Ecology and Metabolism of Plant Lipids 9780841210066, 9780841211629, 0-8412-1006-3

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ACS

SYMPOSIUM

SERIES

Ecology and Metabolism of Plant Lipids Glenn Fuller, EDITOR U.S. Department of Agriculture

W U.S. Department of Agriculture

Developed from a symposium sponsored by the Division of Agricultural and Food Chemistry at the 189th Meeting of the American Chemical Society, Miami Beach, Florida, April 28-May 3, 1985

American Chemical Society, Washington, D C 1987

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

325

Library of Congress Cataloging-in-Publication Data Ecology and metabolism of plant lipids. (ACS symposium series, ISSN 0097-6156; 325) "Developed from a symposium sponsored by the Division of Environmental and Food Chemistry at the 189th Meeting of the American Chemical Society, Miami Beach, Florida, April 28-May 3, 1985." Includes bibliographies and indexes 1. Plant lipids—Congresses. 2 Metabolism—Congresses. 3. Botany—Ecology— Congresses. I. Fuller, Glenn, 1929. II. Nes, W. David, 1953. III. American Chemical Society. Division of Agricultural and Food Chemistry. IV. American Chemical Society. V. Series. QK898.L56E26 1987 ISBN 0-8412-1006-3

581.19'247

86-26559

Copyright © 1987 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 108 of the 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 THE UNITED STATES OF AMERICA

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ACS Symposium Series M . Joan Comstock, Series Editor Advisory Harvey W. Blanch

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University of California—Berkeley

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In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Foreword The A C S

SYMPOSIU

SERIES was founded in 1974 to provide a

medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that, in order to save time, the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Preface PLANTS

PROVIDE APPROXIMATELY 90% of human caloric intake and a major part of protein in the diet. They are important as the ultimate source of nutrition for animal species because they have the unique capability of synthesizing proteins, carbohydrates, and fats from carbon dioxide, water, and inorganic chemicals by using sunlight as an energy source. Despite the importance of plants, knowledge of plant biochemistry has lagged behind that of mammalian biochemistry, although a renewed interest in photosynthetic processes and chemistry of plant enzymes is overcoming that lag. The study of plant lipids has also accelerated with increased understanding of the importance of plant lipids in cell regulatory functions. Now, investiga­ tors are establishing the species as well as in the specie The symposium from which this book was developed was designed to review and present current research on the biosynthesis and metabolism of lipids in plants together with the chemistry and biochemistry of lipid interactions; however, some presentations reported input from disciplines other than chemistry. This 22-chapter volume presents a loose classification of the papers presented at the symposium into four sections: (1) Introduc­ tion, (2) Plant Lipid Metabolism and Plant-Plant Interactions, (3) PlantInsect and Plant-Nematode Interactions, and (4) Plant-Microbial Inter­ actions. We still have much to learn about the lipid metabolism and ecology of higher plants. Especially intriguing is research concerning the genetics, mechanisms, and substrates involved in the formation of multiple double bonds in fatty acids. Results of research may allow us to tailor the triglyceride composition of vegetable oils. Further research on lipid interactions of plants with other organisms can show us how to enhance the resistance of plants to disease and insect infestation or to improve symbiotic relationships. Regulation of isopentenoid pathways may affect the control of vegetative and reproductive processes of both plants and the pathogens that attack them and may enhance the ability of crops to produce chemical raw materials. We hope that this volume will stimulate more interdisciplin­ ary research that will help us understand and modify the reactions of plant lipids.

GLENN FULLER W. DAVID N E S

Western Regional Research Center Agricultural Research Service U.S. Department of Agriculture Albany, C A 94710

September 24, 1986 vii

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 1

Plant Lipids and Their Interactions Glenn Fuller and W. David Nes Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710

Lipids are distinguished by their high solubility in non-polar organic solvents and their low solubility in water. Lipids ca b classified derivative f fatty acids or othe acid moiety. Quantitatively, acy glycerides, polar and non-polar, make up the bulk of plant lipids. These include the neutral triglycerides, found in seed storage lipids, and the polar acyl glycerides such as the phosphatides, glycolipids and sulfolipids. There are a variety of non-glyceride lipids which embrace waxes, sterols, terpenes and their derivatives, hydrocarbons, and even some phenolics. Many organisms do not synthesize all their required lipids de novo, but obtain them from other species. Some plants are able to synthesize lipids which modify behavior in and help to protect against pests and pathogenic organisms. Hence, a variety of interactions have evolved which are the subject matter of this volume. The symposium leading to t h i s book was designed to bring together s c i e n t i s t s working i n the f i e l d of interactions between various species based on l i p i d biochemistry. This area of research i s important to a g r i c u l t u r e because i t can lead to b i o l o g i c a l control of b e n e f i c i a l or deleterious species. Many organisms have evolved requiring l i p i d nutrients from other organisms, e.g., c e r t a i n insects do not synthesize cholesterol de novo, but they can e i t h e r use plant steroids without modification or convert these steroids to cholesterol (Svoboda et a l . , Chap. 11). Other species have evolved protective compounds. Harborne (8) has c l a s s i f i e d the plant protective compounds as p r o h i b i t i n s , i n h i b i t i n s (pre-infect i o n a l ) , p o s t - i n h i b i t i n s , and phytoalexins ( p o s t - i n f e c t i o n a l ) . A high proportion of these compounds are l i p i d s , often functioning by changing membrane permeability i n the invading species. Although there i s overlap, the chapters of the book have been loosely grouped to cover general l i p i d metabolism and function, plant-plant This chapter not subject to U.S. copyright. Published 1987, American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1.

FULLER AND NES

Plant Lipids and Their Interactions

3

interactions, plant-insect interactions and plant-nematode i n t e r a c t i o n s , and e f f e c t s of microbes and plants on one another. Since the book i s a symposium volume, there i s heavy emphasis on plant-microbial r e l a t i o n s h i p s and somewhat less coverage of other areas. However, i t i s representative of current research i n the field. L i p i d s are one of the four major categories of compounds which are involved with growth and reproduction of crop plants and t h e i r pathogens, the others being carbohydrates, proteins and nucleic acids. Bioregulatory processes of l i p i d s have been l a r g e l y ignored. Acyl l i p i d s and s t e r o l s were assumed to play a nonmetabolic r o l e i n the maintenance of c e l l membrane physicochemical properties, while t r i g l y c e r i d e s were important i n energy reserve and isopentenoid hormones influenced reproduction. However, l a t e r chapters of t h i s volume show that l i p i d s may have acted as e c o l o g i c a l determinants of plant interactions with other organisms. Sterols hav l i f e h i s t o r y of fungi an which i s f a m i l i a l l y and temporarily expressed. In nature t h e i r influence i s dependent on a v a i l a b i l i t y of s t e r o l s from the host plant i n which structure and quantity of s t e r o l s are important to the host-pathogen r e l a t i o n s h i p . Fatty acids or a c y l l i p i d s play p h y s i o l o g i c a l roles which are s i m i l a r l y related to t h e i r structures and t h e i r compartmentalization within the c e l l (Mudd, Chapter 2; F u l l e r and Stumpf, Chapter 4 ). The aim of t h i s monograph i s to attempt to cover f o r the f i r s t time t h i s diverse area of research on l i p i d i n t e r a c t i o n s . We have emphasized the s t e r o l and f a t t y acid f i e l d s and plant i n t e r a c t i o n s , rather than those of mammals since t h i s r e f l e c t s the e d i t o r s ' i n t e r e s t i n these subjects. In addition, a few chapters are devoted to structure-occurrence and structure-biosynthesis of l i p i d s since physiology i s the basis f o r the interactions described. L i p i d s are distinguished from other classes of b i o l o g i c a l l y important compounds by the f a c t that they contain large non-polar moieties, which make them poorly soluble i n aqueous media but soluble i n organic solvents such as chloroform, a l c o h o l , hexane or mixtures of these solvents. This c h a r a c t e r i s t i c enables one to extract l i p i d s from fresh t i s s u e ; mixtures of chloroform - methanol or hexane - isopropanol are the most commonly used solvent systems. A f t e r e x t r a c t i o n , the l i p i d s may be separated by t h e i r chemical properties. The techniques of l i q u i d chromatography, gas chromatography and t h i n - l a y e r chromatography have been e s p e c i a l l y useful i n separating classes of l i p i d s for a n a l y t i c a l purposes. I t i s now generally accepted that two major l i p i d biosynthetic pathways e x i s t - the so-called isopentenoid and f a t t y acid pathways. While the two pathways have been assumed to be biochemically independent of one another, carbon-flow v i a the mevalonic acid shunt into the f a t t y acid pathway has been demonstrated i n a crop plant and insect (2, 3). Because some l i p i d s are very h y d r o p h i l l i c and remain i n aqueous media, we sometimes group l i p i d s according to t h e i r biosynthetic rather than chemical r e l a t i o n s h i p s .

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

4

ECOLOGY AND METABOLISM OF PLANT LIPIDS

L i p i d Classes Fatty acids and t h e i r d e r i v a t i v e s . Fatty acids are characterized by the presence of a carboxylic acid function attached to a hydrocarbon chain. Because the biosynthesis of f a t t y acids involves the combination of a series of two-carbon fragments, the common f a t t y acids are unbranched chains with even numbers of carbon atoms. Many are saturated, but biochemical interest centers p r i n c i p a l l y around the unsaturated f a t t y acids containing up to f i v e double bonds. Common unsaturated acids have t h e i r double bonds i n the c i s - c o n f i g u r a t i o n rather than the thermodynamically more stable trans form. M u l t i p l e double bonds are u s u a l l y methylene-interrupted, rather than conjugated. Table I indicates the common names and structures of some of the p r i n c i p a l f a t t y acids found i n plant and animal l i p i d s . Though acyl glycerides of the r e l a t i v e l y few acids l i s t e d make up the bulk of l i p i d s i n l i v i n g organisms, small amount are found i n nature. Thes (>C ) saturated and unsaturated acids, ones with very high degrees of polyunsaturation (found i n marine o i l s ) , epoxy and hydroxy f a t t y acids and acids with cyclopropane moieties i n the chain. The chemistry and biochemistry of the f a t t y acids and t h e i r derived l i p i d s have been reviewed by Gurr and James (4). lg

Table I.

Major Fatty Acids i n Plants and Animals

Chemical Structure

Common Name Saturated Acids Laurie Acid

CH (CH ) COOH

M y r i s t i c Acid

CH (CH ) COOH

P a l m i t i c Acid

CH (CH ) COOH

Stearic Acid

CH (CH ) COOH

3

2

3

1()

2

3

2

3

2

12

14

16

Unsaturated Acids Oleic Acid

CH (CH ) CH=CH(CH ) COOH 3

2

?

2

7

cis L i n o l e i c Acid

CH (CH ) CH=CHCH CH=CH(CH ) COOH 3

2

4

2

cis a-Linolenic Acid

2

?

cis

CH CH CH=CHCH CH=CHCH CH=CH(CH ) COOH 3

2

2

cis

2

cis

2

7

cis

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1.

FULLER AND NES

Plant Lipids and

Their Interactions

5

Fatty Acid Derived L i p i d s The f a t t y acids occur most commonly i n nature as a c y l glycerides (Table I I ) . T r i g l y c e r i d e s are the predominant neutral l i p i d s i n most l i v i n g organisms. T r i g l y c e r i d e s are the storage l i p i d s i n animal f a t and i n plant seeds, and because of t h e i r p h y s i c a l properties as w e l l as high energy content, they are components of many food products. They are also the raw materials f o r making soaps and other surface a c t i v e compounds. Animal t r i g l y c e r i d e s are made up of s i g n i f i c a n t amounts of saturated f a t t y acids and thus tend to be s o l i d at ambient temperature, while vegetable o i l s are u s u a l l y l i q u i d s and have e i t h e r shorter chain saturated acids or acids with higher polyunsaturation. Waxes have s i m i l a r p h y s i c a l properties to those of t r i g l y c e r i d e s , but they occur as saturated acids e s t e r i f i e d to long chain monohydric alcohols rather than to g l y c e r o l , and as minor components, long chain alcohols and alkanes. L i p i d s with high l e v e l considered desirable b help to maintain low l e v e l s of blood c h o l e s t e r o l and favorable l e v e l s of serum high density l i p o p r o t e i n s . Many e f f o r t s are now directed at modification of the f a t t y acid composition i n plants, e s p e c i a l l y the composition of seed o i l s (5). Genetic improvement of soybean o i l i s an e s p e c i a l l y desirable goal since the small amount of ct-linolenic acid present i n the o i l causes f l a v o r instability. Polar L i p i d s . The polar l i p i d s (Table I I ) are extremely important to the l i f e processes of l i v i n g organisms, since glycerophosphol i p i d s are p r i n c i p a l components of membranes. These membranes are for the most part l i p i d b i l a y e r s i n which the nonpolar hydrocarbon t a i l s point toward one another and the polar groups are on the outside, i n t e r a c t i n g with the aqueous phases inside and outside the region enclosed by the membrane. Various g l y c o l i p i d s , s t e r o l s , proteins, lipopolysaccharides and other compounds are also incorporated i n the membranes and influence t h e i r s e l e c t i v e properties. A s i g n i f i c a n t proportion of cell enzymes are membrane-bound and hence are difficult to isolate and characterize. The biosynthesis and r o l e of phospholipids has been reviewed by Mudd (6). Although g a l a c t o l i p i d s (Table I I ) are found i n the nervous systems of animals, they are present i n very few other animal tissues. On the other hand, g a l a c t o l i p i d s and s u l f o l i p i d s are prominent i n green plants as important constituents of the chloroplast photosynthetic membranes. The g a l a c t o l i p i d f a t t y acids of the chloroplast lamallae are highly polyunsaturated with a - l i n o l e n i c acid making up ca. 90% of the f a t t y acid content (7,). S u l f o l i p i d s are also found i n the photosynthetic tissues of the chloroplast. S u l f o l i p i d s are s i m i l a r to the phospholipids i n f a t t y acid composition, i . e . , they contain s i g n i f i c a n t amounts of p a l m i t i c , o l e i c and l i n o l e i c acids, as w e l l as l i n o l e n i c acid.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ECOLOGY AND METABOLISM OF PLANT LIPIDS

6

TABLE IE PRINCIPAL

LIPID

GROUPS

Siructure:

Name: A.

Fatty

Acids

RCOOH CH OOCR, 2

B.

Triglycerides

R COOCH 2

CH OOCR 2

R COOCH R 1

D.

Waxes

E.

Glycerophospholipids

2

3

2

CH,OOCRi I R,COOCH _ CHoOP-OX

A. • Phosphatidyl cholin •Phosphatidyl ethanolamin • Phosphatidyl serine

X= C H C H N H 2

2

COOH • Phosphatidyl glycerol

X= CH CHOHCH OH 2

2

O C H ( C H ) C H = CHCH-CHCH OPOX 3

F.

2

12

2

Sphingophospholipids

H. Galactolipids • Monogalactosyl diacylglycerol (MGDG)

OH NH I COR H Q J — O . O- -CCIH

O.

2

HOOCRi CH OOCR 2

2

CH OH 2

• Digalactosyl diacylglycerol (DGDG)

\qhJ

CHOC CH

H.

Sulfolipids • Plant Sulfolipid (Sulfoquinovosyl diacyl glycerol)

-CH

I

2°

2

CHOOCRi

I

CH OOCR 2

J.

2

Sterols

• Cholesterol

• Fucosterol

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

FULLER AND NES

1.

Plant Lipids and Their Interactions

7

L i p i d s not derived from f a t t y acids Sterols. Sterols and terpenes are both isopentenoid compounds. Like the f a t t y acids, t h e i r biosynthesis begins with acetate, which undergoes a s e r i e s of reactions forming acetoacetate, hydroxymethylglutarate and f i n a l l y mevalonate. Mevalonic acid i s the precursor to a l l the isopentenoid compounds. Through a further series of reactions mevalonic acid i s converted to isopentenyl pyro-

HOOC

phosphate and then through successive condensations to squalene, a 30 ^ isopentenoid. I n nonphotosynthetic organisms squalene forms an epoxide which c y c l i z e s to l a n o s t e r o l , a precursor of s t e r o l s (8). The p r i n c i p a l s t e r o l synthesized by mammals and red algae i s c h o l e s t e r o l , from which a number of important s t e r o i d a l hormones are derived. A s t e r o i d synthesized by one organism may not possess an analogous r o l e i n another organism i n which i t i s synthesized. I f present a t a l l , c h o l e s t e r o l i s only formed i n minute amounts by crop plants; however, plants synthesize several important s t e r o l s , most of which are characterized by an a l k y l o r alkenyl group a t the p o s i t i o n of the s t e r o l side chain. The 24-alkylated s t e r o l s may be metabolized to hormones f o r which cholesterol cannot serve as a precursor, e.g., a n t h e r i d i o l . I n addition to appearance as free s t e r o l s , these compounds are often found as esters or as glycosides. S t e r o i d a l a l k a l o i d s or azasteroids are nitrogen d e r i v a t i v e s which may be important i n the defense mechanisms of plants (9). C

o p e n

c n a

n

Other isopentenoids. Many l i p i d s other than steroids are formed v i a the isopentenoid pathway. Terpenes and t h e i r d e r i v a t i v e s are very important i n i n t e r a c t i o n s of plants with other organisms. Kuc and coworkers have proposed that fungal elicitors modify isopentenoid pathways i n potato, s h i f t i n g biosynthesis from triterpene a l k a l o i d s which are p r e - i n f e c t i o n i n h i b i t o r s to sesquiterpene lactone stress metabolites (9). A v a r i e t y of insect attractants, insect j u v e n i l e hormones, i n h i b i t o r s and plant hormones are terpene d e r i v a t i v e s . Other l i p i d s . Waxes are major l i p i d s i n a few organisms (e.g., jojoba, sperm whale). Cutins (condensation polymers of hydroxy f a t t y acids) are discussed i n a l a t e r chapter (Kolattukudy et a l . , Chap. 10). Hydrocarbons other than isopentenoid compounds occur i n a v a r i e t y of species.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Interactions of L i p i d s The ecology of plants includes t h e i r interactions with b e n e f i c i a l and harmful organisms—human beings, animals, insects, b a c t e r i a , yeasts and fungi. In many of these interactions l i p i d s are produced which are e i t h e r rquired nutrients f o r other organisms or which are part of the defense mechanisms of plants. Isopentenoid compounds produced by one plant may be harmful to another (Elakovich, Chapter 7), while steroids may i n h i b i t plant growth by exerting or modifying a regulatory function (Roddick, Chapter 18, W. D. Nes, Chapter 19). Some of the most i n t e r e s t i n g defenses of plants are those against insects, including physical b a r r i e r s (Kolattukudy, Chapter 10) and regulatory compounds f o r insect development (Svoboda, Chapter 11, Thompson, Chapter 12). The interactions which have evolved i n nature suggest protective strategies i n which molecular biology and other biotechnological approaches may be used number of such solution i n t h i s volume.

Literature Cited 1. Harborne, J. B., "Introduction to Ecological Biochemistry", 2d Edition; Academic Press: London, 1982, p. 230. 2. Nes, W. D.; Bach, T. J. Proc. R. Soc. Lond. B225. 1975, p. 425-444. 3. Nes, W. D.; Campbell, B. C.; Stafford, A. E.; Haddon, W. F.; Benson, M. Biochem. Biophys. Res. Commun., 1982, 108, 1258-1263. 4. Gurr, M. I.; James, A. T., "Lipid Biochemistry: An Intro­ duction", 3d Edition; Chapman and Hall: New York, 1980, Chap. 2. 5. Ratledge, C.; Dawson, P.; and Rattray, J . , "Biotechnology for the Fats and Oils Industry"; American Oil Chemists Socieity, Champaign, IL., 1984. 6. Mudd, J. B. in "The Biochemistry of Plants, Vol. 4, Lipids: Structure and Function"; Stumpf, P. K., Ed.; Academic Press: New York, 1980; Chap. 9. 7. Harwood, J. L., Ibid., Chapter 1. 8. Nes, W. R.; McKean, M. L., "Biochemistry of Steroids and Other Isopentenoids"; University Park Press: Baltimore, 1977; Chapters 4-7. 9. Kúc, J . ; Tjamos, E.; Bostock, R., in "Isopentenoids in Plants"; Nes, W. D., Fuller, G. and Tsai, L.-S., Eds.; Marcel Dekker: New York; 1984. pp. 103-123. RECEIVED September 5, 1986

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 2

Biosynthesis of Chloroplast Glycerolipids 1

J. B. Mudd, D. G. Bishop , J. Sanchez, K. F. Kleppinger-Sparace, S. A. Sparace, J. Andrews, and S. Thomas ARCO Plant Cell Research Institute, 6560 Trinity Court, Dublin, CA 94568

The glycerolipids of the chloroplast comprise mono­ galactosyldiacylglycerol (MGDG)*, digalactosyldiacyl­ glycerol (DGDG), sulfoquin-ovosyldiacylglycerol (SQDG), and phosphatidylglycero these found outsid synthesis of fatty acids is exclusively in the plastid of higher plants, the synthesis of the unique glycero­ lipids of the chloroplast requires contributions from the cytoplasmic compartment. The fatty acids synthesized in the plastid are exported to the cytoplasmic compartment as acyl- CoAs generated by enzymic activity of the outer membrane of the plastid envelope. These acyl- CoAs are utilized in the synthesis of phospholipids in the mitochondria and the endoplasmic reticulum. The fatty acid specificity in the synthesis of phosphatidyl choline (PC) is such that palmitate (16:0) is never found at the sn-2 position. Thus the predominant molecular species are sn1-18, sn2-18 and sn1-16, sn2-18. These molecular species of PC supply the diacylglycerol (DAG) moiety for synthesis of MGDG, DGDG, and SQDG in the plastid. In some cases ("18:3 plants") the DAG from PC is the sole supplier of DAG moieties to the three glycolipids. The mechanism of transfer of the DAG from PC to the chloroplast moiety is unknown. 1

*Abbreviations: ACP, a c y l c a r r i e r p r o t e i n ; APS, adenosine-5 phosphatosulfate; CDP-DG, c y t i d i n e d i p h o s p h a t e - d i a c y l g l y c e r o l ; DAG, d i a c y l g l y c e r o l ; DGDG, d i g a l a c t o s y l d i a c y l g l y c e r o l ; FdH2, ferredoxin (reduced); LPA, lysophosphatidic a c i d ; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic a c i d ; PC, phosphat i d y l c h o l i n e ; PG, phosphatidylglycerol ; PGP, phosphatidylglycerolphosphate; PSSO3", protein-S-sulfonate; SQDG, sulfoquinovosyld i a c y l g l y c e r o l ; UDP-SQ, uridinediphosphate-sulfoquinovose. 'Current address: CSIRO, Division of Food Research, North Ryde, New South Wales, Australia 0097-6156/87/0325-0010$06.00/0 © 1987 American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2. MUDD ET AL.

11

Chloroplast Glycerolipids

DAG i s also synthesized i n the p l a s t i d , but i n t h i s case the f a t t y a c i d s p e c i f i c i t y i s such that 16 C acids are almost without exception found a t the sn-2 p o s i t i o n . Thus the predominant molecular species are snl-18, sn2-l6, and snl-16, sn2-l6. These molecular species are the sole s u p p l i e r o f DAG moieties i n the synthesis o f PG. In some cases ("16:3 plants") these molecular species contribute to the synthesis o f MDGD, DGDG and SQDG.

L i p i d biosynthesis i n c h l o r o p l a s t s has been extensively studied for 25 years. We now have a good understanding o f the synthesis of f a t t y acids and g l y c e r o l i p i d s . Whereas f a t t y a c i d synthesis i n higher plants i s l o c a l i z e d i n the p l a s t i d , the synthesis o f g l y c e r o l i p i d s o f the p l a s t i d s depends to a large degree on enzymes i n the cytoplasmi This review attempt study o f g l y c e r o l i p i d metabolism i n the c h l o r o p l a s t . Detailed current information may be found i n the recently published proceedings o f a symposium on Structure, Function and Metabolism of Plant L i p i d s (1). Fatty Acid Synthesis The studies on f a t t y a c i d synthesis i n higher plants over the l a s t 25 years have led to a consensus about the i n d i v i d u a l reactions and t h e i r l o c a l i z a t i o n i n the c e l l . This consensus i s that the enzyme system for f a t t y a c i d synthesis i s procaryotic i n nature, that i s the enzymes are soluble and separable, and that the system i s l o c a l i z e d e n t i r e l y i n the p l a s t i d . Thus the membranes o f the mitochondria, the endoplasmic reticulum, the plasmalemma, the tonoplast, the nuclear membrane, and the G o l g i apparatus a l l depend for t h e i r f a t t y a c i d components on the a c t i v i t i e s o f the p l a s t i d s . In o u t l i n e the reactions o f f a t t y a c i d synthesis may be summarized: acetate + ATP + CoASH acetylCoA + HCO3 acetylCoA + ACP malonylCoA + ACP acetylACP + malonylACP P-ketoacylACP + reduced nicotinamide P-hydroxyacylACP enoylACP + reduced nicotinamide +

A T P

-> —> —> -+ -> -> —• ->

acetylCoA malonylCoA acetylACP malonylACP P-ketoacylACP P-hydroxyacylACP enoylACP acylACP

[1] [2] [31 [4] [5] [6] [7] [8]

Although there i s general agreement about the o u t l i n e o f the synthetic r e a c t i o n s , some questions may be raised on s p e c i f i c issues, such as the o r i g i n o f the precursors f o r f a t t y a c i d synthesis and the o r i g i n o f the reductants used. Acetate has been used widely as a precursor f o r f a t t y a c i d synthesis by i s o l a t e d c h l o r o p l a s t s l a r g e l y as a matter o f convenience and economy. Several studies have attempted to determine whether acetate i s the p h y s i o l o g i c a l precursor.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

12

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Roughan etal (2) compared acetate, pyruvate and malonate as p o t e n t i a l precursors and found that acetate was about three times better than pyruvate while malonate was not used a t a l l . Consistent with t h i s r e s u l t i s the report o f Kuhn etal Q ) who found that acetate concentration i n plant t i s s u e i s i n the order of mM and that acetate thiokinase i s l o c a l i z e d i n the p l a s t i d . Nevertheless, a l t e r n a t i v e substrates can not be e n t i r e l y excluded at t h i s stage. Schulze-Siebert etal (4) have reported that when c h l o r o p l a s t s are incubated with bicarbonate, pyruvate accumulates i n the c h l o r o p l a s t . Furthermore Williams and Randall (5) have reported that pyruvate dehydrogenase of pea c h l o r o p l a s t s has an a c t i v i t y o f 6-9 umol/h/mg c h l o r o p h y l l . I t i s therefore conceivable that the a c e t y l CoA used i n the f i r s t steps o f f a t t y a c i d synthesis i s derived from pyruvate. The question as to whether p h o t o s y n t h e t i c a l l y f i x e d carbon dioxide can d i r e c t l y give r i s e to precursors o f f a t t y a c i d synthesis has a l s o receive Rees (6) have reported a c i d to pyruvate i s incomplete i n pea c h l o r o p l a s t s because o f the absence of phosphoglyceric a c i d mutase. This r e s u l t would suggest that the f i r s t product of photosynthesis leaves the c h l o r o p l a s t and the precursor o f f a t t y a c i d synthesis (acetate or pyruvate) i s eventually returned to the c h l o r o p l a s t . The r e s u l t of Schulze-Siebertetal (4) with spinach c h l o r o p l a s t s appears to be consistent with the presence o f the phosphoglyceric a c i d mutase i n these c h l o r o p l a s t s . Furthermore the r e s u l t s o f Journet and Douce (J) obtained by using p l a s t i d s from c a u l i f l o w e r i n f l o r e s c e n c e , i n d i c a t e that they are capable o f the conversion o f 3-phosphoglyceric a c i d to a c e t y l CoA. The studies o f the i n d i v i d u a l enzymes o f f a t t y a c i d synthesis i n higher plants has shown that the two reductive steps, 0-ketoacyl ACP reductase and enoyl ACP reductase have d i f f e r e n t cofactor requirements. As a r e s u l t the synthesis o f f a t t y acids depends on the a v a i l a b i l i t y o f both NADH and NADPH. While the p r o v i s i o n of NADPH can be a t t r i b u t e d to the photosynthetic r e a c t i o n s , the source o f NADH i n the c h l o r o p l a s t i s l e s s c e r t a i n . Takahama etal (8) have demonstrated that the content o f NADPH i n the c h l o r o p l a s t i s influenced by i l l u m i n a t i o n as expected, but there i s no such f l u c t u a t i o n o f the o x i d a t i o n s t a t e o f NAD/NADH. The production o f NADH to be u t i l i z e d i n f a t t y a c i d synthesis would therefore appear to depend on dark reactions. One p o s s i b i l i t y would be by the a c t i o n o f pyruvate dehydrogenase, which would generate not only the NADH required for reduction i n f a t t y a c i d synthesis but a l s o the precursor a c e t y l CoA. Most studies o f f a t t y a c i d synthesis by i s o l a t e d c h l o r o p l a s t s are made under photosynthetic c o n d i t i o n s . I l l u m i n a t i o n o f the c h l o r o p l a s t s generates ATP and reductant necessary f o r the incorporation o f acetate i n t o the f a t t y a c i d s . Other e f f e c t s o f i l l u m i n a t i o n may influence f a t t y a c i d synthesis. For example the pH and the magnesium ion concentration o f the stroma both r i s e when the c h l o r o p l a s t i s i l l u m i n a t e d . I t should be noted that non-photosynthetic p l a s t i d s are a l s o assumed to be the sole s i t e o f f a t t y a c i d synthesis and they must have sources

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

2. MUDD ET AL.

Chloroplast Glycerolipids

13

of ATP and reductant a l t e r n a t i v e to photosynthetic mechanisms. Sauer and Heise (J) have addressed the problem of f a t t y a c i d synthesis by c h l o r o p l a s t s i n the dark. They have used the dihydroxyacetone phosphate s h u t t l e as f i r s t described by Werdan etal(]§), which depends on the conversion of DHAP to glyceraldehyde-3-phosphate which i s o x i d i z e d by GPDH, generating ATP and NADPH. Since DHAP i s taken i n t o the c h l o r o p l a s t by the phosphate t r a n s l o c a t o r i n exchange f o r phosphate i t was necessary to include phosphate as a component of the s h u t t l e to counteract the p o t e n t i a l decrease o f phosphate i n the stroma. The o r i g i n a l purpose of the DHAP s h u t t l e was to obtain carbon d i o x i d e incorporation i n t o 3-PGA i n the dark which required ATP but not reductant. I t was therefore necessary to r e o x i d i z e the NADPH generated by the o x i d a t i o n of phosphoglyceraldehyde otherwise the production o f ATP would have been l i m i t e d by the lack o f NADP. The r e o x i d a t i o n o f NADPH was accomplished by the a d d i t i o n o f OAA which was reduced to malat Sauer and Heise (2) a l necessary to observe f a t t y a c i d synthesis i n the dark. This i s rather puzzling since the OAA would be expected to d r a i n o f f NADPH which one would think i s required f o r the reductive steps of f a t t y a c i d synthesis. Perhaps the components o f the s h u t t l e have other e f f e c t s than those o u t l i n e d above. The r e s u l t s o f Browse etal(11) a l s o bear on the question o f f a t t y a c i d synthesis i n the dark. They have reported rates o f f a t t y a c i d synthesis by l e a f d i s c s o f spinach kept i n darkness which were 12-20% o f the rates i n the l i g h t . Sauer and Heise (2) have a l s o demonstrated that the synthesis o f f a t t y acids i n the dark i s stimulated when an ionophore i s used to increase the magnesium ion concentration i n the stroma. This r e s u l t i n d i c a t e s that the increase i n stroma concentration o f magnesium ion during i l l u m i n a t i o n i s favorable f o r f a t t y a c i d synthesis. The optimum pH f o r f a t t y a c i d synthesis i s a l s o achieved during i l l u m i n a t i o n of the chloroplast. Fatty Acid U t i l i z a t i o n The long chain f a t t y acids synthesized by the c h l o r o p l a s t system are i n the form o f ACP d e r i v a t i v e s . At any point i n the f a b r i c a t i o n o f the chain there are four things that can happen to the a c y l moiety: 1) the a c y l ACP can be u t i l i z e d i n another cycle o f elongation, 2) the a c y l chain can be t r a n s f e r r e d to glycerol-3-phosphate, 3) the a c y l moiety can be exported from the c h l o r o p l a s t to the cytoplasmic compartment, and 4) the a c y l ACP can be desaturated. (Figure 1 ). The f a t t y a c i d chain lengths synthesized by the c h l o r o p l a s t are p r i m a r i l y 16 and 18. This c l e a r l y i m p l i e s ' t h a t f o r a c y l ACP of chain lengths l e s s than 16 the predominant r e a c t i o n i s f u r t h e r elongation, and that for chain lengths o f 18, elongation i s r a r e . For 16:0 ACP,18:0 ACP, and 18:1 ACP, both t r a n s f e r to the cytoplasmic compartment and a c y l a t i o n o f glycerol-3-phosphate are d i s t i n c t p o s s i b i l i t i e s . The concentration o f ACP i n the stroma has been determined to be 8 uM [Ohlrogge etal (12)], so only small

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ECOLOGY AND METABOLISM OF PLANT LIPIDS

14

cytoplasm

•

16:0 ACP

18:0 ACP

18:1 ACP

I©

I®

acylation of G-3-P

stroma

Figure 1. U t i l i z a t i o n of a c y l ACP. The a c y l ACP generated by the f a t t y a c i d synthesising system can be elongated (reaction ® ) , transferred to glycerol-3-phosphate (reaction ,

ft

3

2

HifJ Η

Figure 2.

THE REDUCED OF B > ,

AND F )

Structures of the f r e e - a c i d forms of mevinolin and of related compounds. The correct absolute configuration i s shown using the nomenclature given by Alberts et a l . (38). Note that one region of the molecules closely resembles the mevaIdyl moiety of (3S,5R)-mevaloyl-CoA thiohemiacetal, the enzyme-bound intermediate in the two-step reduction of (S)-HMG-CoA to (R)-MVA.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987. 1.0 4.5

embryonic Drosophila c e l l s (microsomes) Crithidia fasciculata. semi-purified yeast, p a r t i a l l y

ML-236B

ML-236B

ML-236B

Continued on next page

0.9

homogenates of the Cropora tobacco hornworm (Manduca sexta)

ML-236B

0.24

Monger et a l . 1982 (47)

1.1

human f i b r o b l a s t s , detergent-solubilized

ML-236B

purified

Brown et a l . 1978 (46)

2.66

rat l i v e r microsomes, partially purified

ML-236B

Nakamura & Abeles 1985 (49)

Kim & Holmlund 1985 (50)

Brown et a l . 1983 (48)

Tanzawa & Endo 1979 (45)

Endo et a l . 1976 (44)

10

rat l i v e r microsomes, partially purified

ML-236B ( = Compactin)

Endo et a l . 1976 (44)

Reference

220

Ki (nM)

rat l i v e r microsomes partially purified

Enzyme source

Comparison of i n h i b i t i o n constants (K^ values) of compactin/mevino1intype metabolites against HMG-CoA reductase preparations from various enzyme sources

ML-236A

Compound

Table I.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

3.7 ?

rat l i v e r microsomes, partially purified rat l i v e r microsomes, partially purified

4α, 5-Dihydrocompactin

4a»5-Dihydro~ mevinolin

Albers-Schoonberg et a l 1981 (40)

Lam et a l . 1981 (41)

Watson et a l . 1983 (54)

For other compounds ( c f . F i g . 2) no values are a v a i l a b l e as yet. The I50 values reported using r a t l i v e r HMG-CoA reductase (42, 43) appear to be about three magnitudes higher than that of 4ct,5-dihydromevinolin (40).

*50 = 2.7

20

Halobacterium halobium

Mevinolin

Bach & Lichtenthaler 1983 (53)

radish seedlings, microsome-bound

Mevinolin

2.2

yeast, p a r t i a l l y p u r i f i e d

Mevinolin

Bach & Lichtenthaler 1982, 1983 (52, 53)

Endo 1980 (39)

Alberts et a l . 1980 (38)

Reference

3.5

0.50

rat l i v e r microsomes, Mevinolin (= Monacolin Κ) p a r t i a l l y p u r i f i e d

(nM) 0.64

Enzyme source rat l i v e r microsomes, partially purified

Mevinolin

Compound

Table I . Continued

8.

