ann. Rev. Biochem. 1984, 53, 625-63. Hayashi, T.; Wong, Y.; Maclachlan G Plant Physiol 1984 75 605-10 Shinkle, J. R.; Jones Fry, S. C. Phytochemistry 1984, 23, 59-64. Fry, S. C.; Miller, J. G. Food Hydrocolloids 1987, 1, 395-7. Biggs, K. J.; Fry, S. C. In Physiology of Cell Expansion During Plant Growth; Cosgrove, D. J.; Knievel, D. P., Eds.; Am. Soc. Plant Physiol.; 1987; pp. 46-57. Chen, J.; Varner, J. E. EMBO J. 1985, 4, 2145-51. Fry, S. C. Planta 1987, 171, 205-11. Fry, S. C.; Northcote, D. H. Plant Physiol. 1983, 73, 1055-61. Yamamoto, E.; Towers, G. H. N. J. Plant Physiol. 1985, 117, 441-9. Yamamoto, E.; Bokelman, G. H.; Lewis, N. G., this volume. Cooper, J. B.; Varner, J. E. Plant Physiol. 1984, 76, 414-7. Holloway, P. J. In The Plant Cuticle; Cutler, D. F.; Alvin, K. L.; Price, C. E., Eds.; Academic: London, 1982; pp. 45-85. Andersen, S. O. Biochim. Biophys. Acta 1964, 93, 213-5. Lopez-Llorca, L. V.; Fry, S. C. Nematologica 1989, in press. Aeschbach, R.; Amado, R.; Neukom, H. Biochim. Biophys. Acta 1976, 439, 292-301. Fry, S. C. J. Exp. Bot. 1987, 38, 853-62. Ralston, I. M.; Dunford, H. B. Can. J. Chem. 1980, 58, 1270-6.

RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 4 Deposition of C e l l W a l l Components in Conifer Tracheids Keiji Takabe , Kazumi Fukazawa , and Hiroshi Harada 1

1

2

1Faculty of Agriculture, Hokkaido University, Kita 9, Nishi 9, Kita-ku, 2

Sapporo, Japan Rector Office, Kasetsart University, Bangkhen, Bangkok, 10900, Thailand

The cell wal depositio processes, cell wall components, and cell organellae involved in the biosynthesis of polysaccharides and lignin in conifer tracheids were investigated using several chemical and microscopy techniques. The deposition process for cellulose was found to differ from that of hemicelluloses. Cellulose deposited actively between the S and S developmental stages, especially in the middle part of the S stage. On the other hand, mannans and xylans were laid down between the latter part of S and the early part of S development, and between the latter part of the S and S stages. These results suggest that (i) the middle portion of S is rich in cellulose, and (ii) hemicelluloses are abundant in S , and the outer and inner portions of S and S tissue. Lignification was initiated at the outer surface of the primary wall cell corners, and proceeded into the intercellular layers, and the intercellular substances between cell corners. Lignification of the secondary walls was initiated at the S cell corner, then proceeded to the unlignified S layer and toward the lumen, while lagging behind cell wall thickening. Cellulose synthesis occurred at the plasma membrane. The Golgi-body, and a small circular vesicle derived from the rough endoplasmic reticulum, were involved in the biosynthesis and/or transport of hemicelluloses, while the Golgi-body and smooth-endoplasmic reticulum were involved in the biosynthesis and/or transport of monolignols. 1

3

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0097~6156/89/0399-0047$06.00A) © 1989 American Chemical Societv

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PLANT C E L L W A L L POLYMERS

C e l l u l o s e , hemicelluloses, a n d l i g n i n are the m a i n c o m p o n e n t s of cell walls i n w o o d y p l a n t s . For a l o n g t i m e , these p l a n t p o l y m e r s have s t i m u l a t e d the interest o f m a n y p l a n t botanists a n d biochemists i n terms of their b i o s y n thetic pathways, functional interrelationships, and anatomical distribution. T h e cell w a l l of conifer tracheids consists of b o t h p r i m a r y a n d seco n d a r y w a l l s . T h e p r i m a r y w a l l is formed d u r i n g cell d i v i s i o n a n d s u b sequent cell enlargement, whereas secondary w a l l f o r m a t i o n o n l y occurs after cell enlargement has been c o m p l e t e d . Secondary walls are s u b d i v i d e d i n t o three layers, n a m e d S i , S2, a n d S3, respectively. A large effort has been devoted to e l u c i d a t i n g (i) s t r u c t u r a l a n d c h e m i c a l properties o f the polysaccharides a n d l i g n i n i n each cell w a l l layer a n d their f u n c t i o n a l i n t e r r e l a t i o n s h i p s , a n d (ii) the role of cell organellae i n cell w a l l biosynthesis. I n spite of t h i s , our knowledge of the entire process of cell w a l l c o n s t r u c t i o n a n d the i n t e r r e l a t i o n s h i p Deposition of Polysaccharides P i o n e e r i n g w o r k o n p o l y s a c c h a r i d e d i s t r i b u t i o n i n the cell w a l l was carried out b y M e i e r a n d W i l k i e (1) a n d M e i e r (2). T h e y isolated r a d i a l sections f r o m the differentiating x y l e m of Pinus sylvestris, Picea abies, a n d Betula verrucosa, separated each i n t o s u b c e l l u l a r fractions b y a m i c r o m a n i p u l a t o r , a n d a n a l y z e d the monosaccharide c o m p o s i t i o n of the different p o l y s a c c h a r i d e fractions b y paper c h r o m a t o g r a p h y . F r o m these studies i t was concluded t h a t , i n softwood tracheids, the outer p a r t of the S2 layer was richest i n cellulose, whereas the S3 layer was richest i n g l u c u r o n o a r a b i n o x y l a n a n d the g l u c o m a n n a n content g r a d u a l l y increased towards the l u m e n . O n the other h a n d , i n h a r d w o o d fibers, the inner p a r t of S a n d S3 were richest i n cellulose, w h i l e S i a n d the outer p a r t of S2 showed a h i g h g l u c u r o n o x y l a n content. Côté et al. (3) also investigated the p o l y s a c c h a ride d i s t r i b u t i o n i n Abies balsamea tracheids a c c o r d i n g to the m e t h o d of M e i e r a n d o b t a i n e d s i m i l a r conclusions. 2

L a r s o n (4,5) fed C 0 2 p h o t o s y n t h e t i c a l l y to Pinus resinosa, d i v i d e d the differentiating x y l e m i n t o several fractions, a n d c o u n t e d the r a d i o a c t i v i t y of each cell w a l l c o m p o n e n t . F r o m these studies, i t was c o n c l u d e d t h a t as t r a c h e i d m a t u r a t i o n o c c u r r e d , xylose d e p o s i t i o n increased, whereas mannose r e m a i n e d r e l a t i v e l y c o n s t a n t , a n d b o t h arabinose a n d galactose decreased considerably. 1 4

In recent years, H a r d e l l a n d W e s t e r m a r k (6) scratched Picea abies t r a cheids w i t h tweezers, collected i n d i v i d u a l cell w a l l layers, a n d then a n a l y z e d the average monosaccharide c o m p o s i t i o n . S u r p r i s i n g l y , a m o n g the i n d i v i d u a l cell w a l l layers no significant difference i n the mannose:xylose:glucose r a t i o a m o n g i n d i v i d u a l cell w a l l layers was observed. W e have also s t u d i e d polysaccharide d e p o s i t i o n processes d u r i n g cell w a l l f o r m a t i o n (7), b y g a s - l i q u i d c h r o m a t o g r a p h i c a n a l y s i s of fractions sep-

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4.

T A K A B E et A L .

Cell Wall Components in Conifer Tracheids

a r a t e d b y m i c r o f r a c t i o n a t i o n a n d enriched i n cells at different t a l stages. ria japonica

49 developmen-

I n t h a t i n v e s t i g a t i o n , the d i f f e r e n t i a t i n g s y s t e m of

Cryptome-

was separated i n t o twelve fractions a n d each h a d its n e u t r a l

monosaccharide

content a n d t y p e d e t e r m i n e d ( F i g s . 1 a n d 2). ( N o t e t h a t

glucose, m a n n o s e , a n d xylose are m a i n l y d e r i v e d f r o m cellulose, m a n n a n s , a n d x y l a n s , respectively.) T h u s , i t was c o n c l u d e d t h a t cellulose was m a i n l y deposited i n the m i d d l e p a r t of the S2 layer, whereas hemicelluloses

(man-

n a n s , x y l a n ) were f o u n d m a i n l y i n the S i a n d the outer p a r t of the S2 a n d S3 layers ( F i g . 3). T h e s e results were f u r t h e r c o n f i r m e d f r o m o u r t r a n s m i s s i o n electron m i c r o s c o p y

( T E M ) studies o n d i f f e r e n t i a t i n g x y l e m s t a i n e d

w i t h P A T A g . T h i s technique, developed by Thiéry (8), is specific for h e m i celluloses a n d showed h e a v y s t a i n i n g o f the S i , outer S2 a n d S3 layers. Interestingly, the w a r t y layer was also h e a v i l y s t a i n e d b y P A T A g , i n d i c a t i n g i t to be m a i n l y c o m p o s e W e next investigated w a l l p o l y m e r s d u r i n g cell w a l l f o r m a t i o n (9).

A f t e r i n c o r p o r a t i o n o f the

l a b e l l e d sugar, the differentiating x y l e m of Cryptomeria

japonica

was sepa-

r a t e d i n t o 8 f r a c t i o n s , each of w h i c h was subjected to m i l d a c i d h y d r o l y s i s . T h e monosaccharides, so released f r o m the polysaccharides, were t h e n sepa r a t e d b y t h i n - l a y e r c h r o m a t o g r a p h y (tic) a n d the i n d i v i d u a l r a d i o a c t i v i t y content for each sugar was v i s u a l i z e d b y a u t o r a d i o g r a p h y a n d by scintillation counting.

measured

A s can be seen f r o m F i g u r e 4, the d i s t r i b u t i o n

o f r a d i o a c t i v i t y i n t o the i n d i v i d u a l sugars was i n g o o d agreement w i t h o u r previous analyses of the p o l y m e r s , i.e., cellulose d e p o s i t i o n m a i n l y o c c u r r e d i n the m i d d l e p a r t of the S2 to S3 d e v e l o p m e n t a l stage, whereas x y l a n dep o s i t i o n was i n the S i t o e a r l y S2 a n d a g a i n i n the S3 d e v e l o p m e n t a l layers. M a n n a n d e p o s i t i o n o c c u r r e d m a i n l y d u r i n g secondary w a l l f o r m a t i o n r a t h e r t h a n d u r i n g f o r m a t i o n of the p r i m a r y w a l l . Lignification of Tracheids T h e first s t u d y o n l i g n i f i c a t i o n of conifer W a r d r o p (10). radiata

tracheids was c a r r i e d out

F r o m e x a m i n a t i o n of the d i f f e r e n t i a t i n g x y l e m of

by

Pinus

under a n u l t r a v i o l e t microscope, he observed t h a t l i g n i f i c a t i o n was

i n i t i a t e d at the cell corners of the p r i m a r y w a l l , then extended t o the m i d dle l a m e l l a a n d secondary

wall.

I m a g a w a et al.

(11) subsequently

U V - p h o t o m i c r o g r a p h s of the differentiating x y l e m of Larix d e m o n s t r a t e d t h a t l i g n i f i c a t i o n proceeded as follows:

leptolepis

took and

lignin accumulation

begins i n the i n t e r c e l l u l a r layer at the cell corners a n d p i t b o r d e r s ,

then

extends to b o t h r a d i a l a n d t a n g e n t i a l m i d d l e l a m e l l a , a n d t h e n towards the l u m e n . F u j i t a et al. (12) investigated l i g n i f i c a t i o n of Cryptomeria

japonica

compression w o o d b y the same m e t h o d . These a u t h o r s f o u n d t h a t there are two types of l i g n i n d e p o s i t i o n processes: O n e was p r i m a r y w a l l l i g n i f i c a t i o n w h i c h o c c u r r e d f r o m the e a r l y phase of S i d e p o s i t i o n to the early phase of

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

PLANT C E L L W A L L POLYMERS

50

F i g u r e 1. C h a n g e s i n the absolute a m o u n t of sugars w i t h t r a c h e i d m a t u ­ r a t i o n . T h e various components are designated as follows: glucose, • ; m a n n o s e , A ; xylose, Δ ; arabinose, Q î d galactose, φ . a

n

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4.

TAKABE ET A L

Cell Wall Components in Conifer Tracheids

51

F i g u r e 2. T h e a m o u n t of increase i n sugars (the difference o f the a b s o l u t e a m o u n t s of sugar between the n e i g h b o r i n g f r a c t i o n s ) . T h e dashed l i n e shows the s u m of arabinose, galactose, x y l o s e , a n d mannose.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

PLANT C E L L W A L L P O L Y M E R S

52

(%) 100Gal.

80H Ara.

Xyl.

Ëlfllil

60-

Mit

Man.

AOH Glc

20·

1 • p-

2 —

2-3 Si

3-4 1

4-5

5-6 S2

6-7

7-8 1

8-9 S3

—

F i g u r e 3. D i s t r i b u t i o n of polysaccharides t h r o u g h the cell w a l l . C , cellulose; M , galacto-glucomannan; X , arabino-4-O-methylglucuronoxylan.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4.

TAKABE ET A L

u

Cell Wall Components in Conifer Tracheids

1

2

F i g u r e 4. T h e c o m p o s i t i o n

3 4 5 6 Fraction Number

7

53

8

o f r a d i o a c t i v i t y i n n e u t r a l sugars. A r a b i c n u -

merals are the f r a c t i o n n u m b e r s . T h e differentiating stages i n each f r a c t i o n are as follows: F r a c t i o n s 1-2, p r i m a r y w a l l stage; 3, S i stage; 4-6, S2 stage; 7-8, S 3 stage.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

54

PLANT C E L L W A L L P O L Y M E R S

S2 t h i c k e n i n g ; the other was secondary w a l l l i g n i f i c a t i o n w h i c h proceeded after S2 t h i c k e n i n g . A l t h o u g h U V - m i c r o s c o p y has p r o v i d e d m u c h i n f o r m a t i o n o n the l i g n i fication process a n d l i g n i n d i s t r i b u t i o n t h r o u g h the cell w a l l , i t has been o f l i m i t e d value because o f i t s low r e s o l v i n g power as c o m p a r e d t o electron microscopy. C o n s e q u e n t l y , some workers, u s i n g specimens fixed w i t h p o t a s s i u m p e r m a n g a n a t e , s t u d i e d l i g n i f i c a t i o n u s i n g T E M . I n t h i s way, W a r d r o p ( 1 3 , 1 4 ) f o u n d t h a t w i t h Eucalyptus elaeophora, l i g n i n f o r m a t i o n was i n i t i ated at the m i d d l e l a m e l l a o f the cell corners, a n d subsequently at the outer p a r t of the S i layer. It t h e n proceeded a l o n g the m i d d l e l a m e l l a , t h r o u g h the p r i m a r y w a l l , a n d u l t i m a t e l y to the secondary w a l l . K u t s c h a a n d S c h w a r z m a n n (15) also e x a m i n e d the l i g n i f i c a t i o n of Abies balsamea tracheids b y T E M a n d showed t h a t it was i n i t i a t e d i n the m i d d l e l a m e l l a between p i t borders of adjacent t r a c h e i d s cell corners. I n cell corners, i t o c c u r r e d at either the outer p o r t i o n o f the p r i m a r y w a l l or the m i d d l e l a m e l l a . A f t e r t h a t , l i g n i f i c a t i o n t o o k place i n the cell corner region of the S i layer, l e a v i n g the p r i m a r y w a l l u n l i g n i f i e d , a n d t h e n proceeded subsequently t o w a r d the l u m e n . T h i s technique, u s i n g p e r m a n g a n a t e fixation, c a n , however, cause s w e l l i n g of b o t h the cell a n d the cell w a l l , a n d the e x t r a c t i o n of m a n y cell c o m p o n e n t s . Indeed, K i s h i et ai (16) r e p o r t e d t h a t the s t a i n i n g i n t e n s i t y p r o d u c e d b y p e r m a n g a n a t e does not reflect true l i g n i n content, t h u s l e a v i n g the aforesaid results i n some d o u b t . S a k a a n d T h o m a s (17) also investigated the l i g n i f i c a t i o n of Pinus taeda tracheids b y the S E M - E D X A technique, a n d showed t h a t i t was i n i t i a t e d i n the cell corner m i d d l e l a m e l l a a n d c o m p o u n d m i d d l e l a m e l l a regions d u r i n g S i f o r m a t i o n . Subsequently, r a p i d l i g n i n d e p o s i t i o n o c c u r r e d i n b o t h regions. Secondary w a l l l i g n i f i c a t i o n was i n i t i a t e d w h e n the m i d d l e l a m e l l a l i g n i n c o n c e n t r a t i o n approached 5 0 % of its m a x i m u m , a n d t h e n proceeded towards the l u m e n . W e (18-20) have also investigated the l i g n i f i c a t i o n process u s i n g Cryptomeria japonica tracheids. Techniques employed were a d m i n i s t r a t i o n o f t r i t i a t e d p h e n y l a l a n i n e as a l i g n i n precursor, followed b y a c o m b i n a t i o n of U V - m i c r o s c o p y , light microscopic a u t o r a d i o g r a p h y , a n d T E M coupled w i t h a p p r o p r i a t e c h e m i c a l t r e a t m e n t s of u l t r a - t h i n sections. F i g u r e 5 shows densitometer traces o f U V - p h o t o n e g a t i v e s of differentiating x y l e m . T h e U V - a b s o r p t i o n of the c o m p o u n d m i d d l e l a m e l l a was first detected i n the t r a c h e i d at the S i d e v e l o p m e n t a l stage, t h e n increased d u r i n g secondary w a l l t h i c k e n i n g , b e c o m i n g constant after the S 3 stage. O n the other h a n d , U V - a b s o r p t i o n at the secondary w a l l was first observed at the outer p o r t i o n i n the t r a c h e i d of the S2 stage, t h e n spread slowly towards the l u m e n i n subsequent stages. T h e t r a c h e i d i n the final p a r t of cell w a l l f o r m a t i o n showed u n i f o r m a b s o r p t i o n t h r o u g h the secondary w a l l . F r o m F i g u r e 6,

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4.

T A K A B E et A L .

55

Cell Wall Components in Conifer Tracheids

F i g u r e 5. D e n s i t o m e t e r traces of U V - p h o t o n e g a t i v e s . A r a b i c n u m e r a l s i n ­ dicate the cell n u m b e r w h i c h s t a r t s f r o m the cell j u s t before S i f o r m a t i o n .

. 0

5

10

15

20

.?r - -. 25

30

Cell

ι • 0

. 5

Ο

• 15

20

,·,.... 25

30

Number

F i g u r e 6. I n c o r p o r a t i o n of t r i t i a t e d p h e n y l a l a n i n e i n t o the c o m p o u n d

mid­

dle l a m e l l a l i g n i n a n d the secondary w a l l l i g n i n d e t e r m i n e d b y c o u n t i n g the silver g r a i n s . S y m b o l s are as follows: φ , c o m p o u n d m i d d l e l a m e l l a ; 0 > secondary w a l l .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

56

PLANT C E L L W A L L P O L Y M E R S

it is evident t h a t the labelled l i g n i n precursor was r a p i d l y i n c o r p o r a t e d i n t o the c o m p o u n d m i d d l e l a m e l l a l i g n i n , whereas i t entered s l o w l y i n t o secondary w a l l l i g n i n . A d d i t i o n a l l y , i n c o r p o r a t i o n i n t o c o m p o u n d m i d d l e l a m e l l a l i g n i n took place d u r i n g S i a n d S2 d e v e l o p m e n t a l stages, w h i l e i n c o r p o r a t i o n i n t o secondary w a l l l i g n i n o c c u r r e d m a i n l y after the S3 stage. In the l a t t e r case, r a d i o a c t i v i t y was d i s t r i b u t e d t h r o u g h o u t the secondary w a l l . T h i s i n d i c a t e d t h a t m o n o l i g n o l s were c o n t i n u o u s l y s u p p l i e d to a l l a r eas o f the secondary w a l l . T h u s , the l i g n i n content i n the secondary w a l l g r a d u a l l y increased b y repeated l i n k i n g of m o n o l i g n o l r a d i c a l s . These findings were also s u p p o r t e d b y analysis of the l i g n i n skeleton of the d i f f e r e n t i a t i n g x y l e m , o b t a i n e d b y t r e a t m e n t of u l t r a - t h i n sections w i t h h y d r o f l u o r i c a c i d after resin e x t r a c t i o n (20). T h i s removes polysaccharides effectively w i t h o u t a n y s w e l l i n g of the cell w a l l . In t h i s way, we f o u n d t h a t l i g n i f i c a t i o n i n the Cryptomeria surface o f the p r i m a r y w a l Subsequently, i t proceeded to the i n t e r c e l l u l a r layer together w i t h l i g n i n d e p o s i t i o n i n the i n t e r c e l l u l a r substances between the cell corners. W h e n the t r a c h e i d was adjacent to a r a y p a r e n c h y m a , the cell corner region o n the r a y p a r e n c h y m a side was lignified earlier t h a n t h a t o n the opposite one. It was very i n t e r e s t i n g t h a t secondary w a l l l i g n i f i c a t i o n was also i n i t i a t e d at the S i cell corner region d u r i n g the S i d e v e l o p m e n t a l stage. It t h e n proceeded to the u n l i g n i f i e d S i layer. W h e n the t r a c h e i d was adjacent to a r a y p a r e n c h y m a , l i g n i f i c a t i o n of the S i layer was also earlier o n the r a y side. A f t e r t h a t , l i g n i f i c a t i o n g r a d u a l l y spread towards the l u m e n , l a g g i n g b e h i n d cell w a l l t h i c k e n i n g . L i g n i n d e p o s i t i o n t h e n p r e d o m i n a t e d after the S3 stage, w h i l e less active d u r i n g the S i , S2, a n d S3 d e v e l o p m e n t a l stages. T h e l i g n i n content of the secondary w a l l became f a i r l y constant i n the final stage of cell w a l l f o r m a t i o n , t h o u g h the w a r t y layer was more h i g h l y lignified. Changes in C e l l Organellae D u r i n g C e l l W a l l F o r m a t i o n T h e cell organellae i n w o o d y plants are the nucleus, m i t o c h o n d r i o n , r o u g h - e n d o p l a s m i c r e t i c u l u m ( r - E R ) , s m o o t h e n d o p l a s m i c r e t i c u l u m (sE R ) , G o l g i - b o d y , p l a s t i d , vacuole, m i c r o b o d y , etc. T h e i r functions are very c o m p l i c a t e d , a n d some have definite roles i n the biosynthesis o f c e l l - w a l l components. H e n c e , changes i n size of cell organellae are likely to o c c u r , since c e l l - w a l l c o m p o s i t i o n depends u p o n the stage of w a l l development. W e t r i e d to estimate the size of the cell organellae i n the c y t o p l a s m , e x c l u d i n g the vacuolar c o m p a r t m e n t . T o do this, we took at r a n d o m a few h u n d r e d electron m i c r o g r a p h s of cells i n the differentiating x y l e m a n d measured the area o f each cell organelle i n the c y t o p l a s m b y a d i g i t i z e r c o u p l e d to a m i c r o c o m p u t e r . W e f o u n d not o n l y changes i n the area of cell organellae i n the c y t o p l a s m , b u t also i n their s t r u c t u r e d u r i n g cell

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4.

TAKABE ET A L

wall formation.

57

Cell Wall Components in Conifer Tracheids

F i g u r e 7 shows the results o f s e m i - q u a n t i t a t i v e a n a l y s i s

of the areas for each cell o r g a n e l l a s d u r i n g cell w a l l f o r m a t i o n .

It was

s u r p r i s i n g t h a t the G o l g i - b o d y , r - E R , a n d s - E R , showed a p p r e c i a b l e changes i n area d u r i n g cell w a l l f o r m a t i o n . T h e area o f the G o l g i - b o d y was largest at the S i stage, a n d t h e n g r a d u a l l y decreased i n size w i t h m a t u r a t i o n o f the t r a c h e i d , whereas the r - E R was largest i n the p r i m a r y w a l l stage, a n d t h e n g r a d u a l l y decreased w i t h cell w a l l f o r m a t i o n . T h e s - E R , o n the other h a n d , was a m i n o r organelle f r o m the p r i m a r y w a l l stage t o the e a r l y p a r t o f the S2 stage, a n d t h e n showed a g r a d u a l increase i n a r e a t o w a r d the S3 d e v e l o p m e n t a l stage. T h e m o s t s t r i k i n g fact was t h a t the e n l a r g e m e n t o f the s - E R c o i n c i d e d w i t h t h a t o f active l i g n i f i c a t i o n of the secondary w a l l . F i g u r e 8 shows the changes i n the s t r u c t u r e o f cell o r g a n e l l a s .

The

p h o t o g r a p h s are t y p i c a l s t r u c t u r e s i n each d i f f e r e n t i a t i n g stage. N o t e t h a t the G o l g i - b o d y consists o d u r i n g the p r i m a r y w a l l stage t h i s b e i n g a c c o m p a n i e d b y the f o r m a t i o n o f m a n y large vesicles c o n t a i n i n g fibrillar

m a t e r i a l d u r i n g the S i a n d S2 d e v e l o p m e n t a l stages.

After that,

the c e n t r a l cisternae b e c a m e s m a l l i n size, t h o u g h the t h i c k n e s s was s i m i l a r t o t h a t o f p r e v i o u s stages. Interestingly, t h i s stage appears to be a c c o m p a n i e d b y the f o r m a t i o n of o n l y a few vesicles, i n d i c a t i n g depression of G o l g i activity. S e v e r a l r e t i c u l a of the r - E R show a n ordered a r r a n g e m e n t a n d m a n y ribosomes are a t t a c h e d to t h e i r m e m b r a n e d u r i n g the p r i m a r y w a l l d e v e l o p m e n t stage. A s m a t u r a t i o n proceeds, the r - E R ' s t h e n g r a d u a l l y decrease not o n l y i n n u m b e r a n d l e n g t h of r e t i c u l a , b u t also i n the n u m b e r o f r i b o somes. T h e s - E R ' s , o n the other h a n d , b e c o m e largest after the S3 stage, a n d sometimes a t t a c h ribosomes at their t e r m i n a l s . D u r i n g p r i m a r y w a l l f o r m a t i o n the p l a s t i d s c o n t a i n s t a r c h a n d other m a t e r i a l s w h i c h s t a i n h e a v i l y w i t h u r a n y l acetate a n d l e a d c i t r a t e . W h e n the t r a c h e i d s t a r t s to f o r m the S i layer, the p l a s t i d becomes s u r r o u n d e d b y a n e n d o p l a s m i c r e t i c u l u m . W h i l e the fate of these c o m p o u n d s is u n k n o w n , it c a n be envisaged t h a t they are used for generation o f energy a n d / o r a source o f cell w a l l m a t e r i a l s . C e l l Organellae Involved in Biosynthesis of Polysaccharides T h o u g h cellulose is one of the most i m p o r t a n t b i o p o l y m e r s , i t has not yet been possible to c o m p l e t e l y e l u c i d a t e i t s b i o s y n t h e t i c p a t h w a y , or e s t a b l i s h e x a c t l y the cell organellae i n v o l v e d i n its synthesis. H o w e v e r , d u r i n g the last decade, the freeze fracture technique has been a p p l i e d to investigate cell w a l l f o r m a t i o n , a n d t h i s has p r o d u c e d m u c h i n f o r m a t i o n o n the site where cellulose synthesis occurs.

It is now generally accepted t h a t b o t h t e r m i -

n a l a n d rosette complexes are responsible for cellulose synthesis (21). O u r results (19,22) s u p p o r t t h a t v i e w .

In a T E M - a u t o r a d i o g r a p h i c investiga-

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F i g u r e 7. S e m i - q u a n t i t a t i v e measurements of cell organellae i n the c y t o plasm.

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F i g u r e 8. C h a n g e s i n the s t r u c t u r e of cell organellae d u r i n g cell w a l l form a t i o n . U p p e r , m i d d l e , a n d lower p h o t o g r a p h s are G o l g i - b o d y , r - E R , a n d p l a s t i d , respectively. A b b r e v i a t i o n s are as follows: P , p r i m a r y w a l l stage; S2#, e a r l y p a r t of S2 stage; S 2 L , later p a r t of S2 stage.

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t i o n of differentiating x y l e m , p r e v i o u s l y a d m i n i s t e r e d t r i t i a t e d glucose, the r a d i o a c t i v i t y was concentrated at the b o u n d a r y between the n e w l y f o r m e d c e l l - w a l l a n d the c y t o p l a s m i n the m i d d l e a n d l a t t e r p a r t s of the S2 stage. A c o m p a r i s o n w i t h F i g u r e 4 indicates t h a t a m a j o r c o m p o n e n t of the l a b e l l e d m a t e r i a l s i n these areas was i n the cellulose p o l y m e r , a n d hence glucose derived. F r o m a cytochemical investigation, fibrillar materials having a w i d t h of 6-7 m m were sometimes observed, these b e i n g generated f r o m the p l a s m a m e m b r a n e ( F i g . 9). T h e y h a d s i m i l a r dimensions to cellulose m i c r o f i b r i l s f r o m gelatinous layers of Populus euramericana as s h o w n b y S u g i y a m a et ai (23), thereby i n d i c a t i n g the involvement of the p l a s m a m e m b r a n e i n cellulose synthesis. T h e biosynthesis of cell w a l l polysaccharides has also been s t u d i e d b y c y t o c h e m i c a l s t a i n i n g m e t h o d s . P A T A g s t a i n i n g (8) resulted i n e s t a b l i s h i n g cell organellae i n v o l v e P i c k e t t - H e a p s (24) a d a p t e d t h i s s t a i n i n g procedure to the root t i p s a n d coleoptiles of Triticum vulgare seedlings. I n t h i s way, he showed t h a t G o l g i cisternae a n d their vesicles were s t a i n e d positively, whereas E R cisternae s t a i n e d negatively. T h i s led to the conclusion t h a t G o l g i - b o d i e s were i n v o l v e d i n the biosynthesis a n d / o r t r a n s p o r t of polysaccharides. T h e s e results were confirmed a g a i n i n later studies b y Fowke a n d P i c k e t t - H e a p s (25) a n d R y s e r (26). I n recent years, S u g i y a m a et ai (27) observed the Valonia macrophysa c e l l - w a l l b y means of selective v i s u a l i z a t i o n a n d c h e m i c a l a n a l ysis of c e l l - w a l l components. T h e authors f o u n d t h a t the m a t e r i a l s s t a i n e d p o s i t i v e l y w i t h P A T A g , or a c o m b i n a t i o n of u r a n y l acetate a n d lead c i t r a t e , were non-cellulosic polysaccharides. W e have also a p p l i e d the P A T A g s t a i n i n g to the d i f f e r e n t i a t i n g x y l e m of Cryptomeria japonica (22). W h i l e the contents i n the Golgi-vesicles s t a i n e d p o s i t i v e l y at a l l stages of cell w a l l f o r m a t i o n , there were three m a i n observ a t i o n s . D u r i n g p r i m a r y w a l l f o r m a t i o n , the Golgi-vesicles were s m a l l a n d their contents o n l y s t a i n e d weakly. A f t e r t h a t , the vesicles b e c a m e larger a n d the c o m p o n e n t s w h i c h showed f i b r i l l a r s t r u c t u r e s were s t r o n g l y s t a i n e d . F o l l o w i n g the S 3 stage, the contents a g a i n s t a i n e d weakly, a n d h a d a s l i m y appearance. T h i s p r e s u m a b l y (i) reflects changes i n non-cellulosic p o l y s a c charide c o m p o s i t i o n a n d (ii) suggests t h a t the G o l g i - b o d i e s are i n v o l v e d i n the biosynthesis of non-cellulosic polysaccharides. F i g u r e 10 shows s m a l l c i r c u l a r vesicles w h i c h were d i s t r i b u t e d at the e n d o f the r - E R cisternae a n d between the cisternae, a n d w h i c h were sometimes a t t a c h e d to the E R m e m b r a n e . A s the size a n d the shape of these vesicles (75 n m i n m e a n diameter) were different f r o m those of Golgi-vesicles (130 n m i n m e a n d i a m e t e r ) , the s m a l l c i r c u l a r vesicles were p r e s u m a b l y der i v e d f r o m r - E R ; they also s t a i n e d p o s i t i v e l y w i t h P A T A g . These facts suggest t h a t the s m a l l c i r c u l a r vesicles f r o m the E R are i n v o l v e d i n the biosynthesis a n d / o r t r a n s p o r t of non-cellulosic polysaccharides.

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F i g u r e 9. A t r a c h e i d i n the S3 stage. F i b r i l l a r m a t e r i a l s (arrows) are generated f r o m the p l a s m a m e m b r a n e . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 22. ©

1986, J a p a n W o o d Research Society.)

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F i g u r e 10. A differentiating t r a c h e i d stained w i t h P A T A g . S m a l l c i r c u l a r vesicles (arrowheads), d i s t r i b u t e d near the E R are s t a i n e d positively. T h e Golgi-vesicles are also s t a i n e d positively. A b b r e v i a t i o n s are as follows: G V , G o l g i - v e s i c l e ; E R , e n d o p l a s m i c r e t i c u l u m . Scale bar is 5 0 0 n m . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 22. ©

1986, J a p a n W o o d Research Society.)

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63

C e l l Organellae Involved in the Biosynthesis of L i g n i n O n l y a few workers have a t t e m p t e d to i d e n t i f y the cell organellae i n v o l v e d i n the biosynthesis of l i g n i n . P i c k e t t - H e a p s (28) observed l i g n i f i c a t i o n i n the x y l e m w a l l of wheat coleoptiles a n d suggested the i n v o l v e m e n t of b o t h the G o l g i - b o d i e s a n d r - E R , since r a d i o a c t i v i t y was d i s t r i b u t e d o n b o t h o r g a n e l lae. M o r e recently, F u j i t a et al. (29) s t u d i e d l i g n i f i c a t i o n i n c o m p r e s s i o n w o o d cell walls of Cryptomeria

japonica

by T E M - a u t o r a d i o g r a p h y . Like

P i c k e t t - H e a p s , they c o n c l u d e d t h a t the G o l g i - b o d i e s p a r t i c i p a t e d i n l i g n i n biosynthesis. W a r d r o p ( 1 3 , 1 4 ) e x a m i n e d s c l e r e n c h y m a fibers of Liriodendron if era a n d sclereids of Pyrus

communis,

tulip-

p r e v i o u s l y fixed w i t h KMnÛ4, a n d

c o n c l u d e d t h a t the vesicles s u p p l i e d t h e i r contents to the cell w a l l , t h o u g h the o r i g i n of the vesicles was not e s t a b l i s h e d . A s discussed before, however, p e r m a n g a n a t e fixation is u n d e s i r a b l C o n s e q u e n t l y , we a d m i n i s t e r e d t r i t i a t e d p h e n y l a l a n i n e , a l i g n i n p r e c u r sor, t o the d i f f e r e n t i a t i n g x y l e m o f Cryptomeria

japonica,

and determined

the l o c a t i o n of the l a b e l b y T E M - a u t o r a d i o g r a p h y (30). T h e r a d i o a c t i v i t y was l o c a t e d o n the c o m p o u n d m i d d l e l a m e l l a , i n c l u d i n g the cell-corner regions f r o m the final p a r t of the p r i m a r y w a l l stage to the e a r l y p a r t of the S2 d e v e l o p m e n t a l stage. C o r r e s p o n d i n g to these stages, the r a d i o a c t i v i t y was d i s t r i b u t e d on G o l g i - b o d i e s w h i c h secreted m a n y vesicles, a n d r - E R ' s ( F i g . 11). R a d i o a c t i v i t y was a b u n d a n t l y l o c a t e d o n the secondary w a l l f r o m the S3 stage to the c o m p l e t i o n of secondary w a l l l i g n i f i c a t i o n . R a d i o a c t i v i t y was also l o c a t e d o n s - E R ' s ( F i g . 12). T h e r a d i o a c t i v i t y o n the cell w a l l coi n c i d e d w i t h l i g n i n d e p o s i t i o n , as e v i d e n c e d b y U V - m i c r o s c o p y a n d T E M . T h e s e results therefore suggest t h a t the G o l g i - b o d y , r - E R , a n d s - E R are a l l i n v o l v e d i n the biosynthesis of l i g n i n . However, since i t is w e l l k n o w n t h a t the r - E R is a site of p r o t e i n synthesis, some p h e n y l a l a n i n e m a y be used for this purpose.

O n the other h a n d , the G o l g i - b o d i e s secrete m a n y vesicles

d u r i n g active l i g n i f i c a t i o n of the c o m p o u n d m i d d l e l a m e l l a a n d secondary wall.

T h e a d m i n i s t e r e d l i g n i n precursor may, therefore, be i n c o r p o r a t e d

i n t o the G o l g i - b o d y d i r e c t l y or v i a other organellae, a n d t h e n converted i n t o m o n o l i g n o l s v i a e n z y m a t i c conversion. T h e m o n o l i g n o l s c a n t h e n be secreted i n t o the cell w a l l b y exocytosis of the G o l g i - v e s i c l e s . M a n y s - E R ' s also a p p e a r i n the c y t o p l a s m a n d these sometimes fuse to the p l a s m a m e m b r a n e at the S3 stage. T h u s , l i g n i n precursors m a y also be converted i n t o m o n o l i g n o l s at the l u m e n or the m e m b r a n e of s - E R ' s , a n d t h e n secreted i n t o the cell w a l l b y fusion of s - E R to the p l a s m a m e m b r a n e .

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F i g u r e 11. A t r a c h e i d i n the early part of S2 stage. R a d i o a c t i v i t i e s observed on the G o l g i - b o d i e s a n d c o m p o u n d m i d d l e l a m e l l a .

are

F i g u r e 12. A t r a c h e i d after S stage. C y t o p l a s m is filled w i t h s - E R ' s . R a d i o a c t i v i t y is d i s t r i b u t e d on the s - E R ' s a n d secondary w a l l . 3

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Concluding Remarks It is e v i d e n t t h a t d e p o s i t i o n processes for cellulose a n d hemicelluloses i n conifer tracheids are q u i t e different, i.e., cellulose is m a i n l y d e p o s i t e d i n t h e m i d d l e p a r t o f the S2 d e v e l o p m e n t a l stage, whereas h e m i c e l l u l o s e d e p o s i t i o n o c c u r s f r o m the S i t o the e a r l y p a r t o f t h e S2 stage, a n d t h e n d u r i n g the l a t t e r p a r t o f t h e S2 t o S3 stages.

A s a r e s u l t , the secondary

shows a heterogeneous d i s t r i b u t i o n o f p o l y s a c c h a r i d e s .

wall

Lignin deposition

lags b e h i n d the p o l y s a c c h a r i d e f o r m a t i o n . T h e m o s t s t r i k i n g fact is t h a t the m o n o l i g n o l s , w h i c h are synthesized i n the c y t o p l a s m a n d secreted t o t h e i n n e r surface o f the n e w l y f o r m e d cell w a l l , pass t h r o u g h the p r e - e x i s t i n g cell w a l l a n d reach the sites where l i g n i f i c a t i o n is p r o c e e d i n g . T h e r e i s , however, no i n f o r m a t i o n as t o h o w t h i s t r a n s p o r t a c t u a l l y occurs.

Moreover, the

i n t e r m o l e c u l a r r e l a t i o n s h i p s between cellulose, hemicelluloses a n d l i g n i n i n the cell w a l l are s t i l l u n c l e a r O u r electron m i c r o s c o p y observations have revealed some o f the roles o f cell organellae i n v o l v e d i n biosynthesis o f cell w a l l c o m p o n e n t s :

(i) t h e

p l a s m a m e m b r a n e is the site o f cellulose synthesis. T h i s s u p p o r t s the p r o p o s a l t h a t t e r m i n a l a n d rosette complexes at t h e p l a s m a m e m b r a n e are responsible for cellulose synthesis, ( i i ) T h e G o l g i - b o d i e s a n d s m a l l c i r c u l a r vesicles d e r i v e d f r o m the r - E R ' s are i n v o l v e d i n t h e b i o s y n t h e s i s a n d / o r t r a n s p o r t o f the hemicelluloses.

O u r i n v e s t i g a t i o n s , however, c o u l d n o t

d i s t i n g u i s h between w h a t t y p e o f cell organellae c o n t a i n e d w h a t k i n d o f hemicelluloses, a n d h o w these p o l y m e r s were processed i n t h e o r g a n e l l a e . (iii) T h e G o l g i - b o d i e s a n d s - E R ' s p a r t i c i p a t e i n the biosynthesis a n d / o r t r a n s p o r t o f m o n o l i g n o l s . It is expected t h a t new techniques o f b o t h elect r o n m i c r o s c o p y a n d b i o c h e m i s t r y w i l l i m p r o v e o u r knowledge o f the precise sites where e n z y m a t i c reactions l e a d i n g t o l i g n i n f o r m a t i o n o c c u r . Literature Cited

1. Meier, H.; Wilkie, K. C. B. Holzforschung 1959, 13, 177. 2. Meier, H. J. Polym. Sci. 1961, 51, 11. 3. Côté, W. Α., Jr.; Kutscha, N. P.; Simon, B. W.; Timell, T. E. Tappi 1968, 51, 33. 4. Larson, P. R. Holzforschung 1969, 23, 17. 5. Larson, P. R. Tappi 1969, 52, 2170. 6. Hardell, H.-L.; Westermark, U. Proc. 1st Int. Symp. Wood Pulping Chem. 1981, I:32. 7. Takabe, K.; Fujita, M.; Harada, H.; Saiki, H. Mokuzai Gakkaishi 1983, 29, 183. 8. Thiéry, J. J. Microscopie 1967, 6, 987. 9. Takabe, K.; Fujita, M.; Harada, H.; Saiki, H. Mokuzai Gakkaishi 1984, 30, 103. 10. Wardrop, A. B. Tappi 1957, 40, 225.

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11. Imagawa, H.; Fukazawa, K.; Ishida, S. Res. Bull. Coll. Exp. Forests, Hokkaido Univ. 1976, 33, 127. 12. Fujita, M.; Saiki, H.; Harada, H. Mokuzai Gakkaishi 1978, 24, 158. 13. Wardrop, A. B. In Lignins; Sarkanen, Κ. V.; Ludwig, C. H., Eds.; Wiley-Interscience: New York, 1971; p. 19. 14. Wardrop, A. B. Appl. Poly. Symp. 1976, 28, 1041. 15. Kutscha, N. P.; Schwarzmann, J. M. Holzforschung 1975, 29, 79. 16. Kishi, K.; Harada, H.; Saiki, H. Bull. Kyoto Univ. Forests 1982, 54, 209. 17. Saka, S.; Thomas, R. J. Wood Sci. Technol. 1982, 16, 167. 18. Takabe, K.; Fujita, M.; Harada, H.; Saiki, H. Mokuzai Gakkaishi 1981, 27, 813. 19. Takabe, K. Ph.D. Thesis, Kyoto University, Kyoto, 1984. 20. Takabe, K.; Fujita, M. Forests, Hokkaido Univ 21. Brown, R. M., Jr.; Haigler, C. H.; Suttie, J.; White, A. R.; Roberts, E.; Smith, C.; Itoh, T.; Cooper, K. J. Appl. Polym. Sci. (Appl. Polym. Symp.) 1983, 37, 33. 22. Takabe, K.; Harada, H. Mokuzai Gakkaishi 1986, 32, 763. 23. Sugiyama, J.; Otsuka, Y.; Murase, H.; Harada, H. Holzforschung 1986, 40(Suppl.), 31. 24. Pickett-Heaps, J. D. J. Cell. Sci. 1968, 3, 55. 25. Fowke, L. C.; Pickett-Heaps, J. D. Protoplasma 1972, 74, 19. 26. Ryser, U. Protoplasma 1979, 98, 223. 27. Sugiyama, J.; Harada, H. Mokuzai Gakkaishi 1986, 32, 770. 28. Pickett-Heaps, J. D. Protoplasma 1968, 65, 181. 29. Fujita, M.; Harada, H. Mokuzai Gakkaishi 1979, 25, 89. 30. Takabe, K.; Fujita, M.; Harada, H.; Saiki, H. Mokuzai Gakkaishi 1985, 31, 613. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 5

Phenylpropanoid Metabolism in Cell Walls An Overview Etsuo Yamamoto , Gordon H . Bokelman , and Norman G . Lewis 1

1

2

1

Departments of Wood Science and Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, V A 24061 Philip Morris U S A , Research Center, Richmond, V A 23261 2

Cell walls are major sites in which products of the phenylalanine-cinnamate pathway accumulate. These metabolites are found in cell walls in the form of (1) monomers, e.g., wall-esterified and ether-linked hy­ droxycinnamic acids, (2) dimers, e.g., didehydroferulic acid, 4,4'-dihydroxytruxillic acid, and (3) polymers, e.g., lignin, suberin. The distribution of these metabolites is characteristic of a particular group of plants, organs or tissues. There appear to be at leastfivetypes of reactions involved in the further conversion of phenylpropanoids in cell walls: (1) photochemical coupling, (2) E/Z iso­ merizations, (3) free-radical coupling reactions catalyzed by peroxidase, (4) hydrolysis of monolignol glucosides to their aglycone forms by β-glucosidases and (5) esterifi­ cation of hydroxycinnamic acids. These reactions are reviewed in relation to cell wall structure. A f a s c i n a t i n g assortment o f p h e n y l p r o p a n o i d s are c o n t a i n e d w i t h i n vascular p l a n t cell w a l l s a n d their vacuoles (1-6). I n the cell w a l l s , these substances ( m a i n l y l i g n i n s , suberins a n d c o v a l e n t l y - l i n k e d h y d r o x y c i n n a m i c acids) are n o r m a l l y a n i n t e g r a l p a r t o f the c o m p l e x w a l l s t r u c t u r e (7-9). I n terms of f u n c t i o n , l i g n i n s are required p r i n c i p a l l y for m e c h a n i c a l s u p p o r t , b u t b o t h they a n d suberins c a n also act as barriers t o diffusion (9,10) a n d m i c r o b i a l i n v a s i o n (11,12). O n the other h a n d , the s i t u a t i o n for w a l l - b o u n d h y d r o x y c i n n a m i c acids remains unclear. Several roles have been suggested b u t n o t p r o v e n , e.g., i n r e g u l a t i n g c e l l - w a l l extension (3,5,7), as a defense m e c h a n i s m against i n v a d i n g plant pathogens (5), a n d i n l i g n i f i c a t i o n ( 1 3 15). S u r p r i s i n g l y , l i t t l e a t t e n t i o n has been p a i d to (i) the m e c h a n i s m o f p h e n y l p r o p a n o i d t r a n s p o r t f r o m the c y t o p l a s m i n t o the cell w a l l a n d ( i i ) subsequent c h e m i c a l a n d b i o c h e m i c a l modifications w i t h i n the cell w a l l 0097-6156/89/0399-0068$06.00/0 © 1989 American Chemical Society

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s t r u c t u r e . S t u d i e s of t h i s t y p e are u r g e n t l y needed i f we are to define the r e g u l a t o r y processes c o n t r o l l i n g c e l l - w a l l development; such i n v e s t i g a t i o n s m a y , i n t u r n , enhance our u n d e r s t a n d i n g of the s t r u c t u r a l a n d p h y s i o l o g i c a l f u n c t i o n s of these m e t a b o l i t e s . U n f o r t u n a t e l y , o u r knowledge of the t r a n s p o r t m e c h a n i s m is c u r r e n t l y insufficient for a d e t a i l e d discussion. T h e p r i m a r y m e c h a n i s m can p r o b a b l y be e x p l a i n e d i n terms of the " e n d o m e m b r a n e t h e o r y " (16,17), i.e., p h e n y l p r o p a n o i d m o n o m e r s are t r a n s p o r t e d v i a vesicles to the p l a s m a m e m b r a n e , a n d are t h e n released i n t o the cell w a l l f o l l o w i n g m e m b r a n e f u s i o n . H o w ever, more extensive e x p e r i m e n t a l evidence is needed to u n a m b i g u o u s l y e s t a b l i s h the d e t a i l e d mechanism(s) i n v o l v e d . A s regards the second t o p i c , n a m e l y t h a t of p h e n y l p r o p a n o i d reactions w i t h i n p l a n t cell w a l l s , a more comprehensive discussion is possible a n d also t i m e l y , due to the recent increase i n interest i n t h i s area. F o r the purpose of t h i s review, the p h e n y l p r o p a n o i d s present i n p l a n t cell walls are first classified a c c o r d i n g to s t r u c t u r a etc.), f o l l o w i n g w h i c h their m a i n reactions are discussed. 1.

Classification of Phenylpropanoids i n Plant C e l l Walls

T h e p h e n y l p r o p a n o i d s can be classified i n t o two m a j o r groups, the first of w h i c h c o n t r i b u t e to a b r o a d class of c o m p o u n d s described as e x t r a c t i v e s . T h e s e c o m p o u n d s are n o n - s t r u c t u r a l entities of w o o d a n d b a r k tissue, a n d m a n y o r i g i n a t e i n the vacuoles. A s discussed i n some d e t a i l b y H i l l i s (18), some e x t r a c t i v e s (e.g., t a n n i n s ) m a y diffuse i n t o the cell w a l l m a t r i x followi n g r u p t u r e of the t o n o p l a s t . A n o t h e r i m p o r t a n t g r o u p of p h e n y l p r o p a n o i d s are the n o r m a l l y d i m e r i c l i g n a n s . U b i q u i t o u s i n n a t u r e a n d s t r u c t u r a l l y very diverse, i t is i n t r i g u i n g t h a t w i t h i n a given p l a n t species the stereo c h e m i s t r y of most l i g n a n s is n o r m a l l y very w e l l defined. ( T h i s suggests t h a t the m e c h a n i s m for their f o r m a t i o n differs r a d i c a l l y f r o m t h a t n o r m a l l y proposed for the closely-related p o l y m e r i c m a t e r i a l , l i g n i n . ) T h e second group of p h e n y l p r o p a n o i d s , w h i c h is the m a i n e m p h a s i s of t h i s chapter, consists of those c o m p o n e n t s w h i c h are i n t e g r a t e d i n t o the cell w a l l f r a m e w o r k . T h i s g r o u p can be s u b d i v i d e d i n t o three categories; m o n o m e r s , such as h y d r o x y c i n n a m i c acids, d i m e r s , s u c h as d i d e h y d r o f e r u l i c a n d 4 , 4 ' - d i h y d r o x y t r u x i l l i c acids, a n d p o l y m e r s , s u c h as l i g n i n s a n d s u b e r i n s . It is i m p o r t a n t to emphasize, at t h i s j u n c t u r e , t h a t the d i m e r s (4,5) a n d p o l y m e r s (8,9) discussed i n t h i s chapter are considered to be f o r m e d w i t h i n the cell walls f r o m their c o r r e s p o n d i n g m o n o m e r s . 1.1

Monomeric

Phenylpropanoids

(a) Hydroxy cinnamic Acids. M a n y angiosperme, p a r t i c u l a r l y those b e l o n g i n g to the G r a m i n e a e of the m o n o c o t y l e d o n s (2-5,13), a n d the C a r y o p h y l lales of the dicotyledons (19), c o n t a i n h y d r o x y c i n n a m i c acids l i n k e d to cell walls v i a ester-bonds. R e c e n t l y , H a r r i s a n d H a r t l e y c o n d u c t e d a s y s t e m a t i c h i s t o c h e m i c a l survey of c e l l - b o u n d h y d r o x y c i n n a m i c acids of over 350 a n g i o s p e r m p l a n t species, i n c l u d i n g some w o o d y p l a n t s , a n d the reader is referred to these papers for more detailed i n f o r m a t i o n (20,21). S u r p r i s i n g l y , no p u b l i s h e d d a t a for g y m n o s p e r m s is yet available.

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PLANT C E L L W A L L POLYMERS

H y d r o x y c i n n a m i c acids can be specifically v i s u a l i z e d i n p l a n t tissue b y t h e i r characteristic blue fluorescence when exposed to near u l t r a v i o l e t l i g h t , a n d a green fluorescence following exposure of the tissue to a m m o n i a (22-24). T h i s fluorescence is p a r t i c u l a r l y intense i n the e p i d e r m a l layers, vascular tissues a n d the s t o m a t a of grass leaves (24). T h e most c o m m o n h y d r o x y c i n n a m i c acids f o u n d i n cell walls are fer­ u l i c 1 a n d p - c o u m a r i c 2 acids, a n d these are p r e d o m i n a n t l y i n t h e i r E configurations. T h e y can r e a d i l y be released f r o m the cell walls of grasses b y s a p o n i f i c a t i o n . W h i l e the a m o u n t of ferulic a c i d 1 l i b e r a t e d i n t h i s way n o r m a l l y ranges f r o m 2-10 m g per g r a m d r y cell walls (24-29), the a m o u n t s of p - c o u m a r i c a c i d 2 can v a r y even more m a r k e d l y d e p e n d i n g u p o n the tissue (see T a b l e I) or the species under i n v e s t i g a t i o n (26). In a d d i t i o n to these acids, 5-hydroxyferulic a c i d 3 a n d trace a m o u n t s of s i n a p i c a c i d 4 have also been isolated f r o m the cell walls of corn a n d barley seedlings (29). Indeed, the recent i s o l a t i o n of 5-hydroxyferulic a c i d 3 (29), a n d the e n z y m e responsible for its f o r m a t i o to s i n a p i c a c i d 4, i.e., sinapate f o r m a t i o n occurs v i a direct m e t h y l a t i o n of 5 - h y d r o x y f e r u l a t e 3. T a b l e I. W a l l - E s t e r i f i e d H y d r o x y c i n n a m i c A c i d s i n V a r i o u s Tissues of 2Week O l d M a i z e (Zea mays cvs 259) Hydroxycinnamic Acids (μιηοΐ/g d r y cell w a l l a n d their Z / E ratios) Ferulic A c i d s Tissue Roots* Mesocotyls Nodes Coleoptiles P r i m a r y Leaves blade sheath Secondary Leaves blade sheath T e r t i a r y Leaves

Ε

Ζ

Ζ/Ε

— —

p-Coumaric Acids Ζ/Ε

Ε

Ζ 0.0 0.0 0.2 0.3

0,.003 0 .09

56.8 76.2 45.3 31.9

0.0 0.0 1.3 5.4

0 .03 0 .17

69.1 99.0 70.2 3.4

20.9 21.6

9.7 4.8

0,.46 0 .22

6.5 25.7

1.5 1.9

0,.23 0,.07

29.4 36.2 24.9

11.8 3.3 7.0

0 .40 0 .09 0,.28

17.5 34.0 13.7

2.5 1.0 1.6

0 .14 0 .03 0,.12

* C r o w n roots are not i n c l u d e d . E a c h value corresponds to the m e a n of d u p l i c a t e e x p e r i m e n t s . H y d r o x y c i n n a m i c a c i d d e t e r m i n a t i o n s essen­ t i a l l y followed the procedure of Y a m a m o t o a n d Towers (25). T h e cell w a l l p o l y m e r s to w h i c h the h y d r o x y c i n n a m i c acids 1-4 are k n o w n to be covalently b o u n d are the m a t r i x polysaccharides (31-34), l i g n i n (26,35), a n d s u b e r i n (10). A s far as the polysaccharides are concerned, it is

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

5.

YAMAMOTOETAL.

Phenylpropanoid Metabolism in Cell Walls

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b e c o m i n g recognized t h a t these a t t a c h m e n t s are h i g h l y specific. T h i s was established by e n z y m a t i c digestion of entire cell walls or p u r i f i e d m a t r i x polysaccharides to y i e l d either f e r u l o y l a t e d or p - c o u m a r o y l a t e d oligosaccha­ rides. For instance, t r e a t m e n t of the cell walls of b a r l e y s t r a w w i t h Oxyporus cellulase afforded 0-[5-0-(CU-0-C

72c d

>ÇH-0H

(may be two peaks)

0CH

3

105

f

i'! r i

219

Intact Plant Polyesters

STARK ET AL.

y

Ί ? ι "ι ; ι ι ι Ί '| Ί Ί Ί Ι | Ί Ι ι ' ι " | ι ' ι ' ι" ι γ ι f τ ι ι 1

200

100

0

CHEMICAL SHIFT (PPM)

F i g u r e 2. 50.33 M H z C N M R s p e c t r u m of l i m e c u t i n , o b t a i n e d w i t h cross p o l a r i z a t i o n ( c o n t a c t t i m e 1.5 m s , r e p e t i t i o n rate 1.0 s), m a g i c - a n g l e s p i n ­ n i n g (5.0 k H z ) , a n d d i p o l a r d e c o u p l i n g (yB /2w = 48 k H z ) . T h i s s p e c t r u m was the result of 6000 a c c u m u l a t i o n s a n d was processed w i t h a d i g i t a l l i n e b r o a d e n i n g o f 20 H z . C h e m i c a l - s h i f t assignments are s u m m a r i z e d i n T a b l e I. R e p r o d u c e d f r o m Ref. 7 of the A m e r i c a n C h e m i c a l Society. 1 3

2

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

PLANT C E L L W A L L P O L Y M E R S

220

m a y w e l l correspond (1,23).

to the p o s t u l a t e d crosslinks i n the c u t i n s t r u c t u r e

T a b l e II. S p i n - R e l a x a t i o n T i m e s for Intact L i m e C u t i n (ms) 44 k H z 50 k H z 1 /

Carbon Type

37 k H z

a

T (C)(ms) 1

225*

(CH ) D P 2

ni

(CH )„,CP

2.4

2.8

3.3

CH OCOR CHOCOR,C_HOH a r o m a t i c s , alkenes CH2OCOR CHOÇ.OR

3.4 6.7

4.0 8.7

5.3 12.4

2

2

a

b

c

420* 145 c

122 >7000 ~1000 ~1700

c

c

c

F r o m a s t r a i g h t - l i n e fit of C s i g n a l heights vs. T\p(C) h o l d times of 0.0&-1.00 m s . V a l u e s of B ^ C ) were as n o t e d . A c c u r a c y of the measurements was 1 0 % . F r o m d i r e c t - p o l a r i z a t i o n inversion-recovery e x p e r i m e n t s at 305 a n d 356 K , respectively. A c c u r a c y of the measurements was 1 0 % (7). F r o m c r o s s - p o l a r i z a t i o n inversion-recovery e x p e r i m e n t s at 300 Κ a n d 50.33 M H z . A c c u r a c y of the measurements was 1 5 - 2 0 % (7). 1 3

A l s o d i s p l a y e d i n T a b l e II are s p i n - l a t t i c e r e l a x a t i o n d a t a for " l i q u i d l i k e " ( C H ) groups t h a t were observable i n D P M A S e x p e r i m e n t s . B o t h the dependence o n t e m p e r a t u r e a n d the p a r t i c u l a r Τ χ values suggested r a p i d segmental m o t i o n s w i t h i n l o n g runs of m e t h y l e n e groups, quite s i m ­ i l a r to the d y n a m i c b e h a v i o r reported for soft-segment C H 2 ' s i n s y n t h e t i c polyesters (19). F i n a l l y , C P M A S a n d D P M A S results were c o m b i n e d to estimate the n u m b e r s of each c h e m i c a l l y d i s t i n c t carbon t y p e , as presented i n T a b l e I I I . D e s p i t e some u n c e r t a i n t i e s (7), t h i s q u a n t i t a t i v e i n f o r m a t i o n served to a u g ­ m e n t p r i o r hypotheses r e g a r d i n g c u t i n s t r u c t u r e (1). O u r d e t e r m i n a t i o n s of methylenes, c a r b o n y l s , a n d a l i p h a t i c s b o n d e d to oxygen were consistent w i t h a C i 6 polyester framework, a n d a r o m a t i c moieties c o u l d be present a d d i t i o n a l l y as sidechains ( F i g u r e 3). A s u b s t a n t i a l degree of c r o s s l i n k i n g was suggested b y the large n u m b e r of r i g i d s e c o n d a r y - a l c o h o l ester c a r b o n s a n d the h i g h p r o p o r t i o n of i m m o b i l i z e d methylene groups (Tables II a n d III). 2

n

Cutin Depolymenzation Residue. F i g u r e 4 compares the C C P M A S spec­ t r a o b t a i n e d for i n t a c t c u t i n a n d for the i n s o l u b l e residue r e m a i n i n g after transesterification w i t h B F 3 / C H 3 O H . T h e n a r r o w i n g of most s p e c t r a l lines suggested t h a t the d e p o l y m e r i z a t i o n - r e s i s t a n t m a t e r i a l was a less heteroge­ neous p o l y m e r ; d e l a y e d - c o u p l i n g e x p e r i m e n t s confirmed t h a t , a m o n g those 1 3

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

2

4

2

5

2

2

r ° P ] - A T P i n the presence (+) a n d absence (—) of p r o t e i n kinase as described i n M a t e r i a l s a n d M e t h o d s . 32

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

18.

WASSERMAN ET AL.

(l,3)-fi-Glucan Synthase

255

p h o r y l a t i o n . C a u t i o n m u s t b e exercised i n i n t e r p r e t i n g these results. O n o n e - d i m e n s i o n a l gels, several p o l y p e p t i d e s m a y m i g r a t e close together at 54 k D . T w o - d i m e n s i o n a l gels must be r u n t o be c e r t a i n t h a t p h o s p h o r y ­ l a t i o n , C a - b i n d i n g a n d affinity l a b e l - b i n d i n g sites reside o n t h e same p o l y p e p t i d e . O t h e r s u b u n i t s such as the 68 k D p o l y p e p t i d e were present i n R C G S p r e p a r a t i o n s a n d cannot be r u l e d o u t as f u n c t i o n a l s u b u n i t s . W o r k is c o n t i n u i n g o n t h e development o f a d d i t i o n a l probes such as a n ­ t i b o d i e s a n d photoaffinity labels. C o n f i r m i n g t h e s u b u n i t c o m p o s i t i o n o f the / ? - ( l , 3 ) - g l u c a n synthase s h o u l d enable development o f genetic probes a n d hopefully provide cogent answers t o some o f t h e m a n y l o n g s t a n d i n g questions s u r r o u n d i n g t h e e n z y m a t i c m e c h a n i s m o f callose a n d cellulose biosynthesis. 2 +

A cknowledgment s T h i s research was s u p p o r t e A g r i c u l t u r e ( 8 7 - C R C R - 1 - 2 4 1 4 ) , the C h a r l e s a n d J o h a n n a B u s c h F o u n d a ­ t i o n , a n d the N e w Jersey A g r i c u l t u r a l E x p e r i m e n t S t a t i o n w i t h S t a t e a n d H a t c h A c t funds. N e w Jersey A g r i c u l t u r a l E x p e r i m e n t S t a t i o n , P u b l i c a ­ t i o n N o . F-10546-1-88. W e t h a n k M r s . M a r i a n n e B i a n c o for assisting w i t h manuscript preparation. Literature Cited

1. Delmer, D. P. Ann. Rev. Plant Physiol. 1987, 38, 259-90. 2. Wasserman, B. P.; Eiberger, L. L.; McCarthy, K. J. Food Technol. 1986, 40, 90-98. 3. Wasserman, B. P.; Sloan, M. E. In Biosynthesis and Biodegradation of Cellulose and Cellulosic Materials; Weimer, P.; Haigler, C., Eds.; Marcel Dekker: New York, 1988; in press. 4. Sloan, M. E.; Rodis, P.; Wasserman, B. P. Plant Physiol. 1987, 85, 516-522. 5. Eiberger, L. L.; Wasserman, B. P. Plant Physiol. 1987, 83, 982-987. 6. Hayashi, T.; Read, S. M.; Bussell, J.; Thelen, M.; Lin, F.-C.; Brown, R. M.; Delmer, D. P. Plant Physiol. 1987, 83, 1054-1062. 7. Kauss, H.; Jeblick, E. Plant Sci. 1987, 48, 63-69. 8. Eiberger, L. L.; Ventola, C. L.; Wasserman, B. P. Plant Sci. Lett. 1985, 37, 195-198. 9. Wasserman, B. P.; Frost, D. J.; Lawson, S. G.; Mason, T. L.; Rodis, P.; Sabin, R. D.; Sloan, M. E. In Mod. Meth. Pl. Anal.; Linskens, H. F.; Jackson, J. F., Eds.; Springer: Berlin, 1989; in press. 10. Yoshida, S.; Uemura, M.; Niki, T.; Sakai, Α.; Gusta, L. V. Plant Phys­ iol. 1983, 72, 105-144. 11. Laemmli, U. K. Nature 1970, 227, 680-685. 12. Porzio, Μ. Α.; Pearson, A. M. Biochim. Biophys. Acta 1976, 490, 2734. 13. Towbin, H.; Staehelin, T.; Gordon, J. Proc. Natl. Acad. Sci. 1979, 76, 4350-4354.

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14. Moeremans, M.; Daneels, G.; De Mey, J. Anal. Biochem. 1985, 145, 315-321. 15. Tagaya, M.; Nakamo, K.; Fukui, T. J. Biol. Chem. 260, 6670-6676. 16. Delmer, D. P. J. Appl. Polym. Sci. Symp. 1989, in press. 17. Olmsted, J. B. J. Biol. Chem. 1981, 256, 11955-11957. 18. Mishkind, M. L.; Plumley, F. G.; Raikheil, Ν. V. In Handbook of Plant Cytochemistry; Vaughn, K. C., Ed.; CRC Press: Boca Raton, FL, 1987; Vol. 2, p. 65. 19. Desai, Ν. N.; Allen, A. K.; Neuberger, A. Biochem. J. 1983, 211, 273276. 20. Sojar, H. T.; Bahl, O. P. Arch. Biochem. Biophys. 1987, 259, 52-57. 21. Read, S. M.; Delmer, D. P. Plant Physiol. 1987, 85, 1008-1015. 22. Ranjeva, R.; Boudet, A. M. Ann. Rev. Plant Physiol. 1987, 38, 73-93. 23. Poovaiah, B. W.; Reddy, A. S. N. CRC Crit. Rev. Plant Sci. 1987, 6, 47-103. 24. Paliyath, G.; Poovaiah 25. Bidwai, A. P.; Takemoto, J. Y. Proc. Natl. Acad. Sci. 1987, 84, 67556759. RECEIVED March 10, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 19

Biogenesis of Cellulose Microfibrils and the Role of Microtubules in Green Algae Takao Itoh Wood Research Institute, Kyoto University, Uji, Kyoto 611, Japan

This chapter review cellulose depositio cellulose synthesizing particle complexes in green algae are discussed. Secondly, new evidence on the oriention of microtubules in selected giant marine algae and their relationship to the orientation of cellulose microfibrils is presented. Based on this information, a mechanism for the assembly of cellulose microfibrils in giant marine algae is proposed. O u r c u r r e n t u n d e r s t a n d i n g o f cellulose m i c r o f i b r i l biogenesis a n d assemb l y comes f r o m (a) freeze-fracture studies o f the p l a s m a m e m b r a n e o f cells a c t i v e l y p r o d u c i n g cellulose m i c r o f i b r i l s ; (b) observations o f m i c r o t u b u l e s by immunofluorescence m i c r o s c o p y ; (c) direct i m a g i n g o f cellulose m i c r o f i b rils (1-5); a n d (d) in vitro synthesis o f cellulose u s i n g b a c t e r i a l cell m e m brane p r e p a r a t i o n s (6). T h i s chapter examines recent progress i n freezefracture a n d immunofluorescence studies o n the biogenesis o f cellulose m i crofibrils, as w e l l as addressing the role o f m i c r o t u b u l e s i n several green algae. F o r t h e last decade, m u c h o f our knowledge o f the s t r u c t u r e a n d f u n c t i o n o f cellulose f o r m i n g enzyme complexes (so-called T e r m i n a l C o m p l e x e s or T C ' s ) has been based o n results o b t a i n e d f r o m freeze-fracture studies. T h e m a i n discoveries f r o m these studies were (a) the existence o f l i n e a r T C ' s by B r o w n a n d M o n t e z i n o s (7) i n Oocystis apiculata, a n d (b) t h e occurrence o f rosette T C ' s by G i d d i n g s et ai (8) i n Micrasterias denticulata. A s w i l l be discussed later, the occurrence o f these t w o types o f cellulose s y n t h e s i z i n g complexes has some e v o l u t i o n a r y significance as regards c u r rent phylogenetic r e l a t i o n s h i p s w i t h i n the p l a n t k i n g d o m . T h i s is because a l l l a n d p l a n t s , i n c l u d i n g higher p l a n t s , mosses a n d ferns, have rosettes (911), whereas some algae have linear T C ' s a n d others d o n o t . It has been 0097-6156/89/0399-0257$06.00A) © 1989 American Chemical Society

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proposed t h a t the T C ' s move o n the fluid p l a s m a m e m b r a n e b y forces generated d u r i n g c r y s t a l l i z a t i o n of cellulose m i c r o f i b r i l s (12,13). In green algae, the n e w l y synthesized m i c r o f i b r i l s first have a r a n d o m o r i e n t a t i o n d u r i n g the synthesis of the p r i m a r y w a l l a n d then a n ordered o r i e n t a t i o n d u r i n g secondary w a l l f o r m a t i o n . It is t h u s conceivable t h a t the factors c o n t r o l l i n g the o r i e n t a t i o n of T C ' s i n green algae m a y also be responsible for the o r i e n t a t i o n of m i c r o f i b r i l s ; t h i s c o u l d be achieved, for e x a m p l e , by c h a n n e l l i n g c y t o p l a s m i c m i c r o t u b u l e s . T h i s view is presented because i n higher p l a n t s , a n d some algal cells, rosette particles are a p p a r e n t l y channelled b y c y t o p l a s m i c m i c r o t u b u l e s w h i c h r u n p a r a l l e l to one another (14-16). However, as a counter to t h i s a r g u m e n t , some green algae have l i n e a r T C ' s w h i c h do not appear to have m i c r o f i b r i l l a r o r i e n t a t i o n c o n t r o l l e d b y m i c r o t u b u l e s d u r i n g secondary w a l l synthesis (17). T h i s evidence suggests therefore t h a t there m a y be different mechanisms c o n t r o l l i n g m i c r o f i b r i l l a r o r i e n t a t i o n between higher a n d lower plants. Structure of T C ' s in G r e e n Algae T h e green algae i n c l u d e b o t h C h l o r o p h y t a a n d C h a r o p h y t a . T h e T C ' s of C h l o r o p h y t a have been observed i n four orders of C h l o r e l l a l e s , C l a d o p h o rales, S i p h o n o c l a d a l e s , a n d Z y g n e m a t a l e s , a n d those of C h a r o p h y t a i n one order of C h a r ales (Table I). Recent investigations show t h a t 17 genera a n d 23 species out of three orders of C h l o r e l l a l e s , C l a d o p h o r a l e s a n d S i p h o n o cladales a l l have l i n e a r T C ' s , as shown i n T a b l e I. W h i l e linear T C ' s have been observed i n some freshwater algae such as Oocystis (7,18), Eremosphaera (19), a n d Glaucocystis (20) species, most are f o u n d i n m a r i n e - t y p e algae such as Boergesenia (21,22), Boodlea ( F i g . 1) (23,24), Dictyosphaeria ( F i g . 2), Ernodesmis ( F i g . 3), Microdictyon (19), Siphonocladus ( F i g . 4), Struvea ( F i g . 5), Valonia ( F i g . 6) (17,24,25), Valoniopsis ( F i g . 7), a n d Chaetomorpha ( F i g . 8) (19,25) species; these eight genera belong t o the Siphonocladales. A l t h o u g h Chlorellales, Cladophorales and Siphonocladeles have linear T C ' s , there are significant differences i n their l o c a t i o n s ; t h a t is, C h l o r e l l a l e s have T C ' s o n l y on the E - f r a c t u r e face, w h i l e b o t h C l a d o p h o r a l e s a n d Siphonocladales have T C ' s on b o t h E - a n d P - f r a c t u r e faces, thereby m a k i n g t r a n s m e m b r a n e particles. A d d i t i o n a l l y , the T C s t r u c t u r e of Z y g n e m a t a l e s is quite different f r o m those of the other three orders i n C h l o r o p h y t a . F o u r genera of the Z y g n e m a t a l e s , i n c l u d i n g Closterium (16,26), Micrasterias (8,27,28), Mougeotia (29), a n d Spirogyra (19,30) species have been investigated so far. A l l have rosettes o n l y o n the P - f r a c t u r e face. T h e cells can have either r a n d o m a n d / o r u n i d i r e c t i o n a l rosette d i s t r i b u t i o n s d u r i n g active synthesis of the p r i m a r y w a l l , a n d hexagonal arrays d u r i n g the synthesis of the secondary wall. M o r e recently, rosettes have been observed i n Chara sp. (31) a n d Nitella translucens (32), w h i c h belong to another s u b d i v i s i o n , C h a r o p h y t a . T h e rosettes i n t h i s species occur separately w i t h o u t m a k i n g any p o l y g o n a l arrays as f o u n d i n Z y g n e m a t a l e s . T h i s suggests t h a t C h a r a l e s are closer to vascular p l a n t s t h a n the other algae, i f plant phylogenic classifications

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Figure 1. TCs of Boodlea composita on P-fracture face.

Figure 2. TCs of Dictyosphaeria cavernosa on P-fracture face.

Figure 3. TCs of Ernodesmis verticillata on Ε-fracture face.

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Figure 4. TC's of Siphonocladus tropicus on P-fracture face.

Figure 5. TC's of Struvea elegans on P-fracture face.

Figure 6. TC's of Valonia ventricosa on Ε-fracture face.

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Figure 7. T C s of Valoniopsis pachynema on P-fracture face.

Figure 8. T C s of Chaetomorpha

auricoma on P-fracture face.

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T a b l e I. C u r r e n t S u m m a r y of the S t r u c t u r e a n d L o c a t i o n of T C s a m o n g Green Algae

S u b d i v i s i o n , O r d e r , Species Chlorophyta Chlorellales Eremosphaera sp. Glaucocysiis nostochinearum Oocystis apiculata Oocystis solitaria Cladophorales Chaetomorpha sp. Chaetomorpha aerea Chaetomorpha moniligera Siphonocladales Boergesenia forbesii Boodlea coacta Boodlea composita Dictyosphaeria cavernosa Ernodesmis verticillata Siphonocladus tropicus Struvea elegans Valonia macrophysa Valonia ventricosa Valonia ventricosa Zygnematales Closterium acerosum Closterium sp. Micrasterias cruxmelitensis Micrasterias denticulata Mougeotia sp. Spirogyra sp. Charophyta Charales Chara sp. Nitella translucens

TC Structure

TC Location

Linear Linear Linear Linear

EF EF EF EF

TC TC TC TC

only only only only

Linear T C Linear T C

EF k PF EF k PF

Linear Linear Linear Linear Linear Linear Linear Linear Linear Linear

EF EF EF EF EF EF EF EF EF EF

TC TC TC TC TC TC TC TC TC TC

k k k k k k k k k k

PF PF PF PF PF PF PF PF PF PF

Rosettes Rosettes Rosettes Rosettes Rosettes Rosettes

PF PF PF PF PF PF

only only only only only only

Rosettes Rosettes

P F only P F only

Reference

19 20 7 18 19 24

21,22 23 24 T h i s chapter T h i s chapter T h i s chapter T h i s chapter 17 24,25 T h i s chapter 26 16 28 27,8 29 30,19

31 32

can be m a d e based u p o n cellulose s y n t h e s i z i n g complexes. It c o u l d t h u s be argued f r o m these d a t a t h a t the e v o l u t i o n of T C ' s w i l l follow the lines envisaged i n F i g . 9, w h i c h is a m o d i f i c a t i o n of a scheme proposed b y H e r t h (11) for the h y p o t h e t i c a l e v o l u t i o n a r y lines of p u t a t i v e cellulose s y n t h e s i z i n g complexes. B a s i c a l l y , t h i s stems f r o m the fact t h a t b o t h rosettes a n d l i n e a r T C ' s are composed of c o m m o n p a r t i c l e s u b u n i t s s i m i l a r i n size; rosettes consist of s i x particles, each h a v i n g an 8 n m d i a m e t e r of (8) a n d l i n e a r T C ' s consists of three rows w i t h an average d i m e n s i o n of ca. 8 n m for each i n d i v i d u a l p a r t i c l e (25). However, T C ' s have o n l y been f o u n d i n a l i m i t e d

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n u m b e r of species a m o n g the algae. Consequently, a further survey of T C ' s is necessary to develop a detailed phylogenic hypothesis. T C ' s n o r m a l l y occur at the end of m i c r o f i b r i l i m p r i n t s o n either E - or P - f r a c t u r e faces of the p l a s m a m e m b r a n e . E a c h i m p r i n t does not necessarily correspond to a single m i c r o f i b r i l , but often to bundles of t h e m . D u r i n g the d e p o s i t i o n of r a n d o m m i c r o f i b r i l s of the p r i m a r y w a l l , the i m p r i n t s r u n a l o n g the p l a s m a m e m b r a n e , a n d often w i t h c u r v e d t r a i l s . O n the other h a n d , w h e n ordered m i c r o f i b r i l s of the secondary w a l l are s y n t h e s i z e d , the i m p r i n t s r u n straight a n d p a r a l l e l to one another. In Oocystis species, each p a i r e d T C runs i n an opposite d i r e c t i o n (31). In most giant m a r i n e algae, some T C ' s appear to r u n i n one d i r e c t i o n , w h i l e others r u n i n the opposite d i r e c t i o n d u r i n g the synthesis of ordered m i c r o f i b r i l s . T h e l a t t e r case is i l l u s t r a t e d i n F i g u r e 10 w h i c h shows the Ε-fracture face of n e w l y f o r m e d cellulose m i c r o f i b r i l s of ordered o r i e n t a t i o n . T h e T C n u m b e r e d " 1 " is m o v i n g to the right whereas the T C n u m b e r e d " 2 " is m o v i n g to the left. C l e a r l y , i f the i n d i v i d u a of / ? - l , 4 linkages, the cell w a l l s h o u l d have a n a n t i - p a r a l l e l g l u c a n c h a i n orientation. Development of Linear T C ' s i n Selected G r e e n Algae T h e l e n g t h of linear T C ' s is variable a n d depends o n the d e v e l o p m e n t a l phase of cell g r o w t h . T h i s was determined f o l l o w i n g w o u n d i n g of the m o t h e r cells of B. forbesii, where the aplanospores were regenerated w i t h i n 1.5 h , a n d the p r i m a r y a n d the secondary walls were synthesized 2 to 4h a n d 4 to 5h after w o u n d i n g , respectively (33). F i g u r e 11 shows the effect of t i m e o n T C l e n g t h f o l l o w i n g aplanospore i n d u c t i o n i n Boergesenia forbesii. A s can be seen, the T C length increases o n l y d u r i n g f o r m a t i o n of the p r i m a r y w a l l ; no further increase occurs after deposition of the secondary w a l l . However, w h e n we look closer at the freeze-fracture r e p l i c a of the aplanospores i n B. forbesii j u s t before, or at the t i m e of, synthesis of cellulose m i c r o f i b r i l s , m a n y nascent T C ' s can be observed on the P - f r a c t u r e face of the p l a s m a m e m b r a n e ( F i g . 12). T h e smallest T C observed h a d o n l y 10 p a r t i c l e s . M o s t nascent T C ' s d i d not show a d i r e c t i o n a l arrangement of p a r t i c l e s , w h i l e some h a d already organized an elongated cluster (double arrows i n F i g . 12). T h e m e a n length of the T C ' s i n t h i s phase was 114 n m . T h e nascent T C ' s increased i n length d u r i n g the synthesis of the p r i m a r y w a l l , u n t i l the ordered m i c r o f i b r i l s were assembled. T h e l e n g t h of nascent T C ' s contrasted w i t h t h a t of f u l l y elongated T C ' s , w h i c h h a d a m a x i m u m l e n g t h of ca. 1 m (mean l e n g t h : 665 n m ) i n 20h cells of B. forbesii ( F i g . 13). Orientation of C o r t i c a l Microtubules T h e cellulose s y n t h e s i z i n g enzyme complexes i n green algae can be d i v i d e d i n t o rosettes a n d linear T C ' s ; the latter increases i n size d u r i n g c i r c u m f e r ­ e n t i a l e x p a n s i o n of the cells. T h e movement of b o t h g r o w i n g a n d m a t u r e T C ' s i n the p l a s m a m e m b r a n e is controlled by forces generated b y c r y s ­ t a l l i z a t i o n of m i c r o f i b r i l s , leaving the h i g h l y c r y s t a l l i n e m i c r o f i b r i l s i n their wake (7,34).

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HIGHER

PLANTS

MOSSES & FERNS

CHARALES

SIPHONOCLADALES CLADOPHORALES · · · · · · · · ·

ZYGNEMATALES

CHLORELALES

\

ALGAE

\

(ROSETTES)

(LINEAR

\/

....... FUCALES

TCS)

F i g u r e 9. H y p o t h e t i c a l i l l u s t r a t i o n for e v o l u t i o n a r y t r e n d o f p u t a t i v e cel­ lulose s y n t h e s i z i n g e n z y m e complexes f r o m algae t o higher p l a n t s . B o t h rosettes a n d linear T C ' s originate f r o m their c o m m o n s u b u n i t of 8 n m p a r ­ ticle. A s t e r i s k (*) represents a rosette.

F i g u r e 10. Ε-fracture face o f the p l a s m a m e m b r a n e d u r i n g active synthesis of ordered m i c r o f i b r i l s i n secondary w a l l o f Valonia macrophysa. Imprints of m i c r o f i b r i l s r u n p a r a l l e l t o one another. T C ' s numbered " 1 " a n d " 2 " direct opposite ways t o one another.

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F i g u r e 11. T i m e course of T C length i n the aplanospores of forbesii.

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F i g u r e 12. F o u r nascent T C ' s (arrowheads) are s h o w n on P - f r a c t u r e face of the p l a s m a m e m b r a n e i n 2h aplanospore of Boergesenia forbesii after wounding.

F i g u r e 13. T w o f u l l y elongated T C ' s (ca. 1 m ) are s h o w n o n P - f r a c t u r e face of the p l a s m a m e m b r a n e i n 20h cell of Boergesenia forbesii.

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Since the m a t u r e walls of Valonia a n d Boergesenia have a crossed lamellate s t r u c t u r e (17,35-37), the d i r e c t i o n of T C movement m u s t be c o n t r o l l e d i n some u n k n o w n m a n n e r . O n the other h a n d , giant m a r i n e a l gae, w h i c h also have linear T C ' s , but of a p p a r e n t l y r a n d o m o r i e n t a t i o n , o n l y synthesize m i c r o f i b r i l s of r a n d o m o r i e n t a t i o n on the i n n e r m o s t face of the p l a s m a m e m b r a n e d u r i n g the synthesis of the p r i m a r y w a l l . It was, therefore, t i m e l y to e x a m i n e whether c o r t i c a l m i c r o t u b u l e s were r e s p o n sible for o r i e n t a t i o n of m i c r o f i b r i l s , since recent investigations suggested t h a t the d i r e c t i o n of m i c r o f i b r i l d e p o s i t i o n i n higher p l a n t cells (38-40) a n d some algae (Oocystis a n d Micrasterias (8,16,33)), was c o n t r o l l e d b y c o r t i c a l m i c r o t u b u l e s . However, i n the a l g a Closterium, microtubules functioned o n l y to l i m i t cellulose synthesis to a localized region (41). A d d i t i o n a l l y , freeze-fracture studies suggested t h a t newly synthesized m i c r o f i b r i l s i n the spherical cells of the giant m a r i n e algae Valonia macrophysa were not p a r allel to the u n d e r l y i n g m i c r o t u b u l e s (17). In the light of these c o n t r a d i c t o r y findings, the role of m i c r o t u b u l a o r i e n t a t i o n a m o n g green algae was r e - e x a m i n e d u s i n g immunofluorescence microscopy. L l o y d et al. (43) first used this technique to observe c o r t i c a l m i c r o t u b u l e s , following the pioneering work o n p l a n t cells b y F r a n k e (42). Since t h e n , it has been used m a n y times to show m i c r o t u b u l e o r i e n t a t i o n over whole cells (44). T h e m a t e r i a l s used for the immunofluorescent s t a i n i n g were a p l a n o spores of B. forbesii a n d V. ventricosa, where cellulose m i c r o f i b r i l o r i e n t a t i o n c o u l d easily be s h o w n by s t a i n i n g w i t h the fluorescent b r i g h t e n i n g agents C a l c o f l u o r a n d T i n o p a l L P W (34). A s m e n t i o n e d i n the previous section, Boergesenia aplanospores synthesized r a n d o m m i c r o f i b r i l s between 2 a n d 4 h after w o u n d i n g of m o t h e r cells a n d ordered m i c r o f i b r i l s after 4 h . T h e Boergesenia aplanospores i n w h i c h s p h e r a t i o n h a d j u s t been c o m p l e t e d showed r a n d o m l y oriented m i c r o t u b u l e s j u s t under the p l a s m a m e m b r a n e ( F i g . 14), a n d the aplanospores i n 3h p o s t - w o u n d i n g d i d not differ f r o m those at 1.5h. ( F i g . 15). However, after 6h p o s t - w o u n d i n g , d u r i n g w h i c h t i m e the n o r m a l l y synthesized ordered m i c r o f i b r i l s were deposited, o n l y r a n d o m l y oriented m i c r o t u b u l e s were observed under the thickened cell w a l l ( F i g . 16). A t a later phase of cell w a l l regeneration, immunofluorescent s t a i n i n g of m i c r o t u b u l e s b y a n t i g e n - a n t i b o d y reactions became m o r e difficult because of the thickened wall s u r r o u n d i n g the aplanospores. N e v e r theless, we m a n a g e d to observe the arrangement of m i c r o t u b u l e s even after 8, 10, a n d 20h p o s t - w o u n d i n g w i t h o u t h a v i n g to resort to e n z y m a t i c cell w a l l digestion. In a l l cases, o n l y r a n d o m m i c r o t u b u l a r o r i e n t a t i o n p a t t e r n s ( F i g s . 17, 18 a n d 19) were observed. However, after successive c u l t u r e of the spherical aplanospores for m o r e t h a n five days, g e r m i n a t i o n occurred w i t h a t y p i c a l t i p g r o w t h to make r h i zoids (45); the o r i e n t a t i o n of m i c r o f i b r i l s i n a single e l o n g a t i n g r h i z o i d of 10 days p o s t - w o u n d i n g , for e x a m p l e , has been described recently (13). I n our s t u d y , the innermost w a l l lamellae i n the r h i z o i d showed three different orientations of m i c r o f i b r i l s , i.e., transverse, o b l i q u e , a n d l o n g i t u d i n a l to the g r o w i n g cell a x i s . F o l l o w i n g immunofluorescent s t a i n i n g of the m i c r o t u b u l e s

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Figure 14. Immunofluorescence micrograph of microtubule orientation during cell regeneration in Boergesenia forbesii 1.5 h after wounding.

Figure 15. Immunofluorescence micrograph of microtubule orientation during cell regeneration in Boergesenia forbesii 3 h after wounding.

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Figure 16. Immunofluorescence micrograph of microtubule orientation during cell regeneration in Boergesenia forbesii 6 h after wounding.

Figure 17. Immunofluorescence micrograph of microtubule orientation during cell regeneration in Boergesenia forbesii 8 h after wounding.

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Figure 18. Immunofluorescence micrograph of microtubule orientation during cell regeneration in Boergesenia forbesii 10 h after wounding.

Figure 19. Immunofluorescence micrograph of microtubule orientation during cell regeneration in Boergesenia forbesii 20 h after wounding.

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by the fluorescein isothiocyanate-conjugated a n t i b o d y , a n e l o n g a t i n g r h i z o i d of 8 days p o s t - w o u n d i n g showed ordered m i c r o t u b u l e s oriented l o n g i t u d i n a l l y to the g r o w i n g cell a x i s , as w e l l as p a r a l l e l to one another ( F i g . 20). It is n o t e w o r t h y t h a t l o n g i t u d i n a l m i c r o t u b u l e s were also observed i n the r o o t hairs of some higher p l a n t s w h i c h show t i p g r o w t h (46-48). In the case of V. ventricosa, s p h e r a t i o n was c o m p l e t e d w i t h i n 2h of w o u n d i n g , f o l l o w i n g w h i c h r a n d o m m i c r o f i b r i l s were regenerated between 5 a n d 12h a n d ordered m i c r o f i b r i l s w i t h i n 12 to 15h. T h e t i m e course of m i c r o t u b u l e o r i e n t a t i o n i n the aplanospores of V. ventricosa followed a t r e n d s i m i l a r to t h a t observed for B. forbesii; t h a t is, m i c r o t u b u l e s were a l w a y s oriented r a n d o m l y d u r i n g the synthesis of b o t h r a n d o m a n d ordered m i crofibrils. It is w o r t h n o t i n g the s t r u c t u r a l changes of m i c r o t u b u l e s i n the early phase of cell regeneration; the aplanospores p r o d u c e d f r o m V. ventricosa w i t h i n 2 a n d 3h of p o s t - w o u n d i n g d i d not show c o r t i c a l m i c r o t u b u l e s b u t i n s t e a d perinuclear m i c r o t u b u l e arrays ( F i g . 21), a n d the aplanospores after 4 h showed the i n i t i a t i o and 23b show 3h aplanospores of a c t i v e l y s y n t h e s i z i n g p r i m a r y w a l l p o i n t s . B o t h figures were t a k e n f r o m the same cell w i t h different focusing. F i g ure 23a was focused o n the nucleus; note t h a t several m i c r o t u b u l e s r a d i a t e f r o m the nucleus. F i g u r e 23b was focused o n the c o r t i c a l m i c r o t u b u l e s , where some of the perinuclear m i c r o t u b u l e s appeared as p a r t of the c o r t i cal m i c r o t u b u l e s . T h i s evidence suggests t h a t the nuclei m a y play a role as m i c r o t u b u l e o r g a n i z i n g centers ( M T O C ) d u r i n g the regeneration of the cell w a l l i n these green algae. Summary: entation

Microtubule-Independent

C o n t r o l of M i c r o f i b r i l O r i -

F r o m our immunofluorescence e x p e r i m e n t s , we have concluded t h a t (1) o n l y a r a n d o m m i c r o t u b u l a r o r i e n t a t i o n occurs d u r i n g regeneration of p r i m a r y a n d secondary walls a n d (2) the g r o w i n g r h i z o i d s d u r i n g t i p g r o w t h o n l y showed m i c r o t u b u l e s oriented l o n g i t u d i n a l l y to the g r o w i n g cell w a l l axis. However, since three different o r i e n t a t i o n s of m i c r o f i b r i l s i n the i n n e r most lamellae of the cell wall were observed, these findings suggest t h a t the d i r e c t i o n of movement of linear T C ' s was not controlled by c o r t i c a l m i c r o tubules, at least not i n the case of the two giant m a r i n e algae, Boergesenia and Valonia, s t u d i e d . L a C l a i r e (49) recently described a h i g h l y ordered a r r a y of p a r a l l e l a n d l o n g i t u d i n a l m i c r o t u b u l e s i n the coenocytic green algae Ernodesmis verticillata. However, w i t h other filamentous green algae, Boodlea coacta (50) and Chaetomorpha moniligera (51), aligned m i c r o t u b u l e s a n d m i c r o f i b r i l s were not always observed. It was thus suggested t h a t m i c r o f i b r i l o r i e n t a t i o n i n Siphonocladales and p r o b a b l y C l a d o p h o r a l e s m a y be independent of c y t o p l a s m i c m i c r o t u b u l e o r i e n t a t i o n . O n c e T C ' s are i n i t i a t e d i n the p l a s m a m e m b r a n e , they d i s t r i b u t e r a n d o m l y w i t h each T C r u n n i n g s t r a i g h t ahead. M o r e recent investigations showed t h a t the density of T C ' s was more or less constant (52), i n d i c a t i n g t h a t the n u m b e r of T C ' s d i d not increase a p p r e c i a b l y d u r i n g p r i m a r y w a l l

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F i g u r e 20. Immunofluorescence m i c r o g r a p h of m i c r o t u b u l e o r i e n t a t i o n i n 8 d a y o l d cells of Boergesenia forbesii. H i g h l y ordered m i c r o t u b u l e s are oriented l o n g i t u d i n a l l y to the cell axis (double-headed a r r o w ) .

F i g u r e 21. Immunofluorescence m i c r o g r a p h of perinuclear m i c r o t u b u l e s i n the aplanospore of 3h p o s t - w o u n d i n g of Valonia venlricosa.

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F i g u r e 22. Immunofluorescence m i c r o g r a p h of c o r t i c a l m i c r o t u b u l e s i n the aplanospore of 4h p o s t - w o u n d i n g of Valonia ventricosa.

F i g u r e 23. Immunofluorescence m i c r o g r a p h s i n the aplanospore of 3h postw o u n d i n g of Boergesenia forbesii. F i g u r e 23a is focused on the p e r i n u c l e a r m i c r o t u b u l e s , w h i l e F i g u r e 23b is focused on the c o r t i c a l m i c r o t u b u l e s w h i c h are o r i e n t e d r a n d o m l y .

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F i g u r e 24. O r i e n t a t i o n of m i c r o f i b r i l s is s h o w n i n 4h aplanospore of Boergesenia forbesii, stained w i t h fluorescent b r i g h t e n i n g agent T i n o p a l L P W .

F i g u r e 25. Freeze fractured replica. P-fracture face of the p l a s m a m e m b r a n e i n 15h aplanospore of Valonia ventricosa. T h e clustered T C ' s are s h o w n , suggesting the b u i l d - u p of new axis for m i c r o f i b r i l o r i e n t a t i o n .

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synthesis, even t h o u g h a h i g h density o f T C ' s was often l o c a l i z e d i n some areas o f the p l a s m a m e m b r a n e . F i g u r e 24 shows the o r i e n t a t i o n o f m i c r o f i b rils i n aplanospores o f B. forbesii 4 h after w o u n d i n g , a n d s t a i n e d w i t h t h e fluorescent b r i g h t e n i n g agent T i n o p a l L P W . I n some areas, short s t r i a t i o n s of fluorescence merged into a center, suggesting a shift o f m i c r o f i b r i l o r i e n t a t i o n , p r o b a b l y b y localized membrane flow. T C l o c a l i z a t i o n c a n often be observed i n t h e t r a n s i t i o n f r o m p r i m a r y t o secondary w a l l f o r m a t i o n . F i g u r e 25 is taken f r o m such a phase i n the aplanospore o f V. ventricosa, a n d m a y correspond t o a n area i n w h i c h clusters o f T C ' s occur a n d move i n a d i r e c t i o n different f r o m the former o r i e n t a t i o n o f m i c r o f i b r i l s . L i n e a r T C ' s i n giant m a r i n e algae m a y be stable, because (a) t h e T C ' s d o n o t disappear f o l l o w i n g t r e a t m e n t w i t h c y c l o h e x i m i d e , a p r o t e i n synthesis i n h i b i t o r (34), a n d (b) g l u t a r a l d e h y d e t r e a t m e n t i n advance o f freeze fracture d i d n o t destroy the T C ' s ( u n p u b l i s h e d d a t a ) . T h u s , linear T C ' s i n giant m a r i n e algae m a y be involved i n the synthesis o f ordered m i c r o f i b r i l s w i t h m u c h longer lifetimes t h a R e c e n t l y , more evidence for hélicoïdal arrangement o f cellulose m i crofibrils has been reported for a variety o f plant cells (54,55). I n Nitella, the hélicoïdal o r i e n t a t i o n o f cellulose m i c r o f i b r i l s was s h o w n t o arise by a m e c h a n i s m s i m i l a r t o self-assembly o f a cholesteric l i q u i d c r y s t a l (56). T h e c o n t r o l o f m i c r o f i b r i l o r i e n t a t i o n was p r i m a r i l y at the interface between t h e p l a s m a membrane a n d the innermost lamellae o f the n e w l y formed w a l l . T h e hélicoïdal w a l l , characterized b y a successive change o f cellulose m i crofibrils, was supposed t o be synthesized i n the i n n e r m o s t surface o f t h e new w a l l layer. I f t h i s is true, then t he involvement o f m i c r o t u b u l e s i n higher p l a n t cells is m u c h less probable t h a n p r e v i o u s l y believed. T h e selfassembly o f cellulose m i c r o f i b r i l s is related t o those cells w h i c h have such a p a t t e r n o f cell w a l l t h a t show arced o r i e n t a t i o n o f m i c r o f i b r i l s i n a t r a n s verse section (56). B o t h aplanospores a n d t h a l l u s cells o f B. forbesii have an arced p a t t e r n o f cellulose m i c r o f i b r i l s . However, we showed t h a t t h e m i c r o f i b r i l o r i e n t a t i o n i n b o t h spherical cells a n d e l o n g a t i n g r h i z o i d s was independent o f the o r i e n t a t i o n o f m i c r o t u b u l e s . W h i l e t h i s evidence does not c o n t r a d i c t the self-assembly m e c h a n i s m i n hélicoïdal walls o f green a l gae, b o t h hypotheses need a d d i t i o n a l e x p e r i m e n t a l v e r i f i c a t i o n . A cknowledgment s T h e a u t h o r t h a n k s D r . R . M a l c o l m B r o w n , J r . , at the U n i v e r s i t y o f T e x a s at A u s t i n , T e x a s , for the present p a r t o f the present s t u d y .

Literature Cited 1. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N. Mokuzai Gakkaishi 1984, 30, 98-99. 2. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N. Mokuzai Gakkaishi 1985, 31, 61-67. 3. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N. Planta 1985, 166, 161-68.

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4. Kuga, S.; Brown, R. M., Jr. J. Electron Microscop. Tech. 1987, 6, 34956. 5. Kuga, S.; Brown, R. M., Jr. Polym. Commun. 1987, 28, 311-14. 6. Lin, F. C.; Cooper, J.; Delmer, D. P. Science 1985, 230, 822-25. 7. Brown, R. M., Jr.; Montezinos, D. Proc. Natl. Acad. Sci. USA 1976, 73, 143-47. 8. Giddings, T. H., Jr.; Brower, D. L.; Staehelin, L. A. J. Cell Biol. 1980, 84, 327-39. 9. Wada, M.; Staehelin, L. A. Planta 1981, 151, 462-68. 10. Reiss, H. Planta 1984, 160, 428-35. 11. Herth, W. In Botanical Microscopy; Rolands, A. W., Ed.; 1985, 285310. 12. Chafe, S. C. Wood Sci. Technol. 1978, 12, 203-17. 13. Mueller, S. C.; Brown, R. M., Jr. Planta 1982, 154, 489-500. 14. Roberts, K.; Burgess croscopy; Robards, A 15. Giddings, T. H., Jr.; Staehelin, L. A. Planta 173, 22-30. 16. Staehelin, L. Α.; Giddings, T. H., Jr. In Developmental Order: Its Ori­ gin and Regulation; Sabtelny, S., and Green, P. B., Eds.; Liss: New York, 1982; p. 133-47. 17. Itoh, T.; Brown, R. M., Jr. Planta 1984, 160, 372-81. 18. Quader and Robinson, Ber. Deutsch. Bot. Ges. 1981, 94, 75-84. 19. Brown, R. M., Jr. J. Cell Sci. Suppl. 1985, 2, 13-32. 20. Willison, J. H. M.; Brown, R. M., Jr. J. Cell Biol. 1978, 77, 103-19. 21. Itoh, T.; O'Neil, R. M.; Brown, R. M., Jr. Protoplasma 1984, 123, 174-83. 22. Mizuta, S. Plant Cell Physiol. 1985, 26, 53-62. 23. Mizuta, S. Plant Cell Physiol. 1985, 26, 1443-53. 24. Itoh, T. Proc. Intern. Dissolv. Pulps Conf. 1987, 117-20. 25. Mizuta, S.; Okuda, K. Botanica Marina 1987, 30, 205-15. 26. Hogetsu, T. Plant Cell Physiol. 1983, 24, 777-81. 27. Kiermayer, O.; Sleytr, U. B. Protoplasma 1979, 101, 133-38. 28. Noguchi, T.; Tanaka, K.; Ueda, K. Cell Struct. Fund. 1981, 6, 217-29. 29. Hotchkiss, A. T., personal communication. 30. Herth, W. Planta 1983, 159, 347-56. 31. Brown, R. M., Jr. The Third Philip Morris Science Symposium 1978, 52-123. 32. Hotchkiss, A. T.; Brown, R. M., Jr. J. Phycol. 1987, 23, 229-37. 33. Quader, H. J. Cell Sci. 1986, 83, 223-34. 34. Itoh, T.; Legge, R. L.; Brown, R. M., Jr. J. Phycol. 1986, 22, 224-32. 35. Steward, F. C.; Muhlethaler, K. Ann. Bot. N.S. 1953, 17, 295-316. 36. Cranshaw, J.; Preston, R. D. Proc. R. Soc. Β London Ser. 1958, 148, 137-48. 37. Mizuta, S.; Wada, S. Bot. Mag. Tokyo 1981, 94, 343-53. 38. Lloyd, C. W. Intern. Rev. Cytol. 1984, 86, 1-51. 39. Haigler, C. H. In Cellulose Chemistry and its Applications; Nevell, T. P.; Zeronian, S. H., Eds.; Ellis Horwood; 1987, p. 30-83.

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40. Delmer, D. P. Adv. Carbohyd. Chem. & Biochem. 1983, 41, 105-53. 41. Hogetsu, T.; Takeuchi, Y. Planta 1982, 154, 426-34. 42. Franke, W. W.; Seib, E.; Osborn, M.; Weber, K.; Herth, W.; Falk, H. Cytobiol. 1977, 15, 24-48. 43. Lloyd, C. W. Nature 1979, 279, 239-41. 44. Lloyd, C. W. Ann. Rev. Plant Physiol. 1987, 38, 119-39. 45. Ishizawa, K.; Wada, S. Plant Cell Physiol. 1979, 20, 973-82. 46. Emons, A. M. E.; Wolters-Arts, A. M. C. Protoplasma 1983, 117, 6881. 47. Emons, A. M. C. Planta 1985, 163, 350-59. 48. Traas, J. Α.; Braat, P.; Emons, A. M. C.; Meeks, M.; Derksen, J. J. Cell Sci. 1985, 76, 303-20. 49. LaClaire, J. W., II. Planta 1987, 171, 30-42. 50. Mizuta, S.; Okuda, K. Bot. Gaz. 1987, 148, 297-307. 51. Okuda, Κ.; Mizuta, S 52. Itoh, T.; Brown, R. 53. Schneider, B.; Herth, W. Protoplasma 1986, 131, 142-52. 54. Vian, Β.; Roland, J. C. New Phytol. 1987, 105, 345-57. 55. Roland, J. C.; Reis, D.; Vian, B.; Satiat-Jeunemaitre, B.; Mosiniak, M. Protoplasma 1987, 140, 75-91. 56. Neville, A. C.; Levy, S. Planta 1984, 162, 370-84. RECEIVED March 27, 1989

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Chapter 20

Triple-Stranded Left-Hand Helical Cellulose Microfibril in Acetobacter xylinum and in Tobacco Primary Cell Wall 1

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George C. Ruben , Gordon H. Bokelman , and William Krakow 1

Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 Philip Morris Research Center, P.O. Box 26583, Richmond, VA 23261 IBM, T. J. Watson Researc

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Tobacco primary cell wall and normal bacterial Acetobacter xylinum cellulose formation produced a 36.8 ± 3Å triple-stranded left-hand helical microfibril in freeze-dried Pt-C replicas and in negatively stained preparations for transmission electron microscopy (TEM). A. xylinum growth in the presence of 0.25 mM Tinopal disrupted cellulose microfibril formation and produced a 17.8 ± 2.2Å left-hand helical submicrofibril. Models of the triple-stranded left-hand helical microfibril and the left-hand helical submicrofibril were directly compared to TEM images. Computer generated optical diffraction patterns of the models and the images were complex and similar. The submicrofibril appears to have the dimensions of a nine (1-4)-β-D-glucan parallel chain crystalline unit whose long, 23Å, and short, 19Å, diagonals form major and minor left-handed axial surface ridges every 36Å. Synthesis of the left-hand helical submicrofibril appears to be the driving force for self-assembly of a left-hand helical microfibril from three submicrofibrils. T h e g r a m negative b a c t e r i u m Acetobacier xylinum produces a r i b b o n of c r y s t a l l i n e cellulose I whose n e u t r a l sugar content is 9 6 . 8 % glucose a n d 3 . 2 % xylose (1). G r o w t h o f A. xylinum i n a m e d i u m c o n t a i n i n g 4,4-bis(4-anilino-6-bis (2-hydroxyethyl) amino-1,3,5-triazin-2-ylamino) 2 , 2 - stilbene-disulfonic a c i d , m a r k e t e d under c o m m o n names C a l c o f l u o r W h i t e S T or T i n o p a l L P W , c a n reversibly d i s r u p t n o r m a l r i b b o n f o r m a t i o n i n concentrations greater t h a n 0.1 m M a n d c a n increase the rate o f cellulose synthesis u p t o four times i n concentrations 1 m M o r greater (2-5). T h i s c o m p o u n d s t o i c h i o m e t r i c a l l y b i n d s t o glucose residues o f n e w l y p o l y m e r i z e d g l u c a n chains a n d makes cellulose I c r y s t a l l i n i t y i n the w e t state, measured 0097-6156/89/0399-0278$06.00/0 © 1989 American Chemical Society

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b y X - r a y d i f f r a c t i o n , u n d e t e c t a b l e . I n the d r y state cellulose I c r y s t a l l i n i t y is present a n d , d e p e n d i n g o n C a l c o f l u o r c o n c e n t r a t i o n , the c r y s t a l l i t e sizes c a n be reduced f r o m 65 a n d 74À to 28Â ( 2 , 3 ) . Since m i c r o f i b r i l s are ass u m e d to c r y s t a l l i z e f r o m s m a l l filaments to larger ones b y l a t e r a l f a s c i a t i o n , t h i s enhanced g r o w t h rate a n d a n undetectable wet state c r y s t a l l i n i t y have been i n t e r p r e t e d as the s e p a r a t i o n of the p r i m a r y cellulose I p o l y m e r i z a t i o n step f r o m a s e q u e n t i a l c r y s t a l l i z a t i o n step (2-5) w h i c h is b l o c k e d . W e have i n v e s t i g a t e d A. xylinum cellulose p r o d u c t i o n i n the absence a n d presence o f 0.25 m M T i n o p a l a n d r e p o r t o n the freeze-dried s t r u c t u r e of the 3 6 . 8 ± 3 Â m i c r o f i b r i l p r o d u c e d under n o r m a l c o n d i t i o n s a n d the 1 7 . 8 ± 2 . 2 Â s u b m i c r o f i b r i l p r o d u c e d i n the presence o f T i n o p a l (1). W e have also f o u n d s u b m i c r o f i b r i l s a n d m i c r o f i b r i l s i n tobacco p r i m a r y cell w a l l s i m i l a r t o A. xylinum. W e present models of the t r i p l e - s t r a n d e d l e f t - h a n d h e l i c a l m i c r o f i b r i l , the l e f t - h a n d h e l i c a l s u b m i c r o f i b r i l a n d the a p p a r e n t r e l a t i o n s h i p of the four sugar c h a i n fiber d i f f r a c t i o n u n i t cell to the s u b m i c r o f i b r i l ( 1 , 6 ) . C o m p u t e r generated singl models a n d of representative T E M m i c r o g r a p h s reinforce o u r i m p r e s s i o n o f congruency. T h e p a t t e r n s suggest t h a t the 17.8Â cellulose s u b m i c r o f i b r i l generated i n the presence of 0.25 m M T i n o p a l is o r g a n i z e d as a fibrillar u n i t w i t h n i n e p a r a l l e l sugar chains f o r m i n g a l e f t - h a n d e d h e l i c a l s t r u c t u r e (1). T h e prevalent a s s u m p t i o n t h a t the h i g h rate o f cellulose synthesis i n d u c e d i n A. xylinum b y T i n o p a l or C a l c o f l u o r is due to a cellulose p o l y m e r i z a t i o n step u n c o u p l e d f r o m a s e q u e n t i a l r a t e - l i m i t i n g c r y s t a l l i z a t i o n step is not consistent w i t h a n ordered c r y s t a l - l i k e s u b m i c r o f i b r i l . Methods and Materials T h e A. xylinum ( A m e r i c a n T y p e C u l t u r e C o l l e c t i o n 23769) was g r o w n o n 40 m M D-glucose a n d 0.5 M p h o s p h a t e ( p H 7, 2 0 ° C ) u n t i l i t f o r m e d a w h i t e , flocculent surface cap o n the s o l u t i o n . S a m p l e s p r e p a r e d i n t h i s w a y were t h e n g r o w n consecutively o n 0.25 m M T i n o p a l for 1 h , o n 0.25 m M T i n o p a l for 1.5 h , o n .025 m M T i n o p a l for 1 h , a n d t h e n o n 4 0 m M D glucose a n d 0.5 M p h o s p h a t e ( p H 7, 2 0 ° C ) for 1 h . E a c h 1.25 c m p e l l i c l e o f cellulose r i b b o n s w i t h cells g r o w i n g a n d tethered b y t h e i r r i b b o n s at i t s p e r i p h e r y was r i n s e d sequentially, first i n 5 separate dishes of w a t e r , t h e n i n 5 separate dishes of 1:3 e t h a n o l - w a t e r . E a c h p e l l i c l e was t h e n p l a c e d o n a 1.25 c m W h a t m a n 50 filter p a p e r disc, b l o t t e d , a n d frozen i n l i q u i d p r o p a n e . P e l l i c l e f r o m A. xylinum g r o w n n o r m a l l y was freeze-dried for 1.5 h at - 7 8 ° C , t h e n r e p l i c a t e d w i t h 17.3À P t - C (at - 1 7 8 ° C ) , a n d b a c k e d w i t h 90.2Â c a r b o n o n a W i l t e k Industries m o d i f i e d B a l z e r ' s 301 w i t h c r y o p u m p a n d r e b u i l t c o l d stage (7). T h e T i n o p a l - t r e a t e d s a m p l e was freeze-dried for 2.8 h at - 7 0 ° C , r e p l i c a t e d w i t h 16.4Â P t - C (at - 1 7 8 ° C ) , a n d backed w i t h 156Â o f c a r b o n . T h e t o b a c c o lower e p i d e r m a l peels were p r e p a r e d f r o m a C o k e r 319 leaf ( N o . 1 3 o n s t a l k ) . T h e s e peels were i m m e r s e d i n 1:3 e t h a n o l - w a t e r , b l o t t e d t o remove the excess s o l u t i o n a n d t h e n frozen o n 1.25 c m m i c a discs b y r a p i d i m m e r s i o n i n l i q u i d p r o p a n e ( - 1 9 0 ° C ) . T h i s s a m p l e was freeze-dried for 3 h at - 7 0 ° C , a n d t h e n r e p l i c a t e d w i t h 15.9Â P t - C (45° angle) at - 1 7 8 ° C in vacuo ( 6 . 6 7 / i P a ) a n d b a c k e d w i t h 139Â of f

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c a r b o n . A l l of the samples were digested i n 8 0 % s u l f u r i c a c i d . T h e replicas were r i n s e d i n deionized water a n d t h e n picked u p w i t h c a r b o n - c o a t e d , 300 m e s h grids f r o m u n d e r n e a t h a n d e x a m i n e d o n a J E M 1 0 0 C X i n s t r u m e n t as described p r e v i o u s l y (8). I n d i r e c t l y evaporated c a r b o n films of ~ 80Â thickness, suspended o n 300-mesh g r i d s , were used t o s u p p o r t A. xylinum cellulose t h a t h a d been treated w i t h b o i l i n g trifluoroacetic a c i d to remove hemicellulose. T h i s s a m p l e was negatively s t a i n e d w i t h 2 % u r a n y l acetate at p H 3.8, as p r e v i o u s l y described (9). T o contrast-enhance u n i d i r e c t i o n a l 15-18Â t h i c k P t - C - c o a t e d c e l l u lose specimens backed w i t h 100-173Â t h i c k c a r b o n films, m i c r o g r a p h s were contrast-reversed o n K o d a k 7302 fine-grain, p o s i t i v e film (8). I n a d d i t i o n to i n c r e a s i n g the contrast of 10-20Â features, the P t - C coated surfaces b e c a m e w h i t e , a n d the m o l e c u l a r details were m o d u l a t e d o n t h i s b a c k g r o u n d i n b l a c k s a n d shades of grey for easy s t r u c t u r a l i n t e r p r e t a t i o n (10-14). S h o o t i n g a t i l t series at 10° interval 80 k V w i t h a 5 m m foca a 6.6Â r e s o l u t i o n a n d a 2 6 2 5 A d e p t h of field i n the p i c t u r e series (8). T h e t i l t series was generally viewed stereoscopically, a n d t h e n a single i m a g e representing the 3 - D s t r u c t u r e was s h o w n . I n order to e s t i m a t e the r e a l size of a filament u n d e r n e a t h its P t - C c o a t i n g ( u n i d i r e c t i o n a l at a 45° a n gle), the l o n g i t u d i n a l axis of a filament h a d to be w i t h i n 10° of the general s h a d o w - d i r e c t i o n o n the r e p l i c a surface, so t h a t b o t h sides of the filament were P t - C coated. T h e filament s h o u l d be r o u g h l y at a 45° angle w i t h the P t - C source (checked b y stereo-viewing), a l t h o u g h filaments w h i c h were P t - C coated at a p p r o x i m a t e l y a 90° angle were o n l y 1Â s m a l l e r (12). Series of fiber w i d t h measurements, m a d e at i m a g e m a g n i f i c a t i o n s of 2 t o 5 m i l l i o n , a n d u s u a l l y n u m b e r i n g fewer t h a n 100, were averaged, a n d the P t - C film thickness, measured o n the q u a r t z - c r y s t a l m o n i t o r , s u b t r a c t e d f r o m the average w i d t h , to give a n estimate of the real filament d i a m e t e r (11). It was recently f o u n d t h a t t h i s w i d t h - c o r r e c t i o n m e t h o d s h o u l d be reduced b y 1.5Â ( 1 2 , 1 3 ) . I n contrast, the center to center distance between either ridges or grooves a l o n g a P t - C coated m i c r o f i b r i l or s u b m i c r o f i b r i l were assumed to be u n c h a n g e d . C o m p u t e r generated o p t i c a l diffraction p a t t e r n s of single cellulose m i crofibrils a n d s u b m i c r o f i b r i l s were o b t a i n e d f r o m large field m i c r o g r a p h s p r i n t e d at 1 0 x m a g n i f i c a t i o n . T h e images were d i g i t i z e d v i a a t e l e v i s i o n c a m e r a connected to a n image frame store a n d c o n t r o l l e d t h r o u g h a n I B M 3 0 9 D m a i n frame c o m p u t e r ( 1 5 , 1 6 ) . Briefly, a n area of the image c o n t a i n i n g a single molecule was selected b y a c i r c u l a r electronic a p e r t u r e defined b y a graphics overlay cursor under o p e r a t o r c o n t r o l . W h e n the image was s h i p p e d t o the host C P U , the fast fourier t r a n s f o r m ( F F T ) a n d subsequent power s p e c t r u m were c o m p u t e d f r o m the region defined e l e c t r o n i c a l l y , w i t h a n edge g r a d i n g f u n c t i o n to e l i m i n a t e h a r d edge d i f f r a c t i o n . T h e e l e c t r o n i c a l l y s a m p l e d i m a g e region a n d i t s power s p e c t r u m ( o p t i c a l d i f f r a c t i o n p a t t e r n ) were t h e n sent to a h a r d copy s l i d e - m a k i n g device. T h e diffract i o n p a t t e r n was c a l i b r a t e d u s i n g Keuffel & Esser (46 1513) 10 x 10 to c m g r a p h paper w i t h the same setup for d i g i t i z i n g the molecule. T h e o p t i c a l 6

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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d i f f r a c t i o n p a t t e r n s were o r i e n t e d i n the same d i r e c t i o n as the spacings a p p e a r e d i n the molecule ( F i g u r e s 5, 6,7 a n d 10) a n d were generally scaled over 5-6 decades o f i n t e n s i t y so t h a t i n d i v i d u a l spot i d e n t i t y a n d p o s i t i o n at lower intensities c o u l d be c o r r e c t l y identified at the h i g h e r i n t e n s i t y levels w h i c h enhanced fainter spots. T h e transparencies were a l l r e p h o t o g r a p h e d w i t h a l o w c o n t r a s t film developer s y s t e m a n d p r i n t e d at the same m a g n i f i c a t i o n w i t h 0-2 grade I l f o r d m u l t i g r a d e II p r i n t p a p e r . T h e features of these d i f f r a c t i o n p a t t e r n s were recorded o n overlayed t r a c i n g paper o n a l i g h t t a b l e . T h e distance between centers of spots s y m m e t r i c w i t h the d i f f r a c t i o n p a t t e r n center were measured w i t h a vernier caliper a n d t h e n d i v i d e d b y two t o give the spot p o s i t i o n i n r e c i p r o c a l space. T h e exact center of most elongated spots was e s t i m a t e d . T h e p r e c i s i o n o f the d i f f r a c t i o n measurements was n o t better t h a n 8%. T h e r e c i p r o c a l space d i s t a n c e for the molecule was d i v i d e d i n t o the r e c i p r o c a l distance for the 1 m m g r a p h paper a n d m u l t i p l i e d b y a m a g n i f i c a t i o n factor i n À / m m t o c o m p u t e o p t i c a l d i f f r a c t i o n spacings i n A n g s t r o m s Results I m a g i n g A. xylinum's cellulose r i b b o n has p r e v i o u s l y been a c c o m p l i s h e d b y a d h e r i n g cells t o a film coated g r i d , g r o w i n g the cells o n a n u t r i e n t buffer s o l u t i o n , a n d negative s t a i n i n g t h e m for v i s i b i l i t y a n d for t r a n s m i s s i o n electron microscopy. Images show t h a t A. xylinum produces a lefth a n d t w i s t e d r i b b o n n o r m a l l y , a n d i n the presence o f c a r b o x y m e t h y l c e l l u lose ( C M C ) ( 4 , 5 ) . W h e n g r o w n i n the presence of 0.25 m M C a l c o f l u o r , t h i s m o r p h o l o g y is d r a m a t i c a l l y altered to a b r o a d cellulose b a n d c o m p o s e d of 15Â a n d larger filaments (2-5). W e have a p p r o a c h e d s p e c i m e n p r e p a r a t i o n for T E M i m a g i n g differently. A. xylinum n a t u r a l l y forms a pellicle or gel of cellulose r i b b o n s o n the surface o f a s o l u t i o n d u r i n g g r o w t h w i t h the cells t e t h e r e d at the p e l l i c l e ' s p e r i p h e r y b y r i b b o n s . Since sequential g r o w t h c o n d i t i o n s are recorded l i n e a r l y a l o n g a r i b b o n , A. xylinum was g r o w n n o r m a l l y , i n 0.25 m M T i n o p a l , a n d n o r m a l l y a g a i n , t h e n v i s u a l i z e d after f r e e z e - d r y i n g a n d P t - C r e p l i c a t i o n . F i g u r e 1 shows a n o r m a l l y g r o w n pellicle o f A. xylinum cellulose r i b b o n s t h a t a p p e a r l i n e a r a n d u n t w i s t e d . T h e s m a l l a r r o w s p o i n t o u t lefth a n d t w i s t e d m i c r o f i b r i l s w i t h i n the r i b b o n s . W h e n A. xylinum was g r o w n i n the presence o f 0.25 m M T i n o p a l , a n altered cellulose was p r o d u c e d as s h o w n i n F i g u r e 2. T h e same p r e p a r a t i o n s h o w n at h i g h e r m a g n i f i c a t i o n i n F i g u r e 3 revealed 33Â P t - C coated s u b m i c r o f i b r i l s ( s m a l l a r r o w s ) . T h e s e i n d i v i d u a l s u b m i c r o f i b r i l s averaged 1 7 . 8 ± 2 . 2 Â after c o r r e c t i o n for the P t - C coat (1), a n d f r e q u e n t l y t w i s t e d together to f o r m larger fibrils. S u b m i c r o f i b r i l s , p r e v i o u s l y i m a g e d , have been c o r r e l a t e d w i t h the four g l u c a n c h a i n X - r a y fiber d i f f r a c t i o n u n i t cell as o p p o s e d to the two or eight c h a i n u n i t cell b y average d i a m e t e r measurements (1). I n the F i g u r e 4 s u b m i c r o f i b r i l m o d e l the side a n d d i a g o n a l d i m e n s i o n s of the u n i t cell were e s t i m a t e d . B y t r a n s l a t i n g a n d r o t a t i n g t h i s p a r a l l e l n i n e g l u c a n c h a i n cross s e c t i o n , the l o n g d i a g o n a l o f 23Â a n d the short d i a g o n a l of 19Â generated a m a j o r a n d m i n o r surface ridge a n d also generate a s u b m i c r o f i b r i l w i t h a

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

F i g u r e 1. F r e e z e - d r i e d gel of A. xylinum cellulose r i b b o n s d e p o s i t e d d u r i n g n o r m a l g r o w t h . T h e arrows p o i n t to t r i p l e - s t r a n d e d l e f t - h a n d h e l i c a l m i crofibrils averaging 36.8 ± 3 A i n d i a m e t e r (1). T h e s a m p l e was r e p l i c a t e d w i t h 17.3Â P t - C a n d backed w i t h 90.2Â of c a r b o n .