BACH AND LICHTENTHALER

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Plant Growth Regulation

system from rat liver the inhibitory activity of 6a-hydroxycompactin carboxylate against cholesterol synthesis was more potent than that of the 6 B-hydroxy-derivative (60). S t r a i n SANK 32772 of Absidia coerulea c a t a l i z e d the conversion of ML-236B to 3a-hydroxy-iso-ML-236B (6a- i n the numbering system used by the Sankyo group (61)). This l a t t e r compound, with ^4,4a | Δ » , was s i m i l a r to the parent compound i n i t s l e v e l of i n h i b i t i o n of cholesterol synthesis (61). Other microbial s t r a i n s capable of 6 β-hydroxylation of ML-236 were ascomycetes i s o l a t e d from s o i l samples c o l l e c t e d i n A u s t r a l i a and were i d e n t i f i e d as substrains of Nocardia autotrophica (62). Several fungal strains (Circinella muscae, Absidia cylindrospora and A. glauca) have been reported to b i o l o g i c a l l y phosphorylate the hydroxy 1 group at p o s i t i o n δ of the open carboxylic forms of compact i n as w e l l as of monacolin Κ (= mevinolin), and monacolins L and X (63). The Basidiomycete Schizophyllum commune wa mevinolin (monacolin K derivatives (64).The existence of a n t i b i o t i c s , other than the mevinolin-type (65), produced by Cephalosporin caerulens (known i n h i b i t o r s of the synthesis of polyketides and of f a t t y acids (66)) indicates a complex biochemical and p h y s i o l o g i c a l interplay may e x i s t among organisms throughout the rhizosphere. This interplay may include higher plants i n the rhizosphere. We have shown that mevinolin, applied as i t s water-soluble sodium s a l t , i n h i b i t s the root growth of i n t a c t radish and wheat seeedlings by i n h i b i t i n g t h e i r isopentenoid biosynthesis (67.). We have u t i l i z e d mevinolin as a molecular probe to study the importance of MVA and associate products to growth and development of seedlings, bearing i n mind that i n vivo i n h i b i t i o n of an enzyme, expected to be at least close to rate l i m i t i n g f o r the pathway, should r e s u l t i n c l e a r morphological and biochemical responses. A logical consequence was to e s t a b l i s h what type of isoprenoid compound might be affected i n i t s synthesis or accumulation i n the presence of mevinolin (52, 68, 69), thereby y i e l d i n g information also on the i n t r a c e l l u l a r l o c a l i z a t i o n of HMG-CoA reductase a c t i v i t y i n plant c e l l s which i s currently a matter of controversy i n l i t e r a t u r e (70-72). Since i t appeared that the accumulation of phytosterols was p r i m a r i l y affected by mevinolin (52, 68, 69) (which led us to hypothesize that mevinolin can e a s i l y penetrate the plant c e l l w a l l and the plasmalemma, but rather poorly the envelopes of mitochondria and plastids), we tested other i n h i b i t o r s known or expected to i n t e r f e r e with l a t e r steps of s t e r o l biosynthesis, such as squalene epoxidation, squalene-oxide c y c l i z a t i o n , 14a-desmethylation or side chain a l k y l a t i o n , i n order to look for similarities and differences i n the morphological response of radish seedlings upon treatment (73). A side aspect of these l a t t e r studies i s to e s t a b l i s h a v e r s a t i l e screening system f o r hypocholesterolemic drugs or fungicides. 5

6

an(

Materials and Methods Chemicals. Mevinolin was k i n d l y provided by Dr. A. W. (Merck Sharp & Dohme Res. Labs.) and was converted

Alberts to i t s

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

water-soluble sodium s a l t as described by K i t a et a l . (74). Triparanol (MER-29), U 18666 A, A 25822 Β were g i f t s from Merrell Dow Pharmaceuticals I n c . , Dr. Harry Rudney (University of Ohio, Cincinnati, OH), and Eli Lilly G.m.b.H., respectively. 2,3-Epiminosqualene was kindly synthesized by Dr. Lunkenheimer (Bayer AG), N a f t i f i n e was purchased from Dr. Hôgenauer (Sandoz Forschungszentrum Wien). Triarimol and imminium s a l t ("NES") were g i f t s of Dr. W. D. Nes (USDA Berkeley). SC 32561 was a g i f t from G. D. Searle & Co. Clotrimazole, miconazole, and sodium deoxycholate were purchased from Sigma. C u l t i v a t i o n of seedlings and associated test systems. Radish seeds (Raphanus s at i vus, cv. Saxa Knacker) were immersed in an aerated water bath for 30 min. These seeds were then placed onto a sprouting tray containing 1 1 of H2O or H2O supplied with the chemicals and were allowed to germinate and develop for one week i n the l i g h t (Osra 25°C, 65% r e l a t i v e humidity) dissolved i n a system containing DMSO:Triton X-100 i n the r a t i o 3:1 ( v : v ) , maximum 1 ml/1 i n the growth solution. Controls contained the same amount of this mixture without i n h i b i t o r s . In some cases, the addition of a small amount of EtOH was required to dissolve the compounds. Plant growth was measured d a i l y (25-30 plants per condition). Wheat seedlings were cultivated from Triticum aestivum cv. Anza. E t i o l a t e d primary leaf segments from one week-old plants were obtained and used i n the test system as previously described (75. Zi>. C e l l Suspension Cultures of Silybum marianum. Suspension cultures were grown from primary c a l l u s cultures. Submersed c e l l s were kept i n Erlenmayer-flasks (120 rpm; 25° C; P h i l l i p s TL 40W/47, 2000 lux) i n a growth medium as described by Murashige and Skoog (77). Four to s i x flasks per condition were inoculated with 5 ml aliquots of a c e l l suspension from 8 to 10 days old c e l l cutures and then supplied with mevinolin to a f i n a l concentration of 0.625, 1.25, 1.5, 5 and 10 μ ϋ . At day 3 after inoculation the c e l l s were in the early log phase of evelopment and the plateau phase was reached between day 5 and 6. Under the culture conditions employed the c e l l s started to degenerate between day 6 and 9. Thus, c e l l s were analyzed at day 3 and 6. Chemical analysis. For the extraction of lipids and prenylpigments the plant material was macerated in the presence of 100% acetone and the l i p i d s partitioned into p e t r o l ether (b.p. 50-70° C). Chlorophylls and t o t a l carotenoid content were determined spectrophotometrically (78). Prenylquinones were separated by TLC, HPLC and reversed phase HPLC as described i n d e t a i l (69). Free 4-desmethylsterols were quantified a f t e r TLC on s i l i c a gel (solvent 84 ml p e t r o l ether b.p. 50-70° C and 15 ml Me2C0, Rf = 0.25). Plates were sprayed with saturated antimony trichloride i n H20-free CHCI3, and a f t e r a short heating period at 100° C, the pink spots indicating desmethylsterols (with pure stigmasterol as a standard) were

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8. BACH AND LICHTENTHALER

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Plant Growth Regulation

scanned with densimeter (550 nm) being made for each p l a t e .

with separate

standard

curves

Results and Discussion Growth i n h i b i t i o n by mevinolin. To ascertain the s p e c i f i c i t y of mevinolin as an enzyme i n h i b i t o r and to elucidate the r o l e of HMG-CoA reductase a c t i v i t y i n vivo, i t s e f f e c t on plant growth and development i n conjunction with other isopentenoids has been examined. Mevinolin induced a strong growth i n h i b i t i o n of the main root of the dicotyledonous e t i o l a t e d radish seedlings and of the roots of the monocojbyledonous wheat seedlings (67). A s i g n i f i c a n t drop i n elongation growth was obtained between 10 and 100 ppb (2.5 χ 1 0 " and 2.5 χ 10~ M). We suspected that mevinolin inhibited root elongation growth v i a the interference with HMG-CoA reductase a c t i v i t y i n vivo; therefore, the e f f e c t shoul exogenously supplied MV enzyme reaction. Indeed, increasing concentrations of exogenous MVA i n the presence of mevinolin gave nearly the same growth rate of roots of e t i o l a t e d radish seedlings as found for the control plants (68). Exogenous MVA at an intermediate concentration of 2 mM did not stimulate root elongation growth but rather led to weakly decreased values, probably due to a secondary pH e f f e c t . A main branch point within the isoprenoid pathway i s located at the s i t e of f a m e s y l pyrophosphate (Figure 1). This key p o s i t i o n of C ^ - p r e n y l t r a n s f e r a s e makes i t a l i k e l y candidate for f i n e tuning or rate l i m i t a t i o n of MVA flux into the various end-products, some of which might contribute to normal root (and hypocotyl) growth. Famesol was revealed to enhance root growth i n barley (79, 80). This accelerated root elongation, however, was correlated with increased cytokinin a c t i v i t y as a secondary e f f e c t (79.). This appeared to be to some extent contradictory to the results of Buschmann and Lichtenthaler (81) who demonstrated an inhibitory effect of exogenously supplied kinetin or benzylaminopurine on root elongation of dark- and light-grown radish seedlings. Other related isoprenoid alcohols, however, f a i l e d to enhance the growth of barley roots (80). Furthermore, other plant species tested for t h i s f a r n e s o l - e f f e e t did not exhibit any p o s i t i v e reaction (80). It i s possible that mevinolin might interfere with the synthesis of other isopentenoid phytohormones such as the g i b b e r e l l i n s or the brassinosteroids which are known to induce strong growth promotion ((82-87) and elsewhere i n t h i s volume). We have determined that the growth i n h i b i t i o n of the roots of e t i o l a t e d radish seedlings induced by mevinolin could not be reversed by the addition of g i b b e r e l l i c acid ( G A 3 ) . Exogenously supplied concentrations of GA3 up to 37 uM could not overcome the e f f e c t of mevinolin at 2.5 yM or at 0.25 μΜ (52, 68). Gibberellic acid itself seemed to slightly stimulate root elongation growth of etiolated radish seelings, e s p e c i a l l y i n the l a t e r stages of development [68]. To gain further information about the mode of action of mevinolin, we extended our experiments to light-grown radish 8

7

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

seedlings. In t h i s system, mevinolin proved to be a highly potent i n h i b i t o r of root elongation growth (Figures 3, 4). In e t i o l a t e d seedlings the elongation growth of hypocotyls needs comparably high concentrations of mevinolin to be affected, but in l i g h t - c u l t i v a t e d seedlings i t induces a c l e a r dwarfing growth response. Radish seeds were incubated i n P e t r i dishes on f i l t e r paper with 10 ml solutions of 0, 2.5 μΜ, 0.25 μΜ and .025 μΜ mevinolin (68). Even i n the presence of the i n h i b i t o r at i t s highest concentration, there was no c l e a r e f f e c t on the germination rate (68). The fact that even very high i n h i b i t o r concentrations could not prevent a minimal root growth i n radish seedlings (Figure 3) led us to conclude that t h i s minimal root growth i s a function of MVA-derivatives already present i n the seeds and therefore independent from de novo mevalonate biosynthesis. I t i s evident tha i n h i b i t the elongatio light-grown radish seedlings and also block the formation and development of l a t e r a l roots (Figure 4). This might r e f l e c t , as already discussed, an i n t e r a c t i o n of mevinolin with the synthesis of c e r t a i n isopentenoid phytohormones. In addition to the examples c i t e d above, Geuns (88) demonstrated that, besides the stimulation of hypocotyl growth of e t i o l a t e d mung bean seedlings, the l a t e r a l root number could be increased to 50 and/or 250%, respectively, by rather high concentrations (25 to 50 mg.l""*) of corticosterone and C o r t i s o l . C o r t i s o l was found to stimulate root elongation by about 100% and hypocotyl elongation by ' when added to the growth medium at a concentration of 0.41 μ,η (89). Due to the low uptake of C o r t i s o l by the roots, the concentration i n the plant i t s e l f was thought to be i n the range of 0.1 to 1 nM. The growth stimulation was found to be a r e s u l t of c e l l elongation rather than the production of more c e l l s (89). E f f e c t of mevinolin on isoprenoid accumulation i n radish seedlings. Under the conditions i n which mevinolin i s u s u a l l y applied to the seedlings (see Materials and Methods) i t would f i r s t be taken up by the roots exposed to the watery s o l u t i o n of the i n h i b i t o r and then d i s t r i b u t e d to the remaining seedling parts. The e f f e c t of mevinolin on fresh and dry weights, s t e r o l content, and ubiquinone content i n d i f f e r e n t parts of the seedlings upon treatment has been determined (Table I I ) . At a maximum concentration of 5 μΜ, mevinolin reduces the content of free 4-desmethylsterols i n roots to about 20 per cent of the c o n t r o l . At 0.625 μΜ, s t e r o l content i s reduced by about 20 per cent, thus i n d i c a t i n g a very fast response of s t e r o l accumulation to i n h i b i t i o n of MVA biosynthesis. These e f f e c t s are less dramatic i n hypocotyls, and e s p e c i a l l y i n cotyledons. This r e s u l t implies that there might e x i s t a gradient i n the concentration of mevinolin present i n upper parts of the seedlings. The p o s s i b i l i t y of a low transport rate of mevinolin w i t h i n the t i s s u e was indicated when excised dark-grown radish seedlings were used to study the e f f e c t of i n h i b i t o r on the light-induced accumulation of isoprenoid compounds (68); the e f f e c t on s t e r o l synthesis was

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

BACH AND LICHTENTHALER

Plant Growth Regulation

1

Figure 3. Radish seedlings (4 days old) grown i n the dark (upper part) or i n the l i g h t (lower part) i n the presence of mevinolin from onset of germination. Note the lack of l a t e r a l root growth at the high i n h i b i t o r concentration.

Figure 4. Mevinolin-induced growth i n h i b i t i o n of the main root of l i g h t grown radish seedlings. Mean values + SD from 30 to 50 plants per condition. (Reprinted with permission from Ref. 68. Copyright 1983 Physiologia Plantarum.) In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

Table II.

Fresh/dry weight, s t e r o l content and accumulation of the l i g h t for 6 days in the presence of mevinolin 0

Plant Part, Treatment Cotyledons 0 0.25 0.625 2.5 5.0

Ο 0.25 0.625 2.5 5.0 a D c

Free Sterols (Ug/100 parts)

0

0.62 0.66

1640 1647

3.9 3.4

0.63 0.63

1453 1091

μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μ Mevinolin μΜ Mevinolin

3.9 4.2 4.1 3.3 2.2

0.23 0.25 0.26 0.25 0.18

593 571 521 426 276

μΜ μΜ μΜ μΜ μΜ

2.8 2.5 2.2 1.8 1.3

0.15 0.14 0.12 0.11 0.09

522 416 274 200 112

Mevinolin Mevinolin Mevinolin Mevinolin Mevinolin

Hypocotyls

Roots

g DW (100 parts)

4.7 4.9

μΜ μΜ μΜ μΜ μΜ

0 0.25 0.625 2.5 5.0

g FU (100 parts)

0

0

Mevinolin Mevinolin Mevinolin Mevinolin Mevinolin

Mean values of three independent experiments 100 Cotyledon pairs analyzed per single experiment 100 Hypocotyls per analysis

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8. BACH AND LICHTENTHALER

Plant Growth Regulation

t o t a l ubiquinone (Q-9 + Q-10) i n radish seedlings grown in

% of controls

Q-9 + Q-10 (ug/100 parts)

% of controls

% Q-9 of t o t a l Q-9 + Q-10

100 97 8 77 39

8.6 8.6

100 100 96 89 67

61.1 59.2 52.7 46.8 23.7

100 96 88 72 47

12.2 11.6 10.3 8.5 4.8

100 95 84 69 39

7.2 8.6 10.5 12.8 14.3

100 79 52 39 22

17.0 15.9 11.8 8.0 7.0

100 94 69 47 41

6.3 9.5 11.4 13.0 16.6

11.2 12.5

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

121

122

ECOLOGY AND METABOLISM OF PLANT LIPIDS

p a r a l l e l e d by an i n h i b i t i o n of elongation of hypocotyl segment growth. Total ubiquinone content was about 40% of the control at the maximal i n h i b i t o r concentration regardless of what part of the seedling was analyzed (Table II). However, in radish seedlings mevinolin treatment did cause a s h i f t towards the synthesis of Q-9 at the expense of Q-10, the predominant homologue i n radish (Table II) . Interestingly, only the fresh weight, but not the dry weight, of radish cotyledons was affected, whereas i n hypocotyls, and more c l e a r l y i n roots, fresh weight and dry weight were diminished upon mevinolin treatment (Table II). Even though a clear i n h i b i t i o n of s t e r o l accumulation in cotyledons by mevinolin can be achieved at higher concentrations as compared to the roots, there was no i n h i b i t i o n of chlorophyll and carotenoid biosynthesis under these conditions ((68) and Table III) . At low mevinolin concentrations chlorophyll a+b and carotenoid content was even enhanced as compared to untreated controls (Table III). Th compounds of ρlastidi plastoquinone appeared to be s l i g h t l y enhanced at low i n h i b i t o r concentrations and hardly inhibited even at 5 μΜ (Table III). α - T o c o p h e r o l (Table III), which was considered e a r l i e r to be synthesized i n the chloroplast as well as in the cytoplasm (90. 91), does not react l i k e sterols (Table II), which are c l e a r l y cytoplasmic products, but rather l i k e phylloquinone, known to be exclusively synthesized in the ρ l a s t i d (92, 93). This result agrees with recent findings (94), demonstrating the capacity for α-tocopherol synthesis in envelope membranes from spinach chloroplasts. It appears that s t e r o l s , as they are mainly affected, might be involved in physiological processes governing water uptake and c e l l elongation growth (cf. (95)). Evidently, the diminished synthesis of s t e r o l s , needed for biomembrane formation (28, 29). makes a strong contribution to the growth-retardant e f f e c t of mevinolin. The drastic i n h i b i t i o n of s t e r o l biosynthesis by mevinolin indicates HVA biosynthesis to be r a t e - l i m i t i n g , as has already been demonstrated i n the case of animal c e l l s (cf. 1, 3-7). This may also be true for ubiquinone, but apparently not for other compounds investigated here. Even though conclusive evidence i s available to indicate an independent MVA-synthesizing machinery being present in plant mitochondria (10, 14), possibly regulated d i f f e r e n t i a l l y from that assayed in the ER (96, 97.), HMG-CoA reductase a c t i v i t y i s c l e a r l y i n h i b i t e d , which led us to conclude that mevinolin can penetrate the mitochondrial envelope. Of course, we do not exclude the p o s s i b i l i t y that mitochondrial MVA u t i l i z a t i o n - and thereby ubiquinone biosynthesis - might a d d i t i o n a l l y be linked to cytoplasmic MVA-synthesis, depending on the need of the organelle for additional IPP units (cf. 72). The mevinolin-induced shift in the Q-pattern toward homologues containing shorter isopentenoid side chains (Table II) might r e f l e c t the a b i l i t y of mitochondria to adjust the usage of isopentenyl units to the available substrate inside or outside of the organelle, thereby maintaining a basic rate of synthesis needed for a functional respiratory electron transport. The s l i g h t increase of phylloquinone and, to a somewhat lower extent,

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

c

0

a

μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin

0

-

830 830 840 770

-

124 118 120 113

-

4930 4670 4770 4470

740

100

3970

-

113 113 114 104

100

Carotenoids X+c (ug/100) (%)

Mean values of three independent experiments. 100 Cotyledon pairs analyzed per single experiment 100 Hypocotyls per analysis

0 0.25 0.625 2.5 5.0

0

Chlorophylls a+b (ug/100) (%)

14.0 16.8 19.0 15.5 13.5

158 137

-

154 157 192

-

100 120 135 113 98

2 .2 2..6 3 .1 3..5 2..1

21.,6 20..2

103 89

23,.9 26. .7 25..0

100 118 141 159 96

85

-90

100 112 104

Phylloquinone (ug/100) (%)

a

100 102 124

Plastoquinone (ug/100) (%)

E f f e c t of mevinolin on ρ l a s t i d i c p r e n y l l i p i d s and prenylquinones

μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin μΜ Mevinolin

Hypocotyls

0 0.25 0.625 1.25 2.5 5.0

Cotyledons

Plant Part, Treatment

Table III.

252 259 268 _ 240 212

84

-95

100 103 106

α-Tocopherol (ug/100) (%)

ECOLOGY AND METABOLISM OF PLANT LIPIDS

124

of plastoquinone, upon mevinolin treatment at concentrations below 2.5 μΜ might be explained by an increased a v a i l a b i l i t y of acetate to be routed towards p l a s t i d i c isopentenoid biosynthesis. This would require that the acetate from the cytoplasm penetrates the chloroplast envelope. E f f e c t of mevinolin on isoprenoid synthesis i n primary leaves of wheat. Mevinolin can to some extent i n h i b i t the biosynthesis and accumulation of chlorophylls and carotenoids when i t s u f f i c i e n t l y penetrates the l e a f , as shown i n experiments with leaf segments of e t i o l a t e d wheat seedlings floated on a mevinolin s o l u t i o n of 2 mm thickness i n P e t r i dishes and then exposed to l i g h t . Despite the extreme i n v i t r o e f f i c a c y of mevinolin i n i n h i b i t i n g microsomal plant HMG-CoA reductase (see Table I ) , high concentrations were needed to a f f e c t the light-induced i n vivo formation of cartenoids and chlorophylls (Table I I ) . The chlorophyll b accumulation was i n h i b i t e d to a higher degre be explained by assumin chlorophyll-protein complex containing chlorophyll a present i n reaction centers of photosystem I and I I followed by the formation of (under these experimental conditions where no supply of storage products from the seeds i s possible) rudimentary l i g h t harvesting complexes containing chlorophyll b (12, 98). The light-induced change i n percent composition of carotenoids ( e s p e c i a l l y the increase of β-carotene levels) appeared to be p a r t i a l l y blocked only at the higher mevinolin l e v e l . Because 6-carotene i s mainly located i n the photosynthetic reaction centers (99) and known to protect chlorophyll a molecules from photooxidation; t h i s lower β-carotene l e v e l may also account f o r the apparently diminished accumulation of chlorophylls. In contrast to pigments, the s t e r o l accumulation (defined as increase over dark control) i s completely blocked by the lower mevinolin concentration (Table IV). With primary leaves of wheat, incubated under comparable conditions but supplied with [^C]-acetate and [^Hl-mevalonate, precursors able to enter the isoprenoid pathway before or a f t e r the HMG-CoA reductase step, respectively, i t was shown (75, 76.) that mevinolin could completely prevent acetate incroporation into phytosterols. Incorporation of t r i t i u m from labeled MVA was unaffected. This e l i m i n a t i o n of [ C]-acetate incorporation into phytosterols was observed at a mevinolin concentration which had no e f f e c t on chlorophyll and carotenoid accumulation i n controls; v i r t u a l l y i d e n t i c a l accumulation i n bands of TLC-plates i d e n t i f e d as pheophytins or β-carotene was found f o r controls and mevinolin treatment (Bach & Nes, unpublished). The l i m i t e d a b i l i t y of mevinolin to prevent pigment accumulation i n chloroplasts favors the assumption that ρlastids contain t h e i r own independent enzyme system f o r MVA production. The p l a s t i d i c envelope i s apparently not at a l l or only poorly permeable to mevinolin. The a b i l i t y of p l a s t i d s to synthesize MVA has been questioned (70, 71). Our observations, together with i n v i t r o measurements of enzyme a c t i v i t y (16, 21), support the view that p l a s t i d s possess t h e i r own HMG-CoA reductase. 14

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8. BACH AND LICHTENTHALER

Table IV.

125

Plant Growth Regulation

D i f f e r e n t i a l i n h i b i t i o n of s t e r o l accumulation and of the light-induced p r e n y l l i p i d accumulation at high mevinolin concentration i n excised 9-day-old e t i o l a t e d primary Wheat leaves during 18 h of continuous white light. P r e n y l l i p i d s i n yg per 40 leaf segments. Mean of 2 runs with SD < 10%. X = Xanthopylls; c = β-carotene; x+c = sum of carotenoids. Chlorophylls and carotenoids were separated by TLC (1) and determined photometrically.

Parameter

Initial

F i n a l value

F i n a l value 125

500

a) P r e n y l l i p i d content Chlorophyll a Chlorophyll b Ratio a/b Carotenoids Ratio x/c Ratio a+b/x+c Free desmethylsterols

0 0 134 29 230

273 24 11 165 7.1 1.8 302

134 6.6 20 114 11.3 1.2 232

72 1.5 48 90 19.4 0.8 229

3.3 18.6 17.8

12.4 17.7 11.2

8.1 17.8 13.1

4.9 17.0 15.2

58.2 3.7

54.8 3.9

57.3 3.7

59.5 3.4

b) % composition of carotenoids β-carotene Violaxanthin Antheraxanthin + Luteinepoxide Lutein Neoxanthin

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

126

ECOLOGY AND METABOLISM OF PLANT LIPIDS

E f f e c t of mevinolin on the growth and chemical composition of c e l l suspension cultures of Silybum marianum. In order to t e s t the i n h i b i t o r i n a system where e x t r a c e l l u l a r transport from the s i t e of a p p l i c a t i o n to the expected s i t e of action i s not an important f a c t o r , we used c e l l suspension cultures of Silybum marianum (100). Cell suspension cultures were i n i t i a t e d i n flasks containing growth medium supplemented with mevinolin at various concentrations. After 3 and 6 days the c e l l s were analyzed (Table V). The data demonstrate that the e f f e c t of mevinolin becomes more evident at a l a t e r stage of growth. Also, i n t h i s system the i n h i b i t i o n by mevinolin of isoprenoid synthesis follows a gradient: S t e r o l s > ubiquinones > plastoquinone ^ carotenoids « cholorophylls, thereby emphasizing the r e s u l t s obtained with radish and wheat seedlings. Besides an o v e r a l l i n h i b i t i o n of ubiquinone accumulation to about 50% (on the basis of dry weight), mevinolin induced a s h i f t i n the homologue pattern towards a shorter side-chain, e.g. Q-10 (with Q-9 being th Silybum marianum c e l l s ) . Ryder and Goad (101) have demonstrated using suspension cultures of sycamore (Acer pseudoplatanus L.) that i n the presence of 5 mg/1 compact i n the incorporation of [^C]- leucine and [^C]-acetate into 4-desmethylsterols was inhibited to 95% and 99%, respectively, while C-MVA incorporation was unaffected. However, s t e r o l synthesis from endogenous precursors, measured by incorporation of [Me-I^C]-methionine into the side chain, continued at a reduced rate f o r at least 6 h a f t e r a d d i t i o n of the i n h i b i t o r . This r e s u l t suggested the presence of a pool of precursors which was presumably gradually depleted by the process of sterol biosynthesis but could not be replenished from acetate i n the presence of compactin (101). Compactin was also revealed to i n h i b i t growth of c a l l u s cultures of tobacco (102). At 5 μΜ the average i n h i b i t i o n (fresh weight of c a l l u s ) was between 45 and 80%, at ΙΟμΜ around 95%. Cytokinins such as N -isopentenyladenine or k i n e t i n at concentrations from 0 to 1.0 and 0 to 5μΜ, respectively, could not s u b s t a n t i a l l y counteract the growth i n h i b i t i o n by compact i n (102). Mevinolin as w e l l as compactin were recently tested using expiants of Helianthus tuber ο sus (103). The e f f e c t s on growth parameters (fresh and dry weight) were w e l l w i t h i n the range determined with c e l l cultures of Silybum. 14

6

Aspects of secondary p h y s i o l o g i c a l responses of seedlings and c e l l cultures upon mevinolin treatment. Some observations that were made by the use of our radish growth system provide further support f o r the importance of MVA synthesis f o r normal development of seedlings. In e a r l i e r work (81) i t was documented that k i n e t i n , an a r t i f i c i a l cytokinin, induced i n radish seedlings a growth response comparable to mevinolin, e.g., shortened main roots. Cytokinins have been considered to be senescence regulation factors i n many plant systems. By measuring fast &nd slow fluorescence k i n e t i c s , i t was revealed that mevinolin can i n h i b i t radish seedling senescence, e.g., as indicated by the maintainence of a functional photosynthetic apparatus (68). Thus,

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

0 (control) 0.625 1.25 2.5 5.0 10.0

day 6:

0 (control) 0.625 1.25 2.5 5.0 10.0

day 3:

I n i t i a l value

400 340 300 260 140 110

277 290 310 247 173 180

11

100 85 75 65 35 28

100 105 110 89 63 65

(%)

38.0 34.4 23.3 21.9 19.3 16.4

55.6 50.0 41.0 32.8 24.3 23.3

364

100 90.5 61.3 57.6 50.8 43.2

100 89.3 73.7 59.0 43.7 41.9

(%)

mg dw mg protein 100 mg susp. g dw

3.8 2.9 2.0 1.3 0.8 0.3

3.4 3.0 1.7 1.2 1.1 0.9

2.9

100 76 53 34 21 8

100 88 50 35 33 26

(%)

mg s t e r o l s g dw

92.4 80.3 73.2 69.9 60.3 58.5

94.1 87.3 84.2 85.7 81.5 76.5

100 86.9 79.2 75.6 65.3 63.3

100 92.8 89.5 91.1 86.6 81.3

65.1 48.3 42.1 40.7 35.7 34.0

63.9 54.6 55.4 52.7 44.8 45.2

100 74.2 64.7 62.5 54.8 52.2

100 85.4 86.7 82.5 70.1 70.7

(%)

232 210 234 207 183 178

229 209 227 216 174 155

100 88 101 86 72 66

100 91 99 94 73 65

(%)

199

99.4

146. 9 (%)

ug PQ-9 g dw

ug carot. g dw

ug Chl.a+b g dw

E f f e c t of mevinolin on a c e l l suspension culture of Silybum marianum

μΜ Mevinolin

Table V.

216 204 194 155 116 105

211 203 192 173 144 133

57

100 94 89 71 54 49

100 96 89 82 67 53

(%)

ug t o t a l Q-n g dw

1 1

1 1 1

1 1 1 1 1 1

/

/

/

/

/

/

/

/

/

/

/

0.7 0.4

5.9 2.9 1.7

6 3 1.8 1.6 1.4 1.4

molar r a t i o Q~8 / Q-10

128

ECOLOGY AND METABOLISM OF PLANT LIPIDS

the root growth i n h i b i t i n g a f f e c t by c y t o k i n i n was perplexing since, i n p l a n t s , roots are regarded to be the main source of cytokinins (104, 105)» cytokinins are then transported to the shoot v i a the xylem (106). However, f o r the complete i n h i b i t i o n of,say, N^-isopentenyladenine or zeatin synthesis, concentrations of mevinolin might be required that are magnitudes higher than that needed to completely knock out de novo s t e r o l synthesis. Thus root growth may be i n h i b i t e d by a lack of s t e r o l synthesis i n the treated roots while the c y t o k i n i n synthesis continues i n d i c a t i n g d i f f e r e n t i a l i n h i b i t i o n by mevinolin of the accumulation of various end-products of the multibranched isoprenoid pathway. I t has been reported (84) e f f e c t of b r a s s i n o l i d e treatment on senescence of dark-maintained discs of Xanthium leaves was opposite to equimolar concentrations of k i n e t i n , promoting senescence (loss of chlorophyll) as compared to the c o n t r o l . I t may w e l l s t e r o i d hormones require as has been suggested by work that has been done i n Dr. W. David Nes* lab at Berkeley (107) which supports the t h e s i s of a s c r i b i n g d i f f e r e n t f u n c t i o n a l or metabolic roles f o r d i f f e r e n t s t e r o l s by measuring indivudual turnover rates. In t h i s regard i t i s i n t e r e s t i n g to note that a p p l i c a t i o n of s t e r o i d hormones to e t i o l a t e d mung beans (88) resulted, i n the formation of longer main roots and i n a strongly increased number of side roots. In radish, at very high concentrations of mevinolin (> 1 mg per l i t e r ) the formation of l a t e r a l roots (see Figure 4) was completely prevented, an e f f e c t that could be observed over the f u l l c u l t i v a t i o n period of 10 days. Mevinolin may induce changes i n the endogenous phytohormone balance: a small amount of b i o l o g i c a l l y synthesized MVA might be s u f f i c i e n t f o r the supply of the isopentenyl derived moiety of isopentenyl-adenine or z e a t i n (-riboside?) or the isopentenylation of t-RNA but not for that of steroid phytohormones, thereby leading to a promotion of k i n e t i n - l i k e growth responses. The increased accumulation of anthocyanins i n hypocotyls of mevinolin-treated radish seedlings (69) c e r t a i n l y does not simply r e f l e c t the routing away of acetate u n i t s from s t e r o l s ( c f . Figure 1) but also r e f l e c t s a more general response of plants upon treatment with chemicals (95). I n h i b i t o r s (see also below) a f f e c t i n g l a t e r steps i n phytosterol synthesis seem to a f f e c t various other products of the isoprenoid pathway before and a f t e r squalene formation (95). Secondary e f f e c t s of such growth regulators l i k e d i s r u p t i o n of the f u n c t i o n a l i n t e g r i t y of the endoplasmic reticulum or the plasmalemma, thus leading to changes i n water and ion transport capacity (108) or i n t e r a c t i o n with isoprenoid c a r r i e r proteins, as suggested by Nés et a l . (95), may a d d i t i o n a l l y account for t h e i r mode of action. Since both s t e r o l s and GA3 can independently reverse the retardant action of these biocides on stem growth (109. 110) i t was suggested by Nés et a l . (95) that several end-products of the isoprenoid pathway may act independently on developmental processes, but produce the same end response. This emphasizes the more general concept of a shade-type or sun-type growth response of plants upon biocide t n a t

t n e

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

8. BACH AND LICHTENTHALER

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129

treatment as proposed by Lichtenthaler (111). Treatment of yeast c e l l cultures with systemic fungicides such as c l o t r i m a z o l and triadimefon resulted i n an accumulation of l a n o s t e r o l that, i n a secondary process, proved to be a highly s p e c i f i c feedback i n h i b i t o r of HMG-CoA reductase a c t i v i t y w i t h i n the yeast c e l l s (23). Since there i s some evidence that plant HMG-CoA reductase a c t i v i t y might also be regulated by feedback mechanisms, as demonstrated with pea seedlings (13, 15), s i m i l a r e f f e c t s may account f o r the i n h i b i t o r y potency of several biocides which cause an accumulation of intermediary products of the brached isopentenoid pathway, a problem which needs further i n v e s t i g a t i o n . E f f e c t of i n h i b i t o r s a f f e c t i n g l a t e steps i n phytosterol synthesis on growth of radish seedlings. HMG-CoA reductase and enzymes catalyzing late steps of phytosterol synthesis are membrane-bound. I n c e l l s , membrane-bound enzymes w i t h i n a biosynthetic pathway, suc abundant than solubl regulating t h e i r a c t i v i t y include membrane e f f e c t s such as induced changes i n the l i p i d composition i n the microspheres around various enzyme molecules. The r e s u l t s obtained with mevinolin support the view that HMG-Coa reductase plays a key r o l e a t l e a s t i n the coarse control of phytosterol synthesis. However, t h i s does not exclude the p o s s i b i l i t y that membrane-bound enzymes catalyzing later steps of phytosterol synthesis might be responsible f o r fine-tuning substrate flux. Therefore, s i t e - s p e c i f i c i n h i b i t o r s of such regulating enzymes should e x h i b i t i n s i t u , a c l e a r e f f e c t on growth. The compounds (Figure 5) tested i n the radish system are arranged to indicate some s t r u c t u r a l r e l a t i o n s h i p s . However, t h i s does not indicate per se what enzyme i s the target of these chemicals. I t appears reasonable to c l a s s i f y them according to the enzyme(s) i n h i b i t e d . Most of these compounds, i n p a r t i c u l a r the series of substituted imidazoles Clotrimazole, Miconazole and Bifonazole ( t y p i c a l systemic fungicides) are reported to i n t e r f e r e with the oxidative 14 α-desmethylation by binding nitrogen to the haem i r o n of cytochrome P-450 (112. 113. 114). I n susceptible fungi the accumulation of erogsterol precursors r e t a i n i n g the 14 α-methyl group may have l e d to t h e i r being unsatifactorily packed with the f a t t y a c y l chains of the phospholipids of the fungal membranes (115). This could have l e d to an a l t e r e d membrane f l u i d i t y (116) which then causes a decreased a c t i v i t y of the membrane-bound desaturase, r e s u l t i n g i n an increase of saturated f a t t y acids (115). This group of 14 a-desmethylation i n h i b i t o r s also includes T r i a r i m o l and Triadimefon. The 14 α-desmethylation involves several reactions, including reduction (28). Since the azasterol A 25822 B, a natural a n t i b i o t i c o r i g i n a l l y i s o l a t e d from Geotrichum flavobrunneum and characterized by Chamberlain e t a l . (117), was revealed to a f f e c t -reductase i n yeast thereby causing the accumulation of ignosterol (118), t h i s compound may be included i n the same group. I t has also been shown that i n bramble c e l l s , cultured i n the presence of A 25822 B, A » -sterols accumulated at the expense of A - s t e r o l s (119). Another group (U 18666 A, 8

14

5

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HO" 0

Epiminolanosterol

SC 32561

H

HO 7-Deoxycholate

H5C2N-CH

H C 5

2

-CH

2

-0

U 18666 A

2

2.3- Epiminosqualene OH H5C2'

N -CH -CH -0-^-Ç-CH H^-Ct 2

2

CH

3

2

H5C2'

CH

3

Naftifine Triparanol (MER-29)

Ç)

C.^Q_CH -0-CH-O-C. 2

CI

CH

Miconazole

2

Ο

—

Q-f-O

-N Bilonozole

Clotrimazole OH

Cl-^-O-CH-C-tC^Hg

CI" 1

Ν /) -Ν Triadimefon

Figure 5.

Ο

ad * rN S^ N. a Triarimol

XH3

^ C H KL

CH

3

3

3

"NES"

Structures of s t e r o l synthesis inhibitors used and arranged to indicate some structural relationships.