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F i g u r e 2. Freeze-dried A. xylinum cellulose g r o w n i n the presence of 0.25 m M T i n o p a l a n d r e p l i c a t e d w i t h 16.4Â P t - C a n d backed w i t h 156Â of c a r b o n . A t a n g l e d mass o f 33Â P t - C coated s u b m i c r o f i b r i l s was f o r m e d i n s t e a d o f n o r m a l r i b b o n cellulose.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

F i g u r e 3. H i g h e r m a g n i f i c a t i o n of A. xylinum cellulose g r o w n i n the presence of 0.25 m M T i n o p a l . T h e arrows p o i n t to single P t - C coated s u b m i c r o f i b r i l s averaging 33Â i n d i a m e t e r (17.8 ± 2.2Â after c o r r e c t i o n for the 16.4Â P t C c o a t i n g ) . M a n y of these s u b m i c r o f i b r i l s were t w i s t e d together f o r m i n g thicker fibers.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

F i g u r e 4. T h e m o d e l c o n t a i n s the four c h a i n X - r a y fiber d i f f r a c t i o n u n i t cell d i m e n s i o n s , l i s t e d at the left of the figure, w h i c h were d r a w n as a cross section t h r o u g h the g l u c a n chains. T h i s u n i t cell has encompassed n i n e g l u c a n chains w i t h exterior side d i m e n s i o n s of 15Â a n d 17Â a n d d i a g o n a l d i m e n s i o n s of 19Â a n d 23Â as s h o w n . B y r o t a t i n g a n d t r a n s l a t i n g the u n i t cell we generated a s u b m i c r o f i b r i l ( S M ) geometry w i t h a m a j o r ridge ( 2 3 Â diagonal) a n d a n i n t e r m e d i a t e m i n o r ridge (19Â d i a g o n a l ) . M a j o r ridges o c c u r every 36Â w i t h a h e l i c a l p i t c h of 72Â. T h e o r i g i n a l s u b m i c r o f i b r i l average measurement f r o m m i c r o g r a p h s was 1 7 . 8 ± 2 . 2 Â , i n agreement w i t h the 18.5Â f o u n d b y averaging the exterior sides a n d d i a g o n a l s . T h e average of these d i m e n s i o n s was s l i g h t l y larger t h a n o r i g i n a l l y r e p o r t e d (1) since the m i n o r d i a g o n a l was increased f r o m 16.5Â to 19Â. G e n e r a t i o n of a n ordered s u b m i c r o f i b r i l b y t h i s a p p r o a c h was consistent w i t h the E l l i s a n d W a r w i c k e r u n i t cell d e r i v a t i o n w h i c h o n l y assumed p a r a l l e l g l u c a n c h a i n axes (6).

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n o n u n i f o r m d i a m e t e r whose m a x i m u m d i m e n s i o n is 23Â. T h i s s u b m i c r o f i b r i l h a d a h e l i c a l p i t c h of 72Â w i t h a m a j o r ridge crossing the axis every 36Â a n d a m i n o r ridge crossing h a l f - w a y between the m a j o r ridges. I n F i g u r e 5 the s u b m i c r o f i b r i l m o d e l ( F i g . 5a) was c o m p a r e d at equivalent m a g n i f i c a t i o n t o a T E M i m a g e of the s u b m i c r o f i b r i l ( F i g . 5c). T h e P t - C coated 36Â m a j o r ridge spacings a n d some of the m i n o r ridges a l o n g the s u b m i c r o f i b r i l surface were preserved b u t the d i a m e t e r increased b y 15.2Â to 33Â. T h e i m a g e o r i e n t a t i o n w i t h the c o m p u t e r generated o p t i c a l d i f f r a c t i o n p a t t e r n s i n F i g u r e s 5-7 were also preserved. T h e m o d e l ' s d i f f r a c t i o n p a t t e r n ( F i g . 5b) was generated f r o m one side of a left-handed h e l i x w i t h p r o m i n e n t spacings ( ± 8 % precision) at 25Â a n d 12Â, w i t h a v e r t i c a l s p a c i n g at 12Â a n d w i t h adjacent s p o t s p a r a l l e l i n g the 25Â a n d 12Â s p o t p o s i t i o n s . T h e o p t i c a l diffraction p a t t e r n generated f r o m the T E M image of the s u b m i c r o f i b r i l ( F i g . 5d) was s i m i l a r to F i g . 5b w i t h left-handed spacings (±8%*) of 26Â a n d 12Â or 14Â a n d w i t h two spots below a n d above the larger s p a c i n g of 26À ( see h o r i z o n t a l arrow 26À a p a r t traverse the s u b m i c r o f i b r i l axis at a 45 ± 15° angle. T h i s angle has g e o m e t r i c a l l y d e t e r m i n e d the m a j o r ridge repeat a l o n g the fiber a x i s of 36Â (26Â / s i n 45° = 3 6 . 8 À ) . T h e P t - C r e p l i c a r e s o l u t i o n i n F i g u r e 5 d is 9 À was o n l y s l i g h t l y better t h a n the 12Â or 13Â p r e v i o u s l y r e p o r t e d ( 1 0 , 1 3 ) . T h e m i c r o f i b r i l m o d e l c o m p o s e d of three s u b m i c r o f i b r i l s l e f t - h a n d t w i s t e d together i n F i g u r e 6 a was c o m p a r e d at the same m a g n i f i c a t i o n to the P t - C coated m i c r o f i b r i l image i n F i g u r e 6c. T h e m a j o r a n d m i n o r ridges ( t h i c k a n d t h i n a r r o w heads) were v i s i b l e a l o n g the s u b m i c r o f i b r i l s i n the m o d e l a n d they c o u l d also be seen a l o n g the c e n t r a l s u b m i c r o f i b r i l i n the T E M i m a g e . T h e c o m p u t e r generated o p t i c a l diffraction p a t t e r n s of the m o d e l s h o w n i n F i g u r e 6b a n d of the P t - C coated m i c r o f i b r i l s h o w n i n F i g u r e 6d were c o m p l e x b u t s i m i l a r . In the diffraction p a t t e r n i n F i g u r e 6b, we c o u l d assign the v e r t i c a l s p a c i n g i n the m i c r o f i b r i l m o d e l at 23Â a n d one l e f t - h a n d e d s p a c i n g at 23Â (—45° ± 1 5 ° angle) to the l o n g d i a g o n a l (see F i g . 4) or m a x i m u m d i a m e t e r of the s u b m i c r o f i b r i l . T h e t h i r d 23Â s p a c i n g at - 1 7 ° ± 15° angle was p r o b a b l y related t o a foreshortened p r o j e c t i o n of the m a j o r ridge center to center distance a l o n g the s u b m i c r o f i b r i l w r a p p e d a r o u n d the m i c r o f i b r i l a x i s . T h e v e r t i c a l s p a c i n g i n F i g u r e 6 d of the T E M i m a g e was 27Â a n d there was also a left-handed s p a c i n g at 27Â at a —45° ± 15° angle. T h i s larger value m a y be due to the s l i g h t l y greater s e p a r a t i o n between the s u b m i c r o f i b r i l s i n the T E M i m a g e . A t h i r d s p a c i n g at 23Â or 21Â at a —17° ± 1 5 ° angle was p r o b a b l y related to a foreshortened m a j o r ridge s p a c i n g a l o n g the s u b m i c r o f i b r i l . T h e negatively s t a i n e d m i c r o f i b r i l s h o w n i n F i g u r e 7 a was first treated to remove hemicellulose before i t was negatively s t a i n e d . T h e c o m p u t e r generated o p t i c a l diffraction p a t t e r n was o n l y of the u p p e r m i c r o f i b r i l w i t h the 33À s p a c i n g . T h e diffraction p a t t e r n i n F i g u r e 7b showed a v e r t i c a l s p a c i n g at 24À, a left-handed s p a c i n g of 23Â at a —45° ± 5° angle t o the h o r i z o n t a l a x i s , a n d a s p a c i n g at 25Â s i m i l a r to the m a j o r ridge s p a c i n g i n F i g u r e 6b. T h e 33Â left-handed a x i a l m i c r o f i b r i l s p a c i n g was g e o m e t r i c a l l y related to the 23Â s u b m i c r o f i b r i l s p a c i n g (23Â / s i n 45° = 3 2 . 5 Â ) .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Figure 5. The submicrofibril model in a and the Pt-C coated submicrofibril image in c were compared at the same magnification. The optical diffraction of the model in b demonstrated a left-handed surface spacing of 25A and 12Â with two spots (horizontal arrows) which appeared on layer lines below and above the 25Â spot. The first layer line occurs at 12Â and was the vertical separation of the minorridgeposition between two majorridges23Â apart. The general features of this lefthanded optical diffraction pattern occurred in the TEM image in d with spacings at 26Â and 12A or 14Â with similar spots indicated by horizontal arrows below and above the major ridge spacing of 26A. (c reproduced with permission from réf. 1. Copyright 1987 Elsevier.)

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Figure 6. This model of the microfibril (M) composed of three submicrofibrils (SM) twisted together in a left-handed fashion (a) was compared at the same magnification to the Pt-C coated microfibril image in c. The major and minor ridges (thick and thin arrowheads) were visible along the submicrofibrils in the model and they could also be seen along a submicrofibril at the center of the TEM image. The optical diffraction pattern of the model shown in b and of the Pt-C coated microfibril shown in d were complex but similar. In d, the submicrofibrils cross the TEM microfibril axis at a 45β ± 15 ° angle, (c reproduced with permission from réf. 1. Copyright 1987 Elsevier.)

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F i g u r e 7. T h i s m i c r o f i b r i l was treated w i t h hot trifluoroacetic a c i d to remove hemicellulose a n d was t h e n negatively s t a i n e d w i t h 2 % u r a n y l acetate i n F i g u r e 7 a . T h e o p t i c a l diffraction p a t t e r n i n F i g u r e 7b was o n l y of the u p p e r m i c r o f i b r i l s h o w i n g the 33Â s p a c i n g . In F i g u r e 7b the s u b m i c r o f i b r i l s cross the T E M m i c r o f i b r i l axis at a 45° ± 5 ° angle. (7a r e p r o d u c e d w i t h p e r m i s s i o n f r o m Réf. 1. © 1987 E l s e v i e r Science P u b l i s h e r s Β. V . )

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It was p r e v i o u s l y p o i n t e d out t h a t filaments the size of s u b m i c r o f i b r i l s exit the cell w a l l of A. xylinum t h r o u g h pores (4). F i g u r e 8 shows how a m i c r o f i b r i l self-assembled f r o m three s u b m i c r o f i b r i l s o n the e x t e r i o r of the cell (1). A r r o w s p o i n t to s u b m i c r o f i b r i l s 1 a n d 2 o n the cell surface. A t their j u n c t i o n s u b m i c r o f i b r i l 2 crosses 1 i n a left-handed m a n n e r v i s i b l e i n stereo-micrographs (not s h o w n ) . S u b m i c r o f i b r i l 3 j o i n s a n d t h e n crosses the twisted p a i r of m i c r o f i b r i l s i n a left-handed m a n n e r near the b o t t o m of the figure. T h e image of s u b m i c r o f i b r i l 3 also showed t h a t i t was not r o d - l i k e b u t was l e f t - h a n d s u p e r - t w i s t e d . A m o d e l of t h i s process i n F i g ure 9 showed three l e f t - h a n d h e l i c a l s u b m i c r o f i b r i l s l a b e l l e d S M 1, 2 a n d 3 emerging f r o m the cell w a l l at the top of the figure. T h i s m o d e l depicts three associated s u b m i c r o f i b r i l s being s p u n together to f o r m a m i c r o f i b r i l , a l t h o u g h i t was unclear how three s u b m i c r o f i b r i l s i n i t i a l l y came together. T h i s s p i n n i n g process m a y be d r i v e n b y the l e f t - h a n d r o t a t i o n a n d e l o n g a t i o n of the s u b m i c r o f i b r i l s d u r i n g cellulose synthesis. T h e left r o t a t i o n of each s u b m i c r o f i b r i l drive m i c r o f i b r i l w h i c h also left r o t a t e d as i t grows longer. If any of the s u b m i c r o f i b r i l s elongated more r a p i d l y t h a n the other p a i r , i t w o u l d become l e f t - h a n d s u p e r - t w i s t e d as p o i n t e d out i n F i g u r e 8 for s u b m i c r o f i b r i l 3. In t o b a c c o p r i m a r y cell w a l l the cellulose m i c r o f i b r i l s observed i n d i v i d u a l l y or associated w i t h bundles were also t r i p l e - s t r a n d e d a n d l e f t - h a n d h e l i c a l . These observations are s h o w n i n F i g u r e 10. Since cellulose is o n l y 1 9 % of the tobacco cell w a l l (17), the task of finding a n d i d e n t i f y i n g cellulose was c o m p l i c a t e d . F o r t h i s reason A. xylinum w h i c h p r o d u c e s a pure r i b b o n of cellulose was used for s t u d y i n g cellulose s t r u c t u r e . Discussion and

Conclusions

S u b m i c r o f i b r i l a n d t r i p l e - s t r a n d e d l e f t - h a n d h e l i c a l m i c r o f i b r i l s are f o u n d i n t o b a c c o p r i m a r y cell w a l l a n d b a c t e r i a l A. xylinum cellulose. W e suspect f r o m our results a n d the l i t e r a t u r e survey o u t l i n e d i n reference (1) t h a t the t r i p l e s t r a n d e d structures are p r o m i n e n t i n the p r i m a r y p l a n t cell w a l l . T h e h i g h l y c r y s t a l l i n e cellulose of p l a n t a n d algae secondary cell w a l l appears b y X - r a y fiber diffraction (18,19) a n d T E M l a t t i c e i m a g i n g (20-23) to be largely c r y s t a l l i n e a r r a y s of p l a n a r s t r a i g h t chains of ( l - 4 ) - / ? - D - g l u c a n chains. T h e s u b m i c r o f i b r i l i n F i g u r e 5c was clearly a left-handed h e l i x w i t h a m a j o r ridge repeat of 26Â a n d m i n o r ridge h a l f s p a c i n g at 12Â or 14Â evident i n the o p t i c a l diffraction p a t t e r n ( F i g . 5d). T h e correspondence between the m o d e l a n d the P t - C coated s u b m i c r o f i b r i l was stronger t h a n expected since i t was k n o w n t h a t evaporated m e t a l coatings do not j u s t adhere where they l a n d at o r d i n a r y r e p l i c a t i o n temperatures (24-27). T h e specimen t e m p e r a t u r e used i n t h i s w o r k (—178°C) was colder b y 110°C t h a n the t e m p e r a t u r e used p r e v i o u s l y (7). T h e greater m e t a l s t i c k i n g coefficient at lower temperatures preserved surface resolution to 9Â i n F i g u r e 5 d . T h e m i c r o f i b r i l m o d e l i n F i g u r e 6a s t r o n g l y resembled the P t - C coated m i c r o f i b r i l i n F i g u r e 6c, w h i c h also c o n t a i n e d a m a j o r a n d m i n o r ridge a l o n g a s u b m i c r o f i b r i l at the center of the image. T h e assignment of s p o t s to molecular features i n the c o m p l e x o p t i c a l diffraction p a t t e r n i n F i g u r e 6b

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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F i g u r e 8. Since the s u b m i c r o f i b r i l s exit the cell w a l l o f A. xylinum t h r o u g h pores (4), the self-assembly of a t r i p l e - s t r a n d e d m i c r o f i b r i l has o c c u r r e d at the e x t e r i o r surface of the cell (1). S u b m i c r o f i b r i l s 1 a n d 2 a p p e a r e d s u p e r t w i s t e d o n the cell surface. A t t h e i r j u n c t i o n s u b m i c r o f i b r i l 2 crossed 1 i n a l e f t - h a n d e d m a n n e r w h i c h is o n l y v i s i b l e w i t h s t e r e o - m i c r o g r a p h s (not s h o w n ) . S u b m i c r o f i b r i l 3, w h i c h was also l e f t - h a n d s u p e r - t w i s t e d , j o i n e d a n d crossed the double fiber i n a l e f t - h a n d e d m a n n e r . T h i s s p e c i m e n was coated w i t h 16.4Â o f P t - C . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Réf. 1. © E l s e v i e r Science P u b l i s h e r s Β. V . )

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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F i g u r e 9. T h i s m o d e l shows three left-handed h e l i c a l s u b m i c r o f i b r i l s ( S M ) 1, 2 a n d 3 w h i c h emerged f r o m the cell w a l l at their t e r m i n i . It was not clear how the s u b m i c r o f i b r i l s first associated w i t h other s u b m i c r o f i b r i l s b u t once associated they were s p u n together. T h i s m o d e l assumed t h a t cellulose synthesis p r o v i d e d the m e c h a n i c a l force t h a t s i m u l t a n e o u s l y extended a n d l e f t - h a n d r o t a t e d the s u b m i c r o f i b r i l s , w h i c h i n t u r n drove the secondary f o r m a t i o n of three s u b m i c r o f i b r i l s into a l e f t - h a n d h e l i c a l m i c r o f i b r i l ( M ) .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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c o u l d not be a c c o m p l i s h e d yet except at a r u d i m e n t a r y l e v e l . T h e v e r t i c a l a n d l e f t - h a n d e d s p a c i n g (—45° ± 15°) at 23Â represented the m a x i m u m w i d t h or l o n g d i a g o n a l ( F i g . 4) of the s u b m i c r o f i b r i l . T h e —17° ± 15° spaci n g at 23Â p r o b a b l y represented a foreshortened m a j o r ridge s p a c i n g a l o n g a s u b m i c r o f i b r i l c u r v i n g a r o u n d the m i c r o f i b r i l a x i s . T h e same spacings also a p p e a r e d i n the o p t i c a l diffraction p a t t e r n ( F i g . 7b) of the n e g a t i v e l y s t a i n e d m i c r o f i b r i l i n F i g u r e 7 a . T h e o p t i c a l d i f f r a c t i o n p a t t e r n o f the freeze-dried P t - C r e p l i c a t e d m i c r o f i b r i l ( F i g . 6d) was far m o r e c o m p l e x , res o l v i n g m o r e s p o t s t h a n its negatively s t a i n e d c o u n t e r p a r t i n F i g u r e 7b. T h e v e r t i c a l a n d l e f t - h a n d e d spacings at —45° ± 1 5 ° i n the P t - C coated m i c r o f i b r i l were 27Â, due either t o a t r u l y greater space between s u b m i crofibrils i n F i g u r e 6c or m e r e l y to the 8% p r e c i s i o n o f measurement w h i c h m a d e 27À a n d 23Â of d o u b t f u l d i s c r i m i n a b i l i t y . A 21À or 23Â s p a c i n g was also l o c a t e d at —17° ± 15°, w h i c h c o u l d be a t t r i b u t e d t o the s u b m i c r o f i b r i l . W e believe t h a t the T E M a n d m o d e l images of the cellulose h e l i x i n c o n j u n c t i o n w i t h the c o m p u t e v i d e s t r o n g evidence for a s u b s t r u c t u r e t h a t includes a l e f t - h a n d h e l i c a l m i c r o f i b r i l c o m p o s e d of three s u b m i c r o f i b r i l s ( F i g . 8) a n d a l e f t - h a n d h e l i c a l s u b m i c r o f i b r i l . E v i d e n c e t h a t the four g l u c a n c h a i n fiber d i f f r a c t i o n u n i t cell h a d the same size as the s u b m i c r o f i b r i l came f r o m the correspondence between the u n i t cell's average diameter of 18.5Â a n d the measured s u b m i c r o f i b r i l d i a m e t e r of 17.8 ± 2.2Â (1), the m a j o r a n d m i n o r ridges v i s i b l e i n T E M ( F i g . 5c) a n d the m a x i m u m d i a m e t e r of 23Â for the s u b m i c r o f i b r i l as d e t e r m i n e d f r o m the m i c r o f i b r i l ' s o p t i c a l diffraction p a t t e r n . T h i s s t r o n g c o r r e l a t i o n has s u p p o r t e d the hypothesis t h a t the p a r a l l e l n i n e g l u c a n c h a i n u n i t represents the s u b m i c r o f i b r i l cross s e c t i o n . T h e m e t h o d we have advanced for g e n e r a t i n g the s u b m i c r o f i b r i l f r o m the n i n e sugar u n i t cross section i n F i g u r e 4 provides a m o d e l for s u b m i c r o f i b r i l synthesis. A f t e r each nine g l u c a n c h a i n cross section has been assembled a n d m o v e d to make r o o m for the n e x t , the s u b m i c r o f i b r i l e l o n gates a n d s i m u l t a n e o u s l y rotates. M i c r o f i b r i l self-assembly i n F i g u r e 8 was based o n a cellulose synthesis-powered m e c h a n i s m w h i c h extends a n d r o tates each s u b m i c r o f i b r i l i n the F i g u r e 9 m o d e l . O n e obvious p r e d i c t i o n of the m o d e l , besides the f o r m a t i o n of a l e f t - h a n d h e l i c a l m i c r o f i b r i l , was t h a t the m i c r o f i b r i l w o u l d also rotate i n a l e f t - h a n d e d d i r e c t i o n as i t e l o n g a t e d . W e w i l l r e t u r n to t h i s p o i n t i n the last p a r a g r a p h . O n e i m p o r t a n t consequence of the n i n e p a r a l l e l g l u c a n c h a i n u n i t , t r a n s l a t e d a n d r o t a t e d to generate a l e f t - h a n d h e l i c a l s u b m i c r o f i b r i l , was t h a t a l l the ( l - 4 ) - / ? - D - g l u c a n chains were not c o n f o r m a t i o n ally equivalent i n the F i g u r e 4 s u b m i c r o f i b r i l m o d e l . F o r instance, the g l u c a n c h a i n at the center of t h i s m o d e l w o u l d f o r m a l e f t - h a n d e d h e l i x 7 cellobiose u n i t s l o n g w i t h a 72À p i t c h , b u t a l l other g l u c a n chains w o u l d require m o r e cellobiose u n i t s to reach the same a x i a l p o s i t i o n . Recent t h e o r e t i c a l c a l c u l a t i o n s i n d i c a t e t h a t a f a m i l y of l e f t - h a n d h e l i c a l ( l - 4 ) - / ? - D - g l u c a n chains 7-9 cellobiose u n i t s l o n g w i t h pitches of a b o u t 72Â to 93Â exist near a s i m i l a r c o n f o r m a t i o n a l energy m i n i m u m (28). T h e nine p a r a l l e l g l u c a n c h a i n , t w i s t e d c r y s t a l m o d e l thus cannot be r u l e d out o n either the basis of g l u c a n

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Figure 10. a, Bundled cellulose microfibrils in the lower epidermal cell wall (facing mesophyll cells) of Coker 319 tobacco leaves. This epidermal peel was freeze-dried, Pt-C replicated (15.9 Â thick) and carbon film backed (133 Â thick), b, A cellulose microfibril is seen connecting two bundles of microfibrils (similar to a). This Pt-C coated microfibril averages 51 Â in width, shows left-handed surface striations, and splits into three smaller submicrofibrils. c, Tobacco primary cell wall Pt-C coated microfibril averaging 50 Â shows left-handed surface striations. d, Three 16-18-Â submicrofibrils in adjacent ridges wrap (see arrows) in a left-handed fashion around the microfibril axis. Bar, 100 A . (a-d reproduced with permissionfromréf. 1. Copyright 1987 Elsevier.)

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Figure 10. e, Optical diffraction pattern of c. Its important features were similar to those in the A . xylinum microfibril diffraction pattern in Figure 6d: left-handed spacings at roughly 24 ± 3 A , a vertical spacing at 26 Λ and right-handed spacings at 36 Â and 28 Â The left-handed pattern at 47 Â and 48 A , not previously seen, was probably caused by an artificial bunching of the submicrofibrils (d) when the microfibril was lifted above the cell wall surface by peeling the lower epidermal cell layer from the tobacco leaf.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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c h a i n energy considerations or the lack of left-handed g l u c a n c h a i n conform a t i o n s . O t h e r s have also suggested t h a t cellulose g l u c a n chains f o r m a left-handed h e l i x of 72Â (29) a n d can also be left-handed h e l i c a l i n s o l u t i o n (30). T h e nonequivalence o f g l u c a n chains i n a t w i s t e d c r y s t a l w o u l d l i m i t its m a x i m u m l a t e r a l dimensions. Since chains farthest f r o m the axis center are less t w i s t e d , it is not s u r p r i s i n g t h a t i n larger secondary cell w a l l c r y s t a l l i n e cellulose a l l the g l u c a n chains can energetically assume a flat l i n e a r c o n f i g u r a t i o n w i t h each cellobiose u n i t related to the next b y a 180° r o t a t i o n (18-23,31). O u r observations suggest t h a t the s u b m i c r o f i b r i l s t r u c t u r e is a consequence of its s m a l l size a n d of ( l - 4 ) - / ? - D - g l u c a n c h a i n s ' n a t u r a l tendency t o assume a left-handed h e l i x (21). L a r g e r cellulose c r y s tals c a n u n t w i s t ( l - 4 ) - / ? - D - g l u c a n chains because of the favorable energetics of f o r m i n g p l a n a r s t r a i g h t c h a i n crystals (31). T h e m o d e l i n F i g u r e 9 predicts t h a t each m i c r o f i b r i l w o u l d rotate i n the process of cellulose r i b b o n f o r m a t i o n . If the A. xylinum cell were h e l d s t a t i o n a r y , then the r i b b o the r i b b o n were h e l d s t a t i o n a r y , then the cell w o u l d rotate (32). T h e l a t t e r case e x p l a i n s w h y r i b b o n s appear u n t w i s t e d i n the pellicle of r i b b o n s s h o w n i n F i g u r e 1. M o r e o v e r , i t has been d e m o n s t r a t e d t h a t a n A. xylinum cell ceased r o t a t i o n w h e n C a l c o f l u o r ( > 0.1 m M ) was added to the s o l u t i o n (32). P r e v i o u s w o r k has s h o w n t h a t the presence of C a l c o f l u o r or T i n o p a l c o u l d d r a m a t i c a l l y increase A. xylinum cellulose synthesis. T h i s observat i o n was the basis for the hypothesis t h a t cellulose p o l y m e r i z a t i o n can be u n c o u p l e d f r o m a slower sequential c r y s t a l l i z a t i o n step (2-5). W e believe the hypothesis is not consistent w i t h our observations. A t the very least, the presence of a n ordered a n d c r y s t a l - l i k e s u b m i c r o f i b r i l p r o d u c e d i n the presence of 0.25 m M T i n o p a l w o u l d relegate T i n o p a l ' s or C a l c o f l u o r ' s effects to a n event o c c u r r i n g after the i n i t i a l cellulose p o l y m e r i z a t i o n - c r y s t a l l i z a t i o n step or steps. T h e d a t a we present show t h a t A. xylinum cellulose m i c r o f i b r i l s are f o r m e d b y s u b m i c r o f i b r i l s b e i n g s p u n together, as s h o w n i n F i g u r e s 8 a n d 9, r a t h e r t h a n associated t h r o u g h a m e c h a n i s m of l a t e r a l f a s c i a t i o n ( 1 , 4 , 5 ) . W e have d e m o n s t r a t e d t h a t T i n o p a l d i s r u p t s r i b b o n f o r m a t i o n a n d the m i c r o f i b r i l f o r m a t i o n process i n F i g u r e s 2 a n d 3. Since the m i c r o f i b r i l s p i n n i n g process either rotates the r i b b o n or the c e l l , its d i s r u p t i o n w o u l d u n c o u p l e a r a t e - l i m i t i n g r o t a t i o n , e l o n g a t i o n process f r o m cellulose synthesis. T h u s , u n c o u p l i n g of a cell's or a r i b b o n ' s m e c h a n i c a l r o t a t i o n w i t h T i n o p a l or C a l c o f l u o r w o u l d result i n a n increased rate of cellulose synthesis, a n i n t e r p r e t a t i o n w h i c h is consistent b o t h w i t h our findings a n d w i t h previous work (1-5,32). Acknowledgments W e acknowledge P h i l i p M o r r i s ' financial support for t h i s w o r k , N . J . J a cobs ( D a r t m o u t h M e d i c a l School) for c u l t u r i n g the A. xylinum, a n d the D a r t m o u t h R i p p e l E M F a c i l i t y for the use of their e q u i p m e n t .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Literature Cited 1. Ruben, G. C.; Bokelman, G. H. Carbohydr. Res. 1987, 160, 434-43. 2. Haigler, C. H.; Brown, R. M., Jr.; Benziman, M. Science 1980, 210, 903. 3. Benziman, M.; Haigler, C. H.; Brown, R. M., Jr.; White, A. R.; Cooper, Κ. M. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 6678-82. 4. Haigler, C. H.; Benziman, M. In Cellulose and Other Natural Polymer Systems; Brown, R. M., Jr., Ed.; Plenum Press: New York, 1982; Chap. 14; pp. 273-97. 5. Brown, R. M., Jr.; Haigler, C. H.; Suttie, J.; White, A. R.; Roberts, E.; Smith, C.; Itoh, T.; Cooper, Κ. M. J. Appl. Polym. Sci., Appl. Polym. Symp. 1983, 37, 33-78. 6. Ellis, K. C.; Warwicker, J. O. J. Polym. Sci. 1962, 56, 339-57. 7. Ruben, G. C. J. Elect Microsc Tech 1985 2 253-57 8. Ruben, G. C.; Marx 9. Ruben, G. C.; Harris, ; Nagase, , , 2861-69. 10. Marx, Κ. Α.; Ruben, G. C. J. Biomol. Str. and Dynamics 1984, 1, 1109-32. 11. Ruben, G. C.; Marx, K. A. Proc. 42nd Ann. Mtg. Elect. Microsc. Soc. Amer.; 1984; p. 684. 12. Ruben, G. C.; Shafer, M. W. In Better Ceramics through Chemistry II; Brinker, C. J.; Clark, D. E.; Ulrich, D. R., Eds.; Materials Research Society: Pittsburgh, PA, 1986; V. 73, 207-12. 13. Ruben, G. C.; Bokelman, G. H. Proc. 45th Ann. Mtg. Elect. Microsc. Soc. Amer.; 1987, p. 966. 14. Yurchenco, P. D.; Ruben, G. C. J. Cell Biol. 1987, 105, 2559-68. 15. Krakow, W. Mat. Res. Soc. Symp. Proc. 1984, 31, 39-55. 16. Krakow, W. Ultramicroscopy 1985, 18, 197-210. 17. Bokelman, G. H.; Ryan, W. S., Jr.; Oakley, E. Agric. Food Chem. 1983, 31, 897-901. 18. Gardner, Κ. H.; Blackwell, J. Biopolymers 1974, 13, 1975-2000. 19. Sarko, H.; Muggli, R. Macromolecules 1974, 7, 486-94. 20. Knapek, E. Ultramicroscopy 1982, 10, 71. 21. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N. Mokuzai Gakkaishi 1984, 30, 98. 22. Harada, H. Mokuzai Gakkaishi 1984, 30, 513. 23. Revol, J.-F. J. Mat. Sci. Let. 1985, 4, 1347. 24. Ruben, G. C.; Telford, J. N. J. Microsc. 1980, 118, 191-216. 25. Ruben, G. C. Proc. 39th Ann. Mtg. Elect. Microsc. Soc. Amer. 1981, p. 566. 26. Ruben, G. C. Proc. 39th Ann. Mtg. Elect. Microsc. Soc. Amer. 1981, p. 568. 27. Ruben, G. C. Proc. 39th Ann. Mtg. Elect. Microsc. Soc. Amer. 1981, P. 570. 28. Simon, I.; Scheraga, Η. Α.; Manley, R. St.J. Macromolecules 1988, 21, 983-90.

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29. Viswanathan, Α.; Shenouda, S. C. J. Appl. Polymer Sci. 1971, 15, 519-35. 30. Zugenmaier, P. In Wood and Cellulosics; Kennedy, J. F.; Phillips, G. O.; Williams, P. Α., Eds.; Ellis Harwood: Chichester, 1987; 231-38. 31. Simon, I.; Glasser, L.; Scheraga, Η. Α.; Manley, R. St.J. Macro­ molecules 1988, 21, 990-98. 32. Roberts, E.; Legge, R.; Lin, F. C.; Brown, D.; Brown, R. M., Jr. Video Microscopy movie of Cellulose Synthesis shown at 24th Ann. Mtg. of Amer. Soc. Cell Biol. 1984, 880a. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 21 Structural Characterization and Visualization In Situ and After Isolation of Tobacco Pectin 1

George C. Ruben and Gordon H. Bokelman

2

1

Department of Biology, Dartmouth College, Hanover, NH 03755 Philip Morris USA, Research Center, Richmond, VA 23234 2

Recently two differen elucidation and transmission electron microscopy, were utilized in the study of pectin, with particular empha­ sis on tobacco pectin. The goal was to help bridge the gap between knowledge of their chemical structures to understanding the complex physical structures re­ vealed by microscopy. To provide background on chem­ ical structure, a study established that tobacco pectin was present as a series of related rhamnogalacturonans. All of these polysaccharides had a backbone consist­ ing of 4-linked α-D-galactopyranosyluronic acid residues interspersed with 2-linked L-rhamnopyranosyl residues. However, they varied in content of neutral sugars and extent of methyl-esterification. The presence of rham­ nose in the backbone of pectin was believed to create "kinks" which probably disrupted helical stretches of the 4-linked α-D-galactopyranosyluronic acid residues. In the present study pectin samples were gelled in deionized water, air-dried or freeze-dried, platinum-carbon repli­ cated, carbon-backed and then examined by high reso­ lution transmission electron microscopy. The pectin was found to be present as single chains of 7 ± 3Å diameter that showed helical stretches with a 13Å left-handed surface striation. P e c t i n is the m a j o r c o m p o n e n t f o u n d i n the p r i m a r y cell walls o f dicots a n d m a y p l a y a v i t a l role i n cell g r o w t h . D u r i n g cell g r o w t h , loosening o f the cell w a l l b y a c i d i f i c a t i o n is a n i m p o r t a n t process w h i c h enables the cell t o elongate b y its o w n t u r g o r pressure. It has been suggested t h a t the p r i m a r y a c t i o n o f a c i d i f i c a t i o n is the loosening o f a c a l c i u m pectate gel w i t h i n t h e 0097-6156/89/0399-0300$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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cell w a l l (1). P e c t i n is also f o u n d i n the m i d d l e l a m e l l a of l a n d p l a n t tissues where it is t h o u g h t to f u n c t i o n as a n i n t e r c e l l u l a r b i n d i n g agent (2). P e c t i n constitutes 1 1 - 1 2 % of the t o t a l solids (3) or 3 4 % of the cell w a l l m a t e r i a l s (4) i n t o b a c c o l a m i n a . B y c o n t r a s t , a l l of the f o l l o w i n g c o m ­ ponents are f o u n d to a lesser extent w i t h i n the cell walls of t o b a c c o l a m ­ i n a : p r o t e i n ( 2 1 . 6 % ) , cellulose ( 1 8 . 7 % ) , hemicellulose (11.4%), a n d l i g n i n (4.1%). G a l a c t u r o n i c a c i d is the m a j o r c o n s t i t u e n t of a l l n a t u r a l p e c t i n s . P e c t i n s also c o n t a i n v a r y i n g quantities of n e u t r a l sugars, p r i n c i p a l l y a r a b i nose, galactose a n d rhamnose (5). T h e c a r b o x y l f u n c t i o n o f the g a l a c t u r o n o s y l residues m a y be present as a m e t h y l ester, a c i d or s a l t . P e c t i n s are best k n o w n for t h e i r a b i l i t y to f o r m gels (6), a p r o p e r t y w h i c h often involves i n t e r m o l e c u l a r b i n d i n g m e d i a t e d by c a l c i u m cations (7). T h e p r i n c i p a l c o m m e r c i a l use of p e c t i n is i n the p r e p a r a t i o n of j e l l y a n d j a m p r o d u c t s (8). P e c t i n (9-11). H i s t o r i c a l l y , n a t u r a to prepare r e c o n s t i t u t e d sheets f r o m t o b a c c o b y - p r o d u c t s t h a t are t h e n i n c o r p o r a t e d i n t o cigarette filler or cigar w r a p p e r s (12-14). P e c t i n s have been s t r u c t u r a l l y characterized b y a c o m b i n a t i o n of c h e m ­ i c a l a n d spectroscopic m e t h o d s . C N M R can be used to e x a m i n e the p u ­ r i t y , degree o f e s t e r i f i c a t i o n , a n d n e u t r a l sugar content o f p e c t i n s (15). T h e m o n o m e r i c c o m p o s i t i o n of pectins m a y be d e t e r m i n e d d i r e c t l y b y a c o m b i ­ n a t i o n of m e t h a n o l y s i s (16) a n d s i l y l a t i o n procedures to y i e l d O - s i l y l a t e d m e t h y l g l y c o s i d e s t h a t can be q u a n t i t a t e d by G C (15). T h e linkage p a t ­ tern of the m o n o m e r i c sugars m a y be d e t e r m i n e d b y m e t h y l a t i o n a n a l y s i s . T h i s procedure involves m e t h y l a t i o n of the s t a r t i n g p e c t i n b y the H a k o m o r i m e t h o d (17-19), r e d u c t i o n of the c a r b o x y l i c a c i d f u n c t i o n s (18), h y d r o l y ­ sis, r e d u c t i o n of the aldehyde functions a n d a c e t y l a t i o n to y i e l d p a r t i a l l y m e t h y l a t e d a l d i t o l acetates. T h e p a r t i a l l y m e t h y l a t e d a l d i t o l acetates t h e n can be a n a l y z e d b y G C / M S (20). I n a v a r i a t i o n of the procedures l i s t e d above, p a r t i a l a c i d h y d r o l y s i s m a y be used to generate a series of d i - a n d oligosaccharide derivatives (15). These derivatives can be i d e n t i f i e d b y t h e i r electron i m p a c t mass s p e c t r a l f r a g m e n t a t i o n (21). F r o m a l l of the above i n f o r m a t i o n the c h e m i c a l s t r u c t u r e of the s t a r t i n g p e c t i n t h e n m a y be de­ duced. 1 3

T h e results f r o m s t r u c t u r a l studies on pectins isolated f r o m a n u m b e r of different p l a n t sources have been r e p o r t e d i n several papers a n d review articles ( 1 0 , 2 2 - 2 8 ) . C h e m i c a l investigations o f t o b a c c o p e c t i n ( 1 5 , 2 9 - 3 1 ) have d e m o n s t r a t e d t h a t its s t r u c t u r e is consistent w i t h the basic s t r u c t u r a l elements f o u n d i n pectins f r o m other sources. I n one recent s t u d y (15), i s o l a t i o n a n d p u r i f i c a t i o n o f t o b a c c o p e c t i n y i e l d e d a series of related r h a m n o g a l a c t u r o n a n s . A l l of these p o l y s a c ­ charides were f o u n d to have a backbone c o n s i s t i n g of 4 - l i n k e d a - D g a l a c t o p y r a n o s y l u r o n i c a c i d residues interspersed w i t h 2 - l i n k e d L - r h a m n o p y r a n o s y l residues i n a r a t i o of ~ 16:1 (see F i g . 1). T h e presence of r h a m ­ nose i n the b a c k b o n e of p e c t i n is believed to create " k i n k s " w h i c h p r o b a b l y d i s r u p t h e l i c a l stretches of the 4 - l i n k e d α-D-galactopyranosyluronic a c i d

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

=

=

=

=

Rha

G al A

(5-Me-GalA

R.

2 ) - I l h a - (1 -

- GalA -

^ - -(—4)

- 6 - Me - G a l A - ( l - )

F i g u r e 1. M o d e l c h e m i c a l s t r u c t u r e of t o b a c c o p e c t i n .

ι

^

^

terminal /?-D-galactopyranosyl residue

4-linked /?-D-galactopyranosyl residue

C

(-

2j

Rha -

terminal α-L-arabinofuranosyl residue

^ — 2) -

5-linked α-L-arabinofuranosyl residue

rnethyl-esterified D-galactopyranosyluronic acid residue

D-galactopyranosyluronic acid residue

L-rhamnopyranosyl residue

(—4)

8

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residues (10). T h e s e t o b a c c o r h a m n o g a l a c t u r o n a n s v a r i e d i n content of n e u t r a l sugars a n d extent of m e t h y l - e s t e r i f i c a t i o n . T h e i r average degree of p o l y m e r i z a t i o n was e s t i m a t e d to be 400. X - r a y fiber d i f f r a c t i o n studies have been p e r f o r m e d o n s o d i u m a n d c a l c i u m pectate gels (32). F r o m t h i s research a w o r k i n g m o d e l for the h e l i c a l p o r t i o n s of the p e c t i n c h a i n has emerged, w h i c h is i m p o r t a n t for c o m p a r i s o n to the t r a n s m i s s i o n electron m i c r o s c o p y ( T E M ) studies. T h e fiber d i f f r a c t i o n gel models assume a n t i p a r a l l e l a - ( l —• 4) p o l y g a l a c t u r o n a t e chains w h i c h , w h e n viewed d o w n the c axis of the p e c t i c a c i d u n i t cell, average about 6.8 x 7.2Â a l o n g the a a n d b directions for a single sugar c h a i n , respectively. O n e or t w o waters of h y d r a t i o n can increase t h i s size b y 3À or 6Â ( 3 3 , 3 4 ) i n deep-etched p r e p a r a t i o n s or a n associated c a l c i u m i o n c a n increase its cross-sectional d i m e n s i o n s b y a b o u t 2Â (35). F e w p r e v i o u s a t t e m p t s have been m a d e to v i s u a l i z e p e c t i n at the m o l e c u l a r level (36). I n the present s t u d y , a P t - C r e p l i c a t i o n t e c h n i q u e a n d T E M were used to characterize (facing m e s o p h y l l cells) i n b o t h fresh, green a n d senescing C o k e r 319 t o bacco leaves. These surfaces were c o m p a r e d to freeze-dried a n d a i r - d r i e d calcium-free p e c t i n gels. T h e surface textures a n d e s t i m a t e d d i a m e t e r s of single p e c t i n chains i n these p r e p a r a t i o n s were c o m p a r e d . W i t h h i g h m a g n i f i c a t i o n i m a g i n g we were able to c o n f i r m the presence o f the p o l y g a l a c t u r o n a t e c h a i n h e l i x i n tobacco a n d c i t r u s p e c t i n s . Methods and Materials T h e t o b a c c o p e c t i n used i n t h i s s t u d y was o b t a i n e d f r o m a single grade of h e a v y or b o d i e d , field-grown, flue-cured b r i g h t t o b a c c o h a r v e s t e d at the u p p e r m i d s t a l k p o s i t i o n . C r u d e tobacco p e c t i n was o b t a i n e d b y e x t r a c t i o n w i t h hot w a t e r of the t o b a c c o l a m i n a t h a t p r e v i o u s l y h a d been t r e a t e d w i t h aqueous e t h a n o l t o remove waxes, n i c o t i n e , s i m p l e sugars a n d other low m o l e c u l a r weight c o m p o n e n t s (15). T h i s crude p r o d u c t was p u r i f i e d b y t a n g e n t i a l flow u l t r a f i l t r a t i o n , i o n exchange c h r o m a t o g r a p h y a n d gel p e r m e a t i o n c h r o m a t o g r a p h y (15). T h e p u r i f i e d t o b a c c o p e c t i n h a d a g a l a c t u r o n i c a c i d content of ~ 8 0 % , a degree of esterification of ~ 22 a n d a degree of p o l y m e r i z a t i o n of ~ 400. A separate s a m p l e of deesterified p e c t i n was o b t a i n e d b y s a p o n i f i c a t i o n (15) of the p u r i f i e d tobacco p e c t i n . G e l s were o b t a i n e d i n the f o l l o w i n g m a n n e r f r o m b o t h the p u r i f i e d , s t a r t i n g t o b a c c o p e c t i n a n d the deesterified p e c t i n o b t a i n e d f r o m i t . F i r s t the s a m p l e of p e c t i n was s o l u b i l i z e d i n deionized 100°C water (~ 1% sol u t i o n ) . T h e n the p e c t i n was gelled b y e t h a n o l v a p o r , i n t r o d u c e d s l o w l y (6 hrs) by s u r r o u n d i n g the vessel c o n t a i n i n g aqueous p e c t i n w i t h 1 0 0 % e t h a n o l i n a closed container at 20° C . T h e gel f r o m the p u r i f i e d t o b a c c o p e c t i n was f o r m e d o n 1.3 c m a s h less W h a t m a n 50 filter p a p e r discs w h i c h were frozen i n p r o p a n e at a b o u t - 1 9 0 ° C . These samples were t h e n freeze-dried (90 m i n ) at —70°C, r e p l i cated w i t h 16.9Â P t / C at - 1 7 8 ° C i n a 5 x 1 0 ~ t o r r . v a c u u m a n d backed w i t h 146Â of c a r b o n . F i n a l l y , these samples were digested w i t h 8 0 % s u l furic a c i d , rinsed w i t h deionized w a t e r , p i c k e d u p f r o m u n d e r n e a t h w i t h 8

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c a r b o n - c o a t e d 300 mesh grids (37) a n d e x a m i n e d b y t r a n s m i s s i o n electron microscopy. Unless specifically noted otherwise, the same general p r o c e dures were e m p l o y e d to prepare other samples for T E M e x a m i n a t i o n . A m o r e d e t a i l e d discussion of the T E M procedures used, i n c l u d i n g m i c r o g r a p h reversals, has been p u b l i s h e d p r e v i o u s l y (37). Citrus pectin ( "Polygalacturonic A c i d M e t h y l Ester from Citrus Fruits, G r a d e I") was o b t a i n e d f r o m the S i g m a C h e m i c a l C o m p a n y . It h a d a g a l a c t u r o n i c a c i d content of ~ 8 9 % a n d a degree of esterification of ~ 57. Separate aqueous s o l u t i o n s of c i t r u s p e c t i n were freeze-dried a n d a i r - d r i e d i n deionized w a t e r . These samples were r e p l i c a t e d w i t h 9.8Â P t / C a n d backed w i t h 148Â of c a r b o n . T h e replicas for these samples were p i c k e d u p w i t h o u t a c a r b o n s u p p o r t film (38). T h e f o l l o w i n g procedure was used to o b t a i n images of the e p i d e r m a l cell surfaces w h i c h face the m e s o p h y l l cells w i t h i n the leaf i n t e r i o r . B o t h fresh green a n d senescing greenhouse-grown C o k e r 319 t o b a c c o leaves were e x a m i n e d . T h e lower e p i d e r m a leaf, rinsed t w i c e i n a s o l u t i o n of 1 : 3 / e t h a n o l : w a t e r , a n d frozen o n a | - i n . m i c a disc. T h e senescing s a m p l e was freeze-dried at —80°C for 105 m i n , r e p l i c a t e d w i t h 26.6Â P t / C a n d backed w i t h 215Â o f c a r b o n . T h e fresh, green s a m p l e was freeze-dried at — 7 0 ° C for 3 h r , r e p l i c a t e d w i t h 15.9Â P t / C a n d backed w i t h 139Â of c a r b o n . In a separate e x p e r i m e n t a s a m p l e of the lower e p i d e r m a l layer f r o m a fresh, green C o k e r 319 tobacco leaf was t r e a t e d w i t h b o i l i n g water for 25 m i n . T h i s s a m p l e was t h e n r i n s e d , freeze-dried for 3.5 h r at —80°C, r e p l i c a t e d w i t h 15.9Â P t / C a n d backed w i t h 133Â of c a r b o n . Results and Discussion Since i t was k n o w n t h a t p e c t i n can be s o l u b i l i z e d w i t h hot w a t e r , a s i m p l e e x p e r i m e n t was p e r f o r m e d to help identify the l o c a t i o n o f p e c t i n o n the n o n c u t i n i z e d surface of tobacco lower e p i d e r m a l cells. T h e n o n c u t i n i z e d surface of the lower e p i d e r m a l cells is the side w h i c h faces the m e s o p h y l l cells. F i g u r e s 2 A a n d 2 B , respectively, show the c o n t r o l a n d treated samples for the n o n c u t i n i z e d lower e p i d e r m a l cell surface of a fresh, green C o k e r 319 tobacco leaf. T r e a t m e n t consisted of i m m e r s i n g the lower e p i d e r m a l peel i n b o i l i n g water for 25 m i n . It m a y be seen i n F i g u r e 2 A t h a t the surface p e c t i n coat was continuous, w i t h n u m e r o u s flat regions. T h i s p e c t i n coat d i s a p p e a r e d f o l l o w i n g hot water e x t r a c t i o n . F i g u r e 2 B shows the exterior surface of the lower e p i d e r m i s for the b o i l i n g - w a t e r t r e a t e d s a m p l e f r o m w h i c h most of the p e c t i n h a d been e x t r a c t e d . Some p e c t i n - l i k e m a t e r i a l r e m a i n e d as s m o o t h globs c o a t i n g the filamentous p r i m a r y cell w a l l c e l l u lose. T h e contrast between F i g u r e s 2 A a n d 2 B i n d i c a t e d t h a t a p e c t i n gel permeates the cell w a l l . F i g u r e s 3 A a n d 3 B show l o w a n d h i g h m a g n i f i c a t i o n images of the n o n c u t i n i z e d lower e p i d e r m a l cell surfaces i n a senescent C o k e r 319 t o bacco leaf. T h e j u n c t i o n s between four different e p i d e r m a l cells c a n be seen i n F i g u r e 3 A . A l s o , m u l t i p l e layers o f p e c t i n were evident o n these lower e p i d e r m a l cells, b u t were not present o n the e p i d e r m a l surface of

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F i g u r e 2 A . F r e e z e - d r i e d , P t / C r e p l i c a t e d , u n t r e a t e d n o n c u t i n i z e d lower e p i d e r m a l cell surface of a f r e s h , green C o k e r 319 tobacco leaf. ( B a r = 1,000Â.)

F i g u r e 2 B . Freeze-dried, P t / C r e p l i c a t e d , n o n c u t i n i z e d lower e p i d e r m a l cell surface of a fresh, green C o k e r 319 tobacco leaf treated w i t h b o i l i n g water for 25 m i n u t e s . ( B a r = 5 , 0 0 0 A . )

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F i g u r e 3 A . Freeze-dried, P t / C r e p l i c a t e d , n o n c u t i n i z e d lower e p i d e r m a l cell surfaces i n senescent C o k e r 319 tobacco leaf. ( B a r = 5 , 0 0 0 Â . )

F i g u r e 3 B . S a m e as F i g u r e 3 A , except higher m a g n i f i c a t i o n . ( B a r = 500Â.)

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younger leaves (see F i g u r e 2 A ) . A s m a n y as 6 t o 7 stacked surface layers were seen f r o m w h i c h filaments p r o t r u d e d . T h e h i g h l y bent filaments w i t h some l i n e a r stretches were even m o r e evident i n F i g u r e 3 B . T h e average filament w i d t h was measured at 29.7Â (n = 137, S . D . = 4 . 8 Â ) . C o r r e c t i o n for the P t / C film thickness (39) gave a real size of 4 . 6 ± 4 . 8 À . F i g u r e 4 A shows a r e l a t i v e l y low m a g n i f i c a t i o n m i c r o g r a p h of a gel p r e p a r e d f r o m deesterified t o b a c c o p e c t i n . U n l i k e F i g u r e s 2 A a n d 3 A , i t s surface was smoother a n d d i d not show the l a y e r i n g effect seen o n the lower e p i d e r m a l cell surface. F i g u r e 4 B is a h i g h m a g n i f i c a t i o n m i c r o g r a p h of a gel of p e c t i n filam e n t s p r e p a r e d f r o m p u r i f i e d t o b a c c o p e c t i n . T h e average filament w i d t h w i t h P t / C c o a t i n g was 22.5Â. A f t e r c o r r e c t i n g for the a d d e d size due t o the P t / C c o a t i n g (39), the p e c t i n filament h a d a d i a m e t e r o f 7.1 ± 3Â (n = 112, S . D . = 3 Â ) . W i t h i n the s t a n d a r d d e v i a t i o n o f the m e a s u r e m e n t s , the filament w i d t h s were th t o b a c c o p e c t i n a n d the t o b a c c o e p i d e r m a l cell surface. B o t h of these m e a surements also agreed very w e l l w i t h the x - r a y fiber d i f f r a c t i o n d i a m e t e r , ~ 7À, w h i c h we e s t i m a t e d f r o m the modeled gels of W a l k i n s h a w a n d A r n o t t (32). O n careful i n s p e c t i o n , some of the p e c t i n molecules i n F i g u r e 4 B showed a l e f t - h a n d e d surface s t r i a t i o n o c c u r r i n g every 13Â. W a l k i n s h a w a n d A r n o t t d e m o n s t r a t e d t h a t (citrus) p e c t i n c o n t a i n e d a 13.3Â 3-fold hel i x i n the p o l y g a l a c t u r o n a t e c h a i n (32), b u t x - r a y fiber d i f f r a c t i o n d i d not give the h e l i x handedness. W e report here for the first t i m e t h a t i t is a left-handed helix. Since the x - r a y fiber diffraction measurements based o n c i t r u s p e c t i n (32) were consistent w i t h the T E M measurements of t o b a c c o p e c t i n , we p r e p a r e d a gel f r o m c i t r u s p e c t i n s i m i l a r to the previous x - r a y s a m p l e . T h i s gel was t h e n e x a m i n e d b y T E M . A i r - d r i e d samples of t h i s gel, s h o w n i n F i g u r e 5, d e m o n s t r a t e d l o n g stretches of h e l i x i n the molecules l y i n g o n the surface. (In the freeze-dried g e l s — n o t s h o w n — o n l y short stretches of h e l i x were v i s i b l e . ) T h e average filament w i d t h i n the a i r - d r i e d gel was f o u n d to be 14.2Â. A f t e r c o r r e c t i n g for the added size due to the P t / C c o a t i n g (39), the c i t r u s p e c t i n filament d i a m e t e r was 5.8 ± 2 A (n = 3 7 , S . D . = 2 Â ) . I n F i g u r e 5 the c i t r u s p e c t i n molecules showed a l e f t - h a n d e d surface s t r i a t i o n o c c u r r i n g every 13Â. T h e surface h e l i x p e r i o d f r o m b o t h t o b a c c o a n d c i t r u s p e c t i n samples was i n agreement w i t h the x - r a y fiber d i f f r a c t i o n measurements (32). I n c o n c l u s i o n , we have d e m o n s t r a t e d t h a t h i g h r e s o l u t i o n T E M is a v a l u a b l e complement to x - r a y fiber diffraction a n a l y s i s a n d c h e m i c a l s t r u c t u r a l e l u c i d a t i o n . Its a p p l i c a t i o n p r o v i d e d i n f o r m a t i o n a b o u t the o r g a n i z a t i o n of p e c t i n i n cell walls a n d i n calcium-free gels. U s i n g freeze-dried s a m ples t h a t were P t / C r e p l i c a t e d , we d e m o n s t r a t e d t o b a c c o p e c t i n filaments i n a gel to be of the same d i a m e t e r as the filaments o n the n o n c u t i n i z e d lower e p i d e r m a l surface of senescing C o k e r 319 tobacco leaves. T h e s e filaments were 7.1 ± 3Â a n d 4.6 ± 4.8Â, respectively, a n d r o u g h l y the same d i a m e t e r , ~ 7Â, as fiber-diffraction modeled c i t r u s p e c t i n (32). R e p l i c a t e d

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F i g u r e 4 A . Freeze-dried, P t / C r e p l i c a t e d gel p r e p a r e d f r o m deesterified t o ­ bacco p e c t i n . ( B a r = ΙΟ,ΟΟΟΑ.)

F i g u r e 4 B . F r e e z e - d r i e d , P t / C r e p l i c a t e d gel prepared f r o m t o b a c c o p e c t i n . T h i s h i g h m a g n i f i c a t i o n image shows two molecules w i t h ~ 13Â left-handed h e l i c a l regions. ( B a r = 100A.)

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F i g u r e 5. A i r - d r i e d , P t / C r e p l i c a t e d gel prepared f r o m c i t r u s p e c t i n . T h i s image features two p e c t i n filaments w i t h left-handed surface s t r i a t i o n s h a v i n g ~ 13Â spacings ( E a c h bar — 25Â.)

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c i t r u s p e c t i n filaments were also f o u n d t o have s i m i l a r diameters, 5.8 ± 2 Â . In a d d i t i o n , we d e m o n s t r a t e d images, for t h e first t i m e , o f the l e f t - h a n d e d surface spacings o f ~ 13Â i n single p e c t i n molecules, i n b o t h freeze-dried t o b a c c o a n d a i r - d r i e d citrus p e c t i n . W e believe t h a t single molecule i m a g i n g c a n c o n t r i b u t e t o a m o r e t h o r o u g h u n d e r s t a n d i n g o f the role o f p e c t i n i n t h e cell w a l l . O u r future efforts w i l l be focused o n p e c t i n gels f o r m e d i n the presence o f c a l c i u m . E v e n t u a l l y , it s h o u l d be possible t o v i s u a l i z e side chains o n p e c t i n a n d determine h o w r h a m n o s e residues i n t h e r h a m n o g a l a c t u r o n a n b a c k b o n e o f t o b a c c o p e c t i n d i s r u p t t h e f o r m a t i o n o f h e l i c a l regions.

Literature Cited 1. Baydoun, Ε. A.-H.; Brett, C. T. J. Exp. Bot 1984, 35, 1820. 2. Dey, P. M.; Brinson Κ In Advances in Carbohydrate Chemistry and Biochemistry; Tipson York, 1984; Vol. 42, p 3. Bokelman, G. H.; Ryan, W. S., Jr.; Sun, Η. H.; Ruben, G. C. Recent Adv. Tob. Sci. 1985, 11, 71. 4. Bokelman, G. H.; Ryan, W. S., Jr.; Oakley, Ε. T. J. Agric. Food Chem. 1983, 31, 897. 5. Aspinall, G. O.; Craig, J. W. T.; Whyte, J. L. Carbohydr. Res. 1968, 7, 442. 6. Rees, D. Α.; Welsh, E. J. Angew. Chem. Int. Ed. Engl. 1977, 16, 214. 7. Whistler, R. L.; Smart, C. L. Polysaccharide Chemistry, Academic Press: New York, 1953. 8. Towle, G. Α.; Christensen, O. In Industrial Gums, 2nd edn.; Whistler, R. L., Ed.; Academic Press: New York, 1973; p. 155. 9. Van Buren, J. P.; Peck, Ν. H. J. Food Sci. 1981, 47, 311. 10. Jarvis, M. C. Plant, Cell Environ. 1984, 7, 153. 11. McFeeters, R. F.; Fleming, H. P.; Thompson, R. L. J. Food Sci. 1985, 50, 201. 12. Hind, J. D.; Seligman, R. B. U.S. Patent 3 353 541, 1967. 13. Hind, J. D.; Seligman, R. B. U.S. Patent 3 411 515, 1968. 14. Hind, J. D.; Seligman, R. B. U.S. Patent 3 420 241, 1969. 15. Sun, H. H.; Wooten, J. B.; Ryan, W. S., Jr.; Bokelman, G. H.; Åman, P. Carbohydr. Polym. 1987, 7, 143. 16. Pritchard, D. G.; Todd, C. W. J. Chromatogr. 1977, 133, 133. 17. Hakomori, S. J. Biochem. (Tokyo) 1964, 55, 205. 18. Standford, P. Α.; Conrad, Η. E. Biochemistry 1966, 5, 1508. 19. Philips, L. R.; Fraser, Β. A. Carbohydr. Res. 1981, 90, 149. 20. Jansson, P. E.; Kenne, L.; Liedgren, H.; Lindberg, B.; Lonngren, J. Chem. Commun. (Univ. of Stockholm) 1976, No. 8. 21. Kochetkov, N. K.; Chizhov, O. S. In Advances in Carbohydrate Chem­ istry, Wolfrom, M. L., Ed.; Academic Press: New York, 1966; Vol. 21, p. 39. 22. McNeil, M.; Darvill, A. G.; Albersheim, P. Fortschr. Chem. Org. Naturst. 1979, 37, 191.

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23. Aspinall, G. O. In The Biochemistry of Plants; Preiss, J., Ed.; Aca­ demic Press: New York, 1980; Vol. 3, p. 473. 24. Darvill, A. G.; McNeil, M.; Albersheim, P.; Delmer, D. P. In The Bio­ chemistry of Plants; Tolbert, N. E., Ed.; Academic Press: New York, 1980; Vol. 1, p. 91. 25. Pilnik, W. Proc. Eur. Symp. on Fiber in Human Nutrition, APRIA, 1981, p. 91. 26. Selvendran, R. R. In Dietary Fibre; Birch, G. G.; Parker, K. J., Eds.; Applied Science Publishers: London, 1983; p. 95. 27. Pressey, R.; Himmelsbach, D. S. Carbohydr. Res. 1984, 127, 356. 28. Keenan, M. H. J.; Belton, P. S.; Matthew, J. Α.; Howson, S. J. Carbo­ hydr. Res. 1985, 138, 168. 29. Bourne, E. J.; Pridham, J. B.; Worth, H. G. J. Phytochemistry 1967, 6, 423. 30. Eda, S.; Kato, K. Agric 31. Siddiqui, I. R.; Rosa 32. Walkinshaw, M. D.; Arnott, S. J. Mol. Biol. 1981, 153, 1055, 1075. 33. Marx, Κ. Α.; Ruben, G. C. J. Biomol. Struct. and Dynamics 1984, 1, 1109. 34. Ruben, G. C.; Telford, J. N. J. Micros. 1980, 118, 191. 35. Pauling, L. The Nature of the Chemical Bond; Cornell Univ. Press: Ithaca, NY, 1960; p. 518. 36. Hanke, D. E.; Northcote, D. H. Biopolymers 1975, 14, 1. 37. Ruben, G. C.; Marx, K. A. J. Elect. Microsc. Tech. 1984, 1, 373. 38. Ruben, G. C.; Bokelman, G. H. Proc. 45th Ann. Meet. Elec. Microsc. Soc. Amer., 1987, p. 966. 39. Ruben, G. C.; Bokelman, G. H. Carbohydr. Res. 1987, 160, 434. RECEIVED March 10, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 22

Control of Cell Wall Plasticity Relationship to Pectin Properties 1

1

1

2

Renée Goldberg , Paulette Devillers , Roger Prat , Claudine Morvan , Véronique Michon , and Catherine Hervé du Penhoat 3

3

1Biomembranes et Surfaces Cellulaires Végétales, ENS, 46 rue d'Ulm, 75230 Paris Cedex 05, France 2

SCUEOR, Faculté des Sciences de Rouen, 76130 Mont-Saint Aignan, France 3

Département de Chimie Paris Cedex, France Along the mung bean hypocotyl, the cell wall plasticity represents the limiting factor of cell growth potentials. Pectin molecules, known to control local cell wall pH's and to modulate phenolic cross-linking owing to the number of free acidic domains, were investigated. Young, plastic walls were characterized both by a high level of branched and methylated rhamnogalacturonans which leads to swollen cell walls and by a low level of linear galacturonans which induces a low Cation Exchange Capacity (CEC). In contrast, stiff, mature cell walls are characterized by a low water content and a high CEC which favors cross-linking.

T h e p a r t i c i p a t i o n o f pectins i n c o n t r o l l i n g t h e e x t e n s i b i l i t y o f the p r i m a r y cell walls has often been suggested. M o s t o f the d a t a described were o b t a i n e d w i t h dicots i n w h i c h pectins account for 30 t o 5 0 % i n the cell w a l l m a t e r i a l , whereas m o n o c o t s are k n o w n t o be quite d e v o i d o f p o l y u r o n i d e s . C o r r e l a t i o n s between cell w a l l p l a s t i c i t y a n d frequency o f c a l c i u m bridges, degree o f esterification or m o l e c u l a r size have beeen successively proposed. R e c e n t l y new views about the m a n n e r i n w h i c h pectins m i g h t p a r t i c i p a t e i n the c o n t r o l o f cell w a l l e x t e n s i b i l i t y have been expressed (1-3). O n t h e one h a n d , pectins m i g h t intervene i n cell w a l l stiffening processes as free a c i d i c d o m a i n s act as a t e m p l a t e for c r o s s - l i n k i n g reactions (2). O n t h e other h a n d , pectic molecules c o n t r o l l o c a l cell w a l l p H ' s a n d , consequently, p o s i t i v e or negative feedback systems (3). I t is w i d e l y accepted t h a t w a l l loosening as w e l l as w a l l stiffening processes are e n z y m a t i c a l l y m e d i a t e d . It m a y also be assumed t h a t p e c t i n properties w i l l m o d u l a t e t h e a b i l i t y of cell walls t o develop into more o r less stretchable s t r u c t u r e s . W e have therefore investigated the pectic m a t e r i a l i n fast a n d slow g r o w i n g p a r t s o f M u n g bean h y p o c o t y l s . 0097-6156/89/0399-0312$06.00/0 © 1989 American Chemical Society

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Materials and Methods Vigna radiata ( L . W i l c z e k ) seedlings were raised as p r e v i o u s l y r e p o r t e d (4). Seedlings were g r o w n 3 d at 2 6 ° C i n the d a r k , a n d were used w h e n the h y p o c o t y l s measured 45 ± 5.0 m m . Growth Measurements. E q u i d i s t a n t I n d i a n i n k m a r k s were m a d e a l o n g the h y p o c o t y l a n d their displacements measured after 4 h . D i s p l a c e m e n t vel o c i t y was p l o t t e d as a f u n c t i o n o f the i n i t i a l p o s i t i o n of each m a r k , i.e., the distance f r o m cotyledons. R e l a t i v e e l e m e n t a l rates o f e l o n g a t i o n were t h e n c a l c u l a t e d as the derivatives o f the displacement versus p o s i t i o n (5). G r o w t h measurements of excised segments were recorded w i t h a u x a n o m e ters u s i n g displacement t r a n s d u c e r s . T h e g r o w t h curves were t h e n c a l c u l a t e d w i t h a m i c r o c o m p u t e r ( T R S 80) as p r e v i o u s l y r e p o r t e d (6). E x t e n s i b i l i t y of the successive segments was e s t i m a t e d u s i n g t w o different m e t h o d s . O n the one h a n d , the segments (10 m m long) were p u l l e d at a constant rate of d e f o r m a t i o n (60 m m m i n " " q u i r e d less t h a n 1 sec. Irreversible d e f o r m a t i o n represents the i m m e d i a t e p l a s t i c i t y . O n the other h a n d , the successive segments were s u b j e c t e d to a constant l o a d (40 g) d u r i n g 5 m i n a n d their l e n g t h recorded d u r i n g 10 m i n (5 m i n l o a d i n g followed b y 5 m i n u n l o a d i n g ) . L o n g t e r m p l a s t i c i t y was then e s t i m a t e d b y irreversible d e f o r m a t i o n . S t r a i n values were c a l c u l a t e d for b o t h methods as irreversible deformations per m m . C e l l walls were isolated f r o m 2 parts of the h y p o c o t y l as p r e v i o u s l y des c r i b e d (7). T w o pectic fractions, P F i a n d P F 2 , were sequentially e x t r a c t e d b y b o i l i n g water a n d hot E D T A p H 6.0. A s already r e p o r t e d , the r e s i d u a l cell walls were free of p o l y g a l a c t u r o n i c acids (8). E D T A t r e a t m e n t d i d not significantly degrade the p o l y u r o n i d e s since c o l o r i m e t r i c e s t i m a t i o n s p e r f o r m e d before a n d after d i a l y s i s gave s i m i l a r results. T h e p e c t i c f r a c t i o n e x t r a c t e d b y b o i l i n g water was s u b m i t t e d to ion-exchange c h r o m a t o g r a p h y o n D E A E - S e p h a r o s e C L 6 B ( P h a r m a c i a ) e q u i l i b r a t e d w i t h 0.05 M s o d i u m acetate buffer ( p H 4.7). N e u t r a l polysaccharides were not b o u n d a n d a c i d i c polysaccharides A P F 1 were eluted w i t h 1 M buffer (9). C a l c i u m contents of the pectic fractions were d e t e r m i n e d b y a t o m i c s p e c t r o p h o t o m e t r y w i t h l a n t h a n u m as i n t e r n a l s t a n d a r d . P o t e n t i o m e t r i c measurements a n d e s t i m a t i o n s of s e l e c t i v i t y coefficients were r u n a c c o r d i n g to ref. 8. C a l c i u m a c t i v i t y was measured u s i n g a specific electrode (10). G a l a c t u r o n i c acids were e s t i m a t e d w i t h m - d i p h e n o l (11). For e s t i m a t i o n of cell w a l l water content, the diffusion film was e l i m i n a t e d f r o m i s o l a t e d cell walls o n filter paper a n d the weight of the wet cell walls ( M F ) was e s t i m a t e d (10). T h e w a l l d r y weight ( M S ) was measured after 1 h at 5 0 ° C . T h e cell w a l l water content (t) was o b t a i n e d f r o m the r e l a t i o n t = ( M F — M S ) / M S . T h e r e l a t i v e error was less t h a n 1 0 % . A B r u c k e r A M 400 spectrometer o p e r a t i n g i n the F . t . m o d e at 400.13 M H z for * H a n d 100.57 M H z for C was used for N M R i n v e s t i g a t i o n s . S a m ples were dissolved i n D 2 O at 7 0 ° C . ( C D a ^ S O was the i n t e r n a l reference (6 39.5, 6H 2.72). M e a s u r e m e n t s were p e r f o r m e d as described p r e v i o u s l y (9). 1 3

C

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Results and Discussion Growth Gradient Along the Mung Bean Hypocotyl. A c c u r a t e e s t i m a t i o n of g r o w t h p o t e n t i a l s of cells i n situ is difficult. A l o n g a n a x i s , indeed, d a t a o b t a i n e d b y m e a s u r i n g m a r k displacements a c t u a l l y correspond to the c u m u l a t i v e cell g r o w t h at the successive levels crossed d u r i n g the t i m e of exp e r i m e n t s . T h i s difficulty h a d been overcome b y e s t i m a t i n g the derivatives of displacement velocities (5). These d a t a give the i n s t a n t a n e o u s rate of e l o n g a t i o n at each p o i n t o n the a x i s . A l o n g the M u n g b e a n h y p o c o t y l , t h i s e l o n g a t i o n rate decreases r e g u l a r l y f r o m the hook (segment b) to the base ( F i g . I A ) . In vitro g r o w t h measurement u s i n g excised segments does not i n d i c a t e the i n i t i a l state of g r o w t h b u t the e l o n g a t i o n of the s a m p l e after the e x c i s i o n . These measurements show the same g r o w t h g r a d i e n t ( F i g . I A ) . In order to check whether w a l l properties were the g r o w t h l i m i t i n g factor a l o n g the h y p o c o t y l , w a l l p l a s t i c i t y of successive h y p o c o t y l segments was measured u s i n g two differen diate p l a s t i c i t y as w e l l as l o n g t e r m p l a s t i c i t y o b v i o u s l y decreased a l o n g the M u n g bean h y p o c o t y l . These d a t a suggest t h a t a l o n g t h i s g r o w i n g o r g a n the p l a s t i c properties of the cell walls m i g h t c o n t r o l the g r o w t h p o t e n t i a l of the cells. Composition of Pectin Fractions Extracted from Young and Mature Cell Walls (Table I). Y o u n g cell-walls contained more p o l y u r o n i d e s soluble i n hot water a n d less p o l y u r o n i d e s soluble i n E D T A t h a n m a t u r e cell w a l l s . C a l c i u m ions were m o s t l y b o u n d to water-insoluble p e c t i n s . In t h i s fract i o n the g a l a c t u r o n i c a c i d / C a r a t i o is i d e n t i c a l for y o u n g a n d m a t u r e cell walls w h i c h indicates s i m i l a r affinity for C a ions. M o r e o v e r , P F 2 p o l y u r o n i d e s , i n contrast to P F i , were not m e t h y l a t e d . T h e a c t i v i t y of the c a l c i u m ions was e s t i m a t e d i n each f r a c t i o n . T h e m e a n distance between two free changes, b , c o u l d t h e n be c a l c u l a t e d (8). E x t r a c t s f r o m y o u n g a n d m a t u r e cell walls gave close d a t a . P F i b values were higher t h a n b values of P F b u t deesterified P F i was s i m i l a r to P F , the average distance between two free charges t h e n b e i n g near 4.4 w h i c h corresponds to the value c a l c u l a t e d for p o l y g a l a c t u r o n i c a c i d . T h e p r i n c i p a l chains of P F i a n d P F molecules are t h e n c o n s t i t u t e d b y l i n k e d g a l a c t u r o n i c a c i d molecules, 7 0 % of w h i c h are m e t h y l a t e d i n P F i extracts. In P F i , the average distance between two free charges is n e a r l y three times as great as the value o b t a i n e d after deesterification. It is possible t h a t P F i extracts c o n t a i n o r d e r l y a r r a n g e d molecules w i t h a r e p e a t i n g u n i t c o n s t i t u t e d b y two successive m e t h y l a t e d molecules followed by a n u n m e t h y l a t e d one. Succession of extended sequences of m e t h y l a t e d a n d u n m e t h y l a t e d molecules w o u l d p r o v i d e different values for 7 C a . Indeed, a c c o r d i n g to K o h n (12), occurrence of more t h a n 8 successive u n m e t h y l a t e d molecules inside a p o l y g a l a c t u r o n i c c h a i n e n t a i l s low 7 C a values s i m i l a r to those measured w i t h p o l y g a l a c t u r o n i c a c i d (i.e., n e a r l y 0.18). However, a succession of short sequences ( < 8 u n i t s ) of m e t h y l a t e d a n d u n m e t h y l a t e d molecules cannot be e x c l u d e d . L a s t l y , the h i g h n e u t r a l / a c i d i c sugars r a t i o noted for P F i m i g h t correspond to n e u t r a l sidechains. 2 +

2 +

2

2

2

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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F i g u r e 1. D e v e l o p m e n t of g r o w t h p o t e n t i a l a n d cell w a l l e x t e n s i b i l i t y a l o n g the m u n g b e a n h y p o c o t y l . A , e l o n g a t i o n rates as ^ m / h " / " ) · · relative e l e m e n t a l e l o n g a t i o n rate; · · spontaneous e l o n g a t i o n of excised segments. B , cell w a l l e x t e n s i b i l i t y as / i m / m m " ; · · immediate plasticity; · · l o n g t e r m p l a s t i c i t y . D a t a correspond to irreversible deformations measured as described i n M a t e r i a l s a n d M e t h o d s . 1

1 1 1 1 1 1

1

1

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

316

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T a b l e I. C o m p o s i t i o n of P e c t i c F r a c t i o n s E x t r a c t e d w i t h B o i l i n g W a t e r ( P F i ) and Hot E D T A ( P F ) from Young and Mature Cell Walls 2

Young Cell Walls

Mature Cell Walls PF

PFi

PF

U r o n i c acids ( U A ) Calcium N e u t r a l / a c i d i c sugars U A/Calcium D E (%)

960 50 1.05 19.2 70

340 10 0.35 3.10 0-10

540 45 0.90 12.0 70

610 200 0.12 3.05 0-10

7Ca intact pectins demethylated pectins

0.57 0.22

0.18

0.58 0.20

0.15

2

2

b U r o n i c acids a n d C a as / i e q . g " . D E , degree of esterification. 7 C a = a c t i v i t y coefficient of c a l c i u m ions. b , average distances between two free charges, i n A n g s t r o m s , is c a l c u l a t e d f r o m the r e l a t i o n s : L n 7 C a = —0.5— L n 2ξ a n d ξ — 7.15 Â / b ; ξ is a dimensionless s t r u c t u r a l p a r a m e t e r , the charge density of the p e c t i n s . 2 +

1

W e have also t r i e d to investigate i n t a c t , u n h y d r o l y z e d p e c t i c m a t e r i a l s w i t h N M R spectroscopy. U n f o r t u n a t e l y , h i g h l y m e t h y l a t e d P F i as w e l l as P F were u n s u i t a b l e for h i g h r e s o l u t i o n N M R studies due to low s o l u b i l i t y . N a + or L i + salt forms of the samples gave better results. C s p e c t r a of the N a f o r m of P F i a n d P F are given i n F i g u r e 2. O n these s p e c t r a the m a j o r residues are (1 —• 5) l i n k e d a-arabinofuranose, (9,13,14), (1 —• 4) l i n k e d /3-galactopyranose (9,15,16), a n d (1 —• 4) l i n k e d α-galacturonic a c i d . T h e * H N M R s p e c t r a of these samples were c o m p a t i b l e w i t h the C assign­ ments a n d are i n accord w i t h l i t e r a t u r e d a t a (9,17-19). R h a m n o s e c o u l d not be detected a l t h o u g h G C p e r f o r m e d after a c i d h y d r o l y s i s (8) revealed the presence of s m a l l a m o u n t s , nearly 4%, i n a l l pectic fractions. T h e i n t e n s i t y of the C 5 s i g n a l of arabinofuranose at 68.3-68.5 p p m is consistently weaker, b y about 8 0 % , t h a n t h a t of the m e t h i n e carbons 1-4. T h i s C 5 s i g n a l was absent i n the C N M R s p e c t r u m of P F . These d a t a suggest either t h a t arabino-furanose residues o c c u p y t e r m i n a l positions or t h a t the f u r a n o s y l signals b e l o n g to another residue such as /?-galacto-furanose. G a l a c t u r o n i c a c i d levels were m u c h lower t h a n the values o b t a i n e d f r o m m - d i p h e n y l e s t i ­ m a t i o n s . T h e very weak signals observed for g a l a c t u r o n i c a c i d i n the N M R s p e c t r a m i g h t be due to m u l t i c h a i n aggregation processes w h i c h have a l ­ ready been described for very d i l u t e solutions (17,20-22). D i s a p p e a r a n c e of N M R signals of pectins has already been reported for b o t h s o l i d (23) a n d l i q u i d - s t a t e e x p e r i m e n t s (17). U l t r a s o n i c a t i o n d i d not i m p r o v e the re­ sults. A l l these d a t a show t h a t P F i contains h i g h l y m e t h y l a t e d , h i g h l y b r a n c h e d r h a m n o g a l a c t u r o n a n s . G a l a c t o s e a n d arabinose b u i l d sidechains 2

1 3

+

2

1 3

1 3

2

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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317

QA(i) QA

APR

PF„ 0(4)

EDTA

110

PPM

17S

tOS

100

9S

90

8S PPM

80

7S

70

SS

F i g u r e 2. 100 M H z C s p e c t r a of pectic fractions. I n t e r n a l M e S O , δ 3 9 . 5 G A , g a l a c t u r o n i c a c i d ; G , galactose; A , arabinose. 1 3

2

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L P O L Y M E R S

( a b o u t 3 times more galactose t h a n arabinose) w h i c h c o n t a i n t e r m i n a l a r a binose. I n c o n t r a s t , P F 2 contains m o s t l y h o m o g a l a c t u r o n a n s . T h e n e u t r a l sugars, f r o m 1 0 % t o 3 0 % , detected i n t h i s f r a c t i o n m i g h t represent either c o n t a m i n a t i o n s w i t h P F i or some short sidechains. Physicochemical Properties of Pectins. Solubilized Pectins. Pectins solubil i z e d b y b o i l i n g w a t e r ( P F x ) a n d further e x t r a c t e d b y E D T A ( P F ) f r o m y o u n g a n d m a t u r e cell walls were characterized b y p o t e n t i o m e t r i c measurem e n t s . T i t r a t i o n s were p e r f o r m e d w i t h different c o u n t e r i o n s : K , N a , M g , C a . I n a l l cases the p H curves diverged for monovalent a n d d i v a lent ions for n e u t r a l i z a t i o n degrees > 0.5 ( F i g . 3 A , C , D ) . T h i s divergence is essentially due t o valence differences. N o differences c o u l d be detected w h e n P F i ' s were n e u t r a l i z e d w i t h C a ( O H ) or M g ( O H ) , whereas t i t r a t i o n curves o f P F ' s revealed a higher s e l e c t i v i t y for c a l c i u m ions ( F i g s . 3 B a n d 3 D ) . However, s a p o n i f i c a t i o n of P F i , w h i c h deesterifies the m e t h y l a t e d p o l y m e r s , i n d u c e d a sligh curves ( F i g . 3 C ) . W i t h monovalent counterions, a difference i n affinity for Na+ and K was noted ( F i g . 3 A ) . C u r v e s o b t a i n e d w i t h pectic fractions isolated f r o m y o u n g a n d m a t u r e cell walls were not s i g n i f i c a n t l y different. Pectins i n s i t u . A f t e r b o i l i n g water t r e a t m e n t , the r e s i d u a l cell walls c o n t a i n e d o n l y P F p e c t i n s . C a t i o n exchange c a p a c i t y ( C E C ) of these cell walls was e s t i m a t e d for different p H ' s . In a l l cases, y o u n g cell walls e x h i b i t e d a s m a l l e r C E C t h a n the older ones ( F i g . 4). E x c h a n g e s were then p e r f o r m e d i n order to compare the r e l a t i v e cell w a l l affinities for C a + and M g ions. A f t e r hot water e x t r a c t i o n , the s e l e c t i v i t y coefficients K j ^ g of y o u n g a n d m a t u r e cell walls were almost i d e n t i c a l ( F i g . 5 C ) for a l l p H values. T h e i o n i z a t i o n of m a t u r e walls was higher t h a n t h a t of y o u n g ones ( F i g . 5 D ) . A f t e r E D T A t r e a t m e n t , the cell walls no longer b e h a v e d as c a t i o n exchangers, w h i c h reveals a complete s o l u b i l i z a t i o n of p e c t i n s . C r u d e cell walls w h i c h c o n t a i n e d b o t h P F i a n d P F fractions were also i n v e s t i g a t e d . T h e i r C E C values were higher t h a n those measured for hot water e x t r a c t e d cell walls ( F i g . 4) a n d y o u n g cell walls e x h i b i t e d a smaller C E C t h a n the older ones. T h e differences between the C E C ' s of crude a n d hot water e x t r a c t e d p o l y m e r s can thus be a t t r i b u t e d to the P F i p o l y m e r s . E x c h a n g e e x p e r i m e n t s showed t h a t , at constant ionic s t r e n g t h a n d e q u a l i o n i c fractions of C a a n d M g , the s e l e c t i v i t y coefficients were a l w a y s higher for m a t u r e cell walls t h a n for y o u n g ones. W h a t e v e r the value of the p H i n the i n c u b a t i o n m e d i u m ( F i g . 5 A ) , y o u n g crude cell walls e x h i b i t e d a lower coefficient t h a n y o u n g , hot water e x t r a c t e d ones. T h e P F i f r a c t i o n , m u c h more a b u n d a n t i n y o u n g cell walls ( T a b l e I), h a d a lower affinity for C a ions t h a n P F . These d a t a c o n f i r m the results o b t a i n e d w i t h pectins i n s o l u t i o n . P h y s i c o c h e m i c a l b e h a v i o r of cell walls results t h e n f r o m the P F x / P F r a t i o s , the properties of the pectins embedded i n the p o l y s a c c h a r i d e network b e i n g i d e n t i c a l to those of s o l u b i l i z e d p e c t i n s . W a t e r contents o f i n t a c t a n d e x t r a c t e d cell walls were also e s t i m a t e d ( T a b l e II). T h e a m o u n t of absorbed water was the highest i n y o u n g , i n t a c t cell w a l l s . A f t e r b o i l i n g water t r e a t m e n t , the swelling o f the cell w a l l s was lower a n d q u i t e s i m i l a r for y o u n g a n d m a t u r e cell w a l l s . T h e h i g h water 2

+

2 +

+

2 +

2

2

2

+

2

2

2 +

2

2 +

2 +

2 +

2

2

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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GOLDBERG ET AL.

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Control of Cell Wall Plasticity

F i g u r e 3. T i t r a t i o n of p e c t i n fractions e x t r a c t e d f r o m y o u n g cell w a l l s . A , B , C , pectins e x t r a c t e d w i t h b o i l i n g water; D , e x t r a c t e d w i t h hot E D T A . A , B , i n t a c t P F i ; C , d e m e t h y l a t e d P F i . T i t r a t i o n s were p e r f o r m e d with K O H ( Δ Δ ) , N a O H (A A), C a ( O H ) (· · ) , and M g ( O H ) (o o). 2

2

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

320

PLANT

c

C E L L WALL

POLYMERS

w

-

••

05

• η • ο ° • o° • o • ο • ο •ο •ο •0

°

• ••

u

•aJ-?2_i

3

.

5

.

ι—- ν/ί

7

3

• ο • ο • ο • ο *°ι •

5

•

•

«—

7

F i g u r e 4. C a t i o n exchange c a p a c i t y ( C E C ) of isolated cell walls as a f u n c t i o n of the p H of the i n c u b a t i o n m e d i u m — C E C as meq per g cell walls. T h e t i t r a t i o n s were r u n i n C a C b solutions (I = 100 m M ) . C W , intact isolated cell walls; R C W , residual cell walls after b o i l i n g water t r e a t m e n t . · · m a t u r e cell walls; ο ο y o u n g cell walls.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

22.

G O L D B E R G ET AL.

F i g u r e 5. S e l e c t i v i t y coefficients (A

321

Control of Cell Wall Plasticity

K ^ g of y o u n g ( ·

·)

and

mature

A ) cell w a l l s . V a l u e s of K ^ g p l o t t e d as a f u n c t i o n of p H ( A , C ) a n d

of x , the n u m b e r of ionized c a r b o x y l groups ( B , D ) . A a n d B , i n t a c t i s o l a t e d cell w a l l s ; C a n d D , cell walls p r e v i o u s l y e x t r a c t e d by b o i l i n g water.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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content o f y o u n g cell walls results f r o m t h e h i g h a m o u n t o f P F i i n these walls. These d a t a m i g h t e x p l a i n t h e m i c r o s c o p i c aspects o f b o t h k i n d s o f cell w a l l s , t h e y o u n g ones b e i n g m o r e swollen t h a n t h e m a t u r e ones ( 8 ) . T a b l e I I . W a t e r content o f i n t a c t ( C W ) a n d e x t r a c t e d cell walls ( R C W ) isolated f r o m y o u n g a n d m a t u r e tissues o f the h y p o c o t y l as percent o f cell wall dry matter Y o u n g Tissues

M a t u r e Tissues

CW

R C W

C W

R C W

850

260

425

260

I n c o n c l u s i o n , a l o n g th depends u p o n t h e w a l l e x t e n s i b i l i t y . Y o u n g , p l a s t i c w a l l s are c h a r a c t e r i z e d b y a h i g h level o f b r a n c h e d a n d m e t h y l a t e d r h a m n o g a l a c t u r o n a n s w h i c h leads t o swollen cell walls a n d a low level o f linear g a l a c t u r o n a n s w h i c h induces a low C E C . I n contrast, stiff, m a t u r e cell walls are characterized b y a low water content a n d a h i g h C E C . T h e low water content i n d i c a t e s stronger cohesion o f the polysaccharide network. T h e h i g h C E C is a possible factor for cross l i n k i n g a n d i n t u r n for w a l l stiffening processes. Literature Cited

1. Lamport, D. T. A. In Cellulose: Structure Modification and Hydrolysis; Young, R. Α.; Rowell, R. M., Eds.; John Wiley&Sons: New York, 1986; p. 77-90. 2. Fry, S. C. Ann. Plant Physiol. 1986, 37, 165-86. 3. Nari, J.; Noat, G.; Diamantidis, G.; Woundstra, M.; Ricard, J. Eur. J. Biochem. 1986, 155, 199-202. 4. Goldberg, R.; Prat, R. Physiol. Veg. 1981, 19, 523-32. 5. Prat, R. J. Exp. Bot. 1985, 36, 1150-58. 6. Bouchet, M. H.; Prat, R.; Goldberg, R. Physiol. Plantarum 1983, 57, 95-100. 7. Goldberg, R. Plant Sci. Lett. 1977, 8, 233-42. 8. Goldberg, R.; Morvan, C.; Roland, J. C. Plant Cell Physiol. 1986, 27, 417-29. 9. Hervé du Penhoat, C.; Michon, V.; Goldberg, R. Carbohydr. Res. 1987, 165, 31-42. 10. Morvan, C.; Demarty, M.; Thellier, M. Physiol. Veg. 1985, 23, 333-44. 11. Blumenkrantz, N.; Asboe-Hansen, G. Anal. Biochem. 1973, 54, 484-89. 12. Kohn, R. Pure Appl. Chem. 1975, 42, 371-95. 13. Joseleau, J. P.; Chambat, G.; Lanvers, M. Carbohydr. Res. 1983, 122, 107-13. 14. Capek, P.; Toman, R.; Kardosova, Α.; Rosik, J. Carbohydr. Res. 1983, 117, 133-40.

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15. Eby, R.; Schuerch, C. Carbohydr. Res. 1981, 92, 149-53. 16. Gorin, P. A. J.; Carbohydr. Res. 1982, 101, 13-20. 17. Rinaudo, M.; Ravanat, G.; Vincedon, M. Makromol. Chem. 1980, 181, 1059-70. 18. Joseleau, J. P.; Chambat, G. Physiol. Vég. 1984, 22, 461-70. 19. Nakahara, Y.; Ogawa, T. Carbohydr. Res. 1987, 167, C1-C7. 20. Jordan, R. C.; Brant, D. A. Biopolymers 1978, 17, 2885-95. 21. Davies, M. A. F.; Gidley, M. J.; Morris, E. R.; Powzll, D. Α.; Rees, D. A. Int. J. Biol. Macromol. 1980, 2, 330-32. 22. Grasladen, H.; Kvam, B. J. Macromolecules 1986, 19, 1913-20. 23. Keenan, M. H. J.; Belton, P. S.; Matthew, J. Α.; Howson, S. J. Carbo­ hydr. Res. 1985, 138, 168-77. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 23 Effect of Chemical Structure of Pectins on Their Interactions with Calcium M . Rinaudo University Joseph Fourier of Grenoble, Centre de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique, B.P. 53X-38041, Grenoble Cedex, France

The interaction role of carboxylic acid salt formation and the degree of polymerization are first considered in terms of electrostatic and/or cooperative specific interactions. Then the effect of the degree of esterification and that of the pattern of carboxylic group distribution are discussed; pectin esterase forms blocks which behave as fully hydrolyzed polymers and favor aggregation. Finally, the role of the calcium addition on the degree of aggregation was established. All the data show the important role of molecular structure of the pectins on calcium interactions. P e c t i n s are polysaccharides f o u n d i n the p r i m a r y cell walls o f plants a n d i n the m i d d l e l a m e l l a . T h e y m a i n l y consist o f p o l y (1 —• 4)o>D-galacturonic acids a n d their m e t h y l esters. I n a d d i t i o n , they also c o n t a i n n e u t r a l s u g ars inserted i n t o the m a i n c h a i n ( L - r h a m n o s e units) a n d i n the side c h a i n (nearly 1 0 - 1 5 % o f the n e u t r a l sugar b y weight). P e c t i n s are considered t o have i m p o r t a n t s t r u c t u r a l roles (ion exchange a n d m e c h a n i c a l p r o p e r t i e s ) , a n d t o be f o r m e d m a i n l y o f h o m o g a l a c t u r o n a n blocks w h i c h play a n i m p o r t a n t role i n the mechanisms o f i n t e r a c t i o n i n the cell w a l l s . T h e degree of esterification ( D E ) a n d the degree o f p o l y m e r i z a t i o n ( D P ) o f the pectic substances are d i r e c t l y controlled b y enzymes a n d / o r p H . T h i s i m p l i e s m o d i f i c a t i o n o f their behavior w i t h age, for e x a m p l e , a n d also indicates the necessity for c o n t r o l o f c o n d i t i o n s o f e x t r a c t i o n i n order t o isolate p o l y m e r s representative o f their n a t i v e state. T h e role o f the s t r u c t u r a l properties o f pectins was described recently (1) where i t was s h o w n t h a t interactions o f c a l c i u m w i t h pectic substances were d i r e c t l y related t o the existence o f u n b r a n c h e d a n d non-esterified g a l a c t u r o n i c b l o c k s . C a l c i u m was able t o i o n i c a l l y b i n d two o r more chains 0097-6156/89/0399-0324$06.00/0 © 1989 American Chemical Society

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a n d t o establish a t h r e e - d i m e n s i o n a l network g i v i n g u n i q u e m e c h a n i c a l p r o p e r t i e s . I n t h i s c h a p t e r , we describe the b e h a v i o r o f p e c t i n s i n aqueous s o l u t i o n s . D e p e n d i n g u p o n the e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d , the c a l c i u m ions can i n d u c e a sol-gel t r a n s i t i o n . Materials and Methods P o l y g a l a c t u r o n i c a c i d ( N u t r i t i o n a l B i o c h e m i c a l s C o r p . , U S A ) was p u r i f i e d a n d i s o l a t e d as the s o d i u m salt (2,3) to give a p r e p a r a t i o n c o n t a i n i n g > 9 5 % g a l a c t u r o n i c a c i d . T h e oligogalacturonates were prepared b y p a r t i a l h y d r o l y s i s (4-6). C o m m e r c i a l apple p e c t i n f r o m U n i p e c t i n e (France) was purified a n d p a r t i a l l y deesterified (i) under alkaline c o n d i t i o n s to p r o d u c e r a n d o m l y dispersed c a r b o x y l i c a c i d f u n c t i o n a l i t i e s a l o n g the c h a i n a n d (ii) b y a n esterase t o p r o d u c e blockwise d i s t r i b u t i o n of the c a r b o x y l i c groups. T h e s e t w o series o f samples allowed the d e m o n s t r a t i o n of the effect of the d i s t r i b u t i o n of c a r b o x y l i c o n the properties. These ionic p o l y m e r s can be considered as polyelectrolytes a n d their electrostatic properties can be predicted f r o m the u s u a l theories. In t h a t respect, a p o l y e l e c t r o l y t e is characterized b y a s t r u c t u r a l charge p a r a m e ter i n d e p e n d e n t of the DPÀ = 7 e / D h k T w i t h 7 e q u a l to the n u m b e r of i o n i z e d groups o n a p o l y m e r i c chain w i t h l e n g t h h , D , the dielectric c o n s t a n t , e, the electronic charge a n d k T , the B o l t z m a n t e r m . C o n s i d e r i n g the s t r u c t u r a l l e n g t h of the D - g a l a c t u r o n i c u n i t i n C\ c o n f o r m a t i o n (b = 4 . 3 5 A ( 7 ) ) , the p o l y α-D-galacturonate is characterized b y a λ value e q u a l to 1.65. T h i s p a r a m e t e r controls the t h e r m o d y n a m i c properties as w e l l as the a c t i v i t y coefficient of counterions or p o t e n t i o m e t r i c t i t r a t i o n . T h e i n t e r a c t i o n w i t h c a l c i u m was d e t e r m i n e d f r o m a c t i v i t y measurements of C a w i t h a specific electrode (3,8); the a p p a r e n t p K a of the c a r b o x y l i c acids was o b t a i n e d f r o m p H t i t r a t i o n d u r i n g n e u t r a l i z a t i o n . C o n f o r m a ­ t i o n a l changes were m o n i t o r e d by c i r c u l a r d i c h r o i s m ( C D ) (3,9). I n t e r c h a i n i n t e r a c t i o n s were e s t i m a t e d by viscosity, gel p e r m e a t i o n c h r o m a t o g r a p h y (10), l i g h t s c a t t e r i n g measurements (11,12), a n d C N M R (2). A l l the d a t a were o b t a i n e d at a constant t e m p e r a t u r e ( 2 5 ° C ± 0 . 1 ) . 2

A

2 +

1 3

Results and Discussion Role of DP and ihe Degree of Neutralization. T h e role of the D P o n the a c t i v i t y of c a l c i u m counterions i n the presence of o l i g o - a n d p o l y g a l a c t u r o n a t e s was first investigated b y K o h n (13); the observed b e h a v i o r was confirmed b y R a v a n a t (10), w h o showed t h a t when the D P is larger t h a n 10, the a c t i v i t y coefficient o f c a l c i u m decreases s t r o n g l y due to i n t e r c h a i n c r o s s l i n k i n g i n t e r p r e t e d i n terms of the "egg-box" m o d e l (14,15). T h e ge­ o m e t r y of the c a l c i u m - c a r b o h y d r a t e c o m p l e x was more recently discussed b y D h e u - A n d r i e s a n d Perez (16). F r o m R a v a n a t ' s a n d K o h n ' s d a t a , the t r a n s i t i o n observed for c a l c i u m a c t i v i t y is i n the range of D P 10 to 20, w h i c h means t h a t at least 5 to 10 Ca cations are necessary to f o r m a cooperative s t r o n g i n t e r c h a i n b i n d ­ i n g . U n t i l n o w , f o r m a t i o n of c h a i n dimers (as p r e d i c t e d i n the "egg-box" 2 +

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m o d e l ) was o n l y s h o w n to exist w i t h p o l y m e r s t a k i n g i n t o c o n s i d e r a t i o n the a c t i v i t y coefficient of C a counterions at i n f i n i t e d i l u t i o n (8). I n fact, f r o m t h e r m o d y n a m i c considerations, i t was concluded t h a t p o l y g a l a c t u r onate b e h a v i o r u p to D P = 5 was i n agreement w i t h electrostatic theories c o n s i d e r i n g a n o r m a l single molecule process (3). F r o m C D measurements, t h i s c o n c l u s i o n a v o i d i n g a specific i n t e r a c t i o n w i t h c a l c i u m is c o n f i r m e d ( F i g . 1; curves (1) a n d (2)). F r o m c i r c u l a r d i c h r o i s m , i t appears t h a t for p e c t i n s w i t h a degree of n e u t r a l i z a t i o n ( α ' ) of over 0.4, a specific i n t e r a c t i o n w i t h c a l c i u m occurs (9). A t the same t i m e , the apparent p K a decreases c o r r e s p o n d i n g to a specific b i n d i n g (3). T h i s c r i t i c a l value corresponds to a λ value of 0.64, w h i c h means t h a t the C a m u s t be associated w i t h 2 4 % of the c a r b o x y l groups. 2 +

2 +

T h i s result is d i r e c t l y p r o p o r t i o n a l to the m i n i m u m n u m b e r of c a r ­ b o x y l i c a c i d sites required to f o r m a stable j u n c t i o n zone o n p o l y m e r s . T h e n u m b e r of c a l c i u m cation b i l i t y of the j u n c t i o n a n d its t h e r m o r e v e r s i b i l i t y . F i n a l l y , i t m u s t be also p o i n t e d o u t t h a t i t is not the p H , b u t the degree of n e u t r a l i z a t i o n , α ' , w h i c h controls c a l c i u m b i n d i n g . Effect of the Degree of Esterification and Carboxyl Group Distribution. It is generally agreed t h a t a t r a n s i t i o n i n the b e h a v i o r of p e c t i n s occurs a r o u n d a degree of esterification of 5 0 % . T h i s is i n fact the case for r a n d o m l y dispersed c a r b o x y l i c groups a l o n g the c h a i n . T h e c o r r e s p o n d i n g charge p a r a m e t e r is t h e n the same as for a p o l y g a l a c t u r o n i c a c i d w i t h cx' = 0.5; t h i s value is i n g o o d agreement w i t h our previous discussion. T h e effect of the d i s t r i b u t i o n of i o n i c sites o n the aggregation process has also been e l u c i d a t e d (8,11,12). F o r these investigations, p a r t i a l deesteri f i c a t i o n o n apple pectins was p e r f o r m e d , causing a r a n d o m d i s t r i b u t i o n of the c a r b o x y l i c sites a l o n g the c h a i n ; o n the other h a n d , a c t i o n of a p e c t i n esterase p r o d u c e d a blockwise d i s t r i b u t i o n of the i o n i c groups. T h e s e two series of samples were tested a l l o w i n g the c o m p a r i s o n of t h e r m o d y n a m i c properties for p o l y m e r s h a v i n g the same average charge p a r a m e t e r b u t dif­ ferent p a t t e r n s of d i s t r i b u t i o n of the c a r b o x y l i c groups. It was t h e n f o u n d t h a t the p e c t i n esterase p r o d u c e d blocks of free c a r b o x y l i c group w h i c h behaved j u s t as a f u l l y h y d r o l y z e d p o l y m e r , i.e., each a t t a c k p r o d u c e d a free zone o n a c h a i n w h i c h was able to be chelated i n the presence of C a . A s s o c i a t i o n of chains was clearly d e m o n s t r a t e d by light s c a t t e r i n g measure­ ments (12). 2 +

Effect of Calcium Content. T h e a p p l i c a t i o n of gel p e r m e a t i o n c h r o m a t o g ­ r a p h y c a n also be used to answer the questions of i n t e r a c t i o n of c a l c i u m with sodium polygalacturonate. U s i n g a d i l u t e aqueous s o l u t i o n of the p o l y m e r i n the absence of ex­ t e r n a l s a l t , a m o l e c u l a r weight d i s t r i b u t i o n was o b t a i n e d , a l t h o u g h i t was p e r t u r b e d b y electrostatic e x c l u s i o n . W h e n C a C b was a d d e d , a salt e x c l u ­ sion c o r r e s p o n d i n g to N a C l was f o r m e d , i.e., a n exchange process o c c u r r e d u p to at least 4 0 % of the stoichiometry, where i n each case one C a ex­ p e l l e d two N a + (10). 2 +

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ΔΣ

a ι—• 1

ι

Q5

1

F i g u r e 1. N o r m a l i z e d e l l i p t i c i t y at 210 n m as a f u n c t i o n of the degree of n e u ­ t r a l i z a t i o n ( α ' ) for (1) s o d i u m f o r m of oligogalacturonates a n d p o l y g a l a c t u r o n a t e ; (2) c a l c i u m f o r m of galacturonates ( O D P 2 , Δ D P 4 , • DP5), (3) c a l c i u m f o r m o f the p o l y g a l a c t u r o n a t e (φ). (ΑεΗ taken as reference is the e l l i p t i c i t y of a c i d forms for each p r o d u c t ; o p e n = s o d i u m f o r m ; filled = c a l c i u m form.)

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^

Increasing

M

F i g u r e 2. R e f r a c t o m e t r i c (—) a n d c o n d u c t i m e t r i c ( ) traces for gel p e r m e a t i o n c h r o m a t o g r a p h y of p o l y g a l a c t u r o n a t e s o d i u m w i t h progressive a d d i t i o n of C a C b expressed i n equivalent percentage of C a added (polymer c o n c e n t r a t i o n 5 g/1; S p h e r o s i l porous m a t e r i a l s w i t h average pore d i a m e t e r of 6000Â; e l u t i o n H 0 ) . 2 +

2

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(W%)

%Ca --(eq) 2

F i g u r e 3. Dependence of the i n t r i n s i c v i s c o s i t y Ο (M)> e l Φ a n d gel f r a c t i o n φ (wt. percent W % ) as a f u n c t i o n of equivalent percentage of C a a d d e d , ([η] was d e t e r m i n e d by isoionic d i l u t i o n of a 6 g/1 s o l u t i o n of s o d i u m p o l y g a l a c t u r o n a t e . ) n

m

r

S 1

n a

2 +

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F i g u r e 4. C r o s s l i n k i n g m e c h a n i s m proposed for pectins i n presence of C a ' (10). B l a c k dots represent the c a l c i u m .

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F r o m F i g u r e 2, i t is clear t h a t the a p p a r e n t average m o l e c u l a r weight of the p o l y m e r increases progressively as soon as 2 5 % C a equivalents are a d d e d a n d , at the same t i m e , the i n t r i n s i c v i s c o s i t y o f the samples increased ( F i g u r e 3). T h e f r a c t i o n o f c a r b o x y l i c groups b o u n d w i t h C a agrees w i t h the v a l u e f o u n d i n t h e discussion o n t h e role o f α ' . F r o m the refractometric trace o n t h e c h r o m a t o g r a m s , i t w a s possible t o c a l c u l a t e the f r a c t i o n o f gel f o r m e d . W h e n gel was f o r m e d i n t h e s y s t e m , the C N M R signals o f t h e different c a r b o n a t o m s d i s a p p e a r d u e t o t h e decrease i n t h e i r m o b i l i t y ( F i g . 3 ) . 2 +

2 +

1

3

Conclusions T h e m e c h a n i s m o f i n t e r a c t i o n o f c a l c i u m is d i r e c t l y related t o t h e c h e m ­ i c a l s t r u c t u r e o f p e c t i n s . I t also depends o n t h e p o l y m e r c o n c e n t r a t i o n , the d i s t r i b u t i o n o f c a r b o x y l i c groups a n d t h e a m o u n t o f c a l c i u m present i n the m e d i u m . T o conclude ure 4 w h i c h allows us t o predict progressive aggregation, g e l a t i o n o r phase separation depending on experimental conditions. F r o m t h e different e x p e r i m e n t a l results o b t a i n e d , i t c a n be c o n c l u d e d t h a t t h e average n u m b e r o f C a ions b o u n d is 1 p e r 8 c a r b o x y l i c a c i d g r o u p s . However, f r o m gel c h r o m a t o g r a p h y i t is clear t h a t t h i s d i s t r i b u t i o n is n o t homogeneous because aggregation is p r o d u c e d progressively; t h i s also i m p l i e s a degree o f c o o p e r a t i v i t y i n the i n t e r a c t i o n . T h e f o l l o w i n g questions r e m a i n t o b e answered: 2 +

-

W h a t is t h e r e l a t i o n s h i p between t h e observed b e h a v i o r a n d t h a t i n the cell w a l l ? - W h a t is t h e d i s t r i b u t i o n o f C a a l o n g t h e chains? - H o w m u c h C a + is necessary t o f o r m a stable j u n c t i o n ? T h i s result is directly proportional to the m i n i m u m . 2 +

2

A cknowledgment s T h e a u t h o r wishes t o t h a n k J . - F . T h i b a u l t a n d G . R a v a n a t for t h e i r e x p e r ­ imental contributions. Literature Cited

1. Jarvis, M. C. Plant Cell Environ. 1984, 7, 163. 2. Rinaudo, M.; Ravanat, G.; Vincendon, M. Makromol. Chem. 1980, 181, 1059. 3. Ravanat, G.; Rinaudo, M. Biopolymers 1980, 19, 2209. 4. Kohn, R.; Larsen, B. Acta Chem. Scand. 1972, 26, 2455. 5. Kohn, R.; Luknar, O. Coll. Czech. Chem. Commun. 1975, 40, 959. 6. Kohn, R. Carbohydr. Res. 1971, 120, 351. 7. Atkins, E. T. D.; Isaac, D. H.; Nieduszynski, I. Α.; Philips, C. F.; Sheehan, J. K. Polymers 1974, 15, 263. 8. Thibault, J.-F.; Rinaudo, M. Biopolymers 1985, 24, 2131.

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POLYMERS

9. Thibault, J.-F.; Rinaudo, M. In Chemistry and Function of Pectins; ACS Symp. Ser. No. 310, 1986, p. 61. 10. Ravanat, G. Thesis, Grenoble, 1979. 11. Thibault, J.-F.; Rinaudo, M. Biopolymers 1986, 25, 455. 12. Thibault, J.-F.; Rinaudo, M. Brit. Polym. 1985, 17, 181. 13. Kohn, R.; Luknar, O. Coll. Czech. Chem. Commun. 1977, 42, 731. 14. Grant, G. T.; Morris, E. R.; Rees, D. Α.; Smith, P. J. C.; Thom, D. FEBS Lett. 1973, 32, 195. 15. Powell, D. Α.; Morris, E. R.; Gidlley, M. J.; Rees, D. A. J. Mol. Biol. 1982, 15, 517. 16. Dheu-Andries, M.-L.; Perez, S. Carbohydr. Res. 1983, 124, 324. RECEIVED April 28, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 24

Comparative Studies on the Cell Wall Polymers Obtained from Different Parts of Rice Grains Naoto Shibuya National Food Research Institute, Ministry of Agriculture, Forestry, and Fisheries, Tsukuba, Ibaraki 305, Japan

Cell wall polymer fered in both compositio cellulosic polysaccharides of the cell wall preparations obtained from both outer and inner parts of the grain contained arabinoxylan and xyloglucan. The amount of β-1,3-,1,4-glucan was negligible in the cell walls of the outer part of the grain when compared to that in the en­ dosperm cell wall. The degree of lignification also differed from tissue to tissue. In addition, the arabinoxylan ob­ tained from the outer part of the grain carried side-chains with more complicated structures than endospermic ara­ binoxylan. Similar differentiation on the molecular level is also suggested for other cell wall polymers. In order t o u n d e r s t a n d the process o f differentiation a n d t h e properties o f the r e s u l t i n g cell walls, i t is necessary t o identify the changes t h a t o c c u r i n cell w a l l polymers. Several recent papers suggest t h a t changes i n cell surface carbohydrates i n a n i m a l s a n d p l a n t s m a y be b o t h a cause a n d effect o f differentiation (1-3). O n e possible approach t o the s t u d y o f differentiation is t o c o m p a r e the c o m p o s i t i o n a n d c h e m i c a l s t r u c t u r e o f c e l l - w a l l p o l y m e r s d u r i n g development. C e r e a l grains, such as rice a n d w h e a t , are c o m p o s e d of several tissues w h i c h appear a t different stages o f differentiation. T h u s , s t a r c h e n d o s p e r m originates f r o m fertilized p o l a r n u c l e i , w h i l e the t h i n cell walls are considered t o be p r i m a r y cell walls (4,5). T h e outer p a r t o f the e n d o s p e r m is covered w i t h several layers o f aleuron cells, w h i c h are also derived f r o m the same fertilized p o l a r nucleus b u t w h i c h differentiate i n t o a specific tissue d u r i n g t h e m a t u r a t i o n process. C e l l walls o f the a l e u r o n cells are m u c h thicker t h a n those o f the e n d o s p e r m , a n d are considered t o be secondary walls. T h e o u t e r m o s t p a r t o f the g r a i n is a caryopsis coat consisting o f several compressed cell layers w i t h very t h i c k ( > 1 u m ) w a l l s . 0097-6156/89/0399-0333$06.00/0 Ο 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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G e r m , or e m b r y o , is derived f r o m the f e r t i l i z e d egg a n d consists o f several different tissues. In view of the p r a c t i c a l i m p o r t a n c e of these g r a i n s , for e x a m p l e i n d i e t a r y fibers, i t is useful to s t u d y the cell w a l l p o l y m e r s of their tissues d u r i n g d i f f e r e n t i a t i o n . I n the present w o r k , we used rice grains ( b r o w n rice w i t h o u t husk) as a s t a r t i n g m a t e r i a l . T h e y were f r a c t i o n a t e d i n t o several d i s t i n c t h i s t o l o g i c a l components, a n d the c o m p o s i t i o n a n d d e t a i l e d s t r u c t u r e of cell w a l l p o l y m e r s was c o m p a r e d . Materials and Methods Isolation of Cell Walls from Different Parts of Rice Grain. Rice grains were g r a d u a l l y peeled u s i n g a c o n v e n t i o n a l m i l l i n g i n s t r u m e n t (Satake M o tor O n e - P a s s T y p e Test M i l l , S a t a k e E n g i n e e r i n g C o . , L t d . , T o k y o ) , a n d the fractions r i c h i n specific h i s t o l o g i c a l c o m p o n e n t s were collected. T h e p o t a s s i u m content of these fractions was used as a n i n d e x to identify the f r a c t i o n enriched i n aleuro t a i n e d f r o m the v e r y first m i l l i n g f r a c t i o n . S t a r c h y e n d o s p e r m was o b t a i n e d b y t h o r o u g h l y r e m o v i n g the outer p a r t o f the g r a i n . T h e g e r m f r a c t i o n was separated f r o m the b r a n f r a c t i o n b y s i e v i n g w i t h 10-20 mesh sieves. C e l l walls were o b t a i n e d by the successive e x t r a c t i o n of the defatted tissues w i t h S D S - m e r c a p t o e t h a n o l a n d d i m e t h y l s u l f o x i d e (7). A n e n z y m a t i c m e t h o d usi n g a c o m b i n a t i o n of protease a n d amylase (8) was also used to o b t a i n cell walls f r o m these tissues, a n d the a n a l y s i s of the cell w a l l p r e p a r a t i o n s so o b t a i n e d showed t h a t b o t h methods gave b a s i c a l l y the same p r e p a r a t i o n s . T h e y i e l d of the cell walls ( w / w of defatted tissue) was 0 . 3 % for the e n d o s p e r m , 1 2 % for the g e r m , 2 0 % for the aleuron tissue, a n d 2 9 % for the caryopsis coat. S c a n n i n g electron m i c r o g r a p h s of these cell w a l l p r e p a r a tions ( F i g . 1) showed t h a t b o t h of the cell w a l l p r e p a r a t i o n s o b t a i n e d f r o m the caryopsis coat, a n d the aleuron tissue, were very t h i c k a n d a p p e a r e d of h a r d t e x t u r e . O n the other h a n d , the e n d o s p e r m cell w a l l p r e p a r a t i o n was very t h i n a n d appeared to be of softer t e x t u r e . T h e m a i n p a r t of the cell w a l l p r e p a r a t i o n f r o m the g e r m looked s i m i l a r t o the e n d o s p e r m cell w a l l b u t was somewhat thicker a n d harder. These p r e p a r a t i o n s were free of other c e l l u l a r particles such as s t a r c h or proteins. Results and Discussion Comparison of the Overall Composition of Cell Wall Preparations. A s can be seen f r o m T a b l e I, cell w a l l p r e p a r a t i o n s showed a significant difference i n t h e i r p o l y m e r c o m p o s i t i o n . T h e e n d o s p e r m cell walls resembled p r i m a r y walls, since they were v i r t u a l l y free of l i g n i n b u t r i c h i n pectic substances. O n the other h a n d , the cell w a l l p r e p a r a t i o n s o b t a i n e d f r o m the caryopsis coat a n d the aleuron tissue were h i g h l y lignified, a n d their pectic content was very low. T h e g e r m cell w a l l showed a somewhat i n t e r m e d i a t e c o m p o s i t i o n between these two types, p r o b a b l y reflecting the fact t h a t i t consists of several different tissues. T h e monosaccharide c o m p o s i t i o n of the hemicelluloses, w h i c h were a m a j o r f r a c t i o n of a l l of these cell w a l l p r e p a r a t i o n s , also showed significant differences, especially i n the a m o u n t s of glucose a n d galactose ( T a b l e II).

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

24.

SHIBUYA

Cell Wall Polymers from Different Parts of Rice

335

F i g u r e 1. S c a n n i n g electron m i c r o g r a p h of the cell w a l l p r e p a r a t i o n s ob­ t a i n e d f r o m the different parts of rice g r a i n (7). C a r y o p s i s coat (upper left), a l e u r o n layer (upper r i g h t ) , germ (lower left) a n d s t a r c h y e n d o s p e r m (lower r i g h t ) . B a r s i n the p i c t u r e i n d i c a t e 5 μπι.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

336

PLANT C E L L W A L L P O L Y M E R S

T a b l e I. C o m p o s i t i o n of the C e l l W a l l P r e p a r a t i o n s O b t a i n e d f r o m Differ­ ent H i s t o l o g i c a l F r a c t i o n s

Fraction

Pectic Substances

Hemicellulose

α-Cellulose

Lignin

7 11 27 23

38 42 49 47

28 31 23 21

27 16 1 9

C a r y o p s i s coat A l e u r o n tissue Endosperm Germ

W h i l e the hemicelluloses o b t a i n e d f r o m the g e r m , a l e u r o n , a n d caryopsis coat cell walls a l l showed a s i m i l a r monosaccharide c o m p o s i t i o n , t h i s was not the case for the e n d o s p e r m tissue. T h u s , a m a j o r difference i n the s t r u c t u r e of hemicellulosic polysaccharides exists between the p r e p a r a t i o n s o b t a i n e d f r o m the e n d o s p e r other parts of the g r a i n , i.e., rice b r a n . ( R i c e b r a n consists of the c a r y o p ­ sis coat, aleuron layer a n d germ.) C o m p a r i s o n of the detailed s t r u c t u r a l features of the hemicellulosic polysaccharides of e n d o s p e r m a n d b r a n cell walls w i l l be discussed i n the following sections. T a b l e II. M o n o s a c c h a r i d e C o m p o s i t i o n of P e c t i c Polysaccharides a n d H e m i ­ cellulose O b t a i n e d f r o m Different C e l l W a l l P r e p a r a t i o n s N e u t r a l Sugar C o m p o s i t i o n ( m o l %)

Fraction

Rhamnose

Fucose

Arabinose

Xyl­ ose

Hemicellulose: C a r y o p s i s coat A l e u r o n layer Germ Endosperm

1.0 1.0 1.2 0.9

0.4 0.4 0.5 0.5

35.6 36.7 36.7 26.4

43.6 43.5 38.1 41.1

1.2 1.1 0.7 0.6

43.6 48.9 46.9 33.0

26.8 27.5 20.5 30.4

Pectic Polysaccharides: C a r y o p s i s coat 5.0 A l e u r o n layer 3.6 Germ 2.3 Endosperm 6.1

Uronic

Glu­ cose

Content (wt.%)

7.4 7.4 8.8 1.9

12.0 11.1 14.7 29.1

13.5 12.8 13.8 12.1

11.9 11.1 10.7 11.4

11.3 7.7 19.0 18.5

31.5 24.9 16.4 34.5

Gal­ actose

T h e sugar c o m p o s i t i o n of the pectic polysaccharides o b t a i n e d f r o m these cell w a l l preparations (Table II) also suggested differences i n the s t r u c ­ t u r a l features of the different cell w a l l p r e p a r a t i o n s . D e t a i l e d s t r u c t u r a l i n f o r m a t i o n is o n l y available for the pectic polysaccharides o b t a i n e d f r o m the e n d o s p e r m cell w a l l : T h e m a i n f r a c t i o n of e n d o s p e r m pectic p o l y s a c ­ charide was separated i n t o two fractions, a n e u t r a l s u g a r - r i c h f r a c t i o n a n d a f r a c t i o n w i t h a very h i g h content of D - g a l a c t u r o n i c a c i d (8). S t r u c t u r a l

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

24.