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Triparanol, epiminolanosterol i s comprised of compounds that a f f e c t the reduction of (120-122) or the Οχ-transfer to the Δ of l a n o s t e r o l and cycloartenol (123). A t h i r d group contains i n h i b i t o r s of oxidosqualene c y c l i z a t i o n (epiminosqualene (124. 125). U 18666 A (126, 127). "NES"). A known antimycotic agent, N a t i f i n e , was found to i n h i b i t fungal squalene epoxidase (128). At t h i s point, however, i t has to be noted that most of the compounds mentioned above are able to i n h i b i t more than one enzyme, including HMG-CoA reductase (113), depending on the concentrations used or organisms tested, i n p a r t i c u l a r i n algae and plant c e l l cultures (129, 130 and l i t e r a t u r e c i t e d t h e r e i n ) . Another drug, SC-32561, has been reported to prevent c h o l e s t e r o l ester accumulation and induction of HMG-CoA reductase i n mammalian t i s s u e cultures and intact animals, presumably through i n d i r e c t feedback regulation (131). Among those compounds being completely i n a c t i v e i n the radish root test was SC-32561 s t e r o l s or t h e i r derivative regulators as they are i n mammalian systems ( f o r a recent review see r e f . (132)), even though conclusive data on t h i s topic are lacking. Iminolanosterol was also completely i n a c t i v e (data not shown), but t h i s i n a b i l i t y to i n h i b i t growth might be due to i t s low s o l u b i l i t y i n water, thereby r e f l e c t i n g the main l i m i t a t i o n of the radish root system which works best i f the compounds are a) r e a d i l y water-soluble or b) can be s o l u b i l i z e d and kept i n s o l u t i o n by the a i d of reasonable concentrations of emulsifying agents. Bifonazole (data not shown) was less active than Miconazole, a very hydrophobic compound, and t h i s was less i n h i b i t o r y than Clotrimazole, which induced a s i m i l a r growth reaction (73) as was found f o r Triadimefon; both caused a dwarfing response i n radish (111). U 18666 A, Triparanol, and "NES" (Figure 6) were comparably e f f e c t i v e growth i n h i b i t o r s with 10 mg per l i t e r being the c r i t i c a l concentration where no further root growth over the i n i t i a l value at day 3 of germination was observed. T r i a r i m o l reached t h i s l e v e l at about 50 mg per l i t e r and was more a c t i v e than N a f t i f i n e (75). Iminosqualene was i n h i b i t o r y at > 5 rag per l i t e r , a concentration that i s at the l i m i t of i t s s o l u b i l i t y . I n a d d i t i o n , the preparation used contained impurities (inner epimino groups) and was probably unstable at room temperature. The "best" agent - even though less e f f e c t i v e than mevinolin - was the n a t u r a l l y produced azasterol (A 25822 B) which reached the forementioned l e v e l between 1 and 5 mg per l i t e r (73.). The experiments give further evidence that inhibition of phytosterol synthesis can cause similar morphological changes i n radish, e. g., a decrease i n root elongation growth. However, one difference between the e f f e c t s of mevinolin as compared to that of a l l other compounds tested has to be noted: The complete lack of l a t e r a l root formation i n the presence of high concentrations of mevinolin was never observed with other chemicals. I t frequently appeared that l a t e r a l root growth was even stimulated as compared to control plants. This may i n d i c a t e that at least one further MVA-derivative, other than s t e r o l s , i s involved not only i n regulation of growth but, more b a s i c a l l y , i n 2 4

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NES control

10ppm 20ppm 50 ppm

3A567 10 days of germination

Figure 6. Growth i n h i b i t i o n of the main root of l i g h t cultivated radish seedlings by imminium salt ("NES"). Mean values + SD from 25 to 30 plants per condition.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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c e l l cycle dynamics. The formation of l a t e r a l roots might require p a r t i c u l a r l y raeristematic i n i t i a l s w i t h i n the root cambium where c e l l s are r a p i d l y d i v i d i n g , and mevinolin, at concentrations higher than 1 mg per l i t e r , presumably i n h i b i t s c e l l d i v i s i o n through a r r e s t i n g the c e l l s w i t h i n the c e l l cycle as i t does i n mammalian c e l l s (133-136). I t should be mentioned that i n crown g a l l tumors caused by ARrobacterium high concentrations of isopentenyladenine are synthesized ( f o r l i t e r a t u r e see r e f . (137) and one gene product of the TI plasmid obviously i s coding f o r a dimethylallylphrophosphate transferase which i s involved i n the formation of isopentenyladenosine from 5*AMP (138, 139). I n t h i s t i s s u e the rate of c e l l d i v i s i o n i s high and requires an overproduction of cytokinins (and auxin (137, 140). I n a d d i t i o n to isopentenyl-derived cytokinins, d o l i c h o l s are involved i n the regulation of the c e l l cycle of eukaryotes as cofactors i n the glycosylation cycle of proteins (141) and i n c e l l wall biosynthesis (142). A a f f e c t t h e i r synthesis regulatory events i n the c e l l cycle which are independent from s t e r o l synthesis. Conclusions and Outlook From our r e s u l t s and those of other groups, there i s strong evidence that the key-regulating r o l e of HMG CoA reductase seems not to be confined only to the animal kingdom, but can also be extended to plants and fungi. Since mammalian c e l l s contain fewer classes of isopentenoid compounds and even fewer d i f f e r e n t c e l l compartments, the regulation of mevalonate and isopentenoid biosynthesis a t the step committed by the HMG-CoA reductase reaction i s f a r more extensively studied and might not be so complex as compared to plant c e l l s . Nevertheless, the models of regulation v a l i d f o r mammalian systems might serve as a basis f o r undrstanding the features of regulation of mevalonate biosynthesis and substrate flow into various classes of isoprenoids p o s s i b l y synthesized w i t h i n d i f f e r e n t organelles of the plant c e l l (Figure 1). The possible interference of mevinolin with the synthesis of recently described brassinolide-type s t e r o i d phytohormones (see l i t e r a t u r e c i t e d above) might open new aspects f o r experimental designs to elucidate s t e r o i d phytohormone-dependent regulation processes i n p l a n t s . Moreover, the i n t e r a c t i o n of mevinolin with HMG-CoA reductase a c t i v i t y and p o s s i b l y with the balance of several growth hormones and how these phytohormones i n t e r a c t with the regulation of isprenoid synthesis at the enzyme l e v e l , may account f o r t y p i c a l growth responses of plants upon biocide treatment and needs further i n v e s t i g a t i o n . The s p e c i f i c i t y of the i n h i b i t i o n by mevinolin through i t s high a f f i n i t y f o r HMG-CoA reductase may also serve as a model to develop h i g h l y e f f e c t i v e and s p e c i f i c a r t i f i c i a l biocides and to stimulate research i n t h i s topic. For example, c i t r i n i n , another a n t i b i o t i c having a b i c y c l i c structure produced by Pénicillium citrinum has been shown to i n h i b i t s t e r o l biosynthesis (143) at the s i t e of acetoacetyl-CoA t h i o l a s e (EC 2.3.2.9) and HMG-CoA

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reductase (144), but rather high concentrations were required. This compound was reported to i n h i b i t plant growth (145) which we suppose i s also a result of interference with HMG-CoA reductase activity. This i s further evidence, besides our studies using mevinolin, to propose HMG-CoA reductase to be a good enzymic target for b i o l o g i c a l l y active biocides. In any case, i n our attempt to shed new l i g h t on the physiological r o l e of mevalonate biosynthesis and metabolism in plants, mevinolin has proved to be a very suitable t o o l and can help to further elucidate the role of a functional isopentenoid and p r e n y l l i p i d pathway in the regulation of plant growth and development.

Acknowledgments Part of this work was Forschungsgemeinschaft. We are assistance i n the preparatio

supported by the Deutsche indebted to Mrs. Ute Sieber for

Legend of symbols GA, G i b b e r e l l i c a c i d ; HMG-COA, 3-hydroxy-3-methylglutaryl-coenzyme MVA, mevalonic acid

A;

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92. Schultz, B . ; Ellerbrock, Β. H . ; Soli, J. Eur. J. Biochem. 1981, 117, 329-32. 93. Kaiping, S.; Soil, J.; Schultz, G. Phytochemistry 1984, 23, 89-91. 94. Soil, J.; Schultz, G . ; Joyard, J.; Douce, R.; Block, M. A. Arch. Biochem. Biophys. 1985, 238, 290-99. 95. Nes, W. D.; Douglas, T. J.; L i n , J.-T.; Heftman, E . ; Paleg, L. G. Phytochemistry 1982, 21, 575-79. 96. Bach, T. J.; Lichtenthaler, H. K. Biochim. Biophys. Acta. 1984, 794, 152-61. 97. Lichtenthaler, H. K.; Kuhn, G . ; Prenzel, U . ; Buschmann, C . ; Meier, D. Z. Naturforsch 1982, 37c, 464-75. 98. Anderson, J. M.; Andersson, B. Trends Biochem. Sci. 1982, 7, 288-92. 99. Lichtenthaler, H. K.; Prenzel, U . ; Kuhn, G. Z. Naturforsch. 1982, 37c, 10-12. 100. D ö l l , M.; Schindler S.; Lichtenthaler H K.; Bach T J. In "Structure, Functio Siegenthaler, P.-Α. Amsterdam, 1984. 101. Ryder, N. S.; Goad, L. J. Biochim. Biophys. Acta. 1980, 619, 424-27. 102. Hashizume, T . ; Matsubara, S.; Endo, A. Agric. Biol. Chem. 1983, 47, 1401-3. 103. Ceccarelli, N . ; Lorenzi, R. Plant Sci. Lett. 1984, 34, 269-76. 104. Weiss, C . ; Vaadia, Y. Life Sci. 1965, 4, 1323-26. 105. Kende, H. Proc. Natl. Acad. Sci. USA 1965, 53, 1302-7. 106. Carr, D. J.; Burrous, W. J. Life Sci. 1966, 5, 2061-77. 107. Heupel, R. C . ; Sauvaire, Y . ; Le, P. H . ; Parish, E. J.; Nes, W. D. Lipids. 1985, 21, 69-75. 108. Fabijan, D. M.; Dhinda, P. O.; Reid, D. M. Planta 1981, 152, 481-6. 109. Douglas, T. J.; Paleg, L. G. J. Expt. Bot. 1981, 32, 59-68. 110. Buchenauer, H . ; Röhner, E. Pesticide Biochem. Physiol. 1981, 15, 58-70. 111. Lichtenthaler, Η. Κ. Z. Naturforsch. 1979, 34c, 936-40. 112. Baldwin, B. C. Biochem. Soc. Transact. 1983, 11, 659-63. 113. Berg, D.; Regel, H. E . ; Harenberg, H. E . ; Plempel, M. Arzneim Forsch./Drug Res. 1984, 34, 139-46. 114. Henry, M. J.; Sisler, H. D. Pesticide Biochem. Physiol. 1984, 22, 262-75. 115. Bloch, K. CRC Crit. Rev. Biochem. 1979, 9, 1-5. 116. van den Bossche, H . ; Wilemsens, G . ; Cools, W.; Marichal, P.; Lauwers, W. Biochem. Soc Transact 1983, 11, 665-7. 117. Chamberlain, J. W.; Chaney, M. O.; Chen, S.; Demarco, P. V . ; Jones, N. D.; Occolowitz, J. L. J. Antibiotics 1974, 27, 992-3. 118. Parks, L. W.; Rodriguez, R. J. Biochem. Soc. Transact. 1980, 19, 525-30. 119. Schmitt, P.; Scheid, F . ; Benvenieste, P. Phytochemistry 1980, 19, 525-30.

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8. BACH AND LICHTENTHALER

Plant Growth Regulation

139

120. Avigan, J. Proc. Soc. Exp. Biol. Med. 1963, 112, 233. 121. Volpe, J. J.; Obert, K. A. J. Neurochem. 1983, 38, 931-8. 122. Avigan, J.; Steinberg, D.; Vroman, H. E . ; Thompson, M. J.; Mosettig, E. J. Biol. Chem. 1969, 235, 3123-9. 123. Malhotra, H. C.; Nes, W. R. J. Biol. Chem. 1971, 246, 4934-7. 124. Corey, E. J.; Ortiz de Montellano, P. R.; Lin, K.; Dean, P. D. G. J. Amer. Chem. Soc. 1967, 89, 2797-8. 125. Nes, W. D. In "Isopentenoids in Plants, Biochemistry and Function"; Nes, W. D.; Fuller, G.; Tsai, L.-S., Eds.; Marcel Dekker, Inc.: New York - Basel, 1984, pp. 325-47. 126. Cenedella, R. J. Biochem. Pharmacol. 1980, 29, 2751-54. 127. Sexton, R. C.; Panini, S. R.; Azran, F . ; Rudney, H. Biochemistry USA, 1983, 22, 5687-92. 128. Paltauf, F . ; Daum, G.; Zuder, G.; Högenauer, G.; Schulz, G. Seidel, G. Biochim. Biophys. Acta 1982, 712, 268-73. 129. Chan, J. T.; Patterson, G. W. Plant Physiol. 1973, 52, 246-7. 130. Hosokawa, G.; Patterson 19, 449-56. 131. Bates, S. R.; Jett, C. M.; Miller, J. E. Biochim. Biophys. Acta 1983, 281-93. 132. Gibbons, G. G. Biochem. Soc. Transact. 1983, 11, 649-51. 133. Brown, M. S.; Goldstein, J. L. J. Lipid Res. 1980, 21, 505-17. 134. Quesney-Huneeus, V.; Wiley, M. H.; Siperstein, M. D. Proc. Natl. Acad. Sci. USA 1979, 76, 5056-60. 135. Quesney-Huneeus, V.; Wiley, M. H.; Siperstein, M. D. Proc. Natl. Acad. Sci. USA 1980, 77, 5842-46. 136. Faust, J. R.; Brown, M. S.; Goldstein, J. L. J. Biol. Chem. 1980, 255, 6546-48. 137. Amrhein, N. Progress in Botany 1983, 45, 136-165. 138. Barry, G. F.; Rogers, S. G.; Fraley, R. T; Brand, L. Proc. Natl. Acad. Sci. USA 1984, 81, 4776-80 139. Thomashow, L. S.; Reeves, S.; Thomashow, M. F. Proc. Natl. Acad. Sci. USA 1984, 81, 5071-75. 140. Akiyoshi, D. E . ; Klee, H.; Amasino, R. M.; Nester, E. W.; Gordon, M. P. Proc. Natl. Acad. Sci. 1984, 81, 5994-98. 141. Lehle, L . ; Tanner, W. Biochem. Soc. Trans. 1983, 11, 568-74. 142. Hemming, F. W. Biochem. Soc. Transact. 1983, 11, 497-504. 143. Kuroda, M.; Hazama-Shimada, Y.; Endo, E. Biochim. Biophys. Acta 1977, 486, 254-9. 144. Tanzawa, K.; Kuroda, M.; Endo, A. Biochim. Biophys. Acta 1977, 488, 97-101. 145. Skorobogäotova, R. Α.; Mirchink, T. G. Sel-skokhozyaistven­ naya Biologiyia 1976, 9, 865, cited in: Bauer, K.; Bischoff, E.; von Hugo, H.; Berg, D.; Kraus, P. "Pflanzenschutzpräparate raikrobieller Herkunft"; CHEMIE DERPFLANZENSCHUTZ-UND SCHÄDLINGSBEKÄMPFUNGSMITTEL; Wegler, R., Ed.; Springer-Verlag: Berlin, 1980; Vol. 6, pp. 215-328. RECEIVED May 29, 1986

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 9

Synthesis and Fungistatic Activity of Podocarpic Acid Derivatives 1

1

1

2

Edward J . Parish , Susan Bradford , Victoria J. Geisler , Patrick K. Hanners , Rick C. Heupel , Phu H. Le , and W. David Nes 2

2

2

1

Department of Chemistry, Auburn University, Auburn, A L 36849 Plant and Fungal Lipid Group, Plant Development and Productivity Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, CA 94710 2

As a class, octahydrophenanthrene lactones, podolactones, and related podocarpic acid derivatives have been reported to possess a wide variety of biological activities, including antileukemic activity, inhibition of plant cell growth, insect toxicity and antifungal properties. In the present study, a series of synthetic derivatives of podocarpic acid have been prepared by chemical synthesis and evaluated with respect to their ability to inhibit fungal growth. These compounds were evaluated against the Oomycetes- Phytophthora cactorum , Saprolesnia ferax, and Achlya bisexualis and the Ascomycetes-Gibberella fujikuroi. The results of these studies indicate that several of these new synthetic derivatives possess significant antifungal properties.

Podocarpic acid ( I ) was f i r s t isolated from the r e s i n of Podocarpus cupressins, an important timber tree which i s endemic to Java, and l a t e r from Podocarpus dacrydioides ("Kahikatea") and Dacrydium cupressinum ("Rimu"), trees which are found i n the timber regions of New Zealand (1). Since 1968, more than f o r t y oxygenated metabolites of podocarpic acid have been i s o l a t e d from various species of Podocarpus (2,3). Interest i n these n a t u r a l l y occurring and synthetic lactones, podolactones, and related podocarpic acid derivatives has been mainly due to the novel structures of these compounds and the various types of b i o l o g i c a l a c t i v i t y possessed by them. Octahydrophenanthrene lactones ( I I ) and related podocarpic acid derivatives ( I I I ) have been reported to possess hormonal and anti-inflammatory properties (4). Other s i m i l a r podolactones have been shown to i n h i b i t the expansion and d i v i s i o n of plant c e l l s (IV) (5-10), to have antileukemic a c t i v i t y (V) (11), to have a n t i b a c t e r i a l a c t i v i t y (12), to have insect t o x i c i t y properties (13-15), and to e x h i b i t antitumor a c t i v i t y (16-19). 0097-6156/87/0325-0140$06.00/0 © 1987 American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

9. PARISH ET AL.

Podocarpic Acid Derivatives

141

OH

/

15

IS

C0 H

C0„R 23 I I I a, R =R=CH , R =H b, R7=R2=CH^, R =Br c, R^=CH ,R =R =H

o

o

2

2

R=CH2CH

2

3

CH R 2

2

'"OCH

n

OH

IV

a, R =H, R =OH b, R7=CH , R =OH c, R^=H, R =SOCH

VI

HO VII

VIII

IX

a, R ^ P r , R =R =H 2

3

1

b,

R = Pr, R =R =0

c,

R - ^ P r , R =OH, R =H

d,

R =OH, R =R =H

1

2

3

2

1

2

Figure 1. Structures I-IX.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

3

3

142

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Other reports have indicated that these types of compounds, as a c l a s s , possess s i g n i f i c a n t antifungal properties. The lactone (VI), f i r s t i s o l a t e d as a mold metabolite, was found to have s i g n i f i c a n t a c t i v i t y against a number of fungi (20). The momilactones A (VII) and Β (VIII) have been shown to be fungitoxic towards C. cucumerinum (21,22). In a recent report several oxidized r e s i n acid d e r i v a t i v e s of dehydroabietic acid (IX a-c) and 13-hydroxypodocarpic acid (IX d) were found to be highly f u n g i s t a t i c against P. p i n i . a c o n i f e r pathogenic fungi (23). I t was observed that mature trees were more r e s i s t a n t to fungal i n f e c t i o n and contained a greater quantity of oxidized r e s i n a c i d derivatives i n t h e i r r e s i n suggesting greater resistance. In view of t h e i r documented b i o l o g i c a l properties, i t appeared worthwhile to evaluate a series of synthetic intermediates derived from podocarpic a c i d f o r f u n g i s t a t i c a c t i v i t y against other plant pathogens. This report describes the preparation of these derivatives and the r e s u l t compound with cultures o ascomycetous fungi. Chemical Synthesis of Podocarpic Acid Derivatives Commercial podocarpic acid i s derived from natural sources. Several recent studies have been directed towards the t o t a l synthesis of t h i s r e s i n acid to assure adequate future supplies of t h i s material f o r use i n a g r i c u l t u r e and medicine (24,25). The goal of the present study was to prepare a s e r i e s of derivatives r e l a t e d to podocarpic acid f o r use i n s t r u c t u r e / a c t i v i t y studies designed to reveal functional groups responsible f o r the molecules f u n g i s t a t i c properties. Four s p e c i f i c modifications were planned: 1. 2. 3. 4.

S u b s t i t u t i o n of electron-withdrawing groups onto C (13) of the aromatic C r i n g (Scheme 1). V a r i a t i o n of the halogen at C (6) (Scheme 2). Formation of the lactones from each 6 α - bromo methyl ester d e r i v a t i v e (Scheme 3). S u b s t i t u t i o n of an acetate group f o r the methyl ester group at C (16) (Scheme 4).

The f i r s t modification, s u b s t i t u t i o n of the electron-with­ drawing halogen and n i t r o groups onto C (11) and/or C (13) of the aromatic r i n g , was based upon the well-known observation that the a n t i s e p t i c properties of phenols are enhanced by the introduction of these groups onto the phenolic r i n g (26). N i t r a t i o n was accomplished by reacting podocarpic acid ( I , Scheme 1) with n i t r i c acid i n acetic acid (27-30). The number of n i t r o groups introduced onto the aromatic r i n g was c o n t r o l l e d by the amount of n i t r i c acid used i n the reaction (one or two equivalents). The 13-nitro d e r i v a t i v e X was methylated with dimethyl s u l f a t e under basic condition to y i e l d XIV. A s i m i l a r methylation of I has been shown to produce methyl o-methyl podocarpate (XII) (4,16). Bromine was introduced at C (13) by the e l e c t r o p h i l i c s u b s t i t u t i o n of bromine into the aromatic ring of XII using bromine i n a c e t i c a c i d . The fact that t h i s reaction gives

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

9. PARISH ET AL.

Podocarpic Acid Derivatives

143

only the monosubstituted 13-bromo derivative i s probably due to s t e r i c hindrance r e s u l t i n g from the angular methyl group i n the a x i a l o r i e n t a t i o n at p o s i t i o n 10 and the large s i z e of the bromine atom which would prevent s u b s t i t u t i o n at p o s i t i o n 11, the other ortho p o s i t i o n on the r i n g . In Scheme 2, benzylic oxidation of X I I , X I I I , and XIV using chromium t r i o x i d e produced the corresponding ketone derivatives I t has also recently been shown that X I I may be oxidized to the ketone XV under conditions of ozonolysis (31). Ketones XV and XVI were brominated using an adapted procedure derived from the work of B i b l e and Grove (4,16) to y i e l d the mono-and di-bromoketones XVIII and XIX. In order to e f f e c t the bromination of XVII, an alternate method was u t i l i z e d which gave ample quantities of bromoketone XX (32). The corresponding chloride derivatives (XXI and XXII) of XV and XVI were prepared by reaction with copper chloride and l i t h i u m chloride i n N,N-dimethylformamide (33). The assignments of the α - configuratio were v e r i f i e d using know Si spectra which are correlated to the x-ray structure determination of XVIII (16,34-37)· In Scheme 3, the α-bromoketones XVIII and XIX were converted to lactones XXIII and XXIV by refluxing i n c o l l i d i n e (4,16,38). By-products of t h i s reaction include the a, B-unsaturated ketone XXV which r e s u l t s from the dehydrobromination (38,39) of XVIII. Ketone XXVI r e s u l t s from a one-step dehydrobromination-decarbomethoxylation (40-43) of XIX. The acetate series of compounds (Scheme 4) was synthesized by hydride reduction of methyl 0-methyl podocarpate (XII) followed by a c e t y l a t i o n of the r e s u l t i n g alcohol XXVII (44). The acetate XXVIII was then oxidized at the benzylic p o s i t i o n to ketone XXIX. In contrast to the methyl ester d e r i v a t i v e s , halogenation at p o s i t i o n 6 (using methods described previously) of the corresponding keto acetates resulted i n two epimers, the 6 a- and 6 B- halogenated compounds, as w e l l as the dehydrohalogenation product XXXIV. The assignments of the a- and B-conf iguration to the halogen atoms at C (6) were determined from the Si NMR coupling constants at C (5) and C (16) as described previously. B i o l o g i c a l Evaluation of P o t e n t i a l Fungistatic Agents Podocarpic acid (I) and a number of i t s chemical derivatives (X-XXXIV) were evaluated f o r t h e i r p o t e n t i a l f u n g i s t a t i c a c t i v i t y as measured by t h e i r e f f e c t s on the growth of the fungi on s o l i d media (Table 1). A l l compounds evaluated were of 98% or greater p u r i t y ( t i c and g l c a n a l y s i s ) . Each structure was consistent with i t s ι 13 spectral analysis ( H NMR, C NMR, i r , and ms). Each compound was evaluated against Phytophthora cactorum (both with (B) and without (A) added c h o l e s t e r o l ) , Gibberella f u i i k u r o i (C), Saprolegnia ferax (D), and Achlya b i s e x u a l i s (both male (E) and female (F) s t r a i n s ) . The fungi were cultured as described i n references 45-47. P. cactorum. u n l i k e the other fungi, f a i l s to synthesize s t e r o l s (48) and requires s t e r o l to complete the reproductive phase of i t s l i f e cycle (49). Compounds (10 ug/ml) evaluated were dissolved i n a minimal amount of ethanol and introduced a s e p t i c a l l y into the s t e r i l i z e d X

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

144

ECOLOGY AND METABOLISM OF PLANT LIPIDS Scheme 1 OH

XXI

R =H, R =C1

XXII

R-j=Br, R =C1

2

OH

XX

2

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

9. PARISH ET AL.

Podocarpic Acid Derivatives

Scheme 3

CH 0Ac o

XXXIV

XXX

R =H, R =Br

XXXI

R =H, R =C1

XXXII

R =Br, R =H

XXXIII

R ^ C l , R =H

1

1

1

2

XXIX

2

2

2

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

Table I .

Comparison of the f u n g i s t a t i c properties of podocarpic acid and i t s d e r i v a t i v e s

1

Fungal Species Compound

A

Β

C

D

Ε

F

I

66

82

94

62

68

74

X

75

97

94

96

96

76

XI

47

50

68

27

0

0

XIII

84

110

97

93

100

100

XIV

97

XV

97

XVI

92

104

90

96

88

88

XVII

91

101

100

93

99

—

XVIII

68

83

100

78

78

76

XIX

120

104

94

93

96

88

XX

58

86

102

100

78

80

XXI

72

86

96

91

88

76

93

91

96

88

XXII

107

110

XXIII

94

104

99

73

81

84

XXIV

68

85

94

100

54

74

XXV

115

98

96

8

84

75

XXVI

77

88

90

100

45

56

87

51

49

60

XXVII

125

84

XXVIII

122

101

93

84

71

87

XXIX

90

99

95

62

65

69

XXX

67

73

100

67

61

71

XXXI

143

99

98

91

74

77

XXXII

121

93

97

18

75

78

The values are expressed as a percentage of control's r a d i a l diameter obtained 3 to 12 days (depending on species) following inoculation with a 5mm plug.

A - P. cactorum

without added

c h o l e s t e r o l , Β - P. cactorum with added cholesterol, C Gibberella f u j i k u r o i . D - Saprolegnia ferax, Ε - Achlya b i s e x u a l i s , male s t r a i n , F - Achlya b i s e x u a l i s , female s t r a i n .

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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PARISH ET AL.

Podocarpic Acid Derivatives

147

agar-supplemented culture medium. The i n h i b i t i o n values obtained are expressed as percent of control by measurement of the r a d i a l diameter of fungal growth. Values greater than 100% represent an enhancement or stimulation of growth. Many of these compounds demonstrated varying degrees of s i g n i f i c a n t f u n g i s t a t i c (defined as i n h i b i t i o n of growth on s o l i d media) a c t i v i t y against one or more of the species in the study (Table I). In p a r t i c u l a r , d i n i t r o derivative XI demonstrated potent a c t i v i t y i n a l l assays. This may be due, i n part, to i t s resemblance to p i c r i c acid ( 2 , 4 , 6 - t r i n i t r o p h e n o l ) , a substance which i s known to complex with, and cause the i r r e v e r s i b l e precipation of protein (50,51). The lack of fungistatic properties of a s p e c i f i c compound to some but not a l l fungi tested may be due to the lack of mycelial uptake, a p o s s i b i l i t y which i s currently under study. Interestingly, cholesterol supplemented to P. cactorum was protective to the fungistatic properties resulting from the i n h i b i t i o n induced by som increase, however, i n r a d i a does not necessarily imply a b e n e f i c i a l e f f e c t , since these mycelia appeared abnormal (cf. 49). In conclusion, these studies have indicated that chemical modification of the basic podocarpic acid structure can produce new compounds with antifungal a c t i v i t y . The results derived from t h i s study represent preliminary findings which are subject to further investigation. We anticipate that more detailed studies w i l l reveal information concerning the mechanism and mode of action of many of these compounds. In the meantime, we continue to develop new antifungal agents using selected natural products as model compounds.

Literature Cited 1. Sherwood, I.R.; Sholt, W.F. J. Chem. Soc. 1938, 1006-13, and references therein. 2. Hayashi, Y.; Matsumoto, T. J . Org. Chem. 1982, 47, 3421 29. 3. Cassady, J.M.; Lightner, T.K.; McCloud, T.G.; Hembree, J.R.; Blym , S.R; Chang, C. J. Org. Chem. 1984, 49, 942-45. 4. Bible, R.H.; Grove, M. U.S. Patent 2 753 357, 1956; Chem. Abstr. 1957, 51, 2869. 5. Galbraith, M.N.; Horn, D.H.S.; Sasse, J.M.; Adamson, D. Chem. Commun. 1970, 170-71. 6. Hayashi, Y.; Yokoi, J; Watanabe, Y.; Sakan, T . , ; Masuda, Y.; Yamamoto, R. Chem. Lett. 1972, 759-62. 7. Hayashi, Y.; Sakan, T. Proc. 8th Int. Conf. Plant Growth Substances 1974, p. 525. 8. Sasse, J.M.; Galbraith, M.M., Horn, D.H.S.; Adamson, D.A. In "Plant Growth Substances, 1970". 9. Call, D.J., Ed.; Springer Verlag, Berlin, 1972; p. 430. 10. Galbraith, M.N.; Horn, D.H.S.; Sasse, J.M. Chem. Commun. 1971, 1362-63. 11. Bryan, R.F.; Smith, P.M. J. Chem. Soc. Perkin Trans. II 1975. 148-52.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

Sacki, I.; Sumimoto, M.; Kondo, T. Holzforschung 1970. 24, 83-87. Russell, G.B.; Fenemore, P.G.; Singh, P. Chem. Commun. 1973, 166-67. Singh, P.; Fenemore, P. G.; Russell, G.B. Aust. J. Biol. Sci. 1973, 26, 911-15. Singh, P. Russell G.B.; Hayashi, Y.; Gallagher, R.T.; Fredricksen, S. Entomol. Exp. Appl. 1979. 25, 121-125. Parish, E . J . ; Miles, P.H. J. Pharm. Sci. 1984, 73,694-98. Hayashi, Y.; Matsumoto, T.; Tashiro, T. Gann. 1979. 70, 369-72. Hayashi, Y.; Matsumoto, T.; Sakurai, Y.; Tashiro, T. Gann. 1975, 66, 587-90. Kupchan, S.M.; Baxter, R.L.; Ziegler, M.R.; Smith, P.M.; Bryan, R.F. Experientia 1975, 31,137-41. Ellestad, G.A.; Evans, R.H., Jr.; Kunstmann, M.P. J. Am. Chem. Soc. 1969 Cartwright, D.W. 1981, 78, 323-26. Fuchs, Α.; Davicse, L.C.; de Waard, M.A.; de Wit, P.G.M. Pestic. Sci. 1983, 14, 272-96, and references therein. Franich, R.A.; Gadgil, P.D. Physiol. Plant Path. 1983, 23, 183-95. Snider, B.B.; Mohan, R.J. Kates, S.A. J. Org. Chem. 1985, 50, 3661-63. Welch, S.C.; Hayah, C.P.; Kim, J.H., Chu, P.S. J. Org. Chem. 1977, 42, 2879-82. Gisvold, O. In "Textbook of Organic Medicinal and Pharmaceutical Chemistry"; Wilson, C.O.; Gisvold, O.; Doerge, R.F.; Eds.; J.B. Lippincott; New York, 1971; p. 255. Niedeli, J.B.; Vogel, H.J. J. Am. Chem. Soc. 1949, 71, 2566-69. Werbin, J.; Holoway, C. J. Biol. Chem. 1956, 223, 651-56. Utne, T.; Jobson, R.B.; Babson, R.D. J. Org. Chem. 1968, 33, 2469-72. Santaniello, R.; Ravasi, M.; Ferraboschi, P. J. Org. Chem. 1983, 48, 739-41. Parish, E . J . ; McKeen, G.G.; Miles, D.H. Org. Prep. Proc. Intern. 1985, 17, 143-46. King, L.C.; Ostrum, G.K. J. Org. Chem. 1964, 29, 3459-61. Kosowel, E.M.; Cole, W.J.; Wu, G.-S.; Cardy, D.E.; Meisters, G., J. Org. Chem. 1963, 28, 630-34. Lickei, A.E.; Rieke, A.C.; Wheeler, D.M.S. J. Org. Chem. 1967, 32, 1647-52. Cutfield, J.F.; Waters, T.N.; Clark, G.R. J. Chem. Soc., Perkin Trans.II 1974, 150-55. Cambie, R.C.; Clark, G.R.; Crump, D.R.; Waters, T.N. Chem. Comm. 1968, 183-85. Clark, G.R.; Waters, T.N. J. Chem. Soc., C 1970, 887-90 Wenkert, E.; Beak, P.; Carney, R.W.J.; Chamberlain, J.W.; Johnston, D.B.R.; Roth, C.D., Taharce, A. Can. J. Chem. 1963, 41, 1924-40. Wenkert, E . ; Fuchs, Α.; McChesney, J.M. J. Org. Chem. 1965, 30, 2931-34.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

9.

PARISH ET AL.

40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51.

Podocarpic Acid Derivatives

149

Miles, D.H.; Parish, E.J. Tetrahedran Lett. 1972, 3987-90. Parish, E.J.; Miles, D.H. J. Org. Chem. 1973, 38, 1223-1225. Parish, E.J.; Mody, N.V., Hedin, P.Α.; Miles, D.H. J. Org. Chem. 1974, 39, 1592-93. Parish, E. J.; Haung, B.-S.; Miles, D.H. Synth. Commun. 1975, 5, 341-45. Parish, E.J. Ph.D. Thesis, Mississippi State University, Mississippi, 1984. Nes, W.D.; Heupel, R.C. Arch. Biochem. Biophys. 1985 in press. Nes, W.D.; Le, P.H.; Berg, L.; Patterson, G.W., Kerwin, J. Experientia 1985, in press. Berg, L.; Ph.D. Thesis Univ. of Md. 1983. Nes, W.D.; Stafford, A.E. Proc. Natl. Acad. Sci. 1983, 80, 3227-31 Nes, W.D.; Stafford A.E. Lipids 1984. 19, 544-49. Haurowitz, F. "Chemistr Press: New York, 1950; p.11. White, Α.; Handler, P.; Smith, E.L.; Hill, R.L.; Lehmann, I.R. "Principles of Biochemistry"; McGraw-Hill: New York, 1978; 6th Ed., p. 107.

RECEIVED May 1,1986

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 10

The Role of Cutin, the Plant Cuticular Hydroxy Fatty Acid Polymer, in the Fungal Interaction with Plants P. E . Kolattukudy, Mark S. Crawford, Charles P. Woloshuk, William F. Ettinger, and Charles L . Soliday Institute of Biological Chemistry and Biochemistry/Biophysics Program, Washington State University, Pullman, WA 99164

Cutin, the structural component of plant cuticle, is a polyester composed of ω-hydroxy-C16 and C 1 8 fatty acids, dihydroxy-C acid, 18-hydroxy-9,10epoxy-C18 acid This insoluble polymer constitutes a major physical barrier to the penetration of pathogenic fungi into plants. Pathogenic fungi produce an extracellular cutinase when grown on cutin as the sole source of carbon. Cutinase has been isolated and characterized from many pathogenic fungi. This enzyme is a "serine hydrolase" containing the characteristic catalytic triad. With the use of ferritin-labeled antibodies and electron microscopy it was shown that cutinase is produced by the penetrating fungus during the actual infection of its host. Inhibition of this enzyme prevents fungal penetration into the plant and thus prevents fungal infection. Cutinase cDNA has been cloned from Fusarium f. sp. solani pisi and Colletotrichum capsici. The complete primary structure of the Fusarium enzyme and a partial structure of the Colletotrichum enzyme have been determined. These two enzymes show conservation of certain structural features. Cutinase mRNA synthesis is rapidly induced in fungal spores by the unique monomers of cutin initially generated by the small amount of the enzyme carried by the spores. The induction of cutinase is followed by the induction of polygalacturonase which is presumably used to degrade the pectin barrier which lies under the cuticle. Plant c u t i c l e , the boundary layer at which microbial pathogens come into contact with the plant, i s derived e n t i r e l y from l i p i d s . Obviously, the c u t i c l e can play an important r o l e i n the i n t e r a c t i o n between the microbe and the host plant. The c u t i c l e i s composed of an insoluble s t r u c t u r a l polymer c a l l e d 0097-6156/87/0325-0152$06.75/0 © 1987 American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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c u t i n which i s derived from i n t e r e s t e r i f i e d hydroxy and hydroxy epoxy f a t t y acids. This polymer i s embedded i n a complex mixture of soluble l i p i d s c o l l e c t i v e l y c a l l e d wax which i s also secreted onto the plant surface. The soluble components of the c u t i c l e may play a chemical role i n influencing the i n t e r a c t i o n between microbes and plants whereas c u t i n would play a physical r o l e i n that the organism must penetrate t h i s b a r r i e r before i t can enter the plant. Although there are many reports that various wax components may have i n h i b i t o r y e f f e c t s on microbial growth, the s p e c i f i c roles of such components have not been elucidated. I t i s possible, i f not l i k e l y , that some of the wax components play s p e c i f i c r o l e s i n the i n t e r a c t i o n between pathogens and plants. On the other hand, the r o l e of c u t i n as the f i r s t b a r r i e r which the pathogen must penetrate to i n f e c t the plant i s rather obvious and i n recent years the mechanism by which fungal pathogens penetrate t h i s b a r r i e r has been elucidated. In t h i s pape t h i s area and discus fungal spore senses that i t i s r e s t i n g on the plant surface so that i t can induce the appropriate enzymes required to disrupt the c u t i c u l a r and underlying b a r r i e r layers. Structura of Cutin Cutin i s a polyester composed of lo-hydroxy-Cig a c i d , 9 or 10,16-dihydroxy-Ci6 a c i d , 18-hydroxy-Ci a c i d , 18-hydroxy-9,10epoxy-C acid and 9,10,18-trihydroxy-C acid (1-3). The C acids containing an a d d i t i o n a l double bond at C-12 are also usually present whereas components containing a d d i t i o n a l double bonds at C-12 and C-15 are less common. The double bond at C-12 i s also epoxidized and subsequently hydrated generating 9,10,12,13,18-pentahydroxy-Ci acids i n some plants. In some cases further oxidation products of the above monomers are also found. For example, 16-oxo-9- or lO-hydroxy-C^g acid was found as a major component of V i c i a faba embryo c u t i n (4), and 9,16-dihydroxy-10-oxo-Ci acid was found i n c i t r u s c u t i n C 5 ) . Besides these common major hydroxy and epoxy acids, a v a r i e t y of other f a t t y acids have been found as minor components i n many cutins as indicated i n other reviews (1-3). The hydroxy acids are i n t e r e s t e r i f i e d to generate the insoluble polyester. The precise d e t a i l s of how the monomers are linked are not understood. Indirect chemical studies have have been conducted to determine the nature of the linkages involved and the extent of involvement of the secondary hydroxyl groups i n ester linkages. Mesylation of the free hydroxyl groups of the polymer followed by depolymerization by L1AID4 resulted i n the s u b s t i t u t i o n of a D atom for each free hydroxyl group i n the polymer whereas the hydroxyl groups involved i n ester linkages retained the hydroxyl groups during depolymerization. Thus GC/MS analysis of the depolymerization products revealed the degree of involvement of the alcohol groups i n ester linkages (6). A s i m i l a r approach was recently used on c u t i n from Quercus suber (7). Another approach to determine the amount and nature of free hydroxyl groups present i n the polymer was oxidation of the free hydroxyl groups i n the 8

18

18

8

8

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

1 8

154

ECOLOGY AND METABOLISM OF PLANT LIPIDS polymer followed by depolymerization by hydrolysis or t r a n s e s t e r i f i c a t i o n and analysis of the products by GC/MS (8). Selective reduction of the ester bonds by LiBH^ was used to determine the amount of free carboxyl groups present i n the polymer. The l i m i t e d amount of such i n d i r e c t chemical studies conducted on c u t i n showed that about one-half of the mid-chain hydroxy groups and most of the primary hydroxyl groups and carboxyl groups are involved i n ester linkages, suggesting the presence of branches and/or c r o s s - l i n k s i n the polymer. Most plants contain a mixture of the 16- and 18-carbon family of c u t i n monomers. Even though there appears to be a degree of species s p e c i f i c i t y i n the c u t i n composition, whether the polymer structure can vary s u f f i c i e n t l y to a f f e c t major functional properties of the polymer i s not known. The polyester also contains covalently attached minor components such as phenolic acids and flavanoids (9,10) which, i f released by the attacking pathogen i n t e r a c t i o n between th areas have not been explored adequately to draw firm conclusions. I s o l a t i o n and Characterization of

Cutinase

How pathogenic fungi penetrate the c u t i c u l a r b a r r i e r has been debated for the better part of a century (11,12). The penetration process was considered by some to be mediated e n t i r e l y by the p h y s i c a l force of growth of the germinating spore whereas others argued that the c u t i c u l a r b a r r i e r i s breached by an e x t r a c e l l u l a r enzyme that catalyzes degradation of c u t i n . Electron microscopic examination of the penetration areas suggested that at least i n some cases the penetration involved enzymatic degradation of the c u t i c l e (13-15). Such u l t r a s t r u c t u r a l evidence constituted apparent d i s s o l u t i o n of c u t i c l e with no obvious signs of physical force such as depression of the b a r r i e r i n the penetration area. However, such an approach could not provide d i r e c t proof for the involvement of a c u t i n degrading enzyme and the controversy continued. To determine whether fungal pathogens can generate enzymes capable of degrading the plant c u t i n , Fusarium s o l a n i p i s i , a pathogen of Pisum sativum was provided apple c u t i n as the sole source of carbon. Since the fungus grew on t h i s carbon source, i t became obvious that t h i s fungus had the a b i l i t y to produce e x t r a c e l l u l a r cutinase. This e x t r a c e l l u l a r enzyme was p u r i f i e d from the e x t r a c e l l u l a r f l u i d of cutin-grown cultures of Fusarium s o l a n i p i s i . Since then a v a r i e t y of fungal pathogens have been grown on c u t i n as the sole source of carbon and cutinases have been p u r i f i e d from many of the pathogens (3) · The procedure that y i e l d s pure cutinase from most fungi i s summarized i n Table I. The fungal pathogens so far examined for t h e i r a b i l i t y to produce cutinase are l i s t e d i n Table I I . The properties of cutinase obtained from the various fungal sources appear to be quite s i m i l a r . The molecular weight i s near 25,000 and the amino acid composition appears to be quite s i m i l a r (3,16). Some of the major features shared by the

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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TABLE I P u r i f i c a t i o n of Cutinase from F. s o l a n i p i s i Step

T o t a l Units

% Yield

Culture F i l t r a t e (25%) +

200,00

T r i t o n X-100 (0.1%) Wash (75%) Acetone (75%) P r e c i p i t a t e

160,000

80

QAE-Sephadex

130,000

56

Octyl-Sepharose

110,000

55

Isozyme C (95 mg)

72,000

36

Isozyme D (45 mg)

24,000

12

SP-Sephadex

a

w. R o l l e r , C.L. Soliday and P.E. Kolattukudy, unpublished r e s u l t s .

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

TABLE I I Host-Pathogen Interactions i n Which Cutinase Has Been Detected

Pathogen

Host

Remarks

Fusarium s o l a n i p i s i

Pea

Cutinase p u r i f i e d ; r o l e

Fusarium roseum sambucinum

Squash

Cutinase p u r i f i e d

Fusarium roseum culmorum

Wheat

Cutinase p u r i f i e d

Ulocladium consotiale

Cucumber

Cutinase p u r i f i e d

Helminthosporium sativum

Barley

Cutinase p u r i f i e d

Streptomyces scabies

Potato

Cutinase p u r i f i e d

Colletotrichum gloeosporioides

Papaya

Colletotrichum c a p s i c i

Pepper

Cutinase p u r i f i e d ; r o l e i n i n f e c t i o n conclusively proven Cutinase p u r i f i e d

Colletotrichum graminicola

Corn

Cutinase p u r i f i e d ; i n h i b i tors prevent i n f e c t i o n

Phytophthora cactorum

Apple

Cutinase p u r i f i e d

Botrytis cineria

Wide Host Range

Cutinase p u r i f i e d

Venturia inequalis

Apple

Cutinase i n h i b i t o r s prevent i n f e c t i o n

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

10. KOLATTUKUDY ET AL.

The Role of Cutin

157

various cutinases include the presence of only one tryptophane, one methionine, one or two h i s t i d i n e s and two or four cysteines all of which are involved in disulfide bridges. Immunologically, the various cutinases from d i f f e r e n t organisms appear to be quite d i f f e r e n t (3,16-19). Only the enzyme from very c l o s e l y related organisms show c r o s s - r e a c t i v i t y . A l l fungal cutinases are glycoproteins containing 3 to 6% carbohydrates. The carbohydrates are attached to the protein v i a O-glycosidic linkages (20). The N-terminus of fungal cutinases appear to be blocked i n a novel manner i n that glucuronic acid i s linked by an amide linkage to the N-terminal amino group (21). The c a t a l y t i c properties of fungal cutinases also show similarities. Cutinase shows s p e c i f i c i t y f o r primary alcohol esters; secondary alcohol esters are hydrolyzed at much lower rates (3,16). Phospholipids are not hydrolyzed but c e r t a i n t r i g l y c e r i d e s , such a t r i p a l m i t i n or t r i s t e a r i n cases the enzymes show c l e a r s p e c i f i c i t y f o r the primary alcohol esters. A l l fungal cutinases also catalyze hydrolysis of £-nitrophenyl esters of short chain f a t t y acids. The r e l a t i v e rates of hydrolysis depend upon on the chain length of the a c y l group and the source of the enzyme. In many cases the longer chain esters such as C ^ or Cjg are hydrolyzed extremely poorly when compared to short chain esters such as C^. On the other hand cutinase from some genera, such as Colletotrichum, catalyzes hydrolysis of longer chain esters at rates comparable to that obtained with the short chain acids (16). The mechanism of c a t a l y s i s of ester bonds by fungal cutinases has been studied using protein-modifying reagents that are r e l a t i v e l y s p e c i f i c to f u n c t i o n a l groups. The r e s u l t s of such studies strongly suggest that a c a t a l y t i c t r i a d i n v o l v i n g serine, h i s t i d i n e and a carboxyl group are involved i n c a t a l y s i s by fungal cutinase (22). Active serine directed reagents such as diisopropylfluorophosphate (23) and a v a r i e t y of other organic phosphates (24,25) as w e l l as t r a n s i t i o n state analogs such as a l k y l and phenyl boronic acids (26), i n h i b i t fungal cutinases. M o d i f i c a t i o n of h i s t i d i n e by diethylpyrocarbonate and carboxyl groups by carbodiimide also i n h i b i t the enzyme a c t i v i t y (22). C o r r e l a t i o n of the number of residues modified with the extent of a c t i v i t y l o s s of the enzyme showed that one serine, one h i s t i d i n e and one carboxy group were e s s e n t i a l f o r c a t a l y s i s by cutinase (22). The presence of the c a t a l y t i c t r i a d c h a r a c t e r i s t i c of serine hydrolases suggested by these r e s u l t s also raised the p o s s i b i l i t y of the involvement of an a c y l enzyme intermediate. To test t h i s p o s s i b i l i t y cutinase was f i r s t treated with diethylpyrocarbonate to i n h i b i t acyl-enzyme hydrolysis and this enzyme was then treated with £-nitophenyl[l- C]acetate. Gel f i l t r a t i o n of t h i s reaction mixture yielded [ l - C ] a c e t y l - c u t i n a s e demonstrating the existence of the postulated acyl-enzyme intermediate (22). The mechanism of c a t a l y s i s by cutinase can be depicted as shown i n Figure 1. 1H

14

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ECOLOGY AND METABOLISM OF PLANT LIPIDS

DEACYLATION

Figure 1. Mechanism of c a t a l y s i s by cutinase. (Reproduced with permission from Ref. 16. Copyright 1984 E l s e v i e r / North Holland Biomedical Press.)

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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The Role of Cutin

Structure of Cutinase The complete primary structure of the enzyme has been deduced from the nucleotide sequence of the cDNA which was cloned (Figure 2). The cDNA sequence showed an open reading frame which could be translated into a protein with a molecular weight of 23,951 (27). The enzyme i s o l a t e d from Fusarium solani p i s i had been shown to have a glucuronic acid attached to a glycine at the N-terminus. The f i r s t glycine residue i n the primary t r a n s l a t i o n product would be at p o s i t i o n 32 according to the nucleotide sequence. Thus the 31 amino acid leader peptide must be removed during the processing of the primary t r a n s l a t i o n product into mature enzyme. In f a c t , the primary t r a n s l a t i o n product had been shown to be larger than the mature enzyme by i n v i t r o t r a n s l a t i o n of i s o l a t e d mRNA (28). The leader sequence revealed by the nucleotide sequence did show a f a i r l y t y p i c a l structure except for th region was longer tha other e x t r a c e l l u l a r proteins (29,30). The biochemistry of the processing of the precursor into the mature enzyme has not been studied. In order to v e r i f y the sequence derived from the nucleotide sequence, p r o t e o l y t i c fragments of the mature enzyme were i s o l a t e d and the amino acid sequence of these peptides were determined. The peptide containing the only tryptophane of the enzyme was i s o l a t e d and sequenced. The a c t i v e serine was located by treatment of the enzyme with radioactive diisopropylfluorophosphate followed by i s o l a t i o n and amino acid sequencing of a t r y p t i c peptide containing the r a d i o a c t i v i t y . The carboxyl group involved i n c a t a l y s i s was located by l a b e l i n g t h i s residue by treatment with carbodiimide and [ *C] glycine e t h y l ester followed by i s o l a t i o n and analysis of the labeled t r y p t i c peptide (W.F. Ettinger and P.E. Kolattukudy, unpublished r e s u l t s ) . These amino acid sequences agreed with the sequences deduced from the nucleotide sequence. The h i s t i d i n e residue involved i n c a t a l y s i s was e a s i l y located as the sole h i s t i d i n e residue present i n the enzyme. The three members of the c a t a l y t i c t r i a d are located far apart i n the primary structure of the p r o t e i n . Obviously these three residues are held i n j u x t a p o s i t i o n so that they can function as a c a t a l y t i c t r i a d by the secondary and t e r t i a r y structure. The d i s u l f i d e bridges must play a s i g n i f i c a n t r o l e i n holding the enzyme i n the active conformation because reduction of the d i s u l f i d e i s known to r e s u l t i n i n a c t i v a t i o n of the enzyme (16). The importance of the d i s u l f i d e bridges i s also r e f l e c t e d i n the fact that the positions of the cys residues involved seem to be highly conserved (W.F. Ettinger and P.E. Kolattukudy, unpublished results). In the three cutinases, from which homologous peptides have been sequenced, the conservation of the cys positions i s obvious (Figure 3). The high degree of homology shown by the enzymes from Fusarium and Colletotrichum i s striking. The structure of the cutinase gene has been determined using genomic DNA cloned i n λ-phage. This gene contains one 51 bp i n t r o n which has the t y p i c a l junction and s p l i c i n g signals ll

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

GLY ASN GGC AAT

GLY ASN GGC AAC

ASP GLY GAC GGT

L E U PRO CTC CCT

ASN THR L Y S AAC ACC AAG

ASN AAC

THR ACG

LYS AAG

ALA GCT

ALA GCC

TRP TGG

GLY GGA

CYS TGC

125

7

GLN CAG

LEU CTC

CYS TGC

4

GLU GAG

LEU CTT

PRO CCT

GLY VAL GGC GTT

GLY GGT

ARG A S P CGC GAT

A L A ARG GCG C G C

KC-57

THR THR ACC ACA

PRO CCT

A S P ALA THR GAC GCG A C T

THR S E R SER A L A ACC TCT AGC GCC

ILE ATT

THR ACT

ALA SER GCT TCC

GLU GAG L E U CTT

ALA LEU GCT CTC

ARG GLY C G C GGA

VAL GTC

LEU TTG

SER AGC

GLN CAG

PRO A L A CCT GCC

ASN AAC

PHE TTC

LYS PHE AAA T T C

MET ATG

LEU TTG

130

ALA GCA

GLY GGC

90

SER AGC

70

ILE ATC

ILE ATC

GLY GGT

ILE ATT

ILE VAL GTC ATC

50

LEU GLN CTT CAG

30

ALA LEU GCC CTC

10

ARG AGA

ALA GLY GCC GGT

ARG'GLU AGG GAG

A L A TYR GCC TAC

ALA SER GCC TCC

PHE ILE TTC ATT

GLY GGT

32

A L A THR GCC ACG

LEU CTT

.

GLY GGC

TYR TAC

MET L E U ATG CTC

1

LEU CTG

ARG A S P CGC GAC

ALA GCT

LEU CTC

LEU CTT

SER TCC

SER GLN AGC CAG

136

GLY GGT

THR ACT

GLU GAG

A L A ARG 'GLY GCC CGA GGT

THR ACT

SER TCG

ARG ALA CGA GCC

ASN AAC

TYR TAT

THR ACA

ALA GCT

GLY GGT

PHE TTC

GLY GGA

ALA GCC

SER TCA

ASP GAT

PRO CCT

ALA GCT

GLN CAG

ASP GAC

99

PHE TTC

THR ACA

LEU CTG

THR ACT

ALA GCA

GLN CAG

ASN AAT

GLY GGC

GLU GAG

ILE ATC

SER TCT

AACCACAACTACCTTCACTTCATCAACATTCACTTCAACTTCTTCGCCTCTTCCTTTTCACTCTTTATCATCCTCACC

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ι

150

187

190

.

194

204

210

230

ALA PRO HIS LEU ALA TYR GLY PRO ASP ALA ARG GLY PRO ALA PRO GLU PHE LEU I L E GCA CCT CAC TTG GCT TAT GGT CCT GAT GCT CGT GGC CCT GCC CCT GAG TTC CTC ATC

Figure 2. Nucleotide sequence of the cloned cutinase cDNA and the amino acid sequence deduced from i t . C-4 and C-57 indicate the beginning of the nucleotide sequence of two a d d i t i o n a l cDNA clones. The s o l i d l i n e s represent the regions f o r which the primary structure was confirmed by amino acid sequencing.

TATTGCGAGGTTTCAAGTTTTTCTTTTGGTGAATAGCCATGATAGATTGGTTCAACACTCAATGTACTACAATGCCC

GLU LYS VAL ARG ALA VAL ARG GLY SER ALA β@@ GGAGGATGAGAATTTTAGCAGGCGGGCCTGTTAAT GAG AAG GTT CGG GCT GTC CGT GGT TCT GCT TGA

ALA GCT

ASP ARG THR LYS VAL PHE CYS ASN THR GLY ASP LEU VAL CYS THR GLY SER LEU ILE VAL GAC AGG ACC AAG GTC TTC TGC AAT ACA GGG GAT CTC GTT TGT ACT GGT AGC TTG ATC GTT

,

VAL LEU PHE GLY TYR THR LYS ASN LEU GLN ASN ARG GLY ARG ILE PRO ASN TYR PRO ALA GTT CTG TTC GGC TAC ACC AAG AAC CTA CAG AAC CGT GGC CGA ATC CCC AAC TAC CCT GCC

170

LEU ALA* ALA ALA SER ILE GLU ASP LEU ASP SER ALA ILE ARG ASP LYS ILE ALA GLY THR CTT GCA GCC GCC TCC ATC GAG GAC CTC GAC TCG GCC ATT CGT GAC AAG ATC GCC GGA ACT

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Val

Capsici:

Tyr

Val Phe

Fusarium II:

Cys

Cys

Val Phe Cys

Fusarium I:

Ala

Asn

Gly

Gly

Asp Leu Val Cys

Thr

Ser

Ser

Leu Glu Ser

lie Asn

Gly

Gly

Gly

Ala Ala Pro

Gly

Ser

Ser

Gly^ Asn Ser

Ser Asn

Ala Ser

Cys

Pro

Cys Arg

Phe

His Leu

Leu Phe lie Leu Pro Ala His

Leu He lie

Leu Tyr

Thr

Leu Ile Val Ala Ala Pro His Leu Ala Tyr

Figure 3. Comparison of homologous peptides from the two cutinases i s o l a t e d from Fusarium solani f. sp. p i s i and Colletotrichum c a p s i c i (W. Ettinger and P.E. Kolattukudy, unpublished r e s u l t s ) .

Asn Glu

Capsici:

Asp Leu

Asp

Fusarium I:

Tyr

Thr

Asp Leu Val Cys Thr

Leu Ala Asp Ala Val Cys

Thr

Asn Thr

Pro

Asp

Gin Ala Asp

Gly

Ala Ala

Ala Arg

5

*0

"Ζ

r > Η

"Ο

Ο τι

2

00 ο

>

Η

m

>

ο

Ο r Ο

m η

ON to

10. KOLATTUKUDY ET AL.

The Role of Cutin

that are homologous to those found i n other filamentous fungi and yeast. I t showed a t y p i c a l polyadenylation s i g n a l but the CAT box and TATA boxes i n the 5*-flanking region were not obvious. Role of Cutinase i n Pathogenesis With the a v a i l a b i l i t y of pure cutinase, an immunological approach became possible to test whether cutinase i s secreted by fungi during the penetration of t h e i r hosts. F. solani p i s i spores were placed on pea stem surface i n a moist atmosphere and the progress of germination and penetration was followed by scanning electron microscopy (Figure 4a). At the time when the germinating spores were found to be penetrating the surface, the i n f e c t i o n area was treated with ferritin-conjugated antibodies prepared against the enzyme and the tissue was processed for transmission electro (Figure 4b,c) clearl secreted cutinase at the i n f e c t i o n area (31). Similar experiments were subsequently done with Colletotrichum gloeosporioides and convincing evidence was obtained that t h i s organism also secreted cutinase during penetration (M.B. Dickman, S. Pat i l , C L . Soliday and P.E. Kolattukudy, unpublished r e s u l t s ) . I f the secreted enzyme i s necessary for penetration, inhibition of the enzyme should stop the penetration process and therefore infection. Bioassays performed by placing Fusarium spores on pea stem surface showed that i n c l u s i o n of cutinase i n h i b i t o r s such as antibodies prepared against the enzyme or chemical i n h i b i t o r s such as organic phosphates prevented the l e s i o n formation (32) (Figure 5). Similar experiments were performed also by placing spores of Colletotrichum gloeosporioides on papaya f r u i t s (18). In t h i s case a l s o , i n c l u s i o n of cutinase i n h i b i t o r s such as the antibodies against t h i s enzyme or organic phosphates completely prevented i n f e c t i o n . Subsequently, s i m i l a r experiments were performed by placing spore suspensions of Venturia inequalis on the leaves of apple seedlings, Colletotrichum graminicola on the leaves of corn seedlings and Colletotrichum c a p s i c i on pepper f r u i t s (33). In a l l of these cases, i n c l u s i o n of i n h i b i t o r s of cutinase prevented i n f e c t i o n . These r e s u l t s c l e a r l y showed that at least under the conditions of the bioassay, fungal i n f e c t i o n involves cutinase mediated penetration of the c u t i c u l a r b a r r i e r and that s p e c i f i c i n h i b i t i o n of t h i s enzyme can completely prevent i n f e c t i o n . Whether the cutinase-targeted approach to prevent fungal i n f e c t i o n of plants can be an e f f e c t i v e method i n the f i e l d i s yet to be determined. One f i e l d t r i a l that was done suggests that t h i s approach may be f e a s i b l e . Spraying of an organic phosphate i n h i b i t o r of cutinase on papaya i n the f i e l d protected the f r u i t s from i n f e c t i o n by Colletotrichum gloeosporioides (W. Nishijima, M.B. Dickman, S. P a t i l and P.E. Kolattukudy, unpublished r e s u l t s ) . This experiment conducted for an e n t i r e season was designed only to test the f e a s i b i l i t y of the approach under f i e l d conditions and therefore parameters such as the optimal amount of i n h i b i t o r or the time of a p p l i c a t i o n were not

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

163

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Figure 4. Scanning (a) and transmission (b and c) electron micrographs of the penetration of pea stem c u t i c l e by germinating spores of F. s o l a n i f. sp. p i s i . Figures represent the state of events 12 h a f t e r placing spores on the stem of the pea seedling. The i n f e c t i o n area was treated with f e r r i t i n - c o n j u g a t e d , anticutinase IgG. F e r r i t i n granules can be seen at the s i t e of penetration i n b and c. C, c u t i c l e ; CW, c e l l w a l l ; F, fungus.

10. KOLATTUKUDY ET AL.

The Role of Cutin

Figure 5. Pea e p i c o t y l segments 72 hours a f t e r i n o c u l a t i o n with c o n i d i a l suspension of F. s o l a n i f. sp. p i s i i n water ( c o n t r o l ) , water containing TgG from preimmune rabbit serum (serum), water containing IgG from antiserum prepared against cutinase (antiserum) and water containing 10 UM diisopropylfluorophosphate (DFP).

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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166

ECOLOGY AND METABOLISM OF PLANT LIPIDS

studied. Instead, the spray program was conducted i n alternate weeks for an entire season and r e s u l t s were p o s i t i v e . It i s l i k e l y that antipenetrant chemicals which i n h i b i t cutinase w i l l work e f f e c t i v e l y i f combined with a low l e v e l of a c l a s s i c a l fungicide. In t h i s manner, the bulk of the fungal spores w i l l not be able to penetrate and some which might penetrate through some breach i n the c u t i c u l a r b a r r i e r can be prevented from s e t t i n g up i n f e c t i o n by the fungicide. A b i l i t y to produce cutinase can determine whether a fungus can i n f e c t a plant. An i s o l a t e of Fusarium solani p i s i (T-30) which lacked the a b i l i t y to produce cutinase f a i l e d to i n f e c t pea stem (34) as indicated below. A wound pathogen of papaya, Mycosphaerella, could i n f e c t papaya f r u i t s when exogenous cutinase was provided instead of a wound (18). More recently cutinaseless mutants of Colletotrichum gloeosporioides (M.B. Dickman and S. P a t i l , private communication) and Fusarium s o l a n i p i s i (Anne Dantzig, privat mutagenesis. These mutant the c u t i c u l a r b a r r i e r was i n t a c t . That the mutation did not a f f e c t elements invovled i n pathogenesis other than penetration was shown by the fact that the mutants were as v i r u l e n t as the w i l d type when the c u t i c u l a r b a r r i e r was mechanically breached or when exogenous cutinase was provided i n the inoculation medium (M.B. Dickman and S. P a t i l , private communication). These r e s u l t s c l e a r l y demonstrate that cutinase i s e s s e n t i a l for i n f e c t i o n by the two pathogens. To test whether the a b i l i t y to produce cutinase might determine the virulence of fungal s t r a i n s a large number of i s o l a t e s of Fusarium s o l a n i p i s i were tested using bioassays with pea stem segments. Spore suspensions were placed on i n t a c t stem or, for comparison, on pea stem with mechanically breached c u t i c l e / w a l l b a r r i e r . In t h i s manner, i s o l a t e s which lacked the a b i l i t y to penetrate the b a r r i e r but had a l l the other necessary elements for pathogenesis could be detected. Thus Fusarium s o l a n i p i s i i s o l a t e T-8 was found to i n f e c t pea stems with or without the c u t i c u l a r b a r r i e r whereas i s o l a t e T-30 could i n f e c t only i f the b a r r i e r was mechanically breached (34) (Figure 6). To test whether i s o l a t e T-30 lacked the a b i l i t y to produce cutinase, the spores of i s o l a t e s T-8 and T-30 were washed with buffer and the cutinase a c t i v i t y was measured using a s p e c i a l l y prepared highly radioactive c u t i n . The r e s u l t s c l e a r l y showed that T-30 lacked cutinase (Table I I I ) . I f t h i s defect were the only reason for the observed i n a b i l i t y of T-30 to i n f e c t i n t a c t pea stem, supplementation with exogenous cutinase might make t h i s i s o l a t e as v i r u l e n t as T-8. Exogenous cutinase did i n fact a s s i s t T-30 i n i n f e c t i n g pea stem segments i n the bioassays. However, supplementation with pectinase, c e l l u l a s e and pectin methylesterase were also required to increase the virulence of T-30 to a l e v e l that was equal to that of T-8 (34) (Figure 7). From these observations i t was concluded that the i n f e c t i o n process involved a set of penetration enzymes to disrupt not only the c u t i c u l a r b a r r i e r but also the carbohydrate polymeric b a r r i e r s that l i e under the c u t i c l e . Although these experiments suggested that T-30 probably lacked the a b i l i t y to produce a l l of the penetration enzymes, no d i r e c t evidence was obtained.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

10. KOLATTUKUDY ET AL.

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The Role of Cutin

Figure 6. Pea e p i c o t y l segments 72 h c o n i d i a l suspension of Fusarium s o l a n i and i s o l a t e T-30 ( r i g h t ) . In each case the r e s u l t s with mechanically breached b a r r i e r , respectively.

a f t e r inoculation with p i s i i s o l a t e T-8 ( l e f t ) the top and bottom show and i n t a c t c u t i c l e / w a l l

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

3

2800

50,500

activity

15

Cutinase

2

3

per 1 0

10

3

3100

2,610,000

activity

Cutinase b

10

12

(%)

Germination

4.6

153.3

activity

PNBase 3

3900

3,020,000

activity

Cutinase

3

1

released h

1

per 1 0

10

spores.

3

56

77

(%)

Germination

24 h Germination

spores using p_-nitophenylbutyrate as substrate.

3.0

132.0

activity

PNBase

6 h Germination

^Cutinase a c t i v i t y i s expressed as the amount of H c t min

1

(%)

Germination

PNB a c t i v i t y i s expressed nmol min

3.0

T-30

3

26.4

activity

T-8

Isolate

PNBase

2 h Germination

Esterase and Cutinase A c t i v i t y Released into the E x t r a c e l l u l a r F l u i d During Spore Germination

TABLE I I I

KOLATTUKUDY ET AL.

169

The Role of Cutin

100 -

Cutinase + + Pectinesterase - - - - Pectinase -- + - + Cellulose + - +

+ + +

+ + -

+ + + +

Figure 7. Infection e f f i c i e n c y of i s o l a t e T-30 conidia on pea e p i c o t y l with i n t a c t c u t i c l e / w a l l b a r r i e r after enzyme supplementation. A c o n i d i a l suspension was mixed with (+) or without (-) indicated enzymes. The mixtures were used to inoculate i n t a c t (•) or wounded ( 0 ) surfaces of pea e p i c o t y l segments. Means followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t (P=0.05, Duncan's multiple range t e s t ) . (Reproduced with permission from Ref. 34. Copyright 1982 Academic Press.)

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

Since the exogenous enzymes used to supplement the spore medium were from sources other than the pathogen, the p a r t i c u l a r kind of enzyme mixtures that were required to get e f f e c t i v e fungal penetration cannot be taken as f i r m evidence for the involvement of such enzymes i n a natural i n f e c t i o n . For example, the requirement for pectin methylesterase might simply imply that the exogenous pectinase used worked more e f f e c t i v e l y i n the presence of t h i s methyl esterase. The nature of the enzymes produced by the pathogen to disrupt the w a l l b a r r i e r can be elucidated only by d i r e c t studies on the pathogen i t s e l f . The enzymes used to break the c u t i c l e / w a l l b a r r i e r s might either be present i n the spore that lands on the plant or these enzymes might be induced as a r e s u l t of contact of the spore with the plant surface. Conclusive evidence was obtained that cutinase i s induced i n the spores as a r e s u l t of contact with c u t i n (35). Thus, the presence of c u t i n was required for induction of the e x t r a c e l l u l a of £-nitrophenyl esters immunological techniques. The degree of induction of cutinase depended upon the amount of c u t i n added and the l e v e l of the enzyme a c t i v i t y increased with time and reached a plateau i n a few hours. Cutin hydrolysate composed of a l l of the monomers also induced cutinase synthesis i n the spores (Figure 8). Isolated monomers were also e f f e c t i v e inducers. The most e f f e c t i v e inducers were dihydroxy-Ci6 acid and trihydroxy-Cl8 a c i d , the most unique components of c u t i n . These r e s u l t s suggested that the low l e v e l s of cutinase present i n the spore at the time of i t s a r r i v a l at the plant surface generated small amounts of c u t i n monomers from the plant surface and these monomers a c t u a l l y induced cutinase production. To test whether the induction of the enzyme involved transcriptional control, thf^ °f mRNA produced by the spore was quantitated using P-labeled cDNA as a probe (Figure 9). Such dot b l o t analysis showed that w i t h i n 15 minutes a f t e r the spores came i n t o contact with c u t i n or c u t i n monomers, cutinase gene t r a n s c r i p t s were detectable and the level increased for the next few hours (35). These r e s u l t s are i n agreement with the observation that the cutinase a c t i v i t y l e v e l increased with a s l i g h t lag period. The induction observed by the presence of c u t i n i n the medium was highly stimulated by the addition of exogenous cutinase, strongly supporting the hypothesis that the small amount of cutinase o r i g i n a l l y present i n the spore was responsible for producing c u t i n monomers which then induced the production of the enzyme required for penetration. The fact that the best inducers were the most c h a r a c t e r i s t i c c u t i n components, which are not found anywhere else i n nature, shows that the induction mechanism i s a highly s p e c i f i c method by which the fungus can sense the presence of the host plant and thus induce cutinase when needed for the penetration process. a m o u n t

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

KOLATTUKUDY ET AL.

0

1

171

The Role of Cutin

2

3 0

1

2

3

Figure 8. Time course of appearance of cutinase as measured by immunochemical techniques ( l e f t ) and cutinase a c t i v i t y as measured by £-nitrophenyl butyrate hydrolysis (right) i n the e x t r a c e l l u l a r f l u i d of spore suspensions of F. s o l a n i f. sp. p i s i induced with either c u t i n hydrolysate or p u r i f i e d Ci6 dihydroxy acid. (Reproduced with permission from Ref. 35. Copyright 1986 The National Academy of Sciences.)

Figure 9. Cutinase mRNA content from spores of F. s o l a n i f. sp. p i s i exposed to either c u t i n hydrolysate or p u r i f i e d dihydroxy-Cig acid f o r various periods of time. Cutinase [ P]cDNA was used as a probe i n the dot b l o t analysis. (Reproduced with permission from Ref. 35. Copyright 1986 The National Academy of Sciences.) 32

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

Enzymatic Penetration of the Pectinaceous B a r r i e r The penetrating fungus comes i n t o contact with the carbohydrate polymers when the germinating spore breaches the c u t i n b a r r i e r and these polymers then might t r i g g e r the synthesis of pectinases. In f a c t , when Fusarium s o l a n i p i s i spores were placed i n a pectin-containing medium pectinase production was induced (M.S. Crawford and P.E. Kolattukudy, unpublished r e s u l t s ) . The p e c t i n hydrolase a c t i v i t y l e v e l reached a maximal l e v e l i n about 8 hours a f t e r the spores came i n t o contact with the pectin-containing medium and subsequently decreased (Figure 10). The i s o l a t e T-8 which was highly v i r u l e n t even on i n t a c t pea stem showed a much higher l e v e l of pectin hydrolase when compared with i s o l a t e T-30, which was unable to penetrate i n t a c t stems. The maximal l e v e l of production of p e c t i n hydrolase coincided with the onset of germination. Only much l a t e r a pectate lyase began to appea much higher l e v e l of th evidence suggested that the hydrolase, induced early during germination, produced components which induced the lyase. The pectate lyase from i s o l a t e T-8 has been p u r i f i e d to homogeneity and rabbit antibodies have been prepared. These antibodies protected pea stem sections against attack by Fusarium s o l a n i p i s i under the bioassay conditions indicated above f o r cutinase. However, the protection i n t h i s case was not complete. Presumably the pectin hydrolase also plays an important r o l e i n the enzymatic penetration of the carbohydrate b a r r i e r that l i e s under the c u t i c l e . However, t h i s pectinase has not been purified and therefore definitive experiments about the importance of t h i s enzyme have not been performed. Since c u t i n i s the f i r s t b a r r i e r that comes i n t o contact with the fungal spore, i t i s tempting to speculate that the hydrolysis products generated from t h i s polymer might also t r i g g e r the production of pectin hydrolase which i n turn generates small molecular weight compounds which subsequently induce the synthesis of the lyase. Preliminary experiments have given i n d i c a t i o n s that c u t i n and c u t i n hydrolysate do induce pectin hydrolase synthesis i n spores. I t i s possible that penetration enzymes might be coordinately c o n t r o l l e d and i n the case of T-30 t h i s c o n t r o l i s being manifested by the lack of i t s a b i l i t y to produce cutinase as w e l l as high l e v e l s of the pectin hydrolyzing enzymes. In any case, the enzymes involved i n breaching both the c u t i c u l a r and the carbohydrate b a r r i e r s might be used as targets to develop methods to prevent fungal penetration of plants and thus to prevent i n f e c t i o n . Acknowledgments This i s S c i e n t i f i c Paper No. 7226, Project 2001, College of Agriculture Research Center, Washington State U n i v e r s i t y , Pullman, WA 99164. This work was supported a grant PCM-8306835 from the National Science Foundation.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

KOLATTUKUDY ET AL.

The Role of Cutin

1

GERMINATION

1

1

1

r

TIME (hr)

Figure 10. Pectin hydrolase and lyase a c t i v i t i e s released by spores of F. s o l a n i f. sp. p i s i i s o l a t e s T-8 and T-30, exposed to pectin containing medium. (Reproduced with permission. Copyright 1985 A l l a n R. L i s s , Inc.)