SHIBUYA

Cell Wall Polymersfrom Different Parts of Rice

337

analysis o f the former f r a c t i o n i n d i c a t e d a s t r u c t u r e s i m i l a r t o the so-called r h a m n o g a l a c t u r o n a n - I s t r u c t u r e (9), w h i c h consists of a b a c k b o n e o f 1,4l i n k e d g a l a c t u r o n a n chain i n t e r r u p t e d b y 1,2-linked L - r h a m n o s y l residues a n d side-chains r i c h i n 1,5-linked L - a r a b i n o f u r a n o s y l a n d 1,4-linked D g a l a c t o s y l residues (10). T h e y i e l d o f the pectic p o l y s a c c h a r i d e o f the e n d o s p e r m cell w a l l a n d the g e r m cell w a l l is s i m i l a r , b u t the sugar c o m p o s i t i o n is very different, as s h o w n i n T a b l e II. T h e u r o n i c a c i d content is m u c h higher i n the e n d o s p e r m p r e p a r a t i o n . O n the other h a n d , the a m o u n t of arabinose a n d rhamnose is m u c h higher i n the g e r m p e c t i n . N o t e t h a t these p r e p a r a t i o n s are crude fractions a n d further s t r u c t u r a l studies o n the purified fractions are necessary to reach any definitive conclusions. N e v e r theless, these results suggest possible differences i n their s t r u c t u r a l features, such as a difference i n the r a t i o of the n e u t r a l s u g a r - r i c h d o m a i n s a n d the g a l a c t u r o n a n r i c h d o m a i n s , l e n g t h a n d s t r u c t u r e s of side-chains, etc. M e t h y l a t i o n analysis of whole cell w a l l p r e p a r a t i o n (7) also suggested differences i n the c o m p o s i t i o charides. F o r e x a m p l e , most of the arabinose residues i n the e n d o s p e r m cell w a l l were n o n - r e d u c i n g . O n the other h a n d , a large p a r t o f the a r a b i nose residues i n the g e r m cell w a l l were located i n the inner c h a i n p o r t i o n w i t h various types of linkages. S i m i l a r results were also o b t a i n e d i n the case o f the a l e u r o n cell w a l l . T h e 1,3-linked glucose residues were detected p r a c t i c a l l y o n l y i n the e n d o s p e r m cell w a l l p r e p a r a t i o n . T h e s e results s u g gest a significant difference i n the c o m p o s i t i o n a n d d e t a i l e d s t r u c t u r e o f the c o m p o n e n t polysaccharides of these p r e p a r a t i o n s . Structure of the Hemicellulose Polysaccharides from Endosperm and Bran Cell Wall. A s suggested above, there is a m a j o r difference i n the s t r u c t u r e of h e m i c e l l u l o s i c polysaccharides o b t a i n e d f r o m the e n d o s p e r m a n d f r o m the other parts of the g r a i n . T h e hemicellulose p r e p a r a t i o n s f r o m b o t h the e n d o s p e r m a n d b r a n cell walls were further f r a c t i o n a t e d a n d s u b j e c t e d to s t r u c t u r a l analysis u s i n g c h e m i c a l a n d e n z y m a t i c m e t h o d s . F i g u r e 2 s u m m a r i z e s the s t r u c t u r e of the hemicellulosic polysaccharides o b t a i n e d f r o m the e n d o s p e r m cell w a l l (11,12). O v e r t w o - t h i r d s o f the hemicellulose is c o m p o s e d of a r a b i n o x y l a n s , especially a c i d i c a r a b i n o x y l a n s . T h e y are h i g h l y b r a n c h e d , c a r r y i n g m o s t l y very short side chains o f a single a r a b i n o f u r a n o s y l or ( 4 - 0 - m e t h y l ) - g l u c u r o n o s y l residue. T h e e n d o s p e r m h e m i cellulose also c o n t a i n e d two other polysaccharides as m i n o r c o m p o n e n t s , n a m e l y , x y l o g l u c a n a n d / ? - l , 3 - , l , 4 - g l u c a n . B o t h of these c o m p o n e n t s were firmly associated w i t h a s m a l l a m o u n t of a r a b i n o x y l a n a n d c o u l d not be isolated f r o m each other b y conventional m e t h o d s . T h e s t r u c t u r e s h o w n was d e r i v e d f r o m the analysis of the fragments o b t a i n e d by the e n z y m a t i c d e g r a d a t i o n of t h i s c o m p l e x w i t h the cellulase of T. viride ( x y l o g l u c a n ) , a n d f r o m the a n a l y s i s of the insoluble polysaccharides o b t a i n e d f r o m the controlled S m i t h d e g r a d a t i o n of t h i s f r a c t i o n ( / ? - l , 3 - , l , 4 - g l u c a n ) . F i g u r e 3 shows the s t r u c t u r e of the h e m i c e l l u l o s i c polysaccharides o b t a i n e d f r o m the b r a n cell w a l l p r e p a r a t i o n s . These s t r u c t u r e s were d e d u c e d f r o m the results of the m e t h y l a t i o n a n a l y s i s o f purified fractions (13). In contrast to the e n d o s p e r m cell w a l l , no / ? - l , 3 - , l , 4 - g l u c a n was o b t a i n e d f r o m

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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SHIBUYA

Cell Wall Polymers from Different Parts ofRice

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In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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the b r a n cell w a l l . T h i s result agreed well w i t h the results of the m e t h y l a t i o n analysis of whole cell w a l l preparations i n w h i c h we c o u l d not detect a n y significant amounts of 1,3-linked glucose residues. T h i s is one of the characteristic features of b r a n hemicelluloses. A g a i n , the acidic a r a b i n o x y l a n was the m a i n c o m p o n e n t of t h i s p r e p a r a t i o n representing over 8 0 % b y weight. However, m e t h y l a t i o n analysis of this a r a b i n o x y l a n i n d i c a t e d a difference i n its detailed s t r u c t u r e c o m p a r e d to the endospermic a r a b i n o x y l a n . For e x a m p l e , the b r a n a r a b i n o x y l a n contained a significant a m o u n t of d o u b l y - b r a n c h e d xylose residues, b u t the e n d o s p e r m a r a b i n o x y l a n d i d not. A l s o , the b r a n a r a b i n o x y l a n seemed to c o n t a i n s o m e w h a t longer, a n d m o r e c o m p l i c a t e d , side-chains t h a n the e n d o s p e r m a r a b i n o x y l a n . T h i s was based u p o n m e t h y l a t i o n a n a l y s i s w h i c h showed the presence of appreciable a m o u n t s of inner c h a i n a r a b i n o s y l residues together w i t h t e r m i n a l g a l a c t o s y l a n d x y l o s y l residues of the b r a n p r e p a r a t i o n . S i m i l a r observations were m a d e for the a r a b i n o x y l a n s isolated f r o m the beeswing b r a n o f wheat (14). B r a n hemicellulose also c o n t a i n e associated w i t h the a r a b i n o x y l a n a n d c o u l d not be isolated b y c o n v e n t i o n a l m e t h o d s . T h e x y l o g l u c a n - r i c h f r a c t i o n was finally digested w i t h a purified arabinofuranosidase a n d an endoxylanase to remove c o n t a m i n a t i n g a r a b i n o x y l a n , a n d the r e s u l t i n g pure x y l o g l u c a n was subjected to m e t h y l a t i o n analysis. U n f o r t u n a t e l y , the s t r u c t u r e of the x y l o g l u c a n so o b t a i n e d ( F i g ure 3) was n o t d i r e c t l y c o m p a r a b l e w i t h the s t r u c t u r e of the e n d o s p e r m x y l o g l u c a n . T h i s was because the s t r u c t u r e i n F i g u r e 3 was deduced f r o m m e t h y l a t i o n analysis of the whole molecule, whereas the e n d o s p e r m x y l o g l u c a n s t r u c t u r e was based u p o n analysis of the fragments l i b e r a t e d by cellulase digestion. Detailed Structure of Side Chains of Bran Arabinoxylan. In order to est a b l i s h the c h e m i c a l i d e n t i t y of the side-chains i n the b r a n a r a b i n o x y l a n , i t was subjected to m i l d a c i d h y d r o l y s i s to liberate oligosaccharides o r i g i n a l l y a t t a c h e d to the x y l a n m a i n c h a i n t h r o u g h the a c i d - l a b i l e a r a b i n o f u r a n o s y l residue (15). A m i x t u r e of the oligosaccharides so o b t a i n e d was reduced a n d m e t h y l a t e d to give the corresponding m e t h y l a t e d oligosaccharide a l d i t o l s . A n a l i q u o t of t h i s m e t h y l a t e d oligosaccharide a l d i t o l m i x t u r e was d i r e c t l y a n a l y z e d by G C - M S , a n d the remainder was h y d r o l y z e d , reduced a n d acetyl a t e d . T h e p r o d u c t s were a n a l y z e d by G C - M S . C h r o m a t o g r a m s of the m e t h y l a t e d oligosaccharide a l d i t o l s showed the presence of d i - a n d t r i s a c charides, i n a d d i t i o n to monosaccharides w h i c h were m a i n l y derived f r o m the n o n - r e d u c i n g end of a r a b i n o f u r a n o s y l residues ( F i g u r e 4). T h e s t r u c ture of each oligosaccharide was d e t e r m i n e d by c o m b i n i n g the i n f o r m a t i o n o b t a i n e d f r o m their C I a n d E I mass s p e c t r a , a n d the linkage i n f o r m a t i o n was o b t a i n e d f r o m the analysis of their h y d r o l y z e d p r o d u c t s . In t h i s way, the s t r u c t u r e of the m a i n peak i n the trisaccharide region (peak 9 i n F i g ure 4) was deduced as follows ( F i g u r e 5). F r o m the pseudo-parent i o n of t h i s peak ( Q M + — M e O H , m / z = 556), it is clear t h a t the o r i g i n a l trisaccharide consists of one hexose a n d two pentose u n i t s . A l s o , f r o m the fragment w i t h m / z = 219, 379 a n d 192, the order of their arrangement m u s t be hexose, pentose, pentose. Hence, the reducing end pentose m u s t be s u b s t i t u t e d at

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Cell Wall Polymersfrom Different Parts of Rice

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200

400

600

Scan number F i g u r e 4. T o t a l i o n c h r o m a t o g r a m o f the m e t h y l a t e d m o n o / o l i g o s a c c h a r i d e o b t a i n e d b y the p a r t i a l a c i d h y d r o l y s i s o f the b r a n a r a b i n o g a l a c t o g l u curonoxylan.

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F i g u r e 5. P r o p o s e d s t r u c t u r e of peak 9 i n F i g u r e 4 a n d its f r a g m e n t a t i o n p a t t e r n . D e u t e r i u m at C - l p o s i t i o n was i n t r o d u c e d d u r i n g the r e d u c t i o n step w i t h s o d i u m borodeuteride.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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SHIBUYA

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the 0 - 3 p o s i t i o n , j u d g i n g f r o m the f r a g m e n t a t i o n at the reduced a l d i t o l p o r t i o n ( m / z = 45, 46, 89 a n d 90). T o further i d e n t i f y each sugar, a n d also the mode of linkage, the i n f o r m a t i o n o b t a i n e d f r o m the h y d r o l y z e d p r o d u c t s was used. F i r s t l y , the o n l y hexose w i t h a n o n - r e d u c i n g end i n the m i x t u r e of m e t h y l a t e d oligosaccharide a l d i t o l s was galactose; t h a t is, the first hexose u n i t must be galactose. In a s i m i l a r way, the r e d u c i n g end p e n tose m u s t be arabinose, because the o n l y r e d u c i n g e n d pentose s u b s t i t u t e d at 0 - 3 was arabinose. T h e pentose between these two sugar u n i t s s h o u l d be xylose, because the o n l y inner c h a i n pentose residues detected were 4l i n k e d a n d 2 - l i n k e d xylose. A t t h i s p o i n t i t is difficult to select a n y one o f these two p o s s i b i l i t i e s . However, a n e m p i r i c a l rule r e p o r t e d b y K a r k k a i n e n (16) was used to determine the linkage of t h i s i n n e r - c h a i n xylose residue. In t h i s case, fragments derived f r o m the so-called b c A f r a g m e n t a t i o n ( m / z = 379 a n d 347) was m u c h more extensive t h a n those d e r i v e d f r o m b a A f r a g m e n t a t i o n ( m / z = 352 a n d 320), t h u s suggesting a 1,2- rather t h a n a 1,4-linkage. T h u s , the s t r u c t u r trisaccharide, G a l l , 2 X y l j , l , 3 A r a / . B a s e d u p o n these studies, T a b l e H I s u m m a r i z e s the p a r t i a l l y deduced s t r u c t u r e of the m o n o - , d i - , a n d trisaccharides, o b t a i n e d b y the p a r t i a l d e g r a d a t i o n of the o r i g i n a l a r a b i n o x y l a n . These s t r u c t u r e s m a y not be c o m p l e t e l y a c c u r a t e , because of some u n c e r t a i n t y i n the theory used t o speculate the m o d e of linkage. C o n s e q u e n t l y , each one needs t o be i s o l a t e d to u n a m b i g u o u s l y verify its s t r u c t u r e . These results do, however, p r o v i d e some i n d i c a t i o n of the c o m p l i c a t e d s t r u c t u r e of the side-chains of b r a n arabinoxylan. p

T a b l e I I I . Possible S t r u c t u r e of Oligosaccharides O b t a i n e d by the P a r t i a l A c i d H y d r o l y s i s of R i c e B r a n A r a b i n o g a l a c t o g l u c u r o n o x y l a n Oligosaccharides* 1 2 3 4 5 6 7 8 9 10

Proposed Structure** Pentose ( M a i n l y A r a ) Hexose ( G a l ) Pen-Pen ( X y l - 1 , 3-Ara) Pen-Pen (Xyl-1, 4-Ara) Pen-Pen ( X y l - 1 , 2-Ara) Hex-Pen (Gal-1, 2-Ara) Hex-Pen Hex-Pen Hex-Pen-Pen (Gal-1, 2 - X y l - l , 2-Ara) Pen-Hex-Pen ( X y l - 1 , 3 - G a l - l , 5-Ara)

* See F i g u r e 4. ** P e n = pentose; H e x = hexose.

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Concluding

Remarks

T h e changes associated w i t h t h e t h i c k e n i n g o f the cell walls i n rice g r a i n are s u m m a r i z e d as follows: (1) a decrease o r s h u t d o w n o f the synthesis o f pectic substances a n d / ? - l , 3 - , l , 4 - g l u c a n ; (2) i n i t i a t i o n o f l i g n i n f o r m a t i o n ; a n d (3) synthesis or a c t i v a t i o n o f the g l y c o s y l transferases responsible for t h e e l o n ­ g a t i o n o f the side chains o f t h e a r a b i n o x y l a n . T h u s , t h e results described i n t h i s article give a n e x a m p l e o f how cell w a l l p o l y m e r s change w i t h differ­ e n t i a t i o n o f p l a n t tissues, a l t h o u g h a n u n d e r s t a n d i n g o f t h e p h y s i o l o g i c a l m e a n i n g o f these changes requires further studies. Literature Cited

1. Van, K. T. T.; Toubart, P.; Cousson, Α.; Darvil, A. G.; Gollin, D. J.; Chelf, P.; Albersheim, P. Nature 1985, 314, 615-17. 2. Nojiri, H.; Takaku, F.; Terui, Y.; Miura, Y.; Saito, M. Proc. Natl. Acad. Sci. U.S.A. 1986 3. Albersheim, P.; Darvill 4. Mares, D. J.; Stone, B.A. Aust. J. Biol. Sci. 1973, 26, 793-812. 5. Selvendran, R. R. Dietary Fibre; Birch, G. G.; Parker, K. J., Eds.; Applied Science: London, 1983; Ch. 7. 6. Tanaka, K.; Yoshida, T.; Asada, K.; Kasai, Z. Arch. Biochem. Bio­ phys. 1973, 155, 136-43. 7. Shibuya, N.; Nakane, R.; Yasui, Α.; Tanaka, K.; Iwasaki, T. Cereal Chem. 1985, 62, 252-58. 8. Shibuya, N.: Iwasaki, T. Agric. Biol. Chem. 1978, 42, 2259-66. 9. McNeil, M.; Darvill, A. G.; Fry, S. C.; Albersheim, P. Ann. Rev. Biochem. 1984, 53, 625-63. 10. Shibuya, N.; Nakane, R. Phytochem. 1984, 23, 1425-29. 11. Shibuya, N.; Misaki, A. Agric. Biol. Chem. 1978, 42, 2267-74. 12. Shibuya, N.; Misaki, Α.; Iwasaki, T. Agric. Biol. Chem. 1983, 47, 2223-30. 13. Shibuya, N.; Iwasaki, T. Phytochem. 1985, 24, 285-89. 14. Brillouet, J. M.; Joseleau, J. P. Carbohydr. Res. 1987, 159, 109-26. 15. Shibuya, N., unpublished. 16. Karkkainen, J. Carbohydr. Res. 1971, 17, 11-18. RECEIVED March 17, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 25

Cell Wall Alterations and Antimicrobial Defense in Perennial Plants R. B. Pearce Oxford Forestry Institute, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, England

Cell wall alterations appear particularl anisms in the perennial tissues of plants. Antimicrobial defence in such tissues may entail the durable operation of resistance mechanisms during prolonged host-parasiteinteractions: wall alterations are well suited to this requirement. In the bark of many trees, the suberized phellem forms a constitutive barrier to invasion, and suberization is an early and important part of the wound and defence responses restoring an intact periderm surface. This is exemplified by the response of Sitka spruce to attack by Armillaria obscura. Induced suberization responses also occur in sapwood, conferring enhanced resistance to degradation by decay fungi and contributing to the formation of barriers which limit the spread of pathogens in the living tree. A n t i m i c r o b i a l defence i n plants has been s t u d i e d most extensively i n a n n u a l , herbaceous species, a category i n c l u d i n g the m a j o r i t y o f i m p o r t a n t a g r i c u l t u r a l crop p l a n t s . A range of different disease resistance m e c h a n i s m s have been described for such p l a n t s , i n c l u d i n g b o t h c o n s t i t u t i v e a n d i n d u c e d a n t i m i c r o b i a l c o m p o u n d s a n d cell w a l l a l t e r a t i o n s ( 1 , 2 ) . A s the g l o b a l emphasis i n w o o d p r o d u c t i o n , for t i m b e r , p u l p , fuel a n d c h e m i c a l p r o d u c t s , shifts f r o m the h a r v e s t i n g o f n a t u r a l o r s e m i - n a t u r a l forests t o p r o d u c t i o n i n increasingly intensively m a n a g e d p l a n t a t i o n s , the p a t h o l o g y of these w o o d y perennials is a t t a i n i n g increasing i m p o r t a n c e . T h e a d o p t i o n o f such practices as the creation o f h i g h l y u n i f o r m ( p h y s i c a l l y a n d genetically) m o n o c u l t u r e stands is likely t o increase the p o t e n t i a l for t h e development o f disease epidemics, as c o m m o n l y occur i n a g r i c u l t u r a l crops ( 3 , 4 ) . I n consequence increased a t t e n t i o n has been g i v e n t o disease resistance i n w o o d y species. A l t h o u g h o u r u n d e r s t a n d i n g o f resistance i n 0097-6156/89/0399-0346$06.00/0 © 1989 American Chemical Society

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these w o o d y p e r e n n i a l s s t i l l lags b e h i n d t h a t i n a n n u a l p l a n t s , significant advances have been m a d e d u r i n g the past fifteen years. Sufficient studies have been c a r r i e d o u t t o i n d i c a t e t h a t the m a j o r defence m e c h a n i s m s r e p o r t e d f r o m herbaceous species occur also i n w o o d y p l a n t s . H o w e v e r , i n the m o r e e x t e n s i v e l y developed secondary tissues o f these p l a n t s defence m e c h a n i s m s m a y b e o r g a n i z e d t o f o r m clearly defined barriers ( 5 ) . T h i s chapter w i l l concentrate o n these s t r u c t u r a l b a r r i e r s , w h i c h a p p e a r p a r t i c u l a r l y i m p o r t a n t i n the c o n t a i n m e n t o f pathogens o r p o t e n t i a l pathogens w i t h i n these secondary tissues. Requirements for Defence i n Perennial Plant Tissues In a n n u a l plants t h e d u r a t i o n o f the h o s t - p a t h o g e n i n t e r a c t i o n is genera l l y r e l a t i v e l y brief; i n p e r e n n i a l p l a n t s , however, these i n t e r a c t i o n s m a y continue for g r e a t l y e x t e n d e d periods i n the l o n g - l i v e d secondary tissues. S u c h l o n g - t e r m infection pathogens, i n c l u d i n g m a n y o f the i m p o r t a n t root- a n d b u t t - r o t t i n g forest pathogens s u c h as Armillaria s p p . a n d Heierobasidion annosum. These p e r e n n i a l pathogens colonize ( a n d frequently k i l l ) a v o l u m e o f host tissue, w h i c h provides a f o o d base t h a t c a n s u s t a i n the fungus d u r i n g a p r o l o n g e d i n t e r a c t i o n w i t h the l i v i n g tissues o f the host. I n the case o f Armillaria spp. n u t r i e n t s m o b i l i z e d f r o m one infected host m a y be t r a n s l o c a t e d a l o n g r h i z o m o r p h s , s u p p o r t i n g their g r o w t h a n d the i n f e c t i o n process where they e n counter a new p o t e n t i a l host (6). I n b o t h p e r e n n i a l cankers a n d p a t h o g e n i c w o o d decays there is a n i n t e r n a l interface between the l i v i n g host a n d the p a t h o g e n at the lesion m a r g i n . T h e advance o f the p a t h o g e n at these i n terfaces is t y p i c a l l y very slow. Defences effective i n l i m i t i n g pathogen advance under these c i r c u m stances must be capable o f m a i n t a i n i n g their f u n c t i o n for a n e x t e n d e d t i m e . A l t h o u g h t h i s r e q u i r e m e n t for d u r a b i l i t y does n o t p r e c l u d e the i n v o l v e m e n t of c h e m i c a l defences, s t r u c t u r a l defence m e c h a n i s m s are p a r t i c u l a r l y well s u i t e d t o the p r o t e c t i o n o f the host under these c i r c u m s t a n c e s . W h i l s t a n t i m i c r o b i a l chemicals m a y be l a b i l e or diffusible, s t r u c t u r a l defences, c o m p r i s i n g either w a l l a l t e r a t i o n s t o p r e - e x i s t i n g cells or tissues f o r m e d de novo b y renewed cell d i v i s i o n , are m u c h more stable. S u c h barriers c a n protect the u n d e r l y i n g tissues indefinitely against m i c r o o r g a n i s m s l a c k i n g a specific a b i l i t y t o penetrate or c i r c u m v e n t t h e m . S t r u c t u r a l defences, p a r t i c u l a r l y those r e q u i r i n g the p r o d u c t i o n o f new tissues b y the p l a n t m a y take some t i m e t o f o r m ( 5 , 7 - 9 ) . A n t i m i c r o b i a l c o m p o u n d s m a y be i m p o r t a n t d u r i n g the i n i t i a l stages o f the host-parasite i n t e r a c t i o n , before b a r r i e r development has o c c u r r e d , or m a y occur c o n c o m i t a n t l y w i t h cell w a l l a l t e r a t i o n s . I n S i t k a spruce (Picea sitchensis) the a n t i f u n g a l stilbenes a s t r i n g e n i n a n d i s o rhapontigenin accumulate around bark wounds inoculated w i t h potentially p a t h o g e n i c f u n g i . These are released o n i n f e c t i o n f r o m the c o r r e s p o n d i n g stilbene glucosides a s t r i n g i n a n d r h a p o n t i c i n w h i c h are c o n s t i t u t i v e i n t h e b a r k tissues, a n d provide a r a p i d response t o infection t h a t m a y i n h i b i t p o t e n t i a l pathogens u n t i l more d u r a b l e s t r u c t u r a l barriers have been f o r m e d (10).

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Structural Defence Mechanisms in Plants S t r u c t u r e s interprétable as h a v i n g a defensive role m a y be either n o r m a l features of the p l a n t , or t h e i r f o r m a t i o n m a y be i n d u c e d as a result of m i c r o b i a l challenge or w o u n d i n g . I n the case of i n d u c e d s t r u c t u r a l defences the responses m a y comprise either changes i n p r e - e x i s t i n g cells, or m a y result i n the f o r m a t i o n of specific b a r r i e r tissues de novo b y renewed or m o d i f i e d cell d i v i s i o n . O c c a s i o n a l l y n o n - c e l l u l a r s t r u c t u r a l defences m a y operate. T h e resin flow t h a t accompanies w o u n d i n g or i n f e c t i o n i n m a n y coniferous trees m a y act as a m e c h a n i c a l b a r r i e r , h a r d e n i n g to f o r m a n a m o r p h o u s v a r n i s h i m p e n e t r a b l e to f u n g a l i n v a s i o n (11). M o r e c o m m o n l y , however, s t r u c t u r a l defences are effected b y cell w a l l a l t e r a t i o n s . T h e s e i n c l u d e l i g n i f i c a t i o n (12-14), s u b e r i z a t i o n ( 1 4 , 1 5 ) , callose d e p o s i t i o n (16), and silicification (17,18). S u c h w a l l a l t e r a t i o n s m a y operate i n several ways. P r o b a b l y the most o b v i o u s a n d w i d e l y suggeste p a t h o g e n . F o r a b a r r i e r to prevent the advance of a p a t h o g e n , i t m u s t be m o r e resistant to the cell w a l l - d e g r a d i n g enzymes of the pathogen t h a n u n m o d i f i e d walls. S u c h enhanced resistance to d e g r a d a t i o n has been d e m o n s t r a t e d b o t h for lignified p a p i l l a e a n d halos i n wheat leaf e p i d e r m a l cells (19) a n d for s u b e r i z e d barriers i n oak x y l e m (15). A l t e r n a t i v e l y , s t r u c t u r a l responses of a p l a n t c o u l d e s t a b l i s h a p e r m e a b i l i t y b a r r i e r , i s o l a t i n g the p a t h o g e n f r o m the l i v i n g tissues of its host. S u c h a p e r m e a b i l i t y b a r r i e r c o u l d protect h e a l t h y cells f r o m f u n g a l t o x i n s or enzymes, i t c o u l d reduce the rate o f diffusion of host a n t i f u n g a l c o m p o u n d s f r o m the infection c o u r t , p e r h a p s e n h a n c i n g the efficacy of these c h e m i c a l defences, or i t c o u l d reduce the flow of n u t r i e n t s or water to the p a t h o g e n (20). A test for tissue p e r m e a b i l i t y to ions, u s i n g ferric chloride a n d p o t a s s i u m f e r r i c y a n i d e , t e r m e d the F - F test (21), has d e m o n s t r a t e d the i m p e r m e a b i l i t y of s u b e r i z e d b a r rier tissues at lesion m a r g i n s i n the b a r k o f various w o o d y species ( 7 , 1 4 ) , a l t h o u g h i n i t i a l l y the s u b e r i z a t i o n of these i m p e r v i o u s tissues was not recognized (7). I n a d d i t i o n , the processes l e a d i n g to the f o r m a t i o n of s t r u c t u r a l barriers m a y have a direct effect o n the pathogen itself, either as a result o f c h e m i c a l i n h i b i t i o n by n o n - p o l y m e r i z e d monomers of the w a l l - m o d i f y i n g m a t e r i a l , or b y d e p o s i t i o n of t h i s m a t e r i a l o n t o the w a l l of the p a t h o g e n , w i t h consequent interference w i t h i t s g r o w t h (22). L i g n i f i c a t i o n responses have been i m p l i c a t e d i n disease resistance i n p e r e n n i a l p l a n t s . I n the leaves o f oak species a lignified p a p i l l a response appears to be i m p o r t a n t i n the resistance of older leaves to oak m i l d e w , Microsphaera alphitoides (23). Leaves of a deciduous tree are, however, e p h e m e r a l organs, c o m p a r a b l e to those o f herbaceous species. A s discussed i n t h i s p a p e r , s u b e r i z a t i o n of cell walls (sometimes a c c o m p a n i e d b y l i g n i f i c a t i o n also) is perhaps more t y p i c a l of the barriers f o u n d i n the secondary tissues of w o o d y ( 8 , 1 4 ) a n d herbaceous (24) p l a n t s . S u b e r i n , a l t h o u g h s t i l l i m p e r f e c t l y c h a r a c t e r i z e d , is p r e d o m i n a n t l y a polyester c o m p o s e d of l o n g c h a i n ( C i s — C 3 0 ) h y d r o x y - a n d h y d r o x y e p o x y - f a t t y acids. L i g n i n - l i k e a r o m a t i c d o m a i n s m a y also be associated w i t h t h i s p o l y m e r (25). It therefore differs m a r k e d l y f r o m the a r o m a t i c a n d c a r b o h y d r a t e p o l y m e r s t h a t c o m -

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prise the b u l k of the secondary p l a n t tissues. A l s o , because of its h y d r o p h o ­ bic properties i t is h i g h l y effective as a p e r m e a b i l i t y b a r r i e r to w a t e r . A s water content has been i m p l i c a t e d i n the e n v i r o n m e n t a l e x c l u s i o n of m a n y w o o d - i n h a b i t i n g f u n g i f r o m the l i v i n g secondary x y l e m (sapwood) of trees (26-28), t h i s m a y be p a r t i c u l a r l y significant i n the p r o t e c t i o n of these t i s ­ sues. W h i l e s u b e r i n c a n be degraded b y some f u n g i , i n c l u d i n g Armillaria sp. (29) a n d Rosellinia desmazieresii (30), the rates of b r e a k d o w n were not high. C o n s t i t u t i v e S t r u c t u r a l Defences A l t h o u g h m a n y s t r u c t u r a l features of a p l a n t , such as f o r m a n d a r r a n g e m e n t of s t o m a t a , can be i n t e r p r e t e d i n terms of defence (31), o n l y the secondary surface w i l l be considered i n d e t a i l here. T h i s comprises a p e r i d e r m , or series of sequent p e r i d e r m s f o r m i n g a r h y t i d o m e . P e r i d e r m s are sites of c a m b i a l a c t i v i t y , suberize phellogen. T h e thickness of p h e l l e m a c c u m u l a t i n g varies g r e a t l y between species. Few pathogens are capable of p e n e t r a t i n g the i n t a c t p e r i d e r m surface of w o o d y p l a n t s . M o s t , i f not a l l , of the f u n g i t h a t infect b y t h i s route (rather t h a n b y b y p a s s i n g the surface b a r r i e r , i n f e c t i n g via w o u n d s a n d n a t u r a l d i s c o n t i n u i t i e s i n the p e r i d e r m ) do so f r o m established infections o n a n e i g h b o r i n g f o o d base, p e r m i t t i n g a prolonged a t t a c k . C o m m o n l y (e.g., w i t h Armillaria spp., Rosellinia spp.) p e n e t r a t i o n is effected b y m y c e l i a l aggregations, s u p p o r t e d m e t a b o l i c a l l y b y m y c e l i a l cords or r h i z o m o r p h s ( 3 0 , 3 2 , 3 3 ) . W i t h o u t t h i s c a p a c i t y to m o u n t a prolonged a t t a c k , i n w h i c h b o t h m e c h a n i c a l a n d e n z y m i c a c t i v i t y m a y be i n v o l v e d , p o t e n t i a l pathogens appear u n a b l e to overcome the p e r i d e r m b a r r i e r . P e r h a p s because of its u b i q u i t y , there have been few detailed studies of the role of surface p e r i d ­ erms i n defence: c o m m o n l y the p r o t e c t i o n p r o v i d e d is assumed (e.g., 34), a n d is a t t r i b u t e d to the resistance of the p h e l l e m cells t o d e g r a d a t i o n . P e r i ­ d e r m (or r h y t i d o m e ) surfaces of some tree species persist w i t h o u t erosion for l o n g periods, surface features r e m a i n i n g v i s i b l e for several decades at least (35). T h e suberized p h e l l e m cells c o m p r i s i n g a n i m p o r t a n t c o m p o n e n t of these tissues are n o n - l i v i n g a n d i n c a p a b l e of any active r e p a i r , b u t are clearly more d u r a b l e t h a n other p l a n t tissues. In c e r t a i n instances, however, factors other t h a n the cell w a l l p o l y ­ mers of the p h e l l e m m a y be i m p o r t a n t i n the p r o t e c t i o n p r o v i d e d by the secondary surface. Rosellinia desmazieresii i n o c u l a t e d i n a food base o n t o the u n d e r g r o u n d stems of a resistant Salix repens h y b r i d ( 5 . χ Friesiana) e x h i b i t e d g r e a t l y reduced e p i p h y t i c g r o w t h a n d cord f o r m a t i o n c o m p a r e d w i t h i n o c u l a t i o n s o n t o susceptible S. repens itself. A t t e m p t e d p e n e t r a t i o n was not observed o n the resistant h y b r i d (30). T h i s b e h a v i o u r suggests t h a t diffusible c h e m i c a l i n h i b i t o r s at the s t e m surface m a y be i m p o r t a n t i n resistance to t h i s p a t h o g e n , w h i c h has a d e m o n s t r a t e d a b i l i t y to degrade s u b e r i n a n d penetrate the surface p e r i d e r m (30). A l t h o u g h i n a n u n w o u n d e d , healthy, p l a n t p e r i d e r m s are n o r m a l l y s u ­ p e r f i c i a l , i n t e r x y l a r y cork has been r e p o r t e d f r o m at least 40 species of d i -

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c o t y l e d o n o u s plants (36). These i n t e r x y l a r y p e r i d e r m s are associated w i t h the fission of p e r e n n a t i n g axes a n d the d y i n g back of a n n u a l organs such as a e r i a l shoots. I n Epilabium angustifolium an i n t e r x y l a r y p e r i d e r m is f o r m e d each year over the surface of the x y l e m connected w i t h the previous year's aerial shoots. T h e older, r e d u n d a n t , vascular tissue is thus w a l l e d off f r o m the f u n c t i o n a l tissues of the p l a n t , w h i c h are then protected b y b o t h inner a n d outer suberized barriers. It has been suggested t h a t such i n t e r n a l p e r i d e r m s m a y be i m p o r t a n t i n affording p r o t e c t i o n against the ingress of m i c r o o r g a n i s m s associated w i t h the d y i n g d o w n of the a n n u a l a e r i a l shoots (37), a f u n c t i o n i n accordance w i t h the h a b i t of m a n y of the species f r o m w h i c h they have been recorded. Defence in B a r k

Tissues

S h o u l d the b a r r i e r presented b y the secondary p l a n t surface be breached, defence m e c h a n i s m s m a y p h l o e m ) of w o o d y p l a n t s , l i m i t i n g pathogen development. These i n c l u d e c h e m i c a l defences a n d s t r u c t u r a l barriers r e s u l t i n g f r o m cell w a l l a l t e r a t i o n s (5). B o t h p r o b a b l y act i n concert, a l t h o u g h i n a n o n - w o o d y p l a n t (carrot) it has been concluded t h a t the i m p o r t a n c e of cell w a l l a l t e r a t i o n s was seco n d a r y to t h a t of c h e m i c a l defence (24). O n l y the cell w a l l a l t e r a t i o n s i n v o l v e d i n defence w i l l be considered further here. Cell Wall Alterations in the Bark of Gymnosperms. Non-specific defence responses to w o u n d i n g , insect a n d f u n g a l a t t a c k i n the b a r k of conifers, l e a d i n g to a r e s t o r a t i o n of the p e r i d e r m surface, have been described. In essence, n e c r o p h y l a c t i c ( w o u n d ) p e r i d e r m s are formed continuous w i t h the n o r m a l s t r u c t u r a l surface p e r i d e r m , w a l l i n g off the lesion a n d effectively e x c l u d i n g it f r o m the p l a n t (7). P r i o r to the development of this p e r i d e r m b y dedifferentiation a n d renewed d i v i s i o n of cells i n the bark s u r r o u n d i n g the lesion, cell w a l l alterations take place i n p r e - e x i s t i n g tissues b o r d e r i n g the lesion. P r o m i n e n t a m o n g these a l t e r a t i o n s is the f o r m a t i o n of a n i m p e r v i o u s zone i m m e d i a t e l y o v e r l y i n g the site of p e r i d e r m r e s t o r a t i o n . T h i s was i n i t i a l l y described as non-suberized i m p e r v i o u s tissue ( N I T ) (7). I n a m o r e recent s t u d y of defence responses f o l l o w i n g w o u n d i n g a n d a r t i f i c i a l i n o c u l a t i o n of Picea sitchensis root b a r k w i t h the weakly pathogenic b u t t rot fungus Phaeolus schweinitzii, use of i m p r o v e d h i s t o c h e m i c a l techniques d e m o n s t r a t e d t h a t s u b e r i z a t i o n of cell walls occurred i n these tissues (9). C e l l walls i n the necrotic tissue of these wounds were b r o w n e d . S t a i n i n g w i t h d i a z o t i z e d o-tolidine a n d t o l u i d i n e blue confirmed the p o l y p h e n o ls n a t u r e of these b r o w n depositions, w h i c h m a y have resulted f r o m the p o l y m e r i z a t i o n of the stilbenes present i n large quantities i n spruce b a r k . P h e n o l i c residues were deposited o n the walls of c e r t a i n cells i n t e r n a l to the necrotic tissues b y 10 days after w o u n d i n g . B y 36 days these cells h a d become t h i c k - w a l l e d . T h e precise nature of substances responsible for this t h i c k e n i n g has not been d e t e r m i n e d , v a r i a b l e responses b e i n g o b t a i n e d w i t h h i s t o c h e m i c a l tests for l i g n i n (cf. T a b l e I). S u b e r i n was detectable i n cells i m m e d i a t e l y u n d e r l y i n g the t h i c k walled cells, w h i c h corresponded to the

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( " n o n - " ) suberized i m p e r v i o u s tissue, a n d i n the t h i n w a l l e d p h e l l e m cells f o r m e d later b y the developing n e c r o p h y l a c t i c p e r i d e r m (9). These b a r k responses are i l l u s t r a t e d d i a g r a m m a t i c a l l y i n F i g u r e 1. T a b l e I. H i s t o c h e m i c a l responses of cell walls associated w i t h the necrop h y l a c t i c p e r i d e r m response i n Picea sitchensis challenged w i t h Armillaria obscura Wall Polymer

Stain

R e a c t i o n i n Tissues

a

Healthy Bark Parenchyma Pectic Materials

Ruthenium red

Cellulose

Zinc-chloriodine Toluidine blue

Callose

Resorcinol blue

Necrophylactic Phellem

+

Thick Walled Tissue

Necrotic Tissues

±

+

+

—

—

—

+

-

-

-

±

—

—

—

—

—

+

+

—

±

+

+

— —

+ —

+ —

— —

-

+

-

-

±

(locally) Lignin/ Wall-bound phenolics

Suberin a

Zinc-chloriodine Toluidine blue Phloroglucinol-HCl M a u l e test Lignin pink Sudan I V

-

S p e c i m e n p r e p a r a t i o n a n d s t a i n i n g methods as described p r e v i o u s l y (15).

E s s e n t i a l l y i d e n t i c a l processes occur i n u n wounded spruce b a r k , c h a l lenged by, a n d resisting successfully, at least i n the short t e r m , the root- a n d b u t t - r o t pathogen Armillaria obscura (Secretan) H e r i n k . A t r h i z o m o r p h contact a n d a t t e m p t e d i n f e c t i o n sites o n the b a r k of buttress roots of c 60-year-old trees of Picea sitchensis (Bong.) C a r r . , p e n e t r a t i o n of the s u r face p e r i d e r m h a d o c c u r r e d . S m a l l (c 10 m m . diameter) necrotic regions resulted, confined to superficial b a r k tissues only. M e c h a n i c a l a t t a c h m e n t of these lesions ( w h i c h h a d been i n i t i a t e d some t i m e p r e v i o u s l y so t h a t host responses to the challenge were essentially complete) was weak, a n d they were r e a d i l y detached f r o m the roots as b a r k scales. Cleavage o c c u r r e d i n the plane of a n e c r o p h y l a c t i c p e r i d e r m , f o r m e d at the m a r g i n between tissue colonized b y A. obscura a n d the h e a l t h y bark ( F i g u r e 2). C e l l w a l l

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F i g u r e 1. S t r u c t u r a l responses of the b a r k of Picea sitchensis to w o u n d i n g a n d i n o c u l a t i o n w i t h Phaeolus schweinitzii. I W , inoculated wound; S P , surface p e r i d e r m ; N T , necrotic tissue; T C , thickened cells; S I T , relic of suberized i m p e r v i o u s tissue; N P , n e c r o p h y l a c t i c p e r i d e r m ; P , p h l o e m ; V C , vascular c a m b i u m .

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a l t e r a t i o n s , i d e n t i c a l to those associated w i t h a r t i f i c i a l wounds (9) were detectable i n a n d a r o u n d the lesion u s i n g a range o f h i s t o c h e m i c a l tests ( T a b l e I). T h e u n d e r l y i n g h e a l t h y b a r k was separated f r o m the f u n g a l l y c o l o n i z e d tissues b y a n e c r o p h y l a c t i c p e r i d e r m , essentially s i m i l a r to the n o r m a l root surface p e r i d e r m . T h i s c o m p r i s e d layers of t h i n w a l l e d , s u b e r i z e d p h e l l e m cells a n d t h i c k w a l l e d cells s t a i n i n g p o s i t i v e l y w i t h the p h l o r o g l u c i n o l H C 1 reagent. O u t s i d e the outermost p h e l l e m cells there existed a single layer o f i r r e g u l a r s u b e r i z e d cells, c o r r e s p o n d i n g to the cells f o r m i n g the suberized i m p e r v i o u s tissue p r i o r to phellogen f o r m a t i o n . T h e t h i c k e n e d , p h l o r o g l u c i n o l - p o s i t i v e cells between these a n d the necrotic tissue were not s u b e r i z e d . F u n g a l h y p h a e were v i s i b l e i n these cells ( F i g u r e 3), i n d i c a t i n g t h a t t h i s w a l l t h i c k e n i n g d i d not itself pose a m a j o r i m p e d i m e n t to the g r o w t h of the fungus, a n d were present also i n the necrotic tissues, f r o m w h i c h evidence of w a l l - b o u n d phenolic c o m p o u n d s was also o b t a i n e d . N o h y p h a e were seen, however were c r u c i a l to the effectiveness of t h i s b a r r i e r response. Cell Wall Alterations in the Bark of Woody Angiosperms. Responses to w o u n d i n g a n d i n f e c t i o n closely s i m i l a r to those f o u n d i n G y m n o s p e r m s occur also i n the b a r k of A n g i o s p e r m s . These responses have been described i n d e t a i l f r o m a n u m b e r o f w o o d y species ( 8 , 1 4 , 3 8 , 3 9 ) a n d a p p e a r essentially s i m i l a r i n a l l . F o r m a t i o n o f a suberized i m p e r v i o u s tissue i n u n d a m a g e d tissues b e n e a t h the lesion is followed by the r e s t o r a t i o n of the p l a n t surface b y development of a n e c r o p h y l a c t i c p e r i d e r m . W h e r e a r a p i d l y s p r e a d i n g lesion results f r o m p o p l a r infections by Cytospora chrysosperma, there m a y be c o l o n i z a t i o n of the host b a r k tissues w i t h o u t expression of these defensive responses. However, w h e n s l o w l y e x t e n d i n g p e r e n n i a l cankers are f o r m e d b y t h i s p a t h o g e n a n e c r o p h y l a c t i c p e r i d e r m is p r o d u c e d b y the p l a n t , a n d advance of the fungus appears to take place o n l y where there are d i s c o n t i nuities i n t h i s b a r r i e r , e.g., where i t is traversed b y bundles of p h l o e m fibres (38). Responses in Non-woody Perennial Plants. A l t h o u g h pathogens capable of m o u n t i n g a p r o l o n g e d a t t a c k f r o m a colonized food base are less u s u a l o n n o n - w o o d y species, such a t t a c k s c a n sometimes o c c u r . In these instances p e r i d e r m r e s t o r a t i o n responses c o m p a r a b l e to those described f r o m w o o d y plants m a y operate to restrict pathogen ingress. T h i s m a y be exemplified b y the response of h o r s e r a d i s h , Armoracia rusticana G a e r t i n , M a y & Scherb. to a t t a c k b y a n Armillaria species ( p r o b a b l y A. bulbosa ( B a r l a ) , K i l e a n d W a t l i n g ) . A l t h o u g h not a n i m p o r t a n t p a t h o g e n of t h i s p l a n t , Armillaria r h i z o m o r p h s m a y a t t e m p t t o penetrate a n d colonize its large, fleshy roots. Sections of infected roots show colonized, necrotic tissues b o u n d e d b y suberized p h e l l e m cells. P e n e t r a t i o n of these n e c r o p h y l a c t i c p e r i d e r m s was effected b y r h i z o m o r p h - l i k e aggregations of h y p h a e ( F i g u r e 4). C e l l s i n the necrotic zone s t a i n e d green w i t h t o l u i d i n e blue a n d gave a p o s i t i v e response w i t h the p h l o r o g l u c i n o l - H C l reagent, b u t d i d not color w i t h the M a u l e test, suggesting the presence o f c e l l - w a l l b o u n d phenolics i n these tissues, a l -

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F i g u r e 2. N e c r o p h y l a c t i c p e r i d e r m i n the b a r k of Picea sitchensis f o l l o w i n g a t t e m p t e d infection b y Armillaria obscura. B a r k sectioned a n d s t a i n e d w i t h S u d a n I V , after e x t r a c t i o n w i t h chlorine dioxide to remove a r o m a t i c c o m ­ p o u n d s , essentially as p r e v i o u s l y described (15). N T , necrotic tissue; T C , thickened cells; S I T , relic of suberized i m p e r v i o u s tissue; N P , n e c r o p h y l a c t i c p e r i d e r m . Scale bar = 25 μπι.

F i g u r e 3. H y p h a e (arrowed) of Armillaria obscura i n t h i c k walled cells ( T C ) o v e r l y i n g the n e c r o p h y l a c t i c p e r i d e r m ( N P ) i n Picea sitchensis. B a r k sec­ t i o n e d a n d s t a i n e d w i t h t o l u i d i n e blue as p r e v i o u s l y described (15). Scale b a r = 25 μηα.

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t h o u g h l i g n i n f o r m a t i o n itself m a y n o t have o c c u r r e d . H y p h a e were present i n these necrotic tissues b u t none were seen p e n e t r a t i n g the s u b e r i z e d cells. A l t h o u g h the role o f c h e m i c a l defences i n t h i s i n t e r a c t i o n was n o t e x a m i n e d (cf. 24), i t w o u l d appear t h a t the suberized p e r i d e r m barriers p r o d u c e d i n response to i n f e c t i o n posed a more effective defence against Armillaria t h a n the w a l l - b o u n d phenolics o f the necrotic tissues. T h e p e r i d e r m s were, however, repeatedly breached b y the aggregated m y c e l i u m o f t h e fungus, r e s u l t i n g i n a c o m p l e x lesion c o n t a i n i n g m a n y defeated p e r i d e r m b a r r i e r s e x t e n d i n g i n t o the root x y l e m . Defence i n L i v i n g W o o d T h e s a p w o o d o f a l i v i n g tree, c o m p r i s i n g the x y l e m tissues f u n c t i o n a l i n t r a n s p o r t a n d storage, contains l i v i n g cells a n d is capable of active responses to i n f e c t i o n . I n contrast the h e a r t w o o d ( i n those species t h a t f o r m i t ) is non-living a n d incapable o sponses are thus impossible i n h e a r t w o o d , a l t h o u g h a n t i f u n g a l c o m p o u n d s or cell w a l l a l t e r a t i o n s l a i d d o w n when the w o o d was s t i l l l i v i n g m a y c o n t i n u e t o f u n c t i o n . Indeed there are s t r i k i n g s i m i l a r i t i e s between m a n y o f the defences i n d u c e d i n s a p w o o d b y f u n g a l a t t a c k a n d the changes o c c u r r i n g d u r i n g h e a r t w o o d f o r m a t i o n , suggesting t h a t these processes m a y be related (5). B o t h a n t i m i c r o b i a l c h e m i c a l defences (40) a n d cell w a l l a l t e r a t i o n s have been r e p o r t e d f r o m x y l e m of w o o d y p l a n t s . A s w i t h defences i n t h e b a r k , o n l y cell w a l l a l t e r a t i o n s w i l l be discussed. T w o types o f b a r rier c a n be i d e n t i f i e d — t h o s e c o m p r i s i n g specialized tissues f o r m e d de novo i n response to w o u n d i n g a n d i n f e c t i o n , a n d those f o r m e d i n p r e - e x i s t i n g xylem. Tissue Barriers Formed de novo. M a n y w o o d y perennials r e s p o n d to w o u n d s r e s u l t i n g i n c a m b i a l damage, a n d c o n c o m i t a n t f u n g a l infection o f u n d e r l y i n g x y l e m , b y the p r o d u c t i o n of a d i s t i n c t i v e b a r r i e r tissue. T h i s b a r r i e r , w h i c h has been termed the c o m p a r t m e n t a l i z a t i o n w a l l 4 (41), c o m prises a sheet o f t r a u m a t i c a x i a l p a r e n c h y m a cells, formed b y the c a m b i u m i n t h e v i c i n i t y o f t h e w o u n d , a n d l a i d d o w n as the first x y l e m tissue p r o duced after w o u n d i n g ( 1 5 , 4 2 ) . I n conifers t h i s p a r e n c h y m a is c o m m o n l y a c c o m p a n i e d by resin ducts (43). I n most, b u t n o t a l l , species so far e x a m i n e d t h e p a r e n c h y m a cells o f the w a l l 4 b a r r i e r zone are s u b e r i z e d , at least where they overlie fungally colonized w o o d (5, Pearce, R . B . , u n p u b l i s h e d d a t a ) . E v i d e n c e has been o b t a i n e d f r o m oak (Quercus robur) t o suggest t h a t the s u b e r i z a t i o n response o f these tissues, b u t n o t necessarily their f o r m a t i o n b y the c a m b i u m , is f u n g a l l y elicited (5). S u b e r i z a t i o n o f these cells renders t h e m resistant t o d e g r a d a t i o n b y w o o d d e c a y i n g f u n g i . Sections t h r o u g h the c o m p a r t m e n t a l i z a t i o n w a l l 4 b a r r i e r region i n Q. robur, i n c u b a t e d for several weeks w i t h the w o o d decay p a t h o g e n Stereum gausapatum, were extensively degraded b y the fungus, except for the suberized cells w h i c h r e m a i n e d essentially i n t a c t (15). S u b e r ized x y l e m cells are clearly able t o resist d e g r a d a t i o n b y w o o d decay f u n g i for prolonged periods: the relics o f suberized cells a n d tyloses, f o r m e d as a

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n o r m a l h e a r t w o o d c o m p o n e n t i n Q. robur (44), r e m a i n e d i n w o o d otherwise more or less t o t a l l y decayed b y n a t u r a l infection w i t h the w h i t e - r o t fungus Ganoderma adspersum ( F i g u r e 5). I n some species, however, e.g. a s h , Fraxinus excelsior, cells of the t r a u m a t i c a x i a l p a r e n c h y m a of the c o m p a r t m e n t a l i z a t i o n w a l l 4 m a y show n o evidence of cell w a l l a l t e r a t i o n s , yet appear to act n o r m a l l y as a f u n c t i o n a l b a r r i e r t o decay (Pearce, R . B . , u n p u b l i s h e d d a t a ) . It is t o be p r e s u m e d t h a t the spread of decay f u n g i is arrested either b y c h e m i c a l defences or by e n v i r o n m e n t a l c o n s t r a i n t s (cf. 26-28) i n such species. C l e a r l y , a c o n t r i b u t i o n m a y be m a d e b y these defences i n s u b e r i z i n g species also: p h y t o a l e x i n - l i k e a n t i f u n g a l c o m p o u n d s have been detected i n association w i t h a s u b e r i z e d w a l l 4 b a r r i e r i n Acer saccharinum (42). M o r e work w i l l be required to e l u cidate the l o n g - t e r m effectiveness of the various m e c h a n i s m s m a i n t a i n i n g the f u n c t i o n of these b a r r i e r w a l l s . Cell Wall Alterations in Pre-existing interface w i t h p r e - e x i s t i n g s a p w o o d , responses of the l i v i n g p a r e n c h y m a cells can result i n the f o r m a t i o n of a r e a c t i o n zone (45-47). R e a c t i o n zones present a less w e l l defined b a r r i e r t o i n f e c t i o n t h a n the c o m p a r t m e n t a l i z a t i o n w a l l 4 a n d have been envisaged as a d y n a m i c response, h e a l t h y s a p w o o d being converted to reaction zone tissue ahead of the a d v a n c i n g fungus ( 4 5 , 4 7 ) . E v i d e n c e has recently been o b t a i n e d t h a t t h i s is not a c o n tinuous process, b u t t h a t reaction zones act as s t a t i c b o u n d a r i e s t o decay, w h i c h advance d i s c o n t i n u o u s l y w i t h i n the tree, periods of r a p i d f u n g a l a d vance e l i c i t i n g l i t t l e or no host response b e i n g followed by periods of stasis, a c c o m p a n i e d b y the f o r m a t i o n of clearly defined reaction zones (5, Pearce, R . B . , unpublished data). T h e a c c u m u l a t i o n of p h y t o a l e x i n - l i k e a n t i f u n g a l c o m p o u n d s c o m m o n l y occurs i n r e a c t i o n zone tissues ( 4 0 , 4 2 ) , a n d b r o w n deposits, g i v i n g s t a i n i n g responses p o s i t i v e for phenolic c o m p o u n d s , are frequently present i n cells i n t h i s region. Vessels m a y be o c c l u d e d b y these deposits (gummosis) or b y the f o r m a t i o n of tyloses, b a l l o o n - l i k e o u t g r o w t h s f r o m the walls of vessel p a r e n c h y m a cells (5). C e l l w a l l s u b e r i z a t i o n responses c o m m o n l y o c c u r i n r e a c t i o n zones ( 5 , 4 8 ) , a l t h o u g h they are not always present, even i n species h a v i n g a d e m o n s t r a b l e s u b e r i z a t i o n response i n the w a l l 4 b a r r i e r , e.g. Acer saccharinum (42). Tyloses are u s u a l l y suberized ( 4 4 , 4 9 , Pearce, R . B . , u n p u b l i s h e d d a t a ) . In those species i n w h i c h a b u n d a n t tylosis form a t i o n o c c u r r e d i n r e a c t i o n zones, s u b e r i z a t i o n of x y l e m p a r e n c h y m a cells, i n c l u d i n g the r a y p a r e n c h y m a , was n o r m a l l y observed. In species i n w h i c h vessel o c c l u s i o n was p r e d o m i n a n t l y b y g u m m o s i s , s u b e r i z a t i o n responses were often l a c k i n g (Pearce, R . B . , u n p u b l i s h e d d a t a ) . U n l i k e the c o m p a r t m e n t a l i z a t i o n w a l l 4, the s u b e r i z e d tissues i n rea c t i o n zones do not f o r m a continuous b a r r i e r , p o t e n t i a l l y i m p r e g n a b l e b y pathogens i n c a p a b l e of d e g r a d i n g suberized walls. However, suberized t y loses a n d p a r e n c h y m a cell walls block the easiest routes of f u n g a l spread i n the x y l e m — a x i a l l y a l o n g vessels a n d r a d i a l l y i n the rays ( 5 0 ) — a n d m a y thus g r e a t l y h i n d e r f u n g a l spread. U n d o u b t e d l y these cell w a l l a l t e r a t i o n s n o r m a l l y act i n concert w i t h other defences—antifungal c o m p o u n d s a n d / o r

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F i g u r e 4. P e n e t r a t i o n of n e c r o p h y l a c t i c p e r i d e r m s ( N P ) i n the root of Armoracia rusticana b y r h i z o m o r p h - l i k e aggregations of h y p h a e ( R ) . Scale bar - 100 μτη.

F i g u r e 5. H e a r t w o o d of Qutrcus robur w i t h advanced decay caused by Ganoderma adspersum. A l t h o u g h the wood has been extensively degraded, suberized tyloses a n d vessel linings r e m a i n recognizable. W o o d sectioned a n d s t a i n e d w i t h S u d a n I V as p r e v i o u s l y described (15). Scale bar = 100 μπι.

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e n v i r o n m e n t a l c o n s t r a i n t s o n pathogen g r o w t h (as w i t h the w a l l 4 b a r r i e r also). W h e r e cell w a l l a l t e r a t i o n s are not detectable i t is to be p r e s u m e d t h a t adequate p r o t e c t i o n is afforded b y these a l t e r n a t i v e m e c h a n i s m s . Concluding Discussion A c o m m o n requirement for the successful p r o t e c t i o n of p e r e n n i a l p l a n t s against m a n y of their pathogens is for defence to be d u r a b l e . S e c o n d a r y tissues m a y persist for decades or centuries, a n d pathogens s u p p o r t e d b y a s u b s t a n t i a l food base m a y be capable of m o u n t i n g a prolonged assault o n the p o t e n t i a l host. C e l l w a l l a l t e r a t i o n s , c o m p r i s i n g either the depos i t i o n of new m a t e r i a l o n p r e - e x i s t i n g cell w a l l s , or the f o r m a t i o n of new tissues w i t h a t y p i c a l or specialized cell w a l l s , have been i m p l i c a t e d as defence mechanisms i n a wide range of circumstances i n p e r e n n i a l p l a n t s . A role i n a n t i m i c r o b i a l defence can be ascribed to the s u b e r i z e d p h e l l e m f o r m i n g the surface of the secondar w h i c h m a y have a defensive f u n c t i o n occur i n some species also. W o u n d i n g or i n f e c t i o n of c o r t i c a l a n d p h l o e m ( b a r k ) tissues n o r m a l l y results i n a sequence of cell w a l l a l t e r a t i o n s , c o m m e n c i n g w i t h changes to w a l l s of p r e - e x i s t i n g cells, a n d c u l m i n a t i n g w i t h the r e s t o r a t i o n of the i n t a c t p l a n t surface b y means of the f o r m a t i o n of a n e c r o p h y l a c t i c p e r i d e r m . W o u n d s to the c a m b i u m a n d x y l e m result i n the p r o d u c t i o n of a t r a u m a t i c b a r r i e r tissue, often w i t h suberized w a l l s , w h i c h prevents damage t o the v i t a l c a m b i u m a n d the youngest, m e t a b o l i c a l l y most i m p o r t a n t , secondary tissues b y pathogens b e c o m i n g established i n the exposed x y l e m . S u b e r i z a t i o n responses also often occur i n the reaction zone m a r g i n s of infection a n d decay in living wood. A s defence mechanisms i n plants c o m m o n l y act i n concert, i t w o u l d be w r o n g to ascribe a p r i m a r y role to these various w a l l a l t e r a t i o n s w i t h o u t a d d i t i o n a l i n v e s t i g a t i o n : i n v i r t u a l l y a l l cases evidence for the c o n c o m i t a n t o p e r a t i o n of c h e m i c a l defences is also available. However, cell w a l l a l t e r ations are p a r t i c u l a r l y w e l l s u i t e d to l o n g t e r m defence, p r o v i d i n g stable a n d d u r a b l e barriers t h a t are not dependant o n the c o n t i n u i n g m e t a b o l i c a c t i v i t y of l i v i n g cells to m a i n t a i n t h e m , a n d w h i c h are not subject to c h e m i c a l i n s t a b i l i t y or l e a c h i n g . In the cell w a l l a l t e r a t i o n s discussed s u b e r i z a t i o n has played a p r o m i nent role. T h i s is p r o b a b l y not c o i n c i d e n t a l . B o t h c a r b o h y d r a t e p o l y mers a n d l i g n i n are a b u n d a n t i n the secondary tissues of p e r e n n i a l p l a n t s , especially w o o d y species. Pathogens a d a p t e d to i n h a b i t t h i s h i g h l y l i g nified e n v i r o n m e n t are likely to possess effective e n z y m e systems for the m e t a b o l i s m of t h i s phenolic p o l y m e r , w h i c h is therefore u n l i k e l y to present a serious i m p e d i m e n t to these f u n g i . T h i s is i n m a r k e d contrast to the pathogens of p r i m a r y plant tissues (leaves, etc.) i n w h i c h l i g n i n is not a b u n d a n t l y present, against w h i c h cell w a l l l i g n i f i c a t i o n m a y present a n effective defence (51). Indeed, f u n g a l c o l o n i z a t i o n of cells w i t h walls altered b y l i g n i f i c a t i o n (or at least deposition of w a l l - b o u n d phenolics) was seen i n most of the interactions discussed here. T h e p r e d o m i n a n t l y a l i p h a t i c p o l y mer s u b e r i n is not a p r i m a r y constituent of these secondary tissues a n d is

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thus likely (as has been demonstrated) t o present more o f a n i m p e d i m e n t to s u c h pathogens. A l s o , i f as has been suggested (26-28), t h e m a i n t e n a n c e of a n a p p r o p r i a t e m o i s t u r e content i n f u n c t i o n a l s a p w o o d is i m p o r t a n t i n l i m i t i n g f u n g a l c o l o n i z a t i o n , s u b e r i n m a y provide the necessary p e r m e a b i l ­ i t y b a r r i e r t o m a i n t a i n t h i s a n d m i n i m i z e t h e extent o f d i s r u p t i o n t o x y l e m t r a n s p o r t r e s u l t i n g f r o m w o u n d i n g o r m i c r o b i a l infection. Literature Cited

1. Callow, J.A ., Ed. Biochemical Plant Pathology; John Wiley & Sons: Chichester, 1983. 2. Horsfall, J. G.; Cowling, Ε. B., Eds. Plant Disease: An Advanced Trea­ tise; Vol. V; Academic Press: New York, 1980. 3. Gibson, I. A. S.; Burley, J.; Speight, M. R. C.F.I Occasional Papers, No. 18., 1980. 4. Woodward, S.; Pearce 5. Pearce, R. B. In Funga ; Pegg, ; Ayres, , Eds.; Cambridge University Press: Cambridge, 1987; pp 219-238. 6. Sinclair, W. Α.; Lyon, Η. H.; Johnson, W. T. Diseases of Trees and Shrubs; Comstock Publ. Assoc.: Ithaca, 1987; pp 308-313. 7. Mullick, D. B. In The Structure, Biosynthesis and Degradation of Wood; Loewus, F. Α.; Runeckles, V. C., Eds.; Plenum Press: New York, 1977; pp 395-442. 8. Biggs, A. R. Phytopathology 1985, 75, 1191-1195. 9. Woodward, S.; Pearce, R. B. Physiol. Mol. Plant Pathol. 1988, 33, 151-162. 10. Woodward, S.; Pearce, R. B. Physiol. Mol. Plant Pathol. 1988, 33, 127-149. 11. Prior, C. Ann. Bot. 1976, 40, 261-279. 12. Sherwood, R. T.; Vance, C. P. Phytopathology 1976, 66, 503-510. 13. Ride, J. P.; Pearce, R. B. Physiol. Plant Pathol. 1979, 15, 79-92. 14. Biggs, J.R. Can. J. Bot. 1984, 62, 2814-2821. 15. Pearce, R. B.; Rutherford, J. Physiol. Plant Pathol. 1981, 19, 359-369. 16. Aist, J. R. Ann. Rev. Phytopathol. 1976, 14, 145-163. 17. Kunoh, H.; Ishizaki, H. Physiol. Plant Pathol. 1975, 5, 283-287. 18. Heath, M. C. Physiol. Plant Pathol. 1979, 15, 141-148. 19. Ride, J. P. Physiol. Plant Pathol. 1980, 16, 187-196. 20. Aist, J. R. In The Dynamics of Host Defence; Bailey, J. Α.; Deverall, B. J., Eds.; Academic Press: Sydney, 1983; pp.33-70. 21. Mullick, D. B. Can. J. Bot. 1975, 53, 2443-2457. 22. Hammerschmidt, R.; Kuc, J. Physiol. Plant Pathol. 1982, 20, 61-71. 23. Edwards, M. C.; Ayres, P. G. New Phytol. 1981, 89, 411-418. 24. Garrod, B.; Lewis, B. G.; Brittain, M. J.; Davies, W. P. New Phytol. 1982, 90, 99-108. 25. Kolattukudy, P. E. Can. J. Bot. 1984, 62, 2918-2933. 26. Boddy, L.; Rayner, A. D. M. New Phytol. 1983, 94, 623-641. 27. Cooke, R. C.; Rayner, A. D. M. Ecology of Saprotrophic Fungi; Long­ man: London, 1984; pp. 196-237.

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28. Rayner, A. D. M. In Water, Fungi and Plants; Ayres, P.G.; Boddy, L., Eds.; Cambridge University Press: Cambridge, 1986; pp. 321-341. 29. Zimmermann, W.; Seemüller, E. Phytopath. Z. 1984, 110, 192-199. 30. Ofong, A. U. Ph.D. Thesis, Oxford University, Oxford, UK, 1988. 31. Royle, D. J. In Biochemical Aspects of Plant Parasite Relationships; Friend, J.; Threlfall, D.R., Eds.; Academic Press: London, 1976; pp. 161-193. 32. Thomas, H. E. J. Agric. Res. 1934, 48, 187-218. 33. Tourvieille de Labrouhe, D. Agronomie 1982, 2, 553-560. 34. Campbell, C. L.; Huang, J. S.; Payne, G. A. In Plant Disease: An Advanced Treatise; Vol. V; Horsfall, J. G.; Cowling, Ε. B., Eds.; Academic Press: New York, 1980; pp. 103-120. 35. Hepper, F. N. Arboricultural J. 1981, 5, 39-43. 36. Moss, E. H.; Gorham, A. L. Phytomorphology 1953, 3, 285-294. 37. Moss, E. H. Amer. J Bot 1936 23 114-120 38. Biggs, A. R.; Davis, D 39. Biggs, A. R.; Merrill, W.; Davis, D. D. Can. J. For. Res. 1984, 14, 351-356. 40. Kemp, M. S.; Burden, R. S. Phytochemistry 1986, 25, 1261-1269. 41. Shigo, A. L.; Marx, H. G. USDA For. Serv. Agric. Inf. Bull. No. 405, 1977. 42. Pearce, R. B.; Woodward, S. Physiol. Mol. Plant Pathol. 1986, 29, 197-216. 43. Tippett, J. T.; Shigo, A. L. Eur. J. For. Pathol. 1981, 11, 51-59. 44. Pearce, R. B.; Holloway, P. J. Physiol. Plant Pathol. 1984, 24, 71-81. 45. Shain, L. Phytopathology 1967, 57, 1034-1045. 46. Shain, L. Phytopathology 1971, 61, 301-307. 47. Shain, L. Phytopathology 1979, 69, 1143-1147. 48. Biggs, A.R. Phytopathology 1987, 77, 718-725. 49. Parameswaran, N.; Knigge, H.; Liese, W. IAWA Bull. N.S. 1985, 6, 269-271. 50. Greaves, H.; Levy, J. F. J. Inst. Wood Sci. 1965, 15, 55-63. 51. Ride, J. P. In Biochemical Plant Pathology; Callow, J. Α., Ed.; John Wiley & Sons: Chichester, 1983; pp. 215-236. RECEIVED March 10, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 26

Infection-Induced Lignification in Wheat J. P. Ride, M. S. Barber, and R. E. Bertram School of Biological Sciences, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, England

Lignification occur to challenge by fungi respons fers on the plant cell walls a high degree of resistance to degradation by plant-pathogenic fungi and may thus be important in disease resistance. Physical or chemical stresses which induce defence mechanisms in other plants do not generally elicit lignification in wheat; the response appears to be specific tofilamentousfungi. Chitin, a key component of the cell walls of many fungi, has been identified as one elicitor of the response. The insolubility of this polymer suggests that soluble fragments are released by plant enzymes and then diffuse to the cells that respond. Chitin oligosaccharides (DP > 3) that elicit lignification have been identified and a model is proposed for the release, degradation and binding of these signal molecules in infected plants. C e l l w a l l a l t e r a t i o n s are n o w recognized as a c o m m o n response o f higher p l a n t s t o a t t e m p t e d i n v a s i o n b y f u n g i . M a n y o f the cell w a l l m o d i f i c a t i o n s m a y have a role t o p l a y i n disease resistance. T h i s is p a r t i c u l a r l y t r u e o f l i g n i f i c a t i o n , w h i c h i n r e l a t i o n t o disease resistance h a s i n t h e past been t h o u g h t o f as m a i n l y a w o u n d response, sealing off infected areas b u t n o t a c t i n g as a m a i n line defence. However, t h e frequent r a p i d i t y o f i n d u c e d l i g n i f i c a t i o n , together w i t h the h i g h resistance i t confers t o m i c r o b i a l d e g r a d a t i o n , is l e a d i n g t o its acceptance as a n i m p o r t a n t defensive response—one w h i c h m a y act alone o r i n concert w i t h other m e c h a n i s m s , such as p h y t o a l e x i n p r o d u c t i o n , t o provide s u b s t a n t i a l resistance t o f u n g a l p e n e t r a t i o n (1-3). 0097-6156/89A)399-0361$06.00/0 C 1989 American Chemical Society

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L i g n i f i c a t i o n as a D e f e n c e M e c h a n i s m i n W o u n d e d W h e a t L e a v e s T h e i n o c u l a t i o n of w o u n d s o n wheat leaves w i t h n o n - p a t h o g e n i c f u n g i (i.e., pathogens of other p l a n t species), such as Botrytis cinerea or Mycosphaerella pinodes, results i n l i m i t a t i o n of f u n g a l g r o w t h to the w o u n d s , despite h e a v y g r o w t h w i t h i n the wounded tissue itself (4). T h i s l i m i t a t i o n is associated w i t h cell w a l l a l t e r a t i o n s at the w o u n d m a r g i n s , g i v i n g a c o m plete r i n g of tissue resistant t o cell w a l l d e g r a d i n g enzymes b e i n g p r o d u c e d w i t h i n 18 to 24 h after i n o c u l a t i o n (4). W o u n d i n g alone does not i n d u c e such a response. H i s t o c h e m i s t r y (4,5), a u t o r a d i o g r a p h y following precursor feeding (6), l i g n i n t h i o g l y c o l a t e e x t r a c t i o n (5), i o n i z a t i o n difference s p e c t r a f o l l o w i n g a l k a l i e x t r a c t i o n (4) a n d nitrobenzene o x i d a t i o n p r o d u c t s (4) a l l i n d i c a t e the presence of l i g n i n at the infected w o u n d m a r g i n s . C h a n g e s i n the difference s p e c t r a , i n the r a t i o of nitrobenzene o x i d a t i o n p r o d u c t s a n d i n some h i s t o c h e m i c a l reactions als f r o m the p o l y m e r f o u n d i s y r i n g y l to g u a i a c y l moieties is increased. T h e c h e m i c a l a n d p h y s i c a l evidence for the presence of l i g n i n i n the m a t e r i a l deposited at w o u n d m a r g i n s is s u p p o r t e d b y b i o c h e m i c a l studies o n the enzymes i n v o l v e d i n p h e n y l p r o p a n o i d m e t a b o l i s m . T h u s , the e x t r a c t a b l e a c t i v i t i e s of p h e n y l a l a n i n e a m m o n i a - l y a s e , tyrosine a m m o n i a - l y a s e , c i n n a m a t e - 4 - h y d r o x y l a s e , caffeic a c i d O - m e t h y l t r a n s f e r a s e , 5 - h y d r o x y f e r u l i c a c i d O - m e t h y l t r a n s f e r a s e , h y d r o x y c i n n a m a t e : C o A ligase ( 4 - c o u m a r a t e ligase) a n d peroxidase a l l increase s u b s t a n t i a l l y f o l l o w i n g i n o c u l a t i o n of w o u n d s w i t h Botryiis cinerea (7-9). A u t o r a d i o g r a p h y f o l l o w i n g a d m i n i s t r a t i o n of l a b e l l e d p h e n y l a l a n i n e , c i n n a m a t e or s i n a p a t e i n d i c a t e s t h a t the r a p i d i n d u c t i o n of l i g n i n d e p o s i t i o n b y f u n g i results i n l i g n i f i c a t i o n not o n l y of the cell walls b u t of a l l the cell contents, p a r t i c u l a r l y i n the cells u n d e r g o i n g the most p r o m i n e n t r e a c t i o n at the very edge of the w o u n d (6). P r e s u m a b l y the n o r m a l c o n t r o l mechanisms t h a t prevent p o l y m e r i s a t i o n of the m o n o m e r s before they are e x p o r t e d to the w a l l are i n some way defective i n these r a p i d l y degenerating cells. T h e i m p o r t a n c e of l i g n i f i c a t i o n as a general resistance m e c h a n i s m against f u n g i is s u p p o r t e d b y the r a p i d i t y of the response to n o n - p a t h o g e n i c f u n g i (4), the slower rate of l i g n i f i c a t i o n i n response to p a t h o g e n i c Septoria (4), Fusarium, a n d Pénicillium (10) species a n d the p r o n o u n c e d resistance to cell w a l l d e g r a d i n g enzymes t h a t the response confers o n the host tissue. A l s o , t r e a t m e n t s w h i c h induce the response, such as n o n - p a t h o g e n i c f u n g i or the f u n g a l p o l y s a c c h a r i d e c h i t i n , provide significant p r o t e c t i o n against subsequent i n o c u l a t i o n w i t h pathogens. Conversely, t r e a t m e n t s such as c y c l o h e x i m i d e or U V light w h i c h i n h i b i t the response have been s h o w n to m a k e p l a n t s h i g h l y susceptible to m a n y f u n g i — a l t h o u g h i t is p l a i n l y p o s s i ble t h a t m a n y other u n k n o w n m e c h a n i s m s m a y be also i n h i b i t e d b y these t r e a t m e n t s (10).

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

26.

R I D E ET AL.

L i g n i f i c a t i o n as Leaves

Infection-Induced Lignification in Wheat a

Defence

Mechanism

in Unwounded

363 Wheat

S m a l l q u a n t i t i e s of l i g n i n are deposited i n u n w o u n d e d wheat leaves w h e n challenged b y f u n g i . A t t e m p t e d i n v a s i o n of the e p i d e r m a l cells of m a n y p l a n t s , b u t p a r t i c u l a r l y members of the G r a m i n e a e , results i n the r a p i d f o r m a t i o n of m i n u t e w a l l - l i k e deposits called p a p i l l a e , often a c c o m p a n i e d b y a m o d i f i c a t i o n to the adjacent e p i d e r m a l w a l l c a l l e d a h a l o (11). W e have s h o w n t h a t i n wheat these s t r u c t u r e s c o n t a i n s m a l l , b u t p r o b a b l y s i g n i f i c a n t , a m o u n t s of l i g n i n (12) a n d t h a t t h i s confers o n the s t r u c t u r e s a s u b s t a n t i a l degree of resistance to f u n g a l d e g r a d a t i o n in viiro. Pathogens of wheat are no more capable of d e g r a d i n g the s t r u c t u r e s t h a n f u n g i n o n p a t h o g e n i c o n wheat (13). T h e success of pathogens is a p p a r e n t l y d e p e n dent u p o n a slower response by the host. S o m e pathogens, such as the s t e m rust p a t h o g e n Puccinia graminis f.sp. iritici, do not a t t e m p use the n a t u r a l openings p r o v i d e d b y s t o m a t a . T h e r e a c t i o n of resistant wheat varieties u s u a l l y involves a " h y p e r s e n s i t i v e " response, where the i n v a d e d m e s o p h y l l cells undergo a r a p i d d e t e r i o r a t i o n . Here a g a i n l i g n i f i c a t i o n has a p o t e n t i a l significance (14). Fluorescence microscopy, h i s t o c h e m istry, and autoradiography a l l indicated that lignification occurred d u r i n g the necrosis of the cells u n d e r g o i n g the hypersensitive response i n nearisogenic wheat lines c a r r y i n g the S r 5 or Srè (at 19°C) alleles for resistance t o P. graminis f.sp. tritici. L i g n i f i c a t i o n was not observed i n the s u s c e p t i ble i n t e r a c t i o n s between the fungus a n d the parent c u l t i v a r M a r q u i s ( 5 r 5 , S V 6 ) , or the S r 6 l i n e when tested at 2 6 ° C (14). Specificity of Induction of Lignification i n W o u n d e d W h e a t Leaves L i g n i f i c a t i o n is one of m a n y defensive responses t h a t p l a n t s e m p l o y to counter p o t e n t i a l l y pathogenic f u n g i . P h y t o a l e x i n a c c u m u l a t i o n has received m u c h a t t e n t i o n a n d i t is evident t h a t t h i s response is not r e s t r i c t e d to occasions of f u n g a l a t t a c k , b u t can u s u a l l y be a c t i v a t e d b y a whole range of other stressful a b i o t i c t r e a t m e n t s , e.g., U V l i g h t , h e a v y m e t a l salts, a n t i m e t a b o l i t e s , o x i d i z i n g a n d r e d u c i n g agents, p h y s i c a l d a m a g e , etc. (15). P h y t o a l e x i n p r o d u c t i o n c o u l d therefore be viewed as a response t o g e n e r a l ized damage to the p l a n t , r a t h e r t h a n a specific response to f u n g a l i n v a s i o n . B y c o n t r a s t , the i n d u c t i o n of l i g n i f i c a t i o n i n wheat appears to be h i g h l y specific t o filamentous f u n g i (16). T h u s , a range of a b i o t i c t r e a t m e n t s r e p o r t e d t o i n d u c e p h y t o a l e x i n a c c u m u l a t i o n i n various plants f a i l e d t o i n d u c e l i g n i fication i n p r i m a r y wheat leaves. F i l a m e n t o u s f u n g i were, however, u s u a l l y potent inducers w i t h yeasts a n d b a c t e r i a b e i n g less a c t i v e (16). Elicitors of Lignification T h e h i g h specificity of i n d u c e d l i g n i f i c a t i o n i n wheat for filamentous f u n g i leads i m m e d i a t e l y to s p e c u l a t i o n as to the n a t u r e of the signals i n v o l v e d i n the general r e c o g n i t i o n of f u n g i by wheat. M i c r o b i a l molecules t h a t trigger p l a n t defence responses are u s u a l l y t e r m e d " e l i c i t o r s " (17).

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In order t o s t u d y elicitors of l i g n i f i c a t i o n i n wheat leaves, a q u a n t i t a t i v e assay for i n d u c e d l i g n i f i c a t i o n has recently been developed (5). T h e m e t h o d involves l o c a t i n g l i g n i n b y s t a i n i n g leaves w i t h p-nitrobenzene d i a z o n i u m t e t r a f l u o r o b o r a t e after removal o f soluble a n d wall-esterified phenolics b y p r e - e x t r a c t i o n i n e t h a n o l a n d a l k a l i . T h e l o c a l i n t e n s i t y of l i g n i n is t h e n d e t e r m i n e d b y s c a n n i n g the leaves w i t h a gel-scanning d e n s i t o m e t e r . T h e m e t h o d gives results w b ' c h correlate w e l l w i t h those based o n e x t r a c t i o n w i t h t h i o g l y c o l i c a c i d , b u t has the advantage t h a t a d d i t i o n a l i n f o r m a t i o n o n the l o c a t i o n of the i n d u c e d l i g n i n is o b t a i n e d . U s i n g t h i s assay, a range of f u n g a l a n d p l a n t p r o d u c t s has been screened for l i g n i f i c a t i o n - e l i c i t i n g a c t i v i t y , i n c l u d i n g a n u m b e r t h a t were k n o w n elic­ itors of defence reactions i n other p l a n t s (5). T h e m a j o r i t y of t r e a t m e n t s were i n a c t i v e . T h e m a j o r elicitors identified were c h i t i n , c h i t o s a n , a c o m ­ m e r c i a l "cellulase" a n d " p e c t i n a s e " of f u n g a l o r i g i n , a n d a p a r t i a l h y d r o l y s a t e of the cell w a l l g l u c a n of the fungus Phytophthora megasperma f.sp. glycinea c o n t a i n i n g oligo-/?-glucoside ( D P ) o f 10-13. T h e "cellulase" was the most active a n d its a c t i v i t y was r e t a i n e d after h e a t - i n a c t i v a t i o n . C h i t i n as a n E l i c i t o r o f L i g n i f i c a t i o n A l t h o u g h c h i t i n , a / ? ( l - 4 ) - l i n k e d p o l y m e r of ΛΓ-acetyl-D-glucosamine, was not the most a c t i v e e l i c i t o r discovered, its widespread occurrence i n f u n g i as a core c o m p o n e n t o f the cell w a l l a n d its p r e d o m i n a n c e at h y p h a l t i p s encouraged us t o e x a m i n e its e l i c i t o r a c t i v i t y i n m o r e d e t a i l . A l t h o u g h c h i t o s a n was also e l i c i t o r - a c t i v e , its a c t i v i t y was t h o u g h t to depend o n the a c e t y l a t e d regions of the p o l y s a c c h a r i d e since f u l l y deacetylated c h i t o s a n h a d negligible a c t i v i t y (5). C h i t i n is h i g h l y i n s o l u b l e i n water a n d i n the i n o c u l a t i o n procedure used i t was u n l i k e l y to come i n t o direct contact w i t h the cells t h a t lignify. It was therefore p o s t u l a t e d t h a t p l a n t enzymes, pre­ s u m a b l y chitinases, release soluble fragments t h a t are the signals t h a t pass f r o m pathogen to host. T o investigate t h i s p o s s i b i l i t y , c h i t i n oligosaccharides were first pre­ p a r e d b y a c i d h y d r o l y s i s of c h i t i n a n d s e p a r a t i o n of the p r o d u c t s b y H P L C (18). F o r c o m p a r i s o n , the deacetylated counterparts were also p r e p a r e d b y ion-exchange c h r o m a t o g r a p h y of a c i d h y d r o l y s a t e s of c h i t o s a n . Tests o n the p r e p a r e d oligomers i n d i c a t e d t h a t a m i n i m u m degree of p o l y m e r i s a t i o n of 4 was necessary for e l i c i t o r a c t i v i t y , a n d t h a t the presence of a c e t y l groups was essential since the deacetylated tetramer was i n a c t i v e (18). W h e a t leaf tissue has now been s h o w n to c o n t a i n several chitinases, a l l of w h i c h are endo i n a c t i o n a n d are capable of releasing the e l i c i t o r - a c t i v e t e t r a m e r , a n d higher oligomers, f r o m c h i t i n ( R i d e & B a r b e r , u n p u b l i s h e d d a t a ) . T h e a m o u n t of c h i t i n t e t r a m e r required to elicit the response, w h e n a d m i n i s t e r e d as a single dose, was r e l a t i v e l y h i g h . O n e e x p l a n a t i o n for t h i s p h e n o m e n o n was t h a t c h i t i n oligomers are u n s t a b l e i n wheat leaf t i s ­ sue, a n d t h a t a d m i n i s t r a t i o n as a single dose d i d not p r o p e r l y m i m i c the l o w , b u t c o n t i n u a l , levels t h a t w o u l d be released f r o m the p o l y m e r . T h i s was s u p p o r t e d b y the observation t h a t the t e t r a m e r , w h e n a d m i n i s t e r e d

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t o wheat leaves at a dose w h i c h j u s t failed to e l i c i t a response, was red u c e d i n c o n c e n t r a t i o n b y a p p r o x i m a t e l y 7 0 % w i t h i n 8 hours. T h e loss was believed to be due not o n l y to the a c t i o n of the chitinases, w h i c h w o u l d generate o n l y d i m e r , b u t possibly also to AT-acetyl-/?-D-hexosaminidases, one o f w h i c h has n o w been purified f r o m leaves ( B a r b e r & R i d e , u n p u b l i s h e d d a t a ) . T h e role of these enzymes i n c h i t i n - e l i c i t e d l i g n i f i c a t i o n is c u r r e n t l y being investigated. W h e a t G e r m A g g l u t i n i n : a P o s s i b l e R e c e p t o r for C h i t i n O l i g o mers? T h e i d e n t i f i c a t i o n of c h i t i n oligomers as signals i n v o l v e d i n the r e c o g n i t i o n of f u n g i b y wheat leads to s p e c u l a t i o n o n the n a t u r e of the p r e s u m e d receptor for these molecules i n p l a n t cells. O n e c a n d i d a t e for the role is the l e c t i n wheat g e r m a g g l u t i n i n ( W G A ) . T h i s p r o t e i n , i s o l a t e d o r i g i n a l l y f r o m wheat g e r m , carries b i n d i n g sites t h a t are h i g h l y specific for J V - a c e t y l D-glucosamine ( G l c N A c ) G l c N A c (19). Increasing the oligomer size results i n increased b i n d i n g affinity, at least u p to the t e t r a m e r (19). E a c h b i n d i n g site a p p a r e n t l y has three subsites w i t h different affinities for G l c N A c (19). A p a r t f r o m s i a l y l lactose, where the i V - a c e t y l n e u r a m i n i c a c i d residue m i m i c s G l c N A c a n d b i n d s to subsite 1, o n l y c h i t i n oligomers a n d other G l c N A c - c o n t a i n i n g molecules are k n o w n to b i n d to the l e c t i n (20). It is t h u s possible t h a t t h i s l e c t i n , whose n a t u r a l f u n c t i o n i n wheat is c u r r e n t l y u n k n o w n , is a receptor for e l i c i t o r a c t i v e c h i t i n oligomers derived f r o m f u n g a l cell w a l l s . If t h i s is the case, i t m i g h t be expected t h a t the c h i t i n t r i m e r w o u l d be a good e l i c i t o r , r a t h e r t h a n the weak one revealed i n o u r tests (18), a n d t h a t s i a l y l lactose w o u l d also e l i c i t a response, w h i c h is not the case ( B e r t r a m & R i d e , u n p u b l i s h e d d a t a ) . T h e s e a p p a r e n t anomalies m a y be due t o r a p i d d e g r a d a t i o n of the oligosaccharides i n wheat leaves w h e n a d m i n i s t e r e d as a single dose; t h i s p o s s i b i l i t y is c u r r e n t l y b e i n g assessed. D e s p i t e its a b u n d a n c e i n seeds, the l e c t i n is present i n m u c h smaller q u a n t i t i e s i n seedlings a n d older p l a n t s (21), a n d reports i n d i c a t e t h a t o n l y traces m a y occur i n the leaves (22). Nevertheless, u s i n g specific a n t i s e r u m raised against purified wheat g e r m a g g l u t i n i n , we have n o w d e m o n s t r a t e d the presence of a W G A - l i k e c h i t i n - b i n d i n g p r o t e i n i n p r i m a r y wheat leaves ( B e r t r a m & R i d e , u n p u b l i s h e d d a t a ) . W e are c u r r e n t l y e v a l u a t i n g the poss i b i l i t y t h a t t h i s molecule is the receptor for e l i c i t o r - a c t i v e c h i t i n oligosaccharides. A M o d e l for t h e R e c o g n i t i o n o f F u n g i b y W h e a t T h e s e results allow us to propose a hypothesis for the processes i n v o l v e d i n the general r e c o g n i t i o n o f filamentous f u n g i b y wheat ( F i g . 1). T h e m o d e l does not a t t e m p t to p r o v i d e a n e x p l a n a t i o n for the h i g h l y specific i n t e r a c t i o n s between c e r t a i n wheat c u l t i v a r s a n d races of f u n g a l pathogens, b u t deals w i t h the m u c h more general case of wheat resistance to n o n p a t h o g e n i c f u n g i , i.e., non-host resistance a n d resistance to s a p r o p h y t e s . T h e hypothesis proposes t h a t t h i s basic i n c o m p a t i b i l i t y between f u n g i a n d wheat is governed b y the r e c o g n i t i o n b y the p l a n t of soluble oligosac-

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F i g u r e 1. A proposed m o d e l for the general r e c o g n i t i o n of filamentous f u n g i b y w h e a t r e s u l t i n g i n l i g n i f i c a t i o n of the p l a n t cells. H y d r o l y t i c enzymes present c o n s t i t u t i v e l y i n the apoplast of the p l a n t , such as endo-chitinase a n d / ? ( l - 3 ) glucanase, release oligosaccharides of N - a c e t y l g l u c o s a m i n e ( G l c N A c ) or glucose ( G l c ) f r o m the c h i t i n a n d g l u c a n components of the f u n g a l cell w a l l . O l i g o m e r s of sufficient size, such as the c h i t i n t e t r a m e r , are the a c t i v e signals t h a t pass to the p l a n t cells; the d e t a i l e d s t r u c t u r e s o f the p u t a t i v e glucose oligosaccharide signals are as yet u n k n o w n . L e c t i n s , such as wheat g e r m a g g l u t i n i n ( W G A ) a n d other u n k n o w n lectins ( R E C ) , m a y be the cell surface receptors for these oligosaccharides. T h e active oligomers are r a p i d l y degraded b o t h by the endo enzymes t h a t l i b e r a t e d t h e m a n d b y glycosidases. E l i c i t a t i o n of defence responses t h u s depends o n a c o n t i n u e d b u t low level of oligosaccharides b e i n g l i b e r a t e d f r o m the h y p h a l t i p .

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c h a r i d e signals, w h i c h are released f r o m the cell walls of the fungus b y p l a n t enzymes or perhaps are present o n soluble g l y c o p r o t e i n s . I n the case o f c h i t i n oligosaccharides, these w o u l d be released f r o m the h y p h a l t i p s b y host chitinases. It is i n t e r e s t i n g i n t h i s context t h a t wheat seed c h i t i nase has been s h o w n to release higher oligomers f r o m freshly-synthesized "nascent" c h i t i n , w h i c h is likely to be f o u n d at g r o w i n g h y p h a l t i p s , t h a n f r o m c o l l o i d a l or regenerated c h i t i n (23). T h e k n o w n i n s t a b i l i t y o f c h i t i n oligomers i n w h e a t leaves, p r e s u m e d to be due p r i m a r i l y to the a c t i o n of chitinases a n d i V - a c e t y l - / ? - D hexosaminidases, suggests t h a t the key oligosaccharide signals are r a p i d l y t u r n e d over in vivo a n d t h a t e l i c i t a t i o n of the response p r o b a b l y depends o n the presence of c o n t i n u e d low levels of the active higher oligomers over a p e r i o d o f t i m e . A n obvious question t h a t arises f r o m s u c h a p r o p o s e d m e c h a n i s m is w h y does wheat possess such a r e l a t i v e l y insensitive s y s t e m o f r e c o g n i t i o n . T h e m e c h a n i s m w o u l d be far more sensitive i f there were no d e s t r u c t i o n of the e l i c i t o r - a c t i v be a c t i v e at m u c h lower doses a n d the p l a n t m i g h t r e s p o n d to a t t e m p t e d i n f e c t i o n at a n earlier stage. A possible answer to t h i s question lies i n the fact t h a t l i g n i f i c a t i o n is a n irreversible, e n e r g y - r e q u i r i n g process t h a t freq u e n t l y involves a c e r t a i n a m o u n t of cell d e a t h ; r e s t r i c t i o n of the response to genuine challenges b y p o t e n t i a l pathogens is therefore beneficial t o the p l a n t . T h u s wheat o n l y responds to the c o n t i n u e d release of oligomers w i t h a r e l a t i v e l y h i g h D P , such as t h a t b r o u g h t a b o u t b y the presence of g r o w i n g h y p h a l t i p s , a n d not to the passing presence of other s t i m u l i , such as insect chitin. Since not a l l f u n g i c o n t a i n c h i t i n , a n d even those t h a t do have other w a l l components, i t must be supposed t h a t other general signals exist. T h e h i g h e l i c i t o r a c t i v i t y of a p a r t i a l l y h y d r o l y s e d g l u c a n o f f u n g a l o r i g i n (5), together w i t h the c o m m o n occurrence of / ? ( l - 3 ) a n d / ? ( l - 6 ) l i n k e d g l u c a n s i n f u n g a l cell w a l l s , leads us to propose t h a t the a c t i o n of glucanases l i b erates glucose oligosaccharides f r o m f u n g a l walls a n d t h a t these are also elicitors of the l i g n i f i c a t i o n response. I n t h i s context i t is i n t e r e s t i n g t h a t / ? ( l - 3 ) glucanase a c t i v i t y has been detected i n m a n y p l a n t s (24). L e c t i n s are proposed as the p r i m a r y receptors for the oligosaccharide s i g n a l s . T h e l o c a t i o n o f the W G A - l i k e p r o t e i n i n wheat leaves has not yet been determ i n e d , b u t b y a n a l o g y w i t h other signal-receptor systems i t seems l i k e l y t h a t the receptor w i l l be at the cell surface, p r e s u m a b l y associated w i t h the p l a s m a l e m m a . Since a n a l y s i s of the p u b l i s h e d a m i n o - a c i d sequences o f W G A isolectins 1 a n d 2 (25,26) does not i m m e d i a t e l y reveal a n y l o n g h y d r o p h o b i c , m e m b r a n e - s p a n n i n g regions, t h e n i f t h i s p r o t e i n is a surface receptor i t m u s t p r e s u m a b l y be l i n k e d in vivo to a p l a s m a m e m b r a n e c o m ponent. L e c t i n s have a h i g h degree of specificity for the sugars they b i n d , a n d hence the p r o p o s a l t h a t more t h a n one t y p e of oligosaccharide s i g n a l is i n v o l v e d i n e l i c i t a t i o n necessarily dictates t h a t more t h a n one receptor is present at the cell surface. Since the different signals u l t i m a t e l y result i n the same response, i t is presumed t h a t the s i g n a l t r a n s d u c t i o n p a t h w a y s

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i n v o l v e d i n t h e i n t r a c e l l u l a r t r a n s m i s s i o n o f the different signals a n d t h e i r c o u p l i n g t o the response m u s t meet at some p o i n t . P e r h a p s a c o m m o n seco n d messenger i s a c t i v a t e d b y t h e i n t e r a c t i o n o f the different signals w i t h their c o r r e s p o n d i n g receptors? A n a l t e r n a t i v e hypothesis w o u l d be t h a t the lectins are e x t r a c e l l u l a r a n d act as t r a n s p o r t e r s rather t h a n m e m b r a n e b o u n d receptors. T h e different e l i c i t o r - l e c t i n complexes m i g h t t h e n b e recognized b y a single receptor. Induced l i g n i f i c a t i o n is blocked b y t r e a t m e n t s w h i c h i n h i b i t p r o t e i n synthesis (10), a n d i t seems likely therefore t h a t t h e increases i n a c t i v i ties observed for enzymes i n v o l v e d i n l i g n i n biosynthesis (7,8) d e p e n d o n enhanced t r a n s l a t i o n , a n d p r o b a b l y enhanced t r a n s c r i p t i o n o f t h e a p p r o p r i a t e genes, as has been s h o w n for the p h y t o a l e x i n response o f some p l a n t s (27). However, t h e m o l e c u l a r b i o l o g y o f the response i n wheat awaits i n vestigation. Literature Cited

1. Vance, C. P.; Kirk, T. K.; Sherwood, R. T. ˙˙Annu. Rev. Phytopath.˙˙1980,18, 259-88. 2. Ride, J. P. In Biochemical Plant Pathology; Callow, J. Α., Ed.; John Wiley: Chichester, 1983; pp 215-36. 3. Ride, J. P. In Natural Antimicrobial Systems, Part 1. Antimicrobial Systems in Plants and Animals; Gould, G. W., Rhodes-Roberts, M. E.; Charnley, A. K.; Cooper, R. M.; Board, R. G., Eds.; Bath University Press: Bath, 1986; pp 159-75. 4. Ride, J. P. Physiol. Pl. Path. 1975, 5, 125-34. 5. Barber, M. S.; Ride, J. P. Physiol. Mol. Pl. Path. 1988, 32, 185-97. 6. Maule, A. J.; Ride, J. P. Physiol. Pl. Path. 1982, 20, 235-41. 7. Maule, A. J.; Ride, J. P. Phytochemistry 1976, 15, 1661-64. 8. Maule, A. J.; Ride, J. P. Phytochemistry 1983, 22, 1113-16. 9. Thorpe, J. R.; Hall, J. L. Physiol. Pl. Path. 1984, 25, 363-79. 10. Ride, J. P.; Barber, M. S. Physiol. Mol. Pl. Path. 1987, 31, 349-60. 11. Aist, J. R. Annu. Rev. Phytopath. 1976, 14, 145-63. 12. Ride, J. P.; Pearce, R. B. Physiol. Pl. Path. 1979, 15, 79-92. 13. Ride, J. P. Physiol. Pl. Path. 1980, 16, 187-96. 14. Beardmore, J.; Ride, J. P.; Granger, J. W. Physiol. Pl. Path. 1983, 22, 209-20. 15. Bailey, J. A. In Phytoalexins; Bailey, J. Α.; Mansfield, J. W., Eds; Halsted Wiley: New York, 1982; pp 289-318. 16. Pearce, R. B.; Ride, J. P. Physiol. Pl. Path. 1980, 16, 197-204. 17. Keen, Ν. T. Science 1975, 187, 74-5. 18. Barber, M. S.; Bertram, R. E.; Ride, J. P. Physiol. Mol. Pl. Path. 1988, in press. 19. Allen, A. K.; Neuberger, Α.; Sharon, N. Biochem. J. 1973, 131, 155-62. 20. Wright, C. S. J. Mol. Biol. 1984, 178, 91-104. 21. Mishkind, M.; Keegstra, K.; Palevitz, B. A. Pl. Physiol. 1980, 66, 95055.

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22. Raikhel, Ν. V.; Mishkind, M. L.; Palevitz, B. A. Planta 1984, 162, 55-61. 23. Molano, J.; Polacheck, I.; Duran, A. ; Cabib, E. J. Biol. Chem. 1979, 254, 4901-07. 24. Boiler, T. In Cellular and Molecular Biology of Plant Stress; Key, J.L., Kosuge, T., Eds. Alan R. Liss: New York, 1985; pp 247-62. 25. Wright, C. S.; Gavilanes, F.; Peterson, D. L. Biochemistry 1984, 23, 280-7. 26. Wright, C. S.; Olafsdottir, S. J. Biol. Chem. 1986, 261, 7191-95. 27. Dixon, R. A. Biological Reviews 1986, 61, 239-91. RECEIVED March 10, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 27

Lignin Biosynthesis in Stem Rust Infected Wheat Bruno M . Moerschbacher Institut für Biologie III (Pflanzenphysiologie) der RWTH Aachen, Worringerweg, D-5100 Aachen, Federal Republic of Germany

Highly resistant wheat varietie exhibit typical hyper sensitive respons of the stem rust fungus. Host cells which are penetrated by a fungal haustorium undergo rapid necrotization, thus depriving the biotrophic parasite of its nutritional basis. This rapid cell death is correlated with the deposition of lignin or lignin-like material in the host cell walls and protoplasts. Inhibition of lignin biosynthesis delays necrotization of penetrated host cells and partially breaks resistance of wheat to stem rust. In resistant plants, an increase in the activities of the general phenylpropanoid pathway and of the specific branch pathway of lignin biosynthesis can be detected at the time of the hypersensitive cell death, contrasting to decreased activities in susceptible near-isogenic plants. The participation of both an elicitor and a suppressor in the signal exchange between host and parasite has been suggested. An elicitor of lignification could be isolated from fungal cell walls. An endogenous suppressor of the resistance reaction was found in wheat cell walls. L i g n i n , the second most a b u n d a n t organic c o m p o u n d o n e a r t h , is e x t r e m e l y resistant t o m i c r o b i a l d e g r a d a t i o n (1,2) a n d thus constitutes one o f t h e most effective m e c h a n i c a l barriers against pathogenic i n v a s i o n (3,4). C o n sequently, t h e l i g n i n content o f higher plants has l o n g been recognized as a n i m p o r t a n t factor i n t h e resistance against t h e a t t a c k b y a m y r i a d o f p o t e n t i a l pathogens. In a d d i t i o n t o i t s role as a preformed resistance factor, Hijwegen (5) has also proposed active i n d u c t i o n o f l i g n i f i c a t i o n as a defense m e c h a n i s m of c u c u m b e r against Cladosporium. Subsequently, i n a n u m b e r o f hostp a t h o g e n i n t e r a c t i o n s , i n d u c e d l i g n i f i c a t i o n has been proposed as the a c t i v e 0097-6156/89/0399-0370$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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m e c h a n i s m for resistance. S o m e of the most intensely s t u d i e d i n t e r a c t i o n s are those of resistance of t o b a c c o against t o b a c c o mosaic v i r u s (6-9), p o t a t o against Phytophihora (10-12) or non-pathogens (13), c u c u m b e r (14,15) a n d melons (16-18) against Cladosporium a n d Colletotrichum species. In the f a m i l y of the G r a m i n e a e , w h i c h includes some of m a n ' s most i m p o r t a n t crops, active l i g n i f i c a t i o n seems t o be of s p e c i a l i m p o r t a n c e for i n d u c e d resistance m e c h a n i s m s (19,20). T h i s m a y be correlated w i t h the n e a r l y complete absence of p h y t o a l e x i n s i n t h i s f a m i l y (21). I n spite of a n intensive search for such i n f e c t i o n - i n d u c e d f u n g i toxic substances, n o p h y t o a l e x i n s have been f o u n d i n wheat to date (22). Nevertheless, i n d u c e d l i g n i f i c a t i o n has been s h o w n t o p l a y a n i m p o r t a n t role i n disease resistance of wheat against a v a r i e t y of f u n g a l pathogens (4): T h e f o r m a t i o n of a r i n g of lignified cells at w o u n d m a r g i n s prevents the s p r e a d of n e c r o t r o p h i c f u n g i (23-25). L i g n i f i e d p a p i l l a e p r o d u c e d i n e p i d e r m a l cells j u s t below f u n g a l appressoria prevent the ingression of f u n gal p e n e t r a t i o n pegs d i r e c t l l i g n i f i c a t i o n of p e n e t r a t e d e p i d e r m a l a n d m e s o p h y l l cells a n d c o n c o m i t a n t cell d e a t h arrest further g r o w t h of b i o t r o p h i c parasites (22,30,31). M o d e s o f A c t i o n o f L i g n i f i c a t i o n as a R e s i s t a n c e

Mechanism

Several modes of a c t i o n of l i g n i f i c a t i o n as a resistance m e c h a n i s m have been proposed b y R i d e (32). L i g n i f i c a t i o n enhances the resistance of p l a n t cell walls against m e c h a n i c a l a n d e n z y m a t i c a t t a c k a n d m a y t h u s i m p e d e f u n g a l i n v a s i o n of host cells. I n c r u s t a t i o n of cell w a l l s w i t h l i g n i n m a y slow d o w n diffusion a n d t h u s i m m o b i l i z e f u n g a l toxins or decrease the flow of n u t r i e n t s t o the parasite. T h e phenolic precursors as w e l l as the free r a d i c a l s f o r m e d d u r i n g the process of l i g n i f i c a t i o n m a y be f u n g i t o x i c a n d act as p h y t o a l e x i n s . F u r t h e r m o r e , l i g n i f i c a t i o n m a y spread t o h y p h a l walls and thus impede further fungal growth. O n e or several of these m e c h a n i s m s m a y be i n v o l v e d i n resistance responses w h e n l i g n i f i c a t i o n takes place i n the p l a n t cell w a l l s . I n the case of b i o t r o p h i c parasites w h i c h rely o n f u n c t i o n a l m a t u r e h a u s t o r i a w i t h i n l i v i n g host cells for their development (33-36), l i g n i f i c a t i o n of the w h o l e cell contents l e a d i n g t o r a p i d host cell d e a t h m a y i n itself be a decisive factor i n the expression of resistance. E v i d e n c e for t h e P a r t i c i p a t i o n o f L i g n i f i c a t i o n i n R e s i s t a n c e actions

Re-

Different approaches have been used to p r o d u c e evidence for the p a r t i c i p a t i o n of l i g n i f i c a t i o n i n resistance reactions. W e w i l l confine the discussion t o those techniques a p p l i e d i n investigations o n resistance reactions of w h e a t , one of the best s t u d i e d host p l a n t s to date (4). T h e most convenient way of d e t e r m i n i n g l i g n i n is p r o v i d e d b y a range of h i s t o c h e m i c a l s t a i n i n g reactions (37): A p o s i t i v e o u t c o m e of the p h l o r o g l u c i n o l / H C l or the c h l o r i n e / s u l f i t e test, supposed to be the most specific s t a i n s for l i g n i n (38,39), p o i n t t o the p a r t i c i p a t i o n of l i g n i n i n the i n v e s t i g a t e d resistance p h e n o m e n a (23-25,28-31).

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Y e l l o w autofluorescence of l i g n i n i n U V - l i g h t (40,41) has also been used as a c r i t e r i o n (28,30,31). However, i n v i e w of the countless autofluorescent substances i n p l a n t s (40,42), even the r e c o r d i n g of a t y p i c a l e m i s s i o n spect r u m for l i g n i n (31) cannot be regarded as a specific p r o o f for the presence of l i g n i n . T h e decreased e n z y m a t i c d i g e s t i b i l i t y of p l a n t cell w a l l s u p o n i n c r u s t a t i o n w i t h l i g n i n (1,2,32) has also been used as a n , albeit quite unspecific, d e m o n s t r a t i o n of l i g n i f i c a t i o n (23,24,28-30). T h e b i o c h e m i c a l d e t e r m i n a t i o n of n e w l y synthesized l i g n i n is difficult because of large a m o u n t s of preformed l i g n i n i n t r a c h e a r y a n d s u p p o r t i n g elements of h e a l t h y tissues. Nevertheless, i t has been achieved i n the case of non-host resistance reactions against w o u n d infections b y n e c r o t r o p h i c parasites (23). W h e n r a d i o a c t i v e l i g n i n precursors are a p p l i e d t o resistant host p l a n t s infected w i t h a n a v i r u l e n t p a t h o g e n , the a u t o r a d i o g r a p h i c l o c a l i z a t i o n of r a d i o a c t i v i t y i n resistant p a r t i c i p a t i o n of l i g n i f i c a t i o n i n the resistance response. T h o r o u g h e x t r a c t i o n of n o n - p o l y m e r i z e d precursor w i t h organic solvents a n d the r e m o v a l of esterified phenolics b y alkaline h y d r o l y s i s are i m p o r t a n t steps i n these e x p e r i m e n t s (25,28,30,31). A different a p p r o a c h to investigate active l i g n i f i c a t i o n d u r i n g resistance reactions is p r o v i d e d b y the d e t e r m i n a t i o n of e n z y m e a c t i v i t i e s i n v o l v e d i n l i g n i n biosynthesis. R e s i s t a n t plants are expected t o be more s t r o n g l y a c t i v a t e d d u r i n g or i m m e d i a t e l y preceding the resistance r e a c t i o n c o m p a r e d to susceptible p l a n t s . T h u s , p h e n y l a l a n i n e a m m o n i a - l y a s e ( P A L ) (43-45), c i n n a m i c a c i d 4-hydroxylase (46), O-methyltransferases (44), a n d 4 - c o u m a r a t e : C o A ligase (46) have been investigated i n a n u m b e r of s t u d ies. A s these enzymes are p a r t of the general p h e n y l p r o p a n o i d p a t h w a y , a n d t h u s not j u s t i n v o l v e d i n l i g n i n biosynthesis, t h e i r a c t i v a t i o n is o n l y p r o o f of o v e r a l l p a r t i c i p a t i o n of phenolic substances i n the resistance reactions (47-49). I n order to prove a specific i n d u c t i o n of l i g n i f i c a t i o n , one or several enzymes of the specific b r a n c h p a t h w a y of l i g n i n biosynthesis must be i n v e s t i g a t e d . A l t h o u g h peroxidases catalyze the last e n z y m i c step of t h i s p a t h w a y (50), increased peroxidase a c t i v i t i e s cannot be regarded as a specific c r i t e r i o n for l i g n i f i c a t i o n , as these enzymes p l a y a range of different roles i n p l a n t m e t a b o l i s m (51). Lignification i n the W h e a t - S t e m

Rust

System

W e w i l l n o w consider the evidence t h a t has a c c u m u l a t e d to show the p a r t i c i p a t i o n of l i g n i f i c a t i o n i n the hypersensitive resistance of wheat t o the wheat s t e m rust fungus, Puccinia graminis f. sp. tritici. B e a r d m o r e et al (30) a n d T i b u r z y (31) showed t h a t e p i d e r m a l (31) a n d m e s o p h y l l (30,31) cells of resistant wheat p l a n t s , p e n e t r a t e d b y h a u s t o r i a of a n a v i r u l e n t race of the fungus, can be s t a i n e d w i t h p h l o r o g l u c i n o l / H C l (31) a n d c h l o r i n e / s u l f i t e (30,31). These cells show yellow a u t o f l u o rescence u n d e r U V - l i g h t (30,31), the emission s p e c t r u m is i d e n t i c a l t o t h a t of l i g n i f i e d t r a c h e a r y elements (31).

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T h e a p p l i c a t i o n of r a d i o a c t i v e p h e n o l i c p r e c u r s o r s — q u i n i c a c i d a n d s h i k i m i c a c i d (52), p h e n y l a l a n i n e (30,53), t y r o s i n e (53), a n d c i n n a m i c a c i d ( 3 0 , 3 1 , 5 3 ) — t o infected wheat leaves led to a solvent- a n d a l k a l i - r e s i s t a n t i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n t o h y p e r s e n s i t i v e l y r e a c t i n g host cells s u g gesting lignin formation h a d occurred. I n a recent s t u d y (54), we showed increased a c t i v i t i e s o f t w o enzymes o f the general p h e n y l p r o p a n o i d p a t h w a y , P A L a n d 4 - c o u m a r a t e : C o A l i g ase, as w e l l as one e n z y m e of the specific p a t h w a y o f l i g n i n b i o s y n t h e s i s , c i n n a m y l - a l c o h o l dehydrogenase ( C A D ) , i n resistant p l a n t s at the t i m e of the h y p e r s e n s i t i v e host cell d e a t h . O n the other h a n d , decreased a c t i v i t i e s were observed at the same t i m e w i t h susceptible host p l a n t s (54). Furt h e r m o r e , we showed t h a t the w e l l k n o w n increase i n peroxidase a c t i v i t i e s , w h i c h is s t r o n g i n resistant a n d o n l y weak i n susceptible p l a n t s (55-58), is at least p a r t l y due to the increased a c t i v i t y of the l i g n i n b i o s y n t h e t i c p a t h w a y (54,59). W h e n h i g h l y resistan race of the s t e m rust fungus, f u n g a l g r o w t h is arrested b y the h y p e r s e n s i tive d e a t h o f the first p e n e t r a t e d host cells (30,31.) E v e n i n v e r y densely i n o c u l a t e d leaves, the r e a c t i o n of less t h a n one percent of the host cells is sufficient to s t o p f u r t h e r development o f the p a r a s i t e . T h i s s m a l l percentage m a y be the reason, w h y no increased content o f b i o c h e m i c a l l y d e t e r m i n e d l i g n i n was measured i n infected hypersensitive wheat leaves (60,61). H o w e v e r , w h e n a n e l i c i t o r (see b e l o w ) , i s o l a t e d f r o m the s t e m rust fungus (62), is injected i n t o the i n t e r c e l l u l a r spaces o f wheat leaves almost every cell i n the i n f i l t r a t e d area e x h i b i t s a h y p e r s e n s i t i v e - l i k e r e a c t i o n (6265). I n the e l i c i t o r treated leaves, l i g n i n content as d e t e r m i n e d b y the t h i o g l y c o l i c a c i d procedure clearly increased (66). L i g n i f i c a t i o n as a C a u s a l F a c t o r i n

Resistance

T h e above described studies s t r o n g l y suggest a c o r r e l a t i o n between l i g n i fication a n d resistance. H o w e v e r , they do not allow any c o n c l u s i o n c o n c e r n i n g a c a u s a l r e l a t i o n s h i p between the two p h e n o m e n a . T h e q u e s t i o n r e m a i n s as t o w h e t h e r l i g n i f i c a t i o n is indeed responsible for i m p a i r e d f u n g a l development. W e have a l r e a d y briefly m e n t i o n e d a possible c h a i n o f events d u r i n g the i n c o m p a t i b l e i n t e r a c t i o n between h i g h l y resistant wheat leaves a n d a v i r u l e n t s t r a i n s of the s t e m rust fungus. L i g n i f i c a t i o n of the w h o l e cell contents m a y be regarded as a n active m e c h a n i s m for the p e n e t r a t e d host cell to c o m m i t " s u i c i d e " (30,31). T h i s h y p e r s e n s i t i v e cell d e a t h i n t u r n is t h o u g h t t o be responsible for the arrest of f u n g a l g r o w t h , p o s s i b l y b y d e p r i v i n g the obligate parasite o f its n u t r i t i o n a l basis (67,68.) T h e first step t h u s postulates l i g n i f i c a t i o n as the m e c h a n i s m o f a c t i v e cell d e a t h . C e l l d e a t h , a genetically p r o g r a m m e d event associated w i t h a c t i v e processes (69,70), is closely correlated w i t h l i g n i f i c a t i o n i n a range of different d e v e l o p m e n t a l p r o g r a m s : firstly, senescence is often a c c o m p a n i e d b y increased l i g n i f i c a t i o n (71,72); secondly, l i g n i f i c a t i o n is one o f the m o s t p r o m i n e n t reactions d u r i n g w o u n d h e a l i n g (73-79); a n d t h i r d l y , l i g n i f i c a t i o n o f d y i n g cells i n v a r i a b l y o c c u r s d u r i n g x y l e m d i f f e r e n t i a t i o n (80). A direct

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cause-effect r e l a t i o n s h i p of l i g n i f i c a t i o n a n d cell d e a t h has been described recently (81). T h e second step of the proposed c h a i n of events, resistance as a direct consequence of cell d e a t h , is s t i l l controversial (82-86). I n the w h e a t - s t e m rust s y s t e m , evidence f a v o u r i n g the i d e a of hypersensitive cell d e a t h as the cause of resistance (30,31,87-90) contrasts to results suggesting cell collapse as b e i n g a mere consequence of a yet u n k n o w n p r e c e d i n g resistance m e c h a n i s m (90-97). R e c e n t l y , K o g e l et al. (98) showed t h a t the i n j e c t i o n of galactoseb i n d i n g lectins or the e n z y m e galactose oxidase i n t o resistant wheat leaves p r e v e n t e d the hypersensitive cell d e a t h of p e n e t r a t e d host cells a n d c o n c o m i t a n t l y led t o increased f u n g a l g r o w t h , suggesting a causal r e l a t i o n s h i p between hypersensitive cell d e a t h a n d resistance. T i b u r z y (22,31) o b t a i n e d s i m i l a r results b y a p p l i c a t i o n of the P A L i n hibitor aminooxyacetic aci i n h i b i t P A L (99), a n d P A (100). T h u s , A O A a n d the related i n h i b i t o r a m i n o o x y p h e n y l p r o p i o n i c a c i d ( A O P P ) (101,102) i n h i b i t the biosynthesis of l i g n i n (103,104), a n t h o c y a n i n s (105), other flavonoids (106), a n d conjugates of c i n n a m i c acids (107) v i a P A L , as well as ethylene (108-110) v i a a p y r i d o x a l p h o s p h a t e dependent e n z y m e (110,111). I n v i e w of the possible f u n c t i o n of phenolic c o m p o u n d s as p h y t o a l e x i n s (21,112,113) a n d the w e l l d o c u m e n t e d role of ethylene i n some resistance reactions (114-116), the above c i t e d e x p e r i m e n t s w i t h A O A (22, 31) do not p r o v i d e any conclusive evidence for a causal role of l i g n i f i c a t i o n i n resistance. O u r recent observation (Moerschbacher & N o l l , u n p u b l i s h e d ) t h a t a different P A L i n h i b i t o r , ( l - a m i n o - 2 - p h e n y l e t h y l ) p h o s p h o n i c a c i d ( A P E P ) , is e q u a l l y a c t i v e i n p r e v e n t i n g hypersensitive cell d e a t h a n d p a r t i a l l y l o w e r i n g resistance of wheat to s t e m rust s t r o n g l y suggest t h a t the effects of b o t h i n h i b i t o r s is v i a their i n h i b i t o r y a c t i o n against P A L . Besides these w e l l k n o w n P A L i n h i b i t o r s , h i g h l y specific suicide i n h i b i t o r s of C A D are n o w available (117,118). These i n h i b i t o r s combine two i m p o r t a n t features: they act as s u b s t r a t e analogues a n d are able t o specifi c a l l y c o m p l e x z i n c ions. T h e c o m b i n a t i o n of these properties guarantees the h i g h specificity of these substances. Besides C A D , o n l y c i n n a m o y l : C o A reductase is i n h i b i t e d , b o t h enzymes are o n l y i n v o l v e d i n l i g n i n biosynthesis (119). T h e m e c h a n i s m of i n h i b i t i o n is t h o u g h t to be a pseudo-irreversible i n a c t i v a t i o n of the suicide type (120). S u c h i n h i b i t o r s are i d e a l l y s u i t e d for in-vivo assays, as they are h i g h l y specific a n d generally s l o w l y m e t a b o l i z e d (121). T h e a p p l i c a t i o n o f two o f these i n h i b i t o r s , N ( O - h y d r o x y p h e n y l ) s u l f i n a m o y l - t e r t i o b u t y l acetate a n d N ( O - a m i n o p h e n y l ) s u l f i n a m o y l - t e r t i o b u t y l acetate, to h i g h l y resistant wheat leaves infected w i t h a n a v i r u l e n t s t r a i n of s t e m rust r e s u l t e d i n decreased l i g n i f i c a t i o n a n d decreased necrosis of p e n e t r a t e d host cells a n d c o n c o m i t a n t l y led to increased f u n g a l development, o c c a s i o n a l l y even a l l o w i n g some s p o r u l a t i o n t o occur (60). T h e described i n h i b i t i o n of hypersensitive cell d e a t h b y the C A D i n -

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h i b i t o r s s t r o n g l y suggests a causal r e l a t i o n s h i p between l i g n i f i c a t i o n a n d cell d e a t h . It c a n f u r t h e r be c o n c l u d e d f r o m the increase o f f u n g a l g r o w t h u p o n C A D i n h i b i t i o n t h a t the synthesis of the m o n o m e r i c l i g n i n p r e c u r sors is c a u s a l l y related to resistance of wheat to s t e m r u s t . H o w e v e r , i t r e m a i n s s p e c u l a t i v e whether the p o l y m e r i z a t i o n of these m o n o m e r s to the t h r e e - d i m e n s i o n a l network of l i g n i n itself is a prerequisite for resistance. F u r t h e r m o r e , the proposed causal r e l a t i o n s h i p between cell d e a t h a n d resistance cannot be c o n c l u d e d f r o m these e x p e r i m e n t s ; b o t h p h e n o m e n a m a y be i n d e p e n d e n t results of the l i g n i f i c a t i o n process. Elicitation of Lignification T h e t e r m e l i c i t o r , i n i t i a l l y defined as a f u n g a l m e t a b o l i t e c a p a b l e o f i n d u c i n g p h y t o a l e x i n p r o d u c t i o n w h e n a p p l i e d to host p l a n t s (122, 123), has since been a p p l i e d to p a r a s i t e - d e r i v e d molecules w h i c h i n d u c e a n y facet o f resistance i n a p p r o p r i a t e hos T h e work of R i d e a n d his coworkers (24,125,126) showed t h a t p r o d u c t s o f filamentous f u n g i are able to e l i c i t l i g n i f i c a t i o n at w o u n d m a r g i n s i n wheat leaves. C h i t i n a n d some of its soluble derivatives have been s h o w n to be e l i c i t o r s o f l i g n i f i c a t i o n i n wheat (125), c h i t o s a n b e i n g m o r e a c t i v e t h a n c h i t i n (62,126). M o s t other /?-glucans i n c l u d i n g l a m i n a r i n d i d n o t e x h i b i t e l i c i t o r a c t i v i t y (62, 126). A crude e x t r a c t of Phytophthora megasperma f. sp. glycinea cell w a l l s , capable of i n d u c i n g g l y c e o l l i n a c c u m u l a t i o n i n soybeans, also e l i c i t e d l i g n i f i c a t i o n i n wheat (126), b u t p u r i f i e d e l i c i t o r f r o m the same source (62) a n d the s y n t h e t i c hepta-/?-glucoside e l i c i t o r (126) were ineffective. W e have p r e v i o u s l y r e p o r t e d the p u r i f i c a t i o n o f a n e l i c i t o r active f r a c t i o n f r o m cell walls of g e r m i n a t e d s t e m rust uredospores w h i c h induces l i g n i f i c a t i o n w h e n injected i n t o the i n t e r c e l l u l a r spaces of w h e a t leaves (62,63). Besides l i g n i f i c a t i o n , t h i s e l i c i t o r induces other s y m p t o m s t y p i c a l o f the h y persensitive r e a c t i o n , s u c h as m e m b r a n e d e g r a d a t i o n detected as increased a c t i v i t i e s of lipoxygenase (64) a n d phospholipase (65). T h e h i g h m o l e c u l a r weight w a t e r - s o l u b l e e l i c i t o r has been characterized as a heat s t a b l e g l y c o p r o t e i n w i t h the c a r b o h y d r a t e m o i e t y b e a r i n g the active p a r t ( s ) of the molecule(s) (62). Recent work identified a g l y c o p r o t e i n w i t h a b o u t e q u a l a m o u n t s o f galactose a n d mannose a n d almost no glucose as the m o s t a c t i v e c o m p o n e n t (127). I n i t i a l l y , a gene specific effect of t h i s e l i c i t o r c o n c e r n i n g the 5r«5-gene for resistance o f wheat to s t e m rust was r e p o r t e d (63). However, t h i s was not c o n f i r m e d i n later w o r k (66): E l i c i t o r s p r e p a r e d f r o m t w o races o f the s t e m rust fungus differing i n t h e i r s p e c t r u m o f genes for a v i r u l e n c e were e q u a l l y a c t i v e , a n d near-isogenic wheat lines c o n t a i n i n g different genes for resistance reacted s i m i l a r to b o t h e l i c i t o r s . T h e observed differences i n r e a c t i v i t y to e l i c i t o r s between different wheat c u l t i v a r s t u r n e d o u t to be u n r e l a t e d to a n y k n o w n resistance genes. S i m i l a r results h a d been r e p o r t e d p r e v i o u s l y for a n e l i c i t o r i s o l a t e d f r o m i n t e r c e l l u l a r w a s h i n g fluids of leaf rust infected wheat leaves w h i c h i n d u c e d b r o w n i n g a n d chlorosis (128,129). T h e r e a c t i v i t y of wheat c u l t i v a r s

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t o t h i s e l i c i t o r was dependent o n a gene o n chromosome 5 A w h i c h c o n t a i n s no k n o w n genes for seedling resistance to leaf or s t e m rust (129). It was s h o w n t h a t the same holds true for the s t e m rust e l i c i t o r (60, S u t h e r l a n d & Moerschbacher, unpublished). W h e n injected i n t o p r i m a r y leaves of different cereals, the s t e m rust e l i c i t o r causes s y m p t o m s w h i c h closely resemble the respective resistance reactions of these species against the a t t a c k b y s t e m rust of w h e a t , i.e., l i g n i f i c a t i o n i n b a r l e y a n d rye, b r o w n spots i n oat, a n d no v i s i b l e s y m p t o m s i n m a i z e (66). T h e b i o c h e m i c a l s i m i l a r i t i e s of leaf a n d s t e m rust e l i c i t o r s , the m i s s i n g gene specificity of b o t h e l i c i t o r s , the fact t h a t a n e q u a l l y active e l i c i t o r was i s o l a t e d f r o m g e r m tube cell walls of oat c r o w n rust (60), together w i t h the described reactions of different non-hosts t o the s t e m rust e l i c i t o r led us to speculate t h a t the isolated elicitors m a y p l a y a role i n the i n d u c t i o n of general m e c h a n i s m s o be i n v o l v e d i n the e l i c i t a t i o n of r a c e - c u l t i v a r specific resistance as w e l l , r a c e - c u l t i v a r specificity i n the w h e a t - s t e m rust s y s t e m c l e a r l y cannot b e e x p l a i n e d o n the basis of the specificity of the isolated e l i c i t o r s . O n e possible e x p l a n a t i o n w o u l d be the occurrence of r a c e - c u l t i v a r specific suppressors o f the resistance r e a c t i o n (124,131,132). P o l y g a l a c t u r o n i c a c i d has been s h o w n t o be a p o t e n t suppressor of the e l i c i t o r i n d u c e d l i g n i f i c a t i o n response i n wheat leaves (60). M o r e o v e r , p e c t i c fragments were o b t a i n e d f r o m isolated wheat cell walls b y p e c t o l y t i c digest i o n (36,60) or H F - s o l v o l y s i s ( M o e r s c h b a c h e r , R y a n , K o m a l a v i l a s , M o r t , u n p u b l i s h e d ) w h i c h suppressed the a c t i v a t i o n of l i g n i n biosynthesis w h e n injected s i m u l t a n e o u s l y w i t h e l i c i t o r . A c t i v e c o m p o n e n t s were t e n t a t i v e l y classified as fragments of h o m o g a l a c t u r o n a n a n d r h a m n o g a l a c t u r o n a n I. A n i n d e p t h c h e m i c a l c h a r a c t e r i z a t i o n as w e l l as a n e v a l u a t i o n o f possible r a c e - c u l t i v a r specificity of these "endogenous suppressors" or t h e i r in vivo p r o d u c t i o n d u r i n g c o m p a t i b l e a n d i n c o m p a t i b l e w h e a t - s t e m rust i n t e r a c t i o n s is under way. T h e i n j e c t i o n of s m a l l a m o u n t s of p e c t o l y t i c enzymes c o n c o m i t a n t l y w i t h elicitors suppressed the i n d u c t i o n of l i g n i n b i o s y n t h e t i c e n z y m e act i v i t i e s as w e l l (60). T h e i n v e s t i g a t i o n of pectic enzymes of the s t e m rust fungus (133) a n d the e l u c i d a t i o n of their substrate specificities, c o m b i n e d w i t h a d e t a i l e d a n a l y s i s of their n a t u r a l substrates, the pectic c o m p o n e n t s of p r i m a r y wheat cell w a l l s , are targets for future research. T h e h i g h l y developed r a c e - c u l t i v a r specificity i n the w h e a t - s t e m rust s y s t e m m a y be based o n the differential l i b e r a t i o n of active suppressors d u r i n g the penet r a t i o n of the host cell w a l l b y the h a u s t o r i a l neck of the fungus ( F i g u r e 1). A l t h o u g h s p e c u l a t i v e , a m o d e l can be d r a w n i n w h i c h suppressors w h i c h p o t e n t i a l l y exist i n the walls of a l l wheat varieties are set free o n l y i n c o m p a t i b l e i n t e r a c t i o n s . T h e f a i l u r e of p r o d u c i n g active suppressors d u r i n g i n c o m p a t i b l e interactions w o u l d t h e n lead t o the e l i c i t a t i o n of l i g n i f i c a t i o n as the m e c h a n i s m of hypersensitive cell d e a t h a n d resistance of wheat t o stem rust.