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

Kolattukudy, P.E. Science, 1980, 208, 990-1000. Kolattukudy, P.E. Ann. Rev. Plant Physiol., 1981, 32, 540-567. Kolattukudy, P.E. "The Biochemistry of Plants, Vol. 4;" Stumpf, P.K.; Ed; Academic Press: New York, 1980, Chapter 18. Kolattukudy, P.E. Biochemistry, 1974, 13, 1354-1363. Espelie, K.E.; Köller, W.; Kolattukudy, P.E. Chem. Phys. Lipids, 1983, 32, 13-26. Kolattukudy, P.E. "The Structure, Biosynthesis and Degradation of Wood;" Loewus, F.A.; Runeckles, V.C.; Eds; Plenum Press, New York, 1977, Chapter 6 Agulló, C.; Collar, C.; Seoane, E. Phytochemistry, 1984, 23, 2059-2060. Deas, A.H.B.; Holloway Higher Plants;" Tevini Springer-Verlag: New York, 1977, Chapter 16 Riley, R.G.; Kolattukudy, P.E. Plant Physiol., 1975, 56, 650-654. Hunt, G.M.; Baker, E.A. Phytochemistry, 1980, 19, 1415-1419. van den Ende, G.; Linskens, H.F. Ann. Rev. Phytopathol., 1974, 12, 247-258. Kolattukudy, P.E. Ann. Rev. Phytopathol., 1985, 23, 223-250. Aist, J.R. Encycl. Plant Physiol. New. Ser., 1976, 4, 197-221. Dodman, R.L. "Plant Disease - An Advanced Treatise. Vol. 4. How Pathogens Induce Disease;" Horsefall, J.G.; Cowling, E.B.; Eds; Academic Press: New York, 1979, Chapter Verhoeff, K. "The Biology of Botrytis;" Coley-Smith, J.R.; Verhoeff, K.; Eds; Academic Press, New York, 1980, Chapter Kolattukudy, P.E. "Lipases;" Borgström, B.; Brockman, H.; Eds; Elsevier/North Holland Biomedical Press: Amsterdam, 1984, Chapter C. Soliday, C.L.; Kolattukudy, P.E. Arch. Biochem. Biophys., 1976, 176, 334-343. Dickman, M.B.; Patil, S.S.; Kolattukudy, P.E. Physiol. Plant Pathol., 1982, 20, 333-347. Lin, T.S.; Kolattukudy, P.E. Physiol. Plant Pathol., 1980, 17, 1-15. Lin, T.S.; Kolattukudy, P.E. Eur. J. Biochem., 1980, 106, 341-351. Lin, T.S.; Kolattukudy, P.E. Biochem. Biophys. Res. Commun., 1977, 75, 87-93. Köller, W.; Kolattukudy, P.E. Biochemistry, 1982, 21, 3083-3090. Purdy, R.D.; Kolattukudy, P.E. Biochemistry, 1975, 14, 2832-2840. Köller, W.; Allan, C.R.; Kolattukudy, P.E. Phytopathology, 1982, 72, 1425-1430. Dickman, M.B.; Patil, S.S.; Kolattukudy, P.E. Phytopathology, 1983, 73, 1209-1214.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

10.

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The Role of Cutin

26.

Carvalho, S.C.; Allan, C.R.; Kolattukudy, P.E. Amer. Chem. Soc. 39th NW Regional Meeting, 1984, Abst. 108. 27. Soliday, C.L.; Flurkey, W.H.; Okita, T.W.; Kolattukudy, P.E. Proc. Natl. Acad. Sci. USA, 1984, 81, 3939-3943. 28. Flurkey, H.W.; Kolattukudy, P.E. Arch. Biochem. Biophys. 1981, 212, 154-161. 29. Watson, E . E . Nucl. Acids Res. 1984, 12, 5145-5164. 30. Perlman, D.; Halvorson, H.O. J. Mol. Biol., 1983, 166, 391-409. 31. Shaykh, M.; Soliday, C.L.; Kolattukudy, P.E. Plant Physiol., 1977, 60, 170-172. 32. Maiti, I.B.; Kolattukudy, P.E. Science, 1979, 205, 507-508. 33. Chacko, R.; Kolattukudy, P.E., manuscript in preparation. 34. Köller, W.; Allen, C.R.; Kolattukudy, P.E. Physiol. Plant Pathol., 1982, 20, 47-60. 35. Woloshuk, C.P.; Kolattukudy preparation. RECEIVED September 4, 1986

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 11

Variability in Steroid Metabolism Among Phytophagous Insects James A. Svoboda and Malcolm J. Thompson Insect Physiology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

Members of the class Insecta require an exogenous source of sterol to support normal development and reproduction. Cholestero nearly all specie omnivorous insects thrive on diets containing little or no cholesterol. Most of these species that have been critically examined are able to dealkylate and convert dietary 24-alkyl (C and C ) phytosterols to cholesterol. However, significant variations in the utilization and metabolism of dietary sterols between phytophagous species have been discovered in recent years. Thus, it is becoming increasingly difficult to generalize about sterol metabolism even among members of the same Order. These differences in the utilization of neutral sterols can often be correlated with ecdysteroid (molting hormone) production. Certain of the most significant variations in insect steroid utilization and metabolism in phytophagous insects will be discussed with respect to phylogenetic relationships. 28

29

S i n c e i n s e c t s l a c k the c a p a c i t y to b i o s y n t h e s i z e the s t e r o i d n u c l e u s , they g e n e r a l l y r e q u i r e a d i e t a r y source o f s t e r o l f o r normal development and r e p r o d u c t i o n 11). This i s an important area o f b i o c h e m i c a l d i f f e r e n c e , between i n s e c t s and many o t h e r o r g a n i s m s , t h a t might be e x p l o i t e d t o develop new p e s t c o n t r o l s t r a t e g i e s . C h o l e s t e r o l w i l l s a t i s f y t h i s d i e t a r y requirement i n a l l but two known cases i n which d i e t a r y Δ ' - s t e r o l s a r e e s s e n t i a l (,2,2)· a d d i t i o n , some i n s e c t s may o b t a i n an adequate supply o f s t e r o l from symbionts o r i n t e s t i n a l m i c r o o r g a n i s m s . For many y e a r s , i t was b e l i e v e d t h a t phytophagous i n s e c t s i n general were c a p a b l e o f d e a l k y l a t i n g and c o n v e r t i n g d i e t a r y C28 and C29 p h y t o s t e r o l s t o c h o l e s t e r o l t o s a t i s f y t h e i r need f o r c h o l e s t e r o l ( 4 ) . A l s o , a number o f omnivorous s p e c i e s of i n s e c t s a r e known t o be c a p a b l e o f t h i s c o n v e r s i o n (5). Thus, c h o l e s t e r o l I

This chapter not subject to U.S. copyright. Published 1987, American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

n

11. SVOBODA AND THOMPSON

Steroid Metabolism

111

can be made a v a i l a b l e f o r s t r u c t u r a l needs i n membranes (I) and f o r e s s e n t i a l p h y s i o l o g i c a l purposes such as s e r v i n g as a p r e c u r s o r f o r the C27 m o l t i n g hormones ( e c d y s t e r o i d s , F i g u r e 1 ) , e . g . ecdysone (j>). I t has become i n c r e a s i n g l y e v i d e n t t h a t 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 s t e r o i d u t i l i z a t i o n and metabolism e x i s t s among phytophagous s p e c i e s of i n s e c t s . In r e c e n t y e a r s , we have d i s c o v e r e d s e v e r a l phytophagous s p e c i e s t h a t are unable to c o n v e r t C28 o r C29 p h y t o s t e r o l s to c h o l e s t e r o l . T h i s i n c l u d e s one s p e c i e s t h a t d e a l k y l a t e s the C - 2 4 s u b s t i t u e n t of the s i d e c h a i n but produces mostly s a t u r a t e d s t e r o l s and s e v e r a l s p e c i e s t h a t t o t a l l y l a c k the a b i l i t y to d e a l k y l a t e the s t e r o l s i d e c h a i n . Certain members o f t h i s l a t t e r group are of p a r t i c u l a r i n t e r e s t because they have adapted t o u t i l i z i n g a COQ s t e r o l as an e c d y s t e r o i d p r e c u r s o r and makisterone A (C28) has been i d e n t i f i e d as the major e c d y s t e r o i d of c e r t a i n developmental stages of these s p e c i e s . We w i l l d i s c u s s som s t u d i e s and p r o v i d e s p e c i f i unusual v a r i a t i o n s i n s t e r o i d u t i l i z a t i o n and metabolism i n i n s e c t s , and to show how t h i s i n f o r m a t i o n i s u s e f u l i n p r e d i c t i n g d i f f e r e n c e s i n e c d y s t e r o i d b i o s y n t h e s i s i n c e r t a i n s p e c i e s . We w i l l a l s o p o i n t out i n s t a n c e s i n which these v a r i a t i o n s i n n e u t r a l s t e r o l metabolism can be r e l a t e d to p h y l o g e n e t i c r e l a t i o n s h i p s between s p e c i e s . Phytophagous I n s e c t s That Convert C to C h o l e s t e r o l

0 Q

and C

o 0

Phytosterols

'

Lepidoptera. The most e x t e n s i v e s t u d i e s of the u t i l i z a t i o n and metabolism of d i e t a r y s t e r o l s i n phytophagous i n s e c t s have been c a r r i e d out w i t h two L e p i d o p t e r a , the tobacco hornworm, Manduca s e x t a , i n our l a b o r a t o r y (7) and the s i l k w o r m , Bombyx m o r i , by Ikekawa and coworkers i n Japan ( 8 ) . These were p i o n e e r i n g s t u d i e s u t i l i z i n g a r t i f i c i a l d i e t s , r a d i o l a b e l e d s t e r o l s , and s t a t e - o f - t h e - a r t a n a l y t i c a l t o o l s . Manduca l a r v a e r e a d i l y c o n v e r t C28 and C29 p h y t o s t e r o l s ( e . g . c a m p e s t e r o l , s i t o s t e r o l , and s t i g m a s t e r o l ) to c h o l e s t e r o l ( F i g u r e 2) and desmosterol i s the t e r m i n a l i n t e r m e d i a t e i n the c o n v e r s i o n of each of these s t e r o l s to c h o l e s t e r o l ( 9 ) . This was the f i r s t i n t e r m e d i a t e to be i d e n t i f i e d i n the metaboTic c o n v e r s i o n of p h y t o s t e r o l s to c h o l e s t e r o l i n i n s e c t s . F u c o s t e r o l was l a t e r determined to be an i n t e r m e d i a t e between s i t o s t e r o l and desmosterol a n d , a n a l o g o u s l y , 2 4 - m e t h y l e n e c h o l e s t e r o l was found to be the f i r s t i n t e r m e d i a t e between campesterol and c h o l e s t e r o l ( 4 ) . Stigmasterol i s d e a l k y l a t e d and c o n v e r t e d t o 5 , 2 2 , 2 4 - c h o l e s t a t r i e n - 3 3 - o l which i s r e d u c e d , f i r s t to d e s m o s t e r o l , and then to c h o l e s t e r o l (4·). The A 4 - b o n d i s necessary f o r enzyme s p e c i f i c i t y i n o r d e r to reduce the A22-bond. R e s u l t s from research i n our l a b o r a t o r y a l s o i n d i c a t e t h a t o t h e r L e p i d o p t e r a such as the corn earworm, H e l i o t h i s z e a , the f a l l arrçyworm, Spodoptera f r u g i p e r d a ( 1 0 ) , and the I n d i a n meal moth, P l o d i a i n t e r p u n c t e l l a , (11) m e t a b o l i z e C23 and C29 p h y t o s t e r o l s i n a manner s i m i l a r to Manduca. These pathways have been shown to be s i m i l a r i n B. mori and, i n a d d i t i o n , f u c o s t e r o l 2 4 , 2 8 - e p o x i d e has been i d e n t i f i e d as an i n t e r m e d i a t e between f u c o s t e r o l and desmosterol i n mori ( 8 ) . 2

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

178

ECOLOGY AND METABOLISM OF PLANT LIPIDS

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ACS

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; Symposium Series; American Chemical Society: Washington, DC, 1987. Desmosterol

î

t

5,22,24-Cholestatrienol

24-Methylenecholesterol

Stigmasterol

{*

Fucosterol

Cholesterol

F i g u r e 2 . Pathways o f c o n v e r s i o n o f C - 2 4 a l k y l s t e r o l s t o c h o l e s t e r o l i n the tobacco hornworm and o t h e r phytophagous insects. * F u c o s t e r o l 2 4 , 2 8 - e p o x i d e has been shown t o be an i n t e r m e d i a t e between f u c o s t e r o l and desmosterol i n Bombyx mori and Tenebrio m o l i t o r .

Campesterol

Sitosterol

ξ-

s

8

Ο ξ

Η

< ο 00 Ο α > > ζ α

ECOLOGY AND METABOLISM OF PLANT LIPIDS

180

C o l e o p t e r a . The confused f l o u r b e e t l e , T r i b o l i u m confusum, was the f i r s t phytophagous i n s e c t we found t h a t produces an a p p r e c i a b l e amount of a s t e r o l o t h e r than c h o l e s t e r o l from r a d i o l a b e l e d d i e t a r y C28 and C29 p h y t o s t e r o l s . We found t h i s i n s e c t produced l a r g e q u a n t i t i e s o f 7 - d e h y d r o c h o l e s t e r o l , e q u i v a l e n t to as much as 70% o f the t o t a l t i s s u e s t e r o l s i s o l a t e d ( 1 2 ) . I t was f u r t h e r determined t h a t c h o l e s t e r o l and 7-dehydrocTiôlesterol were i n e q u i l i b r i u m i n t h i s f l o u r b e e t l e . Another new i n t e r m e d i a t e , 5 , 7 , 2 4 - c h o l e s t a t r i e n - 3 B - o l was i d e n t i f i e d as an i n t e r m e d i a t e between desmosterol and 7 - d e h y d r o c h o l e s t e r o l ( F i g u r e 3 ) . We found very s i m i l a r pathways of s t e r o l metabolism to e x i s t i n the c l o s e l y r e l a t e d f l o u r b e e t l e , T r i b o l i u m castaneum ( Γ 3 ) . However, another f l o u r b e e t l e , T e n e b r i o ' m o l i t o r , had only about o n e - t h i r d or l e s s of the l e v e l s o f 7 - d e h y d r o c h o l e s t e r o l as the two T r i b o l i u m s p e c i e s , but s t i l l much h i g h e r l e v e l s of t h i s s t e r o l than has been found i n most species. F u c o s t e r o l 2 4 , 2 8 - e p o x i d e was a l s o i m p l i c a t e d as an i n t e r m e d i a t e i n the s y n t h e s i molitor (14). J T v e r y unique m i x t u r e of s t e r o l s was found i n the Mexican bean b e e t l e , E p i l a c h n a v a r i y e s t i s , when s t e r o l s from i n s e c t s fed soybean l e a v e s were a n a l y z e d (15)"! The s t e r o l s from bean b e e t l e pupae c o n s i s t e d o f >70% s a t u r a t e d s t e r o l s and c h o l e s t a n o l was the major s t e r o l i s o l a t e d from the i n s e c t . M e t a b o l i c s t u d i e s w i t h r a d i o l a b e l e d s t e r o l s demonstrated t h a t the Mexican bean b e e t l e does d e a l k y l a t e C28 and C29 p h y t o s t e r o l s , but reduces the Δ -bond f i r s t ( F i g u r e 4 ) ( 1 6 ) . In a d d i t i o n , the A - b o n d can be i n c o r p o r a t e d i n t o c h o l e s t a n o l , and thus a p p r e c i a b l e amounts (>10% of the t o t a l s t e r o l s ) o f l a t h o s t e r o l ( Δ ' - c h o l e s t e n o l ) o c c u r i n the s t e r o l s of t h i s s p e c i e s . Recent s t u d i e s (13) have c o n f i r m e d t h a t i n the metabolism of s i t o s t e r o l and s t i g m a s t e r o l , the s i d e c h a i n d e a l k y l a t i o n and c o n v e r s i o n to a 2 4 - d e s a l k y l s i d e c h a i n i n the Mexican bean b e e t l e p a r a l l e l s the mechanism i n Manduca. A A 2 4 - s t e r o l i s i n v o l v e d as a t e r m i n a l i n t e r m e d i a t e i n the metabolism of both s t e r o l s , and the s i d e c h a i n of s t i g m a s t e r o l i s f i r s t deal k y l ated to form a A 2 2 , 2 4 . - t r m e d i a t e , and then the A22-bond i s reduced. 7

1

Phytophagous Phytosterols

n

e

I n s e c t s Unable to Convert (^Q and C Q Q to Cholesterol

Hemiptera. The l a r g e milkweed bug, Qncopeltus f a s c i a t u s , was the f i r s t phytophagous i n s e c t d i s c o v e r e d to be i n c a p a b l e of c o n v e r t i n g the major p h y t o s t e r o l s (C28 and C29) t o c h o l e s t e r o l ( Γ 7 ) . D i e t a r y s t e r o l s of s u n f l o w e r seeds were i n c o r p o r a t e d e s s e n t i a l l y unchanged i n t o the t i s s u e s and, when i n j e c t e d , n e i t h e r r a d i o l a b e l e d campesterol nor s i t o s t e r o l was m e t a b o l i z e d to c h o l e s t e r o l . A p p a r e n t l y , t h e r e was some s e l e c t i v e uptake of d i e t a r y c h o l e s t e r o l , i n d i c a t e d by an enrichment of c h o l e s t e r o l i n the i n s e c t s t e r o l s compared to the c h o l e s t e r o l c o n c e n t r a t i o n i n the seed s t e r o l s . A C28 e c d y s t e r o i d , m a k i s t e r o n e A ( F i g u r e 1 ) , was i d e n t i f i e d as the major e c d y s t e r o i d of milkweed bug eggs (18) and, s u b s e q u e n t l y , m a k i s t e r o n e A was i d e n t i f i e d as the major e c 3 y s t e r o i d i n hemolymph of l a s t stage milkweed bug nymphs and two o t h e r phytophagous s p e c i e s o f the Pentatomomorpha group of Hemiptera ( 1 9 ) . M a k i s t e r o n e A was

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Sitosterol

Fucosterol HO

7-Dehydrocholesterol

HO"

5,7,24-Cholestatrienol

F i g u r e 3. D e a l k y l a t i o n and c o n v e r s i o n o f s i t o s t e r o l t o 7 - d e h y d r o c h o l e s t e r o l and c h o l e s t e r o l i n T r i b o l i u m confusum.

Desmosterol

HO'

Cholesterol


Ζ

>

Ο CO Ο D

00

to

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

11.

SVOBODA AND THOMPSON

Steroid Metabolism

183

a l s o c a . 10 times more a c t i v e than 20-hydroxyecdysone i n s t i m u l a t i n g c u t i c l e s y n t h e s i s and i n h i b i t i n g v i t e l l o g e n e s i s i n a d u l t milkweed bugs ( 2 0 ) . I t was l a t e r determined t h a t t h i s a d a p t a t i o n t o u t i l i z i n g a C20, p r e c u r s o r f o r e c d y s t e r o i d b i o s y n t h e s i s c o r r e l a t e d w e l l w i t h n e u t r a l s t e r o l metabolism i n each of t h e s e three species (21). S i m i l a r r e s u l t s on s t e r o l u t i l i z a t i o n and o c c u r r e n c e o f màFisterone A have been r e p o r t e d f o r the c o t t o n s t a i n e r bug, Dysdercus f a s c i a t u s ( 2 2 ) . These s p e c i e s a l l have i n common the i n a b i l i t y t o d e a l k y l a t e the C - 2 4 a l k y l s u b s t i t u e n t s o f C28 and C29 p h y t o s t e r o l s . Coleoptera. S t e r o l metabolism s t u d i e s w i t h another i m p o r t a n t s t o r e d p r o d u c t s p e s t , the khapra b e e t l e , Trogoderma g r a n a r i u m , r e v e a l e d a n o t h e r phytophagous i n s e c t t h a t i s unable t o d e a l k y l a t e and c o n v e r t C28 and C29 p h y t o s t e r o l s t o c h o l e s t e r o l ( 2 3 ) . Similar r e s u l t s were o b t a i n e d whether a d i e t c o n s i s t i n g of c r a c k e d wheat and b r e w e r ' s y e a s t o r an a r t i f i c i a s t e r o l s was used ( 2 4 ) . Ther c h o l e s t e r o l from tfie d i e t a r y s t e r o l s , as i n d i c a t e d by an enrichment of c h o l e s t e r o l i n the pupal s t e r o l s (1.2% of t o t a l ) , compared to the d i e t a r y s t e r o l s (0.5% of t o t a l ) . U n l i k e the p r e v i o u s l y d i s c u s s e d s t o r e d product c o l e o p t e r a n p e s t s , T. confusum and T. castaneum, both o f which had h i g h l e v e l s o f 7 - d e h y ï ï r o c h o i e s t e r o l , no 7 - d e h y d r o c h o l e s t e r o l c o u l d be i d e n t i f i e d i n the s t e r o l s from the khapra b e e t l e . Hymenoptera. While examining the e f f e c t s of v a r i o u s d i e t a r y s t e r o l s on brood p r o d u c t i o n i n honey b e e s , A p i s m e l l i f e r a , we d i s c o v e r e d t h a t the honey bee u t i l i z e d d i e t a r y C28 and C29 p h y t o s t e r o l s unchanged ( 2 5 , 2 6 ) . R e g a r d l e s s of the d i e t a r y s t e r o l added to a chemical 1 y-deTTnëcT d i e t , o r even w i t h no s t e r o l added, 2 4 - m e t h y l e n e c h o l e s t e r o l was always the major s t e r o l of the next g e n e r a t i o n of b e e s , and s i t o s t e r o l and i s o f u c o s t e r o l were a l s o p r e s e n t i n a p p r e c i a b l e amounts. Detailed studies with e i t h e r r a d i o l a b e l e d c a m p e s t e r o l , s i t o s t e r o l , or 2 4 - m e t h y l e n e c h o l e s t e r o l added t o the a r t i f i c i a l d i e t p r o v i d e d no e v i d e n c e f o r the metabolism o f any of these p h y t o s t e r o l s to c h o l e s t e r o l o r o t h e r s t e r o l s ( 2 7 ) . In f a c t , H - 2 4 - m e t h y l e n e c h o l e s t e r o l has been t r a c e d unchanged through two g e n e r a t i o n s of bees ( 2 8 ) . Thus, t h e r e i s a very unusual mechanism t h a t e n a b l e s the w o r k e r T e e to s e l e c t i v e l y t r a n s f e r c e r t a i n d i e t a r y s t e r o l s or s t e r o l s c y c l e d from t h e i r endogenous p o o l s t o the brood food t o m a i n t a i n a c o n s t a n t supply o f c e r t a i n s t e r o l s f o r the brood f o o d . The u t i l i z a t i o n of n e u t r a l s t e r o l s by the honey bee and the i n a b i l i t y t o produce c h o l e s t e r o l from the d e a l k y l a t i o n o f 2 4 - a l k y l C g and C29 p h y t o s t e r o l s i s r e f l e c t e d i n the r e c e n t i s o l a t i o n of m a k i s t e r o n e A as the major e c d y s t e r o i d a t peak t i t e r i n the honey bee pupa ( 2 9 ) . We have a l s o found another phytophagous hymenopteran, the a l f a l f a l e a f c u t t e r bee, M e g a c h i l e rotunda t a , t h a t u t i l i z e s d i e t a r y p h y t o s t e r o l s s i m i l a r l y to the honey bee [SB). As i n the honey bee, 2 4 - m e t h y l e n e c h o l e s t e r o l was a major component ( 3 4 . 1 % o f the t o t a l s t e r o l s ) of the s t e r o l s of newly-emerged a d u l t s . In a d d i t i o n , t h e r e was l i t t l e c h o l e s t e r o l (

20-hydroxyecdysone"--^ 20,26-dihydroxyecdysone

[1]

The f a c t t h a t 2 0 , 2 6 - d i h y d r o x y e c d y s o n e was l e s s a c t i v e than ecdysone suggests t h a t i t was an i n a c t i v a t i o n product and t h a t we c o u l d expect an i n c r e a s e i n the q u a n t i t y of t h i s compound a t a l a t e r p e r i o d of p u p a l - a d u l t development. Indeed, 2 0 , 2 6 - d i h y d r o x y e c d y s o n e was the major e c d y s t e r o i d d u r i n g p u p a l - a d u l t development f i v e days a f t e r peak t i t e r of MH a c t i v i t y , f o l l o w e d by l e s s e r amounts of 3-epi-20-hydroxyecdysone, 20-hydroxyecdysone, 3 - e p i - 2 0 , 2 6 - d i h y d r o x y e c d y s o n e , 3 - e p i e c d y s o n e and ecdysone ( £ ) . W h i l e the i n c r e a s e d c o n c e n t r a t i o n of 2 0 , 2 6 - d i h y d r o x y e c d y s o n e was a n t i c i p a t e d as a r e s u l t o f 20-hydroxyecdysone m e t a b o l i s m , i t cannot be r u l e d out t h a t 2 0 , 2 6 - d i h y d r o * y e c d y s o n e has a p h y s i o l o g i c a l r o l e of i t s own i n i n s e c t development (3). This assumption was supported

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

12. THOMPSON ET AL.

Ecdysteroids in the Tobacco Hornworm

189

by the f a c t t h a t ecdysone, 2 0 - h y d r o x y e c d y s o n e , and 2 0 , 2 6 - d i h y d r o x y e c d y s o n e were a l l e p i m e r i z e d t o the f a r l e s s a c t i v e 3-epi-ecdysterolds (4). F i v e days a f t e r peak t i t e r , the 3 a - e c d y s t e r o i d s q u a n t i t a t i v e l y surpassed t h e i r 3 & - i s o m e r s . 3 - E p i e c d y s o n e and 3 - e p i - 2 0 - h y d r o x y e c d y s o n e are a p p r o x i m a t e l y 1/10 as a c t i v e as ecdysone (4) i n the house f l y assay (5). 3-Epi-20,2T>-dihydroxyecdysone i s 1/20 as a c t i v e as 2 0 , 2 6 - d i h y d r o x y e c d y s o n e and 1/300 as a c t i v e as ecdysone ( 4 ) . Thus, the b i o l o g i c a l a c t i v i t y o f the 3 , presumed c o n v e r s i o n s ; - > - > a s s u m e d two-step r e a c t i o n s ) ,

20,26-dihydroxyecdysone—> - - > 3 - e p i -20,26-dihydroxyecdysone When the f i r s t 3 - e p i e c d y s t e r o i d s were i s o l a t e d (j>,7), we p o s t u l a t e d t h a t the c o n v e r s i o n of 3 f J - e c d y s t e r o i d s to t ï ï e i r 3 a - i s o m e r s c o u l d proceed through t h e i r r e s p e c t i v e 3-dehydroecdysteroids ( F i g . 2). During t h a t y e a r t h e r e were r e p o r t s t h a t l a b e l e d ecdysone and 20-hydroxyecdysone were c o n v e r t e d t o 3-dehydroecdysone and 3 - d e h y d r o - 2 0 - h y d r o x y e c d y s o n e r e s p e c t i v e l y i n C a l 1 i p h o r a v i c i n a R o b i n e a l 1 - D e s v o i d y (8) and L o c u s t a m i g r a t o r i a (L) (9). More r e c e n t l y the f o r m a t i o n and T a t e of 3 - d e h y d r o e c d y s t e r o i d s were s t u d i e d i n v i t r o i n t i s s u e e x t r a c t s from P i e r i s b r a s s i c a e ( L ) ( 1 0 ) . The a u t h o r s showed t h a t t h r e e c y t o s o l i c enzymes are i n v o l v e d : an ecdysone o x i d a s e c o n v e r t s 3 0 - h y d r o x y e c d y s t e r o i d s i n t o 3 - d e h y d r o e c d y s t e r o i d s , and two d i f f e r e n t r e d u c t a s e s , r e q u i r i n g NADPH as c o f a c t o r s , t r a n s f o r m 3 - d e h y d r o e c d y s t e r o i d s i n t o 3a-hydroxy o r the 30-hydroxy f o r m s , r e s p e c t i v e l y . I n t e r e s t i n g l y , the h y d r o x y e c d y s t e r o i d f o r m a t i o n i s only d e t e c t e d i n the gut ( 1 0 ) . It indeed appears t h a t c o n v e r s i o n of 3 f 5 - h y d r o x y e c d y s t e r o i d s to 3). 3 - E p i e c d y s o n e was a l s o i d e n t i f i e d as the p r o d u c t of an i n v i t r o enzyme system from Manduca m i d g u t . These two

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

192

ECOLOGY AND METABOLISM OF PLANT LIPIDS

FEMALE TOBACCO HORNWORM PUPAE (130 g) Homogenized i n MeOH, then i n 70% MeOH

AQUEOUS MeOH EXTRACTS Reduced i n volume under vacuum, p a r t i t i o n e d between hexane and 70% MeOH

AP0LAR STEROLS IN HEXANE

1

4

t

POLAR STEROIDS, CONJUGATES IN 70% MeOH

5.80 Χ 10 dpm

|

6

1.46 Χ 1 0 dpm 6

Dried under vacuum P a r t i t i o n e d between BuOH and water

FREE ECDYSTEROIDS IN BuOH

M • • » CONJUGATED ECDYSTEROIDS IN H 0 2

0.26 X 10° (dpm

1.2 Χ 1 0 dpm (413 mg) 6

Reduced to dryness under vacuum,

RESIDUE (3.74 g)

XAD-16 Column ( 2 . 5 χ 23 cm) 1) H 0 (500 ml) (Discarded) 2

2) EtOH (500 ml) Dried under vacuum

RESIDUE (300 mg; 2.6 χ 1 0 dpm) 5

C Discarded

2

SEP-PAK (150 mg; 1.3 χ 1 0 dpm) 5

1 8

1) 5 ml 10% MeOH j 5

m l

l Q %

M e U H

^

u

3) 5 ml 30% MeOH (14.8 mg; 0.56 χ 1Q dpm) 4) 10 ml 30% MeUH ( 3 . 8 mg; 0.43 χ 1 0 dpm)m) 5) 5 ml 40% MeOH ( 5 . 0 mg; 0.23 χ 1 0 ) 5

b

5

F i g u r e 3. conjugates

Procedure used f o r the i s o l a t i o n of from Manduca pupae.

d p m

Ι ρ πνςτΡοητη ttuibitKUiu Γ

1

CONJUGATES

ecdysteroid

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

THOMPSON ET AL.

Ecdysteroids in the Tobacco Hornworm

5-7

ΓΊ

1

4

J 8

1

12

TIME (min) F i g u r e 4. Ion s u p p r e s s i o n r e v e r s e d - p h a s e HPLC t r a c e i n d i c a t i n g r a d i o a c t i v e peaks o f p a r t i a l l y p u r i f i e d e c d y s t e r o i d c o n j u g a t e s from 8 - d a y - o l d Manduca pupae, on IBM C column ( 4 . 6 mm χ 15 cm) by i s o c r a t i c e l u t i o n w i t h 30% methanol i n 0 . 0 3 M aqueous N a H P 0 s o l u t i o n (pH 5) a t f l o w r a t e of 0 . 8 ml/min. g

2

4

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ECOLOGY AND METABOLISM OF PLANT LIPIDS

194

l i n e s of r e s e a r c h l e d t o the f i r s t i s o l a t i o n and i d e n t i f i c a t i o n of 3a-ecdysteroids. S i n c e the f i r s t s t e r o l c o n j u g a t e from an i n s e c t source was i s o l a t e d from the meconium of the tobacco hornworm (16), we are c u r r e n t l y c o n d u c t i n g q u a l i t a t i v e and q u a n t i t a t i v e comparative s t u d i e s of f r e e and c o n j u g a t e d e c d y s t e r o i d s of the meconium of male and female pupae i n j e c t e d w i t h [ C ] c h o l e s t e r o l on day 13. 1 4

Embryonated Eggs of t h e

Hornworm

Young e g g s , 1- to 4 - h o u r - o l d c o n t a i n n e g l i g i b l e MH a c t i v i t y (17), whereas o l d e r embryonated e g g s , 24- t o 4 4 - h o u r - o l d (18) o r 48- t o 6 4 - h o u r - o l d (17), have r e l a t i v e l y high m o l t i n g hormone t i t e r . In both l i ï e 24- t o 44-hour-group and 48- t o 6 4 - h o u r - g r o u p , 26-hydroxyecdysone was the major e c d y s t e r o i d a c c o u n t i n g f o r n e a r l y 80% of the t o t a l f r e e e c d y s t e r o i d s , whereas the t h r e e MH's i s o l a t e d d u r i n g p u p a l - a d u l t development, ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone e c d y s t e r o i d of eggs. 26 e c d y s t e r o i d i n younger embryonated eggs (4- to 1 8 - h o u r - o l d ) a c c o u n t i n g f o r about 90% o f the t o t a l e c d y s t e r o i d s (19). In a d d i t i o n , e c d y s o n e , 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were a l s o p r e s e n t i n 4- t o 18-hour o l d eggs. These accounted f o r a l l of the MH a c t i v i t y though c o m p r i s i n g l e s s than 2% o f the t o t a l r e c o v e r e d e c d y s t e r o i d s . Interestingly, 26-hydroxyecdysone was shown to be devoid of MH a c t i v i t y i n the house f l y assay and ecdysone was the major M H - a c t i v e e c d y s t e r o i d i n hornworm eggs (19). In a d d i t i o n to the 3 3 - e c d y s t e r o i d s , two 3 a - e c d y s t e r o i d s , 3 - e p i - 2 6 - h y d r o x y e c d y s o n e , and 3 - e p i - 2 0 , 2 6 - d i h y d r o x y e c d y s o n e were i s o l a t e d and i d e n t i f i e d from 4- to 1 8 - h o u r - o l d e g g s . 3 - E p i - 2 6 - h y d r o x y e c d y s o n e comprised 3% of the t o t a l r e c o v e r e d e c d y s t e r o i d s and was the second major component i s o l a t e d from t h i s groups of e g g s . Thus, from these eggs, s i x e c d y s t e r o i d s were c o n c l u s i v e l y or t e n t a t i v e l y i d e n t i f i e d and s i x o t h e r u n i d e n t i f i e d e c d y s t e r o i d s were i s o l a t e d . In our r e c e n t s t u d i e s concerned w i t h e c d y s t e r o i d s i n d e v e l o p i n g o v a r i e s and eggs of t h e THW (20), and the f a t e of r a d i o l a b e l e d s t e r o i d s i n o v a r i e s and eggs ô T the THW (12), we encountered some i n t e r e s t i n g and c o n f l i c t i n g r e s u l t s . In T a b l e I we show t h a t i n 1t o 1 8 - h o u r - o l d eggs more than 63% o f the e c d y s t e r o i d e x i s t e d i n the f r e e form. The p r o p o r t i o n was s i m i l a r i n 48- t o 6 4 - h o u r - o l d eggs, though the sum of the f r e e and conjugated e c d y s t e r o i d was f a r l e s s i n 48- t o 6 4 - h o u r - o l d eggs. In our l a t e s t s t u d y , we showed t h a t f o l l o w i n g i n j e c t i o n i n t o Manduca female pupae (day 16), [ C ] c h o l e s t e r o l was c o n v e r t e d t o l a b e l e d e c d y s t e r o i d c o n j u g a t e s (12), o f which the l a t t e r i s m a i n l y 26-hydroxyecdysone 26-phosphate~Tll)« In t h i s s t u d y , however, 48- t o 6 4 - h o u r - o l d or 72- t o 8 8 - h o u r - o l d eggs were found to c o n t a i n l i t t l e i f any f r e e e c d y s t e r o i d s (Table I I ) . The absence of f r e e e c d y s t e r o i d s i n these egg groups was unexpected and c o m p l e t e l y d i f f e r e n t from r e s u l t s of our e a r l i e r s t u d i e s (20). In our search f o r an e x p l a n a t i o n f o r the d i f f e r e n c e s between tTie" r e s u l t s of t h i s study and those of our e a r l i e r s t u d i e s (20), we determined t h a t , i n t h i s s t u d y , we had 1 4

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

12. THOMPSON ET AL.

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195

Table I. T i t e r of Free and Conjugated E c d y s t e r o i d s o f O v a r i e s and Eggs a t V a r i o u s Developmental Stages of the Tobacco Hornworm (20)

Ovaries from 9 3 - h o u r - o l d A d u l t Females Ecdysone 26-Hydroxyecdysone 0 - t o 1 - h o u r - o l d Eggs** Ecdysone 26-Hydroxyecdysone 1 - t o 1 8 - h o u r - o l d Eggs++ Ecdysone 26-Hydroxyecdysone 4 8 - to 6 4 - h o u r - o l d Eggs+ Ecdysone 26-Hydro ^ e c d y s o n e

Free Ecdysteroids Mg/g f r e s h weight*

Conjugated Ecdysteroids pg/g f r e s h weight**

not d e t e c t e d not d e t e c t e d

0.58 20.20

not d e t e c t e d 0.96

0.73 21.00

0.38

0.22

0.02 6.90

not d e t e c t e d 6.0

Determined by RP-HPLC Expressed as Free E c d y s t e r o i d s Recovered A f t e r H y d r o l y s i s of Conjugates **Tissues S t o r e d i n MeOH AT -20°C T i s s u e s S t o r e d a t -20°C