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cell

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wall

degrading enzymes

cell

wall

host

cell

hypersensitive successful haustorium

cell

death

formation resistance

• susceptibility

F i g u r e 1. H y p o t h e t i c a l scheme of events l e a d i n g t o r a c e - c u l t i v a r specific resistance or s u s c e p t i b i l i t y i n the rust s y s t e m . If the substrate specificity o f the f u n g a l cell w a l l d e g r a d i n g enzymes (e.g., pectinases) is s u i t a b l e for d e g r a d a t i o n o f a specific host cell w a l l c o m p o n e n t (e.g., p a r t l y esterified p e c t i n ) , endogenous suppressors w i l l be p r o d u c e d w h i c h prevent the e l i c i t o r i n d u c e d l i g n i f i c a t i o n response, thus l e a d i n g t o s u s c e p t i b i l i t y .

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A cknowledgment s T h e a u t h o r ' s w o r k r e p o r t e d i n t h i s c o m m u n i c a t i o n was c a r r i e d o u t u n d e r the constant advice a n d encouragement f r o m P r o f . D r . H . J . Reisener, i n c o l l a b o r a t i o n w i t h U . N o l l , B . E . F l o t t , U . W i t t e , D . Kônigs, A . W u s t e f e l d , U . G o t t h a r d t , D r . F . Schrenk, D r . M . Sutherland, D r . J . R y a n , D r . P . K o m a l a v i l a s a n d P r o f . D r . A . J . M o r t . T h e P A L i n h i b i t o r A P E P was generally provided b y Prof. D r . F . J . Schwinn, C i b a Geigy A G , Basel, Switzerl a n d . P r o f . D r . L . G o r r i c h o n , Université P a u l S a b a t i e r , T o u l o u s e , F r a n c e , k i n d l y p r o v i d e d t h e C A D i n h i b i t o r s . T h e work was s u p p o r t e d i n p a r t b y g r a n t s f r o m t h e L a n d N o r d r h e i n - W e s t f a l e n , t h e Deutsche Forschungsgem e i n s c h a f t , a n d t h e Deutscher A k a d e m i s c h e r A u s t a u s c h d i e n s t .

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106. 107. 108. 109. 110.

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In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 28

Virulence-Inducing Phenolic Compounds Detected by Agrobacterium tumefaciens Paul A. Spencer and G. H. N. Towers Department of Botany, University of British Columbia, Vancouver, British Columbia V6T 2B1, Canada

Construction of sequent analysis terium tumefaciens has permitted the discovery of a class of phytochemicals that this pathogen detects and which induce virulence. Preliminary screening of a variety of commercially available phenolics revealed that some were active as vir inducers. Two acetophenones were isolated from transformed tobacco root cultures. The results of a recent study by us indicate that there exists a range of virulence-inducing plant phenolics which are not limited to acetophenones but include chalcones as well as cinnamic acid derivatives. Among the latter are acids, alcohols and esters known to be associated with plant cell walls or implicated in lignin biosynthesis, a discovery which suggests that this wide host range pathogen likely responds to chemicals common to all susceptible hosts. We are currently studying signal compounds and natural inhibitors in relation to the host range of Agrobacterium strains. Activity was detected in extracts from a grapevine tissue culture, grapevine bark, and flavan-containing fractions obtained from grapes. In addition, as yet unidentified compounds inhibitory to vir­ -induction have been discovered. It is n o w k n o w n t h a t the i n i t i a l i n t e r a c t i o n between p l a n t s a n d b a c t e r i a of the R h i z o b i a c e a e is a c h e m i c a l d e t e c t i o n b y the m i c r o b e o f a s u s c e p t i ble host, i . e . , t h e host produces c o m p o u n d s w h i c h act as signals for the m i c r o b i a l p a t h o g e n o r s y m b i o n t . T h e m i c r o b e responds t o these signals b y expression o f genes necessary i n subsequent stages o f the i n t e r a c t i o n . F o r a few o f the R h i z o b i a c e a e some s i g n a l c o m p o u n d s i n v o l v e d have been identified (1-7). 0097-6156/89/0399-0383$06.00A) © 1989 American Chemical Society

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T h e s i g n a l c o m p o u n d s o f p l a n t - Agrobacterium tumefaciens i n t e r a c t i o n s have received m u c h a t t e n t i o n . T h i s g r a m negative s o i l b a c t e r i u m , w h i c h causes c r o w n g a l l disease o f a w i d e variety o f dicotyledonous p l a n t s (8), is responsible for a neoplastic g r o w t h of the p l a n t tissue b y passing T - D N A , a p a r t o f its t u m o r i n d u c i n g p l a s m i d ( p T i ) , into the host p l a n t genome (9-14). T h i s T - D N A includes genes w h i c h encode enzymes of a u x i n (15,16) a n d c y t o k i n i n (17,18) biosynthesis, w h i c h are expressed i n the t r a n s f o r m e d p l a n t cell (19,20). T h e m i c r o b e is a useful vector for genetic engineering i n p l a n t s because c e r t a i n of the n o r m a l T - D N A genes m a y be replaced w i t h new genes o f interest. P l a n t cells infected w i t h the b a c t e r i u m c o n t a i n i n g the m o d i f i e d T i - p l a s m i d are used to generate transgenic p l a n t s . D e t e c t i o n of susceptible host cells a n d e a r l y stages of tumorogenesis are m a i n l y c o n t r o l l e d by a set of p T i genes k n o w n as the v i r u l e n c e (vir) genes (21,22). T h e s e genes are expressed u p o n c o c u l t i v a t i o n of the b a c t e r i a w i t h host p l a n t cells (23,24) . Because of their role i n the early stages of tumorogenesis, a n d therefor p l a n t genomes, research has been directed t o u n d e r s t a n d i n g the m e c h a n i s m i n v o l v e d i n vir gene expression a n d i d e n t i f y i n g the v i r gene p r o d u c t s . T w o o f the vir genes ( A a n d G ) are r e g u l a t o r y i n n a t u r e (25,26). vir A is also a host range d e t e r m i n a n t a n d is thought to be the e n v i r o n m e n t a l sensor of the p l a n t - d e r i v e d inducer molecules (27). A t least one more vir locus (virC) is connected w i t h host range (28-30), a n d another (the virO o p e r o n ) is now k n o w n to encode a n endonuclease w h i c h recognizes a n d cleaves the left a n d right b o r d e r sequences of T - D N A (31). T h e virB region encodes p o l y p e p ­ tides s i m i l a r t o those i n v o l v e d i n b a c t e r i a l c o n j u g a t i o n (32). R e c e n t l y i t was d e t e r m i n e d t h a t virE encodes a single s t r a n d e d D N A - b i n d i n g p r o t e i n (33). A c t i v a t i o n of v i r gene expression is k n o w n to result i n the p r o d u c t i o n o f m u l t i p l e s i n g l e - s t r a n d e d T - D N A molecules w i t h i n the b a c t e r i u m (34). B o l t o n et al. (35) f o u n d t h a t a m i x t u r e of s i m p l e , low m o l e c u l a r weight p h e n o l i c c o m p o u n d s c o u l d be used to i n d u c e expression of most of the vir genes. S t a c h e l et al. (7) i d e n t i f i e d two active s i g n a l c o m p o u n d s , acetosyringone ( A S ) a n d α-hydroxyacetosyringone ( H O - A S ) , f r o m tobacco tissues. In t h a t r e p o r t a few other related c o m p o u n d s were assayed at one or more c o n c e n t r a t i o n s for t h e i r v i r - i n d u c i n g a c t i v i t y . T h i s c o m p r i s e d a very b r i e f s t r u c t u r e - a c t i v i t y s t u d y w h i c h presented some i n f o r m a t i o n about the s t r u c ­ t u r a l features r e q u i r e d to confer a c t i v i t y . A t the concentrations tested, none of these c o m p o u n d s d i s p l a y e d the level of a c t i v i t y observed w i t h acetosyr i n g o n e . It was suggested t h a t Agrobacterium is a t t r a c t e d to susceptible p l a n t tissues b y f o l l o w i n g a c o n c e n t r a t i o n gradient o f these virulence i n d u c ­ i n g substances, a n d some results w h i c h s u p p o r t t h i s i d e a were o b t a i n e d b y A s h b y et al. (36,37). T h e c o m p o u n d s A S a n d H O - A S have come to be regarded as the u n i q u e c h e m i c a l s w h i c h Agrobacterium detects i n n a t u r e a n d w h i c h trigger the i n i ­ t i a l events w i t h i n the b a c t e r i u m , r e s u l t i n g i n t u m o r f o r m a t i o n . However, it has yet t o be s h o w n t h a t these acetophenones, w h i c h i n fact have never pre­ v i o u s l y been r e p o r t e d as n a t u r a l l y o c c u r r i n g p h y t o c h e m i c a l s , are the s i g n a l c o m p o u n d s p r o d u c e d by any other susceptible hosts. A S has been used to

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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boost the t r a n s f o r m a t i o n efficiency (38). H o w e v e r , t r a n s f o r m a t i o n of soyb e a n cells was p r o m o t e d b y a d d i n g either A S or s y r i n g a l d e h y d e [ l b ] to the i n o c u l u m (39). I n a d d i t i o n , v i r u l e n c e i n d u c i n g w o u n d exudates o b t a i n e d f r o m a host p l a n t e x t e n d e d the n o r m a l host range of Agrobacierium to i n clude a m o n o c o t crop p l a n t (40). W e propose t h a t o t h e r p h y t o c h e m i c a l s are i n v o l v e d i n the i n d u c t i o n o f v i r u l e n c e i n Agrobacterium. W e recently r e p o r t e d the v i r - i n d u c i n g a c t i v i t y over a range of concent r a t i o n s of a v a r i e t y of p l a n t - d e r i v e d p h e n o l i c c o m p o u n d s w i t h s t r u c t u r e s related to t h a t of acetosyringone a n d discussed the s t r u c t u r a l features necessary for the a c t i v a t i o n o f v i r genes (41). T h e a c t i v i t i e s of some c i n n a m i c a c i d d e r i v a t i v e s , chalcones, a n d of the l i g n i n precursors s i n a p y l a l c o h o l a n d c o n i f e r y l a l c o h o l were e x a m i n e d . A n u m b e r of these c o m p o u n d s are of w i d e s p r e a d occurrence, a n d others such as the m o n o l i g n o l s are u b i q u i t o u s i n angiosperms a n d g y m n o s p e r m s . In t h i s r e p o r t we review the results of our structure-activity analysi n a r y results o f o u r searc A. tumefaciens. R e s e a r c h has revealed t h a t the v i r A gene p r o d u c t is l o c a t e d at the b a c t e r i a l cell surface where i t l i k e l y acts as the e n v i r o n m e n t a l sensor of p l a n t d e r i v e d s i g n a l c o m p o u n d s (27). T h e v i r A l o c i of l i m i t e d host range ( L H R ) a n d w i d e host range ( W H R ) s t r a i n s of Agrobacterium were sequenced a n d the p r e d i c t e d gene p r o d u c t s c o m p a r e d . T h e gene p r o d u c t s were f o u n d to have diverged most s t r o n g l y i n t h e i r p u t a t i v e p e r i p l a s m i c d o m a i n . T h e r e fore we considered t h a t differences i n these gene p r o d u c t s m i g h t c o r r e s p o n d to differences i n t h e i r specificity for s i g n a l c o m p o u n d s , a n d t h i s i n p a r t m a y e x p l a i n the differences observed i n the host range of these two types of Agrobacterium. T o prove t h i s h y p o t h e s i s , i n d u c t i o n of other vir l o c i i n the presence of L H R host p l a n t cells, or b y w o u n d exudates thereof, h a d t o be d e m o n s t r a t e d . T h e s i g n a l c o m p o u n d s t h e n h a d t o be i s o l a t e d a n d i d e n t i f i e d . I n i t i a l l y , however, o n l y a v i r B : : / a c Z gene-fusion c o n t a i n i n g L H R s t r a i n o f Agrobacterium ( A 8 5 6 / p S M 2 4 3 c d ) was a v a i l a b l e . I n o u r p r e l i m i n a r y e x p e r i m e n t s w i t h t h i s s t r a i n , v i r expression was n o t g r e a t l y i n d u c e d b y c o c u l t i v a t i o n w i t h host p l a n t cells or b y p u r i f i e d w o u n d - i n d u c e d p h e n o l i c c o m p o u n d s ; therefore we w i s h e d to e x a m i n e v i r - i n d u c t i o n i n a different c o n s t r u c t , n a m e l y , the L H R s t r a i n c a r r y i n g a lacL f u s i o n to a different vir gene. W e have p r e p a r e d a v i r E : : / a c Z gene f u s i o n - c o n t a i n i n g L H R s t r a i n , A 8 5 6 / p S M 3 5 8 c d , b y t r i p a r e n t a l m a t i n g a n d have used t h i s s t r a i n , a l o n g w i t h the s t r a i n A 8 5 6 / p S M 2 4 3 c d , to e x a m i n e v i r gene expression i n the l i m i t e d host range (grapevine) s t r a i n A 8 5 6 . P h e n o l i c w o u n d exudates f r o m the leaves a n d stems of Vitis lubrusca, as w e l l as exudates p r o d u c e d by t w o g r a p e v i n e c a l l u s cultures ( Vitis sp. cv. S e y v a l a n d V. lubruscana cv. S t e u b e n ) a n d e x t r a c t s o b t a i n e d f r o m two varieties o f grapes (red a n d green seedless) a n d g r a p e v i n e b a r k (cv. C o n c o r d ) were e x a m i n e d for v i r - i n d u c i n g c o m p o u n d s . W e have established t h a t the specificity of L H R s i g n a l c o m p o u n d s is u n l i k e t h a t p r e v i o u s l y described for W H R A. tumefaciens (41) i n t h a t vir gene expression is not as g r e a t l y i n d u c e d b y acetosyringone.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

386

PLANT C E L L W A L L P O L Y M E R S

T h i s i n d i c a t e s t h a t , u n l i k e the W H R s t r a i n s of Agrobacterium, the L H R s t r a i n s are less sensitive to p h e n y l p r o p a n o i d m e t a b o l i t e s . P r e l i m i n a r y results i n d i c a t e t h a t the v i r u l e n c e of grapevine isolates of Agrobactenum may be influenced b y the presence o f c e r t a i n higher m o l e c u l a r weight p h e n o l i c esters of grape flavans i n a d d i t i o n to less p o l a r , c h l o r o f o r m soluble phenolics present i n aqueous g r a p e v i n e - s t e m w o u n d exudates. A n u n d e r s t a n d i n g of the p h y t o c h e m i s t r y of vir gene expression i n b o t h W H R (7,35,41) a n d i n L H R Agrobacterium s h o u l d p r o v i d e an i n t e r e s t i n g a n d p o t e n t i a l l y useful m o d e l s y s t e m of host range c o n t r o l i n p l a n t - b a c t e r i a l i n t e r a c t i o n s . N e w insights i n t o r e g u l a t i o n of host range are of i m p o r t a n c e i n p l a n t b i o c h e m i s t r y , biotechnology a n d p a t h o l o g y i n t h a t c h e m i c a l clues are p r o v i d e d w h i c h c o u l d allow for extension of t h i s pathogen's host range t o i n c l u d e species w h i c h are refractory to t r a n s f o r m a t i o n . Results and Discussion In o u r a n a l y s i s o f the c h e m i c a l s t r u c t u r e s w h i c h are a c t i v e vir-inducers (41) i t was f o u n d t h a t the c o m p o u n d s fell i n t o four groups: (1) acetophenones a n d related s t r u c t u r e s , (2) m o n o l i g n o l s , (3) h y d r o x y c i n n a m i c acids a n d t h e i r esters, a n d (4) chalcone derivatives ( F i g . 1). E a c h c o m p o u n d h a d either a g u a i a c y l or a s y r i n g y l nucleus, a n d w i t h the exception of the m o n o l i g n o l s , possessed a c a r b o n y l g r o u p . M o s t were of c o m m o n occurrence i n vascular p l a n t s . T h e a c t i v i t y curves w i t h increasing c o n c e n t r a t i o n of a n u m b e r of viri n d u c i n g p h e n o l i c c o m p o u n d s are s h o w n i n F i g u r e s 2 a ( m o n o l i g n o l s , c h a l cones a n d acetophenones) a n d 2b (phenolic acids a n d their m e t h y l esters). R e g a r d i n g the m o n o l i g n o l s , we e m p h a s i z e d the b a c t e r i u m ' s a b i l i t y to res p o n d t o the presence of these l i g n i n precursors. T h i s result established t h a t Agrobactenum m a y be capable of d e t e c t i n g cells w h i c h are u n d e r g o i n g l i g n i n synthesis or cell w a l l repair a n d thereby target those cells for t r a n s f o r m a t i o n . Agrobacterium responded e q u a l l y w e l l to the presence of l i g n i n d e g r a d a t i o n p r o d u c t s (7,35,41) a n d therefore the virulence of the m i c r o b e c a n be considered as sensitive to l i g n i n metabolites i n general. T h e u n i q u e a c t i v i t y curves of the chalcones [ 4 a & 4 b ] represent an i n t e r e s t i n g a d d i t i o n to the list of effective phenolics. T h e m e t h y l esters of ferulic [3d], s y r i n g i c [ I f ] , a n d s i n a p i c acids [3f] e x h i b i t e d s i g n i f i c a n t l y greater a c t i v i t y t h a n the c o r r e s p o n d i n g free acids ( F i g . 2b). Some effects of esterification are discussed below. T h e e t h y l esters tested were less a c t i v e a g a i n ( d a t a not s h o w n ) , p e r h a p s due to altered h y d r o p h i l i c i t y of the c o m p o u n d or some steric h i n d r a n c e at the b a c t e r i a l receptor site not evident w i t h the m e t h y l esters. In a p l a t e assay for viri n d u c t i o n we discovered t h a t the glucose ester of ferulic a c i d was a n effective i n d u c e r of vir gene expression. It is likely t h a t such glucose esters, released u p o n w o u n d i n g of the host p l a n t , act as s i g n a l c o m p o u n d s . P h e n o l i c g l y cosides, such as glucoferulaldehyde, were i n a c t i v e . W e assayed a n u m b e r o f other phenolic c o m p o u n d s , i n c l u d i n g aurones, flavones, flavanones, flavanols, lignans a n d 5 - h y d r o x y c i n n a m i c a c i d d e r i v a tives, b u t they a l l d i s p l a y e d l i t t l e or no a c t i v i t y . T h e lack of a c t i v i t y of

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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R2 H

H

la

OMe

b

H

c

CH

3

d

CH

3

H OMe

e

OH

OMe

f

OMe

OMe

2a b

R H OMe

3a b c d e f

Ri CH OH OH OMe OMe OMe

CH OH 2

OMe

3

Ri Η Η OMe Η OH OMe

OMe .OH HO.

4a b

R Η OMe

OH F i g u r e 1. T h e s t r u c t u r e s of the w r - i n d u c i n g p h e n o l i c c o m p o u n d s e m p l o y e d i n o u r s t r u c t u r e - a c t i v i t y a n a l y s i s (41).

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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CONCENTRATION (μΜ) F i g u r e 2. T h e virulence i n d u c i n g a c t i v i t y o f (a) m o n o l i g n o l s , chalcones, a n d acetophenones, a n d (b) phenolic acids a n d their m e t h y l esters. F o l ­ l o w i n g i n c u b a t i o n w i t h a c o m p o u n d i n aqueous s o l u t i o n , /?-galactosidase a c t i v i t y i n a s t r a i n o f Agrobacterium c a r r y i n g a virE.lacZ fusion p l a s m i d ( A 3 4 8 / p S M 3 5 8 ) was assayed as a n i n d i c a t o r o f v i r gene i n d u c t i o n . Abréviations used: A S = acetosyringone; C O N . A L C O H O L = coniferyl alcohol; S I N . A L C O H O L = sinapyl alcohol; C H A L C O N E A = 2',4',4-trihydroxy3 - m e t h o x y chalcone; C H A L C O N E Β = 2', 4', 4 - t r i h y d r o x y - 3 , 5 d i m e t h o x y chalcone.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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5 - h y d r o x y f e r u l i c a c i d was of interest because i t was recently i d e n t i f i e d as one of the cell w a l l b o u n d acids i n m o n o c o t s (42). A t t h a t t i m e we h a d not d e m o n s t r a t e d i n h i b i t i o n of v i r - i n d u c t i o n b y a p h e n o l i c c o m p o u n d , a l t h o u g h we s p e c u l a t e d a b o u t the occurrence of p h e n o l i c w r - i n h i b i t o r s i n m o n o c o t s . Indeed, we have been i n f o r m e d of u n i d e n t i f i e d w r - i n h i b i t o r s recently iso­ l a t e d f r o m Zea mays ( E . W . N e s t e r , p e r s o n a l c o m m u n i c a t i o n ) , a n d have i n i t i a t e d w o r k o n the i d e n t i f i c a t i o n of w r - i n h i b i t o r s f r o m Vitis species. I n a d d i t i o n to the i n a c t i v e c o m p o u n d s listed above, each of the c o m ­ p o u n d s used b y B o l t o n et ai (35) was assayed i n d i v i d u a l l y , a n d of these o n l y v a n i l l i n [ l a ] p r o d u c e d a n y significant v i r - i n d u c t i o n . Interestingly, at the c o n c e n t r a t i o n s e x a m i n e d by us the r e m a i n i n g c o m p o u n d s (gallic, βr e s o r c y l i c , p y r o g a l l i c , p - h y d r o x y b e n z o i c , a n d p r o t o c a t e c h u i c acids, a n d c a t ­ echol) were essentially i n a c t i v e . N o n e of these i n a c t i v e c o m p o u n d s has a g u a i a c y l or s y r i n g y l nucleus. W e have observed low level i n d u c t i o n b y g a l l i c a c i d at higher c o n c e n t r a t i o n s (e.g., I m M , d a t a not s h o w n ) . T h e possible significance of t h i s result discussed below. T h e results i n d i c a t e d t h a t two basic s t r u c t u r a l features were r e q u i r e d t o confer a c t i v i t y u p o n a c o m p o u n d : (1) g u a i a c y l or ( u s u a l l y c o n f e r r i n g enhanced a c t i v i t y ) s y r i n g y l s u b s t i t u t i o n on a benzene r i n g , a n d (2) a carb o n y l g r o u p o n a s u b s t i t u e n t p a r a to the h y d r o x y s u b s t i t u e n t o n the r i n g . M o n o l i g n o l s , however, are a c t i v e even t h o u g h there is no c a r b o n y l f u n c t i o n i n the side c h a i n i n the p a r a p o s i t i o n . W h e n present, the c a r b o n y l c a r b o n m a y be one or three c a r b o n a t o m s removed f r o m the r i n g . H o w e v e r , to confer m a x i m a l a c t i v i t y , i n the l a t t e r case there m u s t be a double b o n d between the c a r b o n y l c a r b o n a n d the r i n g , as is present i n the chalcones a n d c i n n a m i c a c i d derivatives. F u r t h e r m o r e , the c a r b o n y l g r o u p of a free a c i d is less effective t h a n t h a t of the c o r r e s p o n d i n g ester. E s t e r i f i c a t i o n a l ­ ters the s o l u b i l i t y of the c o m p o u n d . In a d d i t i o n , esterification prevents one o x y g e n of the c a r b o x y l g r o u p f r o m f o r m i n g a p a r t i a l d o u b l e b o n d , thereby r e n d e r i n g the c a r b o n y l group more reactive. In these cases, a n d the case of the aldehydes a n d chalcones, t h i s c a r b o n y l g r o u p forms the t e r m i n u s of a c o n j u g a t e d double b o n d s y s t e m r u n n i n g f r o m the h y d r o x y l g r o u p a n d t h r o u g h the r i n g . T h e presence of a C r i n g i n the flavonoids tested v i r t u ­ a l l y a b o l i s h e d a c t i v i t y , i n d i c a t i n g t h a t the more t y p i c a l flavonoids are not a c t i v e i n t h i s cell-cell s i g n a l l i n g . T h e s e s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s are different f r o m those r e p o r t e d for the a c t i v a t i o n of nod genes i n Rhizobium species (1-6). H y d r o x y l a t e d flavones, isoflavones or flavanones i n n M to μ Μ c o n c e n t r a t i o n s i n d u c e ex­ p r e s s i o n of nod genes. E a c h Rhizobium species is not o n l y h i g h l y specific for its host p l a n t species b u t also d i s p l a y s a h i g h degree of specificity towards its s i g n a l c o m p o u n d . I n c o n t r a s t , the o r i g i n a l s t r a i n of A. tumefaciens, from w h i c h the s t r a i n used i n our s t u d y was d e r i v e d , e x h i b i t e d a w i d e host range ( W H R ) a n d , as we have seen, a c o m p a r a t i v e l y lower degree of s i g n a l c o m ­ p o u n d specificity. F u r t h e r m o r e , some of the very c o m p o u n d s w h i c h induce vir genes i n Agrobacterium s t r o n g l y i n h i b i t nod gene a c t i v a t i o n by these flavonoids (1). A t higher concentrations most of the w r - i n d u c i n g phenolics

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were b a c t e r i o s t a t i c even against Agrobacterium ( d a t a not s h o w n ) , a n d pre­ s u m a b l y they act i n t h i s way against Rhizobium species, or they m a y act m o r e d i r e c t l y b y c o m p e t i t i v e i n h i b i t i o n of n o d - i n d u c t i o n . A n u m b e r o f these a c t i v e c o m p o u n d s are of widespread occurence d i ­ cotyledons. T h e l i g n i n precursors are u b i q u i t o u s i n susceptible hosts. It is t e m p t i n g t o conclude t h a t the presence o f a n y one of these c o m p o u n d s alone w o u l d determine whether a given p l a n t is susceptible to i n f e c t i o n b y Agrobacterium. However, m o n o c o t s also p r o d u c e these c o m p o u n d s , even e x u d i n g t h e m i n t o the rhizosphere f r o m i n t a c t roots (43) a n d yet, w i t h few exceptions (44-47), they lie outside of the n a t u r a l host range of a n y s t r a i n of Agrobacterium. T h i s l i m i t a t i o n of host range r e m a i n s a significant p r o b l e m i n the use o f t h i s o r g a n i s m as a vector for genetic engineering i n m o n o c o t s . T h e a t t a c h m e n t of Agrobacterium to m o n o c o t cells has been r e p o r t e d (48, a n d references t h e r e i n ) . T h e r e f o r e the u n d e r l y i n g m e c h a n i s m of host range d e t e r m i n a t i o n appears t o d e p e n d , at least i n p a r t , o n the p h y t o c h e m i s t r y o f the i n t e r a c t i o n . T h e recentl m o n o c o t a n d , as w i l l be discussed, L H R host exudates s u p p o r t s the concept o f a p h e n o l i c m i l i e u i n w h i c h v i r - i n d u c e r s c o m p e t e w i t h v i r - i n h i b i t o r s . It m a y be t h a t a s o p h i s t i c a t e d a p p l i c a t i o n of inducer c o m p o u n d s w i l l p e r m i t the T i - p l a s m i d - m e d i a t e d t r a n s f o r m a t i o n o f p l a n t species n o r m a l l y resistant to infection. O u r i n i t i a l e x p e r i m e n t s , u s i n g /?-galactosidase a c t i v i t y i n A 8 5 6 / p S M 2 4 3 c d as a n i n d i c a t o r o f v i r - i n d u c t i o n , suggested t h a t w o u n d - i n d u c e d phenolics f r o m the leaves of V. lubrusca d i d not i n c l u d e any L H R s i g n a l c o m p o u n d s . W e considered the f o l l o w i n g p o s s i b i l i t i e s : (1) t h a t the source of the n a t u r a l L H R s i g n a l c o m p o u n d m i g h t be tissue specific (e.g., l i m i t e d t o the roots or c r o w n of the g r a p e v i n e ) , (2) t h a t u n l i k e the W H R s i g n a l c o m p o u n d s , these u n k n o w n chemicals m a y not be low m o l e c u l a r weight phenolics (e.g., p h e n y l p r o p a n o i d s or acetophenones) e x t r a c t able w i t h the solvents used, a n d (3) t h a t the s i g n a l c o m p o u n d c o u l d be a p h y t o a l e x i n p r o d u c e d o n l y after i n f e c t i o n o f the grapevine tissue. In order to c o n f i r m our results w i t h A 8 5 6 / p S M 2 4 3 c d we prepared the s t r a i n A 8 5 6 / p S M 3 5 8 c d as described. T h e p l a s m i d p S M 3 5 8 c d c o n t a i n s a virE::lacZ gene f u s i o n , a n d i t was f o u n d t h a t the higher levels of βgalactosidase a c t i v i t y w h i c h are i n d u c i b l e f r o m t h i s construct (35) p e r m i t ­ ted d e t e c t i o n o f even v a n i s h i n g l y s m a l l a m o u n t s of i n d u c i n g c o m p o u n d . T h e results w i t h t h i s new s t r a i n confirmed our i n i t i a l results; neither acetosy­ ringone i t s e l f nor a n y of the isolated w o u n d - i n d u c e d p h e n o l i c c o m p o u n d s f r o m grape leaves g r e a t l y i n d u c e d the v i r u l e n c e genes of the L H R A. tume­ faciens. O b v i o u s l y either the techniques used to isolate c o m p o u n d s f r o m the host m a t e r i a l were not a p p r o p r i a t e for the i s o l a t i o n of L H R inducers, or the L H R s t r a i n cannot efficiently detect the W H R s i g n a l c o m p o u n d s due to i t s different vir A gene p r o d u c t . A s s u m i n g the former to be the case, we considered t h a t the L H R i n d u c ­ ers m i g h t be m o r e p o l a r i n n a t u r e a n d were therefore e x c l u d e d b y s e p a r a t o r y techniques for c o m p o u n d s such as those k n o w n to i n d u c e W H R A. tumefa­ ciens. P r o a n t h o c y a n i d i n m o n o m e r s (i.e., catechins, flavan-3-ols), oligomers,

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a n d esters s h o u l d be prevalent i n the w o o d y tissue o f the g r a p e v i n e . S u c h c o m p o u n d s have been s t u d i e d i n grapes w i t h respect t o w i n e q u a l i t y (49) a n d recently r e v i e w e d w i t h respect to t h e i r possible p h y s i o l o g i c a l role i n c o n n e c t i o n w i t h l i g n i n (50). I n v i e w of the c h e m i c a l s p e c i f i c i t y of v i r u l e n c e i n d u c t i o n i n W H R Agrobacterium (41), a feature w h i c h i n i t s s e n s i t i v i t y was n o t s h a r e d b y the L H R s t r a i n used i n t h i s s t u d y , i t was n o t e w o r t h y t h a t these flavans have been envisaged as f u n c t i o n a l l y connected w i t h l i g n i n . I n terestingly, i n a review o n p r o a n t h o c y a n i d i n s a n d l i g n i n c h e m i s t r y , Stafford (50) m e n t i o n e d the p h e n o m e n o n of p l a n t p h e n o l i c c o m p o u n d s as m o l e c u l a r signals for Rhizobium a n d Agrobacterium, a n d recognized the p o t e n t i a l " i n f o r m a t i o n a l " f u n c t i o n of b o t h types of c o m p o u n d s . W e a n a l y z e d the flavan-containing e x t r a c t s o b t a i n e d f r o m t w o l o c a l l y available varieties of grapes a n d detected a c t i v i t y i n the f r a c t i o n s c o n t a i n i n g , a m o n g other p h e n o l i c s , flavan m o n o m e r s , d i m e r s a n d esters. T h e s e c o m p o n e n t s were separated b y c h r o m a t o g r a p h y o n S e p h a d e x L H 20. F r a c t i o n s w i t h s i m i l a r t h i n laye a n d at least 3 o u t of 12 s u c h p o o l e d samples c o n t a i n e d substances w h i c h res u l t e d i n w r - i n d u c t i o n i n the L H R s t r a i n . S i m i l a r l y , g r a p e v i n e b a r k flavans were e x a m i n e d , b u t o n l y c o m p o u n d s i n d u c i n g very low levels of vir gene e x p r e s i o n were f o u n d . I n fact, i n a d d i t i o n to vz'r-inducers, a v i r - i n h i b i t o r y e x t r a c t was o b t a i n e d f r o m g r a p e v i n e b a r k . In p l a t e assays, t h i s L H R hostd e r i v e d i n h i b i t o r y substance c o m p l e t e l y prevented W H R w r - i n d u c t i o n by acetosyringone. R e p e a t e d T L C of a c t i v e , p o o l e d fractions f r o m R e d F l a m e grapes revealed m a j o r spots w i t h R / ' s c o r r e s p o n d i n g to those of c a t e c h i n a n d e p i c a t e c h i n , i d e n t i c a l color r e a c t i o n w i t h p-toluenesolfonic a c i d s p r a y reagent, a n d c o e l u t i o n o f t r i m e t h y l s i l a n e ( T M S ) d e r i v a t i v e s b y G C w i t h reference s a m p l e s of these flavans. H o w e v e r c a t e c h i n a n d e p i c a t e c h i n were assayed w i t h the b a c t e r i a l s t r a i n s described a n d no a c t i v i t y was d e t e c t e d . T M S d e r i v a t i z e d samples of a c t i v e grape flavans were e x a m i n e d b y G C - M S , b u t a search for the m o l e c u l a r ions of a n u m b e r of k n o w n v i r - i n d u c i n g phenolics y i e l d e d negative results. Isolated c o m p o u n d s were collected after s e p a r a t i o n by H P L C a n d assayed for w r - i n d u c t i o n i n the s t r a i n s d e s c r i b e d . It b e c a m e a p p a r e n t t h a t the l i m i t e d host range s t r a i n responded to a l l the substances t h a t i n d u c e d vir expression i n the w i d e host range s t r a i n , b u t the L H R s t r a i n was less sensitive to the same substances a n d as a result was considered a less sens i t i v e bioassay o r g a n i s m . Therefore work o n i s o l a t i o n of g r a p e v i n e - d e r i v e d s i g n a l c o m p o u n d s c o n t i n u e d u s i n g the more sensitive W H R s t r a i n a n d p l a t e assay s y s t e m d e s c r i b e d . U s i n g A 3 4 8 / p S M 3 5 8 as o u r bioassay o r g a n i s m w i t h w h i c h to detect the w r - i n d u c i n g substances, we i s o l a t e d b y H P L C the a c t i v e c o m p o u n d present i n a m i x t u r e o b t a i n e d f r o m gel filtration o n S e p h a d e x L H - 2 0 . B e t ter r e s o l u t i o n o f the c o m p o n e n t s of the grape flavan m i x t u r e was achieved b y gel f i l t r a t i o n w i t h S e p h a d e x G 25. I n t h i s w a y f r a c t i o n s e n r i c h e d for the i n d u c i n g c o m p o u n d s m a y be o b t a i n e d . T h i s m a y p r o v i d e samples f r o m w h i c h we c a n efficiently isolate new s i g n a l c o m p o u n d s i n sufficient q u a n -

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t i t y to p e r m i t s t r u c t u r e e l u c i d a t i o n . However, w i t h the d a t a at h a n d , we can m a k e the f o l l o w i n g educated guess as to the n a t u r e of the active s t r u c ture i n the active grape flavan m i x t u r e . C o n s i d e r i n g the facts t h a t g a l l i c a c i d was r e p o r t e d to induce v i r u l e n c e (35), t h a t epicatechin-gallate ( F i g . 3) was p r e v i o u s l y reported f r o m grapes (49), a n d t h a t esters of p h e n o l i c acids e x h i b i t e d enhanced a c t i v i t y , we suggest t h a t a flavan ester such as epicatechin-gallate is the active c o m p o n e n t i n our grape flavan f r a c t i o n s . Esters i n c l u d i n g phenolic acids w i t h g u a i a c y l or s y r i n g y l nuclei s h o u l d exh i b i t enhanced a c t i v i t y . T w o other sources of s i g n a l c o m p o u n d s f r o m L H R host tissues were f o u n d . O n e source was the S e y v a l callus c u l t u r e (see E x p e r i m e n t a l ) a n d the other was the aqueous exudate p r o d u c e d i n a b u n d a n c e u p o n c u t t i n g new grapevine stems i n the s p r i n g , when the sap flow was great. A n other grapevine callus c u l t u r e (obtained f r o m V. lubrascana cv. S t e u b e n , a n a t u r a l host of b o t h L H R a n d W H R Agrobacterium), its exudates, a n d fractions p a r t i t i o n e d t h e r e f r o m glauca p l a n t s , were i n c a p a b l e of i n d u c i n g vir gene expression i n any of the s t r a i n s used. A p p a r e n t l y not a l l callus cultures were e q u a l l y capable of p r o d u c i n g w r - i n d u c i n g m i x t u r e s of substances. P e r h a p s the two cultures differed i n the a m o u n t s of w r - i n h i b i t o r y substances p r o d u c e d . F i n a l l y , i n c o n s i d e r a t i o n of the n a t u r a l s e t t i n g i n w h i c h the L H R strains infect their host, we felt it w o r t h w h i l e to e x a m i n e the copious aqueous exudates of cut g r a p e v i n e stems. T h e c h l o r o f o r m soluble f r a c t i o n of such an e x u d a t e f r o m V. lubrusca was s t r o n g l y active i n a plate assay a n d is b e i n g further characterized. Conclusion In c o n c l u s i o n , b o t h w r - i n d u c i n g a n d w r - i n h i b i t o r y substances were p r o duced f r o m hosts a n d nonhosts of strains of A. tumefaciens. The comp o u n d s i n v o l v e d covered a range of p o l a r i t y a n d m o l e c u l a r weight, a n d t h i s likely reflects ester or other linkages between k n o w n lower m o l e c u l a r weight, w r - i n d u c i n g , c i n n a m i c a c i d derivatives a n d other organic c o m p o u n d s such as sugars a n d p r o a n t h o c y a n i d i n monomers (flavan-3-ols) or oligomers. It has been d e m o n s t r a t e d (7,35,41) t h a t A. tumefaciens is sensitive to the m o n o m e r s i n v o l v e d i n l i g n i n biosynthesis. T h e d a t a presented here suggest Agrobacterium m a y also be sensitive to c o m p o u n d s such as c a t e c h i n - or e p i c a t e c h i n - g a l l a t e , w h i c h l i n k s w r - i n d u c t i o n w i t h the m o n o m e r s of p r o cyanidin polymers. W e are c o n t i n u i n g our efforts to identify b o t h i n d u c i n g a n d i n h i b i t o r y c o m p o u n d s f r o m g r a p e v i n e c u l t i v a r s a n d other hosts of Agrobactenum. W i t h a more complete u n d e r s t a n d i n g of the c h e m i c a l signals i n v o l v e d , it s h o u l d be possible to induce T i - m e d i a t e d t r a n s f o r m a t i o n i n v i r t u a l l y a n y p l a n t species. C l e a r l y such a d i s t a n t goal w i l l also require a synthesis of d a t a f r o m a n u m b e r of fields of research.

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Experimental Bacterial Strains. In order to m o n i t o r w r - i n d u c t i o n i n the L H R s t r a i n A 8 5 6 a virEr.lacZ gene f u s i o n - c o n t a i n i n g p l a s m i d ( p S M 3 5 8 c d ) was i n t r o d u c e d b y t r i p a r e n t a l m a t i n g . T h i s was done so t h a t /?-galactosidase a c t i v i t y i n A 8 5 6 / p S M 3 5 8 c d c o u l d be assayed as an i n d i c a t o r of w r - i n d u c t i o n . T h i s p l a s m i d c o n t a i n e d the virE region of a W H R p T i i n the absence of other vir l o c i , so o n l y the L H R vir A gene p r o d u c t acted as the e n v i r o n m e n t a l sensor of s i g n a l c o m p o u n d s . T h e donor s t r a i n of E. coli ( J C 2 9 2 6 / p S M 3 5 8 c d ) was m a i n t a i n e d o n L B m e d i u m (51) c o n t a i n i n g 100 / i g / m L k a n a m y c i n . T h e s t r a i n o f E. coli w h i c h contained the helper p l a s m i d ( J C 2 9 2 6 / p R K 2 0 1 3 ) was m a i n t a i n e d o n L B m e d i u m c o n t a i n i n g 30 ^ g / m L s p e c t i n o m y c i n . A 8 5 6 was resistant to c h l o r a m p h e n i c o l , r i f a m p i c i n , a n d n a l i d i x i c a c i d (40 μ g / m L , 10 μ g / m L , a n d 20 μ g / m L , r e s p e c t i v e l y ) . Resistance to these a n t i b i o t i c s were used i n a d d i t i o n to k a n a m y c i n resistance to select for A 8 5 6 / p S M 3 5 8 c d . T h i s s t r a i n was m a i n t a i n e k a n a m y c i n . I n a d d i t i o n , the L H R s t r a i n A 8 5 6 / p S M 2 4 3 c d ( p r o v i d e d by D r . E u g e n e N e s t e r , U n i v e r s i t y of W a s h i n g t o n ) was used to m o n i t o r vir gene i n d u c t i o n a n d was also m a i n t a i n e d on A B m e d i u m c o n t a i n i n g 100 μ g / m L kanamycin. Plant Materials. T h e callus cultures used i n t h i s e x p e r i m e n t ( Vitis sp. cv. S e y v a l a n d V. lubrascana cv. Steuben) were also p r o v i d e d by D r . Nester. H e a l t h y , m a t u r e leaves of a n u m b e r of Vitis c u l t i v a r s were o b t a i n e d f r o m the U n i v e r s i t y of B r i t i s h C o l u m b i a B o t a n i c a l G a r d e n s a n d also f r o m l o c a l p r i v a t e l y o w n e d vines. R e d seedless ( F l a m e red) a n d G r e e n seedless grapes, i m p o r t e d f r o m C h i l e , were o b t a i n e d f r o m a l o c a l grocery store. N. glauca seedlings were o b t a i n e d f r o m the A g r i c u l t u r e C a n a d a Research S t a t i o n at U . B . C . , a n d raised i n our greenhouse. Isolation of LHR ν'ιτ-Inducers. C o n d i t i o n e d m e d i u m was o b t a i n e d f r o m 5 varieties of Vitis b y c u t t i n g about 20 fresh leaves a n d stems i n t o 1 c m pieces a n d p l a c i n g t h e m i m m e d i a t e l y into 1.5 L of sterile p H 5.7 M u r a s h i g e a n d S k o o g ( M S ) m e d i u m (52). C o n d i t i o n e d M S m e d i u m f r o m callus cultures of the Vitis c u l t i v a r S t e u b e n was o b t a i n e d by b r e a k i n g up h e a l t h y c a l l i f r o m 6-8 p e t r i plates i n 500 m L of sterile p H 5.7 M S m e d i u m . A f t e r 8-12 hours at r o o m t e m p e r a t u r e , the p l a n t m a t e r i a l was removed a n d the c o n d i t i o n e d m e d i u m was filtered, then processed i m m e d i a t e l y . W o u n d i n d u c e d p h e n o l i c aglycones were p a r t i t i o n e d f r o m the c o n d i t i o n e d m e d i u m u s i n g three v o l ­ umes each of e t h y l acetate or d i e t h y l ether, a n d phenolic glycosides were p a r t i t i o n e d f r o m the c o n d i t i o n e d m e d i u m u s i n g three volumes of n - b u t a n o l . T h e solvents were removed b y r o t a r y e v a p o r a t i o n a n d each e x t r a c t was resuspended i n a s m a l l v o l u m e of 100% m e t h a n o l . M e t h a n o l i c e x t r a c t s were m a d e d i r e c t l y f r o m healthy, m a t u r e c a l l i so t h a t a c o m p a r i s o n between the w o u n d i n d u c e d a n d n a t u r a l l y present c o m p o u n d s c o u l d be m a d e . T h e c o m p o u n d s present i n these m i x t u r e s were separated either by c o l ­ u m n c h r o m a t o g r a p h y on p o l y a m i d e ( S C 6 - A C ) or by b a n d i n g on p r e p a r a ­ tive p o l y a m i d e ( A C 6) T L C plates. E a c h b a n d was collected, the c o m p o u n d

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e l u t e d f r o m the p o l y a m i d e a n d t h e n tested for v i r - i n d u c t i o n i n the Agrobac­ tenum s t r a i n s A 8 5 6 / p S M 3 5 8 c d a n d A 8 5 6 / p S M 2 4 3 c d . G r a p e flavans were i s o l a t e d f r o m the t w o varieties of c o m m e r c i a l l y available grapes a n d f r o m the b a r k of Vitis g r a p e v i n e c u l t i v a r C o n c o r d ac­ c o r d i n g t o the m e t h o d s of C z o c h a n s k a et ai (46). CHCI3, E t O A c , a n d aqueous f r a c t i o n s o f b o t h grape varieties were assayed for v i r - i n d u c i n g ac­ t i v i t y i n b o t h A 8 5 4 / p S M 2 4 3 c d a n d A 8 5 6 / p S M 3 5 8 c d . T h e E t O A c frac­ t i o n s f r o m the flavan e x t r a c t i o n s o f b o t h varieties of grapes a n d b a r k were r e s u s p e n d e d either i n 1 0 0 % e t h a n o l or m e t h a n o l for c h r o m a t o g r a p h y on S e p h a d e x L H 20. T h e f r a c t i o n s were e x a m i n e d b y T L C o n cellulose ( M e r c k , 0 . 1 m m ) developed w i t h s e c - B u O H : A c O H : H 2 0 (14:1:5), a n d s i m i l a r frac­ t i o n s were p o o l e d a n d t h e n assayed for a c t i v i t y or a l t e r n a t i v e l y they were screened for a c t i v i t y a n d groups of active f r a c t i o n s were p o o l e d . G r a p e flavan f r a c t i o n s were also separated o n S e p h a d e x G 25 u s i n g 10 m M N a C l as the d e v e l o p i n g solvent. T L C o n cellulose ( s e c - B u O H : A c O H : H t i o n s revealed at least 5 or m o r e m a j o r c o m p o u n d s . G C showed t h a t there were c o n s i d e r a b l y m o r e c o m p o u n d s also present. H P L C of the active fractions was p e r f o r m e d o n a V a r i a n m o d e l 50200000 e q u i p p e d w i t h a n a n a l y t i c a l W a t e r s C - 1 8 c o l u m n (0.5 x 3 0 c m ) a n d u s i n g solvent A : 5 % acetic a c i d i n H 2 O , a n d B : a c e t o n i t r i l e , u n d e r the f o l l o w i n g c o n d i t i o n s 9 5 % A : 5 % B , 10 m i n . , c h a n g i n g to 2 5 % Β i n 10 m i n . , t h e n 3 0 % Β i n 10 m i n . a n d m a i n t a i n e d for 10 m i n . , a n d finally to 4 0 % i n 10 m i n . U V absorbance was m o n i t o r e d at 254 or 275 n m . T h e i s o l a t e d c o m p o u n d s were t e s t e d for v i r - i n d u c t i o n as described below. v i r - I n d u c t i o n Assay. F o r the s t r u c t u r e - a c t i v i t y s t u d y , /?-galactosidase ac­ t i v i t y was assayed as a measure of vir-gene i n d u c t i o n i n a w i d e host range s t r a i n w h i c h c a r r i e d a virE.JacZ gene f u s i o n . T h e c o m p o u n d s tested were dissolved i n D M S O a n d d i l u t e d i n c i t r a t e - p h o s p h a t e buffered p H 5.70 M S m e d i u m (52) t o a final c o n c e n t r a t i o n of 0 . 1 % D M S O . 100 μΐ, of b a c t e ­ r i a l cells f r o m a n o v e r n i g h t c u l t u r e of A 3 4 8 / p S M 3 5 8 (23) were i n o c u l a t e d i n t o each 25 x 1 5 0 m m c u l t u r e t u b e a n d s u b j e c t e d to c o n t i n u o u s s h a k i n g at 200 R P M a n d at 2 8 ° C for 8 hours to allow for i n d u c t i o n of virEv.lacl ex­ p r e s s i o n . C e l l d e n s i t y was d e t e r m i n e d b y m e a s u r i n g absorbance at 600 n m a n d 1 m l a l i q u o t s were r e m o v e d for /?-galactosidase assay essentially as d e s c r i b e d b y M i l l e r (53). E a c h p o i n t o n the a c t i v i t y curve of a test c o m p o u n d represented the av­ erage o f the results of each c o n c e n t r a t i o n tested i n t r i p l i c a t e . w r - I n d u c t i o n was s t r o n g l y p H dependent (24, o u r r e s u l t s , d a t a not s h o w n ) , so the buffer s y s t e m was used to m i n i m i z e v a r i a t i o n i n p H . S t a n d a r d d e v i a t i o n s r a r e l y reached 1 0 % , the average b e i n g 4 . 7 % (n = 92) for results of 100 M i l l e r u n i t s a n d above. I n the search for L H R w r - i n d u c e r s , each p h e n o l i c aglycone or other f r a c t i o n to be tested was dissolved i n D M S O a n d d i l u t e d i n p H 5.50 or 5.70 M S m e d i u m t o a final c o n c e n t r a t i o n of 0 . 1 % D M S O ( p h e n o l i c glycosides a n d the grape flavan f r a c t i o n s were dissolved d i r e c t l y i n ρ Η 5.50 or 5.70 M S m e d i u m ) . S u b s e q u e n t e x p e r i m e n t s revealed a more a c i d i c p H o p t i m u m for

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w r - i n d u c t i o n i n t h e L H R s t r a i n , so some e x p e r i m e n t s were c o n d u c t e d at p H 5.0. S t a n d a r d s o f acetosyringone, c a t e c h i n , e p i c a t e c h i n , a n d a p r o a n t h o c y a n i d i n p o l y m e r were assayed for L H R w r - i n d u c t i o n . 100 μ L o f cells f r o m a n overnight c u l t u r e o f A 8 5 6 / p S M 3 5 8 c d or A 8 5 6 / p S M 2 4 3 c d was i n o c u l a t e d i n t o each 25 x 1 5 0 m m test t u b e c o n t a i n i n g 10 m L o f M S or 0 . 1 % D M S O - M S s o l u t i o n w i t h various concentrations o f t h e test substances, a n d a l l tubes were s u b j e c t e d t o 200 r p m for 10-24 h a n d at 28° C t o allow for i n d u c t i o n of vir expression. /?-Galactosidase a c t i v i t y was t h e n assayed as described b y M i l l e r (53). A l t e r n a t i v e l y , t h e f o l l o w i n g screening assay was used t o identify v i r - i n d u c i n g f r a c t i o n s . H P L C fractions were collected, reduced t o d r y ­ ness under v a c u u m , resuspended i n a s m a l l a m o u n t o f M e O H a n d a few μΐ, o f each was a p p l i e d t o a filter p a p e r disc. T h e discs were p l a c e d o n a M 9 (51) agar p l a t e c o n t a i n i n g 0 . 1 % 5-bromo-4-chloro-3indolyl-/?-D-galactopyranoside (Xgal) with a lawn of A 8 5 6 / p S M 3 5 8 c d or A 8 5 6 / p S M 2 4 3 c d a n d th blue zones ( i n d i c a t i n g /?-galactosidase a c t i v i t y ) developed s u r r o u n d i n g a n y disc. Gas Chromatography. In our G C analyses, N , 0 - b i s - ( T r i m e t h y l s i l y l ) T r i f l u o r o a c e t a m i d e ( B S T F A ) - d e r i v a t i z e d s t a n d a r d s o f k n o w n w r - i n d u c i n g pheno­ lics f a i l e d t o correspond i n r e t e n t i o n t i m e t o a n y o f the d e r i v a t i z e d samples of t h e most active grape flavan fractions. C a t e c h i n a n d e p i c a t e c h i n were t e n t a t i v e l y identified by G C . Acknowledgments W e w o u l d like t o give s p e c i a l t h a n k s t o D r . Eugene W . Nester w h o p r o v i d e d the Agrobactenum strains A 3 4 8 / p S M 3 5 8 a n d A 8 5 6 / p S M 2 4 3 c d , a n d the b a c t e r i a l s t r a i n s f r o m w h i c h we prepared A 8 5 6 / p S M 3 5 8 c d . W e also t h a n k D o n C h a m p a g n e for useful discussions a n d Felipe B a l z a for c o n d u c t i n g mass spectroscopy. W e are grateful to L a c e y Samuels for p e r m i s s i o n t o use her S E M figures i n o u r s y m p o s i u m p r e s e n t a t i o n . P . A . S . was s u p p o r t e d b y a U n i v e r s i t y G r a d u a t e F e l l o w s h i p at the U n i v e r s i t y o f B r i t i s h C o l u m b i a . T h e research was f u n d e d b y the N a t u r a l Sciences a n d E n g i n e e r i n g Research Council of Canada. Literature Cited

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7. Stachel, S. E.; Messens, E.; Van Montagu, M.; Zambryski, P. Nature 1985, 318, 624. 8. DeCleen, M.; Deley, J. Bot. Rev. 1976, 42, 389. 9. Chilton, M. D.; Montoya, A. L.; Merlo, D. J.; Drummond, M. H.; Nutter, R.; Gordon, M. P.; Nester, E. W. Cell 1977, 11, 263. 10. Thomashow, M. F.; Nuter, R.; Montoya, A. L.; Gordon, M. P.; Nester, E. W. Cell 1980, 19, 729. 11. Yadav, N. S.; Postle, K.; Saiki, R. K.; Thomashow, M. F.; Chilton, M.-D. Nature 1980, 287, 458. 12. Chilton, M.-D.; Saiki, R. K.; Yadav, N.; Gordon, M. P.; Quetier, F. Proc. Natl. Acad. Sci. USA 1980, 77, 4060. 13. Willmitzer, L.; De Beuckeleer, M.; Lemmers, M.; Van Montagu, M.; Schell, J. Nature 1980, 287, 359. 14. Zambryski, P.; Holsters, M.; Kruger, K.; Depicker, Α.; Schell, J.; Van Montagu, M.; Goodman 15. Schroder, G.; Waffenschmidt Biochem. 1984, 138, 387. 16. Thomashow, L. S.; Reeves, S.; Thomashow, M. F. Proc. Natl. Acad. Sci. 1984, 81, 5071. 17. Akiyoshi, P. E.; Monis, R. O.; Hing, R.; Mischke, B. S.; Kosuge, T.; Garfinkel, D. J.; Gordon, M. P.; Nester, E. W. Proc. Natl. Acad. Sci. 1983, 80, 407. 18. Akiyoshi, P. E.; Klee, H.; Amasino, R. M.; Nester, E. W.; Gordon, M. P. Proc. Natl. Acad. Sci. 1984, 81, 5994. 19. Hille, J.; Hoekema, P.; Hoojkaas, P.; Shilperoort, R. In Plant Gene Research. Genes Involved in Plant-Microbe Interactions; Verman, D. P. S.; Hohn, T., Eds.; Springer-Verlag: New York, 1984. 20. Willmitzer, L.; Schmalenbach, W.; Schell, J. Nucleic Acid Res. 1981, 9, 4801. 21. Horsch, R. B.; Klee, H. J.; Stachel, S.; Winans, S. C.; Nester, E. W.; Rogers, S. G.; Fraley, R. T. Proc. Nat. Acad. Sci. USA 1986, 83, 2571. 22. Klee, H. J.; White, F. F.; Iyer, V. N.; Gordon, M. P.; Nester, E. W. J. Bacteriol. 1983, 153, 878. 23. Stachel, S. E.; An, G.; Flores, C.; Nester, E. W. EMBO J. 1985, 4, 891. 24. Stachel, S. E.; Nester, E. W.; Zambryski, P. C. Proc. Natl. Acad. Sci. 1986, 83, 379. 25. Winans, S. C.; Ebert, P. R.; Stachel, S. E.; Gordon, M. P.; Nester, E. W. Proc. Natl. Acad. Sci. 1986, 83, 8278. 26. Stachel, S. E.; Zambryski, P. Cell 1986, 46, 325. 27. Leroux, B.; Yanofsky, M. F.; Winans, S. C.; Ward, J. E.; Ziegler, S. F.; Nester, E. W. EMBO J. 1987, 6, 849. 28. Hille, J.; van Kan, J.; Shilperoort, R. J. Bacteriol. 1984, 158, 754. 29. Hooykaas, P. J. J.; Hofker, M.; Den Dulk-Ras, H.; Shilperoort, R. A. Plasmid 1984, 11, 195. 30. Yanofsky, M.; Lowe, B.; Montoya, Α.; Rubin, R.; Krul, W.; Gordon, M.; Nester, E. W. Molec. Gen. Genet. 1985, 201, 237.

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Chapter 29

Properties of a Cutinase-Defective Mutant of Fusarium solani Anne H . Dantzig Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285

The fungal plan an extracellular enzyme, cutinase, which catalyzes the degradation of the bipolymer, cutin, in the plant cuticle. The enzyme was repressed when the microorganism was grown on a medium containing glucose and induced to high levels by cutin or its hydrolysis products, the true inducers. In the present study, culture filtrates contained basal levels of cutinase when Fusarium was grown on 0.5% acetate as the sole carbon source and high levels of cutinase when grown on cutin. After mutagenesis, a cutinase-defective mutant of Fusarium was identified by screening acetate-grown colonies for a loss of enzyme activity. The mutant exhibited an 80-90% reduction in cutinase activity under several growth conditions due to a quantitative reduction in a qualitatively normal enzyme. The mutant also exhibited a reduction in virulence in the pea stem bioassay. Taken together, these data indicated that a growth condition exists where the cutinase enzyme was neither induced nor repressed and was present in basal levels. This condition may pose the pathogen for rapid enzyme induction when in the proximity of the plant cuticle. The cutinase-defective mutant was either a regulatory mutant with an altered expression of cutinase, or a mutant modified in its ability to excrete the enzyme. T h e b i o p o l y m e r c u t i n is a m a j o r constituent o f the p l a n t cuticle t h a t p r o vides a protective covering for p l a n t s ( 1 , 2 ) . A t the t i m e o f i n f e c t i o n , a n u m b e r o f f u n g a l pathogens secrete a n e x t r a c e l l u l a r h y d r o l y t i c e n z y m e , cutinase, w h i c h facilitates the d e g r a d a t i o n o f c u t i n i n t o i t s constituent C\e~ to C i s - l e n g t h h y d r o x y f a t t y acids ( 3 , 4 ) . Since the e n z y m e is believed t o 0097-6156/89/0399-0399$06.00/0 © 1989 American Chemical Society

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p l a y a n i m p o r t a n t role i n v i r u l e n c e (5), m a n y studies have been c o n d u c t e d to d e t e r m i n e its b i o c h e m i c a l properties a n d p h y s i o l o g i c a l role d u r i n g p a t h o genesis. T o date the most comprehensive studies have been c a r r i e d o u t b y K o l a t t u k u d y a n d coworkers w i t h a f u n g a l p l a n t p a t h o g e n of peas, Fusarium solani f. sp. pisi. W h e n g r o w n o n c u t i n , the fungus p r o d u c e d two isozymes of cutinase of m o l e c u l a r weights 2 2 , 4 0 0 a n d 2 1 , 2 0 0 , w i t h the former b e i n g the precursor of the l a t t e r , m a t u r e f o r m (6-8). T h e c D N A for the c u t i nase gene was i s o l a t e d , sequenced, a n d the p r i m a r y a m i n o a c i d sequence deduced ( 9 , 1 0 ) . D N A h y b r i d i z a t i o n studies i n d i c a t e d t h a t more t h a n one gene was present i n Fusarium solani (9). A l t h o u g h details of the r e g u l a t i o n of the expression of cutinase at the D N A level were not established, p h y s i o l o g i c a l studies i n d i c a t e d t h a t the r e g u l a t i o n of the enzyme was likely to be c o m p l e x . T h e e n z y m e u n d e r w e n t c a t a b o l i t e repression a n d was i n d u c e d to h i g h levels w h e n c u t i n , or its h y d r o l y z e d p r o d u c t s , wer c u t i n has a large m o l e c u l a r weight, it is not likely to penetrate the f u n g a l cell w a l l ; the d e g r a d a t i o n p r o d u c t s are therefore believed to be the true inducers of the enzyme ( 1 1 , 1 2 ) . T h u s , the presence of the cutinase e n z y m e seemed to be required for its o w n i n d u c t i o n i n order to generate the s m a l l m o l e c u l a r weight inducers. C o n s e q u e n t l y , i t seemed p l a u s i b l e t h a t g r o w t h c o n d i t i o n s m i g h t exist i n w h i c h cutinase were present i n b a s a l q u a n t i t i e s , thereby p o s i n g the p a t h o g e n for r a p i d e n z y m e i n d u c t i o n w h e n presented w i t h the c u t i n b i o p o l y m e r . T h e present s t u d y was u n d e r t a k e n t o e x a m i n e the r e g u l a t i o n of cutinase b y a n alternate c a r b o n source t h a t m i g h t p e r m i t b a s a l levels of the e n z y m e t o be synthesized, a n d t h e n to subsequently use t h i s g r o w t h c o n d i t i o n for the i s o l a t i o n o f a cutinase-defective m u t a n t . T h e details of t h i s work have been p r e v i o u s l y p u b l i s h e d i n Ref. 13. Results A c e t a t e is k n o w n to be a g o o d c a r b o n source for f u n g i a n d w o u l d be expected to be the u l t i m a t e d e g r a d a t i o n p r o d u c t of c u t i n (14). T h e effect of acetate o n the p r o d u c t i o n of cutinase b y the T - 8 s t r a i n of F. solani was exa m i n e d a n d c o m p a r e d w i t h t h a t of glucose. Since previous studies showed t h a t h y d r o l y s i s of the a r t i f i c i a l substrate p - n i t r o p h e n y l b u t y r a t e , P N B , was specifically h y d r o l y z e d by cutinase i n the T - 8 s t r a i n , t h i s a c t i v i t y was used to measure cutinase levels (8). F i g u r e 1 i l l u s t r a t e s t h a t b a s a l levels of c u t i nase a c t i v i t y were detected i n the g r o w t h m e d i u m w h e n T - 8 was g r o w n o n 0.5 a n d 1.5% acetate, b u t not 2 . 0 % glucose, as the sole c a r b o n source. A s s h o w n i n F i g u r e 2 A , T - 8 p r o d u c e d h i g h levels of cutinase i n the g r o w t h m e d i u m when g r o w n on apple c u t i n as the sole c a r b o n source, a n d was repressed by i n c r e a s i n g concentrations of glucose; b y c o n t r a s t , F i g u r e 2 B showed t h a t a d d i t i o n of increasing concentrations of acetate was less repressive t h a n glucose. These d a t a suggest t h a t the e n z y m e was present at low concentrations when the o r g a n i s m was g r o w n o n acetate. Since g r o w t h of F. solani o n acetate m e d i u m p e r m i t t e d p r o d u c t i o n o f cutinase, t h i s g r o w t h c o n d i t i o n was used for the i s o l a t i o n of a m u t a n t

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F i g u r e 1. Specific a c t i v i t y of cutinase as a f u n c t i o n of sole c a r b o n source i n the g r o w t h m e d i u m . T h e T - 8 s t r a i n of F. solani was g r o w n o n 0 . 5 % acetate (o), 1.5% acetate ( Δ ) , or 1.5% glucose ( · ) as the sole c a r b o n source. ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. © 1986, A m e r i c a n Society for Microbiology.)

1

2 DAYS

3

F i g u r e 2. Specific a c t i v i t y of cutinase r e s u l t i n g f r o m g r o w t h i n c u t i n c o n t a i n i n g m e d i u m w i t h the a d d i t i o n of a second c a r b o n source. T h e T - 8 s t r a i n of F. solani was grown on m e d i u m c o n t a i n i n g 200 m g of apple c u t i n a n d glucose ( A ) or acetate ( B ) . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. © 1986, A m e r i c a n Society for M i c r o b i o l o g y . )

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defective i n cutinase; the procedure for m u t a n t selection was described i n Ref. 13 a n d is s u m m a r i z e d i n F i g u r e 3. I n i t i a l l y , Fusarium was g r o w n o n m e d i u m c o n t a i n i n g peanut m e a l - p e p t o n e - S t a d e x II t o p r o d u c e m i c r o c o n i d i a needed for genetic studies (13). T h e c o n i d i a were uv i r r a d i a t e d to i n t r o d u c e m u t a t i o n s , r e g r o w n to p e r m i t expression of a n y m u t a t i o n , a n d p l a t e d o n g r o w t h m e d i u m c o n t a i n i n g agarose a n d 0 . 5 % acetate as the sole c a r b o n source. T h e r e s u l t i n g colonies were t h e n overlaid w i t h agarose c o n t a i n i n g P N B . W i t h i n 30 m i n the p a r e n t a l type h y d r o l y z e d the s u b s t r a t e a n d t u r n e d y e l l o w , a n d the p r e s u m p t i v e m u t a n t s r e m a i n e d w h i t e . T h e r e s u l t i n g PNB1 m u t a n t was one out of 4 , 3 0 0 colonies screened. T h e properties of t h i s m u t a n t were further characterized as described below. P r e l i m i n a r y e v a l u a t i o n of the m u t a n t i n d i c a t e d t h a t i t h a d p r o d u c e d low levels of the cutinase w h e n g r o w n o n either acetate or c u t i n as the sole c a r b o n source. A t i m e course of the p r o d u c t i o n of cutinase was e x a m i n e d for the T - 8 p a r e n t a l s t r a i n a n d the PNB-1 m u t a n t s t r a i n when g r o w n o n cutin. A s shown in Figur 8 0 - 9 0 % over the 15-day g r o w t h p e r i o d , w h e n e n z y m e a c t i v i t y was assayed u s i n g the a r t i f i c i a l substrate P N B ( P a n e l A ) or w h e n assayed u s i n g [ C ] l a b e l l e d n a t u r a l s u b s t r a t e c u t i n ( P a n e l B ) . These d a t a suggested t h a t the m u t a n t was defective i n cutinase b u t not i n general esterase. 1 4

N e x t the parent a n d m u t a n t were c o m p a r e d for t h e i r a b i l i t y to i n d u c e cutinase b y c u t i n a n d h y d r o l y z e d c u t i n (consisting of s m a l l m o l e c u l a r weight inducers) after g r o w t h on glucose. A s s h o w n i n F i g u r e 5, cutinase a c t i v i t y increased over the three-day i n d u c t i o n p e r i o d for b o t h s t r a i n s i n the presence of c u t i n ( P a n e l A ) or h y d r o l y z e d c u t i n ( P a n e l B ) ; however, cutinase was i n d u c e d less effectively i n the m u t a n t s t r a i n , as evidenced by a n 8 0 - 9 0 % r e d u c t i o n . H y d r o l y z e d c u t i n was a 10-fold less effective inducer t h a n c u t i n i n b o t h s t r a i n s . These d a t a i n d i c a t e d t h a t lack of i n d u c t i o n i n the m u t a n t was not related to its i n a b i l i t y to h y d r o l y z e c u t i n to s m a l l m o l e c u l a r weight inducers. C o n s e q u e n t l y the defect observed was not related to the i n a b i l i t y of the m u t a n t to produce the i n d u c e r . T o g a i n f u r t h e r insight i n t o the n a t u r e of the defect i n PNB-1 m u t a n t , the c o n c e n t r a t i o n dependence of the cutinase enzyme a c t i v i t y was e x a m i n e d over a wide s u b s t r a t e range a n d c o m p a r e d w i t h t h a t of the p a r e n t a l s t r a i n ( F i g u r e 6). I n b o t h s t r a i n s , the enzyme a c t i v i t y was s a t u r a t e d w i t h i n creasing substrate c o n c e n t r a t i o n , a n d gave s i m p l e M i c h a e l i s - M e n t e n curves w h i c h were subsequently fitted by c o m p u t e r to a single M i c h a e l i s t e r m . T h e K for P N B was d e t e r m i n e d to be 0 . 9 7 ± 0 . 2 0 a n d 0 . 6 4 ± 0 . 0 7 m M , respectively, i n the p a r e n t a l a n d m u t a n t s t r a i n s , i n d i c a t i n g t h a t l i t t l e change i n the affinity for the substrate h a d o c c u r r e d . B y c o n t r a s t , the V was reduced by 9 2 % f r o m 38.86 ± 2.68 to 2.62 ± 0 . 0 9 / i m o l / m i n per m g p r o t e i n i n the p a r e n t a l a n d m u t a n t s t r a i n s , respectively. T h e large r e d u c t i o n i n V m i g h t result f r o m a q u a n t i t a t i v e r e d u c t i o n of a n o r m a l enzyme or f r o m a n aberrant enzyme being produced i n n o r m a l quantities. m

m

a

x

m

a

r

T o find out i f the m u t a n t was m a k i n g less e n z y m e , the two s t r a i n s were i n d u c e d to produce cutinase (as i l l u s t r a t e d i n F i g u r e 5) i n the presence of [ S ] m e t h i o n i n e , a n d the e x t r a c e l l u l a r proteins were separated b y 35

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T-8 parent

Grow colonies on agarose medium containing 0.5% acetate

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3 days

Assay for cutinase production

PNB-1 mutant

F i g u r e 3. S u m m a r y o f the selection procedure for cutinase-defective m u ­ t a n t . T h e T - 8 s t r a i n o f F. solani was m u t a g e n i z e d b y u l t r a v i o l e t i r r a d i ­ a t i o n , g r o w n for 3 days, a n d p l a t e d o n m e d i u m c o n t a i n i n g 0 . 5 % acetate a n d agarose for 5-7 days t o p e r m i t colony f o r m a t i o n . Subsequently, t h e colonies were overlaid w i t h a n agarose s o l u t i o n c o n t a i n i n g 1.26 m M P N B . T h e p a r e n t a l colonies h y d r o l y z e d the substrate a n d t u r n e d yellow w h i l e the p r e s u m p t i v e m u t a n t colonies r e m a i n e d w h i t e a n d were selected for a n a l y s i s . F u r t h e r details are given i n Ref. 13.

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τ

1

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DAYS F i g u r e 4. Specific a c t i v i t y of cutinase for the T - 8 p a r e n t a l s t r a i n a n d PNB-1 m u t a n t s t r a i n after g r o w t h o n m e d i u m c o n t a i n i n g 200 m g c u t i n . E n z y m e a c t i v i t y was assayed w i t h the a r t i f i c i a l substrate P N B ( P a n e l A ) a n d w i t h the n a t u r a l substrate, [ C ] - l a b e l l e d c u t i n ( P a n e l B ) . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. (c) 1986, A m e r i c a n Society for M i c r o b i o l o g y . ) 14

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F i g u r e 5. C o m p a r i s o n of effect of a d d i t i o n of c u t i n or h y d r o l y z e d c u t i n o n the i n d u c t i o n of cutinase. T h e two strains were g r o w n o n m e d i u m c o n t a i n i n g 0 . 1 % glucose for 3 d a y s , a n d then 200 m g of c u t i n ( A ) or 8 m g of h y d r o l y z e d c u t i n ( B ) was a d d e d to the g r o w t h m e d i u m o n day zero. ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. © 1986, A m e r i c a n Society for M i c r o b i o l o g y . )

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F i g u r e 6. K i n e t i c s of the specific a c t i v i t y of cutinase p r o d u c e d b y the p a r e n t a l T - 8 s t r a i n a n d the PNB-1 m u t a n t s t r a i n . T h e d a t a were fitted to a M i c h a e l i s - M e n t e n e q u a t i o n (13). ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. © 1986, A m e r i c a n Society for M i c r o b i o l o g y . )

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electrophoresis o n a 1 5 % s o d i u m d o d e c y l sulfate p o l y a c r y l a m i d e gel. A fluo r o g r a p h of the gel i n d i c a t e d t h a t b o t h s t r a i n s possessed a p r o t e i n b a n d of a b o u t 22,000 i n the same m o l e c u l a r weight range as the cutinase isozymes; however, by densitometer t r a c i n g the m u t a n t e x h i b i t e d about a n 8 0 % red u c t i o n i n this b a n d . I m m u n o b l o t s were conducted w i t h r a b b i t a n t i c u t i n a s e s e r u m w h i c h confirmed t h a t t h i s b a n d corresponded to cutinase ( F i g u r e 7). T h u s , the PNB-1 m u t a n t a p p e a r e d to make less of a " n o r m a l " e n z y m e . A r e d u c t i o n i n cutinase p r o d u c t i o n s h o u l d result i n the PNB-1 m u t a n t b e i n g less v i r u l e n t . T h e pathogenesis of the two s t r a i n s were e v a l u a t e d i n a p e a s t e m bioassay developed b y K o l a t t u k u d y a n d coworkers i n w h i c h i n fection by Fusanum solani results i n w o u n d f o r m a t i o n w i t h i n three days o n the e p i c o t y l of pea sedlings (15). T h e v i r u l e n c e of T - 8 h a d p r e v i o u s l y been s h o w n t o be reduced i n t h i s assay b y the a d d i t i o n of i n h i b i t o r s of cutinase or b y r a b b i t a n t i c u t i n a s e antibodies (15-18), i n d i c a t i n g t h a t cutinase played a n i m p o r t a n t role i n pathogenesis. W h e n the cutinase-defective m u t a n t was e v a l u a t e d i n the bioassay, i n v i r u l e n c e c o m p a r e d w i t h the T - 8 p a r e n t a l s t r a i n a n d the a d d i t i o n of purified cutinase at 1 m g / m l to the m u t a n t enhanced w o u n d f o r m a t i o n to 8 0 % of t h a t of the parent (p > 0.5). These d a t a further s u p p o r t the n o t i o n t h a t the m u t a n t was defective i n cutinase. Discussion T a k e n together, these d a t a i n d i c a t e t h a t Fusarium solani p r o d u c e d low levels of cutinase when g r o w n o n acetate as the sole c a r b o n source a n d t h a t t h i s c a r b o n source was less repressive t h a n glucose. T h e finding t h a t a cutinase-defective m u t a n t c o u l d be isolated u s i n g t h i s g r o w t h c o n d i t i o n p r o v i d e d a d d i t i o n a l s u p p o r t t h a t cutinase was b e i n g p r o d u c e d w h e n g r o w n o n acetate. T h u s there were three discrete g r o w t h c o n d i t i o n s w h i c h affected the synthesis of cutinase b y the m i c r o o r g a n i s m : one i n w h i c h the e n z y m e was repressed (such as glucose); one i n w h i c h the e n z y m e was i n d u c e d to h i g h levels (such as w i t h c u t i n or its h y d r o l y z e d p r o d u c t s ) ; a n d one i n w h i c h i t was neither i n d u c e d nor repressed (such as a c e t a t e ) — r e s u l t i n g i n the p r o d u c t i o n of basal levels of enzyme. Therefore, cutinase p r o d u c t i o n b y a fungus present i n the field i n the presence of a g o o d c a r b o n source w o u l d be expected to be repressed. O n c e t h a t c a r b o n source was depleted, t h e n b a s a l levels of the e n z y m e m a y be s y n t h e s i z e d . If the fungus was i n the p r o x i m i t y of the p l a n t c u t i c l e , t h e n c u t i n m a y be h y d r o l y z e d , r e s u l t i n g i n i n d u c e r f o r m a t i o n a n d r a p i d i n d u c t i o n o f cutinase synthesis a i d i n g the fungus i n the p e n e t r a t i o n of the p l a n t (12). T h e PNB-1 m u t a n t is a " l e a k y " m u t a n t t h a t is p a r t i a l l y defective i n cutinase p r o d u c t i o n , a n d a p p a r e n t l y p r o d u c e d 80 to 9 0 % less " n o r m a l " e n z y m e . T h e r e g u l a t i o n of the r e s i d u a l e n z y m e i n the m u t a n t was u n changed. T h e e n z y m e was repressed by glucose, a n d was i n d u c e d b y c u t i n or h y d r o l y z e d c u t i n after d e p l e t i o n of glucose. C u t i n a s e a c t i v i t y increased over a 15-day t i m e p e r i o d when the m u t a n t was g r o w n o n c u t i n as the sole c a r b o n source. T h e m u t a n t was also less v i r u l e n t t h a n the p a r e n t a l s t r a i n . T h e n a t u r e of the PNB-1 m u t a t i o n r e m a i n s to be e l u c i d a t e d a n d

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F i g u r e 7. C o m p a r i s o n of separated [ S ] - l a b e l l e d e x t r a c e l l u l a r proteins f r o m the c u t i n i n d u c t i o n m e d i u m of the p a r e n t a l a n d m u t a n t s t r a i n s o n 1 5 % s o d i u m d o d e c y l s u l f a t e - p o l y a c r y l a m i d e gels b y fluorography a n d W e s t e r n b l o t t i n g . M y c e l i a were g r o w n a n d i n d u c e d w i t h c u t i n (as i l l u s t r a t e d i n F i g ­ ure 5) w i t h the a d d i t i o n of 115 / i C i of [ S ] m e t h i o n i n e . A 20-fold concen­ t r a t e d s a m p l e c o n t a i n i n g 100,000 c p m for the parent (lanes 1 a n d 3) or the m u t a n t (lanes 2 a n d 4) was a p p l i e d to p a r a l l e l gels. T o t a l p r o t e i n synthesis was a n a l y z e d by fluorography of the gel c o n t a i n i n g lanes 1 a n d 2. I m m u n o r e a c t i v e m a t e r i a l i n the gel c o n t a i n i n g lanes 3 a n d 4 was detected w i t h r a b b i t a n t i c u t i n a s e s e r u m after e l e c t r o b l o t t i n g . M o l e c u l a r weight s t a n d a r d s ( χ 1 0 ) are i n d i c a t e d o n the left. T h e b a n d c o r r e s p o n d i n g to the m o l e c u l a r weight of cutinase is i n d i c a t e d w i t h the a r r o w . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 13. © 1986, A m e r i c a n Society for M i c r o b i o l o g y . ) 35

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m a y reside i n a p r o m o t e r for the s t r u c t u r a l gene o f cutinase or a n as yet u n i d e n t i f i e d r e g u l a t o r y gene. A l t e r n a t i v e l y , the m u t a n t m a y be defective i n t h e secretion o f cutinase. F u r t h e r s t u d y o f t h e PNB-1 m u t a n t s h o u l d p r o v i d e i n s i g h t i n t o the c o m p l e x r e g u l a t i o n o r secretion o f this b i o p o l y m e r h y d r o l y z i n g e n z y m e . W i t h a better u n d e r s t a n d i n g o f these processes, i t m a y be possible t o design agents t h a t c a n intervene i n cutinase p r o d u c t i o n r e s u l t i n g i n the c o n t r o l o f f u n g a l pathogenesis i n t h e field. Acknowledgments S p e c i a l t h a n k s are given t o D r . K o l a t t u k u d y for p r o v i d i n g the r a b b i t c u t i ­ nase a n t i s e r u m a n d purified cutinase used i n t h i s s t u d y . Literature Cited

1. Espelie, Κ. E.; Davis 498-511. 2. Van den Erde, G.; Linskens, H. F. Ann. Rev. Phytopathol. 1974, 12, 247-58. 3. Baker, C. J.; Bateman, D. F. Phytopathology 1978, 68, 1577-84. 4. Dickman, M. B.; Patil, S. S.; Kolattukudy, P. E. Physiol. Plant Pathol. 1982, 20, 333-47. 5. Lin, T. S.; Kolattukudy, P. E. Physiol. Plant Pathol. 1980, 17, 1-15. 6. Purdy, R. E.; Kolattukudy, P. E. Arch. Biochem. Biophys. 1973, 159, 61-9. 7. Purdy, R. E.; Kolattukudy, P. E. Biochemistry 1975, 14, 2824-31. 8. Purdy, R. E.; Kolattukudy, P. E. Biochemistry 1975, 14, 2832-40. 9. Soliday, C. L.; Flurkey, W. H.; Okita, T. W.; Kolattukudy, P. E. Proc. Natl. Acad. Sci. USA 1984, 81, 3939-43. 10. Ettinger, W. G.; Thukral, S. K.; Kolattukudy, P. E. Biochemistry 1987, 26, 7883-92. 11. Lin, T. S.; Kolattukudy, P. E. J. Bacteriol. 1978, 133, 942-51. 12. Woloshuk, C. P.; Kolattukudy, P. E. Proc. Natl. Acad. Sci. USA 1986, 83, 1704-8. 13. Dantzig, A. H.; Zuckerman, S. H.; Andonov-Roland, M. M. J. Bacte­ riol. 1986, 168, 911-6. 14. Cochrane, V. W. In Physiology of Fungi; John Wiley & Sons: New York, 1958; pp. 55-98. 15. Maiti, I. B.; Kolattukudy, P. E. Science 1979, 205, 507-8. 16. Koller, W.; Allan, C. R.; Kolattukudy, P. E. Physiol. Plant Pathol. 1982, 20, 47-60. 17. Koller, W.; Allan, C. R.; Kolattukudy, P. E. Pestic. Biochem. Physiol. 1982, 18, 15-25. 18. Shaykh, M.; Soliday, C.; Kolattukudy, P. E. Plant Physiol. 1977, 60, 170-2. RECEIVED March 10, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 30

Roles of Secondary Metabolism of Wood Rotting Fungi in Biodegradation of Lignocellulosic Materials 1

2

1

1

Mikio Shimada , Akira Ohta , Hiroshi Kurosaka , Takefumi Hattori , Takayoshi Higuchi , and Munezoh Takahashi 1

1

1

W o o d Research Institute, Kyoto University, Uji, Kyoto 611, Japan 2

Shiga Forest Research Center, Yasu, Shiga 520—23, Japan

The brown-rot fungu ferent phenylpropanoids and methyl p-anisate as sec­ ondary metabolites in both high and low nitrogen nutri­ ent-containing cultures. A new metabolite, p-methoxy­ phenylpropanol, was also identified. The white-rot fun­ gus Phanerochaete chrysosporium produces veratrylglyc­ erol and veratryl alcohol as secondary metabolites in the extracellular culture fraction. The "ligninase" of this white-rot fungus catalyzes Cα-Cβ cleavage of vera­ trylglycerol, yielding glycolaldehyde and veratraldehyde. Both a synthetic lignin model substrate and the natu­ ral metabolites of the white-rot fungus were oxidized by this extracellular peroxidase. The possible roles of this nitrogen recycling system and the cinnamate pathway, which are involved in the secondary metabolism of L­ -phenylalaninein brown-rot and white-rot fungi, are dis­ cussed in relation to wood decay processes. It is t i m e l y t o a t t e m p t t o f o r w a r d a n u n i f y i n g hypothesis for the processes of: (i) l i g n i n biosynthesis i n higher p l a n t s a n d l i g n i n biodégradation b y w h i t e - r o t f u n g i ; (ii) cellulose a n d l i g n i n d e g r a d a t i o n b y w h i t e - r o t f u n g i ; ( i i i ) the d e g r a d a t i o n o f p l a n t l i g n i n s a n d m o n o m e r i c f u n g a l m e t a b o l i t e s d u r i n g w o o d decay; a n d ( i v ) differences i n L - p h e n y l a l a n i n e - c i n n a m a t e p a t h w a y s between w h i t e - r o t a n d b r o w n - r o t f u n g i . A s F i g u r e 1 depicts, p h e n y l a l a n i n e a m m o n i a - l y a s e ( P A L ) , w h i c h occurs u b i q u i t o u s l y i n higher plants a n d the w o o d - r o t t i n g B a s i d i o m y c e t e s (1-3), seems t o play a c o m m o n c e n t r a l role i n the conversion o f p h e n y l a l a n i n e ( b y d e a m i n a t i o n ) t o a w i d e v a r i e t y o f secondary m e t a b o l i t e s . These i n c l u d e lignins i n higher plants (4), v e r a t r y l a l c o h o l i n the w h i t e - r o t fungus Phanerochaete chrysosporium (4a), a n d m e t h y l p-anisate i n the b r o w n - r o t fungus 0097-6156/89/0399-0412$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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413

Lentinus lepideus (5). Interestingly, P A L is absent f r o m b o t h b a c t e r i a l a n d animal kingdoms. T h e t r u e b i o c h e m i c a l significance o f t h i s d e a m i n a t i o n , a n d t h e f u n c t i o n o f t h e secondary m e t a b o l i c p a t h w a y o r i g i n a t i n g f r o m L - p h e n y l a l a n i n e (69), are n o t f u l l y u n d e r s t o o d . It i s , t h o u g h , n o t e w o r t h y t h a t w o o d - r o t t i n g Basidiomycetes preferentially attack nitrogen-poor wood substrates i n their n a t u r a l e n v i r o n m e n t s . [Note t h a t the average C / N r a t i o s for h a r d w o o d s a n d softwoods are 300 a n d 1000, respectively (10).] T h i s preference is s u r p r i s i n g since t h e a v a i l a b i l i t y o f n i t r o g e n is c r u c i a l for the g r o w t h o f w o o d - d e s t r o y i n g m i c r o o r g a n i s m s . T h i s fact, together w i t h t h e a b u n d a n t a c c u m u l a t i o n o f nitrogen-free secondary m e t a b o l i t e s i n b o t h p l a n t s a n d w o o d - r o t t i n g f u n g i , attracts our attention to the phenylalanine-cinnamate pathway i n relation to carbon and nitrogen economy during growth. C o n s e q u e n t l y , t h i s review p r i n c i p a l l y focuses o n three p o i n t s : (i) a p o s s i b l e f u n c t i o n o f n i t r o g e n r e c y c l i n g , i n w h i c h P A L p l a y s a c o m m o n role for L - p h e n y l a l a n i n e - c i n n a m a t deus) a n d t h e w h i t e - r o t (P. chrysosporium) f u n g i ; ( i i ) possible m e t a b o l i c connections between l i g n i n biodégradation a n d v e r a t r y l a l c o h o l b i o s y n t h e sis c a r r i e d o u t b y t h e same w h i t e - r o t f u n g i ; a n d ( i i i ) t h e means w h e r e b y secondary m e t a b o l i c p a t h w a y s f u n c t i o n t o s u p p o r t l i g n i n d e g r a d a t i o n . Comparison of the Phenylalanine-Cinnamate Pathway in B r o w n Rot and White-Rot Fungi Brown-Rot Fungi. T h e b r o w n - r o t fungus L. lepideus was chosen as a m o d e l m i c r o o r g a n i s m , since i t has l o n g received a t t e n t i o n as a p i n e t i m b e r - d e g r a d e r (11). F r o m L - p h e n y l a l a n i n e , i t p r o d u c e s m e t h y l pm e t h o x y c i n n a m a t e a n d p - m e t h o x y b e n z o a t e (p-anisate) esters as m a j o r m e t a b o l i t e s (11-13). T h e p h e n y l a l a n i n e - c i n n a m a t e p a t h w a y o f t h i s f u n gus has been e s t a b l i s h e d , as s h o w n i n F i g u r e 2, b y S h i m a z o n o et ai (13) a n d Towers (14). T h e r e l a t i o n s h i p , i f any, between t h e secondary m e t a b o l i s m o f L p h e n y l a l a n i n e a n d c a r b o h y d r a t e d e g r a d a t i o n d u r i n g b r o w n - r o t w o o d decay processes has n o t yet been d e t e r m i n e d . However, we suspect t h a t t h e seco n d a r y m e t a b o l i s m o f t h i s a r o m a t i c a m i n o - a c i d plays a n i m p o r t a n t role i n c o n v e r t i n g m o n o m e r i c sugars t o nitrogen-free m e t a b o l i t e s ( S h i m a d a , M . , a n d T a k a h a s h i , M . , I n Handbook of Wood and Cellulosic Materials; H o n , D . N . S. a n d S h i r a i s h i , N . , E d s . ; M a r c e l D e k k e r , i n press). T a b l e I shows t h e a m o u n t s o f secondary m e t a b o l i t e s f o r m e d o n d a y s 11 a n d 33 ( d u r i n g a n i n c u b a t i o n o f 63 days) for b o t h n i t r o g e n - p o o r ( H C / L N ) a n d n i t r o g e n - r i c h ( H C / H N ) cultures (15). T h e i n i t i a l C / N r a t i o s o f the t w o c u l t u r e s were 240 a n d 24, respectively. F i g u r e 3 shows t h e v a r i a t i o n s i n t h e t o t a l a m o u n t s o f t h e secondary m e t a b o l i t e s p r o d u c e d , t h e weights o f t h e f u n g a l m y c e l i u m , a n d t h e n i t r o g e n a n d glucose c o n c e n t r a t i o n s r e m a i n i n g i n the H C - L N c u l t u r e m e d i a d u r i n g the i n c u b a t i o n p e r i o d s h o w n . T h e m a j o r m e t a b o l i t e s f o r m e d i n the H C / L N a n d H C / H N c u l t u r e s were m e t h y l p - m e t h o x y c i n n a m a t e (11), m e t h y l p - m e t h o x y b e n z o a t e (11),

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

414

PLANT C E L L W A L L P O L Y M E R S

CH OH 2

CH

COOMe

OMe

OMe ( L. lepideus )

F i g u r e 1. P h e n y l a l a n i n e a m m o n i a - l y a s e ( P A L ) involvement i n t h e b i o s y n thesis o f p h e n y l p r o p a n o i d - d e r i v e d secondary m e t a b o l i t e s i n p l a n t s a n d B a sidiomycetes.

COOH I CHNH I

2

COOH I CH II

COOH I CH II

COOMe I CH II

COOMe I CH II

F i g u r e 2. T h e p h e n y l a l a n i n e - c i n n a m a t e p a t h w a y i n t h e b r o w n - r o t fungus Lentinus lepideus.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

F i g u r e 3. R e l a t i o n s h i p between secondary m e t a b o l i t e p r o d u c t i o n , c o n s u m p t i o n o f n i t r o g e n a n d c a r b o n sources, a n d g r o w t h of the b r o w n - r o t fungus Lentinus lepideus ( H C - L N m e d i u m ) .

416

PLANT C E L L W A L L P O L Y M E R S

T a b l e I. Secondary M e t a b o l i t e s P r o d u c e d by the B r o w n - R o t F u n g u s nus lepideus (15)

Lenti-

A m o u n t s ( m g / 1 0 m l culture) HC-LN Metabolites M e t h y l p-methoxybenzoate trans-Methyl p-methoxycinnamate cis-Methyl p-methoxycinnamate M e t h y l iso-ferulate M e t h y l p-coumarate p-Methoxyphenylpropanol

HC-HN

a

b

a

b

0.72 0.98 0.40 0.00 0.00 0.00

0.23 1.98 0.20 0.03 0.00 0.01

1.38 0.93 0.05 0.15 0.08 0.51

0.18 0.00 0.03 0.00 0.00 0.00

T o t a l amounts a a n d b i n d i c a t e the 11-day-old a n d 3 3 - d a y - o l d cultures used, respectively, for analyses. H C - L N = high carbondow nitrogen ratio. H C - H N = high carbomhigh nitrogen ratio. a n d p - m e t h o x y p h e n y l p r o p a n o l ; the l a t t e r was a p r e v i o u s l y u n k n o w n seco n d a r y m e t a b o l i t e f r o m this source, a n d was p r o d u c e d i n even greater a m o u n t s i n H C / H N c u l t u r e . W h i l e the t o t a l a m o u n t of these secondary m e t a b o l i t e s f o r m e d i n H C / H N c u l t u r e was s l i g h t l y greater t h a n t h a t observed for the H C / L N c u l t u r e , the relative a m o u n t per n i t r o g e n u n i t i n the H C / L N c u l t u r e was 8-fold greater t h a n t h a t o f the H C / H N c u l t u r e . T h e results ( F i g u r e 3) i n d i c a t e t h a t j u s t before complete c o n s u m p t i o n of n i t r o g e n , the q u a n t i t y of secondary metabolites increases, r e a c h i n g a first m a x i m u m o n day 11. A second m a x i m u m then appears o n d a y 33, at a b o u t the t i m e of 6 0 % glucose c o n s u m p t i o n . A l t h o u g h the reason for the appearance of two m a x i m a is not clear, these results were r e p r o d u c i b l e . It is n o t e w o r t h y , t h o u g h , t h a t n i t r o g e n s t a r v a t i o n accelerates the b i o s y n t h e sis of m e t a b o l i t e s derived f r o m L - p h e n y l a l a n i n e i n H C / L N c u l t u r e . T h u s , P A L m a y c o n t r i b u t e to secondary m e t a b o l i t e a c c u m u l a t i o n under n i t r o g e n l i m i t i n g c o n d i t i o n s , perhaps i n a n effort to economize the use of a v a i l a b l e nitrogen. White-Rot Fungi. T h e w h i t e - r o t fungus P. chrysosporium was chosen for c o m p a r i s o n , since i t has been extensively investigated for l i g n i n biodégrad a t i o n d u r i n g t h i s decade. V e r a t r y l a l c o h o l was first r e p o r t e d (16) to be b i o s y n t h e s i z e d f r o m p h e n y l a l a n i n e as a secondary m e t a b o l i t e i n the l i g n i n o l y t i c c u l t u r e of t h i s w h i t e - r o t fungus. Recently, other w h i t e - r o t f u n g i have been r e p o r t e d to produce the same secondary m e t a b o l i t e (17). H o w ever, the b i o c h e m i c a l significance of v e r a t r y l alcohol has r e m a i n e d unclear for some t i m e (18). T h e p r e v i o u s l y proposed m e t a b o l i s m of L - p h e n y l a l a n i n e to v e r a t r y l a l cohol (19-22) is now s l i g h t l y m o d i f i e d , as s h o w n i n F i g u r e 4. T h i s p a t h w a y

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

30.

Roles ofSecondary Metabolism of Fungi

SHIMADA E T A L

Glucose

417

L - Methionine 4 (SAM)

COOH

COOH

!

CΗ·Ν Η

x

11

2

-

J CH

COOH

I

I

CH

CH

II

HC

2

HC

OH

A

-ΟH

. OH

OMe

^OMe

OMe

OMe

OMe

OMe OMe

1 CH OH 2

ii ,011 COOH

C -ί unit

COOH

il

OMe

CHO I

I CHOH

OMe

OMe

OMe

CHOH

πιο

OMe

^OMe OMe

0 CH OH

0 Η

2

Η

OMe

MeO \ 0

OMe Ό

OMe OMe

F i g u r e 4. T h e L - p h e n y l a l a n i n e - c i n n a m a t e p a t h w a y for b i o s y n t h e s i s a n d biodégradation of v e r a t r y l a l c o h o l i n the w h i t e - r o t fungus Phanerochaete chrysosporium.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

418

PLANT C E L L W A L L POLYMERS

i n d i c a t e s t h a t p h e n y l a l a n i n e serves as a p r i m a r y a m i n o a c i d precursor, bei n g converted t o the a c c u m u l a t i n g m e t a b o l i t e v e r a t r y l a l c o h o l v i a caffeic a c i d , ferulic a c i d , 3 , 4 - d i m e t h o x y c i n n a m i c a c i d , 3 , 4 - d i m e t h o x y c i n n a m y l a l c o h o l , a n d v e r a t r y l g l y c e r o l intermediates. V e r a t r y l a l c o h o l t h e n seems t o be degraded, v i a v a n i l l i c a c i d or r i n g cleavage p r o d u c t s (23,24). T h e two m e t h o x y l carbons of v e r a t r y l a l c o h o l are p r o b a b l y derived f r o m m e t h i o nine v i a S - a d e n o s y l m e t h i o n i n e ( S A M ) (19), w h i c h is also i n v o l v e d i n the biosynthesis of p - m e t h o x y c i n n a m a t e i n L. lepideus (25,26). Interestingly, the f u n g a l h y d r o x y c i n n a m a t e p a t h w a y s i n b o t h the b r o w n - r o t a n d w h i t e rot f u n g i r e m i n d s us o f the h y d r o x y c i n n a m a t e p a t h w a y s i n the biosynthesis of p l a n t l i g n i n s (27). T a b l e II shows (28) a c o r r e l a t i o n between the biosynthesis of v e r a t r y l a l c o h o l a n d P A L a c t i v i t i e s , b o t h of w h i c h are affected b y i n i t i a l glucose a n d a m m o n i u m salt levels. T h e H C - L N c u l t u r e w i t h a C / N r a t i o of 240, w h i c h is a l m o s t c o m p a r a b l e to t h a t of w o o d , shows the greatest a m o u n t of v e r a t r y l a l c o h o l biosynthesis a n d seen, d e p e n d i n g u p o n the C : N balance of the m e d i a used, the a m o u n t s of secondary m e t a b o l i t e s formed a n d P A L activités c a n v a r y g r e a t l y (Tables I a n d II). T a b l e I I . P A L A c t i v i t y of the W h i t e - R o t F u n g u s P. chrysosporium i n the Different C u l t u r e M e d i a (28)

Grown

Veratryl alcohol ( n m o l e s / 1 0 m l culture) Culture" HC-LN HC-HN LC-HN LC-LN α

b

C/N Ratio 240 24 6 60

7 days

14 days

PAL Activity

1786 0 250 1595

7142 294 0 857

131,529 19,176 20,450 35,496

4

% 100 15 16 27

H C a n d L C i n d i c a t e 2 % a n d 0 . 5 % glucose c o n t a i n e d i n the m e d i u m , respectively. H N a n d L N i n c i d a t e 24 m M a n d 2.4 m M a m m o n i u m salt n i t r o g e n , respectively, used for the cultures. P A L a c t i v i t i e s / 6 - d a y o l d c u l t u r e (20 m l ) are expressed as r a d i o a c ­ t i v i t i e s ( d p m ) of c i n n a m a t e formed f r o m L - p h e n y l a l a n i n e - U - C (2 n m o l e s / μΟΊ). 1 4

B i o c h e m i c a l Significance o f t h e P h e n y l a l a n i n e - C i n n a m a t e way i n P l a n t s a n d F u n g i i

Path­

W o o d y p l a n t s c a n synthesize anywhere f r o m 15-30% of a l l biomass as l i g n i n . H e n c e , equivalent a m o u n t s of p h e n y l a l a n i n e are required at some p o i n t , i.e., d u r i n g l i g n i n f o r m a t i o n , large a m o u n t s of a m m o n i a are recycled as a conse­ quence of P A L a c t i v i t y . W h i l e p l a n t s have no serious p r o b l e m s i n o b t a i n i n g glucose as a c a r b o n source, s u p p l i e d a b u n d a n t l y t h r o u g h photosynthesis,

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

30.

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the same c a n n o t be s a i d for n i t r o g e n n u t r i e n t a v a i l a b i l i t y . T h i s underscores the i m p o r t a n c e o f a m m o n i u m r e c y c l i n g d u r i n g l i g n i n f o r m a t i o n . A s i m i l a r a n a l o g y c a n be a p p l i e d t o the r a t i o n a l i z a t i o n o f the b i o s y n ­ thesis o f secondary m e t a b o l i t e s p r o d u c e d b y w o o d - r o t t i n g f u n g i i . T h e B a sidiomycetes i n h a b i t i n g w o o d hosts have no t r o u b l e o b t a i n i n g glucose, b u t have p r o b l e m s i n o b t a i n i n g significant a m o u n t s o f n i t r o g e n - c o n t a i n i n g n u ­ trients. Nitrogen Recycling with P A L T h e t o t a l a m o u n t of m e t a b o l i t e s synthesized b y the b r o w n - r o t fungus after 33 d a y s ( F i g u r e 3, second m a x i m u m ) is a b o u t 1 0 % of the d r y weight of m y c e l i a p r o d u c e d . However, the a c t u a l percentage is m u c h h i g h e r , since m e t a b o l i c t u r n o v e r occurs d u r i n g f u n g a l g r o w t h . C u l t u r e s of Phanerochaete chrysosporium gave s i m i l a r r e s u l t s , since the a m o u n t o f v e r a t r y l a l c o h o l p r o d u c e d was a b o u t 1 0 % m u s t be h i g h e r , since v e r a t r y therefore be reasoned t h a t the a m o u n t s o f f u n g a l m e t a b o l i t e s p r o d u c e d a p p r o x i m a t e those of l i g n i n i n w o o d y p l a n t s . T h e r e f o r e , a s i m i l a r level o f n i t r o g e n r e c y c l i n g ( u s i n g P A L ) operates i n b o t h p l a n t s a n d the w o o d r o t t i n g B a s i d i o m y c e t e s (see F i g u r e 1). I n contrast to l i g n i n s , the b i o l o g i c a l f o r m a t i o n of the B a s i d i o m y c e t e s secondary m e t a b o l i t e s is not clearly u n d e r s t o o d . H o w e v e r , we propose t h a t there m a y be some b i o l o g i c a l significance i n the conversion of n u t r i t i o n a l l y v a l u a b l e glucose a n d a m i n o acids to secondary m e t a b o l i t e s w i t h l i t t l e n u ­ t r i t i o n a l value t o other o r g a n i s m s s h a r i n g the same ecosystems. I n other w o r d s , the a c c u m u l a t i o n o f m o n o m e r i c sugars, p r o d u c e d b y the e n z y m a t i c h y d r o l y s i s of cellulose a n d hemicelluloses, w o u l d j e o p a r d i z e t h e i r h a b i t a ­ t i o n b y a t t r a c t i n g i n t r u d e r s . T h u s , the u n i q u e n u t r i t i o n a l e n v i r o n m e n t o f w o o d s u b s t r a t e s w i t h h i g h C / N r a t i o s (i.e., n i t r o g e n - p o o r ) m a y have forced the B a s i d i o m y c e t e s t o create a c o m m o n c i n n a m a t e p a t h w a y . T h i s is one possible e x p l a n a t i o n for the u b i q u i t o u s occurrence o f P A L i n the w o o d destroying Basidiomycetes. C o r r e l a t i o n b e t w e e n V e r a t r y l A l c o h o l Synthesis a n d L i g n i n D e ­ gradation in White-rot Fungi Aromatic Ring and Οα-Οβ Bond Cleavage. L e t us now t u r n o u r a t t e n ­ t i o n t o a r o m a t i c r i n g cleavage of v e r a t r y l a l c o h o l , a n d the C a - C / ? b o n d cleavage o f v e r a t r y l g l y c e r o l , w h i c h are b o t h f o r m e d f r o m p h e n y l a l a n i n e as s h o w n i n F i g u r e 4. B o t h cleavage reactions m a y be r e l a t e d to the corre­ s p o n d i n g d e g r a d a t i o n reactions of l i g n i n (29). Indeed, v e r a t r y l a l c o h o l is c o m m o n l y used as a s u b s t r a t e for a n assay o f " l i g n i n a s e " a c t i v i t y (30), b y m e a s u r e m e n t o f the absorbance due to v e r a t r a l d e h y d e f o r m e d . [However, s m a l l a m o u n t s of 7 - ( 5 - m e m b e r e d ) (23) a n d ^ ( 6 - m e m b e r e d ) lactones (24) are also p r o d u c e d f r o m v e r a t r y l a l c o h o l as r i n g cleavage p r o d u c t s ( F i g u r e 5).] A s c a n also be seen, v e r a t r y l g l y c e r o l undergoes Co>C/? b o n d cleavage, y i e l d i n g v e r a t r a l d e h y d e a n d g l y c o l a l d e h y d e i n the presence o f " l i g n i n a s e "

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CHO

OMe

F i g u r e 5. T h e e n z y m a t i c o x i d a t i o n of a s y n t h e t i c β-Ο-4 l i g n i n m o d e l s u b ­ strate a n d f u n g a l secondary metabolites. B o t h undergo C a - C / ? b o n d a n d a r o m a t i c r i n g cleavages i n reactions c a t a l y z e d by the same " l i g n i n a s e " i n the presence of H 2 O 2 .

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a n d h y d r o g e n peroxide (31). [Isolation of g l y c o l a l d e h y d e f r o m the e n z y m i c r e a c t i o n m i x t u r e as its 2 , 4 - d i n i t r o p h e n y l h y d r a z o n e d e r i v a t i v e was achieved b y t r e a t m e n t of the m i x t u r e w i t h 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e at 3 0 ° C for 30 m i n . ] O x i d a t i v e d e g r a d a t i o n of the β-Ο-4 l i g n i n s u b s t r u c t u r e m o d e l c o m ­ p o u n d (32), c a t a l y z e d b y " l i g n i n a s e " / H 2 O 2 , consists of two types of cleav­ age reactions (routes b l a n d b2) (see F i g . 5). These correspond to C a - C / ? b o n d cleavage (see route a) of v e r a t r y l g l y c e r o l a n d a r o m a t i c r i n g o p e n i n g (see route c) of v e r a t r y l a l c o h o l . T h u s , " l i g n i n a s e " is shared b y the three substrates of different o r i g i n : the s y n t h e t i c l i g n i n m o d e l substrate a n d the two n a t u r a l m e t a b o l i t e s of f u n g a l o r i g i n . It can therefore be proposed t h a t the b i o s y n t h e t i c p a t h w a y of v e r a t r y l a l c o h o l ( F i g u r e 4) is l i n k e d to l i g n i n d e g r a d a t i o n . Hydrogen Peroxide-Generating System. A n o t h e r i m p o r t a n t feature of t h i s secondary m e t a b o l i c p a t h w a substrate for g l y o x a l oxidas fungus to generate h y d r o g e n peroxide required for l i g n i n d e g r a d a t i o n (route d i n F i g . 5). Interestingly, b o t h v e r a t r y l g l y c e r o l a n d v e r a t r y l a l c o h o l o c c u r e x c l u s i v e l y i n the e x t r a c e l l u l a r fluid of the f u n g a l c u l t u r e . F u r t h e r m o r e , f o r m a t i o n of g l y o x a l , w h i c h is also f o u n d i n the e x t r a c e l l u l a r f r a c t i o n of the c u l t u r e , is a secondary m e t a b o l i c event (33). W e therefore suspect t h a t g l y o x a l m i g h t be p r o d u c e d f r o m g l y c o l a l d e h y d e , w i t h the l a t t e r b e i n g f o r m e d b y s i d e - c h a i n cleavage of b o t h "endogenous" v e r a t r y l g l y c e r o l a n d "exogenous" l i g n i n substrates. C o n s e q u e n t l y , the e x t r a c e l l u l a r o x i d a t i o n of g l y c o l a l d e h y d e , d e r i v e d f r o m either the c i n n a m a t e p a t h w a y or s i d e - c h a i n cleavage of l i g n i n , m a y f u n c t i o n t o s u p p o r t l i g n i n d e g r a d a t i o n by p r o d u c i n g H 2 O 2 , (route d i n F i g . 5). T h i s w o u l d t h e n act i n concert w i t h other h y d r o g e n p e r o x i d e p r o d u c i n g systems such as: (i) g l y o x a l / g l y o x a l a s e (33), (ii) g l u c o s e / g l u c o s e oxidase (34), ( i i i ) N A D ( P ) H / p e r o x i d a s e (35), a n d (iv) f a t t y a c y l - c o e n z y m e A oxidase (36). Physiological a n d Biochemical Relationships between L i g n i n B i o degradation a n d V e r a t r y l A l c o h o l Biosynthesis P a r a l l e l p h y s i o l o g i c a l a n d b i o c h e m i c a l r e l a t i o n s h i p s between l i g n i n biodég r a d a t i o n a n d v e r a t r y l a l c o h o l biosynthesis are s u m m a r i z e d below: 1. B o t h l i g n i n biodégradation a n d v e r a t r y l a l c o h o l biosynthesis are seco n d a r y m e t a b o l i c events affected b y several c o m m o n p h y s i o l o g i c a l factors, such as o x y g e n tension, a g i t a t i o n , a n d n i t r o g e n content (16,3739). These C - , N - , a n d S - s t a r v a t i o n s are i m p o r t a n t triggers for i n " l i g n i n a s e " i n d u c t i o n (37-39), since " l i g n i n a s e " is p r o d u c e d c o n s t i t u t i v e l y regardless of the presence or absence of l i g n i n as s u b s t r a t e . 2. B o t h processes are repressed b y a d d i t i o n i n t o the c u l t u r e of n i t r o g e n n u t r i e n t s such as a m m o n i u m salts a n d L - g l u t a m a t e (16, 37-39). 3. T h e level of c y c l i c A M P ( c A M P ) is increased b y n i t r o g e n s t a r v a t i o n ; t h i s triggers expression of l i g n i n o l y t i c a c t i v i t y a n d v e r a t r y l a l c o h o l biosynthesis (40).

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4. Interestingly, peroxidase-less ( P O ) m u t a n t s , w h i c h cannot decompose l i g n i n nor biosynthesize v e r a t r y l a l c o h o l (41,42), l a c k P A L a c t i v i t y (28). H o w e v e r , there are two articles w h i c h report evidence against these r e l a t i o n s h i p s between biosynthesis of t h i s secondary m e t a b o l i t e a n d l i g n i n d e c o m p o s i t i o n (43,44). F o r e x a m p l e , a m u t a n t of P. chrysosporium, which does not produce v e r a t r y l a l c o h o l , has l i g n i n o l y t i c a c t i v i t y (43). A n o t h e r m u t a n t , w h i c h lacks glucose oxidase, is u n a b l e to decompose l i g n i n to C O 2 a n d therefore is "ligninase"-less. It is, however, able to produce a b o u t 3 0 % of the a m o u n t of v e r a t r y l a l c o h o l n o r m a l l y f o u n d i n the fungus (44). T h e s e findings m u s t be carefully i n t e r p r e t e d . T h e v e r a t r y l a l c o h o l negative m u t a n t w h i c h has " l i g n i n a s e " lacks P A L a c t i v i t y . C o n s e q u e n t l y , v e r a t r y l a l c o h o l biosynthesis is shut d o w n ( F i g . 4). H o w e v e r , i f v e r a t r y l a l c o h o l or r e l a t e d a r o m a t i c c o m p o u n d s are a d d e d t o the c u l t u r e , they are d e c o m posed to C O 2 b y the P A L - l e s s m u t a n t (42). F o r the glucose oxidase negative m u t a n t , i t m a y be t h a t th produces v e r a t r y l a l c o h o l tive p a t h w a y , e.g., b y / ? - o x i d a t i o n of 3 , 4 - d i m e t h o x y c i n n a m i c a c i d to v e r a t r i c a c i d , w h i c h is t h e n subsequently reduced to v e r a t r y l a l c o h o l . U n f o r t u n a t e l y , those a u t h o r s d i d not e x a m i n e the p o s s i b i l i t y of the absence of " l i g n i n a s e " i n the g o x " m u t a n t . T h e "ligninase"-less a n d P A L - l e s s m u t a n t is , t h o u g h , capable of c o n v e r t i n g exogenously added 3 , 4 - d i m e t h o x y c i n n a m i c a c i d a n d v e r a t r i c a c i d to v e r a t r y l a l c o h o l (28). T h i s suggests t h a t there is another route for the biosynthesis of v e r a t r y l a l c o h o l , i n a d d i t i o n to the v e r a t r y l g l y c e r o l cleavage p a t h w a y already discussed ( F i g . 4). T a k i n g these findings together, w i t h the above-described p a r a l l e l i s m a n d a p p a r e n t c o n t r a d i c t i o n s , the f o l l o w i n g hypothesis is p r o p o s e d . W e s u g gest t h a t the same key e n z y m e s y s t e m , or " l i g n i n a s e " , couples the b i o s y n thesis of v e r a t r y l a l c o h o l a n d the biodégradation of l i g n i n . It is also notew o r t h y t h a t " l i g n i n a s e " u t i l i z e s not o n l y l i g n i n , b u t a w i d e variety o f x e n o b i o t i c c o m p o u n d s regardless of their c h e m i c a l s t r u c t u r e s . T h e i r i o n i z a t i o n p o t e n t i a l s are r a t h e r i m p o r t a n t for e n z y m a t i c o x i d a t i o n (45). T h i s is the reason w h y c o m p o u n d s such as benzo(a)pyrene (46), d i o x i n (47) a n d a - k e t o 7 - m e t h y l t h i o b u t y r i c a c i d a n d dyes (48) are also o x i d i z e d by " l i g n i n a s e . " C o n c l u d i n g R e m a r k s a n d Perspectives In c o n c l u s i o n , f o c u s i n g o n the s i m i l a r i t i e s a n d differences between w h i t e - r o t a n d b r o w n - r o t f u n g i , these seemingly different b i o l o g i c a l processes can be e x p l a i n e d as follows. 1. B o t h w h i t e - r o t a n d b r o w n - r o t f u n g i have a c o m m o n c i n n a m a t e p a t h way, i n i t i a t e d b y P A L , w h i c h plays a key role i n the biosynthesis of p h e n y l p r o p a n o i d s i n b a s i d i o m y c e t o u s f u n g i . T h e s e f u n g i are able to convert glucose i n t o "scavenged" m e t a b o l i t e s under n i t r o g e n - l i m i t i n g conditions. 2. T h e p h e n y l a l a n i n e - c i n n a m a t e p a t h w a y of the w h i t e - r o t fungus P. chrysosporium is l i n k e d to l i g n i n biodégradation b y two reactions, i.e., b y b o t h C a - C / ? b o n d a n d a r o m a t i c r i n g cleavages. These represent the

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p r e d o m i n a n t features o f l i g n i n biodégradation, a n d therefore operate as p a r t o f the secondary m e t a b o l i s m o f p h e n y l a l a n i n e . T h u s , d u r i n g t h e i r b i o c h e m i c a l e v o l u t i o n , t h e w h i t e - r o t f u n g i have succeeded i n a d a p t i n g e x t r a c e l l u l a r peroxidases t o l i g n i n b r e a k d o w n , whereas t h e b r o w n - r o t f u n g i f a i l e d t o develop s u c h a b i o c a t a l y s t o r m e t a b o l i c p a t h w a y . I n c o n t r a s t , higher p l a n t s m i g h t have created peroxidases for p o l y m e r i z i n g h y d r o x y c i n n a m y l alcohols t o l i g n i n s d u r i n g t h e i r b i o c h e m i c a l e v o l u t i o n . I n c l o s i n g , t h e recent advances i n l i g n i n biodégradation research are r e m a r k a b l e a n d are r e c e i v i n g w i d e s p r e a d interest f r o m m a n y fields (49,50). A t present, " l i g n i n a s e " is k n o w n t o c a t a l y z e a w i d e v a r i e t y o f one-electron o x i d a t i o n s , b u t i t s t i l l cannot d e p o l y m e r i z e t h e l i g n i n i n vitro (51,52). O n the o t h e r h a n d , t h e w h i t e - r o t f u n g i c a r r y o u t a n a l m o s t c o m p l e t e d e c o m p o s i t i o n o f l i g n i n i n w o o d i n t h e i r n a t u r a l e n v i r o n m e n t . T h u s , there m a y be a n o t h e r e n z y m e (or system) i n v o l v e d f u n c t i o n i n g as a d e p o l y m e r i z i n g fact o r c o u p l e d t o l i g n i n d e g r a d a t i o n . F u r t h e r f u n d a m e n t a l research is needed i n order t o e l u c i d a t e t h c l u d i n g t h e n i t r o g e n r e c y c l i n g o f a m i n o acids i n v o l v e d i n f u n g a l secondary m e t a b o l i s m . F u r t h e r m o r e , b i o m i m e t i c systems based o n o u r u n d e r s t a n d i n g of the b i o c h e m i s t r y o f p l a n t s a n d f u n g i (53,54) m a y be m o r e a p p l i c a b l e for conversion o f l i g n o c e l l u l o s i c m a t e r i a l s . Literature Cited

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14. Towers, G. H. N. In Perspectives in Phytochemistry; Harborne, J. B.; Swain, T., Eds.; Academic: London, 1969; p. 179. 15. Ohta, Α.; Shimada, M.; Hattori, T.; Higuchi, T.; Takahashi, M. Proc. 38th Meet. J. Wood Res. Soc. 1988; p. 200. 16. Shimada, M., Nakatsubo, F.; Kirk, T. K.; Higuchi, T. Arch. Microbiol. 1981, 129, 321. 17. Kawai, S.; Umezawa, T.; Higuchi, T.; Wood Research 1984, 73, 18. 18. Kirk, T. K. In Lignin Biodegradation: Microbiology, Chemistry, and Potential Applications; Kirk, T. K.; Higuchi, T. K.; Chang, H.-M., Eds.; CRC Press: Boca Raton, 1980; p. 51. 19. Shimada, M.; Nakatsubo, F.; Higuchi, T.; Kirk, T. K. Proc. ISWPC (Ekman days) 1981, p. 91. 20. Buswell, J. Α.; Ander, P.; Petersson, B.; Eriksson, K.E. FEBS Lett. 1979, 103, 98. 21. Yajima, Y.; Enoki, Α. 22. Ander, P.; Hatakka, 189. 23. Leisola, M. S. Α.; Schmidt, B.; Thaney-Wyss, U.; Fiechter, A. FEBS Lett. 1985, 189, 267. 24. Shimada, M.; Hattori, T.; Umezawa, T.; Higuchi, T., Uzura, K. FEBS Lett. 1987, 221, 327. 25. Shimazono, H. Arch. Biochem. Biophys. 1959, 83, 206. 26. Wat, C.-H.; Towers, G. H. N. Phytochemistry 1975, 14, 663. 27. Higuchi, T. Biosynthesis and Biodegradation of Wood Components; Academic: New York, 1985; p. 141. 28. Shimada, M. Wood Res. Tech. Notes 1983, 17, 21. 29. Tai, D.; Terazawa, M.; Chen, C. L.; Chang, H. M.; Kirk, T. K. In Recent Advances in Lignin Biodegradation; Higuchi, T.; Chang, H.-M.; Kirk, T. K., Eds.; Uni: Tokyo, 1983; p. 44. 30. Tien, M.; Kirk, T. K.; Bull, C.; Fee, J. A. J. Biol. Chem. 1986, 261, 1687. 31. Shimada, M.; Kurosaka, H.; Hattori, T.; Higuchi, T. Proc. 38th Ann. Meet. Wood Res. Soc. Japan; 1988, p. 100. 32. Umezawa, T.; Shimada, M.; Higuchi, T.; Kusai, K. FEBS Lett. 1986, 205, 287. 33. Kersten, P. J.; Kirk, T. K. J. Bacteriol. 1987, 169, 2195. 34. Kelley, R. L.; Reddy, C. A. Arch. Microbiol. 1986, 144, 248. 35. Kuwahara, M.; Ishida, Y.; Miyagawa, Y.; Kawakami, C. F. Ferment. Technol. 1984, 62, 237. 36. Green, R.; Gould, J. M. Biochem. Biophys. Res. Commun. 1984, 118, 437. 37. Kirk, T. K. Proc. ISWPC (Ekman Days) 1981, 3, 67. 38. Fenn, D.; Kirk, T. K. Arch. Microbiol. 1981, 130, 59. 39. Fenn, D.; Kirk, T. K. Arch. Microbiol. 1981, 130, 65. 40. MacDonald, M. J.; Paterson, Α.; Broda, P. J. Bacteriol. 1984, 160, 470.