Enzymatic

+ +

immediately p l a c e d and s t o r e d a l l b i o l o g i c a l m a t e r i a l i n methanol a t -20°C ( T a b l e I I ) . P r e v i o u s l y , t h i s was done o n l y w i t h o v a r i e s and 0 - t o 1 - h o u r - o l d e g g s . O l d e r eggs were r o u t i n e l y p l a c e d i n g l a s s b o t t l e s and kept a t -20°C u n t i l w o r k - u p . Perhaps c e r t a i n phosphatases of these eggs were a c t i v a t e d by the l o w e r i n g of the temperature and subsequent h y d r o l y s i s of the e c d y s t e r o i d c o n j u g a t e s caused an a c c u m u l a t i o n of f r e e e c d y s t e r o i d s . There c o u l d a l s o have been a sudden b u r s t of enzyme a c t i v i t y a t a c r i t i c a l temperature d u r i n g c o o l i n g of the eggs t o -20°C. Undoubtedly, the low temperature d e s t r o y e d the normal embryonic developmental p r o c e s s e s , but d i d not e l i m i n a t e t h i s h y d r o l y t i c a c t i v i t y . E f f o r t s are i n p r o g r e s s to determine the e x a c t c o n d i t i o n s t h a t cause the hydrolysis. We are a l s o a c c u m u l a t i n g l a r g e q u a n t i t i e s of eggs ( 1 t o 1 8 - h o u r - o l d ) , s t o r e d i n m e t h a n o l , from which the f r e e ( i f any) and c o n j u g a t e d e c d y s t e r o i d s w i l l be i s o l a t e d . The c o n j u g a t e s w i l l then be e n z y m a t i c a l l y h y d r o l y z e d to determine i f the s i x e c d y s t e r o i d s p r e v i o u s l y i d e n t i f i e d i n 4 - t o 1 8 - h o u r - o l d eggs (19) are indeed the n a t u r a l e c d y s t e r o i d s of t h i s age group. Our r e s u l t s o f a n a l y s e s of e c d y s t e r o i d s of o v a r i e s i n both s t u d i e s ( 1 2 , 2 0 ) agree i n t h a t no f r e e e c d y s t e r o i d s were d e t e c t e d a l t h o u g h improvements i n the method o f a n a l y s e s of e c d y s t e r o i d c o n j u g a t e s c o u l d account f o r the g r e a t e r amount of the 26-hydroxyecdysone c o n j u g a t e (31 pg/g o f o v a r i e s ) (Table I I ) b e i n g found i n the r a d i o l a b e l e d study compared t o 20 pg/g found p r e v i o u s l y (20)(Table I ) . We would a d v i s e t h a t every e f f o r t be made to d e s t r o y o r e l i m i n a t e any p o t e n t i a l f o r i n i t i a t i o n or c o n t i n u a t i o n of enzyme

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

196

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Table I I . T i t e r of Free and Conjugated E c d y s t e r o i d s of O v a r i e s ^ and Eggs a t V a r i o u s Developmental Stages of the Tobacco Hornworm Conjugated Ecdysteroids Mg/g f r e s h weights*

Free Ecdysteroids Mg/g f r e s h weights" " 1

Ovaries from 9 3 - h o u r - o l d A d u l t hemales ecdysone 26-Hydroxyecdysone 4 8 - t o 6 4 - h o u r - o l d Eggs** Ecdysone 26-Hydroxyecdysone 72- to 88-hour-old-Eggs** Ecdysone 26-Hydroxyecdysone

trace 31

not d e t e c t e d not d e t e c t e d

trace 25

not d e t e c t e d

trace

* C - C h o l e s t e r o l I n j e c t e d I n t o Pupae (Day Determined by RP-HPLC * T i s s u e s S t o r e d i n MeOH a t -20° 1 4

r

not d e t e c t e d not d e t e c t e d

16)

a c t i o n b e f o r e s t o r i n g any b i o l o g i c a l m a t e r i a l . T h i s i s very i m p o r t a n t i f a n a l y t i c a l r e s u l t s or f i n a l c o n c l u s i o n s d e r i v e d from such m a t e r i a l a r e to be c o n s i d e r e d v a l i d . On the o t h e r hand, i f eggs i n i t i a l l y had been s t o r e d i n methanol a t - 2 0 C , the d i s c o v e r y o f 26-hydroxyecdysone i n M. s e x t a eggs ( l ^ ) would have a t l e a s t been delayed. U n l i k e c o n j u g a t e s i n eggs of o t h e r i n s e c t s p e c i e s , the c o n j u g a t e s i n eggs of the THW c o n t a i n , p r i m a r i l y , 26-hydroxyecdysone ( T a b l e s I and I I ) . T h i s enhanced the i s o l a t i o n and i d e n t i f i c a t i o n of the major c o n j u g a t e of eggs of the THW. For t h i s work we used 4 8 t o 6 4 - h o u r - o l d eggs t h a t had been s t o r e d f r o z e n a t - 2 0 C a l t h o u g h t h i s age group o f eggs c o n t a i n e d only about 6 pg of the 26-hydroxyecdysone c o n j u g a t e per gram o f eggs (Table I ) . A p p r o x i m a t e l y 750 pg of a c h r o m a t o g r a p h i c a l l y pure c o n j u g a t e was i s o l a t e d from 120 g of 4 8 - t o 6 4 - h o u r - o l d eggs ( 1 1 ) . Enzymatic h y d r o l y s i s of the c o n j u g a t e w i t h a c i d phosphatase from human seminal f l u i d gave 26-hydroxyecdysone. The c o n j u g a t e was i d e n t i f i e d as 26-hydroxyecdysone 26-phosphate ( F i g . 1) by NMR and f a s t atom bombardment mass s p e c t r o m e t r y . The compound i s a l s o the major c o n j u g a t e of n e w l y - l a i d eggs ( 0 - t o 1 - h o u r - o l d ) . Thus, l a r g e q u a n t i t i e s o f t h i s c o n j u g a t e are r e a d i l y a v a i l a b l e from a r e l a t i v e l y small q u a n t i t y o f 1 - t o 1 8 - h o u r - o l d THW eggs (25 t o 31 pg/g of eggs) p r o v i d i n g t h a t when they are c o l l e c t e d they are immediately s t o r e d i n methanol a t -20°C u n t i l work-up ( T a b l e I I ) . T h i s age group of eggs i s e a s i l y c o l l e c t e d and a c c u m u l a t e d . Though the r a t i o o f 26-hydroxyecdysone to o t h e r e c d y s t e r o i d s i n eggs and o v a r i e s o f the THW can be as h i g h as 2 0 : 1 ( 2 0 ) , i t was not a n t i c i p a t e d t h a t the C - 2 6 hydroxy! would be the o n l y p o s i t i o n involved i n conjugate formation. In a study i n which the c o n j u g a t e was l a b e l e d , RP-HPLC and r a d i o a s s a y a n a l y s e s showed a r a d i o a c t i v e peak p r e c e d i n g the peak of 26-hydroxyecdysone 2 6 - p h o s p h a t e . This e

e

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

12. THOMPSON ET AL.

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peak was a l s o p r e s e n t i n the chromatogram of eggs from which we i s o l a t e d 26-hydroxyecdysone 26-phosphate ( Π ) . We have now accumulated enough of t h i s m a t e r i a l t o determine whether i t i s a phosphate c o n j u g a t e of ecdysone o r 26-hydroxyeccfysone and the p o s i t i o n of conjugation. I t has been surmised t h a t f o r m a t i o n of c o n j u g a t e s a l l o w s f o r s t o r a g e o f l a r g e q u a n t i t i e s of e c d y s t e r o i d s i n i n s e c t eggs to be s u b s e q u e n t l y r e l e a s e d d u r i n g a developmental stage ( i . e . embryogenesis) i n c a p a b l e of s t e r o i d uptake and/or c o n v e r s i o n ( 2 1 - 2 4 ) . Our r e s u l t s w i t h eggs s t o r e d i n methanol a t -20°C (Table ITT suggest t h a t c o n t r o l l e d r e l e a s e of f r e e e c d y s t e r o i d s i n M. s e x t a eggs i s more l i m i t e d than p r e v i o u s l y thought ( 2 0 ) . C e r t a i n l y no s i g n i f i c a n t amounts of f r e e e c d y s t e r o i d s could~ïïe d e t e c t e d . In f a c t , RP-HPLC and r a d i o a s s a y a n a l y s e s of the e c d y s t e r o i d c o n j u g a t e f r a c t i o n o f 7 2 - t o 8 8 - h o u r - o l d eggs show the presence of a d d i t i o n a l and s u b s t a n t i a l q u a n t i t i e s of more p o l a r c o n j u g a t e s ( 1 2 ) . P r e s e n t l y , the f a t e o f 26-hydroxyecdyson w i l l have t o a w a i t the i d e n t i f i c a t i o conjugates. Our i s o l a t i o n of ecdysone, 2 0 - h y d r o x y e c d y s o n e , 2 0 , 2 6 - d i h y d r o x y e c d y s o n e , 3 - e p i - 2 0 , 2 6 - d i h y d r o x y e c d y s o n e , and 26-hydroxyecdysone from THW eggs l e d us to suggest t h a t t h e r e were a t l e a s t two b i o s y n t h e t i c pathways f o r e c d y s t e r o i d s d u r i n g embryonic development of the hornworm: the pathway through 26-hydroxyecdysone as the p r i n c i p a l r o u t e and the f o r m a t i o n o f 20-hydroxyecdysone as a minor pathway, w i t h ecdysone s e r v i n g as an i n t e r m e d i a t e i n both pathways ( J J ) . There s t i l l i s no d i r e c t e v i d e n c e t h a t ecdysone s e r v e s as a p r e c u r s o r f o r 26-hydroxyecdysone. In f a c t , f o l l o w i n g i n j e c t i o n o f [ H ] e c d y s o n e i n t o female pupae (day 1 6 ) , t h e r e was no i n c o r p o r a t i o n of r a d i o a c t i v i t y i n t o o v a r i e s or eggs (.12). On the o t h e r hand [ l C ] c h o l e s t e r o l was r e a d i l y i n c o r p o r a t e d and m e t a b o l i z e d t o 2 6 - [ l C ] h y d r o x y e c d y s o n e 26-phosphate ( 1 2 ) . P e r h a p s , [ H ] e c d y s o n e s h o u l d be i n j e c t e d i n tobacco hornworms a t an e a r l i e r stage o f the l i f e c y c l e (5th i n s t a r ) to determine whether i t i s i n c o r p o r a t e d and m e t a b o l i z e d to 2 6 - h y d r o x y e c d y s o n e , the major e c d y s t e r o i d o f o v a r i e s and eggs of Manduca. The e c d y s t e r o i d c o m p o s i t i o n of eggs i n d i c a t e s t h a t t h e r e a r e a t l e a s t two e c d y s t e r o i d c o n j u g a t e s p r e s e n t i n o v a r i e s and 0 - to 1 - h o u r - o l d eggs and s e v e r a l i n o l d e r eggs ( 7 2 - t o 8 8 - h o u r - o l d ) and very l i t t l e i f any f r e e e c d y s t e r o i d s ( 1 ^ ) · 26-Hydroxyecdysone 26-phosphate i s the major c o n j u g a t e of o v a r i e s and i s by f a r the predominant c o n j u g a t e throughout embryogenesis. Thus, our p r e s e n t knowledge of no known i n t e r m e d i a t e s i n the i n c o r p o r a t i o n of [ 1 4 C ] c h o l e s t e r o l i n t o the o v a r i a n e c d y s t e r o i d s i n Manduca can o n l y be expressed as shown i n [ 3 ] : 3

4

4

3

[l C]cholesterol---> 4

--•>

26-[l C]hydroxyecdysone 26-phosphate 4

[3]

Though 26-hydroxyecdysone i s w i t h o u t m o l t i n g hormone a c t i v i t y i n the house f l y a s s a y , the e x c e p t i o n a l l y high c o n c e n t r a t i o n of 26-hydroxyecdysone c o n j u g a t e i n o v a r i e s and e g g s , t o g e t h e r w i t h the assumption t h a t i t i s u n l i k e l y t h a t o v a r i e s and eggs o n l y serve as a d e p o s i t o r y o f waste f o r i n a c t i v a t e d e c d y s t e r o i d s , c e r t a i n l y i n d i c a t e some p h y s i o l o g i c a l r o l e f o r 26-hydroxyecdysone.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Conclusions The i s o l a t i o n and i d e n t i f i c a t i o n of f r e e e c d y s t e r o i d s from Manduca and i n s e c t s i n general has not been too d i f f i c u l t , because we had p a r t i t i o n systems t h a t e f f e c t i v e l y s e p a r a t e d f r e e e c d y s t e r o i d s from their impurities. For example, from p r o c e s s i n g 130 g of pupae, the e c d y s t e r o i d s which p a r t i t i o n i n t o the butanol phase are now p r e s e n t i n o n l y 413 mg of r e s i d u e ( F i g . 3 ) . T h i s m a t e r i a l c o u l d be f u r t h e r p u r i f i e d by column and t h i n - l a y e r chromatography and c o u n t e r c u r r e n t distribution. On the o t h e r hand, the e c d y s t e r o i d c o n j u g a t e s a r e p r e s e n t i n 3 . 7 4 g of r e s i d u e t h a t i s w a t e r - s o l u b l e which p r e s e n t s a d d i t i o n a l o b s t a c l e s to f u r t h e r p u r i f i c a t i o n . The nature of the c o n j u g a t i o n or the i m p u r i t i e s p r e s e n t q u i t e o f t e n prevented s u c c e s s f u l column or t h i n - l a y e r chromatography of the c o n j u g a t e s . More r e c e n t l y , however, a method was d e s c r i b e d f o r the s e p a r a t i o n of f r e e and c o n j u g a t e d e c d y s t e r o i d s and the i s o l a t i o n of e c d y s t e r o i d conjugates (21). We have now s u c c e s s f u l l XAD-16 r e s i n i n the p u r i f i c a t i o n of our c o n j u g a t e s . After applying the 3 . 7 4 g of r e s i d u e from the aqueous phase to the XAD-16 column b e t t e r than 92% of the i m p u r i t i e s were removed from the column w i t h water ( F i g . 3) and the p a r t i a l l y p u r i f i e d c o n j u g a t e s were e l u t e d w i t h e t h a n o l . The C i s SEP-PAK f r a c t i o n a t i o n removed a d d i t i o n a l impurities. F i n a l p u r i f i c a t i o n can now be a c h i e v e d by i o n s u p p r e s s i o n RP-HPLC f o l l o w e d by d e s a l t i n g on SEP-PAK. Thus, w i t h the p r e s e n t - d a y i n s t r u m e n t a t i o n and t e c h n i q u e s we are now r o u t i n e l y a b l e to i s o l a t e and i d e n t i f y both f r e e and conjugated e c d y s t e r o i d s . The c o n v e r s i o n of [ ^ C ] c h o l e s t e r o l to l a b e l e d f r e e and c o n j u g a t e d e c d y s t e r o i d s i n Manduca d u r i n g v a r i o u s developmental stages f u r t h e r enhances our c a p a b i l i t i e s f o r d e t e r m i n i n g the f a t e of e c d y s t e r o i d s i n Manduca a t any developmental s t a g e . Tremendous p r o g r e s s i s being made i n f o l l o w i n g the f a t e of e c d y s t e r o i d s i n Manduca eggs d u r i n g embryogenesis. The mixture of e c d y s t e r o i d c o n j u g a t e s of Manduca pupae a t peak t i t e r ( F i g . 4) a l s o a w a i t s i d e n t i f i c a t i o n . We are now i n a p o s i t i o n to i n v e s t i g a t e the i n t e r r e l a t i o n s h i p between f r e e and c o n j u g a t e d e c d y s t e r o i d s . C e r t a i n l y , the c o n t i n u e d i d e n t i f i c a t i o n of f r e e and c o n j u g a t e d e c d y s t e r o i d s a t v a r i o u s developmental s t a g e s w i l l enhance our u n d e r s t a n d i n g of the p h y s i o l o g i c a l c o n t r o l of e c d y s t e r o i d s i n Manduca and i n s e c t s i n general as w e l l as our knowledge and u n d e r s t a n d i n g o f the v a r i o u s enzymes i n v o l v e d . With each stage of i n s e c t development r e p r e s e n t i n g a p o s s i b l e t a r g e t f o r s e l e c t i v e d i s r u p t i o n of s t e r o i d m e t a b o l i z i n g pathways, i n h i b i t o r s of these pathways ( m o l t i n g hormone metabolism) o f f e r p o t e n t i a l means of insect control. I t i s a l s o p o s s i b l e t h a t one c o u l d develop i n s e c t r e s i s t a n t c r o p s by i n t r o d u c i n g the c a p a b i l i t y of p r o d u c i n g c e r t a i n of the i n s e c t i n h i b i t o r s i n the p l a n t . 4

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Goodwin, T. W.; Horn, D. H. S.; Karlson, P.; Koolman, J.; Nakanishi, K.; Robbins, W. E . ; Siddall, J. B.; Takemoto, T. Nature 1978, 272, 122. Kaplanis, J. N.; Thompson, M. J.; Yamamoto, R. T.; Robbins, W. E.; Louloudes, S. J. Steroids 1966, 8, 605-623. Thompson, M. J.; Kaplanis, J. N.; Robbins, W. E . ; Yamamoto, R. T. Chem. Commun. 1967, 650-3. Kaplanis, J. N.; Thompson, M. J.; Dutky, S. R.; Robbins, W. E. Steroids 1979, 34, 333-345. Kaplanis, J. N.; Tabor, L. Α.; Thompson, M. J.; Robbins, W. E . ; Shortino, T. J. Steroids 1966, 8, 625-631. Nigg, H. N.; Svoboda, J. A.; Thompson, M. J.; Kaplanis, J. N.; Dutky, S. R.; Robbins, W. E. Lipids 1974, 9, 971-4. Thompson, M. J.; Kaplanis J. N.; Robbins W E.; Dutky S R.; Nigg, H. N. Steroid Karlson, P.; Koolman Hoffmann, J. Α.; Koolman, J.; Karlson, P.; Joly, P. Gen. Comp. Endocrinol. 1974, 22, 90-7. Blais, C.; LaFont, R. Hoppe-Seyler's Z. Physiol. Chem. 1984, 365, 809-817. Thompson, M. J.; Weirich, G. F.; Rees, H. H.; Svoboda, J. Α.; Feldlaufer, M. F . ; Wilzer, K. R. Arch. Insect Biochem. Physiol. 1985, 2, 227-236. Thompson, M. J.; Svoboda, J. Α.; Feldlaufer, M. F.; Lozano, R. Lipids 1985, In press. Weirich, G. F. In "Methods in Enzymology"; Law, J. H.; Rilling, H. C., Eds.; Academic Press: New York, 1985; Vol. 111, pp. 454-458. Weirich, G. F.; Thompson, M. J.; Svoboda, J. A. Arch. Insect Biochem. Physiol., In press. Thompson, M. J.; Kaplanis, J. N.; Robbins, W. E.; Dutky, S. R.; Nigg, H. N. Steroids 1974, 24, 359-366. Hutchins, R. F. Ν.; Kaplanis, J. N. Steroids 1969, 13, 605-614. Kaplanis, J. N.; Robbins, W. E . ; Thompson, M. J.; Dutky, S. R. Science 1973, 180, 307-308. Kaplanis, J. N.; Robbins, W. E.; Thompson, M. J.; Dutky, S. R. Steroids 1976, 27, 675-9. Kaplanis, J. N.; Thompson, M. J.; Dutky, S. R.; Robbins, W. E. Steroids 1980, 36, 321-335. Thompson, M. J.; Svoboda, J. Α.; Weirich, G. F. Steroids 1984, 43, 333-341. Dinan, L. N.; Rees, Η. H. J. Insect Physiol. 1981, 27, 51-8. Lagueux, M.; Sail, C.; Hoffmann, J. A. Am. Zool. 1981, 21, 715-726. Sail, C.; Tsoupras, G.; Kappler, C.; Lagueux, M.; Zachary, D.; Luu, B.; Hoffmann, J. A. J. Insect Physiol. 1983, 29, 491-507. Isaac, R. E . ; Sweeney, F. P.; Rees, Η. H. Biochem. Soc. Trans. 1983, 11, 379-380.

Received May 1,1986

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 13

Metabolism and Function of Sterols in Nematodes David J. Chitwood, Ruben Lozano, William R. Lusby, Malcolm J. Thompson, and James A. Svoboda Insect Physiology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD 20705

Current knowledge of sterol biochemistry and physiology in nematodes is reviewed. Nematodes possess a nutritiona they lack the capacit biosynthesis. The free-living nematode Caenorhabditis elegans has recently been used as a model organism for investigation of nematode sterol metabolism. C. elegans is capable of removal of the C-24 alkyT substituent of plant sterols such as sitosterol and also possesses the remarkable ability to attach a methyl group at C-4 on the sterol nucleus. An azasteroid and several long-chain alkyl amines disrupt the phytosterol dealkylation pathway in C. elegans by inhibiting its ∆ -sterol reductase. These compounds inhibit growth and reproduction in certain parasitic nematodes and provide model compounds for development of novel nematode control agents. 24

Nematodes a r e nonsegmented roundworms which i n c l u d e s e v e r a l d i v e r s e groups. F r e e - l i v i n g nematodes i n c l u d e m i c r o s c o p i c s o i l - d w e l l i n g o r a q u a t i c s p e c i e s t h a t feed on microorganisms and dead o r g a n i c m a t t e r . Of g r e a t e r economic importance i n the s o i l a r e p l a n t - p a r a s i t i c nematodes, which cause an e s t i m a t e d annual l o s s o f s i x b i l l i o n d o l l a r s t o American a g r i c u l t u r e ( 1 ) . Because o f t h e i r f r e q u e n t l y l a r g e r s i z e as w e l l as t h e i r t h r e a t t o human h e a l t h , a n i m a l - p a r a s i t i c nematodes are more f a m i l i a r t o the general p u b l i c and i n c l u d e a s c a r i d s , hookworms, pinworms, the dog heartworm, and the c a u s a l agents o f t r i c h i n o s i s , e l e p h a n t i a s i s , and r i v e r blindness. Readers w i t h f u r t h e r c u r i o s i t y about the l i f e h i s t o r y o r b i o l o g y o f nematodes are r e f e r r e d t o r e c e n t monographs by P o i n a r (2) and Maggenti (3). The p r e s e n t d i f f i c u l t y i n r o u t i n e c u l t u r e of p a r a s i t i c nematodes through t h e i r e n t i r e l i f e c y c l e s away from t h e i r p l a n t o r animal hosts has s e v e r e l y h i n d e r e d i n v e s t i g a t i o n s o f t h e i r b i o c h e m i s t r y and p h y s i o l o g y . C o n s e q u e n t l y , most s t u d i e s of s t e r o l n u t r i t i o n and metabolism i n nematodes have n e c e s s a r i l y i n v o l v e d the This chapter not subject to U.S. copyright. Published 1987, American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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use of f r e e - l i v i n g genera such as C a e n o r h a b d i t i s , T u r b a t r i x and Panagrellus. These can be e a s i l y propagated upon b a c t e r i a or i n s t e r i l e l i q u i d media c o n t a i n i n g s e m i d e f i n e d components such as y e a s t e x t r a c t and soy peptone o r , w i t h g r e a t e r d i f f i c u l t y , i n c h e m i c a l l y d e f i n e d l i q u i d media. H i s t o r i c a l l y , p a r a s i t i c nematodes have been d i f f i c u l t to c o n t r o l f o r s e v e r a l r e a s o n s , i n c l u d i n g the r e s i s t a n c e of the nematode c u t i c l e t o p e n e t r a t i o n by p o t e n t i a l n e m a t i c i d e s , the r e s i s t a n c e of the s o i l to the m i g r a t i o n of n e m a t i c i d e s a p p l i e d t o i t , the h i g h mammalian t o x i c i t y of many a n t h e l m i n t i c s , and the general s i m i l a r i t y of the m e t a b o l i c pathways found i n these p a r a s i t e s and t h e i r h o s t s . However, the e x i s t e n c e of key d i f f e r e n c e s i n s t e r o l metabolism between nematodes and t h e i r animal or p l a n t hosts have presented the p o s s i b i l i t y t h a t nematode s t e r o l metabolism c o u l d be s e l e c t i v e l y i n h i b i t e d . Moreover, the l i k e l y f u n c t i o n of nematode s t e r o i d s i n the hormonal r e g u l a t i o n of i m p o r t a n t l i f e processe w i t h the p o t e n t i a l b e n e f i p r o c e s s e s have p r o v i d e d f u r t h e r impetus f o r the r e c e n t i n t e n s i f i c a t i o n of r e s e a r c h e f f o r t s i n the area of nematode s t e r o i d biochemistry. Nutritional

Requirement f o r S t e r o l

i n Nematodes

I n t e r e s t i n nematode s t e r o l metabolism was s t i m u l a t e d by the d i s c o v e r y t h a t the DD-136 s t r a i n of Steinernema f e l t i a e , an i n s e c t a s s o c i a t e , would not grow and reproduce upon b a c t e r i a l c u l t u r e s u n l e s s a s t e r o l was p r e s e n t ( 4 ) . Several compounds were c a p a b l e of s a t i s f y i n g t h i s n u t r i t i o n a l requirement, i n c l u d i n g c h o l e s t e r o l , cholestanol, s i t o s t e r o l , stigmastanol, 22-dihydrobrassicasterol, c h o l e s t - 4 - e n - 3 - o n e , 7 - d e h y d r o c h o l e s t e r o l , and l a t h o s t e r o l . Growth and r e p r o d u c t i o n d i d not o c c u r i n c u l t u r e s supplemented w i t h s t i g m a s t e r o l o r e r g o s t e r o l , two s t e r o l s c o n t a i n i n g A22-bonds. Hieb and R o t h s t e i n (5) demonstrated a s i m i l a r requirement i n C a e n o r h a b d i t i s propagated upon the b a c t e r i u m E s c h e r i c h i a c o l i ; a d d i t i o n of c h o l e s t e r o l , 7 - d e h y d r o c h o l e s t e r o l , e r g o s t e r o l , s t i g m a s t e r o l or s i t o s t e r o l r e s u l t e d i n e x c e l l e n t growth and reproduction. T u r b a t r i x a c e t i reproduced s u c c e s s f u l l y upon B a c i l l u s s u b t i l i s supplemented w i t h c h o l e s t e r o l , c h o l e s t a n o l , d e s m o s t e r o l , l a t h o s t e r o l , 7-dehydrocholesterol, 25-norcholesterol, c h o l e s t - 4 - e n - 3 - o n e , cholest-5-en-3-one, campesterol, 2 4 - m e t h y l e n e c h o l e s t e r o l , s t i g m a s t e r o l , s i t o s t e r o l , or f u c o s t e r o l ; two s t e r o l s w i t h a c i s - A / B r i n g c o n f i g u r a t i o n , c o p r o s t a n o l and c o p r o s t - 7 - e n o l , were not u t i l i z e d (6). In o t h e r e x p e r i m e n t s , squalene o r l a n o s t e r o l i n c r e a s e d T. a c e t i and C a e n o r h a b d i t i s p o p u l a t i o n s i n c h e m i c a l l y d e f i n e d media supplemented w i t h casamino a c i d s and myoglobin or cytochrome c (7). S i m i l a r l y , some p o p u l a t i o n i n c r e a s e o c c u r r e d i n c u l t u r e s of the s n a i l a s s o c i a t e R h a b d i t i s maupasi i n c h e m i c a l l y d e f i n e d media c o n t a i n i n g l a n o s t e r o l , a l t h o u g h the r e p r o d u c t i v e r a t e was g r e a t e r i n c h o l e s t e r o l or ergosterol-supplemented cultures (8). D i f f i c u l t i e s i n i n t e r p r e t a t i o n of the r e s u l t s of some of these n u t r i t i o n a l experiments are a consequence of p o s s i b l e s t e r o l contaminants i n media i n g r e d i e n t s , p o s s i b l e i m p u r i t i e s i n the added

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s t e r o l s , p o s s i b l e b a c t e r i a l metabolism of the supplemented s t e r o l s , or the p o t e n t i a l a b i l i t y of many s t e r o l s to s u b s t i t u t e f o r c h o l e s t e r o l i n a s t r u c t u r a l r o l e and f r e e small q u a n t i t i e s of endogenous c h o l e s t e r o l i n such c u l t u r a l systems f o r use as a s p e c i f i c p r e c u r s o r of s t e r o i d hormones o r o t h e r m e t a b o l i t e s by a " s p a r i n g " p r o c e s s , as can occur i n many i n s e c t s ( 9 ) . In o r d e r t o m i n i m i z e t h e s e e f f e c t s , we have propagated C a e n o r F a b d i t i s e l e g a n s i n a c h e m i c a l l y d e f i n e d a x e n i c c u l t u r e (CbMM, 10) supplemented w i t h chloroform/methanol ( 2 : 1 , v / v ) - e x t r a c t e d bovine hemoglobin and v a r i o u s h i g h l y p u r i f i e d s t e r o i d s a t 6 \ig/m\ ( u n p u b l i s h e d ) . Nematodes were i n i t i a l l y t r a n s f e r r e d to a s t e r o l - d e f i c i e n t medium, i n c u b a t e d two weeks, and then t r a n s f e r r e d to media supplemented w i t h the a p p r o p r i a t e s t e r o i d . Two s u b c u l t u r e s were performed a t two-week i n t e r v a l s to g r e a t l y minimize the amount of r e s i d u a l s t e r o l . At the c o n c l u s i o n of these e x p e r i m e n t s , t h r i v i n g p o p u l a t i o n s were p r e s e n t i n c u l t u r e s supplemented w i t h c h o l e s t e r o l , l a t h o s t e r o l , d e s m o s t e r o l , 7-dehydrocholesterol, campesterol stigmasterol, stigmastanol I n t e r e s t i n g l y , 2 9 - f l u o r o s t i g m a s t e r o l , a compound which i s t o x i c to the i n s e c t Manduca s e x t a by v i r t u e of the g e n e r a t i o n of f l u o r o a c e t a t e d u r i n g C - 2 4 d e a l k y l a t i o n (11) s a t i s f i e d the s t e r o l n u t r i t i o n a l requirement i n these e x p e r i m e n t s . ( T o x i c i t y d i d appear a t c o n c e n t r a t i o n s of 50 Mg/ml.) In the same e x p e r i m e n t s , r e p r o d u c t i o n and movement e v e n t u a l l y ceased i n media c o n t a i n i n g coprost-7-enol, 4a-methylcholest-8(14)-enol, lanosterol, 2 2 , 2 3 - d i h y d r o x y s i t o s t e r o l , p r o g e s t e r o n e , ecdysone, or 20-hydroxyecdysone. Although a d d i t i o n a l s t e r o i d s s h o u l d be i n v e s t i g a t e d i n t h i s s t e r i l e , h i g h l y d e f i n e d system b e f o r e c o n c l u s i o n s can be drawn, a p p a r e n t l y , 4 a - m e t h y l - , 4 , 4 - d i m e t h y l - , h e a v i l y h y d r o x y l a t e d , or c i s - A / B s t e r o l s cannot s a t i s f y the s t e r o l n u t r i t i o n a l requirement i r T C . e l e g a n s i n c o n t i n u o u s c u l t u r e . Among p a r a s i t i c nematodes, a s t e r o l requirement has been i n v e s t i g a t e d in only Nippostrongylus b r a s i l i e n s i s , a rat parasite whose eggs w i l l develop i n t o t h i r d - s t a g e , i n f e c t i v e l a r v a e i n a c u l t u r e medium c o n t a i n i n g f o r m a l i n - k i l l e d £ . c o l i and c h o l e s t e r o l , 7-dehydrocholesterol, ergosterol, s i t o s t e r o l , stigmasterol, l a n o s t e r o l , or c h o l e s t a n e , but not c o p r o s t a n o l o r coprostanone ( 1 2 ) . In a d d i t i o n , i n v i t r o development of t h i r d - s t a g e j u v e n i l e s of A s c a r i s t o f o u r t h - s t a g e j u v e n i l e s and the s i z e of the r e s u l t a n t j u v e n i l e s are markedly i n c r e a s e d by the a d d i t i o n of c h o l e s t e r o l (13). Lack o f De Novo S t e r o l B i o s y n t h e s i s i n Nematodes. The d i e t a r y requirement f o r s t e r o l r e s u l t s from the l a c k of de novo s t e r o l b i o s y n t h e s i s i n nematodes. S p e c i e s i n which r a d i o l a b e l e d a c e t a t e or mevalonate are not c o n v e r t e d to r a d i o l a b e l e d s t e r o l i n c l u d e T. a c e t i (14, 1 5 ) , C a e n o r h a b d i t i s (15) and the animal p a r a s i t e s A s c a r i s ( 1 6 ) , D i r o f i T a r i a i m m i t i s ( 1 7 ) , and B r u g i a pahangi ( 1 7 ) . On o c c a s i o n , r a d i o l a b e l e d s t e r o l s T ï ï e n t i f i e d by t h i n - l a y e r chromatography have been d e t e c t e d from nematodes i n c u b a t e d w i t h r a d i o l a b e l e d a c e t a t e ; i n the lone case i n which such compounds were f u r t h e r c h a r a c t e r i z e d by g a s - l i q u i d chromatography, the r a d i o l a b e l e d components possessed r e t e n t i o n times much e a r l i e r than c h o l e s t e r o l ( 1 6 ) . I t i s not known which enzymes i n the t y p i c a l de novo

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b i o s y n t h e t i c pathway are absent i n nematodes. Nutritional i n v e s t i g a t i o n r e v e a l e d t h a t i n T. a c e t i and C a e n o r h a b d i t i s the b l o c k o c c u r s subsequent t o f a r n e s o l [7). P a n a g r e l l u s r e d i v i v u s was r e p o r t e d to possess the i n t e r e s t i n g a b i l i t y to c o n v e r t t r i t i a t e d 2 , 3 - o x i d o s q u a l e n e t o l a n o s t e r o l ; t r i t i a t e d C27 s t e r o l s were not detected (18). Experiments w i t h a d d i t i o n a l r a d i o l a b e l e d p r e c u r s o r s a r e necessary t o f u r t h e r i n v e s t i g a t e t h i s i n t e r e s t i n g s u b j e c t . S t e r o l Composition o f P a r a s i t i c Nematodes Because of the p r e v i o u s l y d e s c r i b e d problems i n c u l t u r e of p a r a s i t i c nematodes, i n v e s t i g a t i o n of s t e r o l metabolism i n these organisms has been l a r g e l y l i m i t e d t o comparison of the s t e r o l c o m p o s i t i o n s of host and p a r a s i t e . For example, l a t h o s t e r o l and c h o l e s t e r o l were the major s t e r o l s of H. carpocapsae DD-136 propagated i n wax moth l a r v a e , organisms t h a t c o n t a i n e d c h o l e s t e r o l as t h e i r p r i n c i p a l sterol. Radiolabeled cholestero r e c o v e r e d as r a d i o l a b e l e nematode ( 1 9 ) . Not u n e x p e c t e d l y , c h o l e s t e r o l i s the major s t e r o l of the few v e r t e b r a t e - p a r a s i t i c nematodes examined thus f a r ( 1 6 , 2 0 , 2 1 ) . F l e m i n g and F e t t e r e r (22) have demonstrated r e c e n t l y " v i a o c c l u s i o n of the d i g e s t i v e t r a c t , c o n t i n u o u s p e r f u s i o n of the pseudocoelom and c o l l e c t i o n of p e r i e n t e r i c f l u i d from A s c a r i s i n c u b a t e d i n the presence of t r i t i a t e d c h o l e s t e r o l t h a t t r a n s c u t i c u l a r and t r a n s m u s c u l a r t r a n s p o r t i s the primary means of s h o r t - t e r m cholesterol absorption. The i n t e s t i n e of t h i s animal s e l e c t i v e l y absorbs c h o l e s t e r o l a t about t w i c e the r a t e of s i t o s t e r o l ( 2 3 ) . A b s o r p t i o n of p h y t o s t e r o l s i s undoubtedly r e s p o n s i b l e f o r t f i ? f r e q u e n t o c c u r r e n c e of s u b s t a n t i a l q u a n t i t i e s of these 24-methyl or 2 4 - e t h y l s u b s t i t u t e d compounds i n many p a r a s i t i c nematodes, i n c l u d i n g d i g e s t i v e - t r a c t p a r a s i t e s whose host d i e t s can i n c l u d e s u b s t a n t i a l q u a n t i t i e s of p h y t o s t e r o l s . Because a l l of the p h y t o p a r a s i t i c nematodes examined thus f a r c o n t a i n s u b s t a n t i a l l y g r e a t e r r e l a t i v e p r o p o r t i o n s of c h o l e s t e r o l and/or l a t h o s t e r o l than t h e i r h o s t s (24-27) and because most (but not a l l ) phytophagous i n s e c t s (28) as w e l l as c e r t a i n f r e e - l i v i n g nematodes ( s u b s e q u e n t l y d e s c r i b e d T ~ a r e c a p a b l e of c o n v e r t i n g p h y t o s t e r o l s to c h o l e s t e r o l v i a a C - 2 4 d e a l k y l a t i o n p r o c e s s , i t has been s p e c u l a t e d t h a t p l a n t - p a r a s i t i c nematodes are capable of a s i m i l a r d e a l k y l a t i o n (24, 25, 27). O b v i o u s l y , experiments w i t h r a d i o l a b e l e d 2 4 - a l k y l s t e r o l s are needed to c o n c l u s i v e l y e s t a b l i s h whether the e x i s t e n c e of c h o l e s t e r o l or o t h e r 2 4 - d e s a l k y l s t e r o l s i n p h y t o p a r a s i t i c nematodes i s due to d e a l k y l a t i o n r a t h e r than some s e l e c t i v e uptake mechanism, as i n some i n s e c t s ( 2 9 , 3 0 ) . A s c a r i s a d u l t s d i d not c o n v e r t i n j e c t e d [ C ] s i t o s t e r o l ~ i b any o t h e r s t e r o l ( 1 6 ) ; i t i s unknown i f the C - 2 4 d e a l k y l a t i o n pathway i s s i m i l a r l y not f u n c t i o n a l i n o t h e r stages of t h i s i n t e s t i n a l p a r a s i t e or i n o t h e r a n i m a l - p a r a s i t i c nematodes. 1 4