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41. Ander, P.; Hatakka, Α.; Eriksson, Κ. E. Arch. Microbiol. 1980, 125, 189. 42. Gold, M. H.; Mayfield, M. B.; Cheng, T. M.; Krisnangkura, K.; Shi­ mada, M.; Enoki, A. Arch. Microbiol. 1982, 132, 115. 43. Liwicki, R.; Patterson, Α.; MacDonald, M. J.; Broda, P. J. Bacteriol. 1985, 162, 641. 44. Ramasamy, K.; Kelley, R.L.; Reddy, C.A. Biochem. Biophys. Res. Commun. 1985, 131, 436. 45. Kersten, P. J.; Kalyanaraman, B.; Hammel, Κ. E.; Kirk, T. K. In Lignin Enzymic and Microbial Degradation; Odier, E., Ed.; INRA Publ., 1987; p. 75. 46. Haemmerli, S. D.; Leisola, M. S. Α.; Sanglard, D.; Fiechter, A. J. Biol. Chem. 1986, 261, 6903. 47. Hammel, Κ. E.; Kalyanaraman, B.; Kirk, T. K. J. Biol. Chem. 1986, 261, 16948. 48. Gold, M. H.; Glenn, H. In Recent Advances in Lignin Biodegradation; Higuchi, T.; Kirk, T. K.; Chang, H.-M., Eds.; Uni: Tokyo, 1983; p. 219. 49. Odier, E. Lignin Enzymic and Microbial Degradation; INRA, 1987. 50. Buswell, J. Α.; Odier, E. CRC Crit. Rev. Biotechnol. 1987, 6, 1. 51. Haemmerli, S. D.; Leisola, M. S. Α.; Fiechter, A. FEMS Microbiol. Lett. 1986, 35, 33. 52. Kirk, T. K. Proceedings Tappi Res. Dev. 1986, p. 73. 53. Shimada, M.; Habe, T.; Higuchi, T.; Okamoto, T.; Panijpan, B. Holz­ forschung 1987, 41, 277. 54. Paszcynski, R.; Crawford, R. L.; Blanchette, R. A. Appl. Environ. Mi­ crobiol. 1988, 54, 62. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 31

Ultrastructural Localization of Lignocellulose­ -Degrading Enzymes 1

1

1

2

I. M. Gallagher , M. A. Fraser , C. S. Evans , P. T. Atkey , and D. A. Wood 2

1

School of Biological Sciences, Thames Polytechnic, London SE18 6PF, England AFRC Institute of Horticultural Research, Littlehampton BN16 3PU,

2

Transmission electron microscopy of immuno-gold labelled sections was used to show the localisation of the ligninolytic enzymes, lignin-peroxidase and laccase in ultrathin sections of hyphae of the white-rot fungus Coriolus versicolor. Both enzymes were localized in the fungal cell walls and mucilage layers of both generative and skeletal hyphae, whereas ligninase but not laccase was also evident adjacent to the plasma membrane. The localization of these enzymes was the same in hyphae grown in culture and in sections of beech heartwood infected with C. versicolor. Control experiments showed that no labelling was detected in the absence of primary antibodies, or when antibodies to animal or plant enzymes were substituted but antibodies to fungal proteins from different species were labelled in sections of C. versicolor. The source of antigenicity in these sections was investigated. B a s i d i o m y c e t e f u n g i are the m a j o r organisms responsible for biodégradation of w o o d , w i t h w h i t e - r o t fungi able t o degrade a l l components o f the w o o d cell w a l l . E n z y m e s w h i c h p a r t i a l l y degrade l i g n i n , l i g n i n - p e r o x i d a s e s , were first isolated f r o m Phanerochaete chrysosponum (1-2) a n d more recently f r o m other w h i t e - r o t fungi i n c l u d i n g Coriolus versicolor (3). These enzymes appear t o be s i m i l a r i n a l l w h i t e - r o t f u n g i investigated, a l l c o n t a i n i n g a heme p r o s t h e t i c group a n d r e q u i r i n g trace a m o u n t s o f h y d r o g e n peroxide t o effect t h e b r e a k d o w n o f the l i g n i n p o l y m e r (4-5). T h e p o l y p h e n o l oxidase, laccase, p r o d u c e d as a n e x t r a c e l l u l a r enzyme b y C. versicolor, has been w e l l d o c u m e n t e d as a p o l y m e r i z i n g enzyme b u t recent work has s h o w n t h a t under c e r t a i n c o n d i t i o n s i t c a n also effect d e p o l y m e r i z a t i o n o f l i g n i n (6-8). 0097-6156/89/0399-0426$06.00/0 © 1989 American Chemical Society

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M o r p h o l o g i c a l d a t a o n t h e patterns o f d e g r a d a t i o n o f w o o d cell w a l l s after i n f e c t i o n b y w h i t e - r o t f u n g i showed t h a t i n a d d i t i o n t o w h i t e - r o t s c a u s i n g a l o c a l i z e d d e g r a d a t i o n , characterized b y a deep erosion t r o u g h i n the secondary w a l l o f the w o o d cell, a progressive t h i n n i n g o f the S2 layer of the secondary w a l l also o c c u r r e d (9-12). I d e n t i f i c a t i o n o f o s m i o p h i l i c p a r ticles i n sections o f d e c a y i n g w o o d led t o t e n t a t i v e suggestions t h a t these were either e n z y m e molecules i n v o l v e d i n w o o d decay or t h e p r o d u c t s of lignocellulosic d e g r a d a t i o n (12-13). Studies o f t h e b i o c h e m i c a l m e c h a n i s m of l i g n i n - p e r o x i d a s e have suggested t h a t i t is n o t always essential t h a t t h e e n z y m e s h o u l d be i n direct contact w i t h i t s substrate t o effect l i g n i n b r e a k d o w n . T h e release o f diffusible r a d i c a l cations i n p h e n o l i c substrates c a n enable d e g r a d a t i o n o f t h e l i g n i n p o l y m e r t o proceed at some distance f r o m the e n z y m e , a n d f u n g a l h y p h a (14 ). W h i t e - r o t f u n g i secrete a p o l y s a c c h a r i d e mucilage a r o u n d the h y p h a e w h i c h m a y f u n c t i o n as a m a t r i x for e n z y m e i m m o b i l i z a t i o n a n d enable p r o d u c t s o f reactions t o b E v a n s , C . S., u n p u b l i s h e d d a t a ) . Recently, significant advances were m a d e i n the field o f i m m u n o - l a b e l l i n g w i t h electron m i c r o s c o p y as a m e t h o d o f l o c a l i z i n g proteins a n d other molecules w i t h i n tissue sections, u s i n g c o l l o i d a l g o l d as a m a r k e r o f a n t i b o d y - a n t i g e n i n t e r a c t i o n s (16). I n order t o i d e n tify the site o f a c t i o n o f l i g n i n - d e g r a d i n g enzymes i n w o o d decay, c o l l o i d a l g o l d i m m u n o l o c a l i z a t i o n procedures were used t o locate the e x t r a c e l l u l a r enzymes, laccase a n d l i g n i n - p e r o x i d a s e , f r o m the w h i t e - r o t fungus C. versicolor c u l t u r e d i n beech h e a r t w o o d a n d i n m a l t agar. Methods Organism. Coriolus versicolor s t r a i n 2 8 A P R L ( B u i l d i n g Research E s t a b l i s h m e n t , P r i n c e s R i s b o r o u g h L a b o r a t o r y , A y l e s b u r y , B u c k s . , U . K . ) was m a i n t a i n e d o n a s o l i d m e d i u m o f 3 % ( w / v ) m a l t e x t r a c t , 2 % ( w / v ) agar at 2 0 ° C , or o n n i t r o g e n - l i m i t e d n u t r i e n t m e d i u m (17) solidified w i t h 2 % agar. Growth ofC. versicolor on Wood. S a m p l e s o f beech h e a r t w o o d (Fagus sylvatica) were added t o c u l t u r e plates of C. versicolor after 7 d . g r o w t h , u s i n g the B r a v e r y m i n i a t u r e woodblock technique (12). P l a t e cultures were covered w i t h a 1 m m mesh 6 0 m m diameter sterile n y l o n n e t . S t e r i l e sections of beech (30 x 10 x 3 m m ) were placed aseptically onto the net a n d c u l t u r e d at 2 0 ° C for 4 weeks. T h e e x t e r n a l g r o w t h o f m y c e l i u m was removed a n d the w o o d block c u t i n t o segments o f a p p r o x i m a t e l y 3 x 1 x 1 m m for p r e p a r a t i o n for electron microscopy. U n i n f e c t e d w o o d samples were prepared i n a similar manner. Immunogold Labelling. Tissues were fixed i n excess g l u t a r a l d e h y d e ( 2 . 5 % w / v ) i n l O O m M s o d i u m cacodylate buffer ( p H 7.2) for 3 h , washed twice i n t h e same buffer, followed b y d e h y d r a t i o n i n a graded e t h a n o l series. S a m p l e s were i n f i l t r a t e d w i t h L . R . W h i t e h a r d grade resin ( L o n d o n R e s i n C o . L t d ) . T h i n sections were cut w i t h a n L K B U l t r a m i c r o t o m e II u s i n g a d i a m o n d knife a n d collected o n copper grids (200 mesh). T h e i m m u n o g o l d l a b e l l i n g procedure followed the technique o f B e r g m a n (18). Sections were

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etched i n 5 % H 2 O 2 for 5-10 m i n , washed twice i n P B S ( 2 0 m M p h o s p h a t e buffer at p H 7.4 a n d 0.9% N a C l ) c o n t a i n i n g 0 . 1 % T w e e n 20, i n c u b a t e d for l h i n r a b b i t anti-laccase a n t i s e r u m or r a b b i t a n t i - l i g n i n a s e a n t i s e r u m , d i l u t e d 1:100 ( v / v ) i n P B S c o n t a i n i n g 0 . 1 % T w e e n 20 a n d 1% b o v i n e s e r u m a l b u m e n ( B S A ) , washed twice i n P B S c o n t a i n i n g 0 . 1 % T w e e n 20, i n c u b a t e d for l h i n goat a n t i - r a b b i t I g G conjugated to l O n m g o l d particles ( S i g m a ) , d i l u t e d 1:20 i n P B S c o n t a i n i n g 1% B S A a n d 0 . 1 % T w e e n 20, washed twice i n d i s t i l l e d water a n d air d r i e d . Post-staining Procedure. I m m u n o g o l d l a b e l l e d sections were p o s t - s t a i n e d i n s a t u r a t e d aqueous u r a n y l acetate for 15 m i n a n d lead c i t r a t e for 10 m i n (19) . Transmission Electron Microscopy. T r a n s m i s s i o n electron m i c r o s c o p y was p e r f o r m e d o n a J e o l 100S electron microscope o p e r a t i n g at 8 0 k V . Production of Antibodies. Laccas sicolor a n d p u r i f i e d b y a m o d i f i e d procedure of Fahraeus a n d R e i n h a m m e r (20) . T h e c r i t e r i o n for p u r i t y was a single b a n d o n a h e a v i l y l o a d e d S D S p o l y a c r y l a m i d e electrophoresis gel. A n t i b o d i e s were raised i n r a b b i t s i n response to three intravenous injections each of 2 m g . of laccase A w i t h F r e u n d s a d j u v a n t , at 2 weekly intervals. B l o o d was removed 7d after the last i n j e c t i o n a n d s e r u m collected after c o a g u l a t i o n of the b l o o d cells. T h e a n t i b o d y to laccase A was purified b y affinity c h r o m a t o g r a p h y o n C N - B r a c t i v a t e d Sepharose 4 B . Laccase (15mg) was b o u n d to a c o l u m n of 3 . 5 m l v o l u m e i n a c o u p l i n g buffer of 0 . 1 M N a H C 0 a n d 0 . 5 M N a C l . T h e r e m a i n i n g a c t i v e groups o n the c o l u m n were blocked w i t h 0 . 2 M g l y c i n e , before excess adsorbed p r o t e i n was removed w i t h the c o u p l i n g buffer, res u l t i n g i n 9 5 % c o u p l i n g of the laccase to the c o l u m n . C r u d e a n t i s e r a was b o u n d to the laccase o n the c o l u m n , washed w i t h buffer of 0 . 1 M Na2HP04 a n d 0 . 5 M N a C l , before e l u t i o n w i t h 0 . 2 M g l y c i n e - H C l a n d 0 . 5 M N a C l . T h e purified a n t i b o d y was used as the p r i m a r y a n t i b o d y to laccase A . L i g n i n - p e r o x i d a s e , w h i c h oxidised v e r a t r y l a l c o h o l to v e r a t r a l d e h y d e , was isolated f r o m cultures of Phanerochaete chrysosporium b y the m e t h o d of T i e n a n d K i r k (1). It h a d a m o l e c u l a r weight of 4 4 K d on p o l y a c r y l a m i d e gels, i n the presence of s o d i u m dodecyl s u l p h a t e . A n t i b o d i e s to l i g n i n peroxidase were raised i n r a b b i t s as described for laccase A . 3

Results T h e t i t r e of antibodies i n laccase antisera was 1:16 as measured b y the i m m u n o d i f f u s i o n technique of O u c h t e r l o n y (21). These a n t i b o d i e s were effective i n h i b i t o r s o f catechol oxidase a c t i v i t y , w i t h 100/il of a n t i s e r a red u c i n g a c t i v i t y b y 9 5 % , i n a n assay m i x t u r e of 3 m l o f 0 . 1 M catechol i n 0 . 1 M acetate buffer at p H 5 as described b y E v a n s (7). T a b l e I shows the effect of a d d i n g different volumes of a n t i s e r a to the assay m i x t u r e . I n h i b i t i o n of enzyme a c t i v i t y was not used to assess the a n t i b o d y antigen reaction of l i g n i n - p e r o x i d a s e , as the a d d i t i o n of c o n t r o l s e r u m to e n z y m e reaction m i x t u r e s increased the p H above the p H specificity of the

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T a b l e I. T h e effect of laccase a n t i s e r u m o n e n z y m e a c t i v i t y . T h e r e a c t i o n m i x t u r e c o n t a i n e d 3 m l o f 0.1 M catechol i n 0.1 M acetate buffer at p H 5, 5/ig laccase a n d 0-100/d o f s e r u m V o l u m e o f a n t i s e r a (μ\) a d d e d : Laccase a c t i v i t y (OD440 catechol o x i d a t i o n m i n

- 1

0

10

20

50

100

0.21

0.18

0.15

0.04

0.02

)

e n z y m e , w h i c h has a n o p t i m u m at p H 2.9 a n d is i n h i b i t e d above p H 4 (4). C r o s s r e a c t i v i t y between a n t i b o d y a n d antigen was measured b y i m m u n o p r e c i p i t a t i o n at a d i l u t i o n of 1:16 o f a n t i s e r u m (21). B o t h a n t i b o d i e s showed specificity i n r e a c t i n g w i t h t h e i r respective antigens as d e t e r m i n e d b y W e s t ­ ern b l o t t i n g . T h e d i s t r i b u t i o n of laccase i n h y p h a e o f C. versicolor g r o w n i n beech h e a r t w o o d for 4 week o n m a l t agar. B o t h cultures gave p o s i t i v e tests for laccase w h e n t r e a t e d w i t h g u a i a c o l , p r o d u c i n g y e l l o w / b r o w n colorations (22). Sections o f h y p h a e f r o m b o t h agar a n d w o o d cultures of C. versicolor were treated w i t h either laccase a n t i s e r u m or purified laccase I g G . N o difference i n the p a t t e r n of l a b e l l i n g was observed w h e n crude antisera was used c o m p a r e d w i t h p u r i ­ fied I g G . F i g u r e 1 showed t h a t hyphae g r o w n o n m a l t agar h a d l a b e l l i n g w h i c h was r e s t r i c t e d to the h y p h a l cell w a l l a n d m u c i l a g e layer, w i t h l i t t l e c y t o p l a s m i c l a b e l l i n g a n d a v e r y low level of b a c k g r o u n d l a b e l l i n g . T h e r e was no l a b e l associated w i t h the p l a s m a m e m b r a n e as seen i n F i g u r e 2, a n d the m a j o r site o f l a b e l l i n g was the cell w a l l ( i n most sections the thickness of the mucilage was so s m a l l as t o be i n d i s t i n g u i s h a b l e f r o m the cell w a l l ) . H y p h a e i n sections of infected w o o d showed s i m i l a r m o r p h o l o g y to t h a t of h y p h a e g r o w n o n agar. A s i m i l a r p a t t e r n of l a b e l l i n g was also observed, w i t h most g o l d particles a t t a c h e d to the cell w a l l a n d m u c i l a g e layers a n d m i n i m a l l a b e l l i n g i n the c y t o p l a s m ( F i g u r e s 3 a n d 4). A t higher m a g n i f i ­ c a t i o n i t was seen t h a t mucilage separated f r o m the h y p h a l w a l l was also l a b e l l e d , as was the secondary w a l l of the w o o d cell w a l l . T h e r e was a m o d e r a t e level o f l a b e l i n the S2 w a l l layer whereas the m i d d l e l a m e l l a a n d decayed m a r g i n s of the w a l l were not l a b e l l e d . Sections o f u n i n f e c t e d beech h e a r t w o o d showed no b a c k g r o u n d l a b e l l i n g of cell walls i n d i c a t i n g t h a t there was no non-specific b i n d i n g of the laccase a n t i b o d y t o w o o d cell w a l l c o m p o n e n t s ( F i g . 5). Sections of C. versicolor h y p h a e g r o w n o n N - l i m i t e d n u t r i e n t agar were also treated w i t h antibodies to l i g n i n - p e r o x i d a s e p u r i f i e d f r o m P. chrysospo­ rium. C r o s s reactive m a t e r i a l was detected i n the cell w a l l a n d m u c i l a g e of the h y p h a e as s h o w n i n F i g u r e 6. T h e r e was a c o n s i d e r a b l y lower level of l a b e l l i n g i n the c y t o p l a s m . T h e l a b e l w i t h i n the w a l l o f generative h y ­ phae was l o c a l i z e d i n t o two d i s t i n c t layers w h i c h was especially o b v i o u s i n the thickened w a l l a n d mucilage layer, as s h o w n i n F i g u r e 7. T h i s double layer o f l i g n i n - p e r o x i d a s e was s i t u a t e d adjacent to the p l a s m a m e m b r a n e a n d o n the outer surface of the w a l l w i t h i n the m u c i l a g e layer ( F i g u r e 8). In the skeletal h y p h a e there was intense l a b e l l i n g of the thicker cell w a l l

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F i g u r e 1. Single h y p h a of C. versicolor grown on m a l t agar, labelled w i t h r a b b i t anti-laccase antibodies, localized w i t h g o a t - a n t i r a b b i t g o l d c o n j u ­ gate. T h e label is restricted to the h y p h a l w a l l , w i t h l i t t l e label i n the c y t o p l a s m . M a g n i f i c a t i o n χ 16,000.

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F i g u r e 2. H i g h e r m a g n i f i c a t i o n of laccase-labelling i n the h y p h a l w a l l a n d mucilage layer. N o l a b e l is seen on the p l a s m a m e m b r a n e or w i t h i n the c y t o p l a s m . M a g n i f i c a t i o n χ 59,500.

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F i g u r e 3. Beech h e a r t w o o d , decayed for 4 weeks w i t h C. versicolor, s h o w i n g t h i n n i n g of the secondary w a l l w i t h enlargement of the c a v i t y at the cell corner (the p r o t r u d i n g ends of the m i d d l e l a m e l l a can be seen). A single h y p h a close to the w o o d cell w a l l shows where the secondary w a l l has been degraded. L a b e l for laccase is seen u n i f o r m l y d i s t r i b u t e d i n the secondary w a l l . M a g n i f i c a t i o n χ 12,600.

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F i g u r e 4. H i g h e r m a g n i f i c a t i o n of h y p h a l w a l l i n F i g u r e 3 s h o w i n g l a b e l l i n g of the w a l l a n d mucilage layer. M a g n i f i c a t i o n χ 66,500.

433

intense

F i g u r e 5. A single h y p h a f r o m an agar g r o w n c u l t u r e of C. versicolor s h o w i n g an absence of label after replacing p r i m a r y antisera w i t h r a b b i t I g G g l o b u l i n . M a g n i f i c a t i o n χ 19,800.

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F i g u r e 6. H y p h a e f r o m N - l i m i t e d nutrient agar g r o w n cultures of C. versi­ color s h o w i n g (a) a thick walled skeletal h y p h a a n d (b) a t h i n walled gen­ erative h y p h a labelled for lignin-peroxidase. L a b e l is seen t h r o u g h o u t the h y p h a l walls w i t h l i t t l e l a b e l l i n g i n the c y t o p l a s m . M a g n i f i c a t i o n χ 15,700.

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Figure 7. Higher magnification of generative hyphal walls from N-limited nutrient agar grown cultures labeled margins of the hyphal wall. Magnification χ 11,000.

Figure 8. Higher magnification of generative hyphal walls from N-limited nutrient agar grown cultures labeled for lignin-peroxidase. Label on the hyphal tip where a thickening of the wall and mucilage form a "cap" at the tip. Magnification χ 44,000.

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w i t h v i r t u a l l y no l a b e l l i n g of the c y t o p l a s m , or of the mucilage s u r r o u n d i n g the w a l l . T h e difference i n p a t t e r n , a n d i n the degree of l a b e l l i n g of l i g n i n peroxidase i n sections of generative a n d skeletal h y p h a e are p r e s u m a b l y due t o the difference i n thickness of the w a l l . H y p h a e g r o w n o n m a l t agar showed s i m i l a r l a b e l l i n g of the p l a s m a m e m b r a n e w i t h l i g n i n - p e r o x i d a s e antisera, t h o u g h l a b e l l i n g of the cell w a l l was at a lower i n t e n s i t y t h a n t h a t seen i n hyphae c u l t u r e d o n N - l i m i t e d n u t r i e n t agar. Sections of decayed a n d undecayed beech h e a r t w o o d were also t r e a t e d w i t h l i g n i n - p e r o x i d a s e a n t i b o d i e s . F i g u r e 9 shows t h a t the l a b e l l i n g o f decayed w o o d was far more intense t h a n t h a t seen i n sections t r e a t e d w i t h laccase a n t i b o d i e s w i t h the l a b e l u n i f o r m l y d i s t r i b u t e d t h r o u g h o u t the S2 cell w a l l layer. L i t t l e l a b e l l i n g of the S I layer or m i d d l e l a m e l l a was o b served. O n sections of undecayed w o o d there was o n l y a v e r y slight a m o u n t o f b a c k g r o u n d l a b e l o n the cell walls i n d i c a t i n g t h a t there was l i t t l e n o n specific b i n d i n g to cell w a l l components, as seen i n F i g u r e 10. I n p r e l i m i nary experiments dilution clearest results w i t h m i n i m u m b a c k g r o u n d l a b e l l i n g yet s h o w i n g the highest i n t e n s i t y of specific l a b e l l i n g . L o w e r d i l u t i o n s gave less specific l a b e l l i n g , whereas higher d i l u t i o n s gave more non-specific l a b e l l i n g . C o n t r o l e x p e r i m e n t s to assess the specificity of the l a b e l l i n g were p e r f o r m e d by v a r i a t i o n s i n the procedure. F o r e x a m p l e , w h e n the p r i m a r y a n t i b o d y was o m i t t e d no l a b e l l i n g was observed, i n d i c a t i n g t h a t there was no non-specific b i n d i n g of goat a n t i - r a b b i t I g G - g o l d conjugate t o the sections. W h e n r a b b i t I g G i m m u n o g l o b u l i n was used to replace the p r i m a r y a n t i b o d y , again no l a b e l l i n g was seen i n d i c a t i n g t h a t non-specific b i n d i n g of i m m u n o g l o b u l i n s to sections was not responsible for l a b e l l i n g i n either h y p h a e or decayed w o o d cell w a l l s . S i m i l a r l y no l a b e l l i n g was observed w h e n the p r i m a r y a n t i b o d y was replaced w i t h antisera to p l a n t p h y t o c h r o m e ( F i g u r e 11). W h e n the p r i m a r y a n t i b o d y was replaced w i t h antisera to laccase i s o l a t e d f r o m Agaricus bisporus g r o w n i n m a l t e x t r a c t c u l t u r e s , the l a b e l l i n g p a t t e r n o b served was i d e n t i c a l to t h a t seen w i t h the p r i m a r y a n t i b o d y to laccase f r o m C. versicolor ( F i g . 12). T h i s suggests t h a t laccase f r o m A. bisporus has s i m i l a r antigenic sites to laccase f r o m C. versicolor. W h e n the p r i m a r y a n t i b o d y was replaced w i t h a n t i s e r a raised against the e n z y m e m a n n i t o l dehydrogenase, a c y t o p l a s m i c enzyme f r o m Agaricus bisporus, slight l a b e l l i n g was observed o n sections of the h y p h a e , associated w i t h b o t h the w a l l a n d c y t o p l a s m , a n observation w h i c h is difficult to e x p l a i n as m a n n i t o l dehydrogenase is a c y t o p l a s m i c enzyme i n A. bisporus, not e x t r a c e l l u l a r . W h e n sections of beech w o o d were treated w i t h a n t i s e r a to m a n n i t o l d e h y drogenase no l a b e l l i n g of the w o o d cell walls o c c u r r e d . T h i s i n d i c a t e d t h a t non-specific b i n d i n g was not t a k i n g place. Discussion T r a n s m i s s i o n electron m i c r o s c o p y of i m m u n o g o l d l a b e l l e d sections has s h o w n t h a t the e x t r a c e l l u l a r l i g n i n - d e g r a d i n g enzymes l i g n i n - p e r o x i d a s e a n d laccase were l o c a l i z e d w i t h i n the cell w a l l a n d mucilage of the h y p h a e of C. versicolor. Laccase was present i n the cell w a l l layer whereas l i g n i n -

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F i g u r e 9. Beech heartwood decayed for 12 weeks w i t h C. versicolor l a ­ belled for lignin-peroxidase. T h e label is d i s t r i b u t e d u n i f o r m l y over the secondary w a l l w h i c h shows characteristic t h i n n i n g a n d m a r k e d r e d u c t i o n i n electron density. N o label occurs i n the m i d d l e l a m e l l a a n d cell corners. M a g n i f i c a t i o n χ 22,000.

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F i g u r e 10. Undecayed beech h e a r t w o o d labelled for lignin-peroxidase, w i t h very l i t t l e label o n the secondary w a l l or m i d d l e l a m e l l a . M a g n i f i c a t i o n χ 9,800.

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F i g u r e 11. A single h y p h a f r o m an agar grown c u l t u r e of C. versicolor s h o w i n g an absence of l a b e l after r e p l a c i n g p r i m a r y antisera w i t h antisera to p l a n t p h y t o c h r o m e . M a g n i f i c a t i o n χ 17,400.

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Figure 12. A single hypha from an agar grown culture of C. versicolor showing labeling when the primary antisera was replaced with antisera to laccase from malt agar cultures of Agaricus bisporus. Magnification χ 37,800. All bars, 1 μχη. All tissue was unstained and consequently of low contrast.

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peroxidase was v i s u a l i s e d as a double layer o n the cell w a l l a n d adjacent to the p l a s m a m e m b r a n e . B o t h enzymes were l o c a l i z e d w i t h i n the S2 layer o f the secondary w a l l of beech h e a r t w o o d infected w i t h C. versicolor. Pene­ t r a t i o n o f the cell walls of w o o d was more pronounced for l i g n i n - p e r o x i d a s e t h a n for laccase b u t neither enzyme h a d penetrated to the m i d d l e l a m e l l a or cell corners. T h e w o o d sections were s i g n i f i c a n t l y degraded a n d i t is not k n o w n whether the diffusion o f enzymes i n t o the secondary w a l l o c c u r r e d after w i d e s p r e a d d e g r a d a t i o n of the lignocellulose or preceded secondary w a l l t h i n n i n g . O t h e r workers (23,24) have f o u n d t h a t i n P. chrysosponum, l i g n i n - p e r o x i d a s e was present as a c y t o p l a s m i c enzyme associated m a i n l y w i t h the p l a s m a m e m b r a n e i n h y p h a e g r o w n i n sawdust a n d l i q u i d c u l t u r e . G a r c i a et al. (23) d i d not find the enzyme localised w i t h i n the w o o d cell w a l l , whereas S r e b o t n i k et al. (24) showed ligninase was detected e x t r a c e l l u l a r l y after fixation i n p i c r i c a c i d . It is k n o w n t h a t secretion of l i g n i n peroxidase o n l y occurs d u r i n g the secondary m e t a b o l i c phase of g r o w t h i n l i q u i d cultures (25) a n d i e x p e r i m e n t s , beechwood infected by C. versicolor showed decay p a t t e r n s t y p i c a l of advanced stages o f f u n g a l infection w h i c h w o u l d i m p l y t h a t the secondary m e t a b o l i c phase h a d been reached. F u r t h e r studies are required on the continuous d e g r a d a t i o n patterns r e s u l t i n g f r o m f u n g a l infections of w o o d to e x p l a i n a l l these results. T h e results of the c o n t r o l e x p e r i m e n t s w i t h antisera to f u n g a l proteins have led us to question the specificity of the antibodies to laccase a n d l i g n i n peroxidase. These enzymes are glycoproteins a n d b o t h c a r b o h y d r a t e a n d p r o t e i n moieties w i l l provide antigenic sites for the p o l y c l o n a l a n t i b o d i e s . However, u s i n g W e s t e r n b l o t t i n g techniques, the laccase a n t i s e r a d i d not cross-react w i t h l i g n i n - p e r o x i d a s e , nor d i d the l i g n i n - p e r o x i d a s e a n t i s e r a cross-react w i t h laccase. It is likely t h a t as e x t r a c e l l u l a r f u n g a l proteins are secreted t h r o u g h the mucilage-polysaccharide layer a r o u n d the h y p h a e the c a r b o h y d r a t e moieties of these glycoproteins have c a r b o h y d r a t e s t r u c t u r e s i n c o m m o n w i t h the h y p h a l w a l l layer. T h i s m a y e x p l a i n w h y a l l the a n ­ t i b o d i e s to f u n g a l proteins w h i c h were tested p r o d u c e d l a b e l l i n g p a t t e r n s i n the w a l l , as some of the antigens w o u l d b i n d to c a r b o h y d r a t e c h a r a c t e r ­ istic of f u n g a l cell walls. Unless deglycosylated proteins are used to raise a n t i b o d i e s the l a b e l l i n g patterns o b t a i n e d do not i n d i c a t e specifically the l o c a t i o n of the p r o t e i n moiety. However, this s t u d y has s h o w n t h a t a l l the f u n g a l tissues tested have a c o m m o n antigen w h i c h is not s h a r e d by the p l a n t a n d a n i m a l tissues tested. In order to u n d e r s t a n d the l a b e l l i n g patterns of laccase a n d lignin-peroxidase more fully, i t w i l l be necessary to s t u d y the apoenzymes separated f r o m c a r b o h y d r a t e before conclusions c a n be d r a w n about their d i s t r i b u t i o n i n hyphae d u r i n g p r i m a r y a n d secondary g r o w t h phases i n n a t u r a l substrates. A cknowledgment s W e t h a n k the f o l l o w i n g for gifts of antisera: D r . Β. Z . C h o w d h r y for I g G g l o b u l i n , D r . J . B . W . H a m m o n d for m a n n i t o l dehydrogenase a n t i s e r a , D r . B . T h o m a s for p l a n t p h y t o c h r o m e a n t i s e r a . W e t h a n k S E R C ( U K ) for the award of a p o s t g r a d u a t e s t u d e n t s h i p .

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Literature Cited 1. Tien, M.; Kirk, T . K. Science 1983, 221, 7-12. 2. Kuwahara, M.; Glenn, J . K.; Morgan, Μ. Α.; Gold, M . H. FEBS Letts. 1984, 169, 247)250. 3. Dodson, P. J.; Evans, C. S.; Harvey, P. J.; Palmer, J . M. FEMS Micro. Letts. 1987, 42, 17-22. 4. Schoemaker, Η. E.; Harvey, P. J.; Bowen, R. M.; Palmer, J . M . FEBS Letts. 1985, 183, 7-12. 5. Kersten, P. J.; Tien, M.; Kalyanaran, B.; Kirk, T . K. J. Biol. Chem. 1985, 260, 2609-2612. 6. Bollag, J. M.; Leonowicz, A. Appl.Environ. Micro. 1984, 48, 647-653. 7. Evans, C.S. FEMS Micro Letts. 1985, 27, 339-343. 8. Kawai, S.; Umezawa, T.; Higuchi, T. FEBS Letts. 1987, 210, 61-65. 9. Blanchette, R.A. Can. J. Bot. 1980 1496-1500 10. Blanchette, R. A. Appl. 11. Otjen, L.; Blanchette, Appl. , , 12. Messner, K.; Stachelberger, H. Trans. Brit. Mycol. Soc. 1984, 83, 209216. 13. Messner, K.; Foisner, R.; Stachelberger, H.; Rohr, M. Trans. Brit. My­ col. Soc. 1985, 84, 457-466. 14. Harvey, P. J.; Schoemaker, H. E.; Palmer, J . M . FEBS Letts. 1986, 195, 242-246. 15. Montgomery, R. A. P. In Decomposer Basidiomycetes; Frankland; Hedger; Swift, Eds.; Cambridge Univ. Press: 1982; 3, 51-65. 16. Horisberger, M . In Scanning Electron Microscopy; Johari, O., Ed.; 1981, 2, 9-13. 17. Kirk, T . K.; Schultz, E . ; Connors, W. J.; Lorenz, L.F.; Zeikus, J . G . Arch. Micro. 1978, 117, 277-285. 18. Bergman, B.; Lindblad, P.; Pettersson, Α.; Renstrom, E.; Tiberg, E . Planta 1985, 166, 329-332. 19. Reynolds, E . S. J. Cell Biol. 1968, 17, 208-212. 20. Fahraeus, G.; Reinhammer, B. Acta Chem. Scand. 1967, 21, 2367-2378. 21. Ouchterlony, O. Acta Path. Microbiol. Scand. 1949, 26, 507-515. 22. Westermark, U.; Eriksson, Κ. E . Acta Chem. Scand. 1974, B28, 204208. 23. Garcia, S.; Latge, J . P.; Prevost, M . C.; Leisola, M . Appl. Environ. Micro. 1987, 53, 2384-2387. 24. Srebotnik, E . ; Messner, K.; Foisner, R.; Pettersson, B. Curr. Micro. 1988, 16, 221-227. 25. Keyser, P.; Kirk, T . K.; Zeikus, J . G. J. Bact. 1978, 135, 790-797. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 32 Enzyme Excretion During Wood Cell Wall Degradation by Phanerochaete chrysosporium Jean-Paul Joseleau and Katia Ruel Centre de Recherches sur les Macromolécules Végétales, CERMAV-CNRS, B.P. 53X 38041, Grenoble Cedex, France

Wood degradatio with the hypha the host cell walls, was examined by electron microscopy with immunocytochemical techniques. An anti-ligninase antibody and antiserum raised against a mixture of cellulases and hemicellulases secreted from the fungus were used. The respective enzymes were localized in intracellular vesicles and seemed to be able to diffuse from the hyphae only when at a short distance from the site of degradation. When excreted from the hyphae the enzymes seemed to be associated with the 1-3, 1-6 β-glucan which forms the sheath secreted during secondary growth of the fungus. The limited distance of migration of the enzymes suggested that a direct contact is needed be­ tween the wood and the fungal walls for degradation to occur. The propagation of the degradation might take place by an oxygen radical mechanism as revealed by the use of a specific cytochemical method. A m o n g w o o d - r o t t i n g f u n g i the basidiomycete Phanerochaete chrysosporium is able t o degrade b o t h l i g n i n a n d polysaccharides f r o m w o o d cell walls (1). E x a m i n a t i o n o f w h i t e - r o t decayed w o o d at t h e u l t r a s t r u c t u r a l level reveals t h a t several types o f d e g r a d a t i o n can o c c u r (2,3). I n t h e case o f P. chrysosporium a n d i t s a n a m o r p h o u s f o r m (Sporotrichum pulverulentum), t w o p a t t e r n s o f a t t a c k were observed (4). I n one, t h e h y p h a e were t i g h t l y associated w i t h the degraded walls a n d t h i s t y p e o f image can be s a i d t o be " i n c o n t a c t " ( F i g . I A ) . I n the second case, the secondary w a l l was degraded b u t n o h y p h a e c o u l d be observed i n t h e nearby s u r r o u n d i n g s . T h i s t y p e o f d e g r a d a t i o n c a n be described as " a t a d i s t a n c e " ( F i g . I B ) (4). T h e s e t w o extreme aspects o f lysis o f wood cell walls suggest t h a t P. chrysospo­ rium c a n degrade t h e lignocellulosic c o m p l e x o f the w a l l b y a t least t w o 0097-6156/89/0399-0443$06.00/0 © 1989 American Chemical Society

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F i g u r e 1. T w o extreme aspects o f Populus cell w a l l d e g r a d a t i o n by P. chrysosporium. I A , " i n contact;" I B , "at a distance." ( H = h y p h a ; S i , S2 = w o o d secondary w a l l layers.)

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difTerent b i o c h e m i c a l m e c h a n i s m s . C l a s s i c a l l y , biodégradation of cellulose a n d hemicellulose is ascribed to c e l l u l o l y t i c a n d h e m i c e l l u l o l y t i c enzymes o f the fungus a n d l i g n i n b r e a k d o w n is a t t r i b u t e d to the o x i d a t i v e d e g r a d a t i o n b y l i g n i n - p e r o x i d a s e s (5,7). A l l these enzymes have been s h o w n to be p r o d u c e d b y P. chrysosporium i n v a r y i n g p r o p o r t i o n s , d e p e n d i n g on g r o w t h c o n d i t i o n s (8). T h e i r release b y the h y p h a e at the site of the a t t a c k s h o u l d agree w i t h the p a t t e r n o f w o o d d e g r a d a t i o n s a i d to be " i n c o n t a c t . " H o w e v e r , i t is far m o r e difficult to e x p l a i n the p a t t e r n d e s c r i b e d as " a t a d i s t a n c e , " since i n t h i s m o d e of d e g r a d a t i o n the l y t i c enzymes need to be first t r a n s p o r t e d f r o m the f u n g a l h y p h a e to the w o o d cell w a l l s . T h e y must t h e n diffuse w i t h i n the w a l l , i n order to effect a p a r t i a l a n d l o c a l b r e a k d o w n o f the p o l y s a c c h a r i d e s a n d of l i g n i n . S u c h e n z y m e diffusion i n the c o m p a c t s t r u c t u r e o f the w o o d cell w a l l has never been d e m o n s t r a t e d , a n d does not seem c o m p a t i b l e w i t h the r e l a t i v e l y large size o f these h y d r o l y t i c and o x i d a t i v e enzymes. A n o t h e r p o s s i b i l i t y w h i c h c o u l d e x p l a i n the p a r t i a l d e g r a d a t i o n of the w a l l w o u l able agents of s m a l l size, w h i c h c o u l d be generated at a c e r t a i n phase of the h y p h a l g r o w t h , or at a c e r t a i n stage o f b i o c h e m i c a l a t t a c k . I n t h i s respect, a c t i v a t e d o x y g e n species have been p o s t u l a t e d as p o t e n t d e g r a d a t i v e e x t r a c e l l u l a r agents, w h i c h c o u l d be p r o d u c e d b y the fungus (9-11). T h i s chapter describes the use of electron microscopy, coupled w i t h i m m u n o c y t o c h e m i c a l techniques, to investigate the a b i l i t y of l i g n o l y t i c enzymes f r o m P. chrysosporium to diffuse inside the w o o d cell w a l l . Materials and

Methods

Plant Material. W o o d samples were t a k e n f r o m a 20-year-old aspen tree (Populus tremula) harvested i n F r a n c e . W o o d wafers (4 χ 20 x 50 m m ) were degraded b y the w i l d t y p e s t r a i n K 3 of the w h i t e rot fungus P. chrysospo­ rium at the S T F I ( S t o c k h o l m , Sweden) i n the l a b o r a t o r y o f D r . K . - E . Eriksson. Preparation of Antisera. A n t i s e r a directed against the crude enzymes m i x ­ t u r e secreted b y P. chrysosporium a n d c u l t i v a t e d i n a f e r m e n t o r o n cel­ lulose, were raised i n r a b b i t s . I m m u n o g l o b u l i n s G ( = I g G ) were p u r i f i e d at the I n s t i t u t e P a s t e u r ( L y o n ) a n d used for i m m u n o l a b e l i n g . S e c o n d a r y goat a n t i r a b b i t g o l d m a r k e r was f r o m S i g m a . T h e a n t i - l i g n i n a s e a n t i s e r u m was raised i n r a b b i t f r o m a p u r i f i e d ligninase (gift of D r . E . O d i e r , I N A , Paris-Grignon, France). Tissue Preparation for Electron Microscopy. T i s s u e s were fixed i n 2 % p a r a f o r m a l d e h y d e , 2 . 5 % g l u t a r a l d e h y d e i n p h o s p h a t e buffer (0.1 M , p H 7.4) a n d 0 . 0 2 % p i c r i c a c i d . T h e y were then d e h y d r a t e d i n g l y c o l m e t h a c r y l a t e m o n o m e r a n d e m b e d d e d i n g l y c o l m e t h a c r y l a t e ( G M A ) (24). Immunocytochemical Labeling. A n t i b o d i e s were used as p o s t - e m b e d d i n g m a r k e r s . Sections o f decayed w o o d were first i n c u b a t e d o n a d r o p o f T B S ( T r i s - p h o s p h a t e s a l i n e buffer 0.1 M , p H 7.4, N a C l 0.15 M or 0.5 M ) , g l y c i n e 0.15 M. A f t e r r i n s i n g i n T B S , they were floated o n a d r o p of 1% T B S B S A (bovine s e r u m a l b u m i n ) (or n o n - i m m u n e goat serum) before t r e a t i n g

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either w i t h I g G a n t i - c r u d e enzymes ( 1 9 ^ g / m l d i l u t e d i n T B S - B S A ) (or T B S / n o r m a l goat s e r u m = T B S / N G S ) , or a n t i - l i g n i n a s e a n t i s e r u m d i l u t e d 1:250 i n the same buffer, for 60 m i n at r o o m t e m p e r a t u r e . T h e secondary a n t i s e r a labeled w i t h g o l d (10 n m i n d i a m e t e r ) was a goat a n t i r a b b i t a n ­ tisera purchased f r o m Janssen ( P h a r m a c e u t i c a , Beerse, B e l g i u m ) . It was d i l u t e d 1:30 i n T B S / N G S . T h e sections were e x a m i n e d o n a P h i l i p s 400 Τ electron microscope w i t h o u t a n y c o u n t e r s t a i n i n g . Immunocytochemical Controls. a. S u b s t i t u t i o n of the p r i m a r y a n t i b o d y w i t h p r e i m m u n e r a b b i t s e r u m IgG fraction. b . T r e a t m e n t o f section w i t h g o a t - a n t i r a b b i t g o l d - l a b e l l e d secondary a n ­ t i b o d y alone, o m i t t i n g the p r i m a r y a n t i b o d y step. c. L a b e l i n g w i t h a n t i s e r a p r e a d s o r b e d w i t h t h e i r respective antigens. E q u a l volumes o f a n t i - c r u d e enzymes a n d of the e n z y m a t i c e x t r a c t , o r , a n t i - l i g n i n a s e and Carbonyl Groups Labeling. T h i s was done under the u s u a l c o n d i t i o n s of P A T A g s t a i n i n g (periodic a c i d , t h i o c a r b o h y d r a z i d e , silver proteinate) reac­ t i o n s (2) o m i t t i n g the p e r i o d a t e o x i d a t i o n step ( = T A g ) , as described i n (21). F e n t o n ' s reagent was prepared a n d a p p l i e d as described i n (18). Results and Discussion "Diffusion" of the Fungal Glycohydrolases. A crude e n z y m e m i x t u r e f r o m P. chrysosporium was o b t a i n e d b y a m m o n i u m sulfate p r e c i p i t a t i o n f r o m a c u l ­ t u r e g r o w n o n cellulose and m a i n t a i n e d i n p r i m a r y g r o w t h c o n d i t i o n s . T h i s e n z y m e m i x t u r e , w h i c h was used for p r e p a r i n g a p o l y c l o n a l a n t i s e r u m , c o n ­ t a i n e d inter alia p r e d o m i n a n t l y endo- and exoglucanases, w i t h o n l y traces o f hemicellulases as evidenced b y t h e i r a c t i o n o n the c o r r e s p o n d i n g s u b ­ strates. N o l i g n i n peroxidase a c t i v i t y c o u l d be detected ( K . - E . E r i k s s o n , p e r s o n a l c o m m u n i c a t i o n ) . T h e i m m u n o g l o b u l i n s ( I g G ) raised i n r a b b i t were first used to detect the site of secretion of the enzymes i n the f u n g a l cells. T h e p r i m a r y a n t i b o d i e s were l o c a l i z e d w i t h a goat a n t i r a b b i t sec­ o n d a r y a n t i b o d y a d s o r b e d to c o l l o i d a l g o l d . T h e d i a m e t e r of g o l d p a r t i c l e s was 10 n m . C o n t r o l s w i t h the anti-glycohydrolases I g G , first i n c u b a t e d w i t h the e n z y m i c e x t r a c t before b e i n g a p p l i e d i n t h i n sections, showed n o l a b e l i n g ( F i g . 2 B ) . O t h e r controls p e r f o r m e d , i.e., replacement of the p r i m a r y a n t i ­ b o d y b y n o r m a l r a b b i t s e r u m or p r e i m m u n e s e r u m , suppression o f the first step c o r r e s p o n d i n g t o the p r i m a r y a n t i b o d y , or l a b e l i n g o f uninfected w o o d s p e c i m e n , were also negative. T h e h y p h a e p h o t o g r a p h e d i n a h i g h l y decayed w o o d e x h i b i t e d several aspects of their c y t o p l a s m i c content. T h e l o c a l i z a t i o n of the glycohydrolases v a r i e d a c c o r d i n g l y as s h o w n i n F i g u r e 2. In F i g u r e 2 A , they are l o c a l i z e d i n t r a c e l l u l a r ^ i n s m a l l dense vesicles (arrows). In F i g u r e 2 C they appeared c o n c e n t r a t e d a l o n g the p l a s m a l e m m a a n d dispersed t h r o u g h the c y t o p l a s m ( a r r o w s ) . In F i g u r e 3 A , they have been secreted out of the h y p h a ( a r r o w s ) . T h e s e observations o f the presence o f a n i n t r a c e l l u l a r c e l l u l o l y t i c a c t i v i t y

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Figure 2. Labeling with anti-crude enzyme mixture. A and Β show the specificity of the antibodies compared to the preimmune-treated control; C, plasmatic and cytoplasmic localization of the enzymes. No labeling in the fungal wall. (Pm, plasmalemma; W, hyphal wall.)

American Chemical Society Library 1155 16th St., N.W. In Plant Cell Wall Polymers; Lewis, N., et al.; Washington, D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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i n the h y p h a agree w i t h the results of M u r m a n i s et al. (12). I n zones of less advanced d e g r a d a t i o n ( F i g . 3 B ) , h y p h a e " i n c o n t a c t " w i t h p a r t i a l l y degraded w o o d cell walls h a d no enzymes i n their o w n w a l l s , b u t some glycohydrolases c o u l d be observed p e n e t r a t i n g a short distance i n t o the w o o d cell w a l l . T h e g o l d p a r t i c l e s i n w o o d are o n l y seen i n lightened areas where w o o d is a l r e a d y degraded. Nevertheless, this was a n i n d i c a t i o n t h a t some glycohydrolases c o u l d pass t h r o u g h the f u n g a l cell w a l l a n d penetrate a short distance i n t o w o o d cell w a l l s . In areas of the degraded w o o d where no h y p h a e are v i s i b l e i n the s u r r o u n d i n g s ( p a t t e r n "at a d i s t a n c e " ) , no g o l d particles were evident i n the decayed w o o d cell w a l l s . T h i s suggested t h a t i n t h i s case the enzymes r e m a i n associated w i t h the h y p h a e (or the h y p h a l sheath). "Diffusion" of Lignin-peroxidase (Ligninase). A n anti-ligninase antiserum was raised i n a r a b b i t u s i n g a p u r i f i e d ligninase f r a c t i o n (13). In t h i s s t u d y , the g o l d l a b e l i n g was p e r f o r m e i n F i g u r e 4, g o l d particles were l o c a l i z e d i n the c y t o p l a s m , a l o n g the p l a s m a l e m m a ( F i g . 4 A ) a n d i n t o the h y p h a l w a l l i n F i g u r e 4 C . T h i s l o c a l i z a t i o n o f l i g n i n a s e differs f r o m t h a t r e p o r t e d by M e s s n e r et al. (15). Some m i n o r l a b e l i n g was also present i n t r a c e l l u l a r l y i n some cells ( F i g . 4 A ) as described b y G a r c i a et al. (16). Interestingly, some hyphae d i d not show any affinity for the ligninase a n t i b o d y ; these m i g h t correspond to those h y p h a e w h i c h d i d not secrete ligninase (14). [ F i g . 4 B corresponds to a c o n t r o l , where the a n t i b o d y was p r e i n c u b a t e d w i t h the ligninase before b e i n g a p p l i e d o n the section.] In a l l cases e x a m i n e d , a n d regardless of the relative s i t u a t i o n of the h y p h a t o the w o o d cell w a l l , no e x t r a c e l l u l a r a c c u m u l a t i o n of l i g n i n a s e was detectable at the site of d e g r a d a t i o n . However, a n o r i e n t e d secretion of ligninase o u t w a r d s , across the hyphae w a l l , was observed i n F i g u r e 4 D . In t h i s p h o t o g r a p h the p h y s i o l o g i c a l state of the h y p h a c o u l d not be ascert a i n e d a n d i t is therefore difficult to correlate the e x c r e t i o n of ligninase to a given state of the h y p h a . However, it c o u l d be possible t h a t the e x c r e t i o n o f ligninase c o u l d o c c u r i n s u b l e t h a l h y p h a e i n w h i c h the c y t o p l a s m i c m e m brane c o u l d have a m o d i f i e d p e r m e a b i l i t y . In F i g u r e 4 E , there is a burst of l i g n i n peroxidase outside a h y p h a w h i c h is o b v i o u s l y d e a d . F r o m the above results, a n d i n agreement w i t h recent reports (15,16), i t appears t h a t P. chrysosporium does not secrete the b u l k of its l i g n o l y t i c enzymes d u r i n g n a t u r a l d e g r a d a t i o n of w o o d ; t h i s is i n contrast to its secretion i n b a t c h cultures, w h i c h bears l i t t l e resemblance to the n a t u r a l d e g r a d a t i o n o f w o o d tissues. These findings, however, provide no e x p l a n a t i o n for the d e g r a d a t i o n w h i c h takes place at some distance f r o m the h y p h a e . If enzymes are not d i r e c t l y i n v o l v e d i n t h i s process, the diffusion of n o n - e n z y m a t i c agents, such as r a d i c a l cations or a c t i v a t e d oxygen species m a y be o c c u r r i n g (16,18). In t h i s r e g a r d , i t is k n o w n t h a t oxygen species p r o d u c e d v i a h y d r o g e n peroxide, e.g., the h y d r o x y l r a d i c a l ( * O H ) , are able to a t t a c k b o t h the l i g n i n a n d polysaccharides (9,19,20). Action

of Hydroxyl

Radicals

on Wood Cell

Walls.

In order to investigate

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Figure 3. Labeling with anti-crude enzyme mixture. A , some labeling was observed on the electron-dense sheath (arrow). No post-staining. In B, some gold markers were localized on the outer part of the degraded S layer. No labeling of the hyphal wall. (Pm, plasmalemma; W, hyphal wall.) 2

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F i g u r e 4. L a b e l i n g w i t h a n t i - l i g n i n a s e . 4 A : M o s t of the l a b e l i n g was e v i d e n t i n the p l a s m a t i c area a n d also i n the w a l l . T h e l a b e l i n g is s t i l l seen o n the e m p t y hyphae ( 4 A a n d 4 C ) . 4 B corresponds t o a c o n t r o l ; the a n t i b o d y was p r e i n c u b a t e d w i t h the ligninase before b e i n g a p p l i e d o n the section. 4 D : A g r o u p of h y p h a e at different p h y s i o l o g i c a l states. In some h y p h a e ligninase was clearly s h o w n i n the w a l l a n d crossing it o u t w a r d s (arrows). 4 E : A n e m p t y h y p h a is clearly e x c r e t i n g the b u l k of its ligninase (arrows). ( P m = plasmalemma; W = hyphal wall; H = hypha.)

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the possible d e g r a d a t i o n of w o o d c o m p o n e n t s v i a * O H , s o u n d w o o d s a m p l e s were s u b m i t t e d to the a c t i o n o f Fen t o n ' s reagent w h i c h is a g o o d s y s t e m for p r o d u c i n g h y d r o x y l r a d i c a l s . P r e v i o u s results (18) showed t h a t a c t i v a t e d o x y g e n species generated i n situ created p a t t e r n s o f d e g r a d a t i o n h i g h l y c o m p a r a b l e w i t h those created b y the fungus. T h i s o x i d a t i v e a c t i o n o f the fungus c a n be v i s u a l i z e d w i t h electron m i c r o s c o p y i n degraded w o o d s a m ples, u s i n g a specific m e t h o d designed to detect the c a r b o n y l a n d c a r b o x y l groups created i n decayed w o o d b y the fungus (21). F o l l o w i n g c o m p a r i s o n t o s o u n d w o o d ( F i g s . 5 A a n d 5 B ) , i t was c l e a r l y e v i d e n t t h a t degraded w o o d h a d undergone o x i d a t i o n . T h i s o x i d a t i v e p r o cess c o u l d take place o n b o t h polysaccharides a n d l i g n i n (22,23), a n d c o u l d therefore represent a general d e g r a d a t i o n m e c h a n i s m o f the w o o d cell w a l l polymers. Conclusion D i f f u s i o n of enzymes f r o h y p h a e behave differently d e p e n d i n g u p o n t h e i r p h y s i o l o g i c a l state. I m m u n o c y t o c h e m i s t r y allowed the v i s u a l i z a t i o n of p o l y s a c c h a r i d e - d e g r a d i n g enzymes a n d l i g n i n peroxidases d u r i n g t h e i r secretion b y the fungus. P e n e t r a t i o n o f cellulases, hemicellulases a n d l i g n i n a s e was l i m i t e d a n d no g o l d

F i g u r e 5. C a r b o n y l group l a b e l i n g . A p p l i c a t i o n of the T A g sequence. 5 A : C o n t r o l = T A g o n s o u n d w o o d . 5 B : s i l v e r g r a i n deposits c o r r e s p o n d to the c a r b o n y l groups created b y the fungus. ( M L + P W = m i d d l e l a m e l l a + p r i m a r y w a l l ; S i a n d S2 = outer a n d m i d d l e layers of the secondary w a l l , respectively.)

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labeling could be seen in sound wood. This means that enzymes invaded the wood cell walls only in places where a predegradation had already oc­ curred. The oxidative action of Fenton's reagent which could be followed by specific cytochemistry suggested that activated oxygen species could partic­ ipate in the propagation of the degradation initiated by lignin peroxidase. This might be an explanation of the type of decay "at a distance." Acknowledgments Thanks are expressed to Dr. K.-E. Eriksson (STFI, Stockholm, Sweden) for the gift of the enzyme extract and inoculation of the wood samples; to Professor R. Guinet (Institut Pasteur, Lyon Lentilly, France) for the preparation of the IgG directed against the crude protein extracted; and to Dr. E . Odier (INA P.G., France) for providing the anti-ligninase antiserum. Literature Cited 1. Eriksson, K.-E. Pure Appl. Chem. 1981, 53, 33-43. 2. Ruel, K.; Barnoud, F. In Biosynthesis and Biodegradation of Wood Components; Higuchi, T . , Ed.; Academic: New York, 1985, p. 441. 3. Otjen, L.; Blanchette, R. A. Can. J. Bot. 1982, 60, 2770-94. 4. Ruel, K.; Joseleau, J . P. Symbiosis 1986, 2, 355-61. 5. Murmanis, L.; Highley, T.; Palmer, J . G . Holzforschung 1984, 38, 1118. 6. Tien, M.; Kirk, T . K. Science 1983, 221, 661-63. 7. Eriksson, K.-E.; Wood, T . M . In Biosynthesis and Biodegradation of Wood Components; Higuchi, T . , Ed.; Academic: New York, 1985; p. 469. 8. Ander, P.; Eriksson, K . - E . Physiol. Plant. 1977, 41, 239-248. 9. Nakatsubo, F.; Reid, I. D.; Kirk, T . K. Biochem. Biophys. Res. Com­ mun. 1981, 102, 484-91. 10. Forney, L. J.; Reddy, Α.; Tien, M.; Ausi, S. D. J. Biol. Chem. 1982, 257, 11455-62. 11. Schoemaker, Η. E.; Harvey, P. J.; Bowen, R. M.; Palmer, J. M . FEBS Lett. 1985, 183, 7-12. 12. Murmanis, L.; Highley, T . L.; Palmer, J . G. Sci. Technol. 1987, 21, 101-09. 13. Tonon, F.; Odier, E.; Asther, M.; Lesage, L.; Corrieu, G . In Lignin En­ zymic and Microbial Degradation; E . Odier, Ed.; INRA Publications: 1987; pp. 165-70. 14. Keyser, P.; Kirk, T . K.; Zeikus, J . G . J. Bact. 1978, 135, 3, 790-97. 15. Messner, K.; Strebotnik, E . ; Erler, G.; Foisner, R.; Pettersson, B.; Stachelberger, H. In Lignin Enzymic and Microbial Degradation; Odier, E . , Ed.; INRA Publications: 1987; 243-48. 16. Garcia, S.; Latgé, J . P.; Prevost, M . C.; Leisola, M . Appl. Environ. Microbiol. 1987, 53, 2384-87. 17. Gupta, D. P.; Heale, J . B. J. Gen. Microbiol. 1971, 63, 163-73. 18. Ruel, K.; Joseleau, J . P. Food Hydrocolloids 1987, 1, 515-17. 19. Koenigs, J . W. Wood Fibers 1974, 6, 66-79.

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20. Weldock, D. J.; Parsons, B. J.; Phillips, G . O.; Thomas, B. In Cellulose and its Derivatives; Kennedy, J . F., Ed.; John Wiley and Sons: New York, 1985; 46, pp. 503-10. 21. Joseleau, J . P.; Ruel, K. Biol. Cell. 1985, 53, 61-66. 22. Isbell, H. S.; Frush, H. L. Carbohydr. Res. 1987, 161, 181-93. 23. Gilbert, B. C.; King, D. M.; Thomas, C. B. Carbohydr. Res. 1984, 125, 217-35. 24. Spaur, C. R.; Moriarty, G . C. J. Histochem. Chem. 1977, 25, 163-74. RECEIVED March 17, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 33

Oxidation and Reduction in Lignin Biodegradation 1

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DSM Research, Bio-organic Chemistry Section, P.O. Box 18, 6160 MD Geleen, Netherlands Department of Biotechnology, Swiss Federal Institute of Technology, ETH-Hönggerberg, 8093 Zürich, Switzerland Givaudan Forschungsgesellschaft AG 8600 Dübendorf Switzerland 2

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Reductive activities of Phanerochaete chrysosporium were studied using monomeric aromatic acids, aldehydes and three different types of quinones as substrates. All of the tested substrates were rapidly reduced by ligninolytic cultures of P. chrysosporium. Reduction experiments carried out with intact fungal cells, cell extracts and partially purified enzymes gave evidence for the presence of at least three different types of reductases: viz. acid, aldehyde and quinone reductases. Further, comparison of relative quinone reduction rates indicated that possibly two or three different quinone reductases were operating. Based on these results and an extensive literature survey, it is postulated that lignin degradation by P. chrysosporium involves an array of oxidative and reductive conversions. Rapid metabolism of quinone type intermediates represents one possible way of shifting the polymerization-depolymerization equilibrium, induced by lignin peroxidases and phenoloxidases, towards degradation. Moreover, it is postulated that aldehyde and acid reductases play a role in the biodegradation of lignin by P. chrysosporium. L i g n i n biodégradation research has extended over several decades, b u t the progress since t h e late 1970's has been r e m a r k a b l e . T h e d i s c o v e r y — i n d e p e n d e n t l y b y t h e groups o f K i r k (1) a n d G o l d ( 2 ) — o f a l i g n i n o l y t i c e n z y m e , capable o f o x i d i z i n g n o n - p h e n o l i c a r o m a t i c c o m p o u n d s , c a n b e considered as a b r e a k t h r o u g h i n t h i s area. T h e e n z y m e , isolated f r o m l i g n i n o l y t i c cultures o f the w h i t e - r o t basidiomycete Phanerochaete chrysosporium, was o r i g i n a l l y described as a novel t y p e o f oxygenase. Subsequent research, however, showed t h a t t h e enzyme s h o u l d be classified as a p e r oxidase (3-6). Its discovery triggered a m a j o r w o r l d w i d e research effort, 0097-6156/89/0399-0454$06.00/0 © 1989 American Chemical Society

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r e s u l t i n g i n the f o r m u l a t i o n of a u n i f y i n g t h e o r y r a t i o n a l i z i n g m o s t o f the reactions observed i n l i g n i n biodégradation (7-9). R e c e n t l y v a r i o u s review articles o n l i g n i n biodégradation have focused o n the l i g n i n peroxidases f r o m Phanerochaete chrysosporium. I n those a r ticles the m i c r o b i o l o g y , physiology, ( b i o ) c h e m i s t r y , genetics a n d m o l e c u l a r biology of the s y s t e m were discussed (10-12). L i g n i n biodégradation has been recognized as a n e x t r a c e l l u l a r , m a i n l y o x i d a t i v e process, c h a r a c t e r ized by cleavage of the a r y l p r o p y l s i d e - c h a i n s , d e m e t h o x y l a t i o n a n d other ether b o n d b r e a k i n g reactions, a r o m a t i c r i n g cleavage, a r o m a t i c h y d r o x y l a t i o n a n d a r o m a t i c c a r b o x y l i c a c i d f o r m a t i o n (13). W i t h the possible e x c e p t i o n of the l a t t e r r e a c t i o n , most reactions are a d e q u a t e l y r a t i o n a l i z e d by a l i g n i n p e r o x i d a s e - i n d u c e d a r o m a t i c r a d i c a l c a t i o n f o r m a t i o n . H o w ever, a c o r o l l a r y of the l a t t e r process is the occurrence of p h e n o l c o u p l i n g reactions in vitro, r e s u l t i n g i n l i g n i n peroxidase c a t a l y z e d p o l y m e r i z a t i o n of m i l l e d w o o d l i g n i n (14). In vivo, however, l i g n i n is d e p o l y m e r i z e d by l i g n i n o l y t i c cultures o f P. d i t i o n o f l i g n i n peroxidase (15). O b v i o u s l y , to degrade l i g n i n the w h i t e - r o t f u n g i have some c u r r e n t l y u n k n o w n m e c h a n i s m to prevent p o l y m e r i z a t i o n . W e p o s t u l a t e d (16) t h a t since low m o l e c u l a r weight d e g r a d a t i o n p r o d u c t s are r a p i d l y m e t a b o l i z e d b y the fungus, t h i s represents one m e c h a n i s m to shift the e q u i l i b r i u m f r o m spontaneous p o l y m e r i z a t i o n to d e g r a d a t i o n . In t h i s p a p e r , we propose t h a t i n a d d i t i o n to a r o m a t i c r i n g - c l e a v e d p r o d u c t s , quinones a n d h y d r o q u i n o n e s are obvious c a n d i d a t e s to f u n c t i o n as those low m o l e c u l a r weight c o m p o u n d s . I n t h i s r e g a r d , E r i k s s o n a n d coworkers (17,18) suggested t h a t q u i n o n e r e d u c t i o n was a n essential step. In recent reviews h a r d l y a n y a t t e n t i o n was g i v e n to the fact t h a t the l i g n i n o l y t i c s y s t e m of P. chrysosporium possessed a s t r o n g r e d u c i n g a c t i v i t y . H o w ever, as p o i n t e d o u t by E r i k s s o n (19) a n d C h e n a n d C h a n g (20), l i g n i n biodégradation by w h i t e - r o t f u n g i is a c o m b i n a t i o n of a n a r r a y of o x i d a t i v e a n d r e d u c t i v e reactions, r a t h e r t h a n o x i d a t i v e reactions alone. H o w e v e r , the n a t u r e a n d the role of t h i s r e d u c i n g a c t i v i t y is not clear at present. In a d d i t i o n to the quinone reductase s y s t e m , t h i s r e d u c i n g a c t i v i t y is also manifested i n the conversion of a r o m a t i c aldehydes a n d c a r b o x y l i c acids to the c o r r e s p o n d i n g b e n z y l i c alcohols. F o l l o w i n g a brief l i t e r a t u r e survey of some n o t e w o r t h y (reductive) c o n versions of l i g n i n m o d e l c o m p o u n d s by P. chrysosporium, i n c l u d i n g our o w n results o n the m e t a b o l i s m of v e r a t r y l a l c o h o l , we w i l l focus o n the different r e d u c i n g properties of P. chrysosporium. O u r p r e l i m i n a r y results have led us to f o r m u l a t e a h y p o t h e t i c a l scheme for l i g n i n biodégradation based o n b o t h o x i d a t i v e a n d r e d u c t i v e conversions, where the p i v o t a l role o f a r o m a t i c r i n g opened p r o d u c t s a n d q u i n o n e s / h y d r o q u i n o n e s as l i g n i n m e t a b o l i t e s is discussed. Literature Survey Reduction. T h e r e d u c t i v e c a p a c i t y of P. chrysosporium has been k n o w n for a l o n g t i m e . F o r e x a m p l e , v e r a t r a l d e h y d e , v e r a t r i c a c i d , v a n i l l i n , v a n i l lic a c i d a n d analogous s t r u c t u r e s were converted i n t o the c o r r e s p o n d i n g

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b e n z y l i c alcohols (21,22). A l s o the i n i t i a l p r o d u c t s ( m o s t l y v e r a t r a l d e h y d e analogs) i n C a - C / ? cleavage studies o n l i g n i n models w i t h P. chrysosporium are reduced t o the c o r r e s p o n d i n g alcohols(23). A d d i t i o n a l l y , studies b y E r i k s s o n a n d coworkers (17,18) showed t h a t quinones p r o d u c e d were r a p i d l y reduced t o the c o r r e s p o n d i n g h y d r o quinones a n d t h e n f u r t h e r degraded. V a r i o u s a u t h o r s have also described the r e d u c t i o n of d i m e r i c l i g n i n m o d e l s t r u c t u r e s ; b o t h a r o m a t i c aldehydes a n d a r o m a t i c acids were reduced b y l i g n i n o l y t i c cultures of P. chrysosporium, as s h o w n b y the r e d u c t i o n of the / ? - 0 - 4 m o d e l 1 ( a c i d , l a c k i n g the C - s u b s t i t u e n t ) t o the c o r r e s p o n d i n g a l c o h o l 2 (24) a n d the p h e n y l c o u m a r a n models 3 (acid) a n d 4 (aldehyde) to the c o r r e s p o n d i n g a l c o h o l 5 (25). 7

Oxidation. M o s t of the o x i d a t i v e reactions observed i n l i g n i n biodégradat i o n c a n be r a t i o n a l i z e d b y one-electron o x i d a t i o n s o f phenols (e.g., w i t h l a c case or peroxidase) to y i e l peroxidase t o y i e l d r a d i c a l c a t i o n i c i n t e r m e d i a t e s . A s a consequence o f these one-electron o x i d a t i o n s , phenolic l i g n i n a n d l i g n i n models frequently undergo C a - a r e n e cleavage r e s u l t i n g i n q u i n o n e / h y d r o q u i n o n e f o r m a t i o n (26). S u p p o r t i n g evidence for the r a d i c a l c a t i o n m e c h a n i s m was o b t a i n e d for C a - C / ? cleavage, d e m e t h o x y l a t i o n a n d other ether b o n d b r e a k i n g react i o n s , a r o m a t i c h y d r o x y l a t i o n , a r o m a t i c r i n g cleavage, b e n z y l i c o x i d a t i o n , h y d r o x y l a t i o n of b e n z y l i c m e t h y l e n e groups, styrene h y d r o x y l a t i o n , specific oxygen i n c o r p o r a t i o n , p h e n o l c o u p l i n g , etc. (7-12). So far, a r o m a t i c carb o x y l i c a c i d f o r m a t i o n , a r a t h e r c h a r a c t e r i s t i c reaction i n l i g n i n biodégrad a t i o n (20,27), has not been d e m o n s t r a t e d for l i g n i n peroxidase c a t a l y z e d reactions. However, a r o m a t i c c a r b o x y l i c acids were observed i n h e m i n c a t a l y z e d o x i d a t i o n of l i g n i n models (28). Recently, we o b t a i n e d evidence for a r o m a t i c c a r b o x y l i c ester f o r m a t i o n i n the l i g n i n peroxidase c a t a l y z e d o x i d a t i o n of the m e t h y l ether of v e r a t r y l a l c o h o l . T h e ester f o r m a t i o n was m o r e p r o n o u n c e d at higher (4-5) p H - v a l u e s ( S c h m i d t et ai, Biochemistry, i n press). H i g u c h i a n d coworkers (27) have also s h o w n t h a t i n l i g n i n o l y t i c cultures of P. chrysosporium d i m e r i c l i g n i n models of the / J - O - 4 a n d p h e n y l c o u m a r a n t y p e w i t h a l l y l a l c o h o l side-chains (see s t r u c t u r e s 1 respectively 3, X = C H = C H - C H 2 0 H ) were converted t o c a r b o x y l i c acids; due to the r e d u c i n g c a p a c i t y of P. chrysosporium, alcohols a n d aldehydes were also obtained. Quinone/Hydroquinone Formation. In studies on biodégradation of l i g n i n models w i t h l i g n i n o l y t i c cultures of P. chrysosporium ( a n d other f u n g i ) , a n u m b e r o f quinones a n d h y d r o q u i n o n e s were i s o l a t e d . In other studies their f o r m a t i o n has been i m p l i e d f r o m the i s o l a t i o n of their s t r u c t u r a l c o u n terparts (29,30), where r a p i d f u n g a l d e g r a d a t i o n of the quinones prevented their i s o l a t i o n . V a r i o u s routes to these m e t a b o l i c intermediates exist, w h i c h have been extensively reviewed (26,27). F o r the present discussion, a n u m b e r of these conversions are of relevance. P h e n o l i c d i m e r i c m o d e l c o m p o u n d s o f the / ? - l , β-Ο-4 a n d p h e n y l c o u m a r a n t y p e are degraded v i a C a - a r e n e cleavage y i e l d i n g m e t h o x y -

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s u b s t i t u t e d q u i n o n e s / h y d r o q u i n o n e s . T h e m o n o m e r i c l i g n i n models v a n ­ i l l i n , v a n i l l i c a c i d a n d v a n i l l y l a l c o h o l (22) a n d i s o v a n i l l y l a l c o h o l (16) were s h o w n t o be o x i d i z e d t o m e t h o x y - q u i n o n e s / h y d r o q u i n o n e s . M o r e recently, i t was s h o w n t h a t v e r a t r i c a c i d , v e r a t r a l d e h y d e a n d v e r a t r y l a l c o h o l were degraded v i a quinone i n t e r m e d i a t e s (15,31), w i t h the aldehyde (15) a n d the a c i d (21) b e i n g first reduced to v e r a t r y l a l c o h o l . Degradation of Non-Phenolic Lignin Models with a Cct-Carbonyl Sub­ stituent. L i g n i n peroxidase w i l l oxidize a wide v a r i e t y of l i g n i n models. However, n o n - p h e n o l i c a r o m a t i c rings c o n t a i n i n g C a - c a r b o n y l groups are not substrates for t h i s e n z y m e ; e.g., v e r a t r a l d e h y d e a n d the β-0-Α d i m e r i c m o d e l 6 a are not substrates (32) a n d no cleavage of the B - r i n g i n the l a t t e r c o m p o u n d occurs. H o w e v e r , studies (33) w i t h the analogous s t r u c t u r e 7 a , u s i n g l i g n i ­ n o l y t i c cultures of P. chrysosporium as before, resulted i n d e g r a d a t i o n products derived from oxidatio et al. (33) have suggested t h a t the f o r m y l s u b s t i t u e n t is first converted to a less e l e c t r o n - w i t h d r a w i n g g r o u p . R e d u c t i o n to the a l c o h o l or o x i d a t i o n to the a c i d are two obvious possibilities. K a w a i et ai (34) s t u d i e d the l i g n i n peroxidase c a t a l y z e d o x i d a t i o n of the related d i m e r i c a c i d 8 w h i c h c o n t a i n e d an e x t r a m e t h o x y - s u b s t i t u e n t i n the B - r i n g a n d f o u n d o x i d a t i o n to the q u i n o n e - h e m i - k e t a l 9. E n o k i a n d G o l d (35) s t u d i e d the d e g r a d a t i o n of a β-l m o d e l w i t h a C / ? - h y d r o x y s u b s t i t u e n t . O n e of the p r o d u c t s was the k e t o l 1 0 . E n o k i a n d G o l d have p o s t u l a t e d t h a t 1 0 was further degraded to a n i s y l a l c o h o l v i a r e d u c t i o n to the g l y c o l 1 1 , one-electron o x i d a t i o n a n d Ca-C β cleavage followed b y r e d u c t i o n of the i n i t i a l l y formed a n i s a l d e h y d e to the observed p r o d u c t a n i s y l a l c o h o l . However, t h i s v i e w was countered a g a i n b y K i r k a n d N a k a t s u b o (23). T h i s reaction m e r i t s further i n v e s t i g a t i o n . In our studies o n the l i g n i n peroxidase c a t a l y z e d o x i d a t i o n of v e r a ­ t r a l d e h y d e a n d v e r a t r i c a c i d no d e g r a d a t i o n was observed under the reac­ t i o n c o n d i t i o n s used. However, l i g n i n o l y t i c cultures of P. chrysosporium degraded v e r a t r a l d e h y d e a n d veratric a c i d v i a r e d u c t i o n to v e r a t r y l a l c o h o l (15,21). In F i g u r e 1 the isolated reaction p r o d u c t s of the v e r a t r y l a l c o h o l o x i d a t i o n are d e p i c t e d . A i m of the Present

Investigation

L i g n i n peroxidases, M n ( I I ) - d e p e n d e n t peroxidases a n d other a c t i v e oxygen species w i l l catalyze the o x i d a t i v e d e p o l y m e r i z a t i o n of l i g n i n re­ s u l t i n g i n the f o r m a t i o n of ring-opened p r o d u c t s a n d s m a l l a r o m a t i c f r a g ­ m e n t s . T h e l a t t e r fragments can p o l y m e r i z e a g a i n under those c o n d i t i o n s . W e propose t h a t r a p i d m e t a b o l i s m of the s m a l l d e g r a d a t i o n p r o d u c t s is one of the mechanisms b y w h i c h the d e p o l y m e r i z a t i o n - r e p o l y m e r i z a t i o n e q u i ­ l i b r i u m can be shifted towards d e g r a d a t i o n . E v i d e n t l y , f u r t h e r m e t a b o l i s m of the r i n g - o p e n e d p r o d u c t s constitutes a m a j o r d e g r a d a t i o n p a t h w a y . In this p a p e r , however, we w i l l focus o n the m e t a b o l i s m of the a r o m a t i c fragments. W e propose t h a t these a r o m a t i c c o m p o u n d s are degraded v i a

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q u i n o n e / h y d r o q u i n o n e t y p e i n t e r m e d i a t e s . W e also propose t h a t r e d u c t i v e enzymes are i n v o l v e d i n the d e g r a d a t i o n o f the q u i n o n e t y p e s t r u c t u r e s . I n a d d i t i o n , we w i s h t o propose t h a t a r o m a t i c a l d e h y d e reductases a n d a r o m a t i c a c i d reductases p l a y a role also. T h e p a r t i a l p u r i f i c a t i o n of these r e d u c t i v e enzymes is described a n d t h e i r relevance t o the l i g n i n biodégrad a t i o n process is discussed. Materials and

Methods

Cell Growth. M y c e l i a l pellets of P. chrysosponum ( A T C C 24725) were p r o d u c e d under n i t r o g e n l i m i t e d c u l t u r e c o n d i t i o n s at 3 7 ° C u s i n g a spore i n o c u l u m (36). T h e pellets were g r o w n for 72 h r at 150 r p m after w h i c h they were c o n c e n t r a t e d f o u r f o l d b y d e c a n t i n g excess m e d i u m . T h e y were used for r e d u c t i o n e x p e r i m e n t s after a 24 h r a c t i v a t i o n p e r i o d ( 3 7 ° C , 50 r p m , under 1 0 0 % O 2 atmosphere). Reduction Experiments. Th 50 m l o f a c t i v a t e d pellets. T h e cultures were a g i t a t e d at 50 r p m a n d 3 7 ° C . Q u i n o n e s were a d d e d at a c o n c e n t r a t i o n o f 0.2 m M . S a m p l e s of the s u p e r n a t a n t were t a k e n at defined i n t e r v a l s a n d t h e i r a b s o r p t i o n measured at 288 n m a n d 360 n m o n a P e r k i n - E l m e r 557 P h o t o s p e c t r o m e t e r . H i g h pressure l i q u i d c h r o m a t o g r a p h y ( H P L C ) was used for the q u a n t i t a t i v e m e a s u r e m e n t of quinones a n d h y d r o q u i n o n e s i n the c u l t u r e s . 20 μ\ o f s u p e r n a t a n t were injected i n a M e r c k - H i t a c h i H P L C s y s t e m 6 5 5 A - 1 2 e q u i p p e d w i t h a 4.6 x 250 m m N u c l e o s i l C 1 8 c o l u m n (5 μπι, R P 18). T h e s y s t e m was r u n at a flow rate of 1 m l m i n " w i t h a m e t h a n o l / w a t e r g r a d i ­ ent (10 to 2 0 % m e t h a n o l i n 15 m i n , t h e n 20 to 1 0 0 % m e t h a n o l i n 5 m i n ) . T h e U V detector was o p e r a t e d at 281 n m or 275 n m to follow the r e d u c t i o n of quinones 13 a n d 14, respectively (37). 1

Cell Disruption. F u n g a l pellets f r o m 5-6 d o l d , N - l i m i t e d cultures were w a s h e d 3 times w i t h 20 m M T r i s / H C l buffer, p H 7.4, c o n t a i n i n g 2 0 % g l y c ­ erol a n d 1 m M E D T A a n d c o o l e d o n ice. 75 m l o f wet pellets (equals the a m o u n t f r o m t w o a g i t a t e d flasks) a n d 125 m l glass beads (0.4 m m d i a m e ­ ter; cooled to —18°C) were p o u r e d i n t o the 150 m l glass vessel of D y n o m i l l . B r e a k i n g of the cells was c a r r i e d o u t at + 1 ° C for 75 s at 2000 r p m . T h e glass beads were removed b y filtration t h r o u g h a coarse sintered glass fil­ ter a n d washed twice w i t h 50 m l of the T r i s buffer. C e l l fragments were removed b y c e n t r i f u g a t i o n at 30,000 g for 30 m i n . Partial Purification of the Reductases. 4 M C a C b was a d d e d to the crude e x t r a c t t o give a c o n c e n t r a t i o n o f 16 m M . A f t e r m i x i n g for 10 m i n the so­ l u t i o n was centrifuged at 30,000 g for 30 m i n . T h e s e d i m e n t was d i s c a r d e d . T o the s u p e r n a t a n t 4 M a m m o n i u m sulfate was s l o w l y a d d e d t o give a final s a t u r a t i o n of 4 0 % . A f t e r c e n t r i f u g a t i o n at 30,000 g for 30 m i n , 4 M a m m o ­ n i u m sulfate was a d d e d to the s u p e r n a t a n t to a s a t u r a t i o n o f 5 5 % . T h e s e d i m e n t o b t a i n e d b y c e n t r i f u g a t i o n at 30,000 g for 30 m i n was dissolved i n 20 m l of 20 m M T r i s / H C l buffer w i t h 2 0 % g l y c e r o l a n d 1 m M E D T A , p H 7.4 a n d d i a l y s e d over night against 2 1 of t h i s buffer.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Reductase Activity Measurement. Reductase a c t i v i t y was measured b y the r e d u c t i o n of 200 μ Μ v e r a t r a l d e h y d e t o v e r a t r y l a l c o h o l i n the presence of 250 μ Μ N A D P H . T h e r e a c t i o n was c a r r i e d out i n 20 m M T r i s buffer p H 7.4 ( o p t i m a l p H = 6) w i t h 2 0 % glycerol a n d 1 m M E D T A . T h e decrease i n absorbance of N A D P H at 365 n m was measured. N A D P H was s l o w l y o x i d i z e d i n those m i x t u r e s w h i c h were not purified b y i o n exchange even i n the absence of v e r a t r a l d e h y d e . T h i s unspecific r e a c t i o n was considered i n the c a l c u l a t i o n of the reductase a c t i v i t y . O n e u n i t of reductase reduced 1 μτηοΐ m i n of v e r a t r a l d e h y d e t o v e r a t r y l a l c o h o l at r o o m t e m p e r a t u r e (25°C). R e d u c t i o n o f the quinones was measured i n the same way as t h a t of v e r a t r a l d e h y d e , at 365 n m . S u b s t r a t e c o n c e n t r a t i o n was also 200 μ Μ a n d the c o n c e n t r a t i o n of N A D P H or N A D H was 250 μ Μ . In some of the reductions of v e r a t r i c a c i d or v a n i l l i c a c i d 250 μ Μ A T P was a d d e d to the r e a c t i o n m i x t u r e . - 1

Results P r e v i o u s l y , the q u a n t i t a t i v e r e d u c t i o n of v e r a t r a l d e h y d e a n d v e r a t r i c a c i d to v e r a t r y l a l c o h o l b y active l i g n i n o l y t i c cultures of P. chrysosporium had been described by L e i s o l a a n d F i e c h t e r (21). It has n o w been f o u n d t h a t the quinones 1 3 , 1 4 a n d 1 5 f r o m the aerobic o x i d a t i o n of v e r a t r y l a l c o h o l b y l i g n i n peroxidase were also reduced b y f u n g a l m y c e l i u m t o y i e l d the c o r r e s p o n d i n g h y d r o q u i n o n e s . F o r q u i n o n e 1 4 t h i s r e d u c t i o n h a d a l r e a d y been r e p o r t e d b y B u s w e l l et al. (17) i n a study of vanillic acid metabolism. T h e r e d u c t i o n of quinones 1 3 a n d 1 4 by i n t a c t cells of P. chrysospo­ rium is s h o w n i n F i g u r e 2. T h e concentrations of the quinones a n d h y ­ droquinones were c a l c u l a t e d f r o m H P L C d a t a a n d corrected for t h e i r spe­ cific absorbance at given wavelengths. P e a k s were identified by c o m p a r i s o n w i t h r e t e n t i o n times a n d U V s p e c t r a of reference c o m p o u n d s (37, see also M a t e r i a l s a n d M e t h o d s section). F i g u r e 3 shows the change of a b s o r p ­ t i o n at 288 n m a n d 360 n m i n c u l t u r e s u p e r n a t a n t d u r i n g the r e d u c t i o n of q u i n o n e 1 3 . T h i s quinone h a d a n absorbance m a x i m u m at 360 n m w h i l e the h y d r o q u i n o n e 1 3 a h a d one at 288 n m . Q u i n o n e 1 5 was also re­ duced b y whole cells as i n d i c a t e d b y the change of a b s o r p t i o n i n c u l t u r e s u p e r n a t a n t ( d a t a not s h o w n ) . However, the r e s u l t i n g h y d r o q u i n o n e was not detected b y H P L C p r o b a b l y because i t was a u t o x i d i z e d r a p i d l y . S t i l l , q u i n o n e 1 5 was m e t a b o l i s e d b y P. chrysosporium as can be seen i n F i g ­ ure 2. T h e rate of quinone r e d u c t i o n b y the cells was highest j u s t after a d d i t i o n of the c o m p o u n d s to the cultures. H y d r o q u i n o n e f o r m a t i o n was not q u a n t i t a t i v e ( F i g . 2). I n t r a c e l l u l a r d e g r a d a t i o n of the h y d r o q u i n o n e s is a possible e x p l a n a t i o n for t h i s observation (18). F r o m the d a t a i n T a ­ ble I i t can be concluded t h a t the a l d e h y d e - r e d u c i n g e n z y m e ( w h i c h has an absolute requirement for N A D P H ) differs f r o m the a c i d reductase (no a c i d reductase present i n the crude e x t r a c t s ) a n d the quinone reductases ( b o t h N A D H a n d N A D P H can be used as co-substrates at s i m i l a r rates). M o r e o v e r , the d a t a also i n d i c a t e d t h a t more t h a n one quinone reductase

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Time

463

(h)

F i g u r e 2. R e l a t i v e c o n c e n t r a t i o n s o f quinones 13 (·), 14 ( A ) , a n d 15 (*) a n d h y d r o q u i n o n e s 13a (o) a n d 14a ( Δ ) v s . t i m e after a d d i t i o n o f quinones t o l i g n i n o l y t i c c u l t u r e s o f P. chrysosporium.

Ε

Time

(h)

F i g u r e 3. R e l a t i v e absorbance o f c u l t u r e s u p e r n a t a n t a t 360 n m ( · ) a n d 288 n m (o) v s . t i m e after a d d i t i o n o f q u i n o n e 13 t o l i g n i n o l y t i c c u l t u r e s o f P. chrysosporium.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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was o p e r a t i n g , as j u d g e d f r o m differences i n relative rates o f q u i n o n e re­ d u c t i o n for the different p r e p a r a t i o n s . T h e r a t i o of the r e d u c t i o n rates for q u i n o n e 1 4 / q u i n o n e 15 was 0.75 i n b o t h the crude e x t r a c t a n d the 5 0 % sediment f r a c t i o n . I n c o n t r a s t , the r a t i o o f the r e d u c t i o n rates for q u i n o n e 1 3 / q u i n o n e 14 was a p p r o x i m a t e l y 1.0 i n the crude e x t r a c t a n d 0.27 i n the d i a l y z e d 5 0 % sediment f r a c t i o n , i n d i c a t i n g t h a t quinone 13 reductase differs f r o m b o t h quinone 14 reductase a n d f r o m quinone 15 reductase. Q u i n o n e 14 reductase also appears to be different f r o m quinone 15 reductase, as can be inferred f r o m i n h i b i t i o n studies w i t h 1 m M of D T T ( d i t h i o t h r e i t o l ) . D T T c o m p l e t e l y i n h i b i t e d the r e d u c t i o n o f q u i n o n e 13 a n d q u i n o n e 14, b u t D T T d i d not i n h i b i t the r e d u c t i o n o f v e r a t r a l d e h y d e a n d q u i n o n e 15. Research is i n progress t o p u r i f y a n d characterize the r e d u c t i v e e n z y m e systems m e n t i o n e d above.

(ΔΕ365

T a b l e I. R e l a t i v e r e d u c t i o quinones 13-15 b rium Compound crude e x t r a c t

ΔΕ

3 6

5 min

v e r a t r a l d e h y d e 12 quinone 13 quinone 14 quinone 15 veratric a c i d vanillic a c i d

1.0 4.4 4.5 6.0 0.0 0.0

CaCl supernatant

v e r a t r a l d e h y d e 12 quinone 14

1.0 5.0

50%-sediment (dialyzed)

v e r a t r a l d e h y d e 12 quinone 13 quinone 14 quinone 15

1.0 0.4 1.5 2.0

1

1

2

1

1

CofactorDependence NADPH NAD(P)H NAD(P)H NAD(P)H

W i t h added A T P .

Discussion Metabolism of Monomeric Lignin Models. L e i s o l a a n d coworkers (31,37) have r e p o r t e d o n the p r o d u c t s of the l i g n i n peroxidase c a t a l y z e d o x i d a t i o n o f v e r a t r y l a l c o h o l . V e r a t r a l d e h y d e was the m a j o r p r o d u c t ( > 7 0 % y i e l d ) , together w i t h a n u m b e r of m i n o r p r o d u c t s , the quinones 13 a n d 14 a n d the r i n g o p e n e d lactones 16 a n d 17. In a d d i t i o n , S h i m a d a et ai (38,39) showed t h a t the 6-lactone 18 was also f o r m e d . R e c e n t l y we o b t a i n e d evidence t h a t the o r t h o - q u i n o n e 15 a n d the ^-lactone 19 were also p r o d u c t s of the l i g n i n peroxidase c a t a l y z e d o x i d a t i o n o f v e r a t r y l a l c o h o l ( S c h m i d t et al, Biochemistry, i n press). M e c h a n i s m s for the f o r m a t i o n o f those c o m p o u n d s

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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have been discussed (16,31,38,39), a l t h o u g h the r e a c t i o n m e c h a n i s m s for q u i n o n e f o r m a t i o n require further i n v e s t i g a t i o n . L e i s o l a et al. (15) have s t u d i e d the m e t a b o l i s m of v e r a t r y l a l c o h o l b y l i g n i n o l y t i c cultures of P. chrysosporium. F r o m studies w i t h C r i n g labeled c o m p o u n d s , the m e t a b o l i c scheme depicted i n F i g u r e 4 was p o s t u lated. In t h i s scheme v e r a t r y l a l c o h o l was viewed to be m e t a b o l i z e d b y the c o m b i n e d a c t i o n o f o x i d a t i v e systems (the l i g n i n peroxidase a n d p o s s i b l y other active o x y g e n species) a n d r e d u c t i v e conversions (aldehyde a n d q u i n o n e r e d u c t i o n s ) . A possible route v i a v e r a t r i c a c i d was d i s c o u n t e d because b o t h v e r a t r a l d e h y d e a n d veratric a c i d were not substrates for the l i g n i n peroxidase u n d e r the c o n d i t i o n s t u d i e d . However, b o t h v e r a t r a l d e h y d e a n d v e r a t r i c a c i d were r a p i d l y a n d q u a n t i t a t i v e l y reduced b y l i g n i n o l y t i c cultures o f P. chrysosporium (21). 1 4

V e r a t r a l d e h y d e was reduced b y a N A D P H dependent oxidoreductase, w h i c h m o s t p r o b a b l y wa cohol (40), a secondary m e t a b o l i t e of P. chrysosporium. T h e C labeled quinones 1 3 a n d 1 4 , prepared b y l i g n i n peroxidase c a t a l y z e d o x i d a t i o n of [ C] v e r a t r y l a l c o h o l , were also m e t a b o l i z e d by l i g n i n o l y t i c cultures of P. chrysosporium (15). E r i k s s o n a n d coworkers (17,18,22) s t u d i e d the m e t a b o l i s m of r i n g labeled quinone 1 4 , derived f r o m C r i n g - l a b e l e d v a n i l lic a c i d . F r o m t h e i r w o r k i t c o u l d be inferred t h a t quinone reductases played an i m p o r t a n t role. In a d d i t i o n , the d a t a i n F i g u r e s 2 a n d 3 i n d i c a t e t h a t the h y d r o q u i n o n e s are further m e t a b o l i z e d b y the fungus. T h e present s t u d y i n d i c a t e d t h a t the quinones o b t a i n e d f r o m l i g n i n peroxidase c a t a l y z e d oxi d a t i o n o f v e r a t r y l a l c o h o l indeed were first reduced to the c o r r e s p o n d i n g h y d r o q u i n o n e s . N o t e also t h a t these quinone reductions can occur b o t h e x t r a c e l l u l a r l y a n d i n t r a c e l l u l a r l y . T h e e x t r a c e l l u l a r cellobiose-quinone oxidoreductase (41) was o n l y involved i f cellulose is present i n the c u l t u r e s . Q u i n o n e r e d u c t i o n appears to be m a i n l y a n i n t r a c e l l u l a r process, i n v o l v i n g a n u m b e r of different enzymes w h i c h used N A D P H or N A D H as cosubstrates. In a d d i t i o n t o the q u i n o n e reductase observed b y B u s w e l l et al. (17) i n the s t u d y of v a n i l l i c a c i d m e t a b o l i s m , at least one a n d possibly two other q u i n o n e reductases w i t h different c a t a l y t i c properties were present. 1 4

14

i 4

Metabolism of Lignin. H o w can we relate the already c o m p l e x results o b t a i n e d i n d e g r a d a t i o n studies of s i m p l e m o n o m e r i c l i g n i n models like vera t r y l a l c o h o l , v e r a t r i c a c i d , v a n i l l i c a c i d , quinones, etc., to the studies on the m e t a b o l i s m o f the l i g n i n p o l y m e r ? A s p o i n t e d out by K i r k (42), few m o n o m e r i c c o m p o u n d s have been s t u d i e d i n d e t a i l because of the uncert a i n t y o f their relevance to l i g n i n p o l y m e r d e g r a d a t i o n . O f these m o n o m e r s , v a n i l l i c a c i d has been the best s t u d i e d since i t is a well k n o w n d e g r a d a t i o n p r o d u c t o f f u n g a l degraded w o o d . V e r a t r y l a l c o h o l is also b e i n g a c t i v e l y s t u d i e d since i t is a secondary m e t a b o l i t e of P. chrysosporium, a n d its role i n l i g n i n biodégradation is under active i n v e s t i g a t i o n . T h e d e g r a d a t i o n of v e r a t r y l a l c o h o l seems to involve b o t h o x i d a t i v e a n d r e d u c t i v e conversions a n d m e t a b o l i s m v i a r i n g opened a n d quinone t y p e s t r u c t u r e s (15). W e believe t h a t t h i s m e t a b o l i c p a t h w a y can serve i n p a r t as a m o d e l for l i g n i n biodégradation.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Figure nolytic ox red a red b

red a

OMe

OMe Hydroquinones

Ring opened products

red Quinones - H >

-> c o

2

CO2

4. H y p o t h e t i c a l scheme for v e r a t r y l a l c o h o l m e t a b o l i s m b y l i g n i c u l t u r e s o f P. chrysosponum: = o x i d a t i o n b y L i P ( l i g n i n peroxidase) = r e d u c t i o n by a N A D P H dependent aldehyde reductase = r e d u c t i o n by N A D ( P ) H dependent q u i n o n e reductases

OMe

CHO

ox L i P

OMe

2

CH OH

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E v i d e n t l y , further m e t a b o l i s m of r i n g opened p r o d u c t s (43-45) c o n s t i tutes a m a j o r m e t a b o l i c p a t h w a y for the d e g r a d a t i o n of the l i g n i n p o l y m e r . In a d d i t i o n , we w i s h t o postulate a p a t h w a y for the m e t a b o l i s m of s m a l l a r o m a t i c fragments, w h i c h are prone to undergo p o l y m e r i z a t i o n reactions under the o x i d a t i v e c o n d i t i o n s of the biodégradation process. W e propose t h a t r a p i d f u n g a l m e t a b o l i s m of q u i n o n e / h y d r o q u i n o n e i n t e r m e d i a t e s w i l l shift the d e p o l y m e r i z a t i o n - r e p o l y m e r i z a t i o n e q u i l i b r i u m m e n t i o n e d above towards d e g r a d a t i o n . T h i s hypothesis is i n p a r t based o n the fact t h a t i n n u m e r o u s studies (17-19,22,29,30) o n the e n z y m a t i c o x i d a t i o n a n d the biodégradation of a wide v a r i e t y of l i g n i n m o d e l c o m p o u n d s quinones a n d h y d r o q u i n o n e s were p r o d u c e d , or their f o r m a t i o n was i m p l i e d f r o m the isol a t i o n of s t r u c t u r a l c o u n t e r p a r t s . M o r e o v e r , we w i s h to p o s t u l a t e t h a t i n the f o r m a t i o n a n d the d e g r a d a t i o n of the q u i n o n e s / h y d r o q u i n o n e s b o t h o x i d a t i v e a n d r e d u c t i v e conversions play a n i m p o r t a n t role. In the l i g n i n d e g r a d a t i o n scheme depicted i n F i g u r e 5 i t is proposed t h a t l i g n i n peroxidase c a t a l y z e l i g n i n p o l y m e r . T h e n the q u i n o n e s / h y d r o q u i n o n e s f o r m e d , b o t h as the result of C a - a r e n e cleavage i n the o x i d a t i o n of phenolic l i g n i n s u b s t r u c t u r e s b y phenoloxidases (laccases, M n ( I I ) - d e p e n d e n t peroxidases), a n d l i g n i n peroxidase c a t a l y z e d o x i d a t i o n of non-phenolic l i g n i n s u b s t r u c t u r e s , w i l l be m e t a b o l i z e d v i a the reductive s y s t e m described. A l s o depicted i n F i g u r e 5 are the a r o m a t i c r i n g cleavage p a t h w a y a n d the d e g r a d a t i o n of C i , C 2 a n d C 3 fragments derived f r o m the p h e n y l p r o p a n e side c h a i n of the l i g n i n b u i l d i n g blocks. In the d e p o l y m e r i z a t i o n process, however, a c o m p l i c a t i o n can arise. L i g n i n peroxidase c a t a l y z e d o x i d a t i o n w i l l result i n the f o r m a t i o n of C a c a r b o n y l c o m p o u n d s (e.g., u p o n C a - C / ? cleavage or C a - o x i d a t i o n ) . If these d e g r a d a t i o n p r o d u c t s are phenolics, they w i l l be further degraded v i a the p a t h w a y s discussed above. However, i f these c o m p o u n d s are non-phenolics i n w h i c h a l l of the a r o m a t i c rings have a C a - c a r b o n y l s u b s t i t u e n t (most p r o b a b l y , these c o m p o u n d s w i l l be either m o n o m e r i c or d i m e r i c , see for an e x a m p l e c o m p o u n d 6 a ) they are not substrates for the l i g n i n peroxidase. However, we postulate t h a t P. chrysosponum uses its r e d u c t i v e s y s t e m to convert these m o n o m e r i c or d i m e r i c substructures to the c o r r e s p o n d i n g b e n z y l i c alcohols, w h i c h again are substrates for l i g n i n peroxidase. In fact, b o t h r e d u c t i o n of m o n o m e r i c a n d of d i m e r i c l i g n i n s u b s t r u c t u r e s have been des c r i b e d (17,18,21-25,37). Subsequently, e n z y m a t i c o x i d a t i o n either results i n r i n g o p e n i n g or q u i n o n e / h y d r o q u i n o n e f o r m a t i o n a n d further m e t a b o l i s m of the p r o d u c t s as discussed above. It s h o u l d be noted t h a t i f the non-phenolic C a - c a r b o n y l c o m p o u n d c o n t a i n s a n a r o m a t i c r i n g w i t h o u t C a - c a r b o n y l group (i.e., the s t r u c t u r e s h o u l d c o n t a i n at least two a r o m a t i c rings), then the c o m p o u n d w i l l be a s u b s t r a t e for l i g n i n peroxidase and w i l l be degraded a c c o r d i n g to the m e c h a n i s m s discussed above. D e g r a d a t i o n of this type of c o m p o u n d s can result, for e x a m p l e , i n the f o r m a t i o n of v a n i l l i n a n d vanillic a c i d derivatives (see c o m p o u n d s 7 a a n d 8 ) . A s has been s h o w n by E r i k s s o n a n d coworkers

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

phenol oxidases Mn(II)-peroxidase a c i d and aldehyde oxidoreductases

ί

quinone reductases

ι

quinones + hydroquinones—r> C 0 o

*

1

aromatic aldehydes and a c i d s

aromatic r i n g cleavage — ^ COo

F i g u r e 5. H y p o t h e t i c a l scheme for l i g n i n d e g r a d a t i o n b y l i g n i n o l y t i c cultures of P. chrysosporium.

ILIGNINr

•active oxygen* l i g n i n peroxidase

- > C l , C2, C3-fragments — ^ COfrom phenylpropane s i d e c h a i n

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(17-19), these structures are degraded via quinone/hydroquinone intermediates. Thus, by a combination of oxidation by lignin peroxidases, Mn(II)dependent peroxidases and other active oxygen species and reductions of some aromatic aldehydes, acids and ketones to the corresponding benzylic alcohols, all aromatic rings in the lignin polymer can be either converted to ring opened products or to quinones/hydroquinones. These products are then further metabolized to CO2 by a currently unknown mechanism. Conclusion In conclusion, we believe that lignin biodégradation by P. chrysosporium involves a combination of both oxidative and reductive conversions. Oxidative attack on the polymer is the predominant reaction. We postulate that rapid metabolism of ring d product d quinone/hydroquinon intermediates is one possibl depolymerization equilibrium towards degradation. Reduction of the quinones to the hydroquinones appears to be the first step in this process. In addition, depending on specific conditions, the fungus can use the reduction of aromatic ketone, formyl and carboxyl groups in order to convert certain low molecular weight intermediates to compounds that are better substrates for the lignin peroxidase. Subsequent oxidation and degradation again can involve ring cleaved and quinone type intermediates. However, since this type of reduction has to be invoked only in the metabolism of nonphenolic compounds in which all aromatic rings have a Ca-carbonyl substituent (most probably, monomeric and dimeric structures), its relevance to the overall process of lignin biodégradation has yet to be established. In this context also the relevance of reductive conversions in the metabolism of vanillin and vanillic acid requires further investigation. This working hypothesis now serves as the basis for future research. An important aspect to determine is what type of culture conditions influence the reductive system of P. chrysosporium. Is rapid metabolism of quinone type intermediates the only mechanism to prevent repolymerization, or do other factors play a role also? It has been suggested that binding of lignin fragments to the fungal mycelium is an important factor in lignin biodégradation (46,47). Additionally, are acids formed as intermediates on the metabolic pathway, or do they accumulate because their degradation is a relatively difficult process? In conjunction with this, the transport-mechanism of low molecular weight fragments through the cell membrane should be investigated. In our opinion, the answers to these and related questions will help us in understanding the complicated mechanism of lignin biodégradation. Research is in progress in our laboratories which addresses those topics. Literature Cited 1. Tien, M.; Kirk, T . K. Science 1983, 221, 661-663. 2. Glenn, J . K.; Morgan, Μ. Α.; Mayfield, M . B.; Kuwahara, M.; Gold, M. H. Biochem. Biophys. Res. Comm. 1983, 114, 1077-1083.

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3. Harvey, P. J.; Schoemaker, H. E.; Bowen, R. M.; Palmer, J . M . FEBS Lett. 1985, 183, 13-16. 4. Kuila, D.; Tien, M.; Fee, J . Α.; Ondrias, M . R. Biochemistry 1985, 24, 3394-3397. 5. Renganathan, V.; Gold, M . H. Biochemistry 1986, 25, 1626-1631. 6. Hammel, Κ. E.; Kalyanaraman, B.; Kirk, T . K. Proc. Natl. Acad. Sci. USA 1986, 83, 3708-3712. 7. Schoemaker, Η. E.; Harvey, P. J.; Bowen, R. M.; Palmer, J . M . FEBS Lett. 1985, 183, 7-12. 8. Kersten, P. J.; Tien, M.; Kalyanaraman, B.; Kirk, T . K. J. Biol. Chem. 1985, 260, 2609-2612. 9. Hammel, Κ. E.; Tien, M.; Kalyanaraman, B.; Kirk, T . K. J. Biol. Chem. 1985, 260, 8348-8353. 10. Tien, M . CRC Crit. Rev. Microbiol. 1987, 15, 141-168. 11. Buswell, J. Α.; Odier 12. Kirk, T . K.; Farrell, R 13. Biosynthesis and Biodegradation of Wood Components; Higuchi, T . , Ed.; Academic Press: New York, 1985. 14. Haemmerli, S. D.; Leisola, M . S. Α.; Fiechter, A. FEMS Microbiol. Lett. 1986, 35, 33-36. 15. Leisola, M . S. Α.; Haemmerli, S. D.; Waldner, R.; Schoemaker, Η. E . ; Schmidt, H. W. H.; Fiechter, A. Cell. Chem. and Tech. 1988, 22, 267277. 16. Schoemaker, Η. E.; Leisola, M . S. A. Proc. 31st IUPAC Congress of Pure and Appl. Chem. 1987, Section 4, 267-280. 17. Buswell, J . Α.; Hamp, S.; Eriksson, Κ. E . FEBS Lett. 1979, 108, 229232. 18. Ander, P.; Eriksson, Κ. E.; Yu, H.-S. Arch. Microbiol. 1983, 136, 1-6. 19. Eriksson, Κ. E.; Ander, P.; Pettersson, B. Proc. 3rd. Int. Conf. on Biotechn. in Pulp & Paper Ind. 1986, 24-27. 20. Chen, C. H.; Chang, Η. M. In Biosynthesis and Biodegradation of Wood Components; Higuchi, T., Ed.; Academic Press: New York, 1985; Chap­ ter 19. 21. Leisola, M . S. Α.; Fiechter, A. In Advances in Biotechn. Processes 5; Mizrahi, Α.; Van Wezel, A. L., Eds.; Alan R. Liss: New York, 1985, 59-89. 22. Ander, P.; Hatakka, Α.; Eriksson, K . E . Arch. Microbiol. 1980, 125, 189-202. 23. Kirk, T . K.; Nakatsubo, F. Biochem. Biophys. Acta 1983, 756, 376-384. 24. Enoki, Α.; Goldsby, G. P.; Krisnangkura, K.; Gold, M . H. FEMS Mi­ crobiol. Lett. 1981, 10, 373-377. 25. Nakatsubo, F.; Kirk, T . K.; Shimada, M.; Higuchi, T . Arch. Microbiol. 1981, 128, 416-420. 26. Higuchi, T . Wood Res. 1986, 73, 58-81. 27. Higuchi, T . In Biosynthesis and Biodegradation of Wood Components; Higuchi, T., Ed.; Academic Press: New York, 1985; Chapter 20.

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28. Shimada, M.; Habe, T.; Higuchi, T.; Okamoto, T.; Panijpan, Β. Holz­ forschung 1987, 41, 277-285. 29. Higuchi, T . Wood Res. 1981, 67, 47-58. 30. Kamaya, Y.; Higuchi, T . Wood Res. 1984, 70, 25-28. 31. Haemmerli, S. D.; Schoemaker, Η. E.; Schmidt, H. W. H.; Leisola, M . S. A. FEBS Lett. 1987, 220, 149-154. 32. Kirk, T . K.; Tien, M.; Kersten, P. J.; Mozuch, M . D.; Kalyanaraman, B. Biochem. J. 1986, 236, 279-287. 33. Umezawa, T.; Kawai, S.; Yokota, S.; Higuchi, T . Wood Res. 1986, 73, 8-17. 34. Kawai, S.; Umezawa, T.; Higuchi, T . FEBS Lett. 1987, 210, 61-65. 35. Enoki, Α.; Gold, M . H. Arch. Microbiol. 1982, 132, 123-130. 36. Janshekar, H.; Haltmeier, T.; Brown, C. Eur. J. Appl. Biotechn. 1982, 14, 174-181. 37. Haemmerli, S. D. Ph.D of Technology, Zürich 38. Shimada, M.; Hattori. T.; Umezawa. T.; Higuchi. T.; Uzura. K. FEBS Lett. 1987, 221, 327-331. 39. Hattori, T.; Shimada, M.; Umezawa, T.; Higuchi, T.; Leisola, M. S. Α.; Fiechter, A. Agric. Biol. Chem. 1988, 52, 879-880. 40. Shimada, M.; Nakatsubo, F.; Kirk, T. K.; Higuchi, T . Arch. Microbiol. 1981, 129, 321-324. 41. Westermark. U.; Eriksson. Κ. E . Acta Chem. Scand. 1974, B28, 209214. 42. Kirk, T . K. In Microbial Degradation of Organic Compounds; Gibson D. T., Ed.; Marcel Dekker: New York, 1984; Chapter 14. 43. Leisola, M . S. Α.; Schmidt, B.; Thanei-Wyss, U.; Fiechter, A. FEBS Lett. 1985, 189, 267-270. 44. Umezawa, T.; Higuchi, T . FEBS Lett. 1985, 182, 257-259. 45. Miki, K.; Renganathan, V.; Mayfield, M . B.; Gold, M . H. FEBS Lett. 1987, 210, 199-203. 46. Janshekar, H.; Brown, C.; Haltmeier, T.; Leisola, M . S. Α.; Fiechter, A. Arch. Microbiol. 1982, 132, 14-21. 47. Chua, M . G . S.; Choi, S.; Kirk, T . K. Holzforschung 1983, 37, 55-61. RECEIVED March 10, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 34

Oxidative Enzymes from the Lignin-Degrading Fungus Pleurotus sajor-caju Robert Bourbonnais and Michael G. Paice Pulp and Paper Research Institute of Canada, 570 St. John's Boulevard, Pointe Claire, Quebec H9R 3J9, Canada

Two extracellula role in lignin depolymerisation, laccase (polyphenol oxidase) and veratryl alcohol oxidase (VAO), were isolated from ligninolytic cultures of Pleurotus sajor-caju. The enzymes were produced in agitated, mycological broth cultures and were isolated after 12 days from supernatants by precipitation and chromatography. Two purified VAO enzymes had very similar physical and biochemical properties. They oxidised a variety of aromatic primary alcohols to aldehydes with reduction of oxygen to hydrogen peroxide. Sequential treatment of the laccase substrate ABTS with laccase and then VAO and veratryl alcohol producedfirstappearance and then disappearance of characteristic colors. A reduction-oxidation cycle is proposed for the two enzymes in depolymerisation of phenolic substructures of lignin. W h i t e - r o t f u n g i p l a y a key role i n biodégradation o f w o o d y m a t e r i a l s . T h e y are often the i n i t i a l colonizers, a n d are p r o b a b l y the o n l y m i c r o o r g a n i s m s t h a t extensively degrade l i g n i n (1). T h e m e c h a n i s m o f l i g n i n c a t a b o l i s m b y Phanerochaete chrysosporium has been s t u d i e d intensively, a n d a p i c ture is n o w emerging o f the e n z y m o l o g y i n v o l v e d . L i g n i n peroxidase, first recognized i n 1983 (2,3), abstracts a n electron f r o m l i g n i n s u b s t r u c t u r e s a n d subsequent free-radical t r a n s f o r m a t i o n s result i n Co>C/?, /?-ether a n d a r o m a t i c r i n g cleavage (4,5). T h e reactions w h i c h are dependent o n h y drogen peroxide c a n consume oxygen b y a d d i t i o n o f m o l e c u l a r oxygen t o r a d i c a l intermediates (6). M a n g a n e s e peroxidase (7,8) is also p r o d u c e d b y P. chrysosporium a n d abstracts electrons f r o m l i g n i n s u b s t r u c t u r e s w i t h lower redox p o t e n t i a l s such as phenolic u n i t s . T h e e n z y m e d i r e c t l y o x i dised Μ η II t o Μ η I I I as t h e i n i t i a l step i n l i g n i n a t t a c k . O t h e r enzymes 0097-6156/89/0399-0472$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

34.

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473

i m p l i c a t e d i n l i g n i n c a t a b o l i s m b y P. chrysosporium are glucose oxidase (9,10) a n d g l y o x a l oxidase (11) for h y d r o g e n peroxide p r o d u c t i o n , a n d c e l ­ lobiose quinone oxidoreductase (12) for q u i n o n e r e d u c t i o n . Since l i g n i n s c a n be b o t h d e p o l y m e r i z e d a n d p o l y m e r i z e d b y l i g n i n peroxidase (2,13), other enzymes are p r o b a b l y r e q u i r e d for c a t a b o l i s m in vivo. T h e question arises as t o whether other w h i t e - r o t f u n g i e m p l o y the same c a t a b o l i c p a t h w a y s as P. chrysosporium. Also, lignin degradation by P. chrysosporium occurs o n l y d u r i n g secondary m e t a b o l i s m , a n d e m p l o y s v e r a t r y l a l c o h o l , w h i c h is synthesized de novo b y the fungus, as a m e d i a t o r (14) or enzyme i n d u c e r (15). A r e these c o n d i t i o n s required b y other f u n g i ? It n o w appears t h a t l i g n i n peroxidase is p r o d u c e d b y Coriolus versicolor (16) a n d perhaps b y Pleurotus ostreatus (17). However these two w h i t e rot f u n g i produce laccase isoenzymes w h i c h , like manganese peroxidase, c a n a b s t r a c t electrons f r o m p h e n o l i c s u b s t r u c t u r e s of l i g n i n . T h u s there appears to be no obvious requirement for manganese peroxidase i n these f u n g i . I n a d d i t i o n , i t appear d u r i n g p r i m a r y m e t a b o l i s m , i.e. i n a h i g h n i t r o g e n m e d i u m (18), a l t h o u g h t h i s has been d i s p u t e d (19). Pleurotus sajor-caju differs f r o m P. ostreatus i n not r e q u i r i n g c o l d shock for f r u c t i f i c a t i o n (20). P. sajor-caju selectively degrades l i g n i n i n w h e a t - s t r a w under c e r t a i n c o n d i t i o n s (21,22), b u t t h i s s e l e c t i v i t y is lost w h e n f r u i t i n g bodies f o r m (23). C h l o r o l i g n i n c a n be m e t a b o l i s e d b y P. sajor-caju (24). W a l d n e r et al. (17) observed o x i d a t i o n o f v e r a t r y l a l c o h o l to v e r a t r a l d e h y d e b y P. ostreatus. W e have recently r e p o r t e d the discovery of a v e r a t r y l a l c o h o l oxidase f r o m P. sajor-caju (25). W e now propose a role for the e n z y m e , i n c o m b i n a t i o n w i t h laccase, i n the d e p o l y m e r i z a t i o n o f p h e n o l i c moieties o f l i g n i n . Experimental Enzyme Production and Isolation. T h e production and isolation of veratryl a l c o h o l oxidase ( V A O ) was described earlier (25). Laccase p r o d u c e d f r o m the same 12-day c u l t u r e (8 litres) was isolated f r o m the s u p e r n a t a n t b y p r e ­ c i p i t a t i o n at 0 ° C w i t h a m m o n i u m sulfate ( 8 0 % s a t u r a t i o n ) . T h e p r e c i p i t a t e was suspended i n 0.05 M N a acetate buffer, p H 5.0 a n d d i a l y s e d overnight against 4 litres of buffer. T h e soluble m a t e r i a l was c o n c e n t r a t e d b y u l t r a ­ filtration ( A m i c o n P M 1 0 ) to a b o u t 60 m L a n d a p p l i e d to a D E A E - B i o - g e l A c o l u m n (2.5 c m χ 35 c m ) . T h e c o l u m n was washed w i t h 20 m L o f the same buffer, t h e n e l u t e d w i t h a linear g r a d i e n t f r o m 0 to 0.6 M N a C l ( t o t a l v o l u m e 550 m L ) . F r a c t i o n s were m o n i t o r e d for V A O a n d laccase a c t i v i t y as described below. Enzyme Assays. Laccase a c t i v i t y was d e t e r m i n e d b y o x i d a t i o n o f 2,2azinobis-(3-ethylbenzthiazoline-6-sulfonate) ( A B T S ) . T h e reaction of suit­ a b l y d i l u t e d e n z y m e was d e t e r m i n e d at 420 n m i n the presence of 0.03% A B T S a n d 100 m M s o d i u m acetate buffer, p H 5.0. T h e e x t i n c t i o n coeffi­ cient o f A B T S is ε ο = 3.6 χ 1 0 M " c m ' (26). V A O a c t i v i t y was measured w i t h a m i x t u r e of 1 m M v e r a t r y l a l c o h o l , 250 m M s o d i u m t a r t r a t e buffer, p H 5.0 a n d e n z y m e . O x i d a t i o n to v e r a 4 2

4

1

1

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t r a l d e h y d e was d e t e r m i n e d at 310 n m (£310 = 9300 M " c m ) . L i g n i n peroxidase was measured b y v e r a t r y l a l c o h o l o x i d a t i o n at p H 3.0 i n the presence of 0.4 m M H 2 O 2 (2). A l l e n z y m e u n i t s are / / m o l e of p r o d u c t formed/min. T h e c o m b i n e d effect of laccase a n d V A O o n A B T S was d e t e r m i n e d as follows. T h e reagent (0.03%) was oxidised w i t h p u r i f i e d laccase f r o m P. sajor-caju (0.004 U / m L ) . A f t e r the appearance of the green ( A B T S ) colour, V A O (0.07 U / m L ) a n d v e r a t r y l a l c o h o l (1 m M ) was added a n d the decrease i n colour was observed v i s u a l l y . O x y g e n u p t a k e d u r i n g substrate o x i d a t i o n was measured w i t h a C l a r k oxygen electrode ( R a n k B r o t h e r s , C a m b r i d g e , U . K . ) at r o o m t e m p e r a t u r e w i t h 1 m M substrate i n 0.25 m M s o d i u m t a r t r a t e buffer, p H 3.0 (3 m L ) . R a t e s are expressed relative to v e r a t r y l a l c o h o l o x i d a t i o n . 1

- 1

+ e

Results and Discussion Enzyme Production. P. sajor-caju, under aerated, a g i t a t e d c o n d i t i o n s , was able to m i n e r a l i z e p a r t i a l l y CD H P l i g n i n , as s h o w n i n F i g u r e 1 (25). L i g n i n peroxidase a c t i v i t y , (i.e., peroxide-dependent o x i d a t i o n of ver a t r y l a l c o h o l at p H 3) was not detected over the 30 days tested, w h i l e laccase appeared at day 7. C u l t u r e m e d i u m f r o m d a y 7 onwards c o u l d also oxidize v e r a t r y l a l c o h o l to aldehyde w i t h c o n c o m i t a n t conversion o f oxygen to hydrogen peroxide. T h i s a c t i v i t y , w h i c h was o p t i m a l at p H 5.0, was n a m e d v e r a t r y l a l c o h o l oxidase ( V A O ) . T h e e x t r a c e l l u l a r o x i d a t i v e e n z y m e a c t i v i t i e s (laccase a n d v e r a t r y l a l c o h o l oxidase) c o u l d be separated b y ion-exchange c h r o m a t o g r a p h y ( F i g u r e 2). F u r t h e r c h r o m a t o g r a p h y o f the coincident laccase a n d v e r a t r y l a l c o h o l oxidase (peak 2), as described elsewhere (25) resulted i n the s e p a r a t i o n of two v e r a t r y l a l c o h o l oxidases f r o m the laccase. 1 4

Enzyme Properties. T h e two isolated v e r a t r y l a l c o h o l oxidases h a d very s i m i l a r properties ( T a b l e I). T h e difference i n isoelectric p o i n t s m i g h t be accounted for b y aspartate content; a l l other a m i n o a c i d contents except g l y c i n e were the same w i t h i n e x p e r i m e n t a l error (5%). T h e specific a c t i v i ties ( v e r a t r y l a l c o h o l as substrate) were significantly different, b u t b o t h e n zymes contained a flavin prosthetic group (25) a n d converted one molecule of oxygen to one molecule of hydrogen peroxide d u r i n g a l c o h o l o x i d a t i o n . T a b l e I. C o m p a r i s o n of V A O I a n d II ( D a t a s u m m a r i z e d f r o m ref. 25)

M o l e c u l a r weight Isoelectric p o i n t Glycosylated Asp/mole Gly/mole Specific a c t i v i t y , I U / m g

VAO I

V A O II

71,000 3.8 yes 83 77 35

71,000 4.0 yes 92 66 31

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

34.

Oxidative Enzymes from Fungus

BOURBONNAIS & PAICE

0

5

10

15

20

25

475

30

Time, days F i g u r e 1. T i m e course of of l i g n i n o l y t i c a c t i v i t y (conversion of r i n g - l a b e l l e d C - D H P to C C > 2 ) , b i o m a s s c o n c e n t r a t i o n , V A O a c t i v i t y a n d laccase act i v i t y d u r i n g g r o w t h o f Pleurotus sajor-caju i n agitated mycological broth c u l t u r e s . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 25, © 1988, B i o c h e m i c a l Society). 1 4

14

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

476

PLANT C E L L W A L L P O L Y M E R S

Fraction no. F i g u r e 2. E l u t i o n profile of P . sajor-caju s u p e r n a t a n t f r o m D E A E - B i o - g e l c o l u m n , s h o w i n g resolution of laccase a n d V A O peaks of a c t i v i t y .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

34.

477

Oxidative Enzymes from Fungus

BOURBONNAIS & PAICE

p-Methoxybenzyl alcohol was oxidised fastest of the substrates tested. Some relative rates of oxidation, measured as oxygen consumption, are shown in Figures 3 and 4. Methoxy substituted benzyl alcohols showed wide variations in their relative rates of oxidation (Figure 3) which are not easily explained by electron availability. Para-hydroxyl substitution severely inhibited oxidation (Figure 4), even in α, β unsubstituted aryl al­ cohols. Lignin model dimers, both phenolic and non-phenolic, and kraft lignin were not oxidised to any detectable extent. The enzyme properties reported above are similar to those of an aro­ matic alcohol oxidase from Polystictus versicolor (27). However, the latter enzyme had a different substrate specificity and the cultures did not pro­ duce laccase. Possible Role in Lignin Biodégradation. Oxidised products of veratryl alcohol have been proposed to play a key role in lignin peroxidase-mediated oxidation of lignin, eithe reaction which generates idases do not directly oxidize lignin, but they have a broad substrate range for monomeric aromatic alcohols and could therefore be involved in the final cat abolie steps following depolymerization. It seems unlikely however, that the resulting aromatic aldehydes would per se oxidize lignin itself. A possible reductive role for veratryl alcohol oxidase is proposed in Figure 5. Laccases from C. versicolor cam produce both polymerization and depolymerization of lignin (29). In phenolic lignin model dimers, laccase can perform the same electron abstraction and subsequent bond cleavage as found for lignin peroxidase (30). The phenolic radical is however likely to polymerize unless the quinoid-type intermediates can be removed, for example by reduction back to the phenol. Veratryl alcohol oxidase, in

CH OH 2

200 CH2OH

CH OH 2

100 CH OH 2

CH OH

CH OH

50

20

2

CH OH 2

2

CH OH 2

OCH3

Figure 3. Relative oxidation rates of various methoxyl-substituted benzyl alcohols, measured by oxygen consumption/min. (veratryl alcohol = 100). No oxidation of the lower four alcohols was detected.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

478

PLANT C E L L W A L L

POLYMERS

Figure 4. Relative oxidation rates of various aromatic alcohols. Para-hydroxyl substitution eliminates substrate oxidation.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

34.

BOURBONNAIS & PAICE

Oxidative Enzymes from Fungus

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

479

480

PLANT CELL WALL POLYMERS

the presence of veratryl alcohol or other cosubstrate, could provide this reducing power, as has been proposed previously for glucose oxidase (31) and cellobiose quinone oxidoreductase (12). In support of this hypothesis, we find that veratryl alcohol oxidase in the presence of veratryl alcohol will decolorize the oxidised products produced by laccase from ABTS. A cknowledgment s We thank F. Lafortune for excellent technical contribution. This work was supported in part with financial assistance from the National Research Council of Canada under Contribution number 949-6-0011. Literature Cited 1. Kirk, T . K.; Cowling, Ε. B. The Chemistry of Solid Wood; Amer. Chem. Soc.: Washington, D.C., 1984, p. 455-87. 2. Tien, M.; Kirk, T . K. Science 1983, 221, 661-63. 3. Glenn, J . ; Morgan, Μ H. Biochem. Biophys. 4. Schoemaker, H. E.; Harvey, P. J.; Bowen, R. M.; Palmer, J . M . FEBS Lett. 1985, 183, 7-12. 5. Kersten, P. J.; Tien, M.; Kalyanaraman, B.; Kirk, T . K. J. Biol. Chem. 1985, 260, 2609-12. 6. Miki, K.; Renganathan, V.; Gold, M . H. Biochemistry 1986, 26, 479096. 7. Kuwahara, M.; Glenn, J . K.; Morgan, Μ. Α.; Gold, M . H. FEBS Lett. 1984, 169, 247-50. 8. Paszczynski, Α.; Huynh, V. B.; Crawford, R. Arch. Biochem. Biophys. 1986, 244, 750-65. 9. Ramasamy, K.; Kelley, R. L.; Reddy, C. A. Biochem. Biophys. Res. Commun. 1985, 131, 436-41. 10. Eriksson, Κ. E.; Pettersson, B.; Vole, J.; Musilek, V. Appl. Microbiol. Biotechnol. 1986, 23, 257-62. 11. Kersten, P. J.; Kirk, T . K. J. Bacteriol. 1987, 169, 2195-2201. 12. Westermark, U.; Eriksson, Κ. E. Acta Chem. Scand. 1987, B29, 41924. 13. Haemmerli, S. D.; Leisola, M . S. Α.; Fiechter, A. FEMS Microbiol. Lett. 1986, 35, 33-36. 14. Harvey, P. J.; Schoemaker, Η. E.; Palmer, J . M . FEBS Lett. 1986, 195, 242-46. 15. Faison, B. D.; Kirk, T . K.; Farrell, R. L. Appl. Environ. Microbiol. 1986, 52, 251-54. 16. Dodson, P. J.; Evans, C. S.; Harvey, P. J.; Palmer, J . M . FEMS Mi­ crobiol. Lett. 1987, 42, 17-22. 17. Waldner, R.; Leisola, M.; Fiechter, A. Proceeding Biotechnology in Pulp and Paper Industry; 3rd Intl. Conf.; Stockholm 1986, 50-153. 18. Leatham, G . F.; Kirk, T . K. FEMS Microbiol. Lett. 1982, 16, 65-67. 19. Commanday, F.; Macy, J . M . Arch. Microbiol. 1985, 142, 61-65. 20. Mueller, J. C.; Gawley, J . R. Mushroom Newsletter Tropics 1983, 4, 3-12.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

34. BOURBONNAIS & PAICE

Oxidative Enzymesfrom Fungus

481

21. Mueller, J. C.; Troesch, W. Appl. Microbiol. Biotechnol. 1986, 24, 18085. 22. Zadrazil, F. Eur. J. Appl. Microbiol. 1975, 1, 327-335. 23. Tsang, L. J.; Reid, I. D.; Coxworth, E . C. Appl. Environ. Microbiol. 1987, 53, 1304-06. 24. Bourbonnais, R.; Paice, M . G . J. Wood Chem. Tech. 1987, 7, 51-64. 25. Bourbonnais, R.; Paice, M . G . Biochem. J. 1988, 255, 445-50. 26. Wolfenden, B. S.; Willson, R. L. J. Chem. Soc. Perkin Trans. II 1982, 805-12. 27. Farmer, V.; Henderson, Μ. Ε. K.; Russell, J . D. Biochem. J. 1960, 257-62. 28. Haemmerli, S. D.; Schoemaker, H. E.; Schmidt, H. W. H.; Leisola, M . S. A. FEBS Lett. 1987, 220, 279-87. 29. Morohoshi, N.; Nakamura, M.; Katayama, Y.; Haraguchi, T . Pulping and Wood Chemistry 30. Kawai, S.; Umezawa Nishida, T.; Morohoshi, N.; Haraguchi, T . Mokuzai Gakkaishi 1987, 33, 792-97. 31. Green, T . R. Nature 1977, 268, 78-80. RECEIVED March 17, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 35

Mechanisms of Lignin Degradation by Lignin Peroxidase and Laccase of White-Rot Fungi Takayoshi Higuchi Wood Research Institute, Kyoto University, Uji, Kyoto 611, Japan

The main cleavage mechanisms of side-chains and aro matic rings of lignin (DHP) by lignin peroxidase and laccase of white­ -rot fungi have been elucidated. Tracer studies using H-, C- and O-labeled arylglycerol-β-aryl ethers and diarylpropane-1,3-diols with O and H 18O indicated that side-chains and aromatic rings of these substrates were cleaved via aryl radical cation and phenoxy radi­ cal intermediates, in reactions mediated only by lignin peroxidase/HO and laccase/O. 2

13

18

18

2

2

2

2

2

The process of lignin biodégradation by microbes has evoked much interest in recent years (1-3). Several o f these studies have been d i r e c t e d t o w a r d s e s t a b l i s h i n g the exact c h e m i c a l m e c h a n i s m s i n v o l v e d i n l i g n i n sidec h a i n cleavage a n d a r o m a t i c r i n g - o p e n i n g reactions, w h i c h are c a t a l y z e d b y l i g n i n peroxidase (4,5) a n d laccase (6). A l m o s t a l l o f these studies n o r m a l l y employ, as l i g n i n - l i k e substrates, various d i m e r i c phenolic o r n o n phenolic m o d e l c o m p o u n d s w h i c h contain b o n d i n g p a t t e r n s c h a r a c t e r i s t i c of s u b s t r u c t u r e s k n o w n t o be present i n isolated l i g n i n p r e p a r a t i o n s . F o r e x a m p l e , u s i n g β A a n d β-ΟΆ m o d e l c o m p o u n d s , i t has been established t h a t b o t h l i g n i n peroxidase a n d laccase c a t a l y z e one electron o x i d a t i o n s (5-7). I n p a r t i c u l a r , l i g n i n peroxidase converts b o t h phenolic a n d n o n phenolic moieties t o their corresponding p h e n o x y r a d i c a l a n d a r y l r a d i c a l c a t i o n i n t e r m e d i a t e s , whereas laccase o n l y catalyzes p h e n o x y r a d i c a l for­ m a t i o n f r o m phenolic substrates; n o n p h e n o l i c c o m p o u n d s are n o t o x i d i z e d (8). T h e a r y l r a d i c a l cations (formed f r o m n o n p h e n o l i c m o d e l c o m p o u n d s by l i g n i n p e r o x i d a s e / ! ^ O 2 ) t h e n undergo n u c l e o p h i l i c a t t a c k (e.g., b y H 2 O , or h y d r o x y l groups o n adjacent functionalities) t o generate a r y l r a d i c a l i n ­ termediates. S u c h reactive species, as w e l l as t h e p h e n o x y r a d i c a l s afore­ m e n t i o n e d , c a n t h e n react w i t h dioxygen r a d i c a l s o r a l t e r n a t i v e l y they c a n

0097-6156/89/0399-0482$06.00A) © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

35.

HIGUCHI

483

Lignin Degradation by Peroxidase & Laccase

undergo free r a d i c a l c o u p l i n g reactions. T h u s t w o c o m p e t i n g reactions c a n o c c u r d u r i n g l i g n i n biodégradation: (1) r e a c t i o n w i t h d i o x y g e n r a d i c a l s res u l t i n g i n l i g n i n d e g r a d a t i o n a n d (2) free r a d i c a l c o u p l i n g l e a d i n g t o r e p o l y m e r i z a t i o n . T h e s e findings help e x p l a i n the current difficulties e x p e r i e n c e d i n efficiently d e g r a d i n g the l i g n i n p o l y m e r in vitro w i t h i s o l a t e d e n z y m e p r e p a r a t i o n s (1). A t t h i s j u n c t u r e , i t is p e r t i n e n t to briefly describe o u r c u r r e n t u n d e r s t a n d i n g of l i g n i n f o r m a t i o n i n x y l e m cell w a l l s (9). I n i t i a l l y , free r a d i c a l c o u p l i n g o f i n t e r m e d i a t e s , f o r m e d f r o m the c o r r e s p o n d i n g m o n o l i g n o l s v i a m e d i a t i o n o f cell w a l l peroxidases a n d H 2 O 2 , affords d i m e r i c q u i n o n e m e t h i d e s ; subsequent n u c l e o p h i l i c a t t a c k (e.g., b y H 2 O or adjacent h y d r o x y l groups) results i n r e a r o m a t i z a t i o n to afford d i l i g n o l s , s u c h as deh y d r o d i c o n i f e r y l a l c o h o l , d , l - p i n o r e s i n o l , g u a i a c y l g l y c e r o l - / ? - c o n i f e r y l ether, etc. T h e s e phenols c a n t h e n be reconverted i n t o t h e i r free r a d i c a l f o r m s , w h i c h t h e n ( i n a n analogous m a n n e r ) undergo c o u p l i n g a n d r e a r o m a t i z a t i o n to give the c o r r e s p o n d i n results i n l i g n i n f o r m a t i o n ; t h i s process is often described as d e h y d r o g e n a s e p o l y m e r i z a t i o n ( F i g . 1). I n o u r o p i n i o n , the reactions i n v o l v e d i n l i g n i n biodégradation (as c a t a l y z e d b y l i g n i n peroxidase a n d laccase) c a n be c o n sidered as a n e x t e n s i o n of the reactions encountered i n l i g n i n f o r m a t i o n . W e propose t h i s i d e a since, as discussed l a t e r , i t appears t h a t d e g r a d a t i o n of the l i g n i n p o l y m e r involves s i m i l a r reactions. In t h i s c h a p t e r , some o f the m a i n reactions i n l i g n i n biodégradation (i.e., s i d e - c h a i n cleavage a n d a r o m a t i c r i n g - o p e n i n g ) are d e s c r i b e d , a n d the effects of b o t h laccase a n d l i g n i n peroxidase c o m p a r e d . T h e schemes p r o p o s e d are a l l based o n identified p r o d u c t s f r o m m o d e l c o m p o u n d s a n d s u p p o r t e d wherever possible b y a p p r o p r i a t e l a b e l l i n g s t u d i e s . F i n a l l y , the processes of l i g n i n biosynthesis a n d biodégradation are c o m p a r e d . Results and Discussion Side-Chain Cleavage Reactions of β-l and β-0-4 Lignin Model Compounds Catalyzed by Lignin Peroxidase. β-l Model Compounds. T h e s e can be separated i n t o two b r o a d c a t ­ egories: n o n p h e n o l i c a n d p h e n o l i c . A s can be seen f r o m F i g u r e 2, the n o n p h e n o l i c β-l l i g n i n m o d e l c o m p o u n d 1 is first converted i n t o i t s a r y l r a d i c a l c a t i o n i n t e r m e d i a t e . T h i s t h e n undergoes C a - C / ? h o m o l y t i c c l e a v ­ age to afford 3 , 4 , 5 - t r i m e t h o x y b e n z a l d e h y d e 2, a n d the d i o l 3, w i t h the l a t t e r t r a p p i n g oxygen as s h o w n b y a p p r o p r i a t e l a b e l l i n g e x p e r i m e n t s w i t h C>2 ( F i g . 2) (4,10). T h e g e n e r a l i t y of t h i s m e c h a n i s m was d e m o n s t r a t e d u s i n g a n u m b e r of methoxybenzenes a n d d i a r y l e t h a n e d i m e r s as s u b s t r a t e s (7,11). R e a c t i o n progress was m o n i t o r e d b y E S R s p e c t r o m e t r y (11); m e c h a n i s m s were f u r t h e r established by u s i n g m o d e l c o m p o u n d s specifically d e u t e r a t e d at C a a n d C/? as r e q u i r e d (12). W h i l e the c o r r e s p o n d i n g p h e n o l i c β-l m o d e l c o m p o u n d s are also s u b ­ j e c t to C a - C / ? cleavage, other reactions can also occur (13). F i g u r e 3 s u m ­ marizes the m a i n reactions i d e n t i f i e d to t h i s p o i n t . These i n c l u d e (i) C a - C / ? cleavage of 4 to afford s y r i n g a l d e h y d e 6 a n d the d i o l , 7, (ii) C a o x i d a t i o n a n d d e h y d r a t i o n t o give the ketone 5 a n d (iii) a l k y l - a r y l cleavage t o give the d e g r a d a t i o n p r o d u c t s 8 a n d 9 respectively (13). 18

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Figure 1. Formation of guaiacyl lignin and lignin-carbohydrate complexes ( L C C ) via dehydrogenase polymerization of coniferyl alcohol.

35.

HIGUCHI

Lignin Degradation by Peroxidase & Laccase

485

Figure 2. Side-chain cleavage of a nonphenolic β A model compound 1 by lignin peroxidase.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

C a - Ο β CLEAVAGE

9

IALKYL-ARYL CLEAVAGE

F i g u r e 3. P o s s i b l e d e g r a d a t i o n p a t h w a y s o f the p h e n o l i c 0-\ m o d e l c o m ­ p o u n d 4 b y l i g n i n peroxidase.

OCH.

4

S3

35.

HIGUCHI

Lignin Degradation by Peroxidase & Laccase

487

β-0-4 Model Compounds. Investigations were c o n d u c t e d u s i n g b o t h n o n p h e n o l i c a n d phenolic l i g n i n m o d e l s u b s t r a t e s . S t u d i e s (12,14,15) w i t h the n o n p h e n o l i c d i m e r 10 revealed t h a t four m a i n r e a c t i o n p a t h w a y s were possible: (i) b e n z y l i c o x i d a t i o n ( C a ) to give the c o r r e s p o n d i n g ketone (not s h o w n ) ; (ii) f o r m a t i o n of the a r y l r a d i c a l c a t i o n i n t e r m e d i a t e as before, f o l ­ lowed b y f r a g m e n t a t i o n to afford g u a i a c o l 11, the g l y c o l i c aldehyde 12 a n d 4 - e t h o x y - 3 - m e t h o x y b e n z a l d e h y d e 13 ( F i g . 4, p a t h w a y i i ) ; ( i i i ) a r y l r a d i c a l c a t i o n f o r m a t i o n , subsequent i n t e r m o l e c u l a r n u c l e o p h i l i c a t t a c k b y the 7 h y d r o x y l g r o u p a n d r e a r r a n g e m e n t , to give the isomeric d i o l 14 followed b y h o m o l y t i c cleavage to afford the aldehyde 15 a n d 2 - ( 2 - m e t h o x y p h e n o l ) ace t a l d e h y d e 16. These u n u s u a l reactions were confirmed b y l a b e l l i n g studies as s h o w n ( F i g . 4, p a t h w a y i i i ) , a n d (iv) d i s p l a c e m e n t a n d / o r a r o m a t i c r i n g d i s p l a c e m e n t t o y i e l d the t r i o l 19 a n d o-quinone 20 ( F i g . 4, p a t h w a y i v ) (3). /

Studies (16) e m p l o y i n g p h e n o l i c β-0-4 m o d e l substrates revealed three m a j o r reactions, reminiscen (i) b e n z y l i c o x i d a t i o n , (ii) C a - C / ? cleavage to give either the c o r r e s p o n d i n g C a aldehyde or a c i d ; a n d ( i i i ) a l k y l - a r y l b o n d r u p t u r e to give the h y d r o ­ q u i n o n e (cf. 8, F i g . 3) a n d the c o r r e s p o n d i n g g l y c e r a l d e h y d e - 2 - a r y l ether. Side Chain

Cleavage

of β-l

and β-0-4

Model Compounds

by

Laccase.

β-l Model Compounds. O n l y substrates w i t h p h e n o l i c f u n c t i o n a l i t i e s were e x a m i n e d , since n o n p h e n o l i c substrates were unaffected. T h u s , the two p h e n o l i c diols 4 a n d 21 were synthesized (6), a n d b o t h were i n d i v i d ­ u a l l y exposed to laccase f r o m Coriolus versicolor (6). F o r d i o l 4, a l k y l a r y l cleavage gave 2 , 6 - d i m e t h o x y h y d r o q u i n o n e 8, the c o r r e s p o n d i n g b e n z o q u i n o n e 23 a n d the aldehyde 24 ( F i g . 5, p a t h w a y B ) ; C a o x i d a t i o n also afforded the ketone 22 ( p a t h w a y A ) . A d d i t i o n a l l y , other reactions were also observed ( F i g . 6), n a m e l y C a - C / ? cleavage to give s y r i n g a l d e h y d e 6, a n d the a r y l k e t o - a l c o h o l 25 ( F i g . 6, p a t h w a y A ) . O n the other h a n d , d i o l 21 o n l y u n d e r w e n t C a - C / ? cleavage, g e n e r a t i n g the s u b s t i t u t e d b e n z a l d e h y d e 27 a n d the p h e n y l g l y c o l 26 ( F i g . 6, p a t h w a y B ) . N o t e t h a t these m e c h a ­ n i s m s were based u p o n a p p r o p r i a t e l a b e l l i n g studies u s i n g 0 (6). T h u s , reactions i n v o l v i n g p h e n o l i c substrates, a n d u s i n g b o t h l i g n i n peroxidase a n d laccase are s i m i l a r ; b o t h encompass C a - C / ? , a l k y l - a r y l b o n d cleavages a n d o x i d a t i v e reactions. 1

8

2

β-Ο-4 Model Compounds. I n t h i s i n v e s t i g a t i o n , the s u b s t r a t e used for i n c u b a t i o n w i t h laccase was the m o d e l c o m p o u n d , s y r i n g y l g l y c e r o l - / ? g u a i a c y l ether 28 (18). It was i n i t i a l l y r e p o r t e d t h a t t h i s u n d e r w e n t conver­ sion i n t o g l y c e r a l d e h y d e - 2 - g u a i a c y l ether a n d 2 , 6 - d i m e t h o x y b e n z o q u i n o n e (17). A d d i t i o n a l l y , the h y d r o x y l group of the h y d r o q u i n o n e was l a b e l l e d with 0 , w h e n the e x p e r i m e n t was c a r r i e d o u t i n the presence of H 2 0 . H o w e v e r , more comprehensive studies (18) have d e m o n s t r a t e d t h a t the d i m e r 28 also undergoes b e n z y l i c o x i d a t i o n to give the ketone 29, a n d Ca-C β cleavage to afford g u a i a c o l 11 a n d s y r i n g i c a c i d 30 ( F i g . 7). T h u s , once a g a i n , the reactions c a t a l y z e d b y laccase (for these p h e n o l i c substrates) show considerable resemblance to t h a t a l r e a d y noted for l i g n i n peroxidase. 1 8

1 8

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

PLANT C E L L W A L L

POLYMERS

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

35.

HIGUCHI

Lignin Degradation by Peroxidase & Laccase

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

489

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

F i g u r e 5. P o s s i b l e m e c h a n i s m s for C a o x i d a t i o n ( A ) a n d a l k y l - a r y l cleavage ( B ) reactions of a phenolic β-l m o d e l c o m p o u n d 4 b y laccase.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

F i g u r e 6. P o s s i b l e m e c h a n i s m s for Ca-C β cleavage o f p h e n o l i c β-l c o m p o u n d s 4 a n d 2 1 b y laccase.

model

492

PLANT C E L L W A L L POLYMERS

30

F i g u r e 7. S i d e - c h a i n cleavage o f a phenolic β-0-4 laccase.

model compound 28 by

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

35.

HIGUCHI

493

Lignin Degradation by Peroxidase & Laccase

Aromatic Ring Cleavage of Nonphenolic Lignin Substructure Model Compounds and Veratryl Alcohol by Lignin Peroxidase. β-Ο-4 Model Compounds. T h r e e m o d e l c o m p o u n d s 31-33 were s y n ­ thesized (19,20), a n d treated w i t h l i g n i n peroxidase as before. T h i s r e ­ s u l t e d i n t h e i r conversion i n t o t h e a r y l g l y c e r o l c y c l i c c a r b o n a t e s 37-39, the O - f o r m a t e s 40,41, a n d t h e m e t h y l o x a l a t e dérivâtes 34-36. A f o u r t h m o d e l c o m p o u n d 42 was also s y n t h e s i z e d , a n d t h i s u n d e r w e n t d e g r a d a t i o n t o give t h e m e t h y l o x a l a t e d e r i v a t i v e 44 a n d cis-cis m u c o n a t e d e r i v a t i v e s 43 (19,20). L a b e l l i n g e x p e r i m e n t s , w i t h C>2 a n d H 2 0 , respectively, d e m o n s t r a t e d t h a t o n l y one o f t h e oxygens o n t h e c a r b o n y l groups o f t h e m e t h y l o x a l a t e 34 a n d m u c o n a t e s 43 were C>2 d e r i v e d , w i t h t h e other b e i n g f o r m e d f r o m H 2 0 ( F i g . 8) (19). T o account for t h e f o r m a t i o n o f 43 as i n i t i a l r i n g cleavage p r o d u c t s , t h e h y p o t h e t i c a l scheme s h o w n i n F i g u r e 9 is p r o p o s e d ; i n i t i a l f o r m a t i o n o f t h e a r y l r a d i c a l c a t i o n i n t e r m e d i a t e o c curs w h i c h c a n t h e n react w i t h H 2 O , O 2 a n d h y d r o g e n r a d i c a l s as s h o w n . T h e m e c h a n i s m s for t h e v i a a r y l r a d i c a l c a t i o n i n t e r m e d i a t e s are s h o w n i n t h e c h a p t e r b y U m e z a w a and Higuchi. Veratryl Alcohol. L e i s o l a et ai (21,22) recently r e p o r t e d t h a t t r e a t m e n t o f v e r a t r y l a l c o h o l 45 w i t h l i g n i n peroxidase resulted m a i n l y i n t h e f o r m a t i o n o f v e r a t r y l aldehyde 49, the t w o 7 - l a c t o n e s 46 a n d 47 ( F i g . 10) a n d several other quinones n o t s h o w n . W e (23,24) have e s t a b l i s h e d t h a t t h e 6-lactone 48 was also f o r m e d . W h e n e x p e r i m e n t s were c o n d u c t e d i n t h e presence o f C>2 a n d H 2 0 , regiospecific i n c o r p o r a t i o n i n t o p r o d u c t s 46 a n d 47 was observed ( F i g . 11). T h i s regiospecificity d i d n o t o c c u r d u r i n g 6-lactone 48 f o r m a t i o n , a l t h o u g h t h e reasons for t h i s are n o t clear. [Note also t h a t w h e n v a n i l l y l a l c o h o l w a s used as s u b s t r a t e , t h e m a i n p r o d u c t s were the b i p h e n y l C5-C5 a d d u c t s , together w i t h s m a l l a m o u n t s o f i - l a c t o n e s (25).] 18

1 8

18

1 8

18

1 8

Aromatic Ring Cleavage of Phenolic β-0-4 Substructure Model Compounds by Laccase. W h e n v a n i l l y l a l c o h o l was used as a s u b s t r a t e , o n l y b i p h e n y l f o r m a t i o n ( C 5 - C 5 l i n k e d ) o c c u r r e d a n d n o evidence for t h e f o r m a t i o n o f a n y r i n g - o p e n e d p r o d u c t s w a s o b t a i n e d (26). H e n c e , we also e x a m i n e d t h e effect o f laccase o n t h e s t e r i c a l l y h i n d e r e d 4,6-di-2, a n d not H 0 , w a s i n c o r p o r a t e d i n t o t h i s p r o d u c t ( F i g . 13) (28). 18

2

1 8

Aromatic Ring Cleavage of Artificial (DHP) Lignin by Lignin Peroxidase. A s discussed, o u r p r e v i o u s studies established t h a t t h e a r o m a t i c r i n g s o f n o n p h e n o l i c / ? - 0 - 4 m o d e l c o m p o u n d s u n d e r w e n t o p e n i n g t o give cis,cism u c o n a t e derivatives as t h e i r i n i t i a l p r o d u c t s ( F i g . 9 ) . H o w e v e r , these studies p r o v i d e d n o i n f o r m a t i o n as t o whether higher oligomers, o r i n d e e d , the l i g n i n p o l y m e r itself, w o u l d undergo such reactions. S i n c e i t is generally v i e w e d t h a t coniferous l i g n i n has a l m o s t 4 0 % o f its linkages connected v i a / ? - 0 - 4 bonds, these aforementioned results s u g ­ gest t h a t a l i g n i n p o l y m e r connected p r i m a r i l y v i a such linkages s h o u l d

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

A O

4

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ι

.18, •: 0

Ε ι α

2

3

Ο

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3

3

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o

r

^

2

0

C

Eta.

Z

43

3

9

3

D: H.

H

2

3

OC

T

and/or

R'=H

0

36R=R'=CH CH

3

3SR=CH ,

34R=R'=H

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38 R=CH

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44

0 "OCH, OEt

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41R=CH

40R=H

F i g u r e 8. A r o m a t i c r i n g cleavage o f n o n p h e n o l i c β-0-4 m o d e l c o m p o u n d s 31-33 b y l i g n i n peroxidase.

Of H

2

3

R'=H

J

CH

33R=R'=CH CH

3

32R=CH ,

31R-R'=H

OEl

ROV- Ο

f

C D

3

35.

HIGUCHI

495

Lignin Degradation by Peroxidase & Laccase

EtO-Ht^-v

EtOv

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V*OCH OEl

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43 F i g u r e 9. M e c h a n i s m for the f o r m a t i o n o f a r y l g l y c e r o l cis-cis m u c o n a t e 4 3 b y l i g n i n peroxidase f r o m the n o n p h e n o l i c β-Ο-4 m o d e l c o m p o u n d 4 2 . ο

ο

^ O C H

3

0 47

F i g u r e 10. D e g r a d a t i o n o f v e r a t r y l a l c o h o l 4 5 b y l i g n i n peroxidase.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

496

PLANT C E L L W A L L

POLYMERS

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

35.

HIGUCHI

497

Lignin Degradation by Peroxidase & Laccase

^ i r

OH

Ο

50

MH

^

+

m/z

269

51

AUTHENTIC

D-LABELED

ENZYMIC

COMPOUND

NON-ENZYMIC

AUTHENTIC

DEGRADATION

DEGRADATION

COMPOUND

PRODUCT

PRODUCT

' 4 3

m/z 269 272

V

3.5

Time (min.) F i g u r e 12. M a s s c h r o m a t o g r a m s b u t y l g u a i a c o l 50 b y laccase.

of d e g r a d a t i o n

product

51

of

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

4,6-di-t-

498

PLANT C E L L WALL POLYMERS

F i g u r e 13. P r o p o s e d m e c h a n i s m s for a r o m a t i c r i n g cleavage of 4 , 6 - d i - t b u t y l g u a i a c o l 50 by laccase.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

35.

HIGUCHI

Lignin Dégradation by Peroxidase & Laccase

499

undergo s i m i l a r degradative reactions. L i g n i n is often represented b y v a r ious s y n t h e t i c d e h y d r o g e n a t i v e l y p o l y m e r i z e d ( D H P ) p r e p a r a t i o n s , p r o duced v i a the p e r o x i d a s e / H 2 0 2 i n d u c e d p o l y m e r i z a t i o n o f m o n o l i g n o l ( s ) (29). H o w e v e r , d e p e n d i n g u p o n the p o l y m e r i z a t i o n process e m p l o y e d ( Z u l a u f or Z u t r o p f ) the / ? - 0 - 4 linkage frequency can v a r y f r o m 10-40% (29). C o n s e q u e n t l y , we prepared a s y n t h e t i c D H P l i g n i n p o l y m e r f r o m c o n i f e r y l a l c o h o l v i a the Z u t r o p f m e t h o d , hence m a x i m i z i n g the / ? - 0 - 4 l i n k age frequency. F o l l o w i n g i n c u b a t i o n of t h i s s y n t h e t i c p o l y m e r , w i t h l i g n i n p e r o x i d a s e / H 2 0 2 , the degraded p r o d u c t s were s u b j e c t e d to gel p e r m e a t i o n c h r o m a t o g r a p h y u s i n g Sephadex L H 2 0 . T h e e l u t i o n profile revealed t h a t p a r t i a l d e p o l y m e r i z a t i o n h a d o c c u r r e d (30). G C - M S a n a l y s i s also revealed t h a t a s m a l l a m o u n t of v a n i l l i n was f o r m e d v i a C a - C / ? cleavage. S u r p r i s ingly, no a r o m a t i c r i n g cleavage p r o d u c t s were observed. T h u s another D H P , prepared f r o m a m i x t u r e o f 4 - e t h o x y - 3 - m e t h o x y p h e n y l g l y c e r o l - / ? - s y r i n g a r e s i n i n o l ether 51 a n d c o n i f e r y l a l c o h o l 52 was s y n thesized (31). T h i s p o l y m e r e a d i l y cleavable / ? - 0 - 4 s y r i n g y l linkages. F o l l o w i n g D H P f o r m a t i o n , the p o l y m e r s were e t h y l a t e d w i t h diazoethane. T h e r e s u l t i n g e t h y l a t e d p o l y mer p r o d u c t 53 was t h e n a p p l i e d to a Sephadex L H 2 0 c o l u m n , w i t h D M F as eluent. T h e higher m o l e c u l a r weight f r a c t i o n e l u t e d ( M W > 2200) was t h e n used as a substrate for l i g n i n p e r o x i d a s e / ^ O 2 t r e a t m e n t . F o l l o w i n g t r e a t m e n t w i t h l i g n i n p e r o x i d a s e / H 2 0 2 , the degraded D H P p o l y m e r so o b t a i n e d was a c e t y l a t e d a n d the r e s u l t i n g p r o d u c t s p a r t i a l l y p u r i f i e d b y t h i n layer c h r o m a t o g r a p h y a n d a n a l y z e d b y gas c h r o m a t o g r a p h y - m a s s spectroscopy ( G C - M S ) . F r o m t h i s m i x t u r e , the a r y l g l y c e r o l c y c l i c c a r b o n ates 37, 39, a n d formate 40 were i d e n t i f i e d , together w i t h 4 - e t h o x y - 3 m e t h o x y p h e n y l g l y c e r o l ( F i g . 14). These results therefore established t h a t l i g n i n p e r o x i d a s e / H 2 0 2 catalyzes s i d e - c h a i n cleavage a n d a r o m a t i c r i n g o p e n i n g reactions of e t h y l a t e d D H P l i g n i n p o l y m e r s , i n agreement w i t h the results p r e v i o u s l y o b t a i n e d f r o m the m o d e l s y s t e m s . Conclusions O v e r recent years, considerable progress has been m a d e i n e l u c i d a t i n g the b i o c h e m i c a l processes of l i g n i n f o r m a t i o n (biosynthesis) a n d l i g n i n biodégradation. T a b l e I s u m m a r i z e s , a n d compares, the m a i n reactions i n v o l v e d i n b o t h processes. I n o u r o p i n i o n , the o v e r a l l c h e m i c a l p r i n c i p l e s l e a d i n g to b o t h f o r m a t i o n a n d d e g r a d a t i o n are s i m i l a r . O u r investigations i n d i c a t e t h a t l i g n i n can be degraded i n t o s m a l l e r fragments v i a a r y l r a d i c a l c a t i o n a n d p h e n o x y r a d i c a l i n t e r m e d i a t e s , i n a reaction mediated by only lignin p e r o x i d a s e / ! ^ O 2 and laccase/02- H o w ever, i t has been s h o w n t h a t in vitro some of the l i g n i n is p o l y m e r i z e d , p r o b a b l y v i a c o u p l i n g reaction of p h e n o x y r a d i c a l s of p h e n o l i c moieties o f l i g n i n , d u r i n g t r e a t m e n t w i t h l i g n i n peroxidase (32) or laccase (33) i n aqueous s o l u t i o n . P r o t o l i g n i n , o n the other h a n d , w h i c h is present i n the p l a n t cell walls i n association w i t h polysaccharides, seems t o be degraded s m o o t h l y to lower m o l e c u l a r weight fractions. T h u s , the r e a c t i o n c o n d i t i o n s p r e v e n t i n g r e p o l y m e r i z a t i o n d u r i n g in vivo l i g n i n biodégradation need t o be identified.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

F i g u r e 14. A r o m a t i c r i n g - o p e n i n g reactions of a D H P 53 b y l i g n i n p e r o x idase. ( T h e D H P p o l y m e r 53 was prepared f r o m 4 - e t h o x y - 3 - m e t h o x y p h e nylglycerol-/?-syringaresinol ether 51 a n d c o n i f e r y l a l c o h o l 52.)

35.

HIGUCHI

Lignin Degradation by Peroxidase & Laccase

501

Table I. Comparison of Lignin Biosynthesis and Biodégradation Mechanisms Biodégradation

Biosynthesis Enzymatic Reaction: 1.

Cell wall peroxidase/H202 — • Formation of phenoxy radicals of monolignols

Non-enzymatic Reaction: 1.

Enzymatic Reaction: 1.

Lignin peroxidase/H202 — • Formation of aryl radical cations of nonphenolic units

2.

Laccase/02 — • Formation of phenoxy radicals of phenolic units

Non-enzymatic Reaction:

Coupling of phenoxy to give dimeric quinone methides

(Ca-C/?, alkyl-phenyl) and aromatic rings

2.

Dioxygen radical attack on carbon-centered radicals

2.

Dioxygen radical attack on carbon-centered radical intermediates

3.

Nucleophilic attack on quinone methides by H 2 O and R-OH to give dilignols and higher oligomers

3.

Nucleophilic attack on aryl cations and C a cations by H 0 and R-OH to give degradation products 2

A cknowledgment s This paper was prepared based on our recent work on lignin biodégradation in the Research Section of Lignin Chemistry. The author is indebted to Dr. M. Shimada, Dr. T . Umezawa, Messrs. S. Kawai, T . Habe, S. Yokota and T. Hattori in this section for their cooperation for this investigation. Literature Cited 1. 2. 3. 4. 5. 6.

Kirk, T . K.; Farrell, R. L. Ann. Rev. Microbiol. 1987, 41, 465-505. Higuchi, T . Wood Res. 1986, 73, 58-81. Buswell, J . Α.; Odier, E . CRC Rev. Biotechnol. 1987, 6, 1-60. Tien, M.; Kirk, T . K. Proc. Natl. Acad. Sci. USA 1984, 81, 2280-84. Umezawa, T.; Higuchi, T . FEBS Lett. 1988, 218, 255-60. Kawai, S.; Umezawa, T.; Higuchi, T . Arch. Biochem. Biophys. 1988, 262, 111-17. 7. Kersten, P. J.; Tien, M.; Kalyanarama, B.; Kirk, T . K. J. Biol. Chem. 1985, 260, 2609-12. 8. Kawai, S.; Umezawa, T.; Shimada, M.; Higuchi, T.; Koide, K.; Nishida, T.; Morohoshi, N.; Haraguchi, T . Mokuzai Gakkaishi 1987, 33, 792-97. 9. Higuchi, T . In Biosynthesis and Biodegradation of Wood Components; Higuchi, T . , Ed.; Academic Press: Orlando, F L , 1985; Ch. 7.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT CELL WALL POLYMERS

10. Kuwahara, M.; Glenn, J . K.; Morgan, Μ. Α.; Gold, M . H. FEBS Lett. 1984, 169, 247-50. 11. Hammel, Κ. E.; Tien, M.; Kalyanarama, B.; Kirk, T . K. J. Biol. Chem. 1985, 260, 8348-53. 12. Habe, T.; Shimada, M.; Umezawa, T.; Higuchi, T . Agric. Biol. Chem. 1985, 49, 3505-10. 13. Yokota, S.; Umezawa, T.; Kawai, S.; Higuchi, T . Abst. 38th Mtg. Japan Wood Res. Soc. 1988, Asahikawa, Japan. 14. Umezawa, T.; Higuchi, T . FEBS Lett. 1985, 192, 147-50. 15. Umezawa, T.; Higuchi, T . FEMS Microbiol. Lett. 1985, 26, 123-26. 16. Yokota, S.; Umezawa, T.; Higuchi, T . , unpublished. 17. Higuchi, T . In Biosynthesis and Biodegradation of Wood Components; Higuchi, T., Ed.; Academic Press: Orlando, FL, 1985; Ch. 20. 18. Kawai, S.; Umezawa, T.; Higuchi, T., unpublished. 19. Umezawa, T.; Higuchi 20. Umezawa, T.; Higuchi 21. Leisola, M . S. Α.; Schmidt, B.; Thanei-Wyss, U.; Fiechter, A. FEBS Lett. 1985, 189, 267-90. 22. Leisola, M. S. Α.; Haemmerli, S. D.; Smit, J. D. G.; Troller, J.; Waldner, R.; Schoemaker, Η. E.; Schmidt, H. In Lignin Enzymic and Microbial Degradation, INRA Ed.; INRA: Paris, 1987; pp. 81-86. 23. Shimada, M.; Hattori, T.; Umezawa, T.; Higuchi, T.; Uzura, K. FEBS Lett. 1987, 221, 327-31. 24. Hattori, T.; Shimada, M.; Umezawa, T.; Higuchi, T.; Uzura, K. Agric. Biol. Chem. 1988, 52, 879-80. 25. Hattori, T., et al., unpublished. 26. Kawai, S.; Umezawa, T.; Higuchi, T. Abst. 38th Ann. Mtg. Japan Wood Res. Soc. 1988, Asahikawa, Japan. 27. Gierer, J.; Imsgard, F. Acta Chem. Scand. 1977, 31, 546-50. 28. Kawai, S.; Umezawa, T.; Shimada, M.; Higuchi, T . FEBS Lett. 1988, 236, 309-11. 29. Sarkanen, Κ. V. In Lignins: Occurrence, Formation, Structure and Re­ actions; Sarkanen, Κ. V.; Ludwig, C. H., Eds.; Wiley-Interscience: New York, 1971; Ch. 4. 30. Umezawa, T.; Higuchi, T . Abst. 38th Ann. Mtg. Japan Wood Res. Soc. 1988, Asahikawa, Japan. 31. Umezawa, T.; Higuchi, T . FEBS Lett. 1989, 242, 325-29. 32. Haemmerli, S. D.; Leisola, M . S. Α.; Fiechter, A. FEMS Microbiol. Lett. 1986, 35, 33-36. 33. Ishihara, T.; Miyazaki, M . Mokuzai Gakkaishi 1972, 18, 416-19. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 36

Aromatic Ring Cleavage by Lignin Peroxidase Toshiaki Umezawa and Takayoshi Higuchi Research Section of Lignin Chemistry, Wood Research Institute, Kyoto University, Uji, Kyoto 611, Japan

Aromatic ring cleavages by white-rot fungi and by the enzyme lignin peroxidas of white-rot fung structure model compounds as well as polymeric lignin. Lignin peroxidase of Phanerochaete chrysosponum cat­ alyzes the ring cleavage of β-0-4 lignin substructure model compounds and synthetic lignin (DHP). A mech­ anism for the ring cleavage by the enzyme is described. Lignin biodégradation has been s t u d i e d by two c o m p l e m e n t a r y approaches: (i) d e g r a d a t i o n o f p o l y m e r i c lignins (such as the d e h y d r o g e n a t i o n p o l y m e r of coniferyl a l c o h o l ( D H P ) , m i l l e d w o o d l i g n i n , or w o o d per se) (1); a n d (ii) d e g r a d a t i o n o f l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s (2,3). I n the early 1980's, analysis o f l i g n i n isolated f r o m w o o d decayed b y w h i t e - r o t b a s i d iomycetes h a d p r o v i d e d a general o u t l i n e o f l i g n i n biodégradation. F o r e x a m p l e , cleavage o f side chains a n d a r o m a t i c rings o c c u r r e d d u r i n g degrad a t i o n o f p o l y m e r i c l i g n i n b y the f u n g i (1). L i g n i n is a c o m p l e x a n d heterogeneous p o l y m e r consisting o f p h e n y l propane u n i t s connected v i a m a n y C - C a n d C - O - C linkages. T h u s , the e l u c i d a t i o n o f specific reactions i n v o l v e d i n l i g n i n biodégradation has been p e r f o r m e d m a i n l y w i t h l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s as s u b s t r a t e for f u n g a l d e g r a d a t i o n . B y the early 1980's, s u b s t r u c t u r e m o d e l studies identified m a n y o f the degradative reactions suggested f r o m p o l y m e r i c l i g n i n biodégradation such as C a - C / ? cleavage o f p r o p y l s i d e - c h a i n a n d cleavage of β-0-4 bonds (2,3). O n the other h a n d , a r o m a t i c r i n g cleavage p r o d u c t s o f l i g n i n s u b s t r u c ­ ture models b y w h i t e - r o t basidiomycetes were n o t identified u n t i l 1985, a l t h o u g h earlier studies o f the p o l y m e r i c l i g n i n d e g r a d a t i o n suggested t h e i n v o l v e m e n t o f r i n g cleavage reactions (1). W e identified for t h e first t i m e a r o m a t i c r i n g cleavage p r o d u c t s o f a β-0-4 l i g n i n s u b s t r u c t u r e m o d e l d i m e r

0097-6156/89/0399-0503$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

PLANT C E L L W A L L

504

POLYMERS

p r o d u c e d b y i n t a c t cells of the w h i t e - r o t b a s i d i o m y c e t e , Phanerochaete chrysosporium (4,5). Subsequently, we showed t h a t the a r o m a t i c r i n g cleav­ age of the d i m e r was c a t a l y z e d by a n e x t r a c e l l u l a r e n z y m e of the fungus, l i g n i n peroxidase (ligninase) (6-8), a n d proposed a m e c h a n i s m o f the r i n g cleavage b y the e n z y m e (9). T h e purpose of the present p a p e r is to describe the a r o m a t i c r i n g cleav­ age o f l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y w h i t e - r o t basidiomycetes a n d b y l i g n i n peroxidase of P. chrysosponum. T h e a r o m a t i c r i n g cleavage of s y n t h e t i c l i g n i n ( D H P ) b y the e n z y m e w i l l also be d e s c r i b e d . A r o m a t i c R i n g Cleavage iomycetes

b y Intact

Cells of W h i t e - R o t

Basid­

E a r l i e r studies of fungus-degraded l i g n i n i s o l a t e d f r o m decayed w o o d s u g ­ gested cleavage of a r o m a t i c rings of l i g n i n (1,10-15). However, the p r o d ­ ucts o f a r o m a t i c r i n g cleavag w h i t e - r o t basidiomycetes were not identified u n t i l 1985 (4), w h i l e p r o d ­ ucts of s i d e - c h a i n cleavage of l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y the f u n g i were identified earlier (2,3). D u r i n g t h i s s t u d y , we i d e n t i f i e d for the first t i m e a p r o d u c t o f a r o m a t i c r i n g cleavage o f β-0-4 l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d 1 b y P. chrysosponum, n a m e l y the ^ , γ - c y c l i c c a r b o n a t e of a r y l g l y c e r o l 8 (4) ( F i g . 1). Subsequently, several esters o f a r y l g l y c e r o l were identified as p r o d u c t s of a r o m a t i c r i n g cleavage of β-0-4 l i g n i n s u b s t r u c t u r e m o d e l d i m e r s 2 a n d 3 b y the fungus: a , / ? - c y c l i c c a r b o n a t e of a r y l g l y c e r o l 9, 7 - f o r m a t e of a r y l ­ g l y c e r o l 11, a n d m e t h y l oxalate of a r y l g l y c e r o l 10 ( F i g . 1) (5). C - t r a c e r experiments w i t h l,3-dihydroxy-l-(4-ethoxy-3-methoxyphenyl)-2-[U-ring- C](2-methoxyphenoxy)propane and l,3-dihydroxy-l-(4ethoxy-3-methoxyphenol)-2-[U-ring- C](2,6-dimethoxyphenoxy)propane as substrates confirmed t h a t the p r o d u c t s were r i n g cleavage p r o d u c t s . 1 3

13

13

F o r m a t i o n of these a r o m a t i c r i n g cleavage p r o d u c t s was not l i m i t e d to P. chrysosporium, b u t c o u l d be m e d i a t e d b y other w h i t e - r o t b a s i d ­ iomycetes, Coriolus versicolor (16) a n d Coriolus hirsutus (17). I n the d e g r a d a t i o n of 1, 2 a n d 4 b y C. versicolor, 8, 9, a n d 11 were i d e n t i ­ fied as r i n g cleavage p r o d u c t s . A s for C. hirsutus, 8 a n d 9 were identified as r i n g cleavage p r o d u c t s f r o m d e g r a d a t i o n of 1 ( F i g . 1). A r o m a t i c R i n g C l e a v a g e o f /?-0-4 L i g n i n D i m e r s by L i g n i n Peroxidase

Substructure

Model

L e i s o l a et al. r e p o r t e d the a r o m a t i c r i n g cleavage of a m o n o m e r i c a r o m a t i c c o m p o u n d , v e r a t r y l a l c o h o l , b y l i g n i n peroxidase of P. chrysosporium (18), a l t h o u g h they s t a t e d t h a t the p r o d u c t s were o n l y t e n t a t i v e l y i d e n t i f i e d . W e showed, o n the basis of firm i d e n t i f i c a t i o n of the p r o d u c t s w i t h s y n t h e t i c a u t h e n t i c samples, t h a t l i g n i n peroxidase i s o l a t e d f r o m the fungus b y a m o d i f i c a t i o n of the m e t h o d of T i e n a n d K i r k (19), c a t a l y z e d the a r o m a t i c r i n g cleavage of β-0-4 l i g n i n s u b s t r u c t u r e m o d e l d i m e r s (6-8). A r o m a t i c r i n g cleavage p r o d u c t s formed i n the i n t a c t c u l t u r e of the fungus ( F i g . 1)

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

36.

UMEZAWA & HIGUCHI

R

Aromatic Ring Cleavage by Peroxidase

505

2

OEt ^OCH: OEt R =H

R =H

1

0

2

R =OCH 2

3

R =CHO

R =H

R =CHO

R =OCH

1

1

OCH-

2

2

X/0CH3

3

10

F i g u r e 1. β-0-4 l i g n i n s u b s t r u c t u r e m o d e l d i m e r s 1-4 a n d t h e i r d e g r a d a t i o n b y w h i t e - r o t f u n g i , Phanerochaete chrysosporium, Coriolus versicolor, a n d Coriolus hirsutus. T h e ether b o n d between t h e C/? a n d t h e B - a r o m a t i c nucleus is referred t o as "β-0-4 b o n d " i n l i g n i n c h e m i s t r y .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT CELL WALL POLYMERS

were also p r o d u c e d b y t h e e n z y m e . A r o m a t i c r i n g cleavage p r o d u c t s w i t h the e n z y m e were c y c l i c carbonates o f a r y l g l y c e r o l s 8 a n d 9 , f o r m a t e o f a r y l g l y c e r o l 11, m e t h y l oxalates o f arylglycerols 10 a n d 1 0 - E t , a n d a novel p r o d u c t , muconate o f a r y l g l y c e r o l 1 2 - E t ( F i g . 2 ) . A l l t h e p r o d u c t s were confirmed t o b e a r o m a t i c r i n g cleavage p r o d u c t s b y u s i n g r i n g - C labeled substrates. Subsequently, M i k i et al. r e p o r t e d f o r m a t i o n o f a r o m a t i c r i n g cleavage p r o d u c t s i n v o l v i n g cyclic carbonates s i m i l a r t o 8 a n d 9 i n t h e d e g r a d a t i o n o f a β-0-4 l i g n i n m o d e l c o m p o u n d b y t h e e n z y m e (20). T h e muconate o f a r y l g l y c e r o l 1 2 - E t retains a l l s i x c a r b o n a t o m s o f the B - r i n g o f the substrate 1 - E t . Hence, t h i s p r o d u c t seemed t o be a p p r o ­ p r i a t e t o e x a m i n e m e c h a n i s m s for t h e r i n g cleavage. B a s e d o n t h e results of tracer e x p e r i m e n t s , we proposed a r i n g cleavage m e c h a n i s m for t h e e n ­ z y m e (7,9), as now described. D e g r a d a t i o n o f l , 3 - d i h y d r o x y l - l - ( 4 - e t h o x y 3 - m e t h o x y p h e n y l ) - 2 - ( 2 - [ O C H ] m e t h o x y p h e n o x y ) propane 1 - D b y t h e e n ­ z y m e showed t h a t t h e m e t h y l group o f m e t h y l ester o f oxalate 1 0 - D w a s d e r i v e d f r o m the m e t h o x y (7). T h i s result i n d i c a t e d t h a t d e m e t h y l a t i o n (or d e m e t h o x y l a t i o n ) w h i c h was p r e v i o u s l y p o s t u l a t e d for the r i n g cleavage b y t h e fungus (13) is n o t a prerequisite for the a r o m a t i c r i n g cleavage b y the enzyme, a n d t h a t the r i n g cleavage b y t h e enzyme is completely different f r o m the r i n g cleavage c a t ­ a l y z e d b y c o n v e n t i o n a l dioxygenases (21). β-0-4 l i g n i n m o d e l c o m p o u n d s 2, 2 - M e , 2 - E t a n d 1 - E t were degraded under H 0 or 0 ( F i g . 4 ) , a n d G C - M S analysis o f the products showed t h a t one o f t h e c a r b o n y l oxygen a t o m s o f muconate 1 2 - E t a n d oxalates 10, 1 0 - M e a n d 1 0 - E t was d e r i v e d f r o m H 2 O a n d t h e other f r o m O 2 . A s for cyclic carbonates 8, 8 - M e a n d 9 a n d formates 11 a n d 1 1 - M e , the c a r b o n y l oxygen a t o m s were d e r i v e d f r o m H 2 O ( F i g . 4 ) . B a s e d o n these results, we proposed a m e c h a n i s m for a r o ­ m a t i c r i n g cleavage by the enzyme w h i c h involves single electron o x i d a t i o n of the B - r i n g t o the corresponding c a t i o n r a d i c a l , followed b y n u c l e o p h i l i c a t t a c k o f H 2 O a n d r a d i c a l c o u p l i n g w i t h O2 (or a r a d i c a l species d e r i v e d f r o m 0 ( F i g . 5) (9). 1 3

2

3

2

1 8

1

8

2

2

S i m i l a r m e c h a n i s m s were also proposed for the r i n g cleavage o f v e r a t r y l a l c o h o l (22,23) a n d a β-0-4 l i g n i n s u b s t r u c t u r e m o d e l (24) b y the e n z y m e . A s described i n the previous section, the f o r m a t i o n o f the r i n g cleavage p r o d u c t s 8, 9 a n d 11 f r o m β-0-4 l i g n i n s u b s t r u c t u r e models were also m e d i a t e d b y C. versicolor (16) a n d C. hirsutus (17). It is most l i k e l y t h a t the a r o m a t i c r i n g cleavage b y b o t h f u n g i is m e d i a t e d b y a n e n z y m e s i m i l a r to l i g n i n peroxidase o f P. chrysosporium. Recently, i t w a s s h o w n t h a t C. versicolor produces a n enzyme s i m i l a r t o P. chrysosporium l i g n i n peroxidase (25), a l t h o u g h concrete evidence t h a t the enzyme catalyzes the r i n g cleavage has n o t been s h o w n . A r o m a t i c R i n g Cleavage o f a (/?-0-4)-(/?-0-4) L i g n i n S u b s t r u c t u r e M o d e l T r i m e r b y L i g n i n Peroxidase M e c h a n i s m s w h i c h involve t h e c a t i o n r a d i c a l i n t e r m e d i a t e were also p r o ­ posed for t h e cleavage o f C a - C / ? a n d β-0-4 b o n d s o f β-0-4 l i g n i n s u b s t r u c ­ ture models b y t h e enzyme (26,27). T h u s , m e c h a n i s m s for most o f t h e

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

E

t

o

ï

M

OEt

OCHj

F i g u r e 2. β-0-i l i g n i n s u b s t r u c t u r e m o d e l d i m e r s 1, 2, 1 - E t a n d 2 - E t , a n d their d e g r a d a t i o n b y l i g n i n peroxidase of Phanerochaete chrysosporium.

ο

508

PLANT

CELL

WALL

POLYMERS

F i g u r e 3. M e t h y l g r o u p of m e t h y l oxalate was derived f r o m m e t h o x y l g r o u p of the B - r i n g of β-0-4 l i g n i n m o d e l d i m e r 1-D.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

36.

UMEZAWA & HIGUCHI

Aromatic Ring Cleavage by Peroxidase

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

509

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3

L

3

OCH3

3

R OJ^>0 Λ OCH V^OCHo OEt

^

V-OCH OEl

J

^OCH-> OEl

J ^ C H OEt 3

C

^ OEl

3

V^OCH OEt

H

3

^

3

CH 0

CH3O 9(JH Η,·

3

CH3O f

OE

3

H

,CH 3 O H

V-OCH3 OEt ,

OEt

3

CH 0^

2

3 3

OCHo

C H

Ç^OCH, OEl

J

R =R =H

OCH

οΛτ'

OCH, ° 3 OEl 12-Et

OEt

F i g u r e 5. P r o p o s e d mechanisms for a r o m a t i c r i n g cleavage o f β-0-4 l i g n i n s u b s t r u c t u r e m o d e l dimers b y l i g n i n peroxidases.

CH-.

•• 1 - E t

: 2

•• 1

10

10-Ne

10-Et

("5

36.

UMEZAWA & HIGUCHI

Aromatic Ring Cleavage by Peroxidase

511

d e g r a d a t i v e reactions of β-0-4 l i g n i n s u b s t r u c t u r e models b y the e n z y m e can be e x p l a i n e d o n the basis o f single electron o x i d a t i o n of a r o m a t i c r i n g s . O n the other h a n d , a r o m a t i c r i n g cleavage o f p o l y m e r i c l i g n i n by the e n z y m e has not been r e p o r t e d . T h e f o r m a t i o n o f B - r i n g cleavage p r o d u c t s of β-0-4 l i g n i n s u b s t r u c t u r e models b y the e n z y m e ( F i g . 2) was e x p e c t e d t o be a n i n d i c a t o r of a r o m a t i c r i n g cleavage i n p o l y m e r i c l i g n i n b y the e n z y m e . However, the β-0-4 l i g n i n m o d e l c o m p o u n d s used i n the m o d e l studies have no p r o p y l s i d e - c h a i n o n the B - r i n g as s h o w n i n F i g u r e s 1 a n d 2, w h i l e the B - r i n g s present i n p o l y m e r i c l i g n i n have p r o p y l s i d e - c h a i n s . F u r t h e r m o r e , p r e v i o u s i n v e s t i g a t i o n s showed t h a t the a r o m a t i c s u b s t i t u e n t s s u c h as f o r m y l or m e t h o x y l groups influenced d r a s t i c a l l y the d e g r a d a b i l i t y of the β-0-4 l i g n i n s u b s t r u c t u r e models b y the e n z y m e (6,26,27). H e n c e , we e x a m i n e d the effect of the p r o p y l s i d e - c h a i n of the B - r i n g o n the B - r i n g cleavage b y the e n z y m e u s i n g a l i g n i n s u b s t r u c t u r e m o d e l t r i m e r c o m p o s e d of two β-0-4 s u b s t r u c t u r e s 5 ( F i g . 6) (28). Identified d e g r a d a t i o n p r o d u c t s b y the e n z y m e wer f o r m a t e of a r y l g l y c e r o l as B - r i n g cleavage p r o d u c t s 8 , 9 a n d 11; a r y l g l y c e r o l 13 a n d i t s α - c a r b o n y l d e r i v a t i v e 14 as cleavage p r o d u c t s of the β-0-4 b o n d between A - a n d B - r i n g s ; 4 - e t h o x y - 3 - m e t h o x y b e n z a l d e h y d e 15 as a C a - C / ? cleavage p r o d u c t between A - a n d B - r i n g s ; a n d l , 3 - d i h y d r o x y - l - ( 4 - e t h o x y 3 - m e t h o x y p h e n y l ) - 2 - ( 4 - f o r m y l - 2 - m e t h o x y p h e n o x y ) p r o p a n e 3 as a p r o d u c t of C a - C / ? cleavage between B - a n d C - r i n g s . T h e results showed t h a t the p r o p y l s i d e - c h a i n o f the B - r i n g of 5 d i d not influence, at least q u a l i t a t i v e l y , the a r o m a t i c r i n g cleavage b y the e n z y m e . T h e results suggested t h a t p o l y m e r i c l i g n i n is degraded t h r o u g h the cleavage r e a c t i o n of a r o m a t i c rings by the e n z y m e . I n fact, a r o m a t i c r i n g cleavage o f D H P ( s y n t h e t i c l i g n i n ) b y the e n z y m e has now been p r o v e n as discussed i n the f o l l o w i n g s e c t i o n . A r o m a t i c R i n g Cleavage of D H P by L i g n i n Peroxidase R e a c t i o n m e c h a n i s m s for d e g r a d a t i o n of β-0-4 l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y l i g n i n peroxidase are b e i n g clarified r a p i d l y , w h i l e the ac­ t i o n of the e n z y m e o n p o l y m e r i c l i g n i n is not f u l l y e l u c i d a t e d . T i e n a n d K i r k (29) r e p o r t e d d e g r a d a t i o n of m e t h y l a t e d spruce l i g n i n b y the e n z y m e . T h e y showed f o r m a t i o n of low m o l e c u l a r weight d e g r a d a t i o n p r o d u c t s o n the basis of gel f i l t r a t i o n w i t h Sephadex L H - 2 0 , a n d detected a C a - C / ? cleavage p r o d u c t b y a n isotope t r a p p i n g m e t h o d (29). O n the other h a n d , a c c o r d i n g to H a e m m e r l i et α/., w h e n p o l y m e r i c l i g n i n , p h e n o l i c h y d r o x y l groups of w h i c h are not a l k y l a t e d , were t r e a t e d b y the e n z y m e , p o l y m e r ­ i z a t i o n o f the l i g n i n o c c u r r e d o n the basis o f gel filtration a n a l y s i s o f the d e g r a d a t i o n p r o d u c t s (30). T h e y m o n i t o r e d the c h r o m a t o g r a p h i c profile b y a U V - d e t e c t o r . T h e results were confirmed b y O d i e r et ai (31) b y u s i n g C - l a b e l l e d D H P as s u b s t r a t e . T h u s , for the d e g r a d a t i o n of p o l y m e r i c l i g n i n by the e n z y m e , t w o m a ­ j o r questions were left: (i) C a n l i g n i n peroxidase, b y itself, d e p o l y m e r i z e p o l y m e r i c l i g n i n w i t h o u t r e p o l y m e r i z a t i o n or n o t ? (ii) C a n l i g n i n p e r o x ­ idase cleave a r o m a t i c rings a n d β-0-4 b o n d s o f p o l y m e r i c l i g n i n , or n o t ? 1 4

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989. 15

OCH-3

OEt

HO

OEt J

Ca-C0

Ring

11

cleavage

cleavage

cleavage

^OCH

3

OCHO

H O ^ O H

Γ

F i g u r e 6. (/?-0-4)-(/?-0-4) l i g n i n s u b s t r u c t u r e m o d e l t r i m e r 6 a n d i t s d e g r a ­ d a t i o n p r o d u c t s b y l i g n i n peroxidase.

OEl

è

13

OCHi

CHO

OEt

OEt

Ο

36.

UMEZAWA & HIGUCHI

Aromatic Ring Cleavage by Peroxidase

513

Since we have been i n v e s t i g a t i n g the a r o m a t i c r i n g cleavage of β-0-4 l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y i n t a c t cells o f w h i t e - r o t f u n g i a n d l i g n i n peroxidase of P. chrysosporium as described above, we e x a m i n e d the a r o ­ m a t i c r i n g cleavage a n d β-0-4 b o n d cleavage o f p o l y m e r i c l i g n i n b y l i g n i n peroxidase. W e s y n t h e s i z e d a c o p o l y m e r o f (β-0-4)-(β-β) lignin substructure model t r i m e r 6 a n d c o n i f e r y l a l c o h o l 7, w h i c h w i l l be referred t o as (β-0-4)-(β-β)D H P i n t h i s review ( F i g . 7). T h e (β-0-4)-(β-β)-ΌΕΡ was e t h y l a t e d w i t h d i a z o e t h a n e a n d the e t h y l a t i o n p r o d u c t 16 was s u b m i t t e d to gel filtration ( L H - 2 0 / D M F ) to remove low m o l e c u l a r weight f r a c t i o n s . T h e h i g h m o l e c ­ u l a r weight f r a c t i o n o f the e t h y l a t e d (β-0-4)-(β-β)-ΌΕΡ 16 t h u s o b t a i n e d ( m o l e c u l a r weight > 2200, c a l i b r a t e d w i t h p o l y s t y r e n e , F i g . 8) was de­ g r a d e d b y l i g n i n peroxidase. T h e d e g r a d a t i o n p r o d u c t s were e x t r a c t e d w i t h e t h y l acetate, a c e t y l a t e d a n d p a r t i a l l y p u r i f i e d by t h i n - l a y e r c h r o m a t o g r a ­ p h y ( T L C ) . G a s c h r o m a t o g r a p h y mass spectroscopic ( G C - M S ) a n a l y s i s o f the T L C p u r i f i e d f r a c t i o a r y l g l y c e r o l 8 a n d 9, f o r m a t e o f a r y l g l y c e r o l 11, a r y l g l y c e r o l 13, a n d o> c a r b o n y l - a r y l g l y c e r o l 14 ( F i g . 9) (32). Since the c o m p o u n d s were p r e v i ­ o u s l y i d e n t i f i e d as a r o m a t i c r i n g cleavage p r o d u c t s a n d β-0-4 b o n d c l e a v ­ age p r o d u c t s o f β-0-4 l i g n i n s u b s t r u c t u r e m o d e l c o m p o u n d s b y the e n z y m e ( F i g s . 2 a n d 6), the results show t h a t l i g n i n peroxidase cleaves a r o m a t i c rings a n d β-0-4 bonds of the s y n t h e t i c l i g n i n ( D H P ) . T h u s , m o s t of the d e g r a d a t i v e reactions o f p o l y m e r i c l i g n i n s suggested p r e v i o u s l y b y the a n a l y s i s o f decayed l i g n i n i s o l a t e d f r o m decayed w o o d b y w h i t e - r o t f u n g i were c a t a l y z e d by l i g n i n peroxidase. A r o m a t i c R i n g Cleavage of M o n o m e r i c A r o m a t i c C o m p o u n d s by W h i t e - R o t Basidiomycetes M o n o m e r i c a r o m a t i c c o m p o u n d s s u c h as v a n i l l i c a c i d a n d s y r i n g i c a c i d are k n o w n t o be p r o d u c e d i n the d e g r a d a t i o n of p o l y m e r i c l i g n i n b y w h i t e - r o t basidiomycetes (13,14,33). It is s t i l l u n c e r t a i n to w h a t extent a r o m a t i c r i n g s o f p o l y m e r i c l i g n i n are cleaved b y l i g n i n peroxidase, as opposed to b e i n g degraded v i a s i d e c h a i n cleavages to produce the m o n o m e r i c C 6 - C 1 i n t e r m e d i a t e s s u c h as v a n i l l i c a c i d a n d s y r i n g i c a c i d . F u r t h e r m o r e , the fate o f the m o n o m e r i c d e g r a d a t i o n i n t e r m e d i a t e s is not f u l l y e l u c i d a t e d . A n d e r et ai (34) p r o p o s e d a d e g r a d a t i o n p a t h w a y o f v a n i l l i c a c i d v i a 1,2,4t r i h y d r o x y b e n z e n e b y i n t a c t cells o f Sporotrichum pulverulentum ( = P. chrysosporium). T h i s p a t h w a y involves d e c a r b o x y l a t i o n o f v a n i l l i c a c i d c a t ­ a l y z e d b y v a n i l l a t e h y d r o x y l a s e to p r o d u c e m e t h o x y h y d r o q u i n o n e (35,36), followed b y d e m e t h y l a t i o n a n d subsequent a r o m a t i c r i n g cleavage o f the d e m e t h y l a t e d p r o d u c t , 1,2,4-trihydroxybenzene, c a t a l y z e d b y a d i o x y g e nase o f the fungus (37). O n the other h a n d , the role o f l i g n i n peroxidase i n the m e t a b o l i s m o f these a r o m a t i c m o n o m e r s r e m a i n s u n c e r t a i n , a l t h o u g h a n o n - p h e n o l i c a r o m a t i c m o n o m e r , v e r a t r y l a l c o h o l w h i c h is s y n t h e s i z e d de novo b y P. chrysosporium, is o x i d i z e d b y l i g n i n peroxidase to p r o d u c e m a i n l y v e r a t r a l d e h y d e a n d trace a m o u n t s of a r o m a t i c r i n g cleavage p r o d ­ ucts (18,22).

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989. >

3

CH CHN 2

OEt

J-LH-20/DMF

2

High molecular weight fraction

2

ÇHOH 0CH3 ÇH20H CHOH C H — 0 — C H ÇH-0-^p-CH-OH CHO-i ÇHOHOCH3 (^LocH3

F i g u r e 7. P r e p a r a t i o n of s y n t h e t i c l i g n i n 1 6 , e t h y l a t e d c o p o l y m e r o f c o n i f e r y l alcohol 7 a n d a r y l g l y c e r o l - / ? - s y r i n g a r e s i n o l ether 6. In 1 6 , the r e c t a n g u l a r enclosure represents a n assumed s t r u c t u r e of the m o i e t y der i v e d f r o m 7.

OCH.

v

Horseradish peroxidase

3

CH 0^ 2

CH OH -ÇH ÇHOH

36.

UMEZAWA & HIGUCHI

Aromatic Ring Cleavage by Peroxidase

515

Void

-J

i*2

1

1

1

35

28

21

Ml—ι

ία

0

Elution volume (ml) F i g u r e 8. G e l f i l t r a t i o n o f e t h y l a t e d (/?-0-4)-(/?-/?)-DHP 16. S o l i d l i n e : E t h y ­ l a t e d (/?-0-4)-(/?-/?)-DHP 16 after removal o f low m o l e c u l a r weight f r a c t i o n s . T h e c o l u m n w a s c a l i b r a t e d w i t h (/?-0-4)-(/?-/?) l i g n i n s u b s t r u c t u r e m o d e l t r i m e r 6 ( m o l e c u l a r weight 6 4 2 ) ; /3-0-4 l i g n i n m o d e l d i m e r 1 ( m o l e c u l a r weight 348) a n d polystyrenes o f m o l e c u l a r weight 9000, 4000 ( v o i d ) , 2200 ( i n d i c a t e d b y A ) . C o l u m n : Sephadex L H - 2 0 , 1.1 x 48 c m . E l u e n t : D M F , 13.5-14.4 m l / h r . D e t e c t o r : R e f r a c t i v e i n d e x detector R I - 2 ( J a p a n A n a l y t i ­ cal I n d u s t r y C o . , L t d . ) .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

F i g u r e 9. D e g r a d a t i o n p r o d u c t s of s y n t h e t i c l i g n i n ( D H P ) 1 6 b y l i g n i n peroxidase. 8 , 9 a n d 1 1 : A r o m a t i c r i n g cleavage p r o d u c t s ; 1 3 a n d 1 4 : /?0-4 b o n d cleavage p r o d u c t s .

36. UMEZAWA & HIGUCHI

Aromatic Ring Cleavage by Peroxidase

517

Literature Cited 1. Chen, C.-L.; Chang, H.-m. In Biosynthesis and Biodegradation of Wood Components; Higuchi, T., Ed.; Academic: Orlando, FL, 1985; pp. 53556. 2. Higuchi, T . In Biosynthesis and Biodegradation of Wood Components; Higuchi, T., Ed.; Academic: Orlando, F L , 1985; pp. 557-78. 3. Higuchi, T . Wood Res. 1986, 73, 58. 4. Umezawa, T.; Higuchi, T . FEBS Lett. 1985, 182, 257. 5. Umezawa, T.; Kawai, S.; Yokota, S.; Higuchi, T . Wood Res. 1986, 73, 8. 6. Umezawa, T.; Shimada, M.; Higuchi, T.; Kusai, K. FEBS Lett. 1986, 205, 287. 7. Umezawa, T.; Higuchi, T . FEBS Lett. 1986, 205, 293. 8. Umezawa, T.; Higuchi, T . Agric. Biol. Chem. 1987, 51, 2281. 9. Umezawa, T.; Higuchi 10. Kirk, T . K.; Chang, 11. von Ellwardt, P.-C.; Haider, K.; Ernst, L. Holzforschung 1981, 35, 103. 12. Chua, M . G. S.; Chen, C.-L.; Chang, H.-m.; Kirk, T . K. Holzforschung 1982, 36, 165. 13. Chen, C.-L.; Chang, H.-m.; Kirk, T . K. J. Wood Chem. Technol. 1983, 3, 35. 14. Tai, D.; Terazawa, M.; Chen, C.-L.; Chang, H.-m.; Kirk, T . K. In Re­ cent Advances in Lignin BiodegradationResearch;Higuchi, T.; Chang, H.-m.; Kirk, T . K., Eds.; Uni Publishers: Tokyo, 1983; pp. 44-63. 15. Haider, K.; Kern, H. W.; Ernst, L. Holzforschung 1985, 39. 16. Kawai, S.; Umezawa, T.; Higuchi, T . Appl. Environ. Microbiol. 1985, 50, 1505. 17. Yoshihara, K.; Umezawa, T.; Higuchi, T.; Nishiyama, M. Agric. Biol. Chem. 1988, 52, 2345. 18. Leisola, M . S. Α.; Schmidt, B.; Thanei-Wyss, U.; Fiechter, A. FEBS Lett. 1985, 189, 267. 19. Tien, M.; Kirk, T . K. Proc. Natl. Acad. Sci. USA 1984, 81, 2280. 20. Miki, K.; Renganathan, V.; Mayfield, M . B.; Gold, M . H. FEBS Lett. 1987, 210, 199. 21. Cain, R. B. In Lignin Biodegradation: Microbiology, Chemistry, and Potential Applications; Kirk, T . K.; Higuchi, T.; Chang, H.-m., Eds.; CRC Press: Boca Raton, FL, 1980; Vol. 1, pp. 21-60. 22. Shimada, M.; Hattori, T.; Umezawa, T.; Higuchi, T.; Uzura, K. FEBS Lett. 1987, 221, 327. 23. Haemmerli, S. D.; Schoemaker, H. E.; Schmidt, H. W. H.; Leisola, M . S. A. FEBS Lett. 1987, 220, 149. 24. Miki, K.; Kondo, R.; Renganathan, V.; Mayfield, M . B.; Gold, M . H. Biochem. 1988, 27, 4787. 25. Dodson, P. J.; Evans, C. S.; Harvey, P. J.; Palmer, J . M . FEMS Mi­ crobiol. Lett. 1987, 42, 17. 26. Kirk, T . K.; Tien, M.; Kersten, P. J.; Mozuch, M . D.; Kalyanaraman, B. Biochem. J. 1986, 236, 279.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

518 27. 28. 29. 30. 31.

32. 33. 34. 35. 36. 37.

PLANT CELL WALL POLYMERS Miki, K.; Renganathan, V.; Gold, M . H. Biochem. 1986, 25, 4790. Umezawa, T.; Higuchi, T . Mokuzai Gakkaishi 1988, 34, 929. Tien, M.; Kirk, T . K. Science 1983, 221, 661. Haemmerli, S. D.; Leisola, M . S. Α.; Fiechter, A. FEMS Microbiol. Lett. 1986, 35, 33. Odier, E.; Mozuch, M.; Kalyanaraman, B.; Kirk, T . K. In Lignin En­ zymic and Microbial Degradation; Odier, E . , Ed.; INRA: Paris, 1987; p. 131. Umezawa, T.; Higuchi, T . FEBS Lett. 1989, 242, 325. Chen, C.-L.; Chang, H.-m.; Kirk, T . K. Holzforschung 1982, 36, 3. Ander, P.; Eriksson, K.-E.; Yu, H.-s. Arch. Microbiol. 1983, 136, 1. Buswell, J. Α.; Ander, P.; Pettersson, B.; Eriksson, K . - E . FEBS Lett. 1979, 103, 98. Yajima, Y.; Enoki, Α.; Mayfield, M . B.; Gold, M . H. Arch. Microbiol. 1979, 123, 319. Buswell, J. Α.; Eriksson

RECEIVED March 10,1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 37

Biomimetic Studies in Lignin Degradation Futong Cui and David Dolphin Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia V6T 1Y6, Canada

A redox stable, water-soluble iron porphyrin has been used as a mode compounds catalyze found to be dependent on pH and the solvent being used. Model studies showed that veratryl alcohol could mediate the oxidation of a polymeric lignin model compound under certain conditions but could not mediate the oxidation of small molecule model compounds. L i g n i n is the second most a b u n d a n t renewable o r g a n i c c o m p o u n d o n e a r t h . It composes about 15-25% o f the l a n d - p r o d u c e d biomass. A considerable effort has been m a d e t o u n d e r s t a n d l i g n i n biodégradation d u r i n g the last 20 or 30 years, a n d since the discovery o f l i g n i n d e g r a d i n g enzymes (ligninases) i n 1983 (1,2), progress has been m a d e i n u n d e r s t a n d i n g the m e c h a n i s m s o f l i g n i n biodégradation. M o d e l e n z y m e studies are i m p o r t a n t for b o t h m e c h a n i s t i c e l u c i d a t i o n a n d for p r a c t i c a l a p p l i c a t i o n s . T h i s is p a r t i c u l a r l y true for t h e non-specific l i g n i n d e g r a d i n g enzymes. L i g n i n a s e is synthesized b y P. chrysosponum o n l y under c e r t a i n phases o f g r o w t h a n d is difficult t o o b t a i n i n q u a n t i t y . It is a powerful o x i d a n t w h i c h i n i t i a t e s l i g n i n d e g r a d a t i o n b y one electron o x i d a t i o n s , a n d a m a j o r role o f the ligninase p r o t e i n is t o s t e r i c a l l y p r o t e c t the o x i d i z e d heme prosthetic g r o u p . It seems u n l i k e l y t h a t ligninase w i l l c o n t a i n a n " a c t i v e s i t e " to specifically b i n d the diverse s t r u c t u r a l components of l i g n i n . Instead, electron transfer (either direct or m e d i a t e d ) w i l l take place between t h e enzyme a n d p o l y m e r i c l i g n i n . A s t e r i c a l l y protected, water-soluble s y n t h e t i c i r o n p o r p h y r i n c o u l d p r o v i d e a r e a d i l y available b i o m i m e t i c c a t a l y s t for b o t h basic research a n d p o t e n t i a l i n d u s t r i a l a p p l i c a t i o n s . S u c h a s y n t h e t i c h e m i n m i g h t be s u p e r i o r to t h e e n z y m e , i n t h a t b e i n g a s m a l l molecule i t c o u l d interact, w i t h the p o l y m e r i c l i g n i n molecule more r e a d i l y t h a n c a n ligninase. S h i m a d a et al. (3) first f o u n d t h a t a s y n t h e t i c p o r p h y r i n i r o n mesot e t r a p h e n y l p o r p h y r i n ( F e T P P (I)) c a t a l y z e d C - C b o n d cleavage o f β-l type 0097-6156/89/0399-0519$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

520

PLANT C E L L W A L L P O L Y M E R S

l i g n i n m o d e l c o m p o u n d s , a n d further e x p e r i m e n t s (4) showed t h a t the re­ a c t i o n h a d an o p t i m a l p H of 3.0 a n d was s t i m u l a t e d b y the presence of i m ­ idazole. Isotope l a b e l i n g experiments (5) showed t h a t the i r o n p o r p h y r i n c a t a l y z e d reactions followed the same m e c h a n i s t i c routes as those of the ligninase c a t a l y z e d reactions (6). P r o t o h e m i n ( I I ) was also r e p o r t e d to be able to c a t a l y z e r i n g cleavage reactions of v e r a t r y l a l c o h o l (7). Shi­ m a d a ei al (8) recently r e p o r t e d t h a t p r o t o h e m i n m i m i c k e d ligninase i n most cases i n d e g r a d i n g β-l, β-0-4, a n d β-b m o d e l c o m p o u n d s . T h e s i m ­ ple i r o n p o r p h y r i n s (1,11), however, suffer a great disadvantage i n t h a t they are r a p i d l y destroyed b y o x i d a n t s . S t e r i c a l l y p r o t e c t e d w a t e r - s o l u b l e m e t a l l o p o r p h y r i n s have been used i n our l a b o r a t o r y as ligninase m i m i c s a n d we discuss here some observations o n our most p r o m i s i n g c a t a l y s t i r o n m e s o - t e t r a - ( 2 , 6 - d i c h l o r o - 3 - s u l f o n a t o p h e n y l ) p o r p h y r i n chloride ( T D C S P P F e C l (III)). Experimental T h e o x i d a t i o n of v e r a t r y l a l c o h o l was carried out under air at r o o m t e m p e r ­ a t u r e . T h e r e a c t i o n m i x t u r e contained 30 //moles of v e r a t r y l a l c o h o l , 0.05 //moles of T D C S P P F e C l , 30 //moles of ???-chloroperbenzoic acid ( ? ? i C P B A ) m a d e u p to 6 m l u s i n g phosphate buffer or as otherwise i n d i c a t e d i n T a b l e I. 4-methoxyacetophenone (30 /imoles) was added as a n i n t e r n a l s t a n d a r d . T h e r e a c t i o n was s t o p p e d after 2 hours by p a r t i t i o n i n g the m i x t u r e be­ tween m e t h y l e n e chloride a n d s a t u r a t e d s o d i u m b i c a r b o n a t e s o l u t i o n . T h e aqueous layer was t w i c e e x t r a c t e d w i t h m e t h y l e n e c h l o r i d e a n d the ex­ t r a c t s c o m b i n e d . T h e p r o d u c t s were a n a l y z e d b y G C after a c e t y l a t i o n w i t h excess 1:1 acetic a n h y d r i d e / p y r i d i n e for 24 hours at r o o m t e m p e r a t u r e . T h e o x i d a t i o n s of a n i s y l a l c o h o l , i n the presence of v e r a t r y l a l c o h o l or 1,4d i m e t h o x y b e n z e n e , were p e r f o r m e d as i n d i c a t e d i n T a b l e III a n d I V i n 6 m l of phosphate buffer ( p H 3.0). O t h e r c o n d i t i o n s were the same as for the o x i d a t i o n of v e r a t r y l a l c o h o l described above. T D C S P P F e C l r e m a i n ­ i n g after the reaction was e s t i m a t e d f r o m its Soret b a n d a b s o r p t i o n before a n d after the r e a c t i o n . F o r the d e c o l o r i z a t i o n of P o l y B-411 ( I V ) by T D C ­ S P P F e C l a n d m C P B A , 25 //moles of m C P B A were added to 25 m l 0.05% P o l y B - 4 1 1 c o n t a i n i n g 0.01 //moles T D C S P P F e C l , 25 //moles of manganese sulfate a n d 1.5 mmoles of l a c t i c a c i d buffered at p H 4.5. T h e d e c o l o r i z a t i o n of P o l y B - 4 1 1 was followed b y the decrease i n a b s o r p t i o n at 596 n m . F o r the e l e c t r o c h e m i c a l d e c o l o r i z a t i o n of P o l y B-411 i n the presence of v e r a t r y l a l c o h o l , a t w o - c o m p a r t m e n t cell was used. A glassy c a r b o n p l a t e was used as the anode, a p l a t i n u m p l a t e as the a u x i l i a r y electrode, a n d a silver wire as the reference electrode. T h e p o t e n t i a l was controlled at 0.900 V . P o l y B - 4 1 1 (50 m l , 0.005%) i n p H 3 buffer was added to the anode c o m p a r t m e n t a n d p H 3 buffer was added to the cathode c o m p a r t m e n t to the same level. T h e d e c o l o r i z a t i o n of P o l y B-411 was followed by the change i n absorbance at 596 n m a n d the s i m u l t a n e o u s o x i d a t i o n of v e r a t r y l alcohol was followed at 310 n m . T h e same electrochemical a p p a r a t u s was used for the decol­ o r i z a t i o n of P o l y B-411 adsorbed o n t o filter p a p e r . T e t r a b u t y l a m m o n i u m p e r c h l o r a t e ( T B A P ) was used as s u p p o r t i n g electrolyte when methylene chloride was the solvent.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L P O L Y M E R S

Results S h i m a d a et ai have c a r r i e d out most o f t h e i r studies o n m o d e l ligninases i n o r g a n i c solvents (3-5,8). However, reactions i n o r g a n i c a n d aqueous so­ l u t i o n s m a y take different routes a n d D o r d i c k et ai (9) have r e p o r t e d t h a t horseradish peroxidase a n d other peroxidases are able t o d e p o l y m e r i z e b o t h n a t u r a l a n d s y n t h e t i c l i g n i n s i n organic solvent b u t are u n a b l e t o do so i n aqueous s o l u t i o n , a l t h o u g h these results have recently been reassessed (10). W e have s t u d i e d the solvent effect for T D C S P P F e C l c a t a l y z e d reactions u s i n g the s i m p l e s t l i g n i n m o d e l c o m p o u n d , v e r a t r y l a l c o h o l . T a b l e I shows t h a t under the same concentrations o f o x i d a n t a n d c a t a l y s t the y i e l d o f v e r a t r a l d e h y d e was higher i n aqueous s o l u t i o n t h a n i n o r g a n i c solvent. It was i n t e r e s t i n g t o note t h a t a n a d d i t i o n a l p r o d u c t was f o r m e d at a b o u t the same y i e l d as v e r a t r a l d e h y d e w h e n m e t h a n o l was the solvent. A l t h o u g h the s t r u c t u r e o f t h i s c o m p o u n d has not yet been e l u c i d a t e d , t h i s result suggests t h a t solvent can also change the p r o d u c t d i s t r i b u t i o n . T h i s was also d r a m a t i c a l l y d e m o n s t r a t e age is one of the most a b u n d a n t s u b s t r u c t u r e s of l i g n i n . I n spruce l i g n i n the β-Ο-4 b o n d was e s t i m a t e d t o comprise 4 8 % o f the linkages connect­ i n g the p h e n y l p r o p a n o i d u n i t s (11). W h i l e F e T P P (I) (8) cannot degrade 4 - 0 - e t h y l g u a i a c y l g l y c e r o l - / ? - g u a i a c y l ether i n o r g a n i c solvents b y the same p a t h w a y as the e n z y m i c reactions (12), our i n i t i a l results show t h a t T D C ­ S P P F e C l degraded t h i s c o m p o u n d i n water to give, a m o n g other p r o d u c t s , 4 - e t h o x y - 3 - m e t h o x y b e n z a l d e h y d e a n d g u a i a c o l ( F i g . 1). T a b l e I. O x i d a t i o n o f v e r a t r y l a l c o h o l b y T D C S P P F e C l a n d m C P B A i n various solvents Solvent 6 m l p H 2 phosphate buffer 6 ml D M F 6 m l methanol α

b

% Y i e l d of V e r a t r a l d e h y d e 49.6 12.0 11.0

0

6

Based on veratryl alcohol. A second p r o d u c t was o b t a i n e d .

T h e o p t i m a l p H for T D C S P P F e C l c a t a l y s i s was e x a m i n e d for the p r o ­ d u c t i o n o f v e r a t r a l d e h y d e f r o m v e r a t r y l a l c o h o l . T h e y i e l d was highest at the lowest p H used ( T a b l e II). It can also be seen f r o m T a b l e II t h a t T D C ­ S P P F e C l was f a i r l y stable over a w i d e range o f p H . T h e p H o f the m e d i u m is c r u c i a l for the n a t u r a l d e g r a d a t i o n o f l i g n i n . C a t i o n r a d i c a l s are i n v o l v e d i n b o t h the biosynthesis a n d biodégradation o f l i g n i n . T h e biosynthesis occurs at a r o u n d p H 7 w h i c h favors r a d i c a l c o u p l i n g o f the neutral p h e n o late r a d i c a l w h i l e biodégradation occurs under a c i d i c c o n d i t i o n w h i c h favors c a t i o n r a d i c a l i n d u c e d C - C b o n d cleavage. A l t h o u g h no a t t e m p t was m a d e to o p t i m i z e the t u r n o v e r o f the c a t a l y s t , T a b l e II shows t h a t T D C S P P F e C l was stable under the extreme

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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o x i d i z i n g c o n d i t i o n s , i.e., w i t h a c a t a l y s t to o x i d a n t r a t i o of 1:600. O t h e r s i m p l e h e m i n s are r a p i d l y degraded under these c o n d i t i o n s . T a b l e I I . O x i d a t i o n of v e r a t r y l a l c o h o l b y T D C S P P F e C l a n d m C P B A i n aqueous s o l u t i o n as a f u n c t i o n of p H

a

PH

% Yield of Veratraldehyde"

% T D C S P P F e C l Left After Reaction

2 4 6 8 10

49.6 11.4 5.8 3.7 9.9

98 98 97 74 96

Y i e l d based o n v e r a t r y l a l c o h o l .

P a l m e r et ai (13) p r o p o s e d t h a t ligninase degrades l i g n i n b y single electron o x i d a t i o n s a n d t h a t the c a t i o n r a d i c a l s of s m a l l a r o m a t i c molecules c o u l d serve as diffusible redox m e d i a t o r s d u r i n g l i g n i n d e g r a d a t i o n . V e r a t r y l a l c o h o l was f o u n d to s t i m u l a t e the o x i d a t i o n of a n i s y l c o m p o u n d s (14) by ligninase a n d H a r v e y et ai suggested t h a t the c a t i o n r a d i c a l of v e r a t r y l a l c o h o l f u n c t i o n e d as a redox m e d i a t o r i n t h i s r e a c t i o n . Since the c a t i o n r a d i c a l of v e r a t r y l a l c o h o l has not so far been detected (15), the m e d i a t i n g role o f v e r a t r y l a l c o h o l is s t i l l a n open q u e s t i o n . T h e o x i d a t i o n of a n i s y l a l c o h o l by T D C S P P F e C l a n d m C P B A was c a r r i e d o u t i n the presence of v a r y i n g a m o u n t s of v e r a t r y l a l c o h o l . T h e results i n T a b l e III show t h a t the presence of v e r a t r y l a l c o h o l i n h i b i t e d the o x i d a t i o n of a n i s y l a l c o h o l . These observations are r e a d i l y r a t i o n a l i z e d o n the basis of the o x i d a t i o n p o t e n t i a l s of the two c o m p o u n d s . V e r a t r y l a l c o h o l , w h i c h has a lower o x i d a t i o n p o t e n t i a l t h a n a n i s y l a l c o h o l , (1.52 V vs. 1.76 V as d e t e r m i n e d by c y c l i c v o l t a m e t r y i n a c e t o n i t r i l e ) , is m o r e easily o x i d i z e d t h u s decreasing the y i e l d of a n i s y l a l c o h o l b y c o m p e t i n g w i t h i t for the o x i d a n t ; the same w i l l be true i n the n a t u r a l systems. T h e m e d i a t i n g role of a w e l l k n o w n single-electron transfer agent, 1,4-dimethoxybenzene, was tested for c o m p a r i s o n . It can be seen f r o m T a b l e I V t h a t 1,4-dimethoxybenzene c a n n o t s t i m u l a t e the o x i d a t i o n of a n i s y l a l c o h o l either. T h e r e f o r e , the i n creased y i e l d of a n i s y l a l c o h o l o x i d a t i o n by ligninase c a n n o t be a t t r i b u t e d d i r e c t l y to the m e d i a t i n g role of v e r a t r y l a l c o h o l . However the recent w o r k of H a r v e y et ai (17) provides a g o o d e x p l a n a t i o n of t h e i r e a r l y results (14) a n d confirms the s p e c u l a t i o n of K i r k et ai (16). V e r a t r y l a l c o h o l can protect ligninase f r o m i n a c t i v a t i o n by p r e v e n t i n g the f o r m a t i o n of ligninase c o m p o u n d III (17) ( F i g u r e 2). Indeed, the low y i e l d of v e r a t r a l d e h y d e at h i g h p H , c o u p l e d to the s t a b i l i t y of the h e m i n ( T a b l e I I , p H 10) was due to the facile f o r m a t i o n of the c o m p o u n d III a n a l o g of T D C S P P F e C l w h i c h is r e l a t i v e l y l o n g l i v e d at h i g h p H . O u r i n a b i l i t y to e s t a b l i s h a m e d i a t i n g role for v e r a t r y l a l c o h o l i n the o x i d a t i o n of a n i s y l a l c o h o l does not exclude the p o s s i b i l i t y t h a t i t f u n c t i o n s as a m e d i a t o r i n l i g n i n d e g r a d a t i o n . T h e m e d i a t i n g role of v e r a t r y l a l c o h o l m i g h t be difficult to observe for s m a l l m o l e c u l e

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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CHO

OH

OEt

F i g u r e 1. C o m p a r i s o n of the reactions of 4 - O - e t h y l g u a i a c y l g l y c e r o l - / ? g u a i a c y l ether c a t a l y z e d b y F e T P P / t - B u O O H / C H C l , T D C S P P F e C l / m C P B A / H 2 0 a n d l i g n i n a s e / h y d r o g e n peroxide. 3

0

Resting E n z y m e

Ligningx

Lignin

C o m p o u n d II

H 0 2

2

Compound m

F i g u r e 2. T h e c a t a l y t i c cycles of ligninase.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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525

i n t e r a c t i o n s w i t h the low m o l e c u l a r weight m o d e l c o m p o u n d s . Indeed, as d e s c r i b e d below, the results of e l e c t r o c h e m i c a l studies w i t h a p o l y m e r i c m o d e l c o m p o u n d s h o w n i n F i g u r e 3 suggest s u c h a m e d i a t i n g role. T a b l e I I I . O x i d a t i o n of a n i s y l a l c o h o l b y T D C S P P F e C l i n aqueous s o l u t i o n i n the presence of v e r a t r y l a l c o h o l ( p H 3) Substrates Anisyl Alcohol (//moles) 30 30 30 30 30 a

Veratryl Alcohol (pmoles) 30 15 3 0.3 0

% y i e l d of Anisaldehyde"

% T D C S P P F e C l Left After Reaction

1.3 4.2

> 90 > 90

25.0

> 90

Y i e l d based o n a n i s y l a l c o h o l .

T a b l e I V . O x i d a t i o n of A n i s y l A l c o h o l by T D C S P P F e C l a n d m C P B A i n A q u e o u s S o l u t i o n i n the Presence of 1,4-dimethoxybenzene ( p H 3) Substrates Anisyl Alcohol (/xmoles) 30 30 30 30 30 a

1,4-dimethoxy Benzene (/zmoles)

% Y i e l d of Anisaldehyde"

30 15 3 0.3 0

1.3 3.3 17.5 20.4 25.0

% T D C S P P F e C l Left After Reaction > > > > >

90 90 90 90 90

Y i e l d based o n a n i s y l a l c o h o l .

P o l y B - 4 1 1 , a w a t e r - s o l u b l e , blue dye ( I V ) has been used as a l i g n i n m o d e l b y G l e n n et ai (18). T h e dye was deposited onto a piece of filter p a p e r , w h i c h was t h e n a t t a c h e d d i r e c t l y to a glassy c a r b o n anode. T h e a n o l y t e c o n t a i n e d 10 m M v e r a t r y l a l c o h o l a n d 0.1 M t e t r a b u t y l a m m o n i u m p e r c h l o r a t e ( T B A P ) i n methylene chloride. T h e c a t h o l y t e c o n t a i n e d o n l y 0.1 M T B A P i n m e t h y l e n e chloride. A f t e r s i x hours c o n t r o l l e d p o t e n t i a l o x i d a t i o n at 1.2 V , the blue color of filter p a p e r o n the side f a c i n g the anode t u r n e d to r e d d i s h b r o w n . C o m p l e t e o x i d a t i o n of P o l y B - 4 1 1 i n aqueous s o l u t i o n by T D C S P P F e C l a n d m C P B A gave the same color. T h e filter paper was not decolorized i n a c o n t r o l e x p e r i m e n t l a c k i n g v e r a t r y l a l c o h o l . A s P o l y B - 4 1 1 was not able to m a k e direct contact w i t h the electrode (since

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

PLANT C E L L W A L L P O L Y M E R S

526

A596 0.540 0.520-f 0.500 0.480

2"

0.460

3

0.440 H 0.420 10

20

30

40

50

60

Time (min) F i g u r e 3. E l e c t r o c h e m i c a l o x i d a t i o n of P o l y B-411 i n the presence of v e r a t r y l a l c o h o l at various concentrations ( m M ) . 1, 0; 2, 0.1; 3, 1.0; 4, 10. A596 0.70 Η 0.60 0.500.400.30 0.20

Time (min) F i g u r e 4. D e c o l o r i z a t i o n o f P o l y B - 4 1 1 b y T D C S P P F e C l a n d ? n C P B A i n the presence o f manganese sulfate a n d l a c t i c a c i d (curve 3). C u r v e s 1 a n d 2 show c o n t r o l e x p e r i m e n t s l a c k i n g T D C S P P F e C l a n d manganese sulfate, respectively.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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it is insoluble in methylene chloride) in the above experiment, veratryl alcohol (or one of its oxidation products) must have mediated its oxidation. TDCSPPFeCl can also mimic the function of the Mn(II)-dependent peroxidase isolated from ligninolytic cultures of P. chrysosporium (19,20). The mCPBA-oxidized TDCSPPFeCl can oxidize Mn(II) to Mn(III) which in turn oxidizes Poly B-411 in the presence of various α-hydroxy carboxylic acids. Figure 4 shows that TDCSPPFeCl can rapidly decolorize Poly B411 in the presence of manganese sulfate, lactic acid, and mCPBA. Control experiments lacking either manganese sulfate or TDCSPPFeCl caused inef­ ficient and very slow decolorization of Poly B-411 (Fig. 4). It has been sug­ gested (23) that one function of manganese is that of a diffusible mediator for lignin degradation. The above observations support this mediating role and at the same time emphasize the role of the porphyrin (enzyme). Hy­ drogen peroxide, a two electron oxidant, cannot oxidize Mn(II) to Mn(III) (a one electron process), bu th tim ion unlik iro do not initiate Fenton-lik ferric porphyrin to the compound I oxidation state (O = Fe(IV)Por ) and this can bring about the one electron oxidation of Mn(II) leaving Compound II (O = Fe(IV)Por). Compound II can then oxidize a second equivalent of Mn(II). Indeed this latter step is probably critically important in preventing Compound III formation in the manganese dependent peroxidase. Conclusions We have shown that TDCSPPFeCl ( Π Ι ) is so far the most stable and efficient catalyst among the iron porphyrins used as model ligninases. All the known reactions catalyzed by this porphyrin mimic the ligninases quite well. TDCSPPFeCl can be used in both aqueous and polar organic solvent (such as methanol, DMF) so that solvent effects of lignin degradation can be studied. The catalyst is stable over a wide range of pH so the reactions at different pH can be compared. Under carefully controlled conditions veratryl alcohol can act as a redox mediator but from the experiments described above, we expect that such a role must be of only minor importance in nature. The ability of veratryl alcohol (and Mn(II)) to reduce Compound II (preventing the formation of compound III) is probably of far more importance for the ligninases. Acknowledgment This work was supported by the Natural Sciences and Engineering Research Council of Canada. Literature Cited 1. Tien, M.; Kirk, T . K. Science 1983, 221, 661-63. 2. Glenn, J. K.; Morgan, Μ. Α.; Mayfield, M . B.; Kuwahara, M.; Gold, M. H. Biochem. Biophys. Res. Commun. 1983, 114, 1077-83. 3. Shimada, M . ; Habe, T.; Umezawa, T.; Higuchi, T.; Okamoto, T . Biochem. Biophys. Res. Commun. 1984, 122, 1247-52.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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4. Habe, T.; Shimada, M.; Higuchi, T . Mokuzai Gakkaishi 1985, 31, 54-55. 5. Habe, T.; Shimada, M . ; Okamoto, T.; Panijpan, B.; Higuchi, T . J. Chem. Soc., Chem. Commun. 1985, 1323-24. 6. Tien, M.; Kirk, T . K. Proc. Natl. Acad. Sci. USA 1984, 81, 2280-4. 7. Shimada, M . ; Hattori, T.; Umezawa, T.; Higuchi, T.; Okamoto, T . Proc. Intl. Symp. on Lignin Enzymic and Microbial Degradation; Paris, France, April 23-24, 1987; p. 151. 8. Shimada, M.; Habe, T.; Higuchi, T.; Okamoto, T.; Panijpan, B.; Holz­ forschung 1987, 41, 277-85. 9. Dordick, J. S.; Marletta, Μ. Α.; Klibanov, A. M . Proc. Natl. Acad. Sci. USA 1986, 83, 6255-57. 10. Lewis, N. G.; Razal, R. Α.; Yamamoto, E . Proc. Natl. Acad. Sci. USA 1987, 84, 7925-27. 11. Adler, E . Wood Sci. Technol. 1977, 11, 169-218. 12. Kirk, T . K.; Tien, M. 236, 279-87. 13. Schoemaker, Η. E.; Harvey, P. J.; Bowen, R. M.; Palmer, J . M . FEBS Lett. 1985, 183, 7-12. 14. Harvey, P. J.; Schoemaker, Η. E.; Palmer, J. M . FEBS Lett. 1986, 195, 242-46. 15. Tien, M.; Kirk, T . K.; Bull, C.; Fee, J . A. J. Biol. Chem. 1986, 261, 1687-93. 16. Kirk, T . K.; Farrell, R. L. Ann. Rev. Microbiol. 1987, 41, 465-505. 17. Harvey, P. J.; Schoemaker, Η. E.; Palmer, J . M.; Proc. Intl. Symp. on Lignin Enzymic and Microbial Degradation; Paris, France, April 23-24, 1987; p. 145. 18. Glenn,J. K.; Gold, M . H. Appl. Environ. Microbiol. 1983, 45, 1741-47. 19. Kuwahara, M.; Glenn, J . K.; Morgan, Μ. Α.; Gold, M . H. FEBS Lett. 1984, 169, 247-50. 20. Paszczynski, Α.; Huynh, Van-Ba; Crawford, R. FEMS Microbiol. Lett. 1985, 29, 37-41. 21. Glenn, J. K.; Gold. M . H. Arch. Biochem. Biophys. 1985, 242, 329-41. 22. Paszczynski, Α.; Huynh, Van-Ba; Crawford, R. Arch. Biochem. Bio­ phys. 1986, 244, 750-65. 23. Glenn, J . K.; Akileswaran, L.; Gold, M . H. Arch. Biochem. Biophys. 1986, 251, 688-96. RECEIVED March 17, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 38 Bacterial Degradation of Kraft L i g n i n

Production and Characterization of Water-Soluble Intermediates Derived from Streptomyces badius and Streptomyces viridosporus 1

1

1,2

1

3

P. F. Vidal , J . Bouchard , R. P. Overend , E . Chornet , H . Giroux , and F. Lamy 3

1

2

Department of Chemical Engineering, University of Sherbrooke, Sherbrooke, Quebec J1K 2R1, Canada National Research Council of Canada, Ottawa, Ontario K1A 0R6,

3

Department of Biochemistry Quebec J1K 2R1, Canada Two Streptomyces strains, S. badius and S. viridosporus, were found to be able to grow on kraft lignin (Indulin ATR) as sole carbon source. The resulting APPL (Acid Precipitable Polymeric Lignin) was characterized by FTIR and elemental analysis for C, H and N, and was found to contain proteins in addition to a relatively demethoxylated lignin component. The proteins were further characterized by amino acid analysis, while the lignin component was separated by solvent extraction and its molecular weight distribution determined by HPSEC.

In t h e last t e n years, research o n l i g n i n biodégradation has followed t w o routes: f u n g i a n d b a c t e r i a l d e l i g n i f i c a t i o n . C r a w f o r d a n d co-workers were the first t o s t u d y i n d e t a i l t h e a c t i o n o f two streptomyces, S. badius 252 a n d S. viridosporus T 7 A , o n lignocellulose f r o m different sources. T h e i r w o r k l e d t o the conclusion t h a t the b a c t e r i a l a c t i o n o n aqueous suspensions of these lignocellulosics resulted i n t h e s o l u b i l i z a t i o n o f l i g n i n fragments w h i c h p r e c i p i t a t e u p o n a c i d i f i c a t i o n : A c i d P r e c i p i t a b l e P o l y m e r i c L i g n i n ( A P P L ) (1-10). In t h i s p a p e r , we report o n t h e b a c t e r i a l g r o w t h o f S. badius a n d S. viridosporus w i t h I n d u l i n A T R , a c o m m e r c i a l k r a f t l i g n i n p r a c t i c a l l y free of sugars, as sole carbon source a n d o n t h e c h a r a c t e r i z a t i o n o f the A P P L derived from this degradation. Materials a n d Methods T h e m a t e r i a l s a n d most o f the methods used are described i n previous papers (11,12). T o s u m m a r i z e : - I n d u l i n A T R is a purified f o r m (acidified water wash) o f I n d u l i n A T from Westvaco C o r p . , Charleston Heights, South Carolina. 0097~6156/89/0399-0529$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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-

5. badius a n d S. viridosporus are o b t a i n e d f r o m the A m e r i c a n T y p e C u l t u r e s C o l l e c t i o n , A T C C #39115 a n d 39117, respectively. These s t r a i n s are grown i n I n d u l i n A T R suspensions ( 0 . 5 % w / v ) . N H C 1 , yeast e x t r a c t a n d glucose are used together or i n d e p e n d e n t l y as n u t r i e n t s . T h e D N A content is measured i n order to determine the rate of bac­ t e r i a l g r o w t h a c c o r d i n g to B u r t o n (13). A P P L is d e t e r m i n e d b y a c i d p r e c i p i t a t i o n ( 1 2 M H C 1 ) u s i n g either t u r ­ b i d i t y measurements (nephelometry: abs. at 600 n m ) or g r a v i m e t r y a c c o r d i n g to C r a w f o r d et al. (4). A l l e x p e r i m e n t s r e p o r t e d i n t h i s p a p e r were c a r r i e d out w i t h u n i n o c u l a t e d controls whose values were always s u b s t r a c t e d . M e t h o x y l content of the different samples were d e t e r m i n e d u s i n g a m o d i f i e d Zeisel procedure (14). A q u e o u s Size E x c l u s i o n C h r o m a t o g r a p h y ( A S E C ) : A P h a r m a c i a sys­ t e m w i t h superose T M 1 (flow rate 0.4 m l m i n ) , was used, detection being b y U V at 280 n m . F o u r i e r T r a n s f o r m Infrared ( F T I R ) Spectroscopy: A 5 D X B N i c o l e t s y s t e m w i t h a T G D S detector was used at a r e s o l u t i o n of 4 c m " . T h e samples were m i x e d w i t h pure K B r at a c o n c e n t r a t i o n of 2 % w / w a n d 64 scans were collected. H i g h P e r f o r m a n c e Size E x c l u s i o n C h r o m a t o g r a p h y ( H P S E C ) : A V a r i a n M o d e l 5000 l i q u i d c h r o m a t o g r a p h e q u i p p e d w i t h a v a r i a b l e w a v e l e n g t h U V detector, two c o l u m n s i n series ( P L gel, 300 x 7 . 5 m m , p a r t i c l e size 5μπι, p o r o s i t y of 50 a n d 5 0 0 Â ) , was used, T H F b e i n g the eluent.

-

4

-

-

- 1

-

1

-

Results and Discussion Bacterial Growth and APPL Production. G l u c o s e used as a secondary c a r b o n source increases A P P L p r o d u c t i o n , t h o u g h o n l y after a l l glucose has been c o n s u m e d . T h e increase is greater for S. badius t h a n for S. viridosporus ( F i g . 1). A s p r e v i o u s l y s h o w n by C r a w f o r d (2), we also observed t h a t an o r g a n i c source of n i t r o g e n such as yeast e x t r a c t was m u c h better t h a n an i n o r g a n i c source such as N H C 1 , the S t r e p t o m y c e s p r o d u c i n g 7 to 9 times more A P P L i n the former case ( F i g . 2). T h e increase of i n i t i a l p H f r o m 7.2 t o 8.8 s l i g h t l y increases A P P L y i e l d , w h i l e a n a d d i t i o n of C u + + , Fe+++, M n or Zn++ has no effect ( d a t a not s h o w n ) . T h e s t u d y of the D N A content, a n i n d i c a t i o n of b a c t e r i a l g r o w t h , shows t h a t S. badius reaches its s t a t i o n a r y phase after 5 days, 7 days p r i o r to S. viridosporus ( F i g . 3). In b o t h cases, A P P L p r o d u c t i o n s t a r t s i m m e d i a t e l y (t = 0) a n d increases l i n e a r l y d u r i n g i n c u b a t i o n , S. badius p r o d u c i n g more t h a n S. viridosporus. A f t e r 35 days, the A P P L y i e l d represents 7% a n d 5 % of the i n i t i a l I n d u l i n A T R weight for S. badius a n d S. viridosporus, respectively. 4

+

+

Separation of Bacterial Extracellular, Membranous and Cytosolic Proteins and their Effect on Indulin ATR. W e first assume a l i g n i n s o l u b i l i z a t i o n c a t a l y z e d b y enzymes. In order to localize the enzymes responsible for the I n d u l i n A T R d e g r a d a t i o n , the cells of each s t r a i n were f r a c t i o n a t e d : e x t r a

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

38.

531

Bacterial Degradation of Kraft Lignin

VIDAL E T A L

0.35

0.30

0.25

0.20

0.15

0.10

g

0.05

C g

S
3 0 0 , 0 0 0 ) is a t t r i b u t e d to the p r o t e i n f r a c t i o n o f the A P P L , such large molecules never being found i n Indulin A T R . Dissociation of the Protein-Polyphenolie Complex and Characterization of the Polyphenolic Fraction. Since I n d u l i n A T R is almost c o m p l e t e l y s o l u b l e i n T H F w h i l e the A P P L ' s are q u i t e i n s o l u b l e i n t h i s solvent, b u t are soluble i n D M F , a sequence of different percentage m i x t u r e s of these two solvents was used i n order t o dissociate the p r o t e i n - l i g n i n complexes for f u r t h e r analyses of the l i g n i n p a r t . D e s p i t e the fact t h a t crude A P P L ' s are t o t a l l y soluble i n D M F , a n i m p o r t a n t residue is o b t a i n e d at the end of the solvent sequence (0:100, T H F : D M F ) i n d i c a t i n g t h a t the p r o t e i n - r i c h fractions require a s s o c i a t i o n w i t h the p o l y p h e n o l i c p a r t for t h e i r s o l u b i l i z a t i o n i n D M F ( T a b l e V I ) . B e cause each A P P L has a d i s t r i b u t i o n is also different, b u t i n b o t h cases, the T H F f r a c t i o n is the m o s t l i g n i n - l i k e , w i t h o n l y 1% n i t r o g e n . T h i s is confirmed b y F T I R a n a l y sis ( F i g . 7). A s the f r a c t i o n a t i o n proceeds w i t h i n c r e a s i n g solvent p o l a r i t y , the l i g n i n c h a r a c t e r i s t i c b a n d s at 1515, 1460, 1265, 1095, 1035, a n d 810 c m " d i s a p p e a r , w h i l e the a m i d e c h a r a c t e r i s t i c bands at 3290 a n d 3080 c m " appear. 1

1

T a b l e V I . E l e m e n t a l A n a l y s i s of the F r a c t i o n s O b t a i n e d b y S e q u e n t i a l S o l ubilization Sample

THF:DMF

% of S t a r t i n g Material

% N

%C

%H

%O

APPL S. bad.

100:0 85:15 50:50 0:100 Residue

8.0 21.1 19.0 9.8 42.1

1.0 2.7 4.8 4.3 8.7

67.4 61.7 58.6 57.2 49.6

7.1 6.1 6.4 5.7 6.5

24.5 29.5 30.2 32.8 35.2

APPL S. vir.

100:0 85:15 50:50 0:100 Residue

2.3 4.3 36.3 2.8 54.3

not 2.0 3.3 4.0 9.9

enough s a m p l e 65.8 7.1 25.1 61.7 6.6 28.4 57.2 5.1 33.7 49.6 6.9 33.6

x

Percentage of oxygen is c a l c u l a t e d b y difference. T h e m o l e c u l a r weight d i s t r i b u t i o n o b t a i n e d b y size e x c l u s i o n c h r o m a t o g r a p h y , for b o t h A P P L ' s T H F soluble f r a c t i o n a n d I n d u l i n A T R , show s i m i l a r profiles, the differences b e i n g t h a t the r a t i o of h i g h m o l e c u l a r weight ( D P 5 0 ) to the lower m o l e c u l a r weight ( D P 5 ) is inversed between I n d u l i n A T R a n d b o t h A P P L fractions, the l a t t e r b e i n g richer i n lower m o l e c u l a r weight ( F i g . 8). Because the A P P L ' s represent o n l y 7% of the i n i t i a l m a -

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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F i g u r e 6. A q u e o u s size e x c l u s i o n c h r o m a t o g r a p h y of the A P P L derived f r o m S. badius. F e r r i t i n , aldolase, o v a l b u m i n , c h y m o t r y p s i n o g e n A a n d acetone were used as s t a n d a r d s .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

3ΘΟΟ

2ΘΟΟ CCM-15

1OOO

1700

1SOO

1300 WAVENUMBERS

1 lOO CCM-1>

900

F i g u r e 7. F T I R s p e c t r a o f t h e fractions a n d o f t h e i n s o l u b l e residue o b ­ t a i n e d b y s e q u e n t i a l solvent s o l u b i l i z a t i o n . T h e f r a c t i o n s are i d e n t i f i e d b y the T H F : D M F r a t i o used. ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 12, © 1989, C N R C . )

3400 300D WAVENUMBERS

700

542

PLANT C E L L W A L L P O L Y M E R S

F i g u r e 8. H i g h performance size exclusion c h r o m a t o g r a p h y of I n d u l i n A T R a n d of the T H F soluble f r a c t i o n of each A P P L . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 12, © 1989, C N R C . )

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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VIDAL ET AL.

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543

terial, this technique does not allow us to conclude whether or not APPL consists of Indulin ATR depolymerized fragments. Conclusions The experiments reported were all conducted under the important assump­ tion that enzymes present in the bacteria studied were responsible for the solubilization of the lignin. This assumption is supported by some, but not all of the results obtained, such as the H2O2 activation of certain bacterial protein fractions. Another theory would be to suppose that the solubilizing proteins are, in fact, surfactants. This would explain, for example, the activity of these proteins at high temperatures. We do know, however, that proteins attach to APPL to form a soluble complex. Following the sur­ factant theory we would consider that Indulin ATR is a complex of lignin fragments insoluble only because of the medium's properties. The bacterial proteins would attach to the Indulin ATR increasing the hydrophilicity of the fragments, some able solution. To prove that one theory, the other, or even a combination of both is correct, further studies of the purified secreted and cellular proteins from the bacteria will be part of the next series of experiments planned. Literature Cited 1. Antai, S. P.; Crawford, D. L. Appl. Environ. Microbiol. 1981, 42, 37880. 2. Barder, M. J.; Crawford, D. L. Can. J. Microbiol. 1981, 27, 859-63. 3. Crawford, D. L. Biotechnol. Bioeng. Symp. 1981, 11, 275-91. 4. Crawford, D. L.; Pometto, A. L., III; Crawford, R. L. Appl. Environ. Microbiol. 1983, 45, 898-904. 5. Crawford, D. L.; Pettey, T. M.; Thede, B. M.; Deobald, L. A. Biotech­ nol. Bioeng. Symp. 1984, 14, 241-56. 6. Pettey, T. M.; Crawford, D. L. Appl. Environ. Microbiol. 1984, 47, 439-40. 7. Borgmeyer, J. R.; Crawford, D. L. Appl. Environ. Microbiol. 1985, 49, 273-78. 8. Pometto, A. L., III; Crawford, D. L. Appl. Environ. Microbiol. 1986, 51, 171-79. 9. Pometto, A. L., III; Crawford, D. L. Appl. Environ. Microbiol. 1986, 52, 246-50. 10. Ramachandra, M.; Crawford, D. L.; Pometto, A. L., III. Appl. Environ. Microbiol. 1987, 53, 2754-60. 11. Giroux, H.; Vidal, P. F.; Bouchard, J.; Lamy, F. Appl. Environ. Mi­ crobiol. 1989, 54, 3064-70. 12. Vidal, P. F.; Bouchard, J.; Overend, R. P.; Chornet, E.; Giroux, H.; Lamy, F. Can. J. Chem. 1989, in press. 13. Burton, K. Biochem. J. 1955, 61, 473-83. 14. Haluk, J. P.; Metche, M. Cell. Chem. Technol. 1986, 20, 31-50. 15. Hergert, H. L. In Lignins; Sarkanen, Κ. V.; Ludwig, C. H., Eds.; Wiley: New York, 1971. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 39 M i c r o b i a l Calorimetric Analysis Lignin-Related Compounds in Micromolar Concentrations Rex E . Lovrien , Mark L. Ferry , Timothy S. Magnuson , 1

1

2

and Robert A. Blanchette

2

Biochemistry Department, Gortner Laboratory, University of Minnesota,

1

St. Paul, M N 55108 2

Plant Pathology and Forestr

Microbial combustion of lignin fragments and lignin model compounds produces heat which is measurable via heat conduction calorimetry, even with samples ranging in size from 5-200 nanomoles. The general method is called microbial calorimetric analysis (MCA). First, microbes (Pseudomonas or soil bacteria) are grown on lignin fragments or model compounds as a carbon source. The adapted bacteria, so obtained, then function as rapid-acting, specific reagents for metabolizing such compounds. The technique has the following advantages: (i) cells (1-2 mg) are capable of aerobically combusting many kinds of C -C compounds in 5-10 minutes; (ii) interfering compounds can be removed by stripping; (iii) detection limits are comparable to spectrophotometric methods (e.g., to micromolar levels for sugars and phenols); and (iv) chromogenic groups are not required for detection. The sensitivity of the technique is based on the large aerobic heats of such compounds, and the velocities with which adapted cells can drive metabolism. 1

10

Once

common b a c t e r i a have been adapted t o g r o w i n g o n l i g n i n fragments, these c o m p o u n d s essentially undergo c o m b u s t i o n p r o d u c i n g heat. C u r rent heat c o n d u c t i o n calorimeters c a n easily measure s u c h heats o f aerobic m e t a b o l i s m , even i n q u a n t i t i e s r a n g i n g f r o m 5-100 n a n o m o l e s (1,2). B o m b c o m b u s t i o n c a l o r i m e t r y converts samples c o m p l e t e l y t o c a r b o n d i o x i d e a n d w a t e r . A e r o b i c b a c t e r i a l m e t a b o l i s m o n l y produces h a l f as m u c h heat, e.g., sugars, phenols, alcohols a n d a l i p h a t i c acids l i b e r a t e 100-600 K c a l / m o l , a n d l i g n i n a n d C s - 1 2 p e t r o l e u m s generate ~ 1000 K c a l / m o l " . T h e lower c a l o r i m e t r i c measurement ( ± 3 % precision) is ~ 2-30 m i l l i c a l o r i e s . D i v i d i n g s u c h ranges b y c a . 100-500 K c a l h e a t / m o l e gives ~ 5-200 nanomoles o f - 1

1

0097-6156/89/0399-0544$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

39.

L O V R I E N ET A L .