M e t a b o l i s m of S t e r o l s i n F r e e - l i v i n g

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D e f i n i t i v e e v i d e n c e f o r e x i s t e n c e of d e a l k y l a t i o n and o t h e r m e t a b o l i c pathways i n nematodes has been o b t a i n e d through e x t e n s i v e

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e x p e r i m e n t a t i o n w i t h a x e n i c a l l y propagated f r e e - l i v i n g nematodes. Cole and Krusberg (14) c o n c l u s i v e l y demonstrated C-24 d e a l k y l a t i o n i n T. a c e t i by i t s c o n v e r s i o n of [ - ^ s i t o s t e r o l to t r i t i a t e d c h o T e s t e r o l and 7 - d e h y d r o c h o l e s t e r o l . More r e c e n t l y , our l a b o r a t o r y has undertaken a comprehensive i n v e s t i g a t i o n of the metabolism of e i g h t d i f f e r e n t s t e r o l s by C. e l e g a n s i n s t e r i l e , semi d e f i n e d l i q u i d media c o n t a i n i n g c h l o r o f o r m / m e t h a n o l - e x t r a c t e d i n g r e d i e n t s (31-35). D i e t a r y d e s m o s t e r o l , s i t o s t e r o l and s t i g m a s t e r o l were r a d i o l a b e l e d ; when p o s s i b l e t o measure, a l l s t e r o l s recovered from C. elegans i n such experiments c o n t a i n e d a p p r o x i m a t e l y the same s p e c i f i c a c t i v i t y as the o r i g i n a l supplemented s t e r o l and hence were t r u e m e t a b o l i t e s of the d i e t a r y s t e r o l and not t r a c e media contaminants or the products of de novo s y n t h e s i s . These experiments have demonstrated t h a t £ . e l e g a n s performs s e v e r a l d i f f e r e n t s t e r o l m e t a b o l i c p r o c e s s e s , i n c l u d i n g C-7 d e h y d r o g e n a t i o n , A - r e d u c t i o n , 4a-methylation, Δ - s t e r o l to A ( * 4 ) - s t e r o l i s o m e r i z a t i o n , C-24 d e a l k y l a t i o n , and A I n i t i a l experiments t h a t C. e l e g a n s can i n t r o d u c e a double bond a t C-7 a n d , to a l e s s e r e x t e n T , reduce the A - b o n d o f the r e s u l t i n g A5,7_diene, as 7 - d e h y d r o c h o l e s t e r o l and l a t h o s t e r o l were major and minor m e t a b o l i t e s (Table I ) . U n e x p e c t e d l y , we d e t e c t e d s u b s t a n t i a l q u a n t i t i e s of two d i f f e r e n t 4 a - m e t h y l s t e r o l s i n C. e l e g a n s f e d c h o l e s t e r o l , and t h e s t e r y l e s t e r f r a c t i o n was e s p e c i a l l y r i c h i n these compounds. Because 4 a - m e t h y l s t e r o l s are g e n e r a l l y regarded as i n t e r m e d i a t e s between l a n o s t e r o l (or c y c l o a r t e n o l ) and c h o l e s t e r o l i n organisms w i t h de novo s t e r o l b i o s y n t h e t i c c a p a b i l i t y , our i n i t i a l r e a c t i o n was "Eiïârf t h e s e compounds were endogenous media c o n t a m i n a n t s . However, attempts to i s o l a t e them from i n c u b a t e d , nematode-free media f a i l e d - Subsequent experiments w i t h [ C ] d e s m o s t e r o l and [ 1 4 c ] s i t o s t e r o l r e v e a l e d t h a t the 4 a - m e t h y l c h o l e s t - 8 ( 1 4 ) - e n o l and 4 a - m e t h y l c h o l e s t - 7 - e n o l contained a p p r o x i m a t e l y the same s p e c i f i c a c t i v i t y as the o r i g i n a l d i e t a r y s t e r o l and were produced by a d i r e c t n u c l e a r m e t h y l a t i o n pathway. The b i o s y n t h e s i s of 4 - m e t h y l s t e r o l s from m e t h y l a t i o n of a 4 - d e s m e t h y l s t e r o l p r e c u r s o r has not been suggested to occur i n any other organism. The n u c l e a r m e t h y l a t i o n pathway i s not unique t o elegans. We have r e c e n t l y d i s c o v e r e d s i m i l a r but not i d e n t i c a l pathways i n T. a c e t i and P_. r e d i y i v u s (Chitwood e t a l . , u n p u b l i s h e d ) . Cysts of Heterodera zeae d i d not c o n t a i n 4 - m e t h y l s t e r o l s (27h but p o s s i b l y o t h e r l i f e stages of H. zeae or o t h e r p a r a s i t i c nematodes may contain 4-methylsteroTs. Experiments w i t h s i t o s t e r o l - s u p p l e m e n t e d media demonstrated the C-24 d e a l k y l a t i o n o f a 24a-ethy1 s t e r o l by C. e l e g a n s (Table I ) . The a b i l i t y of t h i s nematode t o produce 2 4 - d e s a l k y l s t e r o l m e t a b o l i t e s from c a m p e s t e r o l , 2 2 - d i h y d r o b r a s s i c a s t e r o l , 24-methylenecholesterol, s t i g m a s t e r o l and s t i g m a s t a n o l (Table I) i n d i c a t e s t h a t 24a-methyl, 243-methyl, and 24-methylene s u b s t i t u e n t s a r e e f f e c t i v e l y removed and t h a t C-24 α - e t h y l group removal i s not dependent upon l a c k of a A - b o n d o r presence o f a A - b o n d . However, the f a c t t h a t s u b s t a n t i a l l y l a r g e r q u a n t i t i e s of campesterol remained unmetabolized i n C. e l e g a n s , as compared to the o t h e r f i v e p h y t o s t e r o l s , i n d i c a t e s e i t h e r t h a t s u b s t r a t e s p e c i f i c i t y f o r the 5

8

5

14

2 2

5

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C-24 d e a l k y l a t i o n enzyme system o c c u r s o r t h a t d i f f e r e n t enzymes are u t i l i z e d f o r d i f f e r e n t substrates (34). S e v e r a l o t h e r i n t e r e s t i n g o b s e r v a t i o n s were noted d u r i n g our comparative i n v e s t i g a t i o n s ( T a b l e I ) . For example, s t i g m a s t a n o l - f e d C. e l e g a n s d i d not c o n t a i n any Δ - or A 5 , 7 _ t e r o l s ; t h e r e f o r e , i t i s l i k e l y t h a t t h i s nematode l a c k s a Δ -dehydrogenase. Although n u c l e a r m o d i f i c a t i o n of d i e t a r y 2 4 - e t h y l s t e r o l s d i d not o c c u r p r i o r to d e a l k y l a t i o n , the nucleus o f d i e t a r y 2 4 - m e t h y l s t e r o l s was d i r e c t l y m e t a b o l i z e d to a s u r p r i s i n g l y l a r g e degree, r e s u l t i n g i n p r o d u c t i o n of s i g n i f i c a n t q u a n t i t i e s o f 2 4 - m e t h y l c h o l e s t a - 5 , 7 - d i e n o l , 2 4 - m e t h y l c h o l e s t - 7 - e n o l , and 4a,24-dimethylcholest-8(14)-enol. A p p a r e n t l y , the n u c l e a r m o d i f i c a t i o n enzymes have l i t t l e a f f i n i t y f o r 2 4 - e t h y l s t e r o l s but might indeed b i n d to s t e r o l s w i t h a l e s s b u l k y 24-methyl group. A l t e r n a t i v e l y , s e p a r a t e enzymes f o r n u c l e a r metabolism of 2 4 - m e t h y l s t e r o l s and 2 4 - d e s m e t h y l s t e r o l s c o u l d e x i s t c o n c u r r e n t l y . R o t h s t e i n (15) o r i g i n a l l [l^C]cholesterol~By Caenorhabditi i n v e s t i g a t i o n s , e s t e r i f i e d s t e r o l s comprised from 7 . 3 % t o 2 1 . 3 % of the t o t a l s t e r o l from C. e l e g a n s and were r a d i o l a b e l e d when r a d i o l a b e l e d d i e t a r y s t e r o l s were employed ( 3 2 , 3 4 ) . Steryl ester f r a c t i o n s c o n s i s t e n t l y c o n t a i n e d l a r g e r p r o p o r t i o n s of 4a-methyl s t e r o l s than f r e e s t e r o l f r a c t i o n s . Speculative e x p l a n a t i o n s f o r the abundance of 4 a - m e t h y l s t e r y l e s t e r s i n c l u d e an e s t e r i f i c a t i o n requirement f o r 4 a - m e t h y l s t e r o l s y n t h e s i s or t r a n s p o r t or a s p e c i f i c hormonal, pheromonal or o t h e r p h y s i o l o g i c r o l e f o r a 4 a - m e t h y l s t e r y l e s t e r or m e t a b o l i t e . 5

s

I n h i b i t i o n o f S t e r o l M e t a b o l i s m i n Nematodes L i k e nematodes, i n s e c t s n u t r i t i o n a l l y r e q u i r e s t e r o l because they l a c k the c a p a c i t y f o r de novo s t e r o l b i o s y n t h e s i s (9). Many a z a s t e r o i d s and n o n s t e r o i d a l a l k y l a m i n e s and amides i n t e r f e r e w i t h s t e r o i d m e t a b o l i s m , growth and development i n i n s e c t s and have p r o v i d e d model compounds f o r development of novel agents f o r i n s e c t control (36). C o n s e q u e n t l y , s e v e r a l i n v e s t i g a t o r s have e v a l u a t e d these as w e l l as r e l a t e d compounds f o r t o x i c i t y or g r o w t h - i n h i b i t o r y a c t i v i t y towards v a r i o u s nematodes. C o l e and Krusberg (14) demonstrated the a c c u m u l a t i o n of [ H ] - d e s m o s t e r o l i n T. a c e t i s t e r i l e l y propagated i n media c o n t a i n i n g [ H ] - s i t o s t e r o l and t r i p a r a n o l s u c c i n a t e , a v e r t e b r a t e h y p o c h o l e s t e r o l e m i c agent by v i r t u e of i t s i n h i b i t i o n of A ^ - s t e r o l r e d u c t a s e , an enzyme t h a t c o n v e r t s desmosterol to c h o l e s t e r o l . Feldmesser e t a l . (37) demonstrated the t o x i c i t y o f many d i f f e r e n t N - s u b s t i t u t e d l o n g ^ c h a i n (Cn t o C15) a l k y l amines and amides to ? . r e d i y i y u s and the r o o t - k n o t nematode Meloidogyne i n c o g n i t a ; 100% l e t h a l i t y o c c u r r e d a t c o n c e n t r a t i o n s of 5-40 Mg/ml. Other r e l a t e d amines possessed i n v i t r o t o x i c i t y a g a i n s t the pinewood nematode, Bursaphelenchus x y l o p h i l u s ( 3 8 ) . Douvres e t a l . (39) found t o x i c i t y of many of these a l k y l a m i n e s and amides to be as low as 1 . 0 t o 2 . 5 pg/ml i n v i t r o a g a i n s t the c a t t l e stomach worm O s t e r t a g i a ostertagi. Less i s known about the e f f e c t s of a z a s t e r o i d s upon nematodes because o f t h e i r u n d e s i r a b l e resemblance to human s t e r o i d s as w e l l 3

3

2

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

206

Table I. sterols

ECOLOGY AND METABOLISM OF PLANT LIPIDS

R e l a t i v e percentages

of s t e r o l s

i n free sterol

Cholesterol Recovered s t e r o l Cholesterol 7-Dehydrocholesterol Lathosterol Cholesta-5,7,9(ll)-trienol Cholest-8(14)-enol Cholestanol Desmosterol Cholesta-5,7,24-trienol Campesterol Dihydrobrassicasterol 24-Methylenechol e s t e r o l 24-Methylcholesta-5,7-dienol 24-Methyl chol e s t - 7 - e n o l 2 4 - M e t h y l e n e c h o l e s t a - 5 , 7 - d i enol 24-Methylchol e s t a - 5 , 7 , 9 ( 1 1 ) - t r i enol Sitosterol Stigmasterol Stigmastanol Fucosterol 4a-Methylcholest-8(14)-enol 4a-Methylchol e s t - 7 - e n o l 4a,24-Dimethylcholest-8(14)-enol 4a,24-Dimethylchol estanol Contained

FS 52.3 40.5 3.6 1.4

-

-

-

2.1

SE 41.2 26.7 5.7 1.5

-

-

-

0.1

23.4 1.3

-

-

(FS) and s t e r y l

Desmosterol FS 26.9 31.2 1.7 1.4

SE 18.7 39.0 1.4 1.4

32.3 2.4

22.8 4.5

-

-

-

-

3.8 0.3

-

-

-

-

10.6 1.6

-

1.5% c a m p e s t e r o l .

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

ester

Campesterol FS 3.9 29.4 3.7 1.9

-

35.8

-

3.1 14.1 0.6 1.3 1.0

-

3.6 0.2 0.7 0.4

SE 3.7 10.9 1.8

53.3 3.7 12.9 1.1

-

0.3

-

9.7 0.2 1.4 0.8

207

Sterols in Nematodes

13. CHITWOOD ET AL.

(SE) f r a c t i o n s from C a e n o r h a b d i t i s e l e g a n s propagated w i t h d i f f e r e n t

Dihydrobrassicasterol FS 5.1 45.0 3.5 6.5

-

SE 8.5 21.5 1.6 3.4

-

Supplemented s t e r o l 24-Methyl e n e cholesterol Sitosterol FS 8.6 49.6 5.0 2.1

-

_

-

24.2 1.3 5.4 0.2 0.3 1.1

31.7 8.8 7.1

6.7

14.7

0.3 0.3

0.6 0.4

-

1.3

-

-

31.0

SE 12.2 43.8 2.5 2.5

0.6

_

_

-

0.7

1.3

-

16.5

_

2.5

6.3

-

-

-

SE 9.3 30.5 3.6 0.3

Stigmasterol FS 8.6 55.6 3.9 5.8

SE 11.3 26.5 9.4 2.4

Stigmastanol FS

SE

_

_

68.3

-

28.6

-

_ _

-

FS 6.7 66.5 4.4 0.8

5

dietary

-

-

_ _ _ _ _ _

16.0

-

1.1 0.1

14.9 0.7

0.1 4.2 0.7

-

-

-

_ _ _ _ _ _

_

_ _ _ _

_

_

_

_

_ _ _ _

_ _

_ _ _ _ _

30.3

-

0.1 23.3 1.4 _

20.5

-

5.4 0.2 _

21.9

-_ 27.5 1.0

_

_ _ _

_

_

-

14.2

. 9.3 0.7 _

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

-

30.1 _

27.0 2.3 _

208

ECOLOGY AND METABOLISM OF PLANT LIPIDS

as t h e i r complex s t r u c t u r e and r e s u l t a n t expensive chemical synthesis. The a z a s t e r o i d 2 5 - a z a c o p r o s t a n e s t r o n g l y i n h i b i t e d r e p r o d u c t i o n i n C. e l e g a n s (32) and i η v i t r o development of f i r s t - s t a g e t o t ï ï i r d - s t a g e j u v e n i l e s o f N. b r a s i l i e n s i s and the mouse p a r a s i t e N e m a t o s p i r o i d e s dubius ( 4 0 ) . Specific biological e f f e c t s i n nematodes of many a l k y l a m i n e s and a l k y l a m i des were d e s c r i b e d i n d e t a i l i n 0 . o s t e r t a g i , where e f f e c t s i n c l u d e d reduced s u r v i v a l , decreased m o t i l i t y or induced p a r a l y s i s , delayed development, lowered y i e l d s o f advanced s t a g e s , d e l a y e d or b l o c k e d or i n c o m p l e t e d t h i r d or f o u r t h m o l t , and decreased or n o n e x i s t e n t egg p r o d u c t i o n ( 3 9 ) . A s i m i l a r p a r a l y s i s and i n h i b i t i o n of m o t i l i t y and r e p r o d u c t i o n o c c u r r e d i n £ . e l e g a n s t r e a t e d w i t h v a r i o u s a l k y l a m i n e s (33) or 2 5 - a z a c o p r o s t a n e [3Z). The l a t t e r compound or 2 5 - a z a c h o l e s t a n e induced H. b r a s i l i e n s i s j u v e n i l e s to develop m o r p h o l o g i c a l a b n o r m a l i t i e s observed i n j u v e n i l e s c u l t u r e d i n s t e r o l - d e f i c i e n t media, i n c l u d i n g d e g e n e r a t i o n of i n t e s t i n a l c e l l s , abnormal d i s p e r s i o n of l i p i E f f e c t s o f i n h i b i t o r s on s t e r o l m e t a b o l i c pathways i n C. e l e g a n s . The most s p e c i f i c e f f e c t s on nematodes of the a z a s t e r o i d s , amines and amides have been o b t a i n e d through our i n v e s t i g a t i o n s of C_. e l e g a n s propagated i n media supplemented w i t h one of s e v e r a l d i f f e r e n t i n h i b i t o r s ( 3 1 , 3 2 , 3 3 , 3 5 ) . Our r e s u l t s have demonstrated t h a t these i n f i T b i t o r s can a c t a t s e v e r a l d i f f e r e n t m e t a b o l i c s i t e s ; moreover, the a c c u m u l a t i o n of many p r e v i o u s l y u n d e t e c t e d s t e r o l s has l e d to the d i s c o v e r y of s e v e r a l key i n t e r m e d i a t e s i n the s t e r o l m e t a b o l i c pathways of t h i s organism (Figure 1). I n i t i a l l y , Z. e l e g a n s was propagated i n media supplemented w i t h 5 . 0 pg/ml 2 5 - a z a c o p r o s t a n e h y d r o c h l o r i d e , a c o n c e n t r a t i o n p r e v i o u s l y shown t o decrease r e p r o d u c t i v e r a t e i n £ . e l e g a n s by about 50%. E x c l u d i n g d i e t a r y s i t o s t e r o l , n e a r l y 96% of the s t e r o l s from such organisms were A 2 4 . t e r o l s n o r m a l l y p r e s e n t i n no more than trace q u a n t i t i e s : c h o l e s t a - 5 , 7 , 2 4 - t r i e n o l , desmosterol, c h o l e s t a - 7 , 2 4 - d i e n o l , and f u c o s t e r o l (Table I I ) . The abundance of these compounds i n d i c a t e d t h a t the a z a s t e r o i d s i g n i f i c a n t l y i n h i b i t e d Δ ^ - s t e r o l r e d u c t a s e i n £ . e l e g a n s and t h a t A - s t e r o l s a r e major i n t e r m e d i a t e s i n the £ . e l e g a n s pathway for sitosterol dealkylation. In a d d i t i o n , the predominance o f A - 4 a - m e t h y l s t e r o l s r e v e a l e d t h a t the a z a s t e r o i d i n h i b i t s the isomerase t h a t c o n v e r t s Δ - t o Δ ( ) - 4 a - m e t h y l s t e r o l s . Four n o n s t e r o i d a l d i m e t h y l amines s i m i l a r l y i n h i b i t e d the A 2 4 - s t e r o l r e d u c t a s e i n s i t o s t e r o l - f e d C_. e l e g a n s , b u t to a l e s s e r extent (Table I I ) . Among these compounds, maximal i n h i b i t i o n o c c u r r e d upon a d d i t i o n of N , N , 3 , 7 , l l - p e n t a m e t h y l d o d e c a n a m i n e , f o l l o w e d by Ν,Ν-dimethyldodecanamine, N,N-dimethyltetradecanamine, and Ν,Ν-dimethylhexadecanamine. A l t h o u g h i n h i b i t o r y to growth and r e p r o d u c t i o n i n C. e l e g a n s , the c o r r e s p o n d i n g C12 d i e t h y l a m i d e possessed l i t t l e e f f e c t on s i t o s t e r o l ( T a b l e I I ) o r s t i g m a s t e r o l ( T a b l e I I I ) m e t a b o l i s m ; perhaps s u b s t i t u t i o n of an amide group f o r the amine group d e s t r o y e d the a b i l i t y of the i n h i b i t o r t o c o m p e t i t i v e l y b i n d to the £ . e l e g a n s A 4 - s t e r o l r e d u c t a s e . The b r a n c h e d - c h a i n a l k y l a m i n e r e s u l t e d i n an a c c u m u l a t i o n of u n m e t a b o l i z e d d i e t a r y s i t o s t e r o l , p o s s i b l y because i t s g r e a t e r s

2 4

7

8

1 4

2

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987. 4a -METHYLCH0LEST-8( 14)-EN0L

i

4a -METHYLCHOLEST-7-ENOL

LATHOSTEROL

CH0LESTA-7.24-DIEN0L

TRIENOL

CHOLESTA-5,7,24-

DESMOSTEROL

FUCOSTEROL

SITOSTEROL

24-METHYLCHOLESTA-5.7-DIENOL

4a ,24-DIMETHYLCHOLEST-8(14)-ENOL

CHOLESTANOL

4a,24-DIMETHYL-

24-METHYLCHOLEST-7-ENOL

CHOLESTA-5.7-DIENOL

24-METHYLENE-

24-METHYLCHOLESTEROL

F i g u r e 1. Major pathways o f s t e r o l metabolism i n C a e n o r h a b d i t i s elegans. Dotted l i n e s r e p r e s e n t p o s s i b l e but unproven s t e p s .

TETRAENOL

CHOLESTA-5.7,22,24-

TRIENOL

CHOLESTA-5.22,24- -

TRIENOL

STIGMASTA-5,22.24(28)-

STIGMASTEROL

ECOLOGY AND METABOLISM OF PLANT LIPIDS

210

Table I I . R e l a t i v e percentages of t o t a l s t e r o l s from C a e n o r h a b d i t i s metabolic i n h i b i t o r s . D i e t a r y s i t o s t e r o l c o n t a i n e d 1.5% c a m p e s t e r o l .

Recovered

sterol

None

Cholesterol 8.1 7-Dehydrochol e s t e r o l 56.4 5.5 Lathosterol 2.0 C h o l e s t a - 5 , 7 , 9 ( 1 1 ) - t r i enol Desmosterol Chol e s t a - 5 , 7 , 2 4 - t r i e n o l Cholesta-7,24-dienol Cholesta-5,7,9(ll),24-tetraeno Cholesta-8,24-dienol Campesterol 0.6 Fucosterol 0.1 Sitosterol 18.2 4

Hydrangea Poinsettia Snapdragon

None None None

Stevia

None

None None Injury t o young leaves Injury t o young leaves Injury to leaves Injury t o leaves None None Injury to leaf t i p s None

Source: Reproduced with permission from Ref. 42. New York Entomological Society.

Injury from 2% Soap Slight injury to leaves Severe injury Injury to stems & leaves None None Severe injury Injury to leaves vSevere i n j u r y

to leaves Severe injury to leaves Slight injury Severe injury Severe i n j u r y None Copyright 1937

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

14. KABARA

233

Fatty Acids and Esters

TABLE XI. Tests With Various Concentrations of Coconut O i l Soap on Orchard and Garden Plants Name of Plant Apple Peach

Injury from 0.25% & 0.5% Soap None None

Injury from 1% Soap None None

Cherry

None

None

Grape

None

None

Beets Cabbage (red) Cabbage (green) Kohlrabi Corn Cucumber Cantaloupe Egg Plant

None Non Non None None None None None

None

Lettuce

None

Lima Beans Pumpkin

None None

Blackberries

None

Sweet Potatoes

None

Squash Rose

None None

S t r i n g Beans

None

Sweet Peas

None

Tomato(young plants) None Tomato None (plant i n blossom) Peppers None

None None None None No injury

Injury t o leaves None Injury t o foliage Injury t o foliage & young shoots No injury None None

Injury from 2% Soap None Slight injury to f o l i a g e . Slight injury to f o l i a g e . Appreciable injury to shoots & foliage None None None None None Injury t o blossoms, none t o leaves Severe injury None Injury to foliage Injury t o foliage & young shoots Slight injury to f o l i a g e None Injury to young leaves & flowers

Slight injury to young leaves Slight injury to young leaves Slight None injury Injury t o None blossoms Injury t o Injury t o leaves leaves

Source: Reproduced with permission from Ref. 42. New York Entomological Society.

Copyright 1937

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

234

ECOLOGY AND METABOLISM OF PLANT LIPIDS

TABLE X I I . Tests With Coconut O i l Soap on Flowering Plants i n Bloom Name o f Plant Canna Chrysanthemum, Hardy Dahlia, Etoarf Daisy Delphinium Geranium Geranium, Sweet Hel iotrope Hollyhock Ice Plant Marigold Petunia Phlox Ragged S a i l o r Roses Gladiolus

0.5% Soap None

0.25% Soap None

II

II II

II

II

II

II II

Injury t o flowers None

It

II

II

II

II II II II II

Injury t o flowers Injury to flowers None Injury to flowers None II

II

II

II II

Injury t o flowers

Source: Reproduced with permission from Ref. 42. New York Entomological Society.

Copyright 1937

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

14.

KABARA

235

Fatty Acids and Esters

From these r e s u l t s i t appears that coconut o i l soap i n concentrations of 0.5 per cent or lower i s safe to apply on a l l kinds of plants with the exception of d e l i c a t e blossoms. Although most studies on structure-function r e l a t i o n s h i p s involving g e r m i c i d a l / i n s e c t i c i d a l a c t i v i t y are i n agreement, esters and potassium s a l t s of f a t t y acids are a c t i v e i n s e c t i c i d e s while remaining i n a c t i v e germicides (45). Rather than postulate d i f f e r e n t mechanisms of a c t i o n , one reasonable but not proven explanation i s that the ester undergoes hydrolysis to the free a c i d i n insects. Ihe p i c t u r e of a c i d versus potassium s a l t soaps (K-soaps) i s not c l e a r . While K-soaps s i g n i f i c a n t l y enhanced the t o x i c i t y of c a p r i c , s t e a r i c and l i n o l e i c , i t reduced the e f f e c t s of caproic and p a l m i t i c ; the other f a t t y acids were unchanged. These observations by Pur i t c h (46) need to be confirmed before any mechanism of action can be postulated since i t i s accepted that th specie. Fatty acids have l e t h a l e f f e c t s on a v a r i e t y of organisms (Table X I I I ) . This wide spectrum of a c t i v i t y against so many pests and i t s safety to humans highly recommend the further a p p l i c a t i o n of natural and synthetic l i p i d s as p o t e n t i a l insecticides. TABLE X I I I . Control of Various Household and Garden Pests by Coconut Fatty Acid Soap Adelgids Aphids Cricket Earwigs F r u i t f l y Adults Fungus Gnats Grasshoppers

Lace Bugs Leaf Hoppers Mealybugs Mites Plant Bugs Psyllids Sawflies

Future Role of Fatty Acids as

Scales Spittlebugs Springtails Tent C a t e r p i l l a r s Thrips Whitefly Woolly Aphids

Germicides/Insecticides

To paraphrase Mark Anthony i n Shakespeare's J u l i u s Ceasar. "Ihe e v i l that insecticides/germicides do l i v e s a f t e r them." The Love Canal and other monuments to man's chemical f o l l i e s may indeed be " i n t e r r e d with our bones". In a report by the World Health Organization (WHO,1971) i t was noted that of 1500 consecutive compounds entering the WHO screening procedure, only f i v e emerged as of p o t e n t i a l value (47). A l l of the above point out the r i s k and hazard of looking f o r new compounds as p o t e n t i a l germicide/insecticide agents. The tremendous investment of time and money makes industry very cautious. Not only are development expenses astronomical, but a l s o the l i a b i l i t y costs must be factored. Figures are hard to generate for i n d i v i d u a l products because of the proprietary nature of such information. A review of the problem (48) showed that i n 1970 the p e s t i c i d e industry claimed an average cost of $5.5 m i l l i o n f o r the development of a

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ECOLOGY AND METABOLISM OF PLANT LIPIDS new p e s t i c i d e . The time required f o r commercialization was 77 months. A s i m i l a r survey of 14 companies reported an average cost of $4 m i l l i o n i n 60 months i n 1969. These f i g u r e s are too low since they do not include the cost of p i l o t p l a n t s , process development, or studies of waste c o n t r o l and other environmental f a c t o r s . Their more r e a l i s t i c estimate leads t o a f i g u r e of $11 m i l l i o n over 10 years, which includes not only the cost of developing unsuccessful compounds, which must be borne by successful ones, but a l s o the a d d i t i o n a l cost involved i n the f a i l u r e to invest $11 m i l l i o n a t 8 percent i n t e r e s t i f the company had instead chosen merely t o deposit the money i n the bank. In 1985, the cost i s probably c l o s e r t o $50 m i l l i o n and the time required f o r commercialization i s 10-12 years. L i t t l e wonder that only a few major companies can a f f o r d the money, much l e s s the gamble on a "new" chemical. The above scenario makes the timing of looking f o r a p p l i c a t i o n s of o l d , saf l i t t l e doubt i n my min and mono-esters) w i l l be the agents of choice i n the future. What i s c u r r e n t l y needed i s how we can best formulate these l i p i d s into e f f e c t i v e , safe and cheap products. With these chemicals i t i s comforting t o know that they can become part of the s o l u t i o n and not part of the problem. Literature Cited 1. Clark, J.R. Bot. Gaz. 1899, 28, 289-327. 2. Bayliss, M. J. Bacteriol. 1936, 31, 489-504. 3. Kodicek, E. Soc. Exp. Biol. Symp. 1949, 3, 217-32. 4. Nieman, C. Bacteriol. Rev. 1954, 18, 147-63. 5. Kabara, J.J.; Swieczkowski, D.M.; Conley, A . J . ; Truant, J.P. Antimicrob. Agents Chemother. 1972, 2, 23-8. 6. Shepard, H.H. "The Chemistry and Toxicology of Insecticides"; Burgess: Minnepolis, 1939; p. 11. 7. Kabara, J.J. "Cosmetic & Drug Preservation", Marcel Dekker: New York, 1984, p. 275-304. 8. Keeny, E.L. Clin. Invest. 1944, 23, 929-34. 9. Asami, Y.; Kusakabe, Α.; Eriguchi, K.; Amemiga, M.; Itabe, Α.; Ueno, G.; Saito, S.; Sakai, Y.; Tanaka, Y.G.; Rikagaku Kenkyusho Hokoku 1965, 41, 259-65; Chem. Abstr., 1966, 64, 18271. 10. Pattison, F.L.M.; Buchanan, R.L.; Dean, F.H. Can. J. Chem. 1965, 43, 1700-9. 11. Gershon, H.; Parmegiani, R. J. Med. Chem. 1967, 10, 186-90. 12. Eggerth, A.H. J. Gen. Physiol. 1926, 10, 147-60. 13. Eggerth, A.H. J. Exp. Med. 1927, 46, 671-88. 14. Eggerth, A.H. J. Exp. Med. 1929, 49, 53-62. 15. Eggerth, A.H. J. Exp. Med. 1931, 53, 27-36. 16. Larson, W.P. Proc. Soc. Exp. Biol. Med. 1921, 19, 62-3. 17. Larson, W.P.; Nelson, E. Proc. Soc. Exp. Biol. Med. 1931, 22, 339-42. 18. Miller, C.P.; Castles, R. J. Bacteriol. 1931, 22, 339-50.

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14. KABARA

Fatty Acids and Esters

19. Violle, H. Rend. Acd. Sci. 1933, 197-204, 714. 20. Barnes, L.A.; Clarke, C.M. J. Bacteriol. 1934, 27, 107-17. 21. Kolmer, J.A.; Rule, A.M.; Madden, B. J. Lb. Clin. Med. 1934, 19, 972-85. 22. Rothman, S.; Smiljanic, M.; Shapiro, A.L.K. Proc. Soc. Exp. Biol, 1945, 60, 394-95. 23. Kabara, J.J.; Conley, A.J.; Swieczkowski, D.J.; Ismail, I.A.; Lie Ken Jie, M.; Gunstone, F.D. J. Med. Chem. 1973, 16, 1060-63. 24. Kabara, J.J.; Vrable, R.; Lie Ken Jie, M. Lipids 1977, 9, 753-59. 25. Conley, A.J.; Kabara, J.J. Antimicrob. Agents Chemother. 1973, 4, 501-6. 26. Kabara, J.J. J. Food Prot. 1981, 44, 633-47. 27. Kato, N.; Shibasaki, I. J. Ferment. Technol. 1975, 53, 793-801. 28. Sands, J.A.; Auperin Cadden, S.P. In "Pharmacological Effect of Lipids"; Kabara, J . J . , Ed.; American Oil Chemists' Society: Champaign, Ill., 1979; pp. 75-95. 29. Shibasaki, I.; Kato, N. In "Pharmcological Effect of Lipids"; Kabara, J . J . , Ed.; American Oil Chemists' Society: Champaign, Ill., 1979; pp. 15-24. 30. Beuchat, L.R. Appl. Environ. Microbiol. 1980, 39, 1178-84. 31. Shibasaki, I. J. Food Safety 1982, 4, 35-58. 32. Kato, Α.; Arima, K. Biochim. Biophy. Res. Commun. 1971, 42, 596-601. 33. Kato, Α.; Shibasaki, I. J. Antibacterial Antifung. Agents 1975, 8, 355-61. 34. Kato, N.; Shibasaki, I. J. Anti-bacterial Antifung. Agents 1976, 4, 254-61. 35. Babayan, V.K.; Kaufman, T.G.; Lehjman, H.; Tkaczuk, R.J. J. Soc. Cosmet. Chem. 1964, 15, 473-79. 36. Fleming, W.E.; Baker, F.E. Jour. Econ. Ent. 1934, 23, 625-30. 37. Van der Moulen, P.A.; VanLeeuwen, E.R. Jour. Econ. Ent. 1929, 23, 812-14. 38. Siegler, E.H.; Popence, C.H. Jour. Econ. Ent. 1925, 18, 292-99. 39. Dills, L.E.; Menusan, H., Jr. Contrib. Boyce Thompson Inst. 1935, 7, 63-82. 40. Ginsburg, J.M.; Kent, C. Jour. New York Ent. Soc. 1937, 45, 109-13. 41. Tattersfield, F. J. Agric. Sci. (Cambridge) 1927, 17, 181-208. 42. Ginsberg, J.M.; Kent, C. Jour. New York Ent. Soc. 1937, 45, 109-13. 43. Bourcart, E. "Insecticides, Fungicides & Weed Killers"; D. Vostrand Co.: New York, 1925. 44. Metcalf, C.L.; Flint, W.P. "Fundamentals of Insect Life"; McGraw-Hill Co.: New York, 1932.