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545

c o m p o u n d needed for m i c r o b i a l c a l o r i m e t r i c a n a l y s i s ( M C A ) . S i n c e s a m ple volumes are 1-2 m l , m i c r o m o l a r c o n c e n t r a t i o n s can be used for t h i s m e t h o d . Besides the c o n c e n t r a t i o n ranges w h i c h a n y a n a l y t i c a l m e t h o d m a y be e x p e c t e d to cover, four f u r t h e r aspects are also i m p o r t a n t : n a m e l y , d e t e c t a b i l i t y of the a n a l y t e ; whether a s a m p l e c a n be used " r a w " or has to be i s o l a t e d ; i n t e r f e r i n g c o m p o u n d s a n d s p e c i f i c i t y ; a n d cost. C o n c e r n i n g d e t e c t a b i l i t y of l i g n i n a n d cellulose d e r i v e d fragments, M C A has considerable advantages. S u c h c o m p o u n d s are often p o o r l y c h r o m o g e n i c ; some have no chromogens at a l l , or are p o o r l y p r o c h r o m o g e n i c , i.e., are difficult t o a t t a c h a c h r o m o p h o r e . B u t these sorts o f c o m p o u n d s " b u r n " w i t h large heats i n M C A , so t h a t a l a c k of c h r o m o p h o r e s for o p t i c a l d e t e c t i o n is not at a l l a h i n d r a n c e . M C A c a n also u t i l i z e t u r b i d , r a w or p i g m e n t e d s a m p l e s , w h i c h are i m p o s s i b l e for s p e c t r o p h o t o m e t r y . M i c r o b i a l c a l o r i m e t r y was recently reviewed b y B a t t l e y (3). W h e r e a s earlier c a l o r i m e t e r s r e q u i r e d c a . 100 m l of m i c r o b i a l s u s p e n s i o n , heat c o n d u c t i o n c a l o r i m e t e r s u s i n g the Seebec 0.2 t o 2 m l of s u b s t r a t e or a n a l y s i s s o l u t i o n a n d of cell s u s p e n s i o n . A 100200 m l overnight c u l t u r e p r o v i d e s enough cells for 10-20 measurements for MCA. M i c r o b i a l s t r i p p i n g adds m u c h to M C A ' s scope, a n d s i m p l i f i e s i t . S t r i p p i n g uses i n d u c e d b a c t e r i a for g e t t i n g r i d of i n t e r f e r i n g c o m p o u n d s . F i g u r e 1 o u t l i n e s direct c a l o r i m e t r y , a n d s t r i p p i n g , together w i t h average p a r a m eters for l i g n i n m o d e l c o m p o u n d s a n a l y s i s . S t r i p p i n g b y E. coli was first used i n c e l l u l o l y s i s , q u a n t i t a t i n g cellobiose vs. glucose (4). It was d e v e l o p e d f u r t h e r for p h e n o l i c m a t e r i a l s , u s i n g Pseudomonas and bacteria from s o i l isolates. M C A takes advantage of b a c t e r i a l a b i l i t y t o synthesize large a m o u n t s of enzymes necessary for c a r r y i n g c a r b o n sources t h r o u g h o x i d a t i v e m e t a b o l i s m , thereby i n d u c i n g the enzymes (5). T h e y are analogous to the classic e x a m p l e , the lac o p e r o n of E. coli. ( G r o w n o n glucose, E. coli have 0.5 t o 5 /?-galactosidase e n z y m e molecules per cell. B u t g r o w n o n lactose, E. coli c a n synthesize 1000 to 1,500 /?-galactosidase m o l e c u l e s / c e l l (6).) T h u s , M. irichosporium produces a l m o s t 1 7 % of i t s soluble p r o t e i n as a m e t h a n e m o n o h y d r o x y l a s e (7). A Pse udom on as synthesizes a b o u t 3 % of i t s p r o t e i n as p r o t o c a t e c h u a t e m o n o h y d r o x y l a s e , w h e n g r o w n o n p - h y d r o x y b e n z o a t e (8). M o s t of the p r o - o x i d a t i v e enzymes of b a c t e r i a are s t a b i l i z e d i n s i d e the c e l l , b u t are v e r y fragile outside the cell. T h e r e f o r e , the v i e w t h a t a n a l y s i s m a y be c a r r i e d out v i a i s o l a t e d enzymes for a r o m a t i c processing, p e r h a p s c o u p l e d t o a n electrode o f some k i n d , appears q u i t e i m p r a c t i c a l . M C A takes advantage of w h a t b a c t e r i a l cells can a c t u a l l y do, n a m e l y to s t a b i l i z e a n d p r o t e c t e n z y m e s , besides the i n i t i a l synthesis. Hence, M C A is l i k e l y to be far m o r e p r a c t i c a l t h a n any " b i o e l e c t r o d e " m e t h o d for a n a l y s i s . Bacterial Growth and Adaptation Pseudomonas putida A T C C 11172 was used for b o t h m i c r o b i a l c a l o r i m e t r y a n d for s t r i p p i n g p h e n o l a n d lower cresols. S e v e r a l isolates f r o m l o c a l soils, w h i c h were able to c o m b u s t larger a r o m a t i c s s u c h as c i n n a m i c a n d s y r i n g i c

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L P O L Y M E R S

acids, were o b t a i n e d by a c i r c u l a t i n g e n r i c h m e n t a p p a r a t u s s i m i l a r t o t h a t described b y A u d u s (9). L i g n i n m o d e l c o m p o u n d s , 0 . 0 3 % , p l u s 0 . 0 1 % g l u ­ cose were i n c u b a t e d w i t h ~ 100 g o f soil for 2 d a y s , w i t h a i r c i r c u l a t i o n at r o o m t e m p e r a t u r e . A f t e r 2 days, enricher i n o c u l a were transferred to plates w i t h 0 . 5 % yeast e x t r a c t a n d the c o m p o u n d . ( F o r c i n n a m i c a n d h y d r o x y c i n n a m i c a c i d c a r b o n sources, the yeast e x t r a c t was left o u t . ) P l a t e isolates were g r o w n at r o o m t e m p e r a t u r e for 12-48 h . C o l o n i e s f r o m plates were used t o i n o c u l a t e shake-flask l i q u i d cultures w i t h A s h w o r t h - R o m b e r g salts (10) c o n t a i n i n g 0 . 0 5 % b y weight o f a c a r b o n source a n d 0 . 0 3 % yeast e x t r a c t . L i q u i d c u l t u r e g r o w t h u s u a l l y o c c u r r e d below 0 . 0 5 % c o m p o u n d , b u t some­ w h a t larger concentrations tended t o k i l l even w e l l a d a p t e d Pseudomonas. A f t e r g r o w t h (at 30°) the o r g a n i s m s were d i l u t e d w i t h either m i n i m a l salts or i s o t o n i c saline a n d c e n t r i f u g a l l y washed t w i c e . C e l l c o n c e n t r a t i o n s were a d j u s t e d u s i n g the s p e c t r o p h o t o m e t r y t u r b i d i t y factor 2.0 x 1 0 x Α β β ο * c m " " p r e v i o u s l y described (1) to measure n u m b e r s of c e l l s / m l , ± 1 0 % for Pseudomonas. 9

1

Stripping A d a p t e d cells able to b i n d u n w a n t e d , interfering c o m p o u n d s were washed t w i c e a n d adjusted i n c o n c e n t r a t i o n . A p p r o x i m a t e l y 1 m l of suspension c o n t a i n i n g 5 χ 1 0 cells was u s u a l l y sufficient to s t r i p 1 m l samples c o n ­ t a i n i n g 50-100 nanomoles of i n t e r f e r i n g c o m p o u n d w i t h over 9 0 % r e m o v a l effectiveness. A f t e r m i x i n g the cells a n d samples b y v o r t e x i n g a n d i n c u b a t ­ i n g for a m i n u t e , the s t r i p p e r cells were s p u n d o w n i n a m i c r o c e n t r i f u g e . A n a l i q u o t o f the s u p e r n a t a n t was t a k e n for c a l o r i m e t r y or other means o f a n a l y s i s , e.g., F o l i n a n a l y s i s . 1 0

Spectrophotometric Analysis; Folin Phenol P h e n o l a n d p h e n o l i c derivatives, cresol a n d related c o m p o u n d s were a n a ­ l y z e d by F o l i n - C i o c a l t e a u reagent at 700 m / i , s t a n d a r d i z i n g each c o m p o u n d separately b y i t s F o l i n response. T h e i r slopes ( m o l a r a b s o r p t i o n coefficients) were 8000-10,000 M cm . Interestingly, m e t h o x y l a r o m a t i c s w i t h no free h y d r o x y l groups were not reactive. _

1

_

1

Heat Conduction Calorimetry, Batch M i x i n g F i g u r e 2 i l l u s t r a t e s the arrangement o f a t h r e e - c h a n n e l b a t c h m i x i n g c a l o r i m e t e r u s i n g L i p s e t t - J o h n s o n - M a a s vessels (11) for a 1 m l a n a l y s i s s a m p l e , 2 m l m i c r o b i a l suspension, a n d 3 m l headspace. T h e f o u r t h vessel i n F i g u r e 2, together w i t h i t s Seebeck thermosensors, is a reference m o d u l e . U s u a l l y i t is loaded w i t h 2 m l m i c r o b i a l suspension a n d 1 m l of solvent ( m i n ­ i m a l s a l t s ) . T h e reference m o d u l e voltage opposes a l l three s a m p l e m o d u l e s so t h a t each measurement is a difference or net power measurement. I n effect, the heat of m i x i n g the s y s t e m , or the heat of r e s i d u a l m i c r o b i a l ac­ t i v i t y w i t h o u t c a r b o n , is s u b t r a c t e d f r o m the samples. T h e voltage signals o f heat c o n d u c t i o n calorimeters a c t u a l l y measure power. P o w e r integrated over t i m e equals heat, w h i c h is d i r e c t l y p r o p o r t i o n a l to the area m e a s u r e d

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

39.

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Direct Calorimetry

Stripping

547

Microbial Calorimetric Analysis

Adapted cells 1-2 mg. able to metabolize the carbon source or analvte Adapted cells, 2-4 mg., able to ^ bind interfering compound.

Mix with 1-2 ml. of ~" sample, aerobic

Measure heat proportional to the amount carbon, 2-100 nanomoles, 1-100 meal, 5-10 min.

Mix with 1-3 ml sample, vortex, centrifuge, retain supernate

Stripped supernate, 1-2 ml. for calorimetry

F i g u r e 1. M e t h o d s o f M C A : D i r e c t c a l o r i m e t r y b y m i c r o o r g a n i s m m e t a b o l i s m o f samples f o r a n a l y s i s ; s t r i p p i n g b y m i c r o o r g a n i s m b i n d i n g o f i n t e r fering compounds.

F i g u r e 2. H e a t c o n d u c t i o n (Seebeck effect) b a t c h m i x i n g c a l o r i m e t e r for three samples a n d one reference c h a n n e l . A f t e r l o a d i n g a n d e s t a b l i s h i n g baselines, t h e assembly is i n v e r t e d t o m i x react ants a n d s t a r t heat p r o d u c t i o n . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 2. © 1983, A l a n R . L i s s , Inc.)

American Chemical Society Library 1155 16th St., N.W. In Plant Cell Wall Polymers; Lewis, N., et al.; Washington, D.C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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u n d e r each t h e r m o g r a m as s h o w n i n F i g u r e 3. Headspaces i n each vessel c o n t a i n 3-6 m l o f a i r , t h a t i s , w i t h a p p r o x i m a t e l y 50-100 f o l d excess o x y g e n for aerobic m e t a b o l i s m o f 5-100 nanomoles of most substrates u p t o C12 sizes. M o r e d e t a i l e d p a r a m e t e r s , response t i m e s , noise g e n e r a t i o n , figure o f m e r i t , etc., have been p u b l i s h e d (2) a n d i n c o r p o r a t e d i n B R I C m a n u f a c t u r e d i n s t r u m e n t s . T h e u n i t s h o w n i n F i g u r e 2 is inside a m e t a l s h e l l , s u r r o u n d e d b y three m e t a l boxes. T h e outer two boxes are P e l t i e r p u m p e d , for p r o t e c t i n g against t h e r m a l fluctuations f r o m outside. T h e s y s t e m c a n be o p e r a t e d at a n y chosen t e m p e r a t u r e between 10-50°. T h e m i c r o b i a l c a l o r i m e t r i c analyses were c a r r i e d out at 25°. H e a t c o n d u c t i o n c a l o r i m e ters derive t h e i r signals f r o m heat flow, generated by the samples after m i x i n g . T h e y do not rely o n measurement of s m a l l t e m p e r a t u r e changes. T h e heat c o n d u c t i o n p r i n c i p l e (12) is far easier a n d more sensitive, also less expensive, t h a n n e a r l y a n y means i n v o l v i n g t h e r m i s t o r s or t h e r m o m e t r y . C a l i b r a t i o n is c a r r i e d out i n two ways: electric resistance h e a t i n g w i t h a s m a l l probe inserted i n n e u t r a l i z a t i o n (2). Microbial Calorimetric Analysis Pseudomonas putida A T C C 11172

(MCA)

of C o m p o u n d s

Using

Pseudomonas g r o w n o n p h e n o l a n d various cresols c o m b u s t s t h e m at 25° i n 300-600 sec i f there are excess cells, ~ 1-2 m g d r y weight, a n d l i m i t e d carb o n , ~ 5-30 nanomoles. F i g u r e 3 shows t h a t w h e n Pseudomonas is a d a p t e d t o o-cresol, i t combusts o-cresol a n d p h e n o l t o c o m p l e t i o n i n a b o u t 6 m i n utes. G r o w n o n v a n i l l i c a c i d , Pseudomonas metabolizes l i m i t e d a m o u n t s of v a n i l l i c a c i d , albeit s o m e w h a t more s l o w l y t h a n i n the o-cresol or p h e n o l u t i l i z i n g s y s t e m . Nevertheless, t h i s occurs i n far shorter times t h a n c a n p o s s i b l y be observed b y a g r o w t h response. T e n to twenty n a n o m o l e s of v a n i l l i c a c i d m e t a b o l i z e c a . 3 0 - 5 0 % as r a p i d l y as o-cresol or p h e n o l . G r o w t h o n glucose allows the b a c t e r i a to combust glucose. B u t g r o w t h o n glucose does not equip t h e m to h a n d l e a n y of the l i g n i n m o d e l c o m p o u n d s as i n d i c a t e d b y the glucose g r o w n cells m i x e d w i t h o-cresol (lower trace o f F i g u r e 3). S u c h t h e r m o g r a m s , besides m e a s u r i n g the n a t u r e of v a r ious o r g a n i s m s ' responses to c a r b o n sources, also measure m e t a b o l i c rates. I n the case of glucose a n d glucose-grown cells, when c o m p a r e d w i t h d a t a f r o m B e r k a et al. w h o used P. cepacia a n d a s p e c t r o p h o t o m e t r i c m e t h o d to follow the disappearance o f sugar (13), i t was f o u n d t h a t our rates of u t i l i z a t i o n agreed w i t h i n a factor o f less t h a n two. S u c h rates f r o m c a l o r i m e t r y are s i m p l y a d i v i s i o n of the m o l a r heat o f aerobic glucose m e t a b o l i s m , 300 K c a l / m o l e glucose (1), by the power averaged over the t i m e i n t e r v a l for c o m b u s t i o n a n d the n u m b e r of moles of glucose c o n s u m e d . T h e quotient is a c o m b i n e d u p t a k e a n d m e t a b o l i c rate. S t a n d a r d i z a t i o n plots for M C A have the same n a t u r e as such p l o t s for other a n a l y t i c m e t h o d s . T h e y are plots of " r e a d o u t " (in t h i s case, heat) vs. c o n c e n t r a t i o n of s t a n d a r d . F i g u r e 4 shows a s t a n d a r d i z a t i o n p l o t for M C A response to o-cresol, c o m p a r e d w i t h a p l o t for F o l i n spect r o p h o t o m e t r y w h i c h is also for o-cresol. T h e question t h a t then arises is

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

F i g u r e 3. M i c r o b i a l c a l o r i m e t r y of 10-40 nanomoles of c o m p o u n d s u s i n g P. puiida a d a p t e d (upper four traces), a n d not a d a p t e d (lower trace) to the c o m p o u n d s , 2 5 ° C , m i n i m a l salts solvent. R M = r e m i x i n g i n m i d - r u n to ensure aerobicity. O r d i n a t e is p r o p o r t i o n a l to power. I n t e g r a t e d areas are p r o p o r t i o n a l to o v e r a l l heat of c o n s u m i n g a l l m e t a b o l i z a b l e c a r b o n .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

0.22

50|

20

USEFUL LOWER MICROBIAL CALORIMETRIC RANGE

60

80

NANOMOLES O-CRESOL

40

100

USEFUL LOWER FOLIN SPECTROPHOTOMETRIC RANGE

120

OS

F i g u r e 4. C o m p a r i s o n o f M C A a n d F o l i n s p e c t r o p h o t o m e t r y for a n a l y s i s o f o-cresol s p a n n i n g t h e lower p r a c t i c a l ranges o f i n s t r u m e n t a l r e a d o u t .

Ε υ 0.18 h-

ο ο

0.25

0.29

39.

LOVRIEN ET AL.

551

Microbial Calorimetric Analysis

the s e n s i t i v i t y o f M C A c o m p a r e d w i t h other m e t h o d s i n a n a l y s i s o f one l i g n i n - r e l a t e d c o m p o u n d . S u c h a c o m p a r i s o n depends l a r g e l y o n the lower h e a t s s u i t a b l e for Seebeck c a l o r i m e t r y , a n d the lower absorbancies s u i t a b l e for s p e c t r o p h o t o m e t r y at o p t i m u m w a v e l e n g t h n o r m a l l y u s i n g a 1 c m c u ­ v e t t e . A b s o r b a n c e s of c a . 0.10-0.30 i n s p e c t r o p h o t o m e t r y a n d heats o f 3-40 m i l l i c a l i n c a l o r i m e t r y were t a k e n as reasonable c r i t e r i a for F i g u r e 4. I m p o r t a n t p a r a m e t e r s w h i c h c o n t r o l s u c h a c o m p a r i s o n are the m o l a r a b s o r p t i o n coefficient for o - c r e s o l - F o l i n color, £700 = 8 1 4 0 M ~ c m ~ , a n d the m o l a r heat o f aerobic m e t a b o l i s m for o-cresol, 520 K c a l / m o l e o-cresol u s i n g aerobic Pseudomonas. T h e p a r a m e t e r s are the slopes o f the p l o t s o f F i g u r e 4. C l e a r l y the c r i t e r i a for two c o m p l e t e l y different m e t h o d s c a n be d r a w n closer together or f u r t h e r a p a r t , d e p e n d i n g o n w h a t i n s t r u m e n ­ t a l " r e a d o u t s " are t a k e n as reasonable lower ranges. However, even w h e n a r a t h e r intensely colored s a m p l e for a n a l y s i s is c o m p a r e d , as F o l i n - c r e s o l conjugates are, M C A is at least as sensitive as F o l i n s p e c t r o p h o t o m e t r y i n the present case. T h e aerobic m e t a b o l i s m , a n d use o f excess cells, w e l l a d a p t e d , a n d l i m i t e d for c a r b o n ( l i m i t e d a n a l y t e ) . I f real samples are v e r y t u r b i d , or l a d e n w i t h F o l i n - r e a c t i v e p i g m e n t s as m a n y l i g n i n c o m p o u n d s are, m i c r o b i a l c a l o r i m e ­ t r y has a f u r t h e r a d v a n t a g e . It is far less sensitive t o s u c h interference t h a n spectrophotometry. 1

1

S t r i p p i n g Efficiency a n d S t r i p p i n g Specificity A s w i t h a n y other a n a l y t i c a l m e t h o d , M C A ' s c a p a c i t y is enhanced i f i t is easy t o remove i n t e r f e r i n g c o m p o u n d s . M i c r o b i a l a d a p t a t i o n confers s p e c i ­ f i c i t y i n b i n d i n g u n w a n t e d c o m p o u n d s so they can be swept o u t w i t h o u t l o s i n g the s a m p l e for a n a l y s i s . F i g u r e s 5 a a n d 5b show the d a t a for p h e n o l a n d v a n i l l i c a c i d s t r i p p i n g u n d e r convenient c o n d i t i o n s , n a m e l y , w i t h easily a c q u i r e d a m o u n t s o f s t r i p p e r cells (5 x 1 0 cells), a n d c o n d i t i o n s where i n ­ terfering c o m p o u n d c o n c e n t r a t i o n s are r e l a t i v e l y large, i.e., u p to 10 t i m e s the a m o u n t o f s a m p l e for a n a l y s i s . 1 0

P h e n o l - g r o w n cells n e a r l y q u a n t i t a t i v e l y b i n d a n d c a r r y d o w n p h e n o l a n d some of the cresols i n c o n c e n t r a t i o n s ~ 8 x 1 0 ~ m g / m l (ca. 6 x 10"" M). P h e n o l a n d ο-, ρ - , a n d m-cresol are s t r i p p e d as s h o w n i n F i g u r e 5 a . I n c o n t r a s t , l i t t l e v a n i l l i c a c i d is removed b y p h e n o l - g r o w n P. putida, i.e., v a n i l l a t e r e m a i n s i n the s u p e r n a t a n t , whereas p h e n o l a n d the cresols are b o u n d a n d s p u n d o w n w i t h the s t r i p p e r cells. 3

5

W h e n the same P. putidaare grown on vanillic acid, Figure 5b, vanil­ late efficiently b i n d s a n d is s t r i p p e d , whereas v e r y l i t t l e p h e n o l is removed (see F i g u r e 5a). I n d e e d , F i g u r e s 5 a a n d 5b i l l u s t r a t e the efficiency a n d s p e c i f i c i t y w i t h w h i c h P. putida A T C C 11172 b i n d s phenols, a n d as c a n be seen is s h a r p l y dependent o n the c a r b o n source used for g r o w t h . W i t h i n cogeners, p h e n o l i t s e l f a n d o-, m - , a n d p-cresol, there is n o t m u c h d i s c r i m i ­ n a t i o n i n the a b i l i t y o f p h e n o l - g r o w n cells t o s t r i p t h e m b e l o w levels o f c a . 8 x 1 0 ~ m g / m l . A b o v e t h i s c o n c e n t r a t i o n , differences between the i n d i v i d ­ u a l c o m p o u n d s are seen b u t even these d i s a p p e a r w h e n cell c o n c e n t r a t i o n s are m a d e even larger. F o r 50 x 1 0 p h e n o l - g r o w n P. putida/ml, up to 3

1 0

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989. 3

VANILLIC ACID (mg/ml χ 10 )

1 0

F i g u r e 5. S t r i p p i n g p h e n o l , cresols a n d v a n i l l i c a c i d , dependent o n Pseu­ domonas a d a p t a t i o n t o c o m p o u n d s t o be s t r i p p e d ; 5 x 1 0 c e l l s / m l d u r i n g s t r i p p i n g . A r r o w i n F i g u r e 5 a : C o n c e n t r a t i o n o f c o m p o u n d s below w h i c h s t r i p p i n g is q u a n t i t a t i v e (see t e x t ) .

3

PHENOL (mg/ml χ 10 )

39.

553

Microbial Calorimetric Analysis

LOVRIEN ET A L

30 χ 1 0 ~ m g / m l of these c o m p o u n d s a l l b i n d a n d therefore get s t r i p p e d . H o w e v e r , for p r a c t i c a l purposes, one prefers not to have to use s u c h a large n u m b e r of cells a n d o r d i n a r i l y there is no need to. A t least for t h i s o r g a n ­ ism, usually 5 χ 1 0 c e l l s / m l are enough to s t r i p c o m p o u n d s o f t h i s k i n d i n the 1 0 ~ t o 1 0 " M range. 3

1 0

6

5

G l u c o s e - g r o w n P . putida s t r i p glucose, b u t g r o w n o n the cresols they do not b i n d glucose efficiently i n m i c r o m o l a r sugar c o n c e n t r a t i o n s n o r m e ­ t a b o l i z e i t r a p i d l y . F r o m results o f diverse e x p e r i m e n t s , we favor E. coli as s t r i p p e r s a n d m e t a b o l i z e r s o f c a r b o h y d r a t e s i n M C A (4). H o w e v e r , as m i g h t have been expected f r o m S t a n i e r , P a l l e r o n i a n d D o u d o r o f f ' s research (14), Pseudomonas provides the best prospects for l i g n i n m o d e l c o m p o u n d a n d p e t r o l e u m c o m p o u n d b i n d i n g a n d m e t a b o l i s m . A n arrow i n F i g u r e 5 a i n d i c a t e s a n abscissal v a l u e , below w h i c h essentially 1 0 0 % b i n d i n g a n d s t r i p p i n g occurs. C a l c u l a t i o n s analogous t o those c a r r i e d o u t earlier (15) leads to the e s t i m a t i o n t h a t , given such a n a m o u n t of p h e n o l a n d s u c h a n u m b e r o f cells (8 x 1 0 ~ 1.0% of m e m b r a n e l i p i d s are r e q u i r e d to a c c o m m o d a t e s u c h a n a m o u n t of p h e n o l . T h a t e s t i m a t i o n does not i n d i c a t e the m e c h a n i s m o f b i n d i n g . B u t i t does i n d i c a t e t h a t once the cells are a d a p t e d , t h e y are a d e q u a t e l y large "sponges" for such a n a m o u n t of c o m p o u n d . S t r i p p i n g is s i m p l e , r a p i d , a n d g r e a t l y e x p a n d s M C A for these k i n d s o f c o m p o u n d s . M i c r o b i a l s t r i p p i n g of t h i s k i n d l i k e l y w o u l d be h e l p f u l i n a n a l y t i c a l m e t h o d s other t h a n c a l o r i m e t r y b u t o d d l y t h i s has not been developed. T a b l e I s u m m a r i z e s the m a i n p o i n t s f r o m n u m b e r s of p l o t s s i m i l a r to F i g u r e s 5 a a n d 5 b . T h e c o n d i t i o n s were: 5 x 1 0 c e l l s / m l , 6 to 8 x 1 0 ~ m g c o m p o u n d to be s t r i p p e d / m l , 30 second m i x i n g . G l u c o s e was s t r i p p e d f r o m 15 x 1 0 ~ m g g l u c o s e / m l . 1 0

3

3

T a b l e I. Efficiency of S t r i p p i n g (Percent R e m o v e d ) C o m p o u n d s D e p e n d e n t o n the C a r b o n Source for G r o w t h , P. putida A T C C 11172 C a r b o n Source o n w h i c h G r o w n ( a d a p t e d ) C o m p o u n d to be S t r i p p e d

o-Cresol

m-Cresol

p-Cresol

Vanillic Acid

Phenol

Glc*

97 100 100 0 100 0

100 100 100 1 100

93 100 100 4 99

73 87 92 84 5

100 100 100 0 100

6

o-Cresol m-Cresol p-Cresol Vanillic Acid Phenol Glucose *Glc =

Glucose.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

7 56

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PLANT C E L L W A L L P O L Y M E R S

Calorimetric Combustion of o-Cresol and Vanillic A c i d Mixtures i n 5-100 N a n o m o l e s R a n g e P. putida A T C C 11172 g r o w n o n o-cresol efficiently s t r i p s a n d also c o m b u s t s o-cresol. F i g u r e 6 shows M C A a n d s t r i p p i n g performance for o-cresol (lower abscissa) w h e n considerable v a n i l l i c a c i d is present ( u p p e r abscissa). T h e d a t a i l l u s t r a t e s h o w a d a p t a t i o n confers specificity. F i r s t , u s i n g o-cresol g r o w n cells for c o m b u s t i n g a n e q u i m o l a r m i x t u r e o f o-cresol a n d v a n i l l i c a c i d y i e l d s v i r t u a l l y the same p l o t as o-cresol alone, d o w n to 8 nanomoles o-cresol. Secondly, after o-cresol s t r i p p i n g , n e a r l y a l l the o-cresol is removed a n d v a n i l l i c a c i d interferes m i n i m a l l y (lower p l o t o f F i g u r e 6). T h i r d l y , a d a p t a t i o n to o-cresol enables the cells to combust a n d t o s t r i p o-cresol. B u t i t disables t h e m f r o m u s i n g v a n i l l i c a c i d at a n y finite rate o n M C A ' s t i m e scale. However, v a n i l l i c a c i d - g r o w n P. putida is f u l l y capable of s t r i p p i n g a n d c o m b u s t i n g v a n i l l a t e i n a few m i n u t e s . I n s h o r t , a d a p t a t i o n confers reasonable specificity d o w the a b i l i t y to s t r i p o-cresol Lignin Model

Compounds

I n order t o o b t a i n r a p i d m e t a b o l i s m a n d r a p i d heat generation f r o m a r o ­ m a t i c c o m p o u n d s related to l i g n i n , several s o i l isolates were used. P. putida A T C C 11172 d i d not grow easily o n c o m p o u n d s such as s y r i n g i c a c i d a n d c e r t a i n other c o m p o u n d s m o r e l i g n i n - l i k e . However, soils collected l o c a l l y y i e l d e d b a c t e r i a , first f r o m e n r i c h m e n t cultures a n d t h e n g r o w n o n plates or defined l i q u i d c u l t u r e , were able t o c o m b u s t the c o m p o u n d s l i s t e d i n T a b l e II. T h e T a b l e r e p o r t s concentrations o f the c a r b o n source used i n M C A , the t i m e intervals needed for c o m p l e t e l y m e t a b o l i z i n g 10-50 nanomoles of c o m p o u n d , a n d the a p p a r e n t m o l a r heats o f m e t a b o l i s m i n low c a r b o n c o n c e n t r a t i o n s ( Δ ° Η ) . C i n n a m i c a c i d a n d 3 , 4 - d i m e t h o x y c i n n a m a t e evolved r a t h e r e x t r a o r d i n a r y heats, over 800 K c a l / m o l e . S u c h heats were not ex­ pected i n t h a t 3 , 4 - d i m e t h o x y c i n n a m a t e is a l r e a d y p a r t l y o x i d i z e d . M i c r o ­ b i a l c o m b u s t i o n o f n a p h t h a l e n e gave 810 K c a l / m o l e , 2 - m e t h y l n a p h t h a l e n e gave 652 K c a l / m o l e , u s i n g P. putida i n earlier w o r k (1). P l a i n l y the r e l ­ ative state o f o x i d a t i o n or r e d u c t i o n o f c a r b o n sources does not p r e d i c t h o w m u c h heat c o m p o u n d s generate i n m i c r o b i a l c o m b u s t i o n , i n c o n t r a s t t o c o n v e n t i o n a l oxygen b o m b c a l o r i m e t r i c c o m b u s t i o n . R a t h e r , there is a dependence o n h o w w e l l , or h o w p o o r l y , catabolites fit e x i s t i n g m e t a b o l i c p a t h w a y s of o r g a n i s m s (16). If there is p o o r fitting, m o r e c a r b o n has to be used i n e x o t h e r m i c p a t h w a y s e m i t t i n g C O 2 to generate needed i n t e r ­ mediates such as N A D H r e q u i r e d t o process w h a t e v e r fragments t h a t c a n be r e t a i n e d . O n c e cells (isolates) are a d a p t e d to these c o m p o u n d s they c a n d r i v e m e t a b o l i s m t h r o u g h i n 10-20 m i n u t e s , i f c a r b o n is l i m i t e d a n d there are excess cells a n d oxygen. T h u s , M C A does not need to complete a g r o w t h cycle as i n g r o w t h assays, b u t s i m p l y takes u p c a r b o n a n d gets t h r o u g h the l i n e a r , early c a t a b o l i c stages.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989. 40

50

60

1

NANOMOLES O-CRESOL/ml

30

1

70

1

80

1

72

F i g u r e 6. C a l i b r a t i o n o f heat generation f r o m o-cresol b y Pseudomonas m e t a b o l i s m as a s t a n d a r d p l o t for a n a l y s i s o f o-cresol alone a n d i n ocresol, v a n i l l i c a c i d m i x t u r e s , dependent o n Pseudomonas adaptation i n both calorimetry and stripping.

20

1

10

36 1

1

Ί

NANOMOLES VANILLIC ACID/ml 18

9

90

1

81

556

PLANT C E L L W A L L P O L Y M E R S

Table II. L i g n i n M o d e l C o m p o u n d Calorimetry w i t h Soil Cultures

Compound p-Hydroxyphenyl acetate Syringic acid C i n n a m i c acid Hydrocinnamic acid 3,4-Dimethoxycinnamic acid

Sample Concentration (micromolar)

T i m e for C o m p l e t e C o m b u s t i o n of 5-50 Nanomoles (min)

M o l a r H e a t of Aerobic Metabolism, K c a l

6-40 21-42 2-30

16 18 10

805 470 830

12-30

15

710

9-36

20

850

Conclusions M C A of o x i d i z e d l i g n i n fragments (such as the h y d r o x y c i n n a m i c acids i n T a b l e II) appears to be as sensitive as s p e c t r o p h o t o m e t r i c analyses f r o m results seen so far, even c o m p a r i n g c o m p o u n d s t h a t are b r i g h t l y colored or w h i c h c a n be m a d e so (chromogenic, prochromogenic c o m p o u n d s ) . M C A ' s specificities are d e t e r m i n e d b y those c o m p o u n d s w h i c h b a c t e r i a can u p t a k e a n d m e t a b o l i z e . A d a p t e d b a c t e r i a are quite selective i n t h i s r e g a r d . A l ­ t h o u g h M C A requires a n excess of cells to drive a n a l y t i c a l reactions r a p i d l y , the r e q u i r e d a m o u n t s u s u a l l y are easily o b t a i n e d once the i n o c u l a are a v a i l ­ able. A b o u t 1-5 m g of cells are needed for each c o m b u s t i o n , a n d 2-10 m g for m o s t s t r i p p i n g o p e r a t i o n s . Therefore, 50-300 m l of overnight c u l t u r e is sufficient to p r o d u c e cells for 10-30 combustions or s t r i p p i n g s . C u r r e n t l y we are d e v e l o p i n g means for freezing cells so they m a y be stored. Because M C A ranges d o w n to 5-50 μΜ analytes a n d a i r s o l u b i l i t i e s are ~ 1 . 3 m M (dissolved O 2 , 0.3 m M (1)), there is plenty of oxygen a v a i l a b l e at 25° t o ensure aerobicity. Nevertheless, we use one or two r e m i x i n g s i n m i d - r u n ( F i g . 3) t o ensure a e r o b i c i t y i f s a m p l e concentrations are above c a . 50 μ Μ . O x y g e n r e s p i r o m e t r y has sometimes been used as a basis for a n a l y s i s . I n i t i a l l y one m i g h t t h i n k the two m e t h o d s , o x y g e n r e s p i r o m e t r y a n d m i c r o ­ b i a l c a l o r i m e t r y , are equivalent. However, there are very large differences between t h e m i n practice w h e n samples are m i c r o m o l a r i n c o n c e n t r a t i o n . T o a n a l y z e a few m i c r o m o l a r concentrations v i a disappearance of o x y g e n , the oxygen baseline concentrations have to be held i n very n a r r o w toler­ ances, i.e., i n 0.1 t o 5 or 10 μ Μ d u r i n g a n a l y s i s . H o l d i n g t h a t against a b a c k g r o u n d w h i c h (i) is sufficient to m a i n t a i n f u l l aerobicity, a n d (ii) l i k e l y to fluctuate for various reasons, is not easy i n p r a c t i c e . M C A operates o n very different grounds. A n y r o u g h l y fluctuating oxygen c o n c e n t r a t i o n w i l l do, as l o n g as there is a n oxygen s u r p l u s . M C A is c a r b o n - l i m i t e d , not oxygen difference-limited. T h e s t r e n g t h of M C A is t h a t i t uses a r o u g h excess of cells, a r o u g h excess of oxygen, a n d produces a s i g n a l d i r e c t l y

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

39.

LOVRIEN ET AL.

Microbial Calorimetric Analysis

557

proportional to "carbon" (the sample). It is not dependent on detection of small changes in oxygen concentration against fluctuating, large, oxygen concentrations. Lignin itself usually requires days, months, or years to biodegrade. However, oxidized lignin fragments can biodegrade rapidly if adapted bac­ teria are available. Over the years, dozens of microbial calorimetric studies of carbon utilization as a correlate of microbial growth have been carried out by Dermoun et al. (17). Many such papers leave the impression that heat generation commonly requires 8-24 hours to peak. In fact, however, any organism having a doubling time from 20-60 minutes must transport and metabolize carbon in 2-20 minutes. Therefore, oxidized fragments of lignin, and perhaps other lignin degradation products, whatever their source, can mostly be expected to "burn" rapidly, if cells are adapted and there are enough of them to bind all available carbon. A disadvantage of M C M C A takes advantage o them, namely bacterial processing in soil, silage, sewage, and digestion. M C A simply measures their heats using organisms that propel these pro­ cesses on a large scale. A cknowledgment This work was supported by the University of Minnesota Agricultural Ex­ periment Station and by the University Bioprocess Technology Institute. Literature Cited 1. Lovrien, R.; Jorgenson, G.; Ma, M.; Sund, W. Biotech. Bioeng. 1980, 22, 1249-69. 2. Hammerstedt, R. H.; Lovrien, R. E . J. Exp. Zool. 1983, 228, 459-69. 3. Battley, Ε. H. Energetics of Microbial Growth; Wiley-Interscience: New York, 1987; 322-51. 4. Lovrien, R.; Williams, Κ. K.; Ferry, M . L.; Ammend, D. A. Appl. En­ viron. Microbiol. 1987, 53, 2935-41. 5. Parke, D. V. Enzyme Induction; Plenum Press: New York, 1975; Ch. 1-3. 6. Zubay, G . Biochemistry; Addison-Wesley Publ: Reading, M A , 1983; Ch. 26. 7. Fox, B. G.; Lipscomb, J. D. Biochem. Biophys. Res. Comm. 1988, 154, 165-70. 8. Fujisawa, H.; Hayaishi, O. J. Biol. Chem. 1968, 243, 2673-81. 9. Audus, L. J . Nature 1946, 158, 149-50. 10. Ashworth, J . M.; Kornberg, H. L. Proc. Roy. Soc. London Sec. Β 1966, 165, 179-88. 11. Lipsett, S. G.; Johnson, F. M . G.; Maas, O. J. Am. Chem. Soc. 1927, 49, 925-43. 12. Barisas, B. G.; Gill, S. J . Ann. Rev. Phys. Chem. 1978, 29, 141-46. 13. Berka. T . R.; Allenza, P.; Lessie, T. G . Curr. Microbiol. 1984, 11, 143-48.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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14. Stanier, R. Y.; Palleroni, N. J.; Doudoroff, M. J. Gen. Microbiol. 1966, 43, 159-79. 15. Lovrien, R.; Hart, G.; Anderson, K. J. Microbios 1977, 20, 153-72. 16. Anderson, J. J.; Dagley, S. J. Bacteriol. 1980, 143, 525-28. 17. Dermoun, Z.; Boussand, R.; Cotten, D.; Belaich, J. P. Biotech. Bioeng. 1985, 996-1004. RECEIVED April 28, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 40 Microbial Degradation of Tannins and Related Compounds A. M. Deschamps Laboratoire de Microbiologie Alimentaire et de Biotechnologie, Université de Bordeaux I, Avenue des Facultés, 33405 Talence, France

While tannins ar numerous microorganism g degradation, they can nevertheless be degraded by a large variety of microorganisms. Because of their labile galloyl ester structures, hydrolysable tannins are more readily degraded than condensed tannins. This chapter reviews the limited progress made in understanding the potentially useful processes of the biodegradation of hydrolysable and condensed tannins. T a n n i n s are water-soluble phenolic c o m p o u n d s w h i c h are u s u a l l y e x t r a c t e d f r o m p l a n t m a t e r i a l by h o t water. A f t e r l i g n i n s , they are t h e second most a b u n d a n t group o f plant phenolics. T h e i r t a n n i n g p r o p e r t y is due t o t h e i r c a p a c i t y t o combine w i t h proteins. However, they c a n also c o m p l e x w i t h other p o l y m e r s such as alkaloids, cellulose, a n d p e c t i n s . T a n n i n s are u s u a l l y concentrated i n b a r k s , galls o r leaves o f w o o d y plants (1). T a n n i n s are classified i n t o two different groups, hydroxyzable o r condensed, d e p e n d i n g o n t h e s t r u c t u r e o f the p o l y m e r (2). H y d r o l y z a b l e t a n n i n s are c o m p o s e d o f esters o f g a l l i c a c i d (gallotannins) a n d / o r ellagic a c i d (ellagitannins) w i t h a sugar core, p r e d o m i n a n t l y glucose (see G r o s s , t h i s v o l u m e ) . Some o f the most c o m m o n s t r u c t u r e s are d i g a l l o y l 3,6 glucose or t r i g a l l o y l 1,3,6 glucose ( F i g . 1); such structures have been discussed i n detailed review papers b y H a s l a m (1) a n d M e t c h e a n d G i r a r d i n (2). T h e m a j o r c o m m e r c i a l h y d r o l y s a b l e t a n n i n s are e x t r a c t e d f r o m C h i n e s e g a l l (Rhus semialata), s u m a c h (Rhus coriara), T u r k i s h g a l l (Quercus infectoria), t a r a (Caesalpina spinosa), m y r o b o l a m (Terminalia chebula), a n d chestnut (Castanea sativa (1). T h e second group o f t a n n i n s are the condensed t a n n i n s , o r p o l y m e r i c p r o a n t h o c y a n i d i n s (2). These are composed o f flavonoid u n i t s , a n d are more r e c a l c i t r a n t t o biodégradation t h a n h y d r o l y s a b l e t a n n i n s . O f these, t h e 0097-6156/89/0399-0559$06.00/0 © 1989 American Chemical Society

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i m p o r t a n t c o m m e r c i a l ones are e x t r a c t e d f r o m w a t t l e (Acacia mollissima a n d A. mearnsii), a n d quebracho (Schinopsis lorenizii a n d S. balansae). C o n d e n s e d t a n n i n s are u s u a l l y more a b u n d a n t i n tree b a r k s a n d woods t h a n their h y d r o l y z a b l e counterparts (1). T a n n i n s have l o n g been considered to be i n h i b i t o r s of most m i c r o o r g a n i s m s . F o r e x a m p l e , they are s t r o n g i n h i b i t o r s of Azotobacter and n i t r o g e n - f i x i n g (3), n i t r i f y i n g (4,5) a n d sulfate-reducing b a c t e r i a (6). S o i l b a c t e r i a such as Rhizobium are also often sensitive to t a n n i n s . F o r t h i s reason, t a n n i n s generally r e t a r d the rate of d e c o m p o s i t i o n of vegetable m a t t e r (8) v i a i n h i b i t i o n of b i o d e g r a d a t i v e enzymes of the a t t a c k i n g o r g a n i s m . (9,10). F u n g i , such as Fusarium, Verticillium, Aspergillus, AUernaria (11,12), as w e l l as yeasts (13) can also be i n h i b i t e d b y t a n n i n s . H o w e v e r , other related p l a n t phenolics, such as a n t h o c y a n i n s , flavonoids, catechol, etc., can also i n h i b i t these m i c r o o r g a n i s m s (14). T a n n i n s , w h e n p r o v i d e d i n very h i g h concentrations over extended t i m e periods, can also be t o x i c to higher organisms such a their levels are regulated i n m a n y vegetable foods (16). S o m e a u t h o r s have a t t r i b u t e d the presence of t a n n i n s i n b a r k s or leaves to a defense s y s t e m against predators or decomposing organisms (14,16). Nevertheless, m a n y fungi or b a c t e r i a are quite resistant to t a n n i n s a n d can also degrade t h e m . M i c r o b i a l Degradation of Hydrolyzable Tannins In 1913, K n u d s o n (17) reported t h a t t a n n i c a c i d (the c o m m e r c i a l n a m e of the C h i n e s e g a l l t a n n i n ) c o u l d be degraded by a s t r a i n of Aspergillus niger; p r e v i o u s l y the F r e n c h scientist P o t t e v i n (in 1900) h a d n a m e d t h i s enzyme tannase (10). Since t h e n , most of the progress o n e l u c i d a t i n g the m e c h a n i s m s of h y d r o l y s a b l e t a n n i n biodégradation has o c c u r r e d since 1960. Tannase. T h e enzyme tannase, or t a n n i n - a c y l hydrolase ( E C 3:1:1:20), was described a n d purified f r o m strains of Aspergillus niger by H a s l a m et al. (19) a n d D h a r a n d Bose (20). T h i s enzyme s p l i t s the ester linkage of pendant g a l l o y l groups f r o m glucose i n g a l l o t a n n i n s . S u r p r i s i n g l y , the e n z y m e is not i n d u c e d by t a n n i c a c i d , a n d i n A. niger i t is m o s t l y c e l l wall b o u n d or o n l y s l i g h t l y exocellular (20,21). T h e enzyme's o p t i m a l p H is 4-4.5 a n d its o p t i m a l t e m p e r a t u r e is 3 0 ° C . T a n n a s e has been isolated f r o m m y c e l i a l extracts (22) of other Aspergillus species, as w e l l as f r o m various Pénicillium strains (Table I). T a n n a s e has also been detected i n a yeast c u l t u r e , a l t h o u g h its enzyme h a d different o p t i m a , i.e., the o p t i m a l p H was 6 a n d the t e m p e r a t u r e was 4 0 ° C (26). In Candida sp., tannase h a d a M W of 250,000 a n d was composed of two s u b - u n i t s of g l y c o p r o t e i n s t r u c t u r e (31); the tannase of Aspergillus was also of h i g h m o l e c u l a r weight (24). T a n n a s e has also been detected i n b a c t e r i a l cultures (29), where its o p t i m u m a c t i v i t y was p H 5.5 w i t h different strains isolated f r o m decayed barks. Degradation of Gallotannins. Different investigations o n tannase revealed t h a t t h i s enzyme was not e q u a l l y efficient o n a l l h y d r o l y z a b l e t a n n i n s . T h i s was p a r t i c u l a r l y true for yeast, whose tannase was effective o n l y o n t a n n i c

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T a b l e I. M i c r o o r g a n i s m s P r o d u c i n g T a n n a s e ( t a n n i n - a c y l hydrolase) FUNGI: Aspergillus niger. H a s l a m et al. (19); P o u r r a t et al. (21) Aspergillus oryzae: I i b u c h i et al. (23) Aspergillus flavus: A d a c h i et al. (24) Aspergillus japonicus: G a n g a et al. (22) Pénicillium chrysogenum: R a j a k u m a r a n d N a n d y (25) Pénicillium notatum: G a n g a et al. (22) Pénicillium islandicum: G a n g a et al. (22) YEASTS: Candida sp.: A o k i et al. (26) Pichia, several species: J a c o b a n d P i g n a l (27) Debaryomyces hansenii: J a c o b a n d P i g n a l (27) BACTERIA: Achromobacter sp: L e w i s a n d S t a r k e y (28) Bacillus pumilus: D e s c h a m p s et al. (29) Bacillus polymyxa: D e s c h a m p s et al. (29) Corynebacterium sp: D e s c h a m p s et al. (29) Klebsiella planticola: D e s c h a m p s et al. (29) Pseudomonas solanacearum: M u t h u k u m a r a n d M a h a d e v a n (30)

a c i d , but not o n n a t u r a l t a n n i n s such as chestnut, oak, m y r o b o l a n or t a r a (27). F u r t h e r , w h i l e A o k i et al. (26,31) were able t o degrade t a n n i c a c i d u s i n g Rhodotorula rubra, i t was o n l y w e a k l y a c t i v e o n chestnut t a n n i n (32). O n the other h a n d , f u n g a l tannases efficiently degrade h y d r o l y s a b l e t a n n i n s . T h i s has been s h o w n by the d e g r a d a t i o n of a m l a (33) a n d m y r o b o l a n (34) t a n n i n s w i t h enzymes f r o m Aspergillus niger, a n d chestnut t a n n i n s w i t h enzymes f r o m different f u n g i (28). T h i s p r o p e r t y is c u r r e n t l y used i n the d e t o x i f i c a t i o n of tea t a n n i n e x t r a c t s u s i n g a n i n d u s t r i a l l y p r o duced e n z y m e f r o m A. niger. B a c t e r i a l tannase l i t e r a t u r e is l i m i t e d to the i s o l a t i o n of a n Achromobacter capable of d e g r a d i n g g a l l o t a n n i n f r o m Chinese g a l l ( L e w i s a n d S t a r k e y , 1969 (28)) a n d our own papers (29,30). I n 1980 (35), we des c r i b e d a collection of t a n n i c a c i d d e g r a d i n g b a c t e r i a , m o s t l y f r o m Bacillus, Corynebacterium a n d Klebsiella s t r a i n s , w h i c h c o u l d degrade n a t u r a l t a n n i n s such as those f r o m chestnut a n d t a r a . Indeed, because of the s i m ple s t r u c t u r e of the g a l l o t a n n i n i n t a r a species, t h i s represents a p o t e n t i a l source of g a l l i c a c i d (36). T h e d e g r a d a t i o n of m y r o b o l a n t a n n i n was also r e p o r t e d for Pseudomonas solanacearum (30). Degradation of Gallic Acid. G a l l i c a c i d , a n o b l i g a t e i n t e r m e d i a t e i n the d e g r a d a t i o n of g a l l o t a n n i n s , is degraded b y some b a c t e r i a such as Pseudomonas (37). In our l a b o r a t o r y , b o t h Klebsiella pneumoniae (38) a n d K. planticola s t r a i n s grew on t a n n i n s a n d u t i l i z e d gallic a c i d as a c a r b o n source (35). T h e same c a p a c i t y was observed for Citrobacter species where p y r o -

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g a l l o l a c c u m u l a t e d as a m e t a b o l i c end p r o d u c t . K o s h i d a a n d Y a m a d a (39) recently patented this m e t h o d for p y r o g a l l o l p r o d u c t i o n . W h i l e gallic a c i d is p r o b a b l y u t i l i z e d b y f u n g i a n d yeasts as a carb o n source, o n l y a few papers have suggested t h i s p o s s i b i l i t y (25,40,41), i n c l u d i n g one for Aspergillus flavus (42). Microbial Degradation of Condensed Tannins C o n d e n s e d t a n n i n s were considered to be h i g h l y r e c a l c i t r a n t to biodégrad a t i o n u n t i l B a s a r a b a (3) r e p o r t e d t h a t some b a c t e r i a l isolates c o u l d u t i l i z e w a t t l e t a n n i n as b o t h a c a r b o n a n d energy source. L a t e r , L e w i s a n d S t a r k e y (28) isolated a s t r a i n of Pseudomonas w h i c h degraded c a t e c h i n , a n d s t r a i n s of Aspergillus a n d Pénicillium w h i c h degraded w a t t l e t a n n i n . Fungal Degradation of Condensed Tannins. C h a n d r a et al. (43) first rep o r t e d the i s o l a t i o n of f u n g i terreus a n d various Pénicillium n i n s e x t r a c t e d f r o m apple w o o d . These observations were e x t e n d e d b y G r a n t (44), u s i n g a s t r a i n of P. adametzi capable of d e g r a d i n g d i - a n d t r i p r o c y a n i d i n s t r u c t u r e s , as well as (+) catechin a n d a crude t a n n i n e x t r a c t . S o m e u n u s u a l w h i t e - r o t f u n g i , identified as Bispora betulina a n d Isaria sp., were also able to degrade condensed t a n n i n s e x t r a c t e d f r o m Douglas-fir bark (45). Recently, S i v a s w a m y a n d M a h a d e v a n (46) r e p o r t e d the d e g r a d a t i o n of wattle t a n n i n by a s t r a i n of Aspergillus niger. Interestingly, i t also p r o d u c e d tannase a n d c o u l d degrade the g a l l o t a n n i n , m y r o b o l a n . T h e edible puffball Calvatia gigantea can also degrade b o t h h y d r o l y z a b l e (chestn u t ) a n d condensed (wattle) t a n n i n s (47), as well as c a t e c h i n . It has been proposed t h a t this o r g a n i s m c o u l d be used for d e t o x i f i c a t i o n purposes, or for the v a l o r i z a t i o n of h i g h - t a n n i n vegetable residues. Yeasts have been described for the d e g r a d a t i o n of w a t t l e t a n n i n (48). T h i s d e g r a d a t i o n was d e t e r m i n e d by the e s t i m a t i o n of l e u c o a n t h o c y a n i d i n a n d flavan-3-ol groups ( F i g . 2) i n the r e m a i n i n g degraded t a n n i n . A m o n g the s t r a i n s (Candida guilliermondii, C. tropicalis, Torulopsis Candida) isolated a n d s t u d i e d , the simultaneous d e g r a d a t i o n of b o t h s t r u c t u r e s was not observed, suggesting different mechanisms of d e g r a d a t i o n . F o r e x a m p l e , a s t r a i n o f C. guilliermondii degraded the flavan-3-ol s t r u c t u r e s b u t d i d not affect the l e u c o a n t h o c y a n i d i n components. M o s t yeasts were efficient degraders of quebracho t a n n i n s (32) a n d reduced the t a n n i n content of pine a n d g a b o o n w o o d b a r k extracts b y 70 to 8 0 % i n five days. Bacterial Degradation of Condensed Tannins. A l t h o u g h B a s a r a b a (5) p r o posed t h a t b a c t e r i a c o u l d degrade condensed (wattle) t a n n i n , t h i s t o p i c has o n l y been investigated i n our l a b o r a t o r y i n C o m p i e g n e . In these studies, our objective was to c o n t r o l the rate of biodégradation of t a n n i n - r i c h b a r k s , such as those f r o m pine a n d g a b o o n - w o o d (Acoumea kleneana). Consequently, we succeeded i n i s o l a t i n g , b y c u l t u r e e n r i c h m e n t , b a c t e r i a capable of d e g r a d i n g a n d u t i l i z i n g these b a r k s (49), as w e l l as quebracho (Schinopsis lorentzii) a n d wattle (Acacia mollissima) t a n n i n s . A collection of various genera a n d species was identified i n these studies (50), w i t h the genera

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F i g u r e 2. (a) L e u c o a n t h o c y a n i d i n a n d (b) R ' a n d R " = H or O H .

flavan-3-ol

s t r u c t u r e s , where R ,

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Bacillus, Klebsiella, Corny bacterium, a n d Pseudomonas b e i n g the most frequently observed. T h e c a p a c i t y of these b a c t e r i a to degrade t a n n i n s , a n d detoxify b a r k chips or b a r k s e x t r a c t s , was further d e m o n s t r a t e d w i t h pine (Pinus maritima) (51), oak (Quercus pedonculata) and gaboon wood (Aucoumea kleneana) b a r k s (52). T h e d e g r a d a t i o n of quebracho a n d w a t t l e t a n n i n s was also confirmed i n pure cultures (53). Microbial Degradation of Catechin. Since (+) catechin is a possible biodégradation p r o d u c t f r o m condensed t a n n i n s , i t s u t i l i z a t i o n a n d b i o c o n version have been extensively e x a m i n e d b y several research groups u s i n g f u n g i , b a c t e r i a a n d yeasts. Fungi. F u n g i - d e g r a d i n g c a t e c h i n have been k n o w n for about twenty years, e.g., Aspergillus niger, A. terreus, A. fumigatus, A. flavus a n d Pénicillium sp. (54) a n d Pénicillium adametzi (44). In the l a t t e r case, the o r g a n i s m grew o n catechin a n d a n t h o c y a n i d i n m o d e l c o m p o u n d s as sole carb o n sources. Interestingly also degraded c a t e c h i n . T o account for its biodégradation, C h a n d r i et al. (54) first suggested t h a t an e x t r a c e l l u l a r enzyme must be i n v o l v e d , f o l l o w i n g w h i c h the enzyme catechin 2,3-dioxygenase was isolated f r o m Chaetomium cupreum (56). T h e molecular weight ( M W ) of t h i s g l y c o p r o t e i n was a p p r o x i m a t e l y 40,000 a n d catechin was cleaved v i a meta-nng fission. Bacteria. M a n y b a c t e r i a degrade catechin a n d use it as a c a r b o n source, as s h o w n first by L e w i s a n d S t a r k e y (28). In 1982 M u t h u k u m a r et al. isolated several strains of Rhizobium a n d Bradyrhizobium (57), i n c l u d i n g B. japonicum w h i c h degraded catechin. T h i s last species p r o d u c e d the same enzyme as C. cupreum ( W a h e e t a a n d M a h a d e v a n , p e r s o n a l c o m m u n i c a t i o n ) . W e also d e m o n s t r a t e d t h a t c e r t a i n t a n n i c - a c i d d e g r a d i n g b a c t e r i a can also degrade catechin (35). R e c e n t l y , B a o m i n a t h a n a n d M a hadevan showed t h a t catechin-degrading m a c h i n e r y was p l a s m i d borne i n Pseudomonas solanacearum (58), w h i c h also produces a catechin oxidase. Yeasts. A n interesting paper p u b l i s h e d i n 1984 (59) c l a i m e d t h a t r a t caecal m i c r o f l o r a degraded c a t e c h i n . T o our knowledge no paper has dealt w i t h yeasts, other t h a n t h a t some yeasts d e g r a d i n g w a t t l e of quebracho t a n n i n s were able to grow weakly w i t h catechin as a sole c a r b o n source (32). Concluding Remarks T h i s review of l i t e r a t u r e on t a n n i n d e g r a d a t i o n shows t h a t our knowledge of t h i s t o p i c is o n l y very slowly i m p r o v i n g . O n l y a h a n d f u l of laboratories are c u r r e n t l y i n v o l v e d i n this area. O f these, the I n d i a n laboratories have m a d e several i n t e r e s t i n g investigations recently, p r e s u m a b l y because they are very active i n leather manufacture a n d need to c o n t r o l the t o x i c i t y of their tannery effluents. Some m i c r o o r g a n i s m s d e g r a d i n g condensed t a n n i n s have been isolated and described, but no reports on the m e c h a n i s m of the d e p o l y m e r i z a t i o n process, or the enzymes involved i n biodégradation, have a p p e a r e d . It must be stressed t h a t condensed t a n n i n d e g r a d a t i o n m a y be associated w i t h other

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detoxification mechanisms, other than those operative for catechin. This assertion is made since many strains which grow on condensed tannins do not grow on catechin. This is unusual since this compound is often viewed as an intermediate of biodégradation. Consequently, catechin degradation should not be confused with condensed tannin degradation. More studies are needed, if we are to understand and elucidate the mechanism of the degradation of condensed tannins. Currently, there is insufficient data available for comparison of this degradation process to that of other polyphenols, such as lignin. Further, while the degradation mechanisms of tannic and gallic acids are quite well understood for bacteria and fungi, few commercial applications have yet resulted, e.g., in the production of the enzyme tannase or the bioconversion of gallic acid (or tannins) to pyrogallol (60). In the case of the condensed tannins, however, their biodégradation has been much less thoroughly studied. Suc ification of tannins will continu (61), as well as in biotechnological processes using barks as lignocellulosic substrates (62). For these reasons, the isolation and identification of tannindegrading enzymes, and the determination of their mechanism of action, are of great importance. Literature Cited 1. Haslam, E . In Biochemistry of Plant Phenolics; Plenum Press: New York, 1979, 475-523. 2. Metche, M.; Girardin, M. In Les PolymeresVegetaux; Monties, B., Ed.; Gauthiers Villars: Paris, 1980. 3. Basabara, J. Can. J. Microbiol. 1966, 12, 787-794. 4. Rice, E. L.; Pancholy, S. K.; Am. J. Bot. 1973, 60, 691-702. 5. Basabara, J. Plant Soil 1964, 21, 8-16. 6. Booth, G . H. J. Appl. Bacteriol. 1960, 23, 125-129. 7. Muthukumar, G.; Mahadevan, A. Leather Sci. 1983, 30, 263-269. 8. Benoit, R. E.; Starkey, R. L. Soil Sci. 1968, 105, 203-208. 9. Goldstein, J. L.; Swain, T . Phytochem. 1965, 4, 185-192. 10. Benoit, R. E.; Starkey, R. L. Soil Sci. 1968, 105, 203-208. 11. Lewis, J. Α.; Papavizas, G . C. Can. J. Microbiol. 1967, 13, 1655-1661. 12. Mahadevan, Α.; Muthukumar, G. Hydrobiol. 1980, 72, 73-79. 13. Jacob, F. H.; Pignal, M . C. Mycopathol. Mycol. Appl. 1972, 48, 121142. 14. Mahadevan, A. J. Sci. Indian Res. 1974, 33, 131-138. 15. Cameron, G. R.; Milton, R. F.; Allen, J. W. Lancet 1943, 14, 179-186. 16. MacLeod, M . N. Nutr. Abs. Rev. 1974, 44, 803-815. 17. Knudson, L. J. Biol. Chem. 1913, 14, 159-184. 18. Pottevin, H. Compt. rend. 1900, 131, 1215-1217. 19. Haslam, E.; Haworth, R. D.; Jones, K.; Rogers, H. J . J. Chem. Soc. 1961, 1821-1835. 20. Dhar, S. C.; Bose, S. M . Leather Sci. 1964, 11, 27-38.

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21. Pourrat, H. Regerat, F.; Pourrat, Α.; Jean, D. Biotechnol. Lett. 1982, 4, 583-588. 22. Ganga, P.S., Nandy, S. C.; Santappa, M . Leather Sci. 1977, 24, 8-16. 23. Iibuchi, S.; Minoda, Y.; Yamada, K. Agr. Biol. Chem. 1972, 36, 15531562. 24. Adachi, O.; Watanabe, M.; Yamada, H. Agr. Biol. Chem. 1968, 32, 1079-1085. 25. Rajakumar, G . S.; Nandy, S. C. Appl. Environ Microbiol. 1983, 46, 525-527. 26. Aoki, K.; Shinke, R.; Nishara, H. Agr. Biol. Chem. 1976, 40, 79-85. 27. Jacob, F. H.; Pignal, M . C . Mycopathol. 1975, 57, 139-148. 28. Lewis, J . Α.; Starkey, R. L. Soil Sci. 1969, 107, 235-241. 29. Deschamps, A. M . ; Otuk, G.; Lebeault, J . M . J. Ferment. Technol. 1983, 61, 55-59. 30. Muthukumar, G.; Mahadevan 1085. 31. Aoki, K.; Shinke, R.; Nishara, H. Agr. Biol. Chem. 1976, 40, 297-302. 32. Deschamps, A. M.; Leulliette, L. Int. Biodeterior. Bull. 1984, 20, 237240. 33. Srivastava, S. K.; Sharma, S. K. Indian J. Plant Physiol. 17, 47-52. 34. Freudenberg, K.; Blummel, F.; Frank, T . Z. Physiol. Chem. 1927, 164, 262. 35. Deschamps A. M.; Mahoudeau, G.; Conti, M.; Lebeault, J . M . J. Fer­ ment. Technol. 1980, 58, 93-97. 36. Deschamps, A. M.; Lebeault, J . M. Biotechnol. Lett. 1974, 6, 237-242. 37. Trevors, J . T.; Basabara, J . FEMS Microbiol. Lett. 1980, 7, 307-309. 38. Deschamps, A. M.; Richard, C.; Lebeault, J . M . Ann. Microbiol. 1983, 134A, 189-196. 39. Yoshida, H.; Yamada, H. Agr. Biol. Chem. 1985, 49, 659-663. 40. Rajakumar, G.; Nandy, S. C. Leather Sci. 1986, 33, 220-227. 41. Dalvesco, G.; Fiusello, N.; Veittiranus, M . Allonis 1972, 17, 25-40. 42. Gurujeyalakshmi, G.; Mahadevan, A. Zentralbl. Mikrobiol. 1987, 142, 187-192. 43. Chandra, T.; Krishnamurthy, V.; Madhavakrishana, W.; Nayudamma, Y. Leather Sci. 1973, 20, 269-273. 44. Grant, W. D. Science 1976, 193, 1137-1139. 45. Ross, W. D.; Corden, M . E . Wood Fibers 1976, 6, 2-12. 46. Sivaswamy, S. N.; Mahadevan, A. J. Environ. Biol. 1985, 6, 271-278. 47. Galiotou-Panayotou, M.; Macris, B. J . Appl. Microbiol. Biotechnol. 1986, 23 , 502-506. 48. Otuk, G.; Deschamps, A. M . Mycopathol. 1983, 83, 107-111. 49. Deschamps, A. M.; Mahoudeau, G.; Leulliette, L.; Lebeault, J. M . Rev. Ecol. Biol. Sol. 1980, 17, 577-581. 50. Deschamps, A. M . Eur. J. Forest Pathol. 1982, 12, 252-257. 51. Deschamps, A. M.; Gillie, J . P.; Lebeault, J . M . Eur. J. Appl. Micro­ biol. Biotechnol. 1981, 13, 222-225. 52. Deschamps, A. M.; Leulliette, L. Phytopathol. Z. 1985, 113, 304-310.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Microbial Degradation of Tannins

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53. Deschamps, A. M . Can. J. Microbiol. 1985, 31, 499-502. 54. Chandra, T.; Madhavakrishna, W. Nayudamma, Y . Can. J. Microbiol. 1969, 15, 303-306. 55. Elkins, J. R.; Pate, W.; Lewis, J.; Porterfield, C. III Int. Congr. Plant Path. (Abstract), 1978. 56. Sivaswamy, S. N., Mahadevan, A. J. Indian Bot. Soc. 1986, 65, 95-100. 57. Muthukumar G.; Arunakumari, Α.; Mahadevan, A. Plant Soil 1982, 69, 163-169. 58. Boominathan, K.; Mahadevan, A. FEMS Microbiol. Lett. 1987, 40, 147-150. 59. Groenewoud, G.; Hundt, H. K. L. Xenobiotica 1984, 14, 711-717. 60. Kikuchi, M.; Katsumo, Y.; Mizusawa, K. Japanese patent JP 61, 108393 (86,108,383), 1986. Kokai Tokkyo Koho. 61. Marakis, S. Cryptog. Mycol. 1986, 6, 293-308. 62. Deschamps, A. M . Biotechnological wastes. Int. Symp. Woo RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 41 Specific A s s a y s , P u r i f i c a t i o n , a n d Study of S t r u c t u r e — A c t i v i t y R e l a t i o n s h i p s o f C e l l u l o l y t i c Enzymes 1

2

P. Tomme, V. Heriban , H. Van Tilbeurgh, and M . Claeyssens

Laboratory for Biochemistry, Faculty of Sciences, State University of Ghent (RUG), K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium

Differentiating activit (all specific for β-1,4 glucosidic linkages) are described using chromogenic derivatives of lactose and the cel­ lodextrins. They proved to be valuable not only in specificity studies of these enzymes, but also as active site probes. An affinity chromatographic method for rapid isolation of some of these enzymes was also de­ veloped. New information on the domain structure of two cellobiohydrolases from Trichoderma reesei (CBH I and CBH II) resulted from structure-function investiga­ tions based on partial proteolysis and physical measure­ ments. A cellulose-binding domain (C- or N-terminal) was separated from the core-enzymes, containing the ac­ tive (hydrolytic) sites. In the "tadpole" structure of the intact enzymes—as deduced from small-angle X-ray scattering—the binding peptides protrudes from their cores as flexible tails. Study of adsorption onto and hydrolysis of microcrystalline cellulose ("Avicel") led to new insights into the functional role of these domains and the synergism observed between CBH I and CBH II. N a t i v e o r processed cellulose (e.g., c o t t o n , A v i c e l , filter p a p e r ) a n d i t s s o l ­ uble derivatives (e.g., C M C , H E C ) are substrates most often used i n t h e s t u d y o f cellulases. T h e classification based o n the use o f these substrates ( l , 4 - / ? - D - g l u c a n cellobiohydrolases ( C B H ) , exo-cellulases, " A v i c e l a s e s " a n d 3

N O T E : A list of abbreviations and symbols is given at the end of the text. O n leave from the Technical University Bratislava, Chemical-Technological Faculty, Bratislava, Czechoslovakia Address correspondence to this author.

1

2

0097-6156/89/0399-0570$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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l , 4 - / ? - D - g l u c a n glucanohydrolases, endo-cellulases, " c a r b o x y m e t h y l c e l l u lases" ) is s o m e w h a t a r b i t r a r y . Since the s t r u c t u r e ( c r y s t a l l i n i t y ) a n d c o m p o s i t i o n (degree o f p o l y ­ m e r i z a t i o n ) of the cellulosic substrates is r e l a t i v e l y u n k n o w n a n d the as­ says ( r e d u c t o m e t r y , v i s c o s i m e t r y ) are r a t h e r unspecific a n d s o m e t i m e s lack s e n s i t i v i t y , we have i n t r o d u c e d a l t e r n a t i v e c h r o m o p h o r i c (fluorophoric) substrates c o n t a i n i n g l i g a n d s of k n o w n m o l e c u l a r p r o p e r t i e s (1), e.g., 2'c h l o r o , 4 - n i t r o p h e n y l a n d 4 - m e t h y l - u m b e l l i f e r y l /?-D-glycosides, o b t a i n e d v i a O - g l y c o s i d a t i o n (2). 1-Thioglycosides were p r e p a r e d f r o m the sugar m e r c a p t a n s a n d the a p p r o p r i a t e halogen derivatives (e.g., 1-chloro, 2 , 4 d i n i t r o b e n z e n e ) (3). /

Materials and

/

Methods

E n z y m i c h y d r o l y s i s ( 2 5 - 4 0 ° C ) at the heterosidic b o n d of the c h r o m o g e n i c substrates was followed eithe n i t r o p h e n o l ) at p H 5.5 ( O . D . 405 n m ) or d i s c o n t i n u o u s l y ( 4 - m e t h y l u m b e l liferone fluorescence at p H 10, emission at λ > 435, e x c i t a t i o n at Λ366 n m ) . R e a c t i o n rates were c a l c u l a t e d f r o m the l i n e a r increase o f O . D . (εM = 9000 M " " c m " ) or fluorescence ( s t a n d a r d i z a t i o n w i t h 4 - m e t h y l u m b e l l i f e r o n e ) versus t i m e . A l t e r n a t i v e l y , a n H P L C m e t h o d was used to follow the f o r m a ­ t i o n of c h r o m o p h o r i c r e a c t i o n p r o d u c t s , phenols a n d glycosides (1). C o n ­ c e n t r a t i o n s were c a l c u l a t e d f r o m peak heights after a p p r o p r i a t e s t a n d a r d ­ ization. G l u c o s e was d e t e r m i n e d b y the glucose oxidase-peroxidase m e t h o d . C e l l o b i o s e ( l i b e r a t e d e n z y m a t i c a l l y f r o m m e t h y l c e l l o t r i o s i d e ) was deter­ m i n e d i n a c o u p l e d assay u s i n g cellobiose dehydrogenase f r o m Sporotrichum thermophile (4). Difference s p e c t r o p h o t o m e t r y or fluorescence q u e n c h i n g techniques were used to measure the b i n d i n g of several c h r o m o p h o r i c l i g a n d s to c e l l u l o l y t i c enzymes (5). A d i a f i l t r a t i o n technique (forced-flow d i a l y s i s ) was a d a p t e d to measure b i n d i n g isotherms a n d constants (6). E q u i l i b r i u m b i n d ­ i n g parameters o f n o n - c h r o m o p h o r i c ligands (cellooligosaccharides) were de­ t e r m i n e d b y d i s p l a c e m e n t ( c o m p e t i t i o n ) e x p e r i m e n t s (5) or b y m e a s u r i n g the p r o t e i n U V - a b s o r b a n c e difference s p e c t r u m (7). 1

1

Results Detection of Cellulolytic Activities in Gels. F l u o r o g e n i c m e t h y l u m b e l l i f e r y l derivatives of lactose a n d several c e l l o d e x t r i n s were used t o detect cellulases, after isoelectric f o c u s i n g o f crude or p a r t i a l l y p u r i f i e d p r e p a r a t i o n s f r o m Trichoderma sp. (8), Pénicillium pinophilum (9) a n d Clostridium thermocellum endoglucanases cloned i n E. coli (10). T h e l i b e r a t i o n o f the s t r o n g l y fluorescing 4 - m e t h y l u m b e l l i f e r o n e b e c a m e r e a d i l y v i s i b l e after flooding the gel w i t h a buffered s o l u t i o n o f the a p p r o p r i a t e s u b s t r a t e . T h e same t e c h n i q u e was also a p p l i c a b l e for development of S D S - P A G E c h r o m a t o g r a m s . T h e results are s u m m a r i z e d i n T a b l e I. B o t h the lactoside a n d the cellobioside were substrates o f C B H I (Tr. r., P. p.), E G I (Tr. r.) a n d o f

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E G C a n d E G D (C. t.). W h i l e C B H I a n d E G I f r o m Tr. r . , showed very s i m i l a r specificities, they c o u l d be differentiated b y a d d i n g s m a l l a m o u n t s o f cellobiose, a s t r o n g l y c o m p e t i t i v e i n h i b i t o r of the cellobiohydrolase ( F i g . 1). T h e cellotrioside allowed detection of E G III a c t i v i t i e s i n the c u l t u r e filtrate of Tr. r. i n spite of other a c t i v i t i e s being present; the a d d i t i o n of s m a l l a m o u n t s of gluconolactone effectively suppressed glucosidase a c t i v i t y . T a b l e I. C o n t i n u o u s A s s a y s for C e l l u l o l y t i c E n z y m e s Enzyme Glycosides

Trichoderma

Clostridium

(Chromophore) CBHI (CNP/MeUmb)

C B H II

EG I

E G III

EGA

EGB

EGC

EGD

Lactoside

-f

—

Lactoside

—

—

-f

—

_

_

_

_

Cellobioside

+

-

+

-

_

-

+

+

Cellotrioside

—

—

—

-f

—

+

+

—

(+

G ) 2

Activity Measurements in Solution. T h e 2'-chloro, 4 ' - n i t r o p h e n y l / ? - D glycosides offer a n a t t r a c t i v e a l t e r n a t i v e to classical r e d u c t o m e t r i c m e t h o d s . T h e s u b s t r a t e is sufficiently stable ( p H 5.5, 5 0 ° C ) a n d the favorable a b s o r p ­ t i o n characteristics o f the l i b e r a t e d p h e n o l ( p K = 5.5, £ M 9 0 0 0 M ~ c m " " , p H 5.5; £M 16000 at p H 6.5) allow sensitive, continuous measurements. K i ­ netic parameters for some of these substrates a n d enzymes were d e t e r m i n e d : Κ values were i n the m M range for the lactoside ( C B H I, E G I, E G D ) a n d were at least 10 times lower for the cellobioside; turnover n u m b e r s r a n g e d f r o m 1 ( C B H I, cellobioside) to 300 m i n " ( E G D , cellobioside) ( 2 5 ° C ) . F u r t h e r differentiation was o b t a i n e d u s i n g the c h r o m o p h o r i c cell o o l i g o d e x t r i n s a n d a r a p i d a n d sensitive H P L C a n a l y s i s of the r e a c t i o n p r o d u c t s (1). Specific d e g r a d a t i o n patterns were o b t a i n e d for several e n ­ zymes such as those f r o m Clostridium cloned i n E. coli ( F i g . 2). A w o r d of c a u t i o n is a p p r o p r i a t e , however. T h i s was because unspecific cleavage at the heterosidic b o n d of these substrates was sometimes n o t i c e d , and the specificity was therefore not always e x a c t l y reflected. 1

1

1

Binding Experiments. Some o f these c h r o m o p h o r i c glycosides proved also to be v a l u a b l e i n cases where no h y d r o l y s i s b y the cellulases o c c u r r e d . T h i s was s h o w n i n the case where the 4 - m e t h y l u m b e l l i f e r y l glucoside a n d cel­ lobioside were not h y d r o l y z e d b y the C B H II f r o m Trichoderma (see above) b u t c o u l d be used as reporter ligands i n a series o f b i n d i n g e x p e r i m e n t s . T y p i c a l l y the fluorescence of the cellobioside was quenched i n the presence of C B H II a n d was restored b y the a d d i t i o n of excess a m o u n t s of n o n c h r o m o p h o r i c l i g a n d s , i.e., cellobiose ( F i g . 3). T h u s association constants

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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F i g u r e 1. A n a l y t i c a l isoelectric f o c u s i n g of cellulases f r o m Trichoderma reesei. D e t e c t i o n of C B H I a n d E G I a c t i v i t i e s u s i n g M e U m b L a c , i n the a b sence ( A ) a n d presence ( B ) o f 10 m M cellobiose. L a n e 1, E G I; lane 2, E G I (iso-components); lane 3, C B H I ( p i 3.9 c o m p o n e n t ) ; lane 4, E G I - C B H I m i x t u r e ) . G e l s were flooded w i t h the fluorogenic s u b s t r a t e ( p H 5.0) a n d after 5-10 m i n ( r o o m t e m p e r a t u r e ) p h o t o g r a p h e d ( P o l a r o i d 57, green filter) o n a l o n g wavelength U V - t r a n s i l l u m i n a t o r (8).

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i:4 g l u c o p y r a n o s y l ; ·, 4 - m e t h y l u m b e l l i f e r y l ) a n d the a r r o w s i n d i c a t e scission p o i n t s as d e t e r m i n e d b y H P L C (1). n

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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340

360

380 X(nm)

400

420

F i g u r e 3. Q u e n c h i n g o f the fluorescence s p e c t r u m of M e U m b G 2 b y C B H II f r o m Trichoderma reesei (5). C u r v e A represents the M e U m b G 2 (2 μΜ) s p e c t r u m i n the absence of C B H II. C u r v e s B , C a n d D show the s p e c t r a after the a d d i t i o n o f several aliquots of 137.7 μΜ C B H II. S p e c t r u m D changes to Ε w h e n s o l i d cellobiose ( ± 2 mg) is a d d e d . W h e n c o r r e c t i o n is m a d e for d i l u t i o n , s p e c t r u m Ε is equivalent t o s p e c t r u m A . C u r v e s F a n d G represent buffer a n d p r o t e i n b l a n k s , respectively. S p e c t r a were measured at p H 5.0 a n d 6.6°C.

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a n d t h e r m o d y n a m i c b i n d i n g parameters of n o n - c h r o m o p h o r e s were c o m ­ p u t e d f r o m displacement t i t r a t i o n s , a n d a h y p o t h e t i c a l b i n d i n g scheme for C B H II was proposed (5). T h e b i n d i n g of c h r o m o p h o r i c 1-thioglycosides f r o m lactose or cellobiose to C B H I ( T r . r.) was followed b y difference s p e c t r o p h o t o m e t r y o f the l i g a n d , or b y the d i a f i l t r a t i o n technique ( F i g . 4). A l t e r n a t i v e l y , p e r t u r b a t i o n o f the p r o t e i n s p e c t r u m c o u l d be used i n the case of n o n - c h r o m o p h o r i c l i g a n d s ( F i g . 5). A n Affinity Chromatographic Some Cellulolytic Enzymes

Method

for t h e

Purification of

A considerable affinity of some c e l l u l o l y t i c enzymes (e.g., C B H I a n d C B H II f r o m Tr. r. a n d the E G ' s f r o m C. i.) for a r y l 1-0- a n d 1-S-cellobiosides was evidenced b y the K a n d Κ,· values (see above). T h i s contrasted w i t h the higher K , values ( t y p i c a l l y f r o m Tr. r . ) . C o u p l i n g o f a n a r y l 1-S-cellobioside to a n affinity carrier was therefore expected to be useful i n the c h r o m a t o g r a p h i c f r a c t i o n a t i o n of " e n d o " a n d "exo" enzymes, e.g., f r o m Tr. r. P r e l i m i n a r y tests i n d i c a t e d t h a t C B H I a n d C B H II (prepurified b y ion-exchange c h r o m a t o g r a p h y ) were c o m p l e t e l y r e t a i n e d b y the affinity s u p p o r t ( 4 ' - a m i n o b e n z y l 1-S-cellobioside c o u p l e d to Affigel-10 f r o m B i o r a d ) . D e s o r p t i o n was achieved differentially b y 0.1 M lactose (elutes C B H I) a n d 0 . 0 1 M cellobiose (elutes C B H I a n d C B H II). A t t e m p t s to elute the enzymes w i t h 1 M K C 1 , e t h y l e n e g l y c o l or glucose s o l u t i o n s were unsuccessful (11). T h u s a biospecific a d s o r b a n t was o b t a i n e d ; selective d e s o r p t i o n s h o u l d therefore p e r m i t successive e l u t i o n of c e l l u l o l y t i c e n z y m e s . It was t h u s f o u n d t h a t the m e t h o d was very useful i n the p u r i f i c a t i o n of the c e l l o b i o ­ hydrolases f r o m Tr. r . , s t a r t i n g f r o m very crude c u l t u r e filtrates. A d d i t i o n of glucose ( 0 . 1 M ) or gluconolactone suppressed the a c t i o n of glucosidases present, p r e v e n t i n g d e t e r i o r a t i o n of the c o l u m n s . T h e capacities exceeded 10 m g C B H I per m l gel (prepared w i t h C N B r a c t i v a t e d Sepharose). S i m i l a r results were o b t a i n e d w i t h crude cellulase p r e p a r a t i o n s f r o m Pénicillium pinophilum (12). T h e general a p p l i c a b i l i t y of t h i s biospecific c h r o m a t o g r a p h y is i l l u s t r a t e d b y the i s o l a t i o n of the E G D f r o m C. cloned i n E. c. ( F i g . 6). m

S t r u c t u r a l - F u n c t i o n a l Investigations B a s e d o n P a r t i a l P r o t e o l y s i s and Physical Measurements T h e p r i m a r y s t r u c t u r e of several c e l l u l o l y t i c enzymes has been e l u c i d a t e d a n d t h i s research has been further encouraged b y the successful c l o n i n g a n d expression of heterologuous genes i n r a p i d g r o w i n g host m i c r o o r g a n i s m s , such as yeasts a n d some b a c t e r i a (e.g., 13,14). C o m p a r i s o n of the k n o w n a m i n o a c i d sequences of several cellobiohydrolases a n d endoglucanases of Trichoderma reesei showed t h a t a l l these enzymes shared a h o m o l o g u o u s sequence of a m i n o acids ( A ) separated f r o m the cores of these enzymes b y

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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T O M M E ET A L

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Cellulolytic Enzymes

F i g u r e 4. D e t e r m i n a t i o n of b i n d i n g constants of 2 ' , 4 ' - d i n i t r o p h e n y l l-S-βcellobioside for C B H II by d i a f i l t r a t i o n ( 4 ° C ) . ( A ) T h e f i l t r a t e is collected i n fractions a n d the c o n c e n t r a t i o n of free l i g a n d (Lj) is d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y (313 n m , SM = 12000 M ~ c m " ) . T h e l i g a n d c o n c e n t r a t i o n i n the stock s o l u t i o n is 200 μ Μ . T h e a m o u n t o f b o u n d l i g a n d is c o m p u t e d f r o m a b l a n k (curve I) a n d a b i n d i n g e x p e r i m e n t (curve II) (6). 1

1

(B) Scatchard plot:

= nK

— vK,

ν is the degree of s a t u r a t i o n as c a l ­

c u l a t e d f r o m the a m o u n t of b o u n d l i g a n d a n d p r o t e i n (170 μ Μ ) i n the d i a l y s i s cell; K, the a s s o c i a t i o n c o n s t a n t ; n , the n u m b e r o f b i n d i n g sites. In t h i s case η -

0.97 ± 0 . 0 6 a n d Κ = 2.7 ± 0 . 1 χ

lO ^- . 5

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aooAr

0.002 h




FUSION

COOH

CATALYTIC DOMAIN CenA

COOH

BINDING DOMAIN

F i g u r e 5. S c h e m a t i c of the s t r u c t u r e of the C e x - C e n A fusion p r o t e i n . s t i p p l e d areas are P r o - T h r boxes.

CATALYTIC DOMAIN Cex

BINDING DOMAIN

CenA

CATALYTIC DOMAIN

Cex

The

COOH

42.

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595

E. coli had a M of 110 kDa. It also could be bound to Avicel, but its structural relatedness to Cex and CenA has not yet been determined. A deletion mutant of cenB encoded a polypeptide of M 70 kDa. The missing segment represented the carboxyl terminal 40 kDa or CenB. The 70 kDa fragment had enzymatic activity and it could still bind to substrate (11). The nature and function of the 40 kDa carboxyl terminus of CenB are being determined. r

r

Similarity of C. fimi Cellulases to Trichoderma reesei Cellulases Analysis of the genes for four cellulases of the basidiomycete T. reesei showed that these proteins had a bifunctional organization remarkably similar to that of Cex and CenA of C. fimi, and again with reversal of domain order in pairs of the four enzymes (16). The T. reesei enzymes could also be cleaved into separate domains by proteolysis, and this was discusse P., this volume). Suffice i lulases in C. fimi and T. reesei appear to have arisen by domain shuffling and that the enzymes they encode appear to interact with cellulose in a comparable manner, i.e., a catalytic domain is held on the substrate by a binding domain. Cellulases from the bacterium Clostridium thermocellum also contained sequences analogous to Pro-Thr boxes as well as highly conserved carboxyl terminal sequences (2,17,18). It remains to be seen if they have functional organizations similar to those of the C fimi and T. reesei enzymes. Acknowledgment We thank the Natural Sciences and Engineering Research Council of Canada for financial support. Literature Cited 1. Aubert, J.-P.; Beguin, P.; Millet, J . , Eds. Biochemistry and Genetics of Cellulose Degradation; FEMS Symp. No. 43; Academic Press: San Diego, 1988. 2. Beguin, P.; Gilkes, N. R.; Kilburn, D. G.; Miller, R. C., Jr.; O'Neill, G. P.; Warren, R. A. J . CRC Crit. Rev. Biotechnol. 1987, 6, 129. 3. Stackenbrandt, E.; Kandler, O. Int. J. Syst. Bacteriol. 1979, 29, 273. 4. Beguin, P.; Eisen, H.; Roupas, A. J. Gen. Microbiol. 1977, 101, 191. 5. Beguin, P.; Eisen, H. Eur. J. Biochem. 1978, 87, 525. 6. Langsford, M . L.; Gilkes, N. R.; Wakarchuk, W. W.; Kilburn, D. G.; Miller, R. C., Jr.; Warren, R. A. J . J. Gen. Microbiol. 1984, 130, 1367. 7. Langsford, M . L. Ph.D. Thesis, University of British Columbia, 1988. 8. Gilkes, N. R.; Langsford, M . L.; Kilburn, D. G.; Miller, R. C., Jr.; Warren, R. A. J . J. Biol. Chem. 1984, 259, 10455. 9. Warren, R. A. J.; Beck, C. F.; Gilkes, N. R.; Kilburn, D. G.; Langsford, M. L.; Miller, R. C., Jr.; O'Neill, G. P.; Scheufens, M.; Wong, W. K. R. Proteins 1986, 1, 335.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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10. Langsford, M . L.; Gilkes, N. R.; Singh, B.; Moser, B.; Miller, R. C., Jr.; Warren, R. A. J.; Kilburn, D. G . FEBS Lett. 1987, 225, 163. 11. Owolabi, J.; Beguin, P.; Kilburn, D. G.; Miller, R. C., Jr.; Warren, R. A. J . Appl. Environ. Microbiol. 1988, 54, 518. 12. Gilkes, N. R.; Warren, R. A. J.; Miller, R. C., Jr.; Kilburn, D. G . J. Biol. Chem. 1988, 263, 10401. 13. Frangione, B.; Wolfenstein-Todel, C. Proc. Nat. Acad. Sci. U.S.A. 1972, 69, 3673. 14. Warren, R. A. J.; Gerhard, B.; Gilkes, N. R.; Owolabi, J . B.; Kilburn, D. G.; Miller, R. C., Jr. Gene 1987, 61, 421. 15. Gilkes, N. R.; Kilburn, D. G.; Langsford, M . L.; Miller, R. C . , Jr.; Wakarchuk, W. W.; Warren, R. A. J.; Whittle, D. J.; Wong, W. K. R. J. Gen. Microbiol. 1984, 130, 1377. 16. Knowles, J.; Lehtovaara, P.; Teeri, T . TrendsBiotechnol.1987, 5, 255. 17. Beguin, P.; Cornet, P. Aubert J.-P J. Bacteriol 1985 162 102 18. Grepinet, O.; Beguin RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 43 β-Glucosidases:

M e c h a n i s m and Inhibition

Stephen G. Withers and Ian P. Street Department of Chemistry, University of British Columbia, Vancouver, British Columbia V6T 1Y6, Canada

The generally accepted mechanism of action of glycosi­ dases which hydrolys of configuration ble displacement. Initial general acid-catalyzed gener­ ation of a glycosyl-enzyme intermediate is followed by its general base-catalyzed hydrolysis. Both the forma­ tion and the hydrolysis of the glycosyl-enzyme can be considered to proceed via oxocarbonium ion-like transi­ tion states. Destabilization of such transition states can be achieved by replacing the C-2 hydroxyl of the sub­ strate by the more electronegative fluorine, thus slow­ ing both steps. Simultaneous incorporation of an ex­ cellent leaving group (fluoride or dinitrophenolate) as the aglycone permits the accumulation of the interme­ diate which is sufficiently stable to be isolated. In­ vestigation of such an intermediate generated on a β­ -glucosidase, by means of F-NMR, allowed its iden­ tification as an α-D-glucopyranosyl-enzyme. Activated 2-deoxy-2-fluoroglycosides therefore act as mechanism­ -based inactivators, thereby representing a new class of "suicide" inactivators for glycosidases. 19

/?-Glucosidases p l a y a n i m p o r t a n t role i n t h e d e g r a d a t i o n o f cellulose b y h y d r o l y z i n g cellobiose t o glucose. I n this way, n o t o n l y is t h e key m e t a b o ­ l i t e glucose p r o d u c e d , b u t also cellobiose, a n i n h i b i t o r o f exoglucanases, is removed. A n u n d e r s t a n d i n g o f the detailed c h e m i c a l m e c h a n i s m o f a c t i o n of t h i s class o f enzymes is therefore i m p o r t a n t b o t h i n terms o f possible c h e m i c a l , o r genetic engineering approaches t o generation o f a r t i f i c i a l e n ­ zymes a n d also i n t h e design o f specific i n h i b i t o r s w h i c h c o u l d be v a l u a b l e i n p r e v e n t i n g cellulose d e g r a d a t i o n .

0097-6156/89/0399-0597$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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A l l glycosidases s t u d i e d to date have been f o u n d to effect h y d r o l y s i s of the g l y c o s i d i c linkage b y cleavage of the b o n d between the a n o m e r i c c a r b o n a n d the g l y c o s i d i c oxygen. However, two different stereochemical o u t c o m e s of such a h y d r o l y t i c m e c h a n i s m are possible. T h e b o n d can be cleaved w i t h r e t e n t i o n of a n o m e r i c c o n f i g u r a t i o n (by a " r e t a i n i n g " glycosidase) (1-3), or w i t h i n v e r s i o n of c o n f i g u r a t i o n (by a n " i n v e r t i n g " glycosidase) (1-3). M a n y e x a m p l e s of each t y p e have been f o u n d . P r o b a b l y the m o s t c o m m o n class, a n d c e r t a i n l y the best defined m e c h a n i s t i c a l l y , is t h a t of the " r e t a i n i n g " glycosidases. T h e most generally accepted m e c h a n i s m of a c t i o n for such enzymes is s h o w n i n F i g u r e 1 for a " r e t a i n i n g " /?-glucosidase, a n d involves a n active site c o n t a i n i n g two m e c h a n i s t i c a l l y i m p o r t a n t residues; a n a c i d c a t a l y s t whose i d e n t i t y i n different systems is thought to be a c a r b o x y l g r o u p or t y r o s i n e , a n d a nucleophile considered to be a c a r b o x y l a t e residue i n essentially a l l cases (see references 1-3 for reviews of glycosidase m e c h a n i s m s ) . B i n d i n g of the glycoside substrate is t h o u g h t to be followed b y p r o t o n d o n a t i o n f r o m the a c i thereby increasing the l a b i l i t y of the g l y c o s i d i c b o n d . B o n d heterolysis e n sues, generating a free (aglyconic) a l c o h o l w h i c h departs, a n d a g l y c o s y l o x o c a r b o n i u m i o n species s t a b i l i z e d by i n t e r a c t i o n w i t h the c a r b o x y l a t e g r o u p . T h e t i m i n g o f t h i s " a t t a c k " of the c a r b o x y l a t e g r o u p is p r o b a b l y such t h a t the r e a c t i o n has considerable Sjv2 character w i t h no true o x o c a r b o n i u m i o n i n t e r m e d i a t e , b u t r a t h e r j u s t a t r a n s i t i o n state w i t h s u b s t a n t i a l o x o c a r b o n i u m i o n character. S u c h " p r e - a s s o c i a t i o n " of the nucleophile has also been suggested to be very c o m m o n i n n o n - e n z y m i c g l y c o s y l transfer reactions (4). T h u s the first step of the p a t h w a y involves the a c i d - c a t a l y z e d f o r m a t i o n of a g l y c o s y l - e n z y m e i n t e r m e d i a t e v i a a n o x o c a r b o n i u m i o n - l i k e t r a n s i t i o n state. C o m p l e t i o n o f the process involves a t t a c k of water at the a n o m e r i c center, w i t h some general base c a t a l y t i c assistance g e n e r a t i n g , i n t h i s case, /?-D-glucopyranose as p r o d u c t . T h i s second g l y c o s y l transfer step, d e g l y c o s y l a t i o n , w i l l p r e s u m a b l y also occur v i a a t r a n s i t i o n state w i t h s u b s t a n t i a l o x o c a r b o n i u m i o n character. A l t e r n a t i v e s to t h i s m e c h a n i s m , w h i c h differ i n some cases o n l y r e l a t i v e l y s u b t l y , b u t i n other cases quite d r a m a t i c a l l y , are not w e l l s u p p o r t e d , b u t i n c l u d e a possible m e c h a n i s m i n v o l v i n g a n i n i t i a l e n d o c y c l i c C - 0 b o n d cleavage (5), a n d one i n v o l v i n g a f u l l o x o c a r b o n i u m i o n i n t e r m e d i a t e . T h e case against such mechanisms is discussed i n some d e t a i l elsewhere (2). T h e work described i n this paper s u m m a r i z e s our recent a t t e m p t s to p r o v i d e s u b s t a n t i a l proof for the m e c h a n i s m of F i g u r e 1. T h i s was achieved b y t r a p p i n g a n d i s o l a t i n g the g l y c o s y l - e n z y m e i n t e r m e d i a t e , thus p r o v i n g its existence, a n d also by d i r e c t l y d e t e r m i n i n g the stereochemistry of the linkage of this sugar to the e n z y m e . S u c h studies, b y t h e i r very n a t u r e , led to the generation of a new class of mechanism-based i n a c t i v a t o r s of glycosidases. Results and Discussion Since b o t h f o r m a t i o n a n d h y d r o l y s i s of the g l y c o s y l - e n z y m e proceed v i a o x o c a r b o n i u m i o n - l i k e t r a n s i t i o n states, i t seemed reasonable to assume,

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F i g u r e 1. M e c h a n i s m of a " r e t a i n i n g " /?-glucosidase. O R = the aglycone; N u = the e n z y m e ' s n u c l e o p h i l e ; H A = the e n z y m e ' s a c i d c a t a l y s t .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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i n l i n e w i t h our m o d e l n o n - e n z y m i c studies (6), t h a t s u b s t i t u t i o n of the sugar h y d r o x y l at C - 2 b y the more electronegative fluorine w o u l d result i n significant i n d u c t i v e d e s t a b i l i z a t i o n of the adjacent p o s i t i v e charge at the t r a n s i t i o n state. T h i s s h o u l d result i n decreased rates of g l y c o s y l - e n z y m e f o r m a t i o n and h y d r o l y s i s , thereby p r o d u c i n g very slow substrates. S u c h i n ­ deed was observed to be the case. However, the i n c o r p o r a t i o n of a r e l a t i v e l y reactive l e a v i n g group ( d i n i t r o p h e n o l a t e or fluoride) as the aglycone i n t o such deactivated substrates m i g h t accelerate the rate of g l y c o s y l - e n z y m e f o r m a t i o n sufficiently, without affecting the rate of g l y c o s y l - e n z y m e h y d r o l ­ ysis, to p e r m i t a c c u m u l a t i o n a n d t r a p p i n g of the 2-deoxy-2-fluoro-glycosyle n z y m e i n t e r m e d i a t e . T h i s c o u l d allow the s t r u c t u r e a n d properties of such an i n t e r m e d i a t e to be i n v e s t i g a t e d . Inactivation Studies. T h i s strategy was tested i n i t i a l l y o n a /?-glucosidase isolated f r o m Alcaligenes faecalis (7,8) a n d since cloned a n d expressed at h i g h levels i n E. coli (9), henceforwar I n the i n i t i a l k i n e t i c c h a r a c t e r i z a t i o substrates (highest V x and V / K ) i n c l u d e d 2 , 4 - d i n i t r o p h e n y l β-Όglucopyranoside a n d / 3 - D - g l u c o s y l fluoride. Therefore the most p r o m i s ­ i n g candidates for t r a p p i n g of a g l y c o s y l - e n z y m e i n t e r m e d i a t e appeared to be 2 , 4 - d i n i t r o p h e n y l - 2 - d e o x y - 2 - f l u o r o - ^ - D - g l u c o p y r a n o s i d e ( 2 F / ? D N P G l u ) a n d 2-deoxy-2-fluoro-/?-D-glucopyranosyl fluoride ( 2 F / ? G l u F ) . These two c o m p o u n d s were synthesized as described p r e v i o u s l y (10,11,12,13) a n d tested w i t h p A B G 5 /?-glucosidase for a c c u m u l a t i o n of a n i n t e r m e d i a t e . S u c h testing was r e l a t i v e l y facile, as the a c c u m u l a t i o n of the i n t e r m e d i a t e resulted i n a n apparent time-dependent i n a c t i v a t i o n of the e n z y m e . T h i s o c c u r r e d because the free enzyme, capable of i n t e r a c t i n g w i t h s u b s t r a t e , was converted i n t o the r e l a t i v e l y inert 2-fluoroglucosyl-enzyme i n t e r m e d i ­ ate. B o t h c o m p o u n d s were indeed f o u n d to be excellent t i m e - d e p e n d e n t i n a c t i v a t o r s (10,14), r e a c t i n g according to the expected pseudo-first-order k i n e t i c s , as s h o w n for 2 F / ? D N P G l u i n F i g u r e 2. T h e rate of i n a c t i v a t i o n was dependent u p o n the c o n c e n t r a t i o n of i n a c t i v a t o r , s h o w i n g s a t u r a t i o n k i n e t i c s as expected for a s i m p l e i n a c t i v a t o r b i n d i n g n o n - c o v a l e n t l y i n i t i a l l y w i t h a dissociation constant, a n d t h e n i n a c t i v a t i n g the enzyme w i t h a rate constant k{ a c c o r d i n g to the scheme below. ma

m

Ki Ε +

a

a

:

m

k Ιτ±Ε.Ι-=±Ε-Ι

T h e f o l l o w i n g constants were d e t e r m i n e d : 2 F / ? D N P G l u , ki m i n " , Ki = 0.05 m M ; 2 F / ? G l u F , ki = 5.9 m i n " , Α\· = 0.4 m M . 1

=

25

1

Evidence for the Proposed Mechanism. A considerable effort was then ex­ pended i n p r o v i n g the mode of i n a c t i v a t i o n . E v i d e n c e t h a t i n a c t i v a t i o n o c c u r r e d by b i n d i n g at the active site was given by the observation t h a t the c o m p e t i t i v e i n h i b i t o r i s o p r o p y l t h i o - / ? - D - g l u c o p y r a n o s i d e ( I P T G ) (Ki - 4 m M ) p r o v i d e d the expected p r o t e c t i o n against i n a c t i v a t i o n as s h o w n i n F i g u r e 2c. I n a c t i v a t i o n of the enzyme was a c c o m p a n i e d by the release of a " b u r s t " of aglycone ( d i n i t r o p h e n o l a t e or fluoride) as required b y such a

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

43.

WITHERS & STREET

ι 1/S

β-Glucosidoses: Mechanism & Inhibition

2 (μΜ-1)

·

ι

Time

(min)

601

*

F i g u r e 2. I n a c t i v a t i o n of p A B G 5 /?-glucosidase w i t h 2 F / ? D N P G l u ( s t r u c t u r e s h o w n ) (a) /?-glucosidase i n c u b a t e d w i t h the f o l l o w i n g c o n c e n t r a t i o n s of 2 F / ? D N P G l u a n d aliquots assayed against p - n i t r o p h e n y l /?-glucopyranoside at the times s h o w n : Ο = 0.5μΜ; CZ| = 1-ΟμΜ, φ = 2.0μΜ; • = 3 . 0 / i M ; A = 4 . 0 / i M ; Δ = 5 . 0 / i M ) . (b) R e p l o t of first-order rate c o n ­ stants f r o m 2a. (c) P r o t e c t i o n against i n h i b i t i o n given b y i s o p r o p y l t h i o /?-D-glucopyranoside ( I P T G ) .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L P O L Y M E R S

m e c h a n i s m . T h e " b u r s t " of d i n i t r o p h e n o l a t e was measured s p e c t r o p h o t o m e t r i c a l l y ( F i g u r e 3), w h i l e the " b u r s t " of fluoride was measured b y b o t h F - N M R a n d b y a d y e - b i n d i n g assay (15). In b o t h cases an essentially s t o i c h i o m e t r i c (0.93-1.0:1) reaction was observed w i t h release of one m o l e o f aglycone per mole of enzyme i n a c t i v a t e d . In a d d i t i o n to h e l p i n g prove the m o d e of a c t i o n of these i n a c t i v a t o r s t h i s procedure provides a very c o n ­ venient a n d accurate way of m e a s u r i n g the c o n c e n t r a t i o n o f active e n z y m e present at any t i m e , s i m p l y b y p e r f o r m i n g a n " a c t i v e site t i t r a t i o n " u s i n g 2F/?DNPGlu. T h e f o r m a t i o n of a 2-fluoroglucosyl-enzyme was d e m o n s t r a t e d b y means of F - N M R . A d d i t i o n of 2 F / ? G l u F ( 1 . 0 5 m M ) to a concentrated s o l u t i o n of p A B G 5 /?-glucosidase ( 0 . 7 3 m M ) i n 5 0 m M s o d i u m p h o s p h a t e buffer, p H 6.8 i n a 5 m m N M R t u b e , i n a c t i v a t e d the e n z y m e very r a p i d l y . T h e F - N M R s p e c t r u m of t h i s s a m p l e is s h o w n i n F i g u r e 4. P e a k s were assigned as follows: T h e peak at 121.4 p p m was due to i n o r g a n i c fluoride re­ leased u p o n i n a c t i v a t i o n o due t o F - l a n d F - 2 , respectively, of the s m a l l excess of u n r e a c t e d 2 F / ? G l u F r e m a i n i n g . T h e r e l a t i v e l y b r o a d peak at 197.3 p p m , Δ ι / = 1 3 0 H z , was due to the 2-fluoroglucosyl-enzyme f o r m e d , b o t h the c h e m i c a l shift a n d the l i n e w i d t h b e i n g consistent w i t h t h i s assignment. D i a l y s i s of such a s a m p l e resulted i n removal o f a l l signals except the b r o a d resonance at 197.3 p p m , d e m o n s t r a t i n g t h a t the sugar was indeed covalently l i n k e d . 1 9

1 9

1 9

Stereochemistry of the Intermediate. These e x p e r i m e n t s therefore d e m o n ­ s t r a t e d t h a t a 2-fluoroglucosyl-enzyme h a d been t r a p p e d . It was t h e n of interest to prove the stereochemistry of the linkage of t h i s sugar residue to the e n z y m e . U n f o r t u n a t e l y , the F - N M R s p e c t r u m s h o w n i n F i g u r e 4 was of no use i n this r e g a r d , since the F - c h e m i c a l shift of 2-deoxy2-fluoro-glucose a n d its derivatives is relatively insensitive to the config­ u r a t i o n of the a n o m e r i c s u b s t i t u e n t (o> a n d /?-2-deoxy-2-fluoro-D-glucose differ i n F c h e m i c a l shift b y o n l y 0.18 p p m a n d t h e i r tetra-0-acetates b y o n l y 1.4 p p m ) . C o u p l i n g constants are not m u c h more sensitive a n d w o u l d , i n any case, be lost i n the large n a t u r a l l i n e w i d t h of the reso­ nance. However, the chemical shifts of 2-deoxy-2-fluoro-D-mannose a n d its derivatives were s h o w n p r e v i o u s l y to be very sensitive to a n o m e r i c c o n ­ figuration (16), e.g., o> a n d /?-2-deoxy-2-fluoro-D-mannose differ i n F c h e m i c a l shift b y some 18.5 p p m . Since i t h a d been s h o w n p r e v i o u s l y (8) t h a t p A B G 5 /?-glucosidase e x h i b i t s considerable /?-mannosidase a c t i v i t y , it seemed t h a t 2-deoxy-2-fluoro-/?-mannosyl fluoride ( 2 F / ? M a n F ) m i g h t be a g o o d i n a c t i v a t o r of the enzyme. If so, this w o u l d allow the generation of a 2 - f l u o r o - m a n n o s y l enzyme w h i c h c o u l d be investigated b y F - N M R . F i g ­ ure 5 shows the result of such an i n v e s t i g a t i o n where 2 F / ? M a n F ( 1 . 5 2 m M ) has been added to p A B G 5 /?-glucosidase ( 0 . 7 4 m M ) i n 5 0 m M s o d i u m phos­ p h a t e buffer, p H 6.8, a n d placed i n a 5 m M N M R t u b e . T h e resonance at 121.0 p p m was due to inorganic fluoride released u p o n i n a c t i v a t i o n , whereas resonances at 149.5 a n d 224.4 p p m corresponded to F - l a n d F - 2 , respec­ tively, of the excess 2 F / ? M a n F e m p l o y e d . T h e b r o a d peak at 201.0 p p m was due to the 2-fluoro-mannosyl-enzyme a d d u c t . T h e c h e m i c a l shift o b 1 9

1 9

1 9

1 9

1 9

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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2A4initroph«nol (nmole) F i g u r e 3. M e a s u r e m e n t of the " b u r s t " of d i n i t r o p h e n o l a t e released o n re­ a c t i o n of 2 F / ? D N P G l u w i t h p A B G 5 /?-glucosidase. P l o t of q u a n t i t y of 2 , 4 - d i n i t r o p h e n o l released ( f r o m absorbance at 4 0 0 n m ) versus q u a n t i t y of e n z y m e t r e a t e d . S o l u t i o n s of different c o n c e n t r a t i o n s of /?-glucosidase i n 5 0 m M s o d i u m phosphate buffer, p H 6.8 were i n c u b a t e d at 3 7 ° C i n a 1 c m p a t h l e n g t h glass cuvette i n a s p e c t r o p h o t o m e t e r a n d the absorbance r e a d ­ i n g zeroed. A s o l u t i o n of 2 F / ? D N P G l u (sufficient to p r o v i d e twice the e s t i ­ m a t e d e n z y m e c o n c e n t r a t i o n ) was added a n d the o p t i c a l d e n s i t y at 4 0 0 n m r e c o r d e d . T h e q u a n t i t y of d i n i t r o p h e n o l released was e s t i m a t e d u s i n g a n e x t i n c t i o n coefficient of l l , 3 0 0 M c m . T h e q u a n t i t y of e n z y m e was e s t i ­ m a t e d u s i n g a n e x t i n c t i o n coefficient at 2 8 0 n m of E° = 2 . 2 0 c m " , itself determined by a quantitative amino acid analysis. _ 1

- 1

1

%

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1

604

PLANT C E L L W A L L

POLYMERS

121.4

120

140 (δ

160 ppm)

180

200

F i g u r e 4. P r o t o n decoupled F - N M R s p e c t r u m of p A B G 5 /?-glucosidase i n a c t i v a t e d w i t h 2 F / ? G l u F (conditions as described i n t e x t ) . T h i s s p e c t r u m was recorded on a 270 M H z B r u k e r / N i c o l e t i n s t r u m e n t using gated p r o t o n d e c o u p l i n g (decoupler on d u r i n g a c q u i s i t i o n o n l y ) a n d a 90° pulse angle w i t h a r e p e t i t i o n delay of 2s. A spectral w i d t h of 40,000 H z was employed a n d s i g n a l accumulated over 10,000 transients. 1 9

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

43.

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WITHERS & STREET

Mechanism

& Inhibition

605

149.5 224.4

201.0

120

140

206.2 224.5 DENATURED ©CYME

F i g u r e 5. P r o t o n decoupled F - N M R s p e c t r u m of p A B G 5 /?-glucosidase i n a c t i v a t e d w i t h 2 F / ? M a n F (conditions as described i n t e x t ) . S p e c t r a were recorded on a 270 M H z B r u k e r / N i c o l e t i n s t r u m e n t u s i n g gated p r o t o n de­ c o u p l i n g (decoupler on d u r i n g a c q u i s i t i o n o n l y ) a n d a 90° pulse angle w i t h a r e p e t i t i o n delay of 2s. A s p e c t r a l w i d t h of 40,000 H z was e m p l o y e d a n d s i g n a l a c c u m u l a t e d over 10,000 transients for the n a t i v e p r o t e i n a n d 30,000 transients for the d e n a t u r e d p r o t e i n i n 8 M u r e a , (a) F u l l s p e c t r u m w i t h e x p a n s i o n below i t ; (b) E x p a n s i o n of s p e c t r u m of d e n a t u r e d / d i a l y z e d e n ­ zyme. 1 9

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served was w e l l w i t h i n the range expected for a n α-linked 2-fluoro-mannose residue a n d some 16-20 p p m downfield of the region expected for a βlinkage. In light of the large c h e m i c a l shift differences concerned, i t was u n l i k e l y t h a t other factors affected the c h e m i c a l shift significantly. H o w ­ ever, as described below, other factors were also considered. Since the F - N M R c h e m i c a l shifts of 2-deoxy-2-fluoro-glycosides a n d - g l y c o s y l es­ ters are r e l a t i v e l y insensitive to the c h e m i c a l identities of the a n o m e r i c s u b s t i t u e n t , i t was u n l i k e l y t h a t a large chemical shift resulted f r o m the replacement of the fluorine s u b s t i t u e n t at C - l by the e n z y m e n u c l e o p h i l e so t h i s was u n l i k e l y t o be the source of the large shift, especially as no s u c h large shift h a d been observed for the 2-fluoro-glucosyl enzyme. T h e b i n d ­ i n g of fluorinated ligands to macromolecules generally produces d o w n f i e l d shifts (17) w h i c h m i g h t confuse this i n t e r p r e t a t i o n . However, since such shifts are generally s m a l l , a n d because the c h e m i c a l shift of the 2-deoxy-2fluoro-glucosyl-enzyme was i n the expected region, t h i s seemed a n u n l i k e l y cause for such a large shift not the case, the 2-fluoro-mannosyl-enzyme a d d u c t was d i a l y z e d overnight against 8 M u r e a to denature i t , a n d the F - N M R s p e c t r u m of the r e s u l t a n t unfolded p r o t e i n , i n w h i c h the fluoromannose residue w o u l d be exposed to solvent, was o b t a i n e d (see F i g u r e 5b). W h i l e a very s m a l l (A6 = 1.6 p p m ) upfield shift of the resonance was observed, the resonance r e m a i n e d well w i t h i n the region a n t i c i p a t e d for the α-linked sugar. It is of interest to note t h a t resonances a r i s i n g f r o m excess 2 F / ? M a n F were removed b y the d i a l y s i s , b u t the h i g h m o l e c u l a r weight 2-fluoromannosyl-enzyme species was r e t a i n e d . Resonances at 206.2 a n d 224.5 p p m arose f r o m a - a n d β-2deoxy-2-fluoro-D-mannose w h i c h h a d h y d r o l y z e d after d e n a t u r a t i o n of the enzyme. 1 9

1 9

Conclusion B y u s i n g 2-deoxy-2-fluoro-glycosides w i t h g o o d l e a v i n g groups at the a n o m e r i c center i t was possible to t r a p the g l y c o s y l - e n z y m e a d d u c t l o n g since p o s t u l a t e d as a n i n t e r m e d i a t e i n glycosidase c a t a l y s i s . F u r t h e r , it was possible to u n e q u i v o c a l l y prove the stereochemistry of this species. T h i s therefore provides very s t r o n g s u p p o r t i v e evidence for the m e c h a n i s m of e n z y m e - c a t a l y z e d glycoside hydrolysis s h o w n i n F i g u r e 1. T h e m e c h a ­ n i s m involves a covalent g l y c o s y l enzyme intermediate w h i c h is formed a n d h y d r o l y z e d via o x o c a r b o n i u m i o n - l i k e t r a n s i t i o n states. T h e 2-deoxy-2fluoro-glycosides used i n this work serve as g o o d , specific mechanism-based i n a c t i v a t o r s of glycosidases, a n d m a y have future a p p l i c a t i o n i n studies of oligosaccharide a n d polysaccharide d e g r a d a t i o n . Acknowledgments W e w i s h to t h a n k D . D o l p h i n ( D e p a r t m e n t of C h e m i s t r y , U B C ) a n d R . A . J . W a r r e n a n d W . W . W a k a r c h u k ( D e p a r t m e n t of M i c r o b i o l o g y , U B C ) for their assistance i n this work.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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We also thank the Natural Sciences and Engineering Research Council of Canada and the British Columbia Health Care Research Foundation for financial support. Literature Cited 1. Sinnott, M . L. In The Chemistry of Enzyme Action; Elsevier: New York, 1984; p. 389. 2. Sinnott, M . L. In Enzyme Mechanisms; Royal Society of Chemistry, 1987; p. 259. 3. Lalegerie, P.; Legler, G.; Yon, J . M . Biochimie 1982, 64, 977. 4. Jencks, W. P. Chem. Soc. Rev. 1981, 10, 345. 5. Post, C. B.; Karplus, M . J. Amer. Chem. Soc. 1986, 108, 1317. 6. Withers, S. G.; MacLennan, D. J.; Street, I. P. Carbohydr. Res. 1986, 154, 127. 7. Han, Y . W.; Srinivasan 8. Day, A. G.; Withers, , , 9. Wakarchuk, W. W.; Kilburn, D. G.; Miller, R. C.; Warren, R. A. J . Mol. Gen. Genet. 1986, 205, 146. 10. Withers, S. G.; Street, I. P.; Bird, P.; Dolphin, D. H. J. Amer. Chem. Soc. 1987, 109, 7530. 11. Adamson, J.; Foster, A. B.; Hall, L. D.; Johnson, R. N.; Hesse, R. M . Carbohydr. Res. 1970, 15, 351. 12. Hall, L. D.; Johnson, R. N.; Adamson, J . B.; Foster, A. B. Can. J. Chem. 1971, 49, 118. 13. Street, I. P.; Armstrong, C. R.; Withers, S. G . Biochemistry 1986, 25, 6021. 14. Withers, S. G.; Rupitz, K.; Street, I. P. J. Biol. Chem. 1988, 263, 7929. 15. Megregian, S. In ColorimetricDetermination of Non-Metals: Chemical Analysis; Boltz, D. F.; Howell, J . Α., Eds.; Wiley: New York, 1978; 8, 109. 16. Phillips, L.; Wray, V. J. Chem. Soc. (B) 1971, 1618. 17. Gerig, J. T . In Biological Magnetic Resonance; Berliner, L. T.; Reuben, J., Eds.; 1978, 1, 139. RECEIVED April 11, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 44 E n v i r o n m e n t a l P o t e n t i a l o f the Trichoderma Exocellular Enzyme System J. M . Lynch AFRC Institute of Horticultural Research, Littlehampton, West Sussex BN17 6LP, England

Trichoderma spp (exo- and endo-β-1-4 hemicellulolytic (especially xylanase and β-xylosidase) complexes which are effective in degrading natural lig­ nocelluloses such as straw. Nitrogen can be provided to the fungus by cooperative nitrogen-fixing anaerobic bacteria (Clostridium butyricum). The fungus also pro­ duces a range of enzymes (chitinase, 1,3-β-D glucanase and proteases) with the capacity to degrade the cell walls of a wide range of fungal plant pathogens. No correlation has been obtained between field biocontrol effectiveness and lytic enzyme production by different strains; this is possibly because antibiotic metabolites are also involved in the action. A target is to produce Trichoderma strains with elevated levels of degradative and lytic exocellular enzymes which also produce antibiotics and grow rapidly in the soil and rhizosphere environments. Lignocelluloses are freely available i n the e n v i r o n m e n t as residues f r o m crop plants a n d trees a n d there has been a great effort i n recent years t o develop effective a n d economic processes for their u t i l i z a t i o n . However, outside t h e m u s h r o o m i n d u s t r y , few cost-effective o p t i o n s have been identified. T h i s led W o o d (1) a n d L y n c h (2) t o echo the c o m m e n t s o f T h a y s e n a n d B u n k e r (3) t h a t the most effective route t o u t i l i z e dead vegetation is i n the n a t u r a l processes o f decay. T h e r e has been a tendency t o develop c y c l i c arguments where the energy d e r i v e d f r o m lignocellulose u t i l i z a t i o n , t o p r o d u c e a l c o h o l for e x a m p l e , is merely recycled i n f a r m m a c h i n e r y t o derive more l i g n o c e l ­ luloses w i t h n o net energy g a i n . Trichoderma species have a powerful set o f w e l l - c h a r a c t e r i z e d e x o e n zymes i n v o l v e d i n t h e c e l l u l o l y t i c p a t h w a y : e x o - / ? - l , 4 - g l u c a n a s e w h i c h h y drolyzes a m o r p h o u s a n d m i c r o c r y s t a l l i n e cellulose; e n d o - / ? - l , 4 - g l u c a n a s e 0097-6156/89/0399-0608$06.00/0 © 1989 American Chemical Society

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w h i c h h y d r o l y z e s cellulose derivatives; a n d /?-glucosidase w h i c h h y d r o l y z e s cellobiose d e r i v e d f r o m the glucanase a c t i o n a n d c e l l o d e x t r i n s t o glucose (1). X y l a n o l y t i c a c t i v i t y c a n also be present t o h y d r o l y z e hemicelluloses (heterogeneous p o l y m e r s o f glucose, mannose, galactose, arabinose a n d x y l o s e ) . E v e n t h o u g h there is n o l i g n i n o l y t i c a c t i v i t y i n t h i s genus, t h e p r e p o n d e r ance o f the p o l y s a c c h a r i d e c o m p o n e n t s i n lignocellulose a n d t h e i n d i c a t i o n t h a t m u c h o f t h e p o l y s a c c h a r i d e is n o t t i g h t l y b o u n d t o t h e l i g n i n a n d therefore r e l a t i v e l y freely available i n n o n - w o o d y p l a n t s (4) m a k e Trichoderma a g o o d c a n d i d a t e for e x p l o r i n g lignocellulose u t i l i z a t i o n . E v e l e i g h (5) has reviewed t h e biology a n d b i o c h e m i s t r y o f the genus; b u t other t h a n its u t i l i t y i n p r o v i d i n g t h e source o f c o m m e r c i a l cellulases, there has been l i t t l e i n d u s t r i a l e x p l o i t a t i o n as y e t . However, Trichoderma species, a n d the closely related Gliocladium species, have s h o w n m a j o r p o t e n t i a l as b i o l o g i c a l c o n t r o l agents o f p l a n t diseases (6) a n d a c o m m e r c i a l p r e p a r a t i o n o f T. viride, B i n a b T , is p r o d u c e d i n Sweden as a b i o l o g i c a l c o n t r o l agent against the silver leaf disease o f f r u i In t h i s m o d e i t is a m y c o p a r a s i t e , c o i l i n g a r o u n d a n d p e n e t r a t i n g t h e cell walls o f p l a n t pathogens, a n a c t i o n w h i c h , at least i n p a r t , m u s t i n v o l v e the release o f l y t i c enzymes. T h e purpose o f t h i s a r t i c l e is t o o u t l i n e the role o f these e n z y m e systems i n the s o i l / p l a n t e c o s y s t e m a n d discuss h o w their useful characters m i g h t be elevated a n d e x p l o i t e d f u r t h e r . Lignocellulolysis O n e o f the favored o r g a n i s m s for s t u d y o f cellulolysis b y Trichoderma is T. reesei. C o n s e q u e n t l y , m a n y m u t a n t s t r a i n s w h i c h h y p e r p r o d u c e cellulase have been o b t a i n e d b y t r e a t m e n t w i t h u l t r a v i o l e t l i g h t , g a m m a i r r a d i a t i o n , the l i n e a r accelerator, d i e t h y l s u l p h a t e a n d N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e (7). W h e r e a s m u c h o f the s t u d y o f T. reesei has been w i t h cellulose as s u b s t r a t e , i t is relevant t o consider t h e other f r a c t i o n s o f n a t u r a l lignocelluloses: hemicellulose a n d holocellulose (the c o m b i n e d cellulose a n d hemicellulose f r a c t i o n ) . W h e n t h e h y p e r - c e l l u l o l y t i c m u t a n t T. reesei Q M 9 4 1 4 was g r o w n o n v a r i o u s substrates d e r i v e d f r o m wheat s t r a w i n s t i r r e d b a t c h c u l t u r e , t h e highest specific g r o w t h rates were o n holocellulose a n d t h e h e m i c e l l u l o s e A defined b y O ' D w y e r (8); i t grew p o o r l y o n lignocellulose. B y c o n t r a s t , T. harzianum I M I 275950 isolated f r o m wheat s t r a w grew better o n l i g nocellulose ( T a b l e I ) . D e s p i t e these differences, the e n z y m e y i e l d s o f the c e l l u l a s e / x y l a n a s e complexes d i d n o t correlate w i t h g r o w t h r a t e . T h e s e o b servations f r o m t h i s e m p i r i c a l s t u d y i n d i c a t e t h a t s t r a i n s i s o l a t e d or d e r i v e d for g r o w t h o n e x t r a c t e d substances m a y n o t necessarily be the most useful strains to exploit natural substrates. W h e n s u i t a b l e s t r a i n s t o e x p l o i t n a t u r a l substrates are a v a i l a b l e , t h e i r e x p l o i t a t i o n o f c e l l u l o l y t i c substrates i n pure c u l t u r e c o u l d be r e s t r i c t e d b y the e n d - p r o d u c t s o f cellulolysis (cellobiose a n d glucose) repressing e n z y m e synthesis or i n h i b i t i o n / i n a c t i v a t i o n o f f u r t h e r e n z y m i c a c t i v i t y ( F i g . 1). O n e o f the s i m p l e s t routes t o relieve t h i s difficulty was t o use n o n - c e l l u l o l y t i c

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enzym inactivatio

Non-cellulolytic organisms

enzyme repression

Cellulolytic organisms

)

F i g u r e 1. Possible i n t e r a c t i o n s between m i c r o - o r g a n i s m s d u r i n g cellulose d e g r a d a t i o n . Source: R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 11. © 1985, L . A . Harrison.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

44.

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611

Trichoderma Exocellular Enzyme System

T a b l e I. G r o w t h of Trichoderma reesei Q M 9 4 1 4 ( T r ) a n d T. harzianum I M I 2 7 5 9 5 0 ( T h ) o n wheat s t r a w lignocellulose (lignocell.) or de­ r i v e d cellulosic m a t e r i a l s f r o m s t r a w (cell., cellulose; h e m i c e l l - A , h e m i c e l l u l o s e - A ; h o l o c e l l . , holocellulose) Organism

Tr

Substrate

CM'-cellulase FP -cellulase /?-glucosidase X y l a n ase /?-xylosidase 2

Th

Tr

Tr

Tr

cell.

hemicell-A

holocell.

lignocell.

Enzyme

Yield (units

χ 10 ~ / g 3

cellulosic

lignocell.

substrate)

2.50 0.26 0.06 14.50 0.20

1.20 0.08 0.06 52.02 0.38

3.00 0.40 0.04 28.80 0.48

2.58 0.30 0.06 24.62 0.16

2.06 0.20 0.06 15.50 0.22

0.029

0.085

0.086

0.016

0.025

C u l t u r e s were g r o w n i n a s t i r r e d b a t c h fermenter, w o r k i n g v o l u m e 1 l i t e r , u s i n g cellulose m a t e r i a l s at 0 . 5 % w / v as c a r b o n s u b s t r a t e s . G r o w t h rate of c u l t u r e s was d e t e r m i n e d b y m e a s u r i n g the c u l t u r e A T P c o n c e n t r a t i o n (aggregate of b i o m a s s a n d e x o c e l l u l a r A T P ) d u r i n g e x p o n e n t i a l g r o w t h (9). Carboxymethylcellulose. Filter paper. Source: R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 10. © 1986, D . M . G a u n t . 1

2

or o t h e r c e l l u l o l y t i c species of f u n g i or b a c t e r i a to u t i l i z e the e n d - p r o d u c t s , as p r e s u m a b l y h a p p e n s i n the n a t u r a l e n v i r o n m e n t . H o w e v e r , r a t h e r t h a n a l l o w i n g the energy so generated i n cellulolysis to go to waste, we have considered routes whereby t h a t energy c o u l d be usefully d i v e r t e d to other n a t u r a l processes. O n e process i n soil h e a v i l y r e s t r i c t e d i n n a t u r e b y a v a i l ­ able energy is d i n i t r o g e n fixation, because a b o u t 15 moles A T P is r e q u i r e d in vitro for nitrogenase to reduce one mole of N2 to 2NH3. A c o n c e p t u a l scheme is o u t l i n e d i n F i g u r e 2 where the cellulase comes f r o m T. harzianum and Closindium butyricum is the anaerobic b a c t e r i u m p r o v i d i n g the source of nitrogenase. I n a n open s y s t e m w i t h o u t forced a e r a t i o n , anaerobic m i c r o e n v i r o n m e n t s w i l l occur such t h a t there w i l l be a e r o b i c / a n a e r o b i c i n t e r ­ faces. T h e a s s o c i a t i o n between the o r g a n i s m s was p r o m o t e d by the a d d i t i o n of s m a l l a m o u n t s of a m m o n i u m - N (13) a n d the species i n v o l v e d c o u l d co­ exist i n a range of oxygen atmospheres (14), p o s s i b l y because the fungus was able to exclude oxygen f r o m the anaerobe w h i c h is n o n - c e l l u l o l y t i c . W h e n C. butyricum was p r o v i d e d w i t h the cellulase e n z y m e f r o m T. viride it i n d i r e c t l y m e t a b o l i z e d cellulose as a c a r b o n source i n the absence of a source of fixed n i t r o g e n under anaerobic c o n d i t i o n s . In s e a r c h i n g for means to enhance t h i s in vitro a c t i v i t y , a range of f u n ­ gal isolates were screened for c e l l u l o l y t i c a c t i v i t y o n s t r a w . T h e tests used to assay a c t i v i t y i n c l u d e d c l e a r i n g of cellulose agar (zone size m e a s u r e d ) , loss of weight f r o m s t r a w , colony r a d i a l e x t e n s i o n rate o n a water-soluble e x t r a c t

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of s t r a w a n d o n internode sections of s t r a w , a n d p e n e t r a t i o n rate i n t o s t r a w pieces (15). T h e s e in vitro tests identified useful a c t i v i t y a n d d i f f u s i b i l i t y of the c e l l u l a s e / h e m i c e l l u l a s e enzyme complexes. In a l l tests Trichoderma spp. p e r f o r m e d most favorably, w i t h Sordaria alcina a n d Fusarium s p p . closely f o l l o w i n g . T h e l a t t e r group of species were pathogenic to p l a n t s a n d therefore s h o u l d be e x c l u d e d f r o m a g r i c u l t u r a l e n v i r o n m e n t s , even t h o u g h their c e l l u l o l y t i c a c t i v i t y was p o t e n t i a l l y useful for u t i l i z a t i o n of p l a n t residues. T h e Trichoderma cellulase was active over a w i d e range of water p o t e n t i a l s ( - 0 . 7 to - 7 M P a ) a n d i t s a c t i v i t y was greater at 2 0 ° C t h a n at 10°C (16). T h e s e properties w o u l d again be useful i f a p p l i c a t i o n i n the e n v i r o n m e n t was to be c o n t e m p l a t e d . T h e Trichoderma spp. clearly f o r m e d a n association w i t h C. butyncum, as d i d the Fusarium s p p . a n d S. alcina, i n d i c a t e d b y the n u m b e r of v i a b l e b a c t e r i a a s s o c i a t i n g w i t h s t r a w a n d the rate of d e g r a d a t i o n of t h i s substrate (17). B y c o n t r a s t , Pénicillium spp. were generally less effective. In order to further protec w h i c h produces polysaccharides useful i n s t a b i l i z i n g s o i l s t r u c t u r e s (18), was a d d e d to the Trichoderma/Clostridium c o n s o r t i u m (19) ( F i g . 3). W h e n wheat s t r a w contained i n glass c o l u m n s was treated w i t h t h i s c o n s o r t i u m , a n d a n N-free n u t r i e n t feed s u p p l i e d at a steady rate, a m a x i m u m Ν g a i n of 12 m g N g s t r a w was measured w i t h about 5 0 % of the s u b s t r a t e u t i ­ l i z e d . A d d e d t o 3 m g N g " n a t u r a l l y present i n the s t r a w , the Ν i n p u t t o t a l s 15 m g N . T h i s rate is a l i t t l e less t h a n 18 m g N g " o r i g i n a l s t r a w w i t h about 2 5 % substrate u t i l i z a t i o n w h i c h has been measured w i t h studies u s i n g Cellulomonas sp. C S 1 - 1 as the cellulase-producer a n d Azospirillum brasilense sp. as the (aerobic) nitrogenase-producer (20). E v e n so, i f the figure c o u l d be e x t r a p o l a t e d to field c o n d i t i o n s , (e.g., s t r a w f r o m a wheat crop y i e l d i n g a b o u t 7 t h a " of g r a i n ) a n d a s i m i l a r a m o u n t of s t r a w , it w o u l d p r o d u c e a t o t a l Ν g a i n to s o i l of 105 K g Ν h a . T h e f u n c t i o n i n g of the associative c e l l u l a s e / n i t r o g e n a s e a c t i o n w i l l be dependent o n soil a n d e n v i r o n m e n t a l c o n d i t i o n s , a n d the p o t e n t i a l for i n o c u l a t i o n w i l l depend o n the level of n a t u r a l associations already e x i s t i n g i n soils. W e successfully i n o c u l a t e d T. harzianum onto s t r a w i n s o i l b u t failed to elevate the p o p ­ u l a t i o n s of C. butyricum/E. cloacae a n d gains of Ν over those o c c u r r i n g n a t u r a l l y (21). C o m m e r c i a l o p p o r t u n i t y u s u a l l y depends o n the effective­ ness of i n o c u l a , even where useful n a t u r a l processes are o p e r a t i n g o p t i m a l l y . H o w e v e r , the e n v i r o n m e n t a l benefit of n a t u r a l processes s h o u l d be c o n s i d ­ ered i n m o r e d e p t h a n d f a r m i n g processes reappraised where, for e x a m p l e , p l a n t residues are destroyed b y b u r n i n g to s i m p l i f y soil c u l t i v a t i o n practices. - 1

1

1

1

- 1

Mycoparasitism It has a l r e a d y been i n d i c a t e d t h a t some Trichoderma a n d Fusarium s p p . have c o m p a r a b l e a c t i v i t i e s i n n a t u r a l substrate u t i l i z a t i o n . W h e n inoc­ u l a t e d o n t o plates of m a l t agar at 20°C, colonies of two species of T. harzianum h a d a r a d i a l e x t e n s i o n rate of 20.1 a n d 26.1 m m d " , whereas the m a x i m u m r a d i a l extension of the Fusarium spp. was 12.8 m m d " " (F. culmorum) (22). However, Trichoderma or Fusarium c o u l d become d o m 1

1

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

44.

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C0

I

0

2

613

N

2

2

l Clostridium butyricum \ t

Cellulose

» Cellobiose + Glucose

F i g u r e 2. I n t e r a c t i o n s between Clostridium butyricum and Trichoderma harzianum. V F A s = v o l a t i l e f a t t y acids. S o u r c e : R e p r o d u c e d w i t h p e r m i s ­ s i o n f r o m Ref. 12. © 1985, D . A . V e a l . Atmospheric N 2

Fixed Ν

Cellulosic substrate '

Cellulolytic fungus

η

Fixed Ν

η

Anaerobic N -fixing bacterium 2

Τ

Polysaccharideproducing bacterium

Simple sugars

Atmospheric 0

2

F i g u r e 3. S c h e m a t i c for the d e g r a d a t i o n o f cellulosic substrates b y a m i c r o ­ bial consortium.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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i n a n t w h e n spore suspensions of m i x t u r e s were i n o c u l a t e d together o n t o agar plates. E i t h e r fungus became d o m i n a n t i f its spore c o n c e n t r a t i o n i n the m i x t u r e was greater b y one order of m a g n i t u d e t h a n t h a t of the other species. T h e p o t e n t i a l therefore exists for these species to c o m p e t e for n a t u r a l substrates i n the e n v i r o n m e n t . T h i s p o t e n t i a l m i g h t be enhanced i f one of the species h a d other useful a t t r i b u t e s , i n a d d i t i o n to a h i g h c a p a c i t y to degrade its substrates. A n " a n t i b i o t i c " i n h i b i t i o n zone often appears a r o u n d Trichoderma spp. i n t e r a c t i n g w i t h other f u n g i . T h e genus c o n t a i n s m a n y species w h i c h p r o duce secondary m e t a b o l i t e s . C l a y d o n et ai (23) have identified a n a n t i b i o t i c f r o m T. harzianum as a v o l a t i l e , 6 - n - p e n t y l - 2 H - p y r a n - 2 - o n e ; t h i s was recently s h o w n to be a n active a n t i b i o t i c f r o m T. koningii (24). T h e v o l a t i l e a p p e a r e d t o be the factor responsible for the " c o c o n u t s m e l l " of some biocontrol-effective s t r a i n s of T. harzianum (25). However, i n a P e t r i p l a t e assay, i t can be difficult to be c e r t a i n t h a t antibiosis is i n v o l v e d . A s w e l l as c o m p e t i t i v e g r o w t h a n d Trichoderma has been s h o w n to p r o d u c e 3-glucanase a n d chitinase (26-29). W e observed the i n t e r a c t i o n between T. harzianum a n d the p a t h o g e n Rhizoctonia solani b y use of s c a n n i n g electron m i c r o s c o p y a n d noticed the h y p h a e of the antagonist c o i l i n g a r o u n d the h y p h a e of the p a t h o g e n a n d p e n e t r a t i n g some of t h e m . W h e n the antagonist was detached f r o m the p a t h o g e n , s c a r r i n g of the p a t h o g e n as well as h y p h a l p e n e t r a t i o n o c c u r r e d . T h i s p h e n o m e n o n of m y c o p a r a s i t i s m must at least i n p a r t be responsible for the b i o c o n t r o l a c t i o n . M y c o p a r a s i t i s m was o n l y observed on t a p water agar a n d not p o t a t o dextrose agar, i n d i c a t i n g t h a t more r e a d i l y a v a i l a b l e c a r b o n sources m a y repress the need to use the c a r b o n substrates c o n t a i n e d i n cell w a l l s . W h e n T. harzianum I M I 2 7 5 9 5 0 was a p p l i e d at v a r i ous p r o p a g u l e n u m b e r s w i t h Pythium ultimum at various doses i n pots of p e a t / g r i t a n d lettuce g r o w n i n each pot, the effectiveness of the antagonist a g a i n depended o n i t b e i n g i n o c u l a t e d at a d o m i n a n t c o n c e n t r a t i o n over the pathogen (Lumsden, R . D . , U S D A , Beltsville, M D ; L y n c h , J . M . , A F R C I n s t i t u t e of H o r t i c u l t u r a l R e s e a r c h , L i t t l e h a m p t o n , E n g l a n d ; u n p u b l i s h e d data). W h e n T. harzianum I M I 2 9 8 3 7 2 was g r o w n on cell walls of R. solani as c a r b o n source, electrophoresis showed t h a t a m u c h more c o m p l e x m i x ture of e x t r a c e l l u l a r proteins was f o r m e d w h e n the antagonist was g r o w n o n glucose (28). W h i l e b o t h l , 3 - / ? - D glucanase a n d c h i t i n a s e were i n d u c e d , irrespective of whether glucose or cell walls were used as the c a r b o n s u b strate, the t o t a l a c t i v i t y a n d specific a c t i v i t y depended o n the s u b s t r a t e (Table II). T h e c o m p l e x p r o t e i n profiles f r o m the fungus g r o w n o n the p a t h o g e n cell walls were resolved i n p a r t b y u s i n g t w o - d i m e n s i o n a l electrophoresis. T h i s gave a more accurate p i c t u r e of the n u m b e r of proteins i n d u c e d . T h e profiles of m a j o r o n cell walls of R. solani b u t for A G I the profile c o m p o s i t i o n or s t r u c t u r e

bands were s i m i l a r when T. harzianum was g r o w n s t r a i n s f r o m anastomosis groups A G 2 a n d A G 4 , was different; t h i s m a y reflect differences i n the of cell walls between anastomosis groups ( 2 8 , 2 9 ) .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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T a b l e II. E n z y m e A c t i v i t y I n d u c e d i n T. harzianum

Glucose C e l l walls of Rhizoctonia

solani

1

Total

1

Specific

IMI298372 Chitinase Activity

1,3-/?-D-glucanase Activity G r o w t h S u b s t r a t e at 5 g 1

615

Total

2

Specific

3

36.7

0.573

0.412

0.0065

26.7

0.111

1.132

0.0047

/ i m o l glucose released f r o m l a m i n a r i n m i n " μπιοί p r o d u c t s released m i n " (μζ p r o t e i n ) " μπιοί N - a c e t y l g l u c o s a m i n e released f r o m c h i t i n m i n " Source: R e p r o d u c e d w i t h p e r m i s s i o n f r o m Ref. 28. © Society for Microbiology. 1

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General

In order to f r a c t i o n a t e the e x t r a c e l l u l a r p r o t e i n f r a c t i o n f u r t h e r , i t was a n a l y z e d b y isoelectric focusing, fast p r o t e i n l i q u i d c h r o m a t o g r a p h y a n d c h r o m a t o f o c u s i n g i n c o n j u n c t i o n w i t h gel filtration a n d p o l y a c r y l a m i d e gel electrophoresis (29). G e l filtration revealed two peaks of protease a c t i v i t y at p H 4.0, c o r r e s p o n d i n g to enzymes of m o l e c u l a r weights 65,000 a n d 23,000. W i t h c h r o m a t o f o c u s i n g i n the ranges p H 7-4 a n d p H 9-6, a large n u m b e r of proteases were separated, some of w h i c h c o u l d be " s a t e l l i t e s " caused by a l t e r a t i o n i n charged a m i n o acids. It s h o u l d also be noted t h a t some of the more p r o m i n e n t p r o t e i n peaks d i d not correspond to any peaks of e n z y m e activity studied. E l a d et al. (26) suggested t h a t s t r a i n s of Trichoderma s p p . m a y be se­ lected for t h e i r b i o c o n t r o l effectiveness by screening for l , 3 - / ? - D glucanase a n d c h i t i n a s e a c t i v i t i e s , w i t h c h i t i n a n d g l u c a n b e i n g p e r h a p s the m o s t o b v i o u s targets to a t t a c k i n the f u n g a l p a t h o g e n cell w a l l s . T h u s , t o t a l p r o ­ t e i n profiles were o b t a i n e d f r o m a range of Trichoderma s p p . (28) a n d no c o r r e l a t i o n between those p a t t e r n s a n d b i o c o n t r o l effectiveness against Rhi­ zoctonia solani was established ( R i d o u t , C . J . ; C o l e y - S m i t h , J . R . ; L y n c h , J . M . , u n p u b l i s h e d d a t a ) . C h i t i n a s e a c t i v i t y c o u l d easily be detected by as­ s a y i n g the release of p - n i t r o p h e n y l - N - a c e t y l - / J - D - g l u c o s a - m a n i d e ( p N N A G ) as w e l l as the s t a n d a r d m e t h o d of assaying release of N - a c e t y l - g l u c o s a m i n e ( N A G ) f r o m c h i t i n (29). T h e assay u s i n g p N N A G h a d the advantage of b e i n g s i m p l e , sensitive a n d r e p r o d u c i b l e . T h e a b i l i t y of the chitinase e n ­ z y m e to release N A G f r o m c h i t i n was c o n s i d e r a b l y reduced after p a r t i a l l y p u r i f y i n g t h i s e n z y m e by gel filtration. T h i s m a y be because other enzymes were also necessary to release N A G f r o m c h i t i n a n d these enzymes were removed b y p u r i f i c a t i o n . T h e other enzymes c o u l d be proteases since these were i n d u c e d i n considerable q u a n t i t i e s by cell walls of R. solani. Indeed, cell w a l l s of m a n y f u n g i c o n t a i n proteins ( 3 2 , 3 3 ) a n d so protease a c t i v i t y c o u l d be i m p o r t a n t i n the b i o c o n t r o l a c t i o n of f u n g i . T h e r e is a p a r a l l e l for t h i s w i t h e n t o m o p a t h o g e n i c f u n g i where Beauveria bassiana p r o d u c e s proteases w h i c h act i n concert w i t h chitinase i n the p e n e t r a t i o n of insect

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cuticles (34). O v e r a l l , however, i t has t o be concluded t h a t the role o f e x oenzymes o f Trichoderma i n the b i o c o n t r o l a c t i o n against p l a n t p a t h o g e n i c f u n g i is quite unclear at present. Concluding Remarks Trichoderma s p p . produce a set o f powerful diffusible exo-enzymes w h i c h can degrade the c a r b o h y d r a t e fractions o f lignocelluloses. T h e y also p r o duce, i n a d d i t i o n t o a n t i b i o t i c s , a series o f l y t i c enzymes w h i c h enable t h e m to compete more r e a d i l y for available substrates as w e l l as d e s t r o y i n g m a n y p l a n t pathogens. A n o t h e r p r o p e r t y ascribed t o t h i s genus recently is the a b i l i t y t o s t i m u l a t e p l a n t g r o w t h directly, p o s s i b l y b y t h e p r o d u c t i o n o f p l a n t g r o w t h regulators (35-37). T h e f o l l o w i n g questions therefore arise: (1) H o w i m p o r t a n t is the n a t u r a l a c t i o n o f Trichoderma spp. i n induci n g p l a n t residue d e c o m p o s i t i o n a n d suppressing pathogens? a n d (2) Is i t possible t o elevate t h i s a c t i o question is unclear becaus the fungus i n soils is r e q u i r e d . M o s t considerations have i n v o l v e d c o u n t i n g v i a b l e propagules a n d we f o u n d t h a t t h i s has l i t t l e r e l a t i o n s h i p t o fungal biomass present ( L u m s d e n , R . D . , U S D A , B e l t s v i l l e , M D ; L y n c h , J . M . , W h i p p s , J . M . , C a r t e r , J . R , A F R C Institute of Horticultural Research, L i t t l e h a m p t o n , E n g l a n d ; u n p u b l i s h e d d a t a ) . T h e second question has been answered more p o s i t i v e l y because we have s h o w n t h a t i t has been p o s s i ble t o elevate the Trichoderma p o p u l a t i o n size i n s o i l at the expense o f Fusarium b y recovering s t r a w f r o m s o i l , c h o p p i n g i t i n t o 1 c m lengths a n d d e t e r m i n i n g the percentages o f pieces g i v i n g rise to g r o w t h o f each fungus w h e n i n c u b a t e d o n plates o f m a l t a n d V - 8 j u i c e agars (21 a n d u n p u b l i s h e d ) . T h u s , the e n v i r o n m e n t a l p o t e n t i a l o f Trichoderma m a y be considerable. However, m u c h needs t o be done yet t o d e t e r m i n e the relative role of its enzymes a n d m e t a b o l i t e s i n useful n a t u r a l processes, a n d i t s n a t u r a l p o p u l a t i o n biology. It s h o u l d t h e n be possible to isolate f r o m n a t u r e , or to clone, a s t r a i n w h i c h w i l l be h i g h l y effective at c o l o n i z i n g crop residues a n d the rhizosphere, h e l p i n g t o regulate p l a n t g r o w t h a n d n u t r i t i o n w h i l e p r o v i d i n g crop p r o t e c t i o n value. T h e prospects for t h i s step appear quite g o o d as knowledge o n the m o l e c u l a r biology o f Trichoderma is a d v a n c i n g r a p i d l y , especially for T. reesei ( 7 , 3 8 ) . G e n e t i c m a n i p u l a t i o n is l i k e l y t o be b y p l a s m i d - m e d i a t e d t r a n s f o r m a t i o n . F o r e x a m p l e , the invertase o f Saccharomyces cerevisiae has been cloned a n d expressed i n T. reesei C L 8 4 7 b y p r o t o p l a s t t r a n s f o r m a t i o n w i t h p l a s m i d s (7). Acknowledgments I a m very grateful t o m y colleagues w h o have c o n t r i b u t e d t o o u r studies o n l i g n o c e l l u l o l y s i s ( J . P . C a r t e r , S. J . C h a p m a n , N . C u r t i s , D . M . G a u n t , P . H a n d , S. H . T . H a r p e r , L . A . H a r r i s o n , I. A . K i r k w o o d , N . M a g a n , A . P . J . T r i n c i , a n d D . A . Veal) and mycoparasitism ( J . R . C o l e y - S m i t h , R . D . L u m s d e n , a n d C . J . R i d o u t ) referred t o i n this p a p e r . T h e f i n a n c i a l a i d o f the C o m m i s s i o n o f the E u r o p e a n C o m m u n i t i e s ( C o n t r a c t R U W 0 3 3 - U K ) , the A g r i c u l t u r a l G e n e t i c s C o m p a n y , the D e p a r t m e n t o f T r a d e

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and Industry, and the Science and Engineering Research Council is also gratefully acknowledged. Literature Cited 1. Wood, D. A. Ann. Proc. Phytochem. Soc. Eur. 1985, 26, 295-309. 2. Lynch, J . M . J. Appl. Bacteriol. Symp. Supp. 1987, 71S-83S. 3. Thaysen, A. C.; Bunker, H. J. The Microbiology of Cellulose, Hemicel­ lulose, Pectin and Gums; Oxford Univ. Press: Oxford, 1927. 4. Harper, S. H. T.; Lynch, J . M . J. Sci. Food Agric. 1981, 32, 1057-62. 5. Eveleigh, D. E . In Biology of Industrial Micro-organisms; Demain, A. L.; Solomon, Ν. Α., Eds.; Benjamin Cummings Publishing Co.: Menlo Park, NJ, 1985; p. 489. 6. Papavizas, G . C. Ann. Rev. Phytopath. 1985, 23, 23-54. 7. Durand, H. Baron, M . Genetics of Cellulose J., Eds.; Academic Press: London, 1988; p. 135. 8. O'Dwyer, M . H. Biochem. J. 1926, 20, 656-64. 9. Gaunt, D. M . ; Trinci, A . P. J.; Lynch, J . M . Exp. Mycol. 1985, 9, 174-78. 10. Gaunt, D. M . Ph.D. Thesis, University of Manchester, 1986. 11. Harrison, L. A . Ph.D. Thesis, University of Warwick, 1985. 12. Veal, D. A. Ph.D. Thesis, University of Reading, 1985. 13. Veal, D. Α.; Lynch, J. M . J. Appl. Bacteriol. 1987, 63, 245-53. 14. Veal, D. Α.; Lynch, J. M . Nature, Lond. 1984, 310, 695-96. 15. Harper, S. H. T.; Lynch, J . M . Trans. Brit. Mycol. Soc. 1985, 85, 655-61. 16. Magan, N.; Lynch, J. M . J. Gen. Microbiol. 1986, 132, 1181-87. 17. Harper, S. H. T.; Lynch, J . M . Curr. Microbiol. 1986, 14, 127-31. 18. Chapman, S. J.; Lynch, J . M . Enzyme Microb. Technol. 1985, 7, 16163. 19. Lynch, J. M.; Harper, S. H. T . Phil. Trans. R. Soc. Lond. 1985, B310, 221-26. 20. Halsall, D. M . ; Goodchild, D. J . Appl. Environ. Microbiol. 1986, 51, 849-54. 21. Magan, N.; Hand, P.; Kirkwood, I. Α.; Lynch, J. M . Soil Biol. Biochem. 1989, 21, 15-22. 22. Lynch, J . M . Curr. Microbiol. 1987, 16, 49-53. 23. Claydon, N.; Allan, M.; Hanson, J. R.; Avent, A. G. Trans. Brit. Mycol. Soc. 1987, 20 , 263-64. 24. Simon, Α.; Dunlop, R. W.; Chisalberti, E . L.; Sivasithamparan, K. Soil Biol. Biochem. 1988, 20, 263-64. 25. Denis, C.; Webster, J . Trans. Brit. Mycol. Soc. 1971, 57, 41-48. 26. Elad, Y.; Chet, I.; Henis, Y . Can. J. Microbiol. 1982, 28, 719-25. 27. Elad, Y.; Barak, R.; Chet, I. Can. J. Microbiol. 1984, 16, 381-86. 28. Ridout, C. J.; Coley-Smith, J . R.; Lynch, J . M . J. Gen. Microbiol. 1986, 132, 2345-52.

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29. Ridout, C. J.; Coley-Smith, J . R.; Lynch, J . M . Enzyme Microb. Tech­ nol. 1988, 10, 180-87. 30. Reynolds, M.; Weinhold, A. R.; Morris, T . J. Phytopathology 1983, 73, 903-06. 31. Kuninagen, S.; Yokosawa, R. Ann. Phytopath. Soc. Japan 1980, 46, 150-58. 32. Bartnicki-Garcia, S. In Handbook of Microbiology; Laskin, A. I.; Lechevalier, Η. Α., Eds.; CRC Press: Cleveland, 1973; p. 201. 33. Rosenberger, P. The Filamentous Fungi; Edward Arnold: London, 1976; p. 328. 34. Ferron, P. Ann. Rev. Entomol. 1978, 23, 409-42. 35. Baker, R.; Elad, Y.; Chet, I. Phytopathology 1984, 74, 1019-21. 36. Chang, Y.; Chang, Y.; Baker, R. Plant Disease 1986, 70, 145-48. 37. Windham, M. T.; Elad, Y.; Baker, R. Phytopathology 1986, 76, 518-21. 38. Knowles, J.; Teeri, T ä In Biochemistry and Beguin, P.; Millet, J., Eds.; Academic Press: London, 1988; p. 153. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 45 B i o d e g r a d a t i o n o f the H e t e r o - 1 , 4 - L i n k e d

Xylans

Robert F. H . Dekker Commonwealth Scientific and Industrial Research Organisation, Division of Biotechnology, Private Bag 10, Clayton 3168, Victoria, Australia

The heteroxylans occur in the plant cell wall of terres­ trial plants, and b t t th extent f 35% depending upon group of complex polysaccharides composed of a back bone chain of 1,4-β-linked D-xylose residues to which are attached various appendages. These may be L­ -arabinose;D-glucuronic acid; various short oligosaccha­ ride chains consisting of D-xylose, L-arabinose, galac­ tose and D-glucuronic acid; O-acetyl groups; feruloyl and p-coumaroyl esters linked via L-arabinose residues; and benzyl ether groups as occur in lignin-carbohydrate complexes. The heteroxylans constitute a renewable feedstock from which many chemicals could be derived. This requires hydrolysis, and fermentation or transfor­ mation steps. While enzymes degrading the heteroxy­ lan are known as xylanases, they also require the ad­ ditional actions ofβ-xylosidases,α-arabinosidases, α­ -glucuronidases and certain esterases for total hydrolysis. This chapter is concerned with their specificity in de­ grading their substrate. The biodegradative pathways of the heteroxylans are also discussed. The hetero-l,4-linked x y l a n s (or heteroxylans) constitute a w e l l - c h a r a c t e r ­ ized group o f polysaccharides w h i c h f o r m t h e m a j o r components o f t h e hemicellulosic fractions o f terrestrial p l a n t s (1-4). Softwoods are a n excep­ t i o n , where t h e heteroxylans can be present as a m i n o r c o m p o n e n t o f the t o t a l hemicelluloses. T h e y have been isolated f r o m grasses, legumes, ferns, softwoods a n d h a r d w o o d s , a n d collectively m a y c o n s t i t u t e u p t o 3 5 % o f the t o t a l d r y weight o f higher l a n d plants (4). A s such t h e h e t e r o x y l a n s rank second t o cellulose i n a b u n d a n c e as n a t u r a l l y o c c u r r i n g o r g a n i c c h e m ­ icals i n t h e biosphere. T h e heteroxylans are closely associated w i t h other 0097-6156/89/0399-0619$06.00/0 Published 1989 American Chemical Society

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h e m i c e l l u l o s i c polysaccharides, cellulose a n d l i g n i n a n d are present i n b o t h p r i m a r y a n d secondary layers o f the p l a n t cell w a l l . C e r t a i n h e t e r o x y l a n s are also f o u n d i n the cell walls o f the aleurone layer (5) a n d e n d o s p e r m (6) o f cereal g r a i n s . T h e p e n t o s a n content (as a r a b i n o x y l a n ) has been re­ p o r t e d to range f r o m 5-9% i n w h e a t , 2 - 3 % i n wheat flour, 4 - 1 3 % i n g e r m a n d 2 0 - 3 5 % i n b r a n (7). H e t e r o x y l a n s can therefore be b o t a n i c a l l y cate­ gorized as either n o n - e n d o s p e r m i c , or endospermic (8). T h e h e t e r o x y l a n s f r o m e n d o s p e r m i c m a t e r i a l s (caryopses) can be e x t r a c t e d under r e l a t i v e l y m i l d c o n d i t i o n s (e.g., water or d i l u t e alkali) as these tissues are generally n o n - l i g n i f i e d . L i g n i f i e d tissues (e.g., grasses a n d w o o d y p l a n t s ) , however, require a d e l i g n i f i c a t i o n step (e.g., acidified chlorine d i o x i d e (9)), a n d the h e t e r o x y l a n s are e x t r a c t e d f r o m the r e s u l t i n g holocellulose u s i n g either d i ­ lute aqueous a l k a l i (10) or D M S O (11). I n t h e i r n a t i v e state, i.e., w i t h i n the p l a n t cell w a l l , the h e t e r o x y l a n s are u s u a l l y i n s o l u b l e , b u t f r e q u e n t l y show water s o l u b i l i t y after i s o l a t i o n . C h e m i c a l Structure of the Heteroxylans T h e classification o f the h e t e r o x y l a n s is based u p o n c h e m i c a l s t r u c t u r e . S t r u c t u r a l l y , the h e t e r o x y l a n s are / ? - l , 4 - l i n k e d D - x y l o p y r a n o s y l ( X y i p ) p o l y m e r s to w h i c h are u s u a l l y a t t a c h e d monosaccharide or s h o r t - c h a i n oligosaccharides. These appendages consist m a i n l y o f 1 , 3 - l i n k e d α - L a r a b i n o f u r a n o s y l residues ( A r a f ) , (e.g., a r a b i n o x y l a n s , a r a b i n o g l u c u r o n o x y l a n s ) , or 1,2-linked α-D-glucopyranosyl u r o n i d e residues ( G l c p A , e.g., g l u c u r o n o x y l a n s ) , where the g l u c u r o n i c a c i d residue m a y c o n t a i n a n O - m e t h y l group at c a r b o n 4. 0 - A c e t y l groups are also present as s u b s t i t u e n t s o n h e t e r o x y l a n s (2) a n d these are u s u a l l y located o n C - 3 of the xylose residues b u t m a y also be present o n C - 2 , or b o t h (12-14). T h e degree o f s u b s t i t u t i o n is h i g h , e.g., i n some h a r d w o o d (e.g., b i r c h ) a n d grass (Lolium sp.) h e t ­ e r o x y l a n s , every second xylose residue m a y be a c e t y l a t e d ( 2 , 1 2 , 1 5 ) , w h i l e i n beech leaves i t has been r e p o r t e d (16) t h a t every xylose residue m a y be s u b s t i t u t e d w i t h a n O-acetyl s u b s t i t u e n t . It s h o u l d be e m p h a s i z e d t h a t the O - a c e t y l groups are r e a d i l y removed when the h e t e r o x y l a n s are e x t r a c t e d w i t h a l k a l i (saponified), a n d the isolated p r o d u c t can therefore be m i s l e a d i n g l y low i n O - a c e t y l content. O t h e r c h e m i c a l groups m a y also be l i n k e d to the h e t e r o x y l a n s . T h e r e is now evidence t o suggest t h a t l i g n i n is cov a l e n t l y a t t a c h e d to heteroxylans a n d other p l a n t cell w a l l polysaccharides (17). T h i s evidence is derived f r o m studies on the l i g n i n - c a r b o h y d r a t e c o m ­ p l e x ( L C C ) of various grasses a n d h a r d w o o d s w h i c h were f o u n d to c o n t a i n arabinose, xylose a n d g l u c u r o n i c a c i d ( a n d its 4 - 0 - m e t h y l derivative) as w e l l as other sugars (18-20). T h e r e is s t i l l controversy, however, over the exact n a t u r e of the L C C b o n d despite evidence of the existence of ether (between arabinose a n d l i g n i n (21)) a n d ester (between g l u c u r o n i c a c i d a n d l i g n i n (22)) linkages. M o r e recently, f e r u l o y l a n d p - c o u m a r o y l esters have been s h o w n to be covalently a t t a c h e d to a r a b i n o x y l o b i o s i d e s ( 2 3 , 2 4 ) . T h e f e r u l o y l esters of these c o m p o u n d s were isolated f r o m crude cellulase d i ­ gests o f b a r l e y s t r a w (23), wheat (25) a n d Zea shoot (26) cell w a l l s , b a r l e y aleurone layers (27) a n d bagasse L C C (24). T h e i d e n t i f i c a t i o n of these c o m -

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p o u n d s p r o v i d e s f u r t h e r evidence t h a t p h e n o l i c s u b s t i t u e n t s s u c h as those c o n s t i t u t i n g l i g n i n are a t t a c h e d to the x y l a n c h a i n . T h e d e t a i l e d c h e m i s t r y of the hemicelluloses i n c l u d i n g the h e t e r o x y lans has been reviewed b y A s p i n a l l ( 1 , 2 8 ) , T i m e l l (2-4) a n d W i l k i e (8). O n the basis of t h e i r sugar c o m p o s i t i o n the h e t e r o x y l a n s are classified as a r a b i n o x y l a n s (found m a i n l y i n e n d o s p e r m i c tissue), a r a b i n o g l u c u r o n o x y l a n s (present i n grasses a n d softwood species) a n d a c e t y l a t e d g l u c u r o n o x y l a n s ( i n h a r d w o o d s ) . T h e r e are r e p o r t s of a h o m o x y l a n o c c u r r i n g i n esparto grass (29) w h i l e g a l a c t o a r a b i n o g l u c u r o n o x y l a n s have been f o u n d i n b a m b o o (30). E n d o s p e r m i c h e t e r o x y l a n s i n c l u d i n g those d e r i v e d f r o m b r a n have been c h a r a c t e r i z e d as h a v i n g h i g h l y b r a n c h e d s t r u c t u r e s ( 3 1 , 3 2 ) . W h e a t a n d c o r n b r a n h e t e r o x y l a n s have been r e p o r t e d t o c a r r y at least one, a n d sometimes t w o , s u b s t i t u e n t s per x y l o s y l residue of the x y l a n c h a i n . T h e s e c o m p l e x s t r u c t u r e s have been referred to as g l u c u r o n o a r a b i n o x y l a n s (31). E n z y m e s Degrading th E n z y m e s w h i c h a t t a c k the h e t e r o x y l a n s are o f two types. T h o s e t h a t h y d r o l y z e the g l y c a n c h a i n a n d its appendage groups are k n o w n as hydrolases [ E C 3.2.1.], w h i l e those enzymes t h a t remove the ester groups are k n o w n as esterases [ E C 3.1.1.]. A l t h o u g h enzymes h y d r o l y z i n g the O - a c e t y l groups o n h e t e r o x y l a n s have o n l y been recently discovered (33), h y d r o l y t i c enzymes d e g r a d i n g the h e t e r o x y l a n s are well recognized, a n d have been the s u b j e c t o f several recent reviews (34-39). T h e r e are generally two k i n d s of hydrolases t h a t degrade the h e t e r o x y l a n s . T h e first i n c l u d e the exo-glycosidases, w h i c h cleave m o n o s a c c h a r i d e a n d short s i d e - c h a i n oligosaccharide linkages, a n d are also i n v o l v e d i n h y d r o l y z i n g the low M W e n d - p r o d u c t s (e.g., oligosaccharides) released d u r i n g d e p o l y m e r i z a t i o n of the m a i n b a c k b o n e c h a i n . T h e second g r o u p o f enzymes i n c l u d e the glycanases, or p o l y s a c c h a r i d e hydrolases, a n d these are responsible for a t t a c k o n the p o l y m e r b a c k b o n e itself. T h e y are classified a c c o r d i n g to the n a t u r e o f the p o l y s a c c h a r i d e c h a i n they act u p o n . I n the case o f the h e t e r o x y l a n s , they are k n o w n as the l , 4 - / ? - D - x y l a n a s e s , or xylanases [ E C 3.2.1.8.]. O n l y one t y p e o f x y l a n a s e , the e n d o - x y l a n a s e , has been i d e n t i f i e d w h i c h a t t a c k i n t e r n a l x y l o s i d i c l i n k ages of the h e t e r o x y l a n c h a i n c a u s i n g m u l t i p l e scission t h a t r e s u l t s i n a decreased D P of the s u b s t r a t e . E x o - e n z y m e s s u c h as those t h a t a t t a c k c e l lulose have not yet been u n e q u i v o c a l l y identified for the x y l a n a s e s . T h i s is i n spite of the fact t h a t there have been reports o f /?-xylosidases t h a t c a n s l o w l y a t t a c k x y l a n p r o d u c i n g x y l o s e (see (40)), a n d a novel exo-cellulase w h i c h also h y d r o l y z e d x y l a n t o xylobiose ( X y l , (41)). H o w e v e r , even i f exox y l a n a s e s e x i s t e d , they w o u l d o n l y be expected t o m a k e a l i m i t e d a t t a c k o n h e t e r o x y l a n s as t h e i r actions w o u l d be t e r m i n a t e d at each b r a n c h p o i n t e n countered. T h e t o t a l h y d r o l y s i s of the h e t e r o x y l a n c h a i n requires the endox y l a n a s e s a c t i n g i n s y n e r g i s m w i t h the exo-glycosidases, w h i c h i n c l u d e the /?-xylosidases, α-arabinosidases, galactosidases a n d or-glucuronidases. E n z y m e s a t t a c k i n g the n o n - g l y c o s i d i c appendages o f the h e t e r o x y ­ lans i n c l u d e several recently discovered enzymes, i.e., a c e t y l - x y l a n esterase (42) a n d ferulic a c i d esterase (33), w h i c h are specific for the h y d r o l y 2

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sis of O - a c e t y l a n d f e r u l o y l groups, respectively. A n o t h e r n e w l y d i s c o v ­ ered e n z y m e is α-glucuronidase (43) w h i c h specifically h y d r o l y z e s the 4 - 0 m e t h y l g l u c u r o n i c a n d g l u c u r o n i c a c i d s u b s t i t u e n t s f r o m the x y l a n c h a i n . M o d e of A c t i o n of the

Xylanases

In general, endo-1,4-/?-xylanases degrade heteroxylans b y a t t a c k i n g i n t e r ­ n a l /3-xylosidic linkages of the x y l a n backbone chain r e s u l t i n g i n m u l t i p l e scission. T h e sites attacked a l o n g the x y l a n c h a i n a n d the frequency of b o n d cleavage is governed b y the s t r u c t u r e of the h e t e r o x y l a n (i.e., i n the degree a n d frequency of s i d e - c h a i n s u b s t i t u e n t s ) , a n d by the n u m b e r of subsites i n the active site of the enzyme w h i c h affects the free energy of b i n d i n g to the g l y c o s y l residues (44). T h e d e g r a d a t i o n p r o d u c t s a r i s i n g d u r i n g the e a r l y course of h y d r o l y s i s are xylooligosaccharides, some of m i x e d c o n s t i t u t i o n , i.e., u s u a l l y c o n t a i n i n g arabinose a n d / o r g l u c u r o n i c a c i d or i t s 4 - O - m e t h y l d e r i v a t i v e . T h e degree of is u s u a l l y > 4. A s h y d r o l y s i s proceeds these oligosaccharides are u s u a l l y progressively converted to xylose a n d xylobiose; a n d i n some cases, the fi­ n a l p r o d u c t m a y be xylobiose a n d x y l o t r i o s e b u t no xylose m a y be f o r m e d . A s is the case of most endo-xylanases, x y l o b i o s e is u s u a l l y not f u r t h e r de­ graded (34,37-40). T h e u l t i m a t e size of the final e n d - p r o d u c t is d e t e r m i n e d b y the specificity of the e n z y m e , a n d t h i s i n t u r n , m a y be affected b y the frequency a n d s p a c i n g of the monosaccharide, or s i d e - c h a i n s u b s t i t u e n t s , o n the x y l a n chain (40). T w o types of endo-xylanases are generally recognized a n d are classified a c c o r d i n g to whether arabinose is cleaved d u r i n g h y d r o l ­ ysis (40). In a l l of the cases r e p o r t e d , the x y l a n a s e p r e p a r a t i o n was h i g h l y purified as d e m o n s t r a t e d b y the u s u a l c r i t e r i a for homogeneity of proteins i n d i c a t i n g t h a t the h y d r o l y s i s of arabinose was not due to a c o n t a m i n a t i n g e n z y m e . T h e d e b r a n c h i n g xylanases removed arabinose f r o m a r a b i n o x y l a n s a n d a r a b i n o g l u c u r o n o x y l a n s b y c l e a v i n g 1 , 3 - a - L - a r a b i n o f u r a n o s y l residues f r o m the x y l a n backbone c h a i n . A n o t h e r feature of some h i g h l y p u r i f i e d endo-xylanases was t h a t they showed t r a n s g l y c o s y l a t i o n a c t i v i t y , i.e., not o n l y d i d these xylanases h y d r o l y z e the x y l a n c h a i n , b u t they were also c a p a ­ ble of s y n t h e s i z i n g oligosaccharides f r o m the low M W h y d r o l y s i s p r o d u c t s . A n e x a m p l e is a xylanase p r e p a r a t i o n isolated f r o m Cryptococcus albidus In discussing the m o d e of a c t i o n of the xylanases o n l y selected fea­ tures w i l l be discussed as there are numerous detailed reviews o n t h i s t o p i c ( 3 4 , 3 7 , 3 8 , 4 0 ) . It is, however, i m p o r t a n t t o emphasize t h a t most of the xylanases r e p o r t e d i n the l i t e r a t u r e d i s p l a y s i m i l a r a c t i o n p a t t e r n s w h e n a t t a c k i n g their substrates, whether they be x y l a n or x y l o o l i g o s a c c h a r i d e s . T w o xylanases have been isolated (46) f r o m the i n d u s t r i a l l y i m p o r t a n t b a c t e r i u m Clostridium acetobutylicum. T h i s o r g a n i s m has the a b i l i t y t o fer­ m e n t heteroxylans to acetone, n - b u t a n o l a n d e t h a n o l . O n e of the x y l a n a s e s isolated (i.e., of M W 65,000) h y d r o l y z e d oat spelt x y l a n to oligosaccharides of D P 2-6 w h i c h were not further degraded (47). X y l o h e x a o s e ( X y l ) was o n l y s l o w l y degraded t o xylotetraose ( X y U ) a n d X y l , w h i l e X y l , x y l o p e n taose ( X y l ) , x y l o t r i o s e ( X y l ) a n d X y l 2 were not degraded f u r t h e r . N o 6

2

5

4

3

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x y l o s e was p r o d u c e d . T h e a c t i o n p a t t e r n o f t h i s e n z y m e suggested t h a t i t required a substrate b i n d i n g site of at least 6-7 xylose residues for b o n d cleavage t o o c c u r as X y l e was the s m a l l e s t x y l o o l i g o s a c c h a r i d e a t t a c k e d . T h e other x y l a n a s e ( M W 29,000) was more t y p i c a l of e n d o - x y l a n a s e s i n t h a t it degraded x y l a n i n t o m a i n l y X y l a n d X y l 3 w i t h some xylose a p p e a r i n g after 24 h h y d r o l y s i s (47). A t h e r m o p h i l i c Clostridium sp., v i z . Ci stercorarium, w h i c h produces e t h a n o l (48), p r o d u c e d three x y l a n a s e s . T h e s e were o p t i m a l l y active at 6 5 ° C a n d degraded x y l a n ( l a r c h w o o d ) y i e l d i n g x y l o o l i g o s a c c h arides of D P 2-3. Xyh a n d X y l 3 were not a t t a c k e d , a n d a r a binose was not detected i n the x y l a n digests. T h i s b a c t e r i u m also p r o d u c e d a c e l l - w a l l b o u n d enzyme(s) w h i c h acted p r e f e r e n t i a l l y o n Xyh a n d X y l 3 to y i e l d x y l o s e , a n d w h i c h was r e p o r t e d t o also a t t a c k x y l a n s l o w l y l i b e r a t i n g x y l o s e (48). A n a k l a l o p h i l i c Bacillus sp. has been i s o l a t e d w h i c h p r o d u c e d xylanases w h e n g r o w n i n a l k a l i n e m e d i u m (49). T h i s b a c t e r i u m p r o d u c e d two x y l a n a s e s : one was active at n e u t r a l p H ( x y l a n a s e N ) , w h i l e the other showed a c t i v i t y at a l k a l i n lanase A ) . B o t h xylanases were o p t i m a l l y a c t i v e at 7 0 ° C , a n d degraded x y l a n to m a i n l y X y l a n d X y l . X y l o s e was not p r o d u c e d i n the course of h y d r o l y s i s , nor were X y l a n d X y l a t t a c k e d . X y l was degraded to X y l , X y l 3 a n d higher oligosaccharides, i n d i c a t i n g t r a n s x y l o s y l a t i o n a c t i v i t y . T h e a l k a l i n e stable x y l a n a s e is o f i n d u s t r i a l interest because o f i t s s t a b i l i t y at h i g h p H . It c o u l d find a p p l i c a t i o n i n r e m o v i n g x y l a n f r o m w o o d cellulose p u l p s for use i n the m a n u f a c t u r e o f r a y o n . 2

2

3

2

3

4

2

T h e xylanases of yeasts have been s t u d i e d i n d e t a i l by B i e l y a n d coworkers u s i n g Cryptococcus albidus as a m o d e l (35). T h e x y l a n a s e f r o m C. albidus also possessed t r a n s g l y c o s y l a t i o n a c t i v i t y , a n d was capable of t r a n s f e r r i n g xylose to cellobiose to f o r m 6 ' - 0 - / ? - D - x y l o s y l c o m p o u n d s of cellobiose. These c o m p o u n d s c o u l d be degraded by the x y l a n a s e t h a t was i n v o l v e d i n t h e i r synthesis (50). I n general, the a c t i o n of yeast x y l a n a s e s i n a t t a c k i n g t h e i r substrates was s i m i l a r to t h a t p r o d u c e d by c e r t a i n b a c t e r i a a n d f u n g i , a n d y i e l d e d m a i n l y X y l a n d X y l 3 as e n d - p r o d u c t s (35). 2

X y l a n a s e s p r o d u c e d b y the a c t i n o m y c e t e Streptomyces are w e l l - c h a r a c t e r i z e d (51). A x y l a n a s e p r o d u c e d by Streptomyces sp. s t r a i n E - 8 6 y i e l d e d some very i n t e r e s t i n g xylose oligosaccharides f r o m c o r n c o b a r a b i n o x y l a n w h i c h revealed the m o d e of a c t i o n of the e n z y m e , a n d the n a t u r e o f some o f the side-group appendages t o the x y l a n c h a i n o f c o r n c o b a r a b i n o x y l a n . T h e m a i n e n d - p r o d u c t s of corncob a r a b i n o x y l a n h y d r o l y s i s b y the Streptomyecete x y l a n a s e was X y l a n d Xyh a n d some x y l o s e , w h i l e the a r a b i n o x y l o o l i g o s a c c h a r i d e s consisted of two p r e d o m i n a n t types as s h o w n below: 2

Xylp-Xylp , Xylp-Xylp-Xyfp. & Xylp-Xytp , Xylp-Xylp-Xyfp . I

Aral

I

Araf

9

I

Araf-Xylp

I

Araf-Xylp

O n e series of x y l o o l i g o s a c c h a r i d e h y d r o l y s i s p r o d u c t s c o n t a i n e d a n a - 1 , 3 l i n k e d L - a r a b i n o f u r a n o s y l g r o u p o n the n o n - r e d u c i n g x y l o s e residue, w h i l e

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

624

PLANT C E L L W A L L P O L Y M E R S

the other series o f xylooligosaccharides l i b e r a t e d contained a n interposed L arabinose residue a t t a c h e d t o the n o n - r e d u c i n g xylose u n i t . T h e interposed arabinose w a s l i n k e d t o xylose v i a 1,3- a n d 2 , 1 - b o n d s w h i c h were o f the Gra n d ^ - c o n f i g u r a t i o n , respectively. T h e p r o d u c t i o n o f these h o m o l o g o u s series o f arabinoxylooligosaccharides d e m o n s t r a t e d t h a t the / ? - l , 4 - D - x y l o s i d e linkage i m m e d i a t e l y t o the left o f the s u b s t i t u e n t group o n the x y l a n c h a i n was h y d r o l y z a b l e b y the x y l a n a s e , b u t the b o n d i m m e d i a t e l y t o the right was resistant t o h y d r o l y s i s (51). T w o other arabinoxylooligosaccharides were also p r o d u c e d b y the Streptomyces x y l a n a s e , v i z . ,

xyip-xyip-xyip & Xylp-Xyip-Xyip , Araf

Araf-Xyip

w h i c h are inconsistent w i t h the p r e d i c t e d m o d e o f a c t i o n o f t h i s x y l a n a s e . However, t h e p r o d u c t i o n o c u r r i n g b y the x y l a n a s e a t t a c k i n g the x y l a n c h a i n where there are n o s u b s t i t u e n t groups. D u r i n g t h e early stages o f h y d r o l y s i s X y l t o X y l were p r o d u c e d w h i c h suggests t h a t there were at least 7 contiguous xylose u n i t s u n b r a n c h e d . Cleavage as i n d i c a t e d below w o u l d result i n t h e p r o d u c t i o n o f these b r a n c h e d arabinoxylooligosaccharides. 4

6

Jxy«pJxy«P^ytoJxyi^ Araf

Araf-Xyip

T h e i s o l a t i o n o f the above oligosaccharides also revealed t h a t corncob a r a b i n o x y l a n contained side-chains o f 2 - O - D - x y l o p y r a n o s y l - L - a r a b i n o f u r a n o sides (51). T h e best characterized a n d most s t u d i e d o f the xylanases have been those o f f u n g a l o r i g i n a n d especially those f r o m Aspergillus niger, Sporotrichum dimorphosporum, Ceratocystis paradoxa, Oxiporus s p . , 7Vametes hirsuta a n d some Trichoderma species ( 3 4 , 3 7 ) . M o r e recently, there has been interest i n e m p l o y i n g x y l a n o l y t i c enzymes t o remove x y l a n s f r o m w o o d cellulose p u l p s for use i n t h e m a n u f a c t u r e o f r a y o n . X y l a n a s e s f r o m Trichoderma harzianum (52), a n d the t h e r m o p h i l i c fungus, Thermoascus aurantiacus (53) have been s t u d i e d for t h i s purpose. I n general, m u l t i p l e xylanases have been p r o d u c e d b y the f u n g i , e.g., five f r o m A. niger (54) a n d nine f r o m S. dimorphosporum (55). W h i l e m u l t i p l e xylanases are p r o b a b l y the result o f p r o t e o l y t i c cleavage f o l l o w i n g t h e i r excretion i n t o t h e e x t r a c e l l u l a r m e d i u m , there are also reports o f isozymes o c c u r r i n g (54). X y l a n a s e s f r o m A. niger w i t h u n u s u a l h y d r o l y s i s specificities were r e cently r e p o r t e d (54), a n d five xylanases were isolated a n d p u r i f i e d . X y lanase 1 a t t a c k e d soluble l a r c h w o o d a r a b i n o g l u c u r o n o x y l a n a n d xylose oligosaccharides o f D P > 3, t o m a i n l y X y l a n d xylose, b u t was i n a c tive towards a n insoluble x y l a n f r a c t i o n (larchwood) prepared b y d i s s o l v i n g the aforementioned x y l a n i n water a n d s e p a r a t i n g the undissolved f r a c t i o n 2

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

45.

Biodégradation of Hetero-l,4-Linked Xylans

DEKKER

625

(56). X y l a n a s e s 2 a n d 3, considered isoenzymes, degraded b o t h s o l u b l e a n d i n s o l u b l e x y l a n s d e r i v e d f r o m l a r c h w o o d at sites near a r a b i n o s e s u b s t i t u e n t s o n the x y l a n c h a i n , a n d y i e l d e d m a i n l y X y l 3 a n d X y U w i t h lesser a m o u n t s o f X y l b u t no x y l o s e . X y l a n a s e 4 showed none o f the affinity o f x y l a n a s e s 2 a n d 3 for b r a n c h p o i n t s , b u t was h i g h l y a c t i v e t o w a r d s l i n e a r x y l o o l i g o s a c c h a r i d e s a n d more a c t i v e o n soluble t h a n i n s o l u b l e x y l a n s . F i n a l l y , x y l a n a s e 5 a t t a c k e d soluble x y l a n , b u t a t t a c k e d i n s o l u b l e x y l a n o n l y i f the arabinose branches were r e m o v e d . N o arabinose was l i b e r a t e d b y any of the five A. niger x y l a n a s e s . Insoluble x y l a n was also c a p a b l e of b e i n g h y d r o l y z e d b y t w o x y l a n a s e s o f T . harzianum (57). A light s c a t t e r i n g m e t h o d was used to m o n i t o r h y d r o l y s i s , a n d b o t h enzymes were f o u n d t o c o m p l e t e l y s o l u b i l i z e the s u b s t r a t e w i t h i n 0.5 h . 2

E n z y m a t i c h y d r o l y s i s of a 4 - O - m e t h y l g l u c u r o n o x y l a n f r o m l a r c h w o o d a n d oat spelt a r a b i n o g l u c u r o n o x y l a n b y a x y l a n a s e f r o m Polyporus tulipiferae p r o d u c e d a n a r r a c o n t a i n i n g either L - a r a b i n o s groups (58). T h e s u b s t i t u e n t groups were l o c a t e d o n the n o n - r e d u c i n g e n d o f the x y ^ o l i g o s a c c h a r i d e c h a i n . S i m i l a r oligosaccharides were observed b y others a n d these have been described i n reference 34. F o u r o l i g o s a c c h a rides were i d e n t i f i e d as ( 4 - 0 - M e ) - G l c p A - X y l ( n b e i n g 3 a n d 4) for the a c i d i c x y l o o l i g o s a c c h a r i d e s , a n d A r a f - X y l ( n b e i n g 2 a n d 3) for the a r a b i n o x y ^ o l i g o s a c c h a r i d e s . A new a r a b i n o x y ^ o l i g o s a c c h a r i d e was i d e n t i f i e d f r o m e n z y m i c digests of spear grass (Heteropogon contortus) h e m i c e l l u l o s e Β ( b r a n c h e d x y l a n ) , a n d was p r o d u c e d b y a x y l a n a s e f r o m Cephalosporium sacchari (59). Its s t r u c t u r e was confirmed by p e r m e t h y l a t i o n a n a l y s i s a n d b y specific e n z y m i c h y d r o l y s i s to be 4 - a - L - A r a f - / ? - D - X y l 4 . T h e x y l a n a s e f r o m C. sacchari w h i c h is a n a r a b i n o s e - d e b r a n c h i n g e n z y m e , h y d r o l y z e d A r a f - X y U to A r a f - X y l 3 , w h i c h was f u r t h e r h y d r o l y z e d to a r a b i n o s e , X y l a n d x y l o s e . F i n a l l y , x y l o o l i g o s a c c h a r i d e s c o n t a i n i n g O - a c e t y l groups have been i s o l a t e d f r o m Schizophyllum commune x y l a n a s e h y d r o l y z a t e s o f b i r c h (steamed) a c e t y l a t e d x y l a n (42). S o m e of these oligosaccharides were i d e n ­ tified as c a r r y i n g several O - a c e t y l groups. n

n

3

2

Specificities o f X y l a n a s e s T h e specificity of a x y l a n a s e p r e p a r a t i o n m a y be d e t e r m i n e d f r o m the iso­ l a t i o n a n d c h a r a c t e r i z a t i o n o f oligosaccharides f o r m e d f r o m x y l a n h y d r o l ­ ysis. W h i l e the oligosaccharides released were m a i n l y dependent o n the specificity of the x y l a n a s e , the c o m p l e x s t r u c t u r e of the h e t e r o x y l a n also determines the extent of h y d r o l y s i s a n d the site of cleavage. T w o types o f x y l o o l i g o s a c c h a r i d e s are u s u a l l y p r o d u c e d : a c i d i c ( f r o m g l u c u r o n o x y l a n s ) and neutral (from arabino- and arabinoglucurono-xylans). T h e specificity of the a c t i o n of several p u r i f i e d x y l a n a s e s o n v a r i o u s h e t e r o x y l a n s is s h o w n below:

-xyii^xyip-xyipjxytp4xy«pAraf

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

626

PLANT C E L L W A L L P O L Y M E R S

e.g., Ceratocysiis porus tulipiferae

paradoxa (58);

(60), Sporotrichum

dimorphosporum

(61),

Poly-

-Xytp^xyp-xy«p|xyip4xyip-(Xyip)34xyip^Xyip-XyipJxytp Araf

Araf-Xyip

e.g. Streptomyces

sp. E - 8 6 (51);

-Xy«p^xyip-xyip-xyip|xyfp4xyipAraf e.g. Cephalosporium

(59);

sacchari

-Xylp^XylpJXylp-XylpfXyl '

e.g. Aspergillus

Λ ——

Araf

niger v a n T i e g h e m (62);

-xyipjxyip-xyfpjxyipteyi-

GfcpA e.g. Trichoderma

viride

(63);

-xy«pjxyip-xy«p-xy«p|xyip+xy«p-

GlcpA e.g. Aspergillus sp. (63), Oxiporus (63), Polyporus sp. (64) a n d Sporotrichum dimorphosporum (61).

tulipiferae

(58),

Porta

(— , h y d r o l y z a b l e l , 4 - / ? - x y l o s i d i c b o n d s ; — • , b o n d s resistant to h y d r o l ­ ysis; j , m a j o r cleavage sites b y the x y l a n a s e ; a n d j , m i n o r cleavage sites). Biodégradation of the Heteroxylans T w o p a t h w a y s can be envisaged b y w h i c h m i c r o b i a l xylanases a t t a c k the h e t e r o x y l a n s : (i) the endo-xylanases a t t a c k u n b r a n c h e d , or r e l a t i v e l y m o d erately b r a n c h e d regions of the h e t e r o x y l a n chain to y i e l d a n array o f x y looligosaccharides, some of m i x e d c o n s t i t u t i o n a n d c o n t a i n i n g either a r a binose, g l u c u r o n i c a c i d , O - a c e t y l or other s u b s t i t u e n t groups. T h e s e are t h e n further degraded b y a c t i o n of the endo-xylanases or exo-glycosidases; or (ii) the actions o f c e r t a i n exo-glycosidases (i.e., the a - L - a r a b i n o s i d a s e s , α-glucuronidases, /?-xylosidases a n d galactosidases) a n d esterases (e.g., O a c e t y l x y l a n esterase, ferulic a c i d esterase) c o u l d precede the xylanases i n a t t a c k i n g the x y l a n c h a i n , t h e r e b y r e m o v i n g s i d e - c h a i n s u b s t i t u e n t s a n d

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

45.

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Biodégradation of Hetero-l,4-Linked Xylans

627

opening, or exposing, the backbone xylan chain. This mode of action would allow the xylan chain to be more easily attacked by the xylanases, reducing steric hindrance by the side-chain groups. An exception to this would be the action of the debranching xylanases which are capable of removing the arabinose substituents. The ultimate end-products of the synergistic actions of these enzymes are xylose, arabinose, glucuronic acid and acetic acid, with some galactose and phenolic acids or benzyl compounds also being formed. There is evidence that /?-xylosidases enhance the rate and extent of hydrolysis of heteroxylans by xylanases (65,66). This synergism resembles that between cellulase and /?-glucosidase in cellulose hydrolysis (67). Synergism was also observed between xylanase, /?-xylosidase and aglucuronidase in degrading beechwood (Fagus sylvatica) xylan (43). Thus, α-glucuronidase plays an important role in the complete hydrolysis of glucuronoxylans. Similarly, drolysis of arabinoxylan lanase and /?-xylosidase (68). Likewise, acetylxylan esterases acted coop­ eratively with xylanases in degrading birch {Betula verrucosa) acetylated xylan (69). Xylanase alone released small quantities of xylooligosaccha­ rides, some of which contained O-acetyl groups (69). When an acetylxylan esterase was added to a xylanase digest of acetylated xylan, the majority of the oligosaccharides produced were non-acetylated, and hydrolysis was accompanied by an increase in the rate and amount of xylose equivalents released. A similar increase in the rate and extent of acetic acid released was also observed. The esterase appeared to show preference for the low MW acetylated xylooligosaccharides rather than for the highly acetylated polysaccharide (69). Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Aspinall, G.O. Adv. Carbohyd. Chem. 1959, 14, 429-468. Timell, T.E. Adv. Carbohyd. Chem. 1964, 19, 247-295. Timell, T.E. Adv. Carbohyd. Chem. 1965, 20, 409-483. Timell, T.E. Wood Sci. Technol. 1967, 1, 45-70. McNeil, M.; Albersheim, P.; Taiz, L.; Jones, R.L. Plant Physiol. 1975, 55, 64-68. Mares, D.J.; Stone, B.A. Aust. J. Biol. Sci. 1973, 26, 793-812. Cerning, J., Guilbot, A. In Wheat—Production and Utilization; Inglett, G.E., Ed.; AVI: Westport, 1972, p.146-185. Wilkie, K.C.B. Adv. Carbohyd. Chem. Biochem. 1979, 36, 215-264. Timell, T.E. Meth. Carbohyd. Chem. 1965, 5, 144-145. Whistler, R.L.; Feather, M.S. Meth. Carbohyd. Chem. 1965, 5, 134-137. Hagglund, E.; Lindberg, B.; McPherson, J. Acta Chem. Scand. 1956, 10, 1160-1164. Bouveng, H.O. Acta Chem. Scand. 1961, 15, 96-100. Lindberg, B.; Rosell, K.G.; Svensson, S. Sven. Papperstidn. 1973, 76, 30-32.

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14. Reicher, F.; Correa, J. B. C.; Gorin, P. A. J. Carbohyd. Res. 1984, 135, 129-140. 15. Bacon, J. S. D.; Gordon, A. H.; Morris, E . J. Biochem. J. 1975, 149, 485-487. 16. Wood, T . M.; McCrae, S. I. Photochemistry 1986, 25, 1053-1055. 17. Richards, G . N. Proc. Intl. Workshop on Plant Polysaccharides, Struc­ ture and Function; Nantes, France, 9-11 July 1984, p.47-53. 18. Atsushi, K.; Azuma, J. I.; Kosijima, T . Holzforschung 1984, 38, 141149. 19. Neilson, M . J.; Richards, G . N. Carbohyd. Res. 1982, 104, 121-138. 20. Eriksson, O.; Goring, D. A. I.; Lindgren, B. O. Wood Sci. Technol. 1980, 267-279. 21. Koshijima, T.; Watanabe, T.; Azuma, J. Chem. Lett. 1984, 1737-1740. 22. Das, Ν. N.; Das, S. C.; Sarkar, A. K.; Mukherjee, A. K. Carbohyd. Res. 1984, 129, 197-207. 23. Mueller-Harvey, I.; Hartley hyd. Res. 1986, 148, 71-85. 24. Koshijima, T.; Kato, Α.; Azuma, J. Proc. Intl. Symp. Wood and Pulp Chem. 1983, 1, 159-163. 25. Smith, M . M.; Hartley, R. D. Carbohyd. Res. 1983, 118, 65-80. 26. Kato, Y.; Nevins, D. J. Carbohyd. Res. 1985, 137, 139-150. 27. Gubler, F.; Ashford, A. E . ; Bacic, Α.; Blakeney, A. B.; Stone, B. A. Aust. J. Plant Physiol. 1985, 12, 307-317. 28. Aspinall, G . O. Polysaccharides; Pergamon Press: Oxford, 1970; 103115. 29. Chanda, S. K.; Hirst, E . L.; Jones, J. Κ. N.; Percival, E . G . V.J. Chem. Soc. 1950, 1289-1297. 30. Wilkie, K. C. B.; Woo, S. L. Carbohyd. Res. 1977, 57, 145-162. 31. Brillouet, J. M.; Joseleau, J. P. Carbohyd. Res. 1987, 159, 109-126. 32. Bradbury, A. G . W.; Medcalf, D. G . Abstr. Papers Ann. Mtg. Amer. Assoc. Cereal Chem., Denver, Colorado, 1981. 33. MacKenzie, C. R.; Bilous, D.; Schneider, H.; Johnson, K . G . Appl. En­ viron. Microbiol. 1987, 53, 2835-2839. 34. Dekker, R. F. H. In Biosynthesis and Biodegradation of Wood Compo­ nents; Higuchi, T . , Ed.; Academic Press: New York, 1985; p. 505-533. 35. Biely, P. Trends Biotech. 1985, 3, 286-290. 36. Woodward, J. Top. Enzyme Ferment. Biotechnol. 1984, 8, 9-30. 37. Reilly, P. J. In Trends in Biology of Fermentations for Fuels and Chem­ icals; Hollaender, Α., Ed.; Plenum Press: New York, 1981; p. 111-129. 38. Dekker, R. F. H. In Polysaccharides in Food; Blanchard, J. M . V.; Mitchell, J. R., Eds.; Butterworth's: London, 1979; p.93-108. 39. Wong, K. K. Y.; Tan, L. U. L.; Saddler, J. N. Microbiol. Rev. 1988, 52, 305-317. 40. Dekker, R. F. H.; Richards, G . N. Adv. Carbohyd. Chem. Biochem. 1976 , 32 , 277-352. 41. Shikata, S.; Nisizawa, K. J. Biochem. 1975, 78, 499-512. 42. Biely, P.; Puls, J.; Schneider, H. FEBS Lett. 1985, 186, 80-84.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

45.

DEKKER

Biodégradation of Hetero~l,4-Linked Xylans

629

43. Puls, J.; Schmidt, O.; Granzow, C. Enzyme Microb. Technol. 1987, 9, 83-88. 44. Meagher, M . M.; Tao, Β. Y.; Chow, J. M.; Reilly, P. J. Carbohyd. Res. 1988, 173, 273-283. 45. Biely, P.; Vrsanska, M.; Kratky, Z. Eur. J. Biochem. 1981, 119, 565571. 46. Lee, S. F.; Forsberg, C. W.; Gibbins, L. N. Appl. Environ. Microbiol. 1985, 50, 1068-1076. 47. Lee, S. F.; Forsberg, C. W.; Rattray, J. B. Appl. Environ. Microbiol. 1987, 53, 644-650. 48. Berenger, J. F.; Frixon, C.; Bigliardi,J.;Creuzet, N. Can.J.Microbiol. 1985, 31, 635-643. 49. Honda, H.; Kudo, T.; Ikura, Y.; Horikoshi, K. Can. J. Microbiol. 1985, 31, 538-542. 50. Biely, P.; Vrsanska, M 51. Kusakabe, I.; Ohgushi 1983, 47, 2713-2723. 52. Tan, L. U. L.; Yu, E. K. C.; Louis-Seize, G . W.; Saddler, J. N. Biotech­ nol. Bioeng. 1987, 30, 96-100. 53. Yu, E . K. C.; Tan, L. U. L.; Chan, M . K. H.; Deschatelets, L.; Saddler, J. N. Enzyme Microb. Technol. 1987, 9, 16-24. 54. Fournier, R.; Frederick, M . M.; Frederick, J. R.; Reilly, P.J.Biotechnol. Bioeng. 1985, 27, 539-546. 55. Comtat, J. Carbohyd. Res. 1983, 118, 215-231. 56. Frederick, M . M.; Frederick, J. R.; Fratzke, A. R.; Reilly, P. J. Carbo­ hyd. Res. 1981, 97, 87-103. 57. Tan, L. U. L.; Wong, Κ. Κ. Y.; Saddler, J. N. Enzyme Microb. Technol. 1985, 7, 431-436. 58. Brillouet, J. M . Carbohyd. Res. 1987, 159, 165-170. 59. Shambe, T . Carbohyd. Res. 1983, 113, 125-131. 60. Dekker, R. F. H.; Richards, G . N. Carbohyd. Res. 1975, 42, 107-123. 61. Comtat, J.; Joseleau, J. P. Carbohyd. Res. 1981, 95, 101-112. 62. Takenishi, S.; Tsujisaka, Y . Agric. Biol. Ehcm. 1973, 37, 1385-1391. 63. Sinner, M.; Dietrichs, H. H. Holzforschung 1976, 30, 50-59. 64. Comtat, J.; Joseleau, J. P.; Bosso, C.; Barnoud, F. Carbohyd. Res. 1974, 38 , 217-224. 65. Dekker, R. F. H. Biotechnol. Bioeng. 1983, 25, 1127-1146. 66. Deshpande, V.; Lachke, Α.; Mishra, C.; Keskar, S.; Rao, M . Biotechnol. Bioeng. 1986, 28, 1832-1837. 67. Sternberg, D.; Vijayakumar, P.; Reese, Ε. T . Can. J. Microbiol. 1977, 23, 139-147. 68. Poutanen, K. J. Biotechnol. 1988, 7, 271-282. 69. Biely, P.; MacKenzie, C. R.; Puls, J.; Schneider, H. Bio/Technology 1986, 4, 731-733. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 46 T h e X y l a n o l y t i c E n z y m e S y s t e m o f Trichoderma

reesei 1

Kaisa Poutanen and Jurgen Puls 1

2

VTT, Biotechnical Laboratory, Tietotie 2, SF-02150 Espoo, Finland BFH, Institute of Wood Chemistry, Leuchnerstrasse 91, D-2050 Hamburg, Federal Republic of Germany 2

The xylanolytic a well-known produce satile and well suited for the total hydrolysis of differ­ ent xylans. It consists of two major, specific and sev­ eral non-specific xylanases, at least one β-xylosidase, α-arabinosidase and α-glucuronidase and at least two acetyl esterases. The hydrolysis of polymeric xylans starts by the action of endoxylanases. The side-group­ -cleaving enzymes have their highest activities towards soluble, short xylo-oligosaccharides, and make the sub­ stituted oligosaccharides again accessible for xylanases and β-xylosidase. X y l a n is a n essential constituent o f h a r d w o o d s , softwoods, a n d a n n u a l p l a n t s . I n e n z y m a t i c processing o f lignocellulosic b i o m a s s , x y l a n o l y t i c e n ­ zymes m a y be used either i n d i v i d u a l l y , i n selected m i x t u r e s for specific effects o n o n l y t h e x y l a n c o m p o n e n t o f t h e r a w m a t e r i a l , o r i n m i x t u r e s w i t h c e l l u l o l y t i c , p e c t i n o l y t i c o r a m y l o l y t i c enzymes. X y l a n s are heteropolysaccharides; accordingly, x y l a n o l y t i c enzymes i n ­ clude different types o f endo- a n d exo-glycosidases: l , 4 - / ? - D - x y l a n a s e ( E C 3.2.1.8), /?-xylosidase ( E C 3.2.1.37), α-arabinosidase ( E C 3.2.1.55) a n d a g l u c u r o n i d a s e . I n a d d i t i o n t o these, some a c e t y l esterases are considered x y l a n o l y t i c because o f their a b i l i t y t o deacetylate x y l a n s . O f the x y l a n o l y t i c enzymes, e n d o - l , 4 - / ? - D - x y l a n a s e s have been most extensively s t u d i e d as re­ viewed recently (1-3). E n d o x y l a n a s e s are by d e f i n i t i o n d e p o l y m e r i z i n g e n ­ zymes, w i t h highest a c t i v i t i e s towards l o n g c h a i n xylo-oligosaccharides o r polysaccharides. T h e h y d r o l y s i s o f the /?-l,4-linkages o f x y l a n s is c o m p l e t e d b y the a c t i o n o f /?-xylosidases, w h i c h generally have highest a c t i v i t i e s w i t h xylobiose as substrate (4-6). M u l t i p l e xylanases a n d /?-xylosidases have been observed i n different m i c r o o r g a n i s m s , e.g., o f the genera Streptomyces (7), Aspergillus (8), a n d 0097-6156/89/0399-0630$06.00/0 © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

46.

POUTANEN & PULS

Trichoderma Xylanolytic Enzyme System

631

Trichoderma (9-10). M u c h less is k n o w n a b o u t t h e c o n c u r r e n t p r o d u c t i o n of the enzymes w h i c h cleave s u b s t i t u e n t groups o f the x y l a n p o l y m e r . T h e presence o f a c e t y l x y l a n esterases (11,12) a n d α-glucuronidases (13-15) i n x y l a n o l y t i c e n z y m e systems has o n l y recently been p o i n t e d o u t . A l t h o u g h α-arabinosidases have m a i n l y been s t u d i e d as a r a b i n a n - d e g r a d i n g enzymes (16), they have also been s h o w n t o release arabinose f r o m x y l a n s (17). W h i l e Trichoderma reesei is best k n o w n as a n efficient p r o d u c e r o f c e l l u l o l y t i c enzymes, i t has also been r e p o r t e d t o p r o d u c e x y l a n a s e a n d /?-xylosidase (18-20). T w o xylanases a n d a /?-xylosidase have been p u r i ­ fied f r o m T. reesei (10), a n d t w o xylanases (21,22) a n d a /?-xylosidase (5) f r o m T. viride. W e have p r e v i o u s l y s h o w n t h a t T. reesei p r o d u c e s a l l t h e enzymes needed for complete h y d r o l y s i s o f n a t i v e s u b s t i t u t e d x y l a n s (23). O n e x y l a n a s e (24), a /?-xylosidase (25), a n a - a r a b i n o s i d a s e (26), a n d a n a c e t y l esterase (27) o f T. reesei have so f a r been p u r i f i e d . I n t h i s c h a p t e r , the m o d e o f a c t i o n o f these enzymes i n the h y d r o l y s i s o f different x y l a n s is discussed. Materials a n d Methods Source of Enzymes. C u l t u r e filtrates o f T. reesei s t r a i n s V T T - D - 7 9 1 2 5 a n d R u t C - 3 0 were used as s t a r t i n g m a t e r i a l for p u r i f i c a t i o n o f the i n d i v i d u a l enzymes a n d also as crude e n z y m e p r e p a r a t i o n s i n t h e h y d r o l y s i s e x p e r i ­ m e n t s . C u l t i v a t i o n s were c a r r i e d o u t i n a l a b o r a t o r y fermentor at 3 0 ° C for 4 d o n m e d i a c o n t a i n i n g S o l k a floe cellulose ( J a m e s R i v e r C o r p . , N e w H a m p ­ shire, U S A ) , o r glucose a n d d i s t i l l e r ' s spent g r a i n ( A l k o , L t d . , K o s k e n k o r v a , Finland). Enzyme Activity Assays. X y l a n a s e was assayed u s i n g 1% beechwood x y l a n (prepared a c c o r d i n g t o t h e m e t h o d o f E b r i n g e r o v a et ai (28)) as s u b ­ strate as described p r e v i o u s l y (25). /?-Xylosidase was assayed u s i n g 5 m M p - n i t r o p h e n y l - / ? - D - x y l o p y r a n o s i d e as substrate (25), a n d a - a r a b i n o s i d a s e was assayed u s i n g 10 m M p - n i t r o p h e n y l - a - L - a r a b i n o f u r a n o s i d e (26). a G l u c u r o n i d a s e w a s assayed u s i n g 2 % 4 - O - m e t h y l - g l u c u r o n o s y l - x y l o b i o s e as s u b s t r a t e (13), a n d a c e t y l esterase was assayed u s i n g 1 m M a - n a p h t h y l acetate (27). Enzyme Purification. T h e p u r i f i c a t i o n o f the x y l a n o l y t i c enzymes began w i t h a d s o r p t i o n o n a c a t i o n exchanger ( C M - S e p h a r o s e F F ) at p H 4.0. T h e final p u r i f i c a t i o n w a s a c c o m p l i s h e d b y another i o n exchange step as de­ s c r i b e d p r e v i o u s l y for x y l a n a s e (24), /?-xylosidase (25), a - a r a b i n o s i d a s e (26) a n d a c e t y l esterase (27). Hydrolysis Experiments. T h e substrate i n the h y d r o l y s i s e x p e r i m e n t s i n c l u d e d a l k a l i - e x t r a c t e d beechwood 4 - O - m e t h y l g l u c u r o n o x y l a n , D M S O e x t r a c t e d a c e t y l a t e d beechwood 4 - O - m e t h y l g l u c u r o n o x y l a n , a l k a l i - e x t r a c t e d wheat s t r a w a r a b i n o x y l a n a n d a c e t y l a t e d or deacetylated x y l o - o l i g o m e r s f r o m s t e a m i n g o f b i r c h w o o d (24). D e a c e t y l a t i o n w a s c a r r i e d o u t b y i n c u ­ b a t i n g the freeze-dried x y l o - o l i g o m e r s i n a m m o n i a vapor o v e r n i g h t . S u b ­ strate c o n c e n t r a t i o n was 10 g l " , t e m p e r a t u r e 45° a n d h y d r o l y s i s t i m e 24 h. 1

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L P O L Y M E R S

Analyses. T h e m o n o - a n d disaccharides were a n a l y z e d b y H P L C (26). G e l c h r o m a t o g r a p h i c a n a l y s i s o f the x y l o - o l i g o m e r s was p e r f o r m e d i n F r a c t o g e l T S K H W - 5 0 (S) ( M e r c k , F R G ) a n d B i o g e l P 4 m i n u s 400 mesh ( B i o - R a d , U S A ) c o l u m n s (23). A c e t i c a c i d was a n a l y z e d e n z y m a t i c a l l y ( B o e h r i n g e r Test C o m b i n a t i o n 148 261). Results a n d Discussion T h e c u l t u r e filtrates o f T. reesei contained a large n u m b e r o f b o t h c e l l u ­ l o l y t i c a n d h e m i c e l l u l o l y t i c enzymes, w h i c h c o u l d be p a r t i a l l y separated b y c h r o m a t o f o c u s s i n g ( F i g . 1). O f t h e c e l l u l o l y t i c enzymes, several e n d o g l u canases a n d t w o cellobiohydrolases have a l r e a d y been i s o l a t e d a n d c h a r a c ­ terized (30). S o m e o f the endoglucanases i s o l a t e d are nonspecific a n d have x y l a n a s e a c t i v i t y (31). T h e t w o m a j o r x y l a n a s e ( X y l ) peaks i n F i g u r e 1 corresponded t o p l - v a l u e s o f above 7.5 a n d 5.5. W h e n t h e former e n z y m e was f u r t h e r p u r i f i e d (24) ( T a b l e I ) . P r e v i o u s l y , the presence o f at least three e l e c t r o p h o r e t i c a l l y dif­ ferent xylanases i n T. reesei c u l t u r e filtrates w a s r e p o r t e d , w i t h t h e m o s t a c i d i c one s h o w n t o have b r o a d substrate specificity (10). T h i s is i n agree­ m e n t w i t h the r e p o r t e d occurrence o f three xylanases ( p i > 7, p i 5.1, p i 4.5) i n T. reesei (32). T h e results o f t h i s s t u d y , together w i t h those o f others (10,20,31-33) suggest t h a t t h e enzyme s y s t e m o f T. reesei c o n t a i n s two m a j o r , specific endoxylanases, w i t h isoelectric p o i n t s o f 8.5-9 a n d 5-5.5, a n d m a n y non-specific endoglycanases w i t h m o r e a c i d i c p l - v a l u e s a n d b o t h 1,4-/?-glucanase a n d l,4-/?-xylanase a c t i v i t i e s . T a b l e I. X y l a n o l y t i c E n z y m e s Isolated f r o m Trichoderma

Enzyme Xylanase /?-Xylosidase a-Arabinosidase A c e t y l esterase a

b

reesei

M W (kDa)

Pi*

pH-Optimum

Reference

20 100 53 45

~9 4.7 7.5 6.8

5.3 4.0 4.0 5.5

24 25 26 27

From S D S - P A G E . F r o m chromatofocussing.

T h e /?-xylosidase o f T. reesei was a r a t h e r large, p r o b a b l y d i m e r i c e n z y m e a n d , like most other /?-xylosidases, h a d a n acidic p i - v a l u e ( T a b l e I ) . A /?-xylosidase purified earlier f r o m T. viride w i t h a m o l e c u l a r weight o f 101 k D a also h a d a n isoelectric p o i n t p i 4.45 (5). I n a d d i t i o n t o p - n i t r o p h e n y l /?-xylopyranoside a n d xylo-oligosaccharides, t h e /?-xylosidase o f T. reesei also showed a c t i v i t y w i t h p - n i t r o p h e n y l - a - a r a b i n o f u r a n o s i d e as s u b s t r a t e , b u t d i d n o t h y d r o l y z e a r a b i n a n o r release arabinose f r o m a r a b i n o x y l a n . T h e purified α-arabinosidase, however, was capable o f effecting b o t h the l a t t e r hydrolyses (26, T a b l e II). T h e isoelectric p o i n t o f the α-arabinosidase o f T. reesei w a s p i 7.5 a n d i t s m o l e c u l a r weight 53 k D a ( T a b l e I ) .

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

46.

POUTANEN & PULS

Trichoderma Xylanolytic Enzyme System

CBH II

633

Xyl, EQ

Xyl, Ara, BQ pH 8

salt elutlon

Elution volume

Figure 1. Fractionation of proteins in the culture filtrate of Trichoderma reesei according to their pi values: Xyl, xylanase; Ara, arabinosidase; AE, acetyl esterase; βΧ, /?-xylosidase; aG, α-glucuronidase; fiG, /?-glucosidase; CBH, cellobiohydrolase; EG, endoglucanase. Chromatofocusing was performed in a PBE-94 anion exchange resin (Pharmacia) with a pH-gradient created by ampholyte buffers (Pharmacia). Solid line, A ^ ; dotted line, pH. (Reproduced with permission from ref. 24. Copyright 1988.)

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L POLYMERS

T a b l e I I . T h e effect o f α-arabinosidase o n the h y d r o l y s i s o f wheat s t r a w a r a b i n o x y l a n . S u b s t r a t e c o n c e n t r a t i o n 10 g l " , i n i t i a l p H 4, t e m ­ p e r a t u r e 45° C , h y d r o l y s i s t i m e 24 h 1

Hydrolysis Products (% of substrate)

Enzyme Activities ( n k a t / g substrate) Xylanase 25000 25000 25000 25000 25000* a

b

0

/?-Xylosidase

e

a - Arabinosidase"

Xylose

Arabinose

0 0 1400 14000 1300*

4 37 43 56 56

0 0 4 7 7

0 160 160 160 160*

P u r i f i e d enzymes o f T. reesei. A c t i v i t i e s o f crude T.

T h e h y d r o l y s i s p r o d u c t s o f three different x y l a n s b y t h e 20 k D a x y ­ lanase o f T. reesei varied a c c o r d i n g t o t h e n a t u r e a n d degree o f s u b s t i t u t i o n of the substrate ( F i g . 2a-c). T h e role o f a c e t y l groups i n t h e accessibility o f beechwood x y l a n was evident ( F i g . 2 a a n d 2 b ) : T h e a c e t y l a t e d s u b s t r a t e was m o r e soluble i n water, more u n i f o r m l y a t t a c k e d b y t h e e n z y m e a n d y i e l d e d a w i d e r range o f h y d r o l y s i s p r o d u c t s ( F i g . 2 a ) . O n t h e other h a n d , the h y d r o l y z a t e o f the n o n - a c e t y l a t e d 4 - O - m e t h y l - g l u c u r o n o s y l - s u b s t i t u t e d beech x y l a n contained some insoluble residue (not v i s i b l e i n the c h r o m a t o g r a m ) a n d large soluble oligomers, w h i c h were eluted i n the v o i d v o l ­ u m e ( X ) ( F i g . 2 b ) . X y l o b i o s e a n d s u b s t i t u t e d oligomers were the m a i n s o l ­ uble h y d r o l y s i s p r o d u c t s . T h e presence o f 4 - 0 - m e t h y l g l u c u r o n o - s u b s t i t u t e d xylo-oligosaccharides was verified b y a n i o n exchange c h r o m a t o g r a p h y (14) (results n o t s h o w n ) . T h e h y d r o l y z a t e o f a r a b i n o x y l a n resembled t h a t o f g l u ­ c u r o n o x y l a n ( F i g . 2c). T h e a c c u m u l a t i o n o f 4 - O - m e t h y l - g l u c u r o n o s y l - a n d a r a b i n o s y l s u b s t i t u t e d xylo-oligosaccharides has also been reported w h e n u s i n g x y l a n a s e s f r o m other sources (34,35). T h e role o f a r a b i n o s y l substituents a n d the need for α-arabinosidase i n the p r o d u c t i o n o f xylose f r o m a r a b i n o x y l a n was also deduced f r o m the results i n T a b l e I I . W h e n the purified 20 k D a x y l a n a s e a n d /?-xylosidase were used i n the h y d r o l y s i s , the xylose y i e l d was o n l y 6 6 % o f t h a t p r o d u c e d by the whole c u l t u r e filtrate at the same a c t i v i t y levels, a n d no arabinose was p r o d u c e d . A d d i t i o n o f α-arabinosidase increased the yields o f b o t h xylose a n d arabinose. n

T h e h y d r o l y s i s o f acetylated x y l o - o l i g o m e r s f r o m the s t e a m i n g e x t r a c t of b i r c h w o o d u s i n g a n enzyme m i x t u r e c o n t a i n i n g o n l y x y l a n a s e a n d βx y l o s i d a s e was very l i m i t e d ( T a b l e I I I ) . C h e m i c a l d e a c e t y l a t i o n showed t h a t the substrate specificities o f x y l a n a s e a n d /?-xylosidase w i t h respect to substrate D P overlapped, a n d t h a t /3-xylosidase also h y d r o l y z e d longer oligosaccharides t h a n xylobiose t o xylose. T h e a d d i t i o n o f a c e t y l esterase to the h y d r o l y s i s o f a c e t y l a t e d x y l o - o l i g o m e r s enhanced x y l o s e p r o d u c t i o n ( T a b l e I I I ) , b u t d e a c e t y l a t i o n was s t i l l i n c o m p l e t e (the a c e t y l content o f

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635

c φ c ο υ Φ Φ

>> .C Ο 13 k.

φ

ϋ

υ Elution volume *2

MGA

c φ c ο υ Φ Φ

•σ >»

ο

-Ω L_

Φ

Ο

Elution volume c Φ c ο ο φ φ

k.

"Ο

>s .c ο φ

ϋ

Elution volume c F i g u r e 2. H y d r o l y s i s p r o d u c t s o f beechwood O - a c e t y l g l u c u r o n o x y l a n ( F i g . 2a), beechwood g l u c u r o n o x y l a n ( F i g . 2 b ) , a n d wheat s t r a w a r a b i n o x y l a n ( F i g . 2c), as a n a l y z e d b y gel p e r m e a t i o n c h r o m a t o g r a p h y . T h e h y d r o l y s i s was c a r r i e d o u t at p H 5 at 4 5 ° C for 24 h u s i n g 10.000 n k a t o f the 20 k D a x y l a n a s e o f T. reesei. X = xylose; X = x y l o b i o s e ; XMGA = 4-O-methylg l u c u r o n o s y l s u b s t i t u t e d xylo-oligosaccharides; X = x y l o - o l i g o s a c c h a r i d e s D P > 20. 2

n

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PLANT CELL WALL POLYMERS

substrate A was 1 0 % o f the d r y weight). T h i s p h e n o m e n o n is b e i n g f u r t h e r s t u d i e d . It is possible t h a t t h e p u r i f i e d esterase specifically removed acetic a c i d f r o m o n l y one p o s i t i o n o n the x y l o p y r a n o s e r i n g ( u n p u b l i s h e d results). T a b l e I I I . H y d r o l y s i s o f a c e t y l a t e d ( A ) a n d c h e m i c a l l y deacetylated ( B ) x y l o - o l i g o m e r s i n t h e s t e a m i n g e x t r a c t o f b i r c h w o o d b y p u r i f i e d enzymes o f T. reesei. S u b s t r a t e c o n c e n t r a t i o n 10 g l ~ \ i n i t i a l p H 5, t e m p e r a t u r e 4 5 ° C , h y d r o l y s i s t i m e 24 h Enzyme Activities (nkat g " ) 1

Acetyl Esterase

Xylose

Xylo biose

Acetic Acid

500 500

— —

7 21

< 1 < 1

0.7

500 500 500

—

9 18 33

0 < 1 0

0.9 3.3

Substrate

Xylanase

/?-Xylosidase

A Β

5000 5000

—

A Β

— —

A A Β

5000 5000 5000

Hydrolysis Products (% o f d r y weight)

500

—

—

—

S y n e r g i s m between the different x y l a n o l y t i c enzymes was f u r t h e r d e m o n s t r a t e d w h e n a p a r t i a l l y purified e n z y m e p r e p a r a t i o n was used as a source o f xylanase a c t i v i t y ( T a b l e I V ) . T h i s p r e p a r a t i o n , s e p a r a t e d f r o m T. reesei c u l t u r e f i l t r a t e b y gel c h r o m a t o g r a p h y (10), c o n t a i n e d b o t h t h e 20 k D a x y l a n a s e , another glycanase w i t h h i g h l a m i n a r i n a s e a n d low x y l a n a s e a c t i v i t y , a n d a h i t h e r t o u n i d e n t i f i e d esterase. W h e n s u p p l e m e n t e d w i t h /?-xylosidase, the xylanases a n d esterase o f t h i s p r e p a r a t i o n released a b o u t h a l f o f t h e xylose a n d acetic a c i d o f the substrate ( T a b l e I V ) . W h e n p u r i ­ fied a c e t y l esterase was a d d e d , the yields o f xylose a n d acetic a c i d increased f u r t h e r a b o u t 1.6-fold. Because the α-glucuronidase o f T. reesei has n o t y e t been p u r i f i e d , a c u l t u r e filtrate o f Agaricus bisporus, w i t h h i g h α-glucuronidase a c t i v i t y (14) w a s used t o complete the s y n e r g i s m . T h e a d d i t i o n o f α-glucuronidase cleaved off 4 - O - m e t h y l g l u c u r o n i c a c i d a n d m a d e the oligomers accessible for /?-xylosidase a n d esterases. U s i n g t h i s m i x t u r e , t h e x y l o s e y i e l d w a s increased t o the same value as t h a t o b t a i n e d w i t h the whole c u l t u r e filtrate. Conclusions T h e x y l a n o l y t i c e n z y m e s y s t e m o f T. reesei consists o f several e n d o x y lanases, at least three different exoglycosidases a n d at least t w o a c e t y l es­ terases. These enzymes c o n t r i b u t e t o the h y d r o l y s i s o f p l a n t cell w a l l s i n m a n y o f the a p p l i c a t i o n s o f c e l l u l o l y t i c T. reesei e n z y m e p r e p a r a t i o n s . A s c h e m a t i c figure o f t h e suggested h y d r o l y s i s m e c h a n i s m o f x y l a n s b y the

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T a b l e I V . E n z y m a t i c H y d r o l y s i s o f the H i g h - M o l e c u l a r F r a c t i o n o f S t e a m e d B i r c h w o o d X y l a n . T h e substrate w a s f r a c t i o n a t e d b y u l t r a f i l t r a ­ t i o n p r i o r t o h y d r o l y s i s t o remove i m p u r i t i e s a n d t h e 1-5 D P oligosaccharides. S u b s t r a t e c o n c e n t r a t i o n 10 g l " , i n i t i a l p H 5, t e m p e r a t u r e 4 5 ° C , h y d r o l y s i s t i m e 24 h 1

Enzyme Activities ( n k a t / g substrate)

Xylanase" 12000 0 0 0 12000 12000 12000 15000 " e

α

6

c

d

Hydrolysis Products (% o f substrate)

β-Xylosidase*

Acetyl Esterase*

α-Glucuro­ nidase

0 500 0 0 500 500 500 500

0 0 500

0 0 0

500 500 500

0 30 20

a

a

c

a

Xylose

Acetic Acid

6 6 0

4.5 0.5 1.2

42 53 53

9.4 11.5 12.1

P a r t l y purified p r e p a r a t i o n f r a c t i o n a t e d f r o m T. reesei c u l t u r e b y gel c h r o m a t o g r a p h y (10). P u r e enzymes o f T. reesei. Agaricus bisporus c u l t u r e filtrate. T. reesei c u l t u r e filtrate.

filtrate

enzymes o f T. reesei is s h o w n i n F i g u r e 3. T h e h y d r o l y s i s starts b y t h e a c t i o n o f endoxylanases, w h i c h decreases t h e average D P o f t h e s u b s t r a t e . T h e s i d e - g r o u p - c l e a v i n g enzymes have t h e i r highest a c t i v i t y t o w a r d s s o l ­ u b l e , short xylo-oligosaccharides, a n d t h e h y d r o l y s i s is c o m p l e t e d b y t h e synergistic a c t i o n o f b o t h side-group a n d b a c k b o n e - c l e a v i n g enzymes. In a d d i t i o n t o c e l l u l o l y t i c a n d x y l a n o l y t i c enzymes t h e hydrolases p r o ­ duced b y T. reesei have also been r e p o r t e d t o i n c l u d e m a n n a n a s e , p e c t i nase, amyloglucosidase a n d protease (36,37). T h e present a n d p o t e n t i a l a p p l i c a t i o n s o f T. reesei enzymes i n c l u d e t o t a l h y d r o l y s i s o f l i g n o c e l l u l o s i c m a t e r i a l s t o glucose a n d / o r x y l o s e , s t i m u l a t i o n o f g e r m i n a t i o n i n m a l t ­ i n g , m o r e c o m p l e t e s a c c h a r i f i c a t i o n o f cereals i n g r a i n a l c o h o l p r o d u c t i o n a n d i m p r o v e m e n t o f t h e storage properties a n d d i g e s t i b i l i t y o f silage feed. I n these a p p l i c a t i o n s s y n e r g i s m is needed n o t o n l y between t h e i n d i v i d ­ u a l x y l a n o l y t i c or c e l l u l o l y t i c enzymes t o h y d r o l y z e x y l a n o r cellulose, b u t p r o b a b l y also between different e n z y m e groups for successive h y d r o l y s i s o f the p l a n t cell w a l l m a t r i x . D u e t o i t s v e r s a t i l i t y , T. reesei is a n excellent source o f enzymes i n these cases. I n a p p l i c a t i o n s where selective h y d r o l y s i s is r e q u i r e d , however, the e n z y m e s p e c t r u m s h o u l d be t a i l o r e d for a p a r ­ t i c u l a r purpose. T h i s c o u l d be achieved b y a d j u s t i n g e n z y m e p r o d u c t i o n c o n d i t i o n s , b y f r a c t i o n a t i n g t h e enzymes or b y m o l e c u l a r c l o n i n g .

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O-Acetyl-Glucuronoxylans Arabinoxylans Arabinoglucuronoxylan

\r

Endo-e-1,4-xylanases

Different soluble (substituted) xylo-oligosaccharides Endo-e-1,4-xylanases £-D-xylosidase a-L-arabinosidase a-D-glucuronidase ^ Acetyl esterases Xylose Arabinose 4-o-methylglucuronic acid Acetic acid

F i g u r e 3. T e n t a t i v e h y d r o l y s i s m e c h a n i s m o f different x y l a n s b y the x y l a n o l y t i c enzymes o f T. reesei.

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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Literature Cited 1. Woodward, J . Top. Enz. Ferment.Biotechnol.1984, 8, 9-30. 2. Dekker, R. F. H. In Biosynthesis and Biodegradation of Wood Compo­ nents; Higuchi, T . , Ed.; Academic Press: Orlando, FL, 1985; p. 505. 3. Wong, K. K. Y.; Tan, L . U. L.; Saddler, J . N. Microbiol. Rev. 1988, 52, 305-17. 4. Rodionova, Ν. Α.; Tavobilov, I. M . ; Bezborodov, A . M . J. Appl. Biochem. 1983, 5, 300-12. 5. Matsuo, M.; Yasui, T . Agric. Biol. Chem. 1984, 48, 1845-60. 6. Matsuo, M . ; Fujie, Α.; Win, M.; Yasui, T . Agric. Biol. Chem. 1987, 51, 2367-79. 7. Marui, M.; Nakanishi, K.; Yasui, T . Agric. Biol. Chem. 1985, 49, 33993407. 8. Fournier, R.; Frederick, M. M.; Frederick, J . R.; Reilly, P. J . Biotechnol. Bioeng. 1985, 27, 539-46 9. Wong, Κ. K. Y.; Tan 1986, 8, 617-22. 10. Lappalainen, A. Biotechnol. Appl. Biochem. 1986, 8, 437-48. 11. Biely, P.; Puls, J.; Schneider, H. FEBS Lett. 1985, 186, 80-84. 12. Biely, P.; MacKenzie, C . R.; Puls, J . ; Schneider, H. Bio/Technology 1986, 4, 731-33. 13. Puls, J . ; Poutanen, K.; Schmidt, O.; Linko, M . Proc. 3rd Intl. Conf. Biotechnol. in the Pulp and Paper Industry; 1986, p. 93. 14. Puls, J.; Schmidt, O.; Granzow, C. Enz. Microb. Technol. 1987, 9, 83-88. 15. Ishihara, M.; Shimizu, K. Mokuzai Gakkaishi 1988, 34, 58-64. 16. Kaji, A. Adv. Carbohydr. Chem. Biochem. 1984, 42, 383-94. 17. Andrewartha, Κ. Α.; Phillips, D. R.; Stone, B. A. Carbohydr. Res. 1979, 77, 191-204. 18. Tangnu, S. K.; Blanch, H. W.; Wilke, C. R. Biotechnol. Bioeng. 1981, 23, 1837-49. 19. Dekker, R. F. H. Biotechnol. Bioeng. 1983, 25, 1127-46. 20. Hrmova, M.; Biely, P.; Vrsanska, M . Arch. Microbiol. 1986, 144, 30711. 21. Sinner, M.; Dietrichs, Η. H. Holzforschung 1975, 29, 207-14. 22. Gibson, T . S.; McCleary, Β. V . Carbohydr. Polym. 1987, 7, 225-40. 23. Poutanen, K.; Rättö, M.; Puls, J.; Viikari, L. J. Biotechnol. 1987, 6, 49-60. 24. Poutanen, K. Ph.D. Thesis, Technical Research Centre of Finland, Publications 47, Espoo, 1988. 25. Poutanen, K.; Puls, J . Appl. Microbiol. Biotechnol. 1988, 28, 425-32. 26. Poutanen, K. J. Biotechnol. 1988, 7, 271-82. 27. Poutanen, K.; Sundberg, M . Appl. Microbiol. Biotechnol. 1988, 28, 419-24. 28. Ebringerova, Α.; Kramav, Α.; Rendos, F.; Domansky, R. Holzforschung 1967, 21, 74-77. 29. Puls, J . ; Poutanen, K. Körner, H.-U.; Viikari, L. Appl. Microbiol. Biotechnol. 1985, 22, 416-23.

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30. Teeri, T . Ph.D. Thesis, Technical Research Centre of Finland, Publi­ cations 38, Espoo, 1987. 31. Biely, P.; Markovic, O. Biotechnol. Appl. Biochem. 1988, 10, 99-106. 32. Esterbauer, H.; Hayn, M.; Tuisel, H.; Mahnert, W. Holzforschung 1983, 37, 601-08. 33. Kolarova, N.; Farkas, V. Biologia (Bratislava) 1983, 38, 721-25. 34. Sinner, M.; Dietrichs, H. H. Holzforschung 1976, 30, 50-59. 35. Kusakabe, I.; Ohgushi, S.; Yasui, T.; Kobayashi, T . Agric.Biol.Chem. 1983, 47, 2713-23. 36. Bailey, M . J.; Nevalainen, Κ. M . H. Enzyme Microb. Technol. 1981, 3, 153-57. 37. Haltmeier, T . ; Leisola, M . ; Ulmer, D.; Waldner, R.; Fiechter, A. Biotechnol. Bioeng. 1983, 25, 1685-90. RECEIVED May 19, 1989

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

Chapter 47 Production and Purification of Xylanases David J . Senior, Paul R. Mayers, and John N. Saddler Biotechnology and Chemistry Department, Forintek Canada Corporation, 800 Montreal Road, Ottawa, Ontario K1G 3Z5, Canada

Various groups have recently indicated that xylanase enzymes can be use it is essential tha enzyme ity and have a high specific activity. Strategies followed to produce xylanases without contaminating cellulase activity include selective inactivation of the cellulase by heat and heavy metals and cloning of the xylanase gene into a cellulase-free host. We have shown that xylanase production by Trichoderma harzanium can be enhanced by selection of a suitable hemicellulase-rich growth substrate. A purified xylanase could be obtained by subsequent ultrafiltration and ion exchange treatment of the culturefiltrate.Xylanase treatment of pulps could significantly decrease the xylan content while leaving the cellulose intact. X y l a n a s e p r o d u c t i o n has been r e p o r t e d t o o c c u r i n a w i d e s p e c t r u m o f o r g a n i s m s . A l t h o u g h absent i n vertebrate a n i m a l s , x y l a n a s e s are p r o d u c e d i n m a n y forms o f b a c t e r i a , f u n g i a n d yeasts, crustaceans, algae a n d p l a n t seeds. C u r r e n t interest i n xylanases has been focused p r i m a r i l y o n t h e enzymes p r o d u c e d b y f u n g i a n d b a c t e r i a a n d , t o a lesser e x t e n t , yeasts. T h e h i g h yields a n d r e l a t i v e ease o f p r o d u c t i o n have m a d e these systems the most p r o m i s i n g for future c o m m e r c i a l i z a t i o n . O n e o f the m a j o r expenses i n c u r r e d i n t h e a p p l i c a t i o n o f enzymes for bioconversion processes is the cost o f e n z y m e p r o d u c t i o n (1). T h e t o t a l cost of p r o d u c t i o n includes the cost o f fermentative p r o d u c t i o n as w e l l as d o w n s t r e a m processing requirements. B o t h o f these factors m u s t be o p t i m i z e d a n d integrated for m a x i m u m cost-effectiveness. Several different approaches have been followed t o p r o d u c e x y l a n a s e s . Highest levels o f e x t r a c e l l u l a r xylanases have been p r o d u c e d i n yeast a n d

0097-6156/89/0399-0641$06.00/0 o 1989 American Chemical Society

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f u n g a l systems u s i n g x y l a n i n d u c i n g g r o w t h substrates. I n c o m p a r i s o n , b a c t e r i a l a n d cloned systems have p r o d u c e d moderate q u a n t i t i e s of cellulasefree xylanases. T w o b r o a d areas of a p p l i c a t i o n for x y l a n o l y t i c enzymes have been i d e n tified (1). T h e first involves the use of xylanases w i t h other h y d r o l y t i c enzymes i n the bioconversion of wastes such as those f r o m the forest a n d a g r i c u l t u r a l i n d u s t r i e s , a n d i n the c l a r i f i c a t i o n a n d l i q u i f i c a t i o n of j u i c e s , vegetables a n d f r u i t s . F o r these purposes, the enzyme p r e p a r a t i o n s need o n l y to be filtered a n d concentrated as essentially no f u r t h e r p u r i f i c a t i o n is r e q u i r e d . Several specific examples of a p p l i c a t i o n s i n v o l v i n g crude x y l a n a s e p r e p a r a t i o n s i n c l u d e : bioconversion of cellulosic m a t e r i a l s for subsequent f e r m e n t a t i o n (2); h y d r o l y s i s of p u l p waste liquors; a n d w o o d e x t r a c t i v e s to m o n o m e r i c sugars for subsequent p r o d u c t i o n of single cell p r o t e i n (3-5). X y l o s e p r o d u c e d b y the a c t i o n of xylanases c a n be used for subsequent p r o d u c t i o n of higher value c o m p o u n d s such as e t h a n o l (6), x y l u l o s e (7) a n d x y l o n i c a c i d (8-9). T h e second area of a p p l i c a t i o n involves the use of cellulase-free x y lanases for removal of hemicellulose f r o m p u l p s (10-20) a n d p l a n t fibres (21). It is essential t h a t these xylanase p r e p a r a t i o n s are free of c o n t a m i n a t i n g cellulase a c t i v i t y or d a m a g e to the cellulose fibres a n d consequently the p r o d u c t q u a l i t y w i l l result. Here we e x a m i n e the recent progress w h i c h has been m a d e i n the p r o d u c t i o n a n d p u r i f i c a t i o n of xylanases. Xylanase P r o d u c t i o n by Yeasts B i e l y et ai (22) have s h o w n t h a t x y l a n a s e p r o d u c t i o n can be i n d u c e d b y use of a p p r o p r i a t e substrates i n the yeast Cryptococcus albidus. W o o d x y l a n s a n d x y l a n oligomers were f o u n d to increase e x t r a c e l l u l a r x y l a n a s e p r o d u c t i o n b y two orders of m a g n i t u d e w h e n the yeast was g r o w n o n these m a t e r i a l s rather t h a n o n a s i m p l e glucose m e d i u m . X y l o b i o s e was identified as the o n l y x y l o o l i g o m e r w h i c h was not degraded e x t r a c e l l u l a r l y a n d i t was concluded t h a t xylobiose is either a n a t u r a l inducer of x y l a n a s e or its i m m e d i a t e precursor. O t h e r x y l o p y r a n o s i d e s have also been investigated for their x y l a n a s e i n d u c i n g p o t e n t i a l i n t h i s yeast. M e t h y l - / ? - D - x y l o p y r a n o s i d e i n d u c e d x y l a n a s e p r o d u c t i o n at levels c o m p a r a b l e to t h a t o b t a i n e d after g r o w t h o n x y l a n (23). M o r o s o l i et al. (24) d e m o n s t r a t e d t h a t the i n d u c t i o n effect was manifested at the t r a n s c r i p t i o n a l level i n t h i s o r g a n i s m . Y a s u i et ai o b t a i n e d a 15-20 f o l d greater increase i n x y l a n a s e p r o d u c t i o n u s i n g m e t h y l - / ? - D - x y l o p y r a n o s i d e t h a n when x y l a n was used as a n i n d u c e r i n Cryptococcus flavus (25). L e a t h e r s et al. (26) showed t h a t xylose, xylobiose a n d arabinose were inducers of xylanases i n Aureobasidium pullulans. F o r x y l a n a s e a p p l i c a t i o n s w h i c h require xylanases of h i g h s e l e c t i v i t y (e.g., b i o p u l p i n g ) , any c o n t a m i n a t i n g cellulases can be d e t r i m e n t a l to the t r e a t m e n t s . T h e c o n s t i t u t i v e levels of cellulases i n yeasts is generally very low i n r e l a t i o n to x y l a n a s e levels t h u s i n d i c a t i n g yeast x y l a n a s e p r e p a r a t i o n s are p r o m i s i n g for the selective h y d r o l y s i s of x y l a n s . However, e x t r a c e l l u l a r yeast xylanases are t y p i c a l l y p r o d u c e d i n the order of 1 u n i t per m i l l i l i t r e

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of c u l t u r e filtrate, w h i c h is very low for cost-effective p r o d u c t i o n . R e c e n t l y , L e a t h e r s has r e p o r t e d the o v e r p r o d u c t i o n of a cellulase-free x y l a n a s e o f very h i g h specific a c t i v i t y f r o m a colour v a r i a n t s t r a i n of A. pullulans (27). T h e level of e x t r a c e l l u l a r x y l a n a s e i n the c u l t u r e filtrate was 373 I U / m L w h i c h is c o m p a r a b l e to the most p r o l i f i c x y l a n a s e - p r o d u c i n g f u n g a l s t r a i n s so far identified. Xylanase P r o d u c t i o n by C l o n e d Systems A n o t h e r a l t e r n a t i v e for p r o d u c t i o n of cellulase-free x y l a n a s e p r e p a r a t i o n s has been to clone the x y l a n a s e genes f r o m x y l a n a s e p r o d u c i n g b a c t e r i a . O n e x y l a n a s e p r o d u c i n g s t r a i n , Bacillus subtilis P A P 1 1 5 , was chosen as a x y l a n a s e gene donor because of its r e l a t i v e l y h i g h level o f e x t r a c e l l u l a r x y l a n a s e p r o d u c t i o n (0.9 I U / m L c u l t u r e filtrate) a n d the t h o r o u g h c h a r a c t e r i z a t i o n of the e n z y m e (28). T h e gene was successfully inserted i n t o Escherichia coli ( W A 8 0 2 ) t i a l l y c h a r a c t e r i z e d . O n l y 2 5 % of the o r i g i n a l e x t r a c e l l u l a r x y l a n a s e a c t i v i t y was recovered a n d t h i s was l o c a l i z e d w i t h i n the cells (29). T h e same gene was i n s e r t e d i n t o E. coli J M I 0 5 a n d the new clones p r o d u c e d m o r e x y lanase t h a n the o r i g i n a l Bacillus (10). U n l i k e the first clone, a b o u t h a l f o f the x y l a n a s e a c t i v i t y was f o u n d e x t r a c e l l u l a r l y a n d y i e l d s were increased significantly. S i m i l a r approaches to c l o n i n g of b a c t e r i a l xylanases have been used for genes f r o m Clostridium acetobutylicum (30), Bacillus polymyxa (31), Bacteroides succinogenes (32), Clostridium thermocellum (33) a n d Pseudomonas fluorescens subsp. cellulosa (34). I n each case the x y l a n a s e s were p r e d o m i n a n t l y l o c a t e d i n t r a c e l l u l a r l y a n d the levels of x y l a n a s e s p r o d u c e d f r o m cloned s y s t e m s were, i n general, very low i n c o m p a r i s o n to yeast a n d f u n g a l s y s t e m s . A c o m p a r i s o n of the p r o d u c t i o n y i e l d s a n d extent o f ext r a c e l l u l a r p r o d u c t i o n for v a r i o u s cloned x y l a n a s e genes is f o u n d i n T a b l e I. U n l i k e most other cloned systems, the x y l a n a s e p r e p a r a t i o n f r o m B. succinogenes was c o n t a m i n a t e d w i t h cellulase a c t i v i t y . H o n d a et al. have r e p o r t e d a x y l a n a s e gene f r o m a n a l k a l o p h i l i c Bacillus sp. C I 2 5 i n E. coli (35-36). A b o u t 8 0 % of the x y l a n a s e a c t i v i t y was secreted i n t o the e x t r a c e l l u l a r m e d i u m a n d the a c t i v i t y was s l i g h t l y h i g h e r t h a n t h a t p r o d u c e d b y the o r i g i n a l Bacillus ( a p p r o x i m a t e l y 0.6 I U / m L ) . A x y l a n a s e was p r o d u c e d f r o m a x y l a n a s e gene cloned f r o m a l k a l o p h i l i c Aeromonas sp. 212 at a level a b o u t 80 f o l d higher t h a n i n the o r i g i n a l b a c t e r i u m . U n f o r t u n a t e l y , none of the e n z y m e was secreted e x t r a c e l l u l a r l y (37). H a m m a m o t o a n d H o r i k o s h i have recently succeeded i n the c o n s t r u c t i o n of a secretion vector c o n t a i n i n g the x y l a n a s e gene f r o m a l k a l o p h i l i c Bacillus sp. C 1 2 5 , w h i c h was t h e n cloned i n t o E. coli (38). T r a n s f o r m a n t s w h i c h c a r r i e d these genes secreted a significant a m o u n t o f e x t r a c e l l u l a r x y lanase i n c o m p a r i s o n to clones w h i c h d i d not c o n t a i n the secretion vector. It has been suggested t h a t the secretion o f the x y l a n a s e is d i r e c t e d b y the characteristics of the p r o t e i n itself. F u t u r e w o r k m a y i d e n t i f y i m p r o v e m e n t s to secretory sequences w h i c h w i l l increase the e x t r a c e l l u l a r q u a n t i t i e s of x y lanases f r o m cloned systems.

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T a b l e I. X y l a n a s e s f r o m C l o n e d Systems

X y l a n a s e G e n e Source Aeromonas sp. 212 Bacillus polymyxa Bacillus subtilis Bacillus subiilus Bacillus sp. C 1 2 5 Bacillus sp. C 1 2 5 (secretion vector) Clostridium acetobutylicum Clostridium thermocellum Streptomyces lividans

Enzyme Yield (IU/ml) 1.63 0.037 0.5 2.2 0.75 0.7 64 11.2

%

Extracellular 0 70 50

Reference (37) (31) (29) (10) (35-36) (38)

0

(30)

—

(33)

A l t h o u g h most cloned xylanases are t o t a l l y free f r o m cellulase c o n t a m i n a t i o n , the recovery y i e l d s r e m a i n i n general r e l a t i v e l y low w h e n c o m p a r e d t o f u n g a l systems. M o r e effort i n the areas of i m p r o v e d x y l a n a s e p r o d u c t i o n y i e l d s a n d secretion m a y make the b a c t e r i a l systems appear m o r e p r o m i s i n g for b i o t e c h n o l o g i c a l a p p l i c a t i o n s . R e c e n t l y a n e x t r a c e l l u l a r , cellulase-free x y l a n a s e e n z y m e was prepared b y homologous c l o n i n g of a x y l a n a s e gene f r o m Streptomyces lividans i n t o a xylanase-cellulase-negative m u t a n t of S. lividans (39). A considerable o v e r p r o d u c t i o n of t h i s e n z y m e (up to 1200 I U / m L i n the c u l t u r e filtrate) has also been d e m o n s t r a t e d w h e n g r o w n o n x y l a n or h e m i c e l l u l o s e - r i c h substrates (40). Fungal Xylanase

Production

T h e use of f i l a m e n t o u s f u n g i for p r o d u c t i o n of xylanases was i n i t i a l l y a t t r a c t i v e because the enzymes are released e x t r a c e l l u l a r l y t h u s e l i m i n a t i n g the need for cell lysis procedures. I n a d d i t i o n , x y l a n a s e levels i n f u n g a l c u l ture filtrates are t y p i c a l l y i n m u c h higher concentrations t h a n f r o m yeasts a n d b a c t e r i a . M a n y examples of xylanases p r o d u c e d f r o m f u n g i are l i s t e d i n the review b y D e k k e r (41). It has been proposed t h a t the p r o d u c t i o n of xylanases a n d cellulases is under separate r e g u l a t o r y c o n t r o l i n some filamentous f u n g i (1). H r m o v a et ai (42) reached a s i m i l a r conclusion after m o n i t o r i n g the d a i l y p r o d u c t i o n of these enzymes i n Trichoderma reesei Q M 9414. X y l a n a s e a n d cellulase a c t i v i t i e s followed independent p r o d u c t i o n profiles d u r i n g f u n g a l g r o w t h . T h e same effect has been observed i n b a t c h cultures o f T. harzianum. We have observed peak x y l a n a s e a c t i v i t y o n the t h i r d d a y of g r o w t h whereas the cellulase a c t i v i t y peaked after d a y five or s i x ( u n p u b l i s h e d ) . T h e r e are m a n y recent examples o f x y l a n a s e i n d u c t i o n i n f u n g i g r o w n o n m e d i a c o n t a i n i n g x y l a n , h e m i c e l l u l o s e - r i c h m a t e r i a l a n d low m o l e c u l a r weight c a r b o h y d r a t e s (42-66). It has generally been observed t h a t higher

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levels of x y l a n a s e are i n d u c e d i n systems c o n t a i n i n g higher x y l a n concent r a t i o n s i n the g r o w t h m e d i u m . A c o m p a r i s o n of x y l a n a s e a n d cellulase p r o d u c t i o n f r o m v a r i o u s f u n g i is s h o w n i n T a b l e I I . M o s t c u l t u r e s g r o w n o n x y l a n y i e l d e d higher levels of x y l a n a s e t h a n w h e n g r o w n o n s i m i l a r concent r a t i o n s of cellulose. W i t h the e x c e p t i o n of Schizophyllum commune (45), the highest p r o d u c t i o n of xylanases by various species has been the result of g r o w t h o n x y l a n . A l t h o u g h a t t e m p t s have been m a d e to s t a n d a r d i z e x y l a n a s e assays (67), some of the v a r i a t i o n i n e n z y m e a c t i v i t i e s r e p o r t e d must be a t t r i b u t e d to v a r i a t i o n s i n assay procedures a m o n g workers. U s i n g a x y l a n - f r e e cellulose f r o m Acetobacter xylinum as a g r o w t h s u b s t r a t e , H r m o v a et al. d e m o n s t r a t e d t h a t s m a l l levels of c o n s t i t u t i v e l y p r o duced x y l a n a s e c o u l d be a t t r i b u t e d to cellulase a c t i v i t y i n T. reesei Q M 9 4 1 4 (42). A s the c o n c e n t r a t i o n of x y l a n i n the m e d i u m was i n c r e a s e d , higher x y l a n a s e p r o d u c t i o n r e s u l t e d . U s i n g a series of s t e a m t r e a t e d aspen w o o d samples, we have observed t h a t the r a t i o of x y l a n a s e to cellulase a c t i v i ties p r o d u c e d o n these m a t e r i a l r a t i o of x y l a n to cellulose i n the m e d i u m (measured as x y l a n to g l u c a n r a tio) ( F i g u r e 1). D e t e r m i n a t i o n of these ratios for A v i c e l , S o l k a F l o e a n d oat spelts x y l a n l a r g e l y s u p p o r t e d t h i s r e l a t i o n s h i p . S o m e d e v i a t i o n e n countered between these g r o w t h substrates was likely due to v a r i a t i o n s i n c o m p o s i t i o n a n d accessibility of the s u b s t r a t e . It is therefore a p p a r e n t t h a t w i t h Trichoderma fungal systems, m a x i m a l levels of xylanases can be achieved b y g r o w i n g the cells i n x y l a n c o n t a i n i n g m e d i a . T h e i n d u c t i o n of xylanases i n f u n g i g r o w n o n x y l a n m e d i u m , however, cannot be realized i n a l l f u n g i w i t h equivalent success. U n l i k e the level of xylanases i n d u c e d i n most f u n g i , the x y l a n a s e a c t i v i t y p r o d u c e d f r o m S. commune g r o w n on cellulose was u n u s u a l l y h i g h (See T a b l e I I ) . B i e l y et al. (68) a n d M a c k e n z i e a n d B i l o u s (69) have observed m a r k e d l y higher p r o d u c t i o n of x y l a n a s e i n S. commune w h e n g r o w n o n cellulose r a t h e r t h a n o n x y l a n . T h e h i g h cost of u s i n g p u r i f i e d x y l a n s as substrates for x y l a n a s e i n d u c t i o n are p r o h i b i t i v e for large-scale x y l a n a s e p r o d u c t i o n . A s a n a l t e r n a t i v e , m a n y groups have investigated the effectiveness of other h e m i c e l l u l o s e - r i c h m a t e r i a l s for t h i s purpose. E x a m p l e s of these i n c l u d e : a g r i c u l t u r a l wastes such as straws (2,45,47,50, 52,56,57,70,71), b r a n (47,56,57,60), sugar cane bagasse (57,61), c o r n cob a n d oak dust (70); p u l p (2,45,52); p u l p m i l l waste (3) a n d s t e a m treated w o o d (53,59). A l t e r n a t i v e m e t h o d s for more cost-effective x y l a n a s e p r o d u c t i o n i n f u n g i i n c l u d e the development of c a t a b o l i t e derepressed m u t a n t s , c e l l u lase negative m u t a n t s , genetically engineered s t r a i n s a n d h i g h - x y l a n a s e p r o d u c i n g c o n s t i t u t i v e m u t a n t s . T h e l a t t e r case c o u l d afford h i g h x y l a n a s e y i e l d s u s i n g soluble sugars as g r o w t h substrates w h i c h are b o t h i n e x p e n s i v e and easy to h a n d l e i n c o n v e n t i o n a l f e r m e n t a t i o n e q u i p m e n t . T h e vast m a j o r i t y of xylanases t h a t have been p r o d u c e d are q u i t e l a b i l e above 45-50°C. It w o u l d be of great advantage to e m p l o y xylanases w h i c h r e t a i n a h i g h specific a c t i v i t y above t h i s t e m p e r a t u r e range. A n u m b e r of t h e r m o s t a b l e xylanases have been p r e p a r e d to v a r y i n g

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ϋ