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45. McEarlane, J.E. Comp. Biochem. Physiol. 1968, 24, 377-84. 46. Puritch, G.S. In "The Pharmacological Effects of Lipids"; Kabara, J . J . , Ed.; American Oil Chem. Soc.: Champaign, IL., 1978, pp. 105-12. 47. Wright, J.W. Wld. Hlth. Org, qech. Rep. Ser. 1971, 513, 8-9. 48. Djerassi, C.; Shih-Coleman, C.; Diekman, J. Operational & Policy Aspects Science 1974, 186, 596-607. Received August 19, 1986

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Chapter 15

Hopanoids: Sterol Equivalents in Bacteria Karl Poralla and Elmar Kannenberg Institute for Biology II, Microbiology I, University of Tübingen, Auf der Morgenstelle 1, D-7400, Tübingen, Federal Republic of Germany

Sterols (e.g., cholesterol and ergosterol) are known to occur nearly exclusively in higher organisms (eukaryotes). Onl utilize sterols. Hopanoids triterpenoids, are found in about 50% of the bacterial strains investigated. These hopanoids mostly contain an extended side chain with polar groups, therefore they resemble the geohopanoids found in crude oil and geological sediments. In model membrane systems hopanoids condense phospholipids, enhance viscosity, and diminish permeability. In certain bacteria, subsequent to an increase in growth temperature or in ethanol concentration, the hopanoid content in the cellular lipid raction is enhanced. In cholesterol-dependent bacteria, hopanoids can substitute for the sterol. From these experiments one can conclude that hopanoids possess a membrane-stabilizing function very similar to sterols. I f one s p e c i f i e s the differences between b a c t e r i a (prokaryotes) and higher organisms (eukaryotes), one point i s always mentioned. Higher organisms contain s t e r o l s whereas b a c t e r i a do not. Only a few exceptions are known f o r the occurrence of s t e r o l s i n b a c t e r i a . Do b a c t e r i a dispense with such an important group of compounds? I t w i l l be shown that b a c t e r i a contain s i m i l a r compounds - namely the hopanoids. These are s t r u c t u r a l and functional equivalents of s t e r o l s . Occurrence of Sterols Sterols comprise a subgroup of steroids normally containing one hydroxy group. The side chain i s not shortened and 4a -, 4£- and 14a methyl groups can be cleaved o f f . Sterols occur i n a l l higher organisms, e.g., protozoa, photosynthetic plants, fungi, and animals [1]. I f they cannot synthesize s t e r o l s by themselves the organisms take them up from the environment. Thus the s t e r o l s 0097-6156/87/0325-0239$06.00/0 © 1987 American Chemical Society

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play the role of a growth f a c t o r . The best known examples f o r such a r o l e are i n insects [2]. Sterols occur i n s i g n i f i c a n t amounts i n a few b a c t e r i a l species. The presence of s i g n i f i c a n t amounts must be stressed, otherwise they could not f u l f i l l a function as a membrane strengthener. Detection i n low amounts i s not always s i g n i f i c a n t because the p o s s i b i l i t y of an impurity from outside the bacterium e x i s t s . However an impurity can be excluded by incorporation of a l a b e l l e d precursor, e.g. mevalonate, squalene or even glucose. The b a c t e r i a l genus Mycoplasma i s w e l l known f o r the occurrence of and dependence on s t e r o l s [3], but these b a c t e r i a without c e l l walls do not synthesize t h e i r s t e r o l s by themselves [4]. B a c t e r i a l species which contain and synthesize s t e r o l s by themselves are Methylococcus capsulatus [5,6], Nannocystis exedens [7] and an Lform of Staphylococcus aureus [8]. I t i s i n t e r e s t i n g to note that Methylococcus capsulatus simultaneously contains hopanoids [6]. Biosynthesis of Sterols Biosynthesis s t a r t s from mevalonic acid to produce squalene i n 6 enzymatic steps. This intermediate i s epoxidized i n a reaction i n which one atom i s incorporated from molecular oxygen [9]. Sterols are c y c l i z e d from epoxysqualene to form cycloartenol i n photosynthetic plants and l a n o s t e r o l i n fungi and animals [10,11]. This circumstance presents a very strong argument f o r biosynthesis of sterols being invented at least twice i n the course of evolution. The invention probably occurred a f t e r the separation of photosyn­ t h e t i c plants from fungi and animals [12]. The connection between biosynthesis i n the large taxonomic groups b a c t e r i a , protozoa, plants, fungi and animals i s shown i n F i g . 1. The c y c l i z a t i o n products of epoxysqualene are further aerobic a l l y processed to obtain s t e r o l s devoid of the methyl groups at C-4 and C-14. The side chain i s conserved i n i t s length but not always i n i t s bulkiness. Furthermore, the degree of unsaturation i n ring Β can d i f f e r . The configuration of the rings A - D i s a l l transantic; thus the s t e r o l molecules are f l a t . A l l s t e r o l s con­ t a i n a 3-3-hydroxy group [1]. Functions of Sterols i n Membranes The main occurrence of free s t e r o l s i s i n the cytoplasmic membrane, where they i n t e r a c t with other l i p i d s and proteins. Two modes of action f o r s t e r o l s i n membranes are proposed. One i s the so c a l l e d bulk membrane function, i . e . , the i n t e r a c t i o n with phospholipids and the s p a t i a l separation of these charged molecules [13]. The other role i s a cofactor function f o r the incorporation of unsatu­ rated f a t t y acids i n t o l i p i d s [14]. The t e t r a c y c l i c , amphipathic s t e r o l s incorporate into a b i l a y e r membrane and through van der Waals forces they i n t e r a c t with the a c y l chains of phospholipids. When the temperature i s below the t r a n s i t i o n temperature of the phospholipids, s t e r o l s introduce a sort of disturbance i n t o the ordered l i p i d s . Thereby the t r a n s i t i o n temperature i s lowered and, depending on the s t e r o l concentration, the phase t r a n s i t i o n i s diminished or even abolished. Above the phase t r a n s i t i o n temperature, s t e r o l s reduce a c y l chain m o b i l i t y , a

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F i g . 1: The d i s t r i b u t i o n of biosynthetic pathways f o r p o l y c y c l i c triterpenoids with membrane function i n the major taxonomic groups.

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phenomenon c a l l e d condensation. In other types of experiments one can observe an increase i n microviscosity and a lower permeability of the membrane f o r small molecules [13,15]. These e f f e c t s can be observed with d i f f e r e n t types of s t e r o l s ; to a low degree with an unprocessed s t e r o l containing a l l methyl groups (e.g. lanosterol) and to a high degree with c h o l e s t e r o l . I t should also be mentioned that s t e r o l s possess the p o t e n t i a l to s t a b i l i z e membranes of negat i v e l y charged l i p i d s merely by separating the charged molecules [14]. By t h i s separation, repulsion of the molecules i s diminished. Furthermore (processed) s t e r o l s can have a t o t a l l y d i f f e r e n t function beside t h e i r influence on bulk membrane function. F i r s t indications f o r a metabolic function were obtained by experiments showing a synergism between two s t e r o l s . Low amounts of a processed s t e r o l and high amounts of precursor s t e r o l produce as much growth as high amounts of the processed s t e r o l alone [17]· In Mycoplasma i t could be shown that cholesterol promotes unsaturated f a t t y uptake from culture mediu e f f e c t i s not accompanie i n the membrane. Such synergism was also shown i n yeast [18,19]. Structures and Biosynthesis of Hopanoids William R. Nes was the f i r s t to propose that b a c t e r i a could have s t e r o l - l i k e pentacyclic molecules [20]. Some years l a t e r these were found to be hopanoids by Ourisson, Rohmer and Albrecht [21,22]. Hopanoids comprise a group of pentacyclic triterpenoids f o r which some structures are shown i n F i g . 2. Hopanoids are r i g i d , f l a t , amphipathic molecules with geometric dimensions s i m i l a r to s t e r o l s . Their main s t r u c t u r a l differences are: 1. The c y c l i c system contains 5 rings. As a consequence, the side chain i s shorter. 2. The hydrophilic part i s the side chain, not the nucleus. 3. The side chain can be extended and further hydroxyl functions or other groups Introduced [23]. By mainly s t r u c t u r a l arguments, i t was surmised by Ourisson and Coworkers that hopanoids have a s t e r o l - l i k e function [21]. Hopanoids ( d i p l o p t e r o l and/or hopene) are c y c l i z e d d i r e c t l y from squalene. Thus the biosynthesis i s anaerobic and a l l methyl groups are conserved [24,25]. The hydroxy group of d i p l o p t e r o l probably originates from water. This mode of biosynthesis was proven f o r another pentacyclic t r i t e r p e n o i d , tetrahymanol [26]. Besides not being removed, methyl groups are not s h i f t e d during the enzymatic c y c l i z a t i o n . Thus, one perceives the hopanoids as a sort of crude s t e r o l . In contrast to the lack of a l t e r a t i o n of the nucleus the v a r i a t i o n s of the extended side chain are numerous (Fig. 2 ) . Occurrence of Hopanoids The b i o l o g i c a l s i g n i f i c a n c e of hopanoids was detected i n d i r e c t l y . F i r s t , they were known to occur e x c l u s i v e l y as secondary metabolites of higher organisms; trees, grasses, ferns, and lichens. These hopanoids contain 30 C-atoms and are, therefore, devoid of an extended side chain [27]. Examples f o r such hopanoids are diplopt e r o l and hopene (Fig. 2 ) .

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987. 1

Figure 2. B a c t e r i a l hopanoids. Compounds I I and I I I occur also i n p l a n t s . It 32,33,34,35-tetrahydroxybacteriohopane (= THBH) I I , hopene; I I I , d i p l o p t e r o l (= hopanol); IV, 35-amino-32, 33,3^-trihydroxybacteriohopane; V, 35-ornithyl-32,33,3 *-trihydroxybacteriohopane; VI, 3 5 - ( 0 - β -N-acylglucosaminyl)-32,33,34-trihydroxybacteriohopane.

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Hopanoids and t h e i r derivatives were detected i n crude o i l s and i n the organic part of geological sediments by the groups of Ourisson, Albrecht and Eglinton [22,28]. The global stock of hopa­ noids and t h e i r derivatives i n soluble organic matter of geological sediments i s estimated by Ourisson to be more than 5%. Therefore, hopanoids comprise more material than a l l l i v i n g matter on earth. The hopanoid derivatives show various a l t e r a t i o n s of t h e i r carbon skeleton, some examples of which are given i n F i g . 3. These soc a l l e d geohopanoids often contain an extended side chain or an a d d i t i o n a l methyl group on the nucleus. For t h i s reason, they cannot have originated from plants. A f t e r the detection of hopanoids i n recent sediments such as coastal muds, i t was consistent to f i n d them also i n b a c t e r i a . However, i t was very s u r p r i s i n g to f i n d them i n as many as 50% of the b a c t e r i a l species studied. These b a c t e r i a l hopanoids often contain an extended side chain [21,19] and resemble therefore, the geohopanoids. Thus i t of b a c t e r i a l o r i g i n . The d i s t r i b u t i o n of hopanoids i n the b a c t e r i a l kingdom does not follow a regular pattern [29]. In some groups every tested s t r a i n contains hopanoids, e.g. methylotrophic b a c t e r i a and Rhodos p i r i l l a c e a e (a group of photosynthetic b a c t e r i a ) . I n other groups a s i g n i f i c a n t part (Cyanobacteria) or only a few representatives (Streptomyces) or none Archaebactera) possess hopanoids. Further examples of b a c t e r i a containing hopanoids are B a c i l l u s acidocaldarius, a b a c t e r i a l group i s o l a t e d from acid volcanic springs and s o i l [30,31] and Zymomonas m o b i l i s , an ethanol producing bacterium [32,33]. The Role of Hopanoids i n Bacteria We f i r s t ask the question can hopanoids subsitute f o r the s t e r o l function i n the s t e r o l dependent bacterium Mycoplasma? In experi­ ments by other groups [3,34], i t has been shown that the s t e r o l requirement f u l f i l l i n g the bulk membrane function i s not very s p e c i f i c . Lanosterol, cycloartenol and d i f f e r e n t demethylated s t e r o l s can support growth. In our experiments d i p l o p t e r o l also supports growth [35]. The much b u l k i e r hopane g l y c o l i p i d (compound VI i n F i g . 2) i s not growth promoting. No attempts were made to demonstrate a synergism between d i p l o p t e r o l and c h o l e s t e r o l . B a c i l l u s acidocaldarius which l i v e s i n acid hot springs grows best at 60°C and pH 3. S i m i l a r species l i v e i n high t i t e r s i n s o i l [31]. This bacterium has hopanoids and l i p i d s containing ω-cyclohexane f a t t y acids i n i t s membrane [30,36]. The most prominent hopanoids i n t h i s organism are tetrahydroxy-bacteriohopane and a hopane g l y c o l i p i d (compounds I and VI i n F i g . 2 ) . We were i n t e r ­ ested to know whether the hopanoid content of the c e l l s changes with d i f f e r e n t environmental conditions. We have observed that the hopanoid content i s dependent on growth temperature [37]; e s p e c i a l l y between 60 and 65°C, the hopanoid content increased dramatically, reaching 16% of the c e l l u l a r l i p i d s ( F i g . 4). The lowest hopanoid content was 4%. The dependence of hopanoids on pH of the growth medium was small [37]. This r e s u l t corresponds to

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Figure 3. Hopanoids and diagenetic d e r i v a t i v e s thereof from crude o i l and sediments. Presumably a l l can be d e r i v a t i z e d from tetrahydroxybacteriohopane, one of the most common b a c t e r i a l hopanoids.

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the f i n d i n g with higher organisms where the s t e r o l content i n membranes of Neurospora [38], carp [39] or Chinese hamster ovary c e l l s [40] i s elevated a f t e r growth at higher temperature. Zymomonas mobilis can produce up to 14% ethanol i n i t s culture f l u i d . Normally these amounts k i l l bacteria due to a d i s i n t e g r a t i o n of the c e l l u l a r membrane [41]. In continuous culture conditions by feeding d i f f e r e n t concentrations of glucose, thereby determining ethanol concentration i n the c e l l , we demonstrated that the hopanoid content i s increased with increasing ethanol concentration (Fig. 5). In t h i s case hopanoids can be viewed as membrane strengtheners [33]. Due to t h e i r strong van der Waals i n t e r a c t i o n with phosphol i p i d s , the membrane d i s s o l v i n g property of ethanol i s counteracted. This r e s u l t i s i n good agreement with r e s u l t s i n yeast where ergos t e r o l has a growth promoting e f f e c t or shows an enhancement of s u r v i v a l i n the presence of ethanol [42,43,44]. The Function of Hopanoid From the experiments with c e l l s as shown i n the previous section we can surmise that hopanoids strengthen membranes as s t e r o l s do. Can t h i s function be proven i n model membrane systems? The best defined membrane i n physical terms i s a monolayer membrane. Such a membrane i s a r t i f i c i a l i n a b i o l o g i c a l sense but very valuable i n providing exact molecular data. Without these data the b i o l o g i c a l importance of a molecule cannot be understood. Hopanoids (compounds I , I I and VI i n Fig. 2) form a monomolecu l a r layer on a water surface. I f one compresses t h i s layer, one observes a low compressibility of the f i l m (Fig. 6). This observat i o n i s i n accordance with the r i g i d structure of the molecules. In contrast a phospholipid (e.g. dipalmitoylphosphatidylcholine) possesses a high compressibility correlated to a high molecular area at low surface pressure. A mixture of a hopanoid and a phosp h o l i p i d shows at low surface pressure a lower area requirement as compared to the i n d i v i d u a l molecules [45,46]. Phospholipid molecules are packed more densely i n combination with a hopanoid. This phenomenon i s known as condensation [13]. With isobars of monolayers (Fig. 6) one can demonstrate the phase t r a n s i t i o n diminishing e f f e c t of hopanoids. The i n d i v i d u a l phospholipid shows a s i g n i f i c a n t phase t r a n s i t i o n , a sudden increase i n molecular area at a c e r t a i n temperature. I f one adds a hopanoid to the phospholipid, t h i s increase i s diminished. Equally w e l l , one can demonstrate t h i s effect by d i f f e r e n t i a l scanning calorimetry (Fig. 7). The phospholipid shows an endothermic t r a n s i t i o n at a c e r t a i n temperature. The enthalpy of t r a n s i t i o n i s lowered by adding a hopanoid to the phospholipid. Furthermore, one can observe a broadening of the peak, meaning a lower cooperat i v i t y of the a c y l chains at the phase t r a n s i t i o n [47]. In black l i p i d membranes containing phospholipid i n combination with hopanoid, the m o b i l i t y of a potassium ion complex can be measured [48]. The m o b i l i t y of t h i s complex i s decreased by increasing the molecular f r a c t i o n of the hopanoid. These experiments demonstrate v i s c o s i t y enhancing property of hopanoids. By a fourth physical method complementing the others one can show that hopanoids diminish the permeability of membranes. Small

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Growth temperature, °C

F i g . 4: Hopanoid content i n B a c i l l u s acidocaldarius measured as the acetate of l-hydroxyethane-29-hopane as a function of growth temperature and pH of the medium.

— • Endogenous ethanol (g · I") 1

F i g . 5: Hopanoid content of Zymomonas mobilis i n continuous cultures under d i f f e r e n t concentrations of ethanol produced. Hopanol - d i p l o p t e r o l , THBH - 32,33,34,35tetrahydroxybacteriohopane.

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45 60 Molecular area (Âr) F i g . 6:

F i g . 7:

Isobars from monolayer films at a surface pressure of 25 dyn/cm. , tetrahydroxybacteriohopane; dipalmitoylphosphatidylcholine; - - -, 1 : 1 molecular mixture of these compounds; , calculated isobar of the 1 : 1 molecular mixture. The arrow indicates the condensation.

D i f f e r e n t i a l scanning calorimetry traces i l l u s t r a t i n g the thermotropic phase behaviour of multilamellar dispersions of dipalmitoyl-phosphatidylcholine cont a i n i n g 0 - 2 0 mol% tetrahydroxybacteriohopane (= THBH).

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molecules such as g l y c e r o l permeate liposomal membranes containing hopanoids more slowly. Thus i t can be concluded that l i p i d s i n liposomes are aligned more densely i n the presence of hopanoids [49]. By following the swelling of phospholipid unilamellar v e s i c l e s by stopped-flow transmittance measurements, i t was shown that hopanoids reinforce the mechanical properties of a membrane [50]. Tetrahydroxybacteriohopane has an e f f e c t s i m i l a r to c h o l e s t e r o l , though not quite as large. By these model experiments, i t was shown that hopanoids act i n a manner s i m i l a r to s t e r o l s on l i p i d membranes. Often the magnitude of the influence i s about the same on condensation, suppression of phase t r a n s i t i o n , enhancement of v i s c o s i t y and reduction of perme­ a b i l i t y . Thus nature has invented at least one a d d i t i o n a l molecular type besides s t e r o l s exerting the above described properties. I t should be mentioned that other molecules, e.g., ω-dihydroxycarotenoids have a s m i l i a r e f f e c Conclusions There exists ample evidence by p h y s i o l o g i c a l experiments and by d i f f e r e n t physico-chemical methods that hopanoids i n bacteria possess membrane properties s i m i l a r to s t e r o l s , e s p e c i a l l y choles­ t e r o l , i n higher organisms. We are aware that one l i n e of evidence i n t h i s context i s s t i l l missing. U n t i l now mutant analysis on hopanoids has not been done. Also the influence on enzymatic membrane processes has not been measured. A more t h e o r e t i c a l problem i s of major importance. Why does one seldom f i n d s t e r o l s i n bacteria and why are hopanoids, as mem­ brane constitutents, hardly ever found i n higher orgamisms? One can enumerate some disadvantages for hoapnoids. There e x i s t s a s o l u b i l i t y problem. Liposomal membranes can be loaded with choles­ t e r o l to a higher molecular f r a c t i o n than hopanoids. Secondly, the value f o r the d i f f e r e n t physical effects on membranes are often s l i g h t l y higher f o r c h o l e s t e r o l . From a purely s t r u c t u r a l viewpoint the s t e r o l molecule i s a combination between a hopanoid and a f a t t y acid. The apolar side chain imparts to the s t e r o l molecule a p a r t i a l f l e x i b i l i t y . This f l e x i b i l i t y can eventually be of value for s p e c i f i c requirements i n a biomembrane of higher organisms. In t h i s context i t i s valuable to consider the r e l a t i o n s h i p of the protozoan Tetrahymena to s t e r o l s . This organism can u t i l i z e s t e r o l s from the medium. But i f the medium i s devoid of s t e r o l s , the biosynthesis of pentacyclic t r i t e r p e n o i d tetrahymanol i s derepressed [51]. This organism has not yet aquired sterol-dependent functions i n i t s membrane. Also a study of the above mentioned bacterium Methylococcus capsulatus would be rewarding. This organism synthe­ sizes both s t e r o l s and hopanoids [24,52]. In t h i s case, auxotrophic mutants f o r s t e r o l s and hopanoids should c l a r i f y f o r which s p e c i a l processes these p o l y c y c l i c triterpenoids are necessary.

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Literature Cited 1. Nes, W. R.; McKean, M. L. "Biochemistry of Steroids and other Isopentenoids"; Unversity Park Press, Baltimore, 1977. 2. Lipke, H.; Fraenkel, G. Ann. Rev. Entomol. 1956, 1,17. 3. Smith, P. F. J. Lipid Res. 1964, 5, 121. 4. Razin, S., pp. 183 in Current Topics in Membranes and Transport, Vol. 17, Academic Press, New York, 1963. 5. Bird, C. W.; Lynch, J. M.; Pirt, F.J.; Reid, W.W.; Middleditch, C. J. W. Nature 1971, 230 473. 6. Bouvier, P.; Rohmer, M; Benveniste, P; Ourisson, G. Biochem. J. 1976, 159, 267. 7. Kohl, W; Gloe, A; Reichenbach, H. J. Gen. Microbiol. 1983, 129, 1629. 8. Hayami, M.; Okabe, Α.; Sasai, K.; Hayashi, H.; Kanemasa, Y. J. Bacteriol. 1979, 140, 859. 9. Ono, T.; Bloch, K. 10. Gibbons, G. F.; Goad J. Biol. Chem. 1971, 246, 3967. 11. Benveniste, P.; Hirth, L.; Ourisson, G. Phytochemistry 1966, 5, 45. 12. Poralla, K. FEMS Microbiol. Lett. 1982, 13, 131. 13. Demel, R.A.; De Kruyff, B. Biochim. Biophys. Acta 1976, 457, 109. 14. Bloch, K. E. CRC Crit. Rev. Biochem. 1983, 14, 47. 15. De Gier, J . ; Mandersloot, J.G.; Van Deenen, L. L. M. Biochim. Biophys. Acta 1968, 150, 666. 16. Dahl, C.E.; Dahl, J.S.; Bloch, K. Biochemistry 1980, 19, 1467. 17. Dahl, C. E.; Dahl, J. S.; Bloch, K. J. Biol. Chem. 1983, 258, 11814. 18. Pinto, W. J . ; Lozano, R.;Sekula, B.C.; Nes, W. R. Biochem. Biophys. Res. Commun. 1983, 12-47. 19. Ramgopal, M.; Bloch, K. Proc. Nat. Acad. Sci. U.S. 1983, 80, 712. 20. Nes, W. R. Lipids 1974, 596. 21. Rohmer, M.; Bouvier, P.; Ourisson, G. Proc. Nat. Acad. Sci. U. S. 1979, 76, 847. 22. Ourisson, G.,; Albrecht, P.; Rohmer, M.; Pure Appl. Chem. 1979, 51, 709. 23. Neunlist, S.; Hoist, O.; Rohmer, M. Eur. J. Biochem. 1985, 147, 561. 24. Rohmer, M.; Bouvier, P.; Ourisson, G. Eur. J. Biochem. 112, 557. 25. Seckler, B. Doctoral Thesis, University of Tuebingen, 1980. 26. Zander, J. M.; Greig, J. B.; Caspi, E. J. Biol. Chem. 1970, 245, 1247. 27. Devon, T. K.; Scott, A. I. "Handbook of Naturally Occurring Compounds", Academic Press, New York, 1972; Vol. 2. 28. Van Dorsselaer, Α.; Ensminger, Α.; Spyckerelle, C.; Dastillung, M.; Sieskind, O.; Arpino, P.; Albrecht, P.; Ourisson, G.; Brooks, P. W.; Gaskell, S. J . ; Kimble, B. J . ; Philip, R. P.; Maxwell, J. R.; Eglinton, G. Tetrahedron Lett. 1974, 1349.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

15.

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52.

PORALLA AND KANNENBERG

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Rohmer, M.; Bouvier-Nave, P.; Ourisson, G. J. Gen. Microbiol. 130, 1137. Langworthy, T. Α.; Mayberry, W. R. Biochim. Biophys. Acta 1976, 431, 570. Hippchen, B.; Roell, Α.; Poralla, K. Arch. Microbiol 1981, 129, 53. Barrow, K. D.; Collins, J. G.; Rogers, P. L.; Smith, G. M. Biochim. Biophys, Acta 1983, 753, 324. Bringer, S., Haertner, T.; Poralla, K.; Sahm, H. Arch. Mirco­ biol. 140-312. Dahl, C. Ε.; Dahl, J. S.; Bloch, K. Biochemistry 1980, 19, 1462. Kannenberg, E.; Poralla, K. Arch. Microbiol. 1982, 133, 100. De Rosa, M.; Gambactorta, Α.; Minale, L.; BuLock, J. D. Chem. Commun. 1971, 1334. Poralla, K.; Haertner, T.; Kannenberg, E. FEMS Microbiol. Lett. 1984, 23, 253. Aaronson, L. Α.; Johnson Biophys. Acta 1982, 713, 456. Wodtke, E. Biochim. Biophys. Acta 1978, 529, 280. Anderson, R. L.; Minton, K. W.; L i , G. C.; Hahn, G. M. Biochim. Biophys. Acta 1981, 641, 334. Ingram, L. O.; Buttke, T. M. Advanc. Microbial. Physiol. 25, 253. Thomas, JD. Α.; Hossack, J. Α.; Rose, A. H. Arch. Microbiol. 117, 239. Ohta, K.; Hayashida, S. Appl. Environ. Microbiol. 1983, 46, 821. Janssens, J. H.; Burris, N.; Woodward, Α.; Bailey, R. B. Appl. Environ. Microbiol. 1983, 45, 598. Poralla, K.; Kannenberg, E.; Blume, A. FEBS Lett. 1980, 113, 107. Kannenberg, E.; Poralla, K.; Blume, A. Naturwissenschaften 1980, 57, 458. Kannenberg, E.; Blume, Α.; McElhaney, R. N.; Poralla, K. Biochim. Biophys. Acta 1983, 733, 111. Benz, R.; Hallmann, D.; Poralla, K.; Eibl, H. Chem. Phys. Lipids 1983, 34, 7. Kannenberg, E.; Blume, Α.; Geckeler, K.; Poralla, K. Biochim. Biophys. Acta 1985, 814, 719. Bisseret, P.; Wolff, G.; Albrecht, A. M.; Tanaka, T.; Nakatami, Y.; Ourisson, G. Biochem. Biophys. Res. Commun. 1983, 110, 210. Conner, R. L.; Landrey, J. R.; Burns, C. H.; Mallory, F. E. Protozool Bird, C. W.; Lynch, J. M.; Pirt, F. J.; Reid, W. W. Tetrahedron Lett. 1971, 3189.

Received June 9, 1986

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 16

Structure-Function Relationships for Sterols in Saccharomyces cerevisiae William R. Nes Department of Bioscience and Biotechnology, Drexel University, Philadelphia, PA 19104

Ergosterol has two kinds of function which can be distin­ guished from each other by differing sensitivities to the 24β-methyl moiety of the sterol's structure. The "bulk membrane" function only slightly influence or size of the substituent at C-24 or by the chirality of C-24, while the "regulatory" function has an absolute requirement for the 24β-methyl group. In agreement with the assignment of most of the ergosterol to an architec­ tural role in membranes, the sterol's overall length is close to the distance from the polar to the nonpolar side of the lipid monolayer when the sterol's side chain is in the staggered conformation with C-22 positioned to the right. However, when the side chain is altered so that it has significantly different spatial character­ istics, S. cerevisiae is unable to utilize the resulting sterol. Deleterious changes are brought about for instance by removal of the carbon atoms on the right by direct chain shortening or by shifting them to the left rigidly with a 17(20)-double bond or conformationally by inversion of C-20. V a r i a b i l i t y of S t e r o l Structure Cholesterol, s i t o s t e r o l , and ergosterol are among the names of s t r u c t u r a l l y d i f f e r e n t s t e r o l s which are not only widely recognized but are also commonly associated with animals, plants, and fungi, respectively. This i l l u s t r a t e s an important point: The structure of s t e r o l s i s v a r i a b l e , and there i s some sort of association between structure and biology. A c t u a l l y , the s t r u c t u r a l v a r i a b i l i t y turns out to be quite large when one examines the problem i n d e t a i l (1-3). More than a hundred s t e r o l s have been found i n l i v i n g systems. They f a l l i n t o two major categories i n terms of amount. There are those which accumulate and presumably play a functional r o l e (1) and secondly those which do not accumulate and remain i n trace amounts because they are only transient precursors to the functional

0097-6156/87/0325-0252$06.00/0 © 1987 American Chemical Society

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16.

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Structure-Function Relationships for Sterols

compounds. On occasion there i s some overlap between the two cate­ gories, e s p e c i a l l y with respect to saturated-unsaturated pairs when one of them i s a precursor to the other. This l a t t e r s i t u a t i o n i s probably represented by accumulation of the pairs i s o f u c o s t e r o l / s i t o s t e r o l and s i t o s t e r o l / s t i g m a s t e r o l , but f o r the most part accumu­ lated or dominant s t e r o l s tend to be end-products rather than i n t e r ­ mediates . Good examples of dominant s t e r o l s are cholesterol and i t s homologs, 24a-methylcholesterol (campesterol) and 24a-ethylcholesterol ( s i t o s t e r o l ) . These along with 243-methylcholesterol and the 22Edehydro d e r i v a t i v e of s i t o s t e r o l (stigmasterol) comprise the main s t e r o l component of most tracheophytes. However, there are many more s t e r o l s to consider than j u s t these three even among higher p l a n t s . I f we concentrate only on the s t e r o l s which accumulate, there are as many as three dozen of them which are known to be present i n t e r r e s ­ t r i a l (i_.e_., non-marine) l i v i n g systems (Table I ) . Most of them vary Table I. Variabl of T e r r e s t r i a l L i v i n g Systems 5

7

Nuclear double bond: Δ or Δ Carbon a d d i t i o n to side chain: 24-CH or 24-C2H5 Configuration of substituent at C-24: α or 3 Ε-Double bond at C-22,C-23 Double bond at C-24,C-28 Double bond at C-25,C-27 (only 243-Series) 3

b

a) The features l i s t e d are added cumulatively (except where i n d i ­ cated by "or" or "only") to 5a-cholestan-33-ol. Thus, a d d i t i o n of Δ gives c h o l e s t e r o l , or cumulative a d d i t i o n of Δ , 24a-ethyl, and Δ gives s p i n a s t e r o l . Thirty-two s t e r o l s (including cholesterol and l a t h o s t e r o l ) can be constructed from t h i s l i s t . Stanols, e s p e c i a l l y 5a-cholestan-33-ol and 24a-ethyl-5a-cholestan-33-ol ( s i t o s t a n o l ) , also occur. In addition, A > - s t e r o l s accumulate e s p e c i a l l y i n many fungi and i n some algae. The most common one i s i n the 243-methyl-22E-dehydro series (ergosterol). Others include e r g o s t e r o l s 243-ethyl analog, 7-dehydroporiferasterol. 5

7

2 2 Ε

5

7

1

b) In the 24-ethylidene series both the E- and the Z-configurations are known. by changes i n unsaturation i n r i n g Β and the side chain and by sub­ s t i t u t i o n at C-24. For instance, we have i s o l a t e d f i f t e e n of the possible Δ - and A - s t e r o l s from the seeds of a single plant (the squash, Cucurbita maxima) (Table II) (4,5). Although i n most plants a smaller number of s t e r o l s accumulate, i t i s i n t e r e s t i n g that as many as f i v e s t e r o l s of the squash comprising 81% of the mixture are each present at l e v e l s > 10.0% of the t o t a l . Six more s t e r o l s are present at l e v e l s from 1.0 to 6.4%. This suggests a function f o r each of the s t e r o l s . The l i s t of f u n c t i o n a l s t e r o l s i s made s t i l l longer by the a d d i t i o n of s t e r o l s from the marine environment where v a r i a t i o n can also be based on lengthening or shortening of the side chain as w e l l as on additions of methyl and cyclopropyl groups to several p o s i t i o n s i n the side chain (6-8). Changes also occur i n the skeleton of r i n g A. 5

7

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Table I I . The 4-Desmethylsterols of Cucurbita maxima (squash) Seeds (Garg and Nes, r e f s . k_ and 5) Stero 24a-Ethyl-5a-cholesta-7,22E-dien-33-ol

10.55

26.2

243-Ethyl-5a-cholesta-7,25(27)-dien-33-ol

6.96

17.3

243-Ethyl-5a-cholesta-7,22E,25(27)-trien-33-ol

6.59

16.4

24a-Ethyl-5a-cholest-7-en-33-ol

4.29

10.7

24Z-Ethylidene-5a-cholest-7-en-33-ol

4.19

10.4

243-Methylcholesta-5,25(27)-dien-33-ol

2.56

6.4

243-Ethylcholesta-5,25(27)-dien-33-ol

1.43

3.6

24a-Ethylcholesta-5-en-33-ol

1.20

3.0

24a-Ethylcholesta-5,22E-dien-33-ol

0.79

2.0

24Z-Ethylidenecholes t-5-en-3 3-ol

0.54

1.3

24a-Methylcholest-5-en-33-ol

0.41

1.0

24ξ-Μβ^1-5α-^οΐ63^7-θη-33-ο1

0.33

0.8

243-Ethylcholesta-5,22E,25(27)-trien-33-ol

0.31

0.7

0.07

0.1

trace

-

243-Methyl-5a-cholesta-7,25(27)-dien-33-ol 243-Methylcholest-5-en-33-ol

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255

Implications of Sterol V a r i a b i l i t y Now, the question which has motivated much of the recent work i n my laboratory, and which I should l i k e to discuss here, i s t h i s : What r e a l l y i s the reason why c e r t a i n s t e r o l s accumulate? Is one s t e r o l as good as another? Are we j u s t observing a more or less random d i s t r i b u t i o n based on chance mutations? Or i s a s e l e c t i o n operative, a mating of structure to function, suggesting, f o r instance, i n squash the existence of multiple functions f o r sterol? One way to get at t h i s problem would be to delineate what s t e r o l s a c t u a l l y do, to f i n d a way to quantitate t h e i r value, and then to determine the extent to which deviations i n structure a f f e c t function. I t was t h i s course that was s e t t l e d on, but the question which arose immediately was: What l i v i n g system should be used experimentally? Early Work With Yeast I t so happens that at the time I was f i r s t thinking seriously about t h i s problem (the early to mid 1970s) the l i t e r a t u r e already revealed an apparent s o l u t i o n . The primary s t e r o l (ergosterol) of the fungus, Saccharomyces cerevisiae (the common yeast of bakers and brewers), has a 243-methyl group, as we have confirmed using ^-NMR (9), and the way t h i s group i s introduced b i o s y n t h e t i c a l l y i s f a i r l y complicated (l.,2_,lû). I t requires preservation (rather than reduction) of the terminal double bond of squalene (Δ of s t e r o l s ) so that trans­ fer of a Ci-group can occur to i t from S-adenosylmethionine with elimination of a proton from the incoming Ci-group (C-28) and 1,2hydride migration (C-24 to C-25). This gives a 24-methylene group which then undergoes a stereospecific reduction to y i e l d the 243methyl group. Other a l t e r n a t i v e s could have occurred, e.g., elimina­ t i o n from C-27 instead of C-28, reduction to the a- rather than 3methyl, or a second Cχ-transfer to the 24-methylene group might have occurred. A l l of the biosynthetic steps which a c t u a l l y produce the 243methyl group are w e l l documented (1,2,10), and much more than a single point mutation i n fungal evolution would be necessary to account f o r t h i s involved sequence of reactions. Despite t h i s , w i l d type S_. cerevisiae seemed, according to the l i t e r a t u r e (11-13) » to have l i t t l e use f o r the 243-methyl group. Ergosterol could be r e ­ placed, f o r instance, by cholesterol which has no substituent at C-24 or by s i t o s t e r o l which has an ethyl group at C-24 and no serious loss of function was observable. In p a r t i c u l a r , growth s t i l l occurred with cholesterol or s i t o s t e r o l and almost at the same rate as with ergosterol. This strongly suggested either that the manner i n which the experiments were carried out l e f t the functional question more or less moot or that biosynthesis and function (and by implication e v o l u t i o n a r i l y surviving mutants and function) are not c l o s e l y correlated. Yeast thus presented an i n t r i g u i n g challenge. I t was chosen f o r study both f o r t h i s reason and because i t has some c h a r a c t e r i s t i c s which make i t very useful as an experimental t o o l . 24

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256

U s e f u l c h a r a c t e r i s t i c s of y e a s t . Y e a s t , jL.e_., S_. c e r e v i s i a e , i s , f i r s t o f a l l , e u k a r y o t i c and a t l e a s t t o some degree s h o u l d be r e p r e s e n t a t i v e of h i g h e r forms o f l i f e i n g e n e r a l . S e c o n d l y , i t i s n a t u r a l l y s i n g l e c e l l e d and can be grown i n a l i q u i d c u l t u r e . I f needed, l a r g e q u a n t i t i e s (100 g) can be o b t a i n e d . A g r e a t d e a l i s known about t h i s m i c r o o r g a n i s m , which i s not p a t h o g e n i c , and i t can be e a s i l y o b t a i n e d i n an a x e n i c c o n d i t i o n , f o r i n s t a n c e from the American Type C u l t u r e C o l l e c t i o n . Perhaps o f most importance i s y e a s t ' s u n u s u a l a b i l i t y among e u k a r y o t e s t o be a b l e to l i v e a n a e r o b i c a l l y and to d e r i v e i t s ATP by nonrespiratory glycolysis. Under these c o n d i t i o n s , as seen under the e l e c t r o n m i c r o s c o p e , the m i t o c h o n d r i a change m o r p h o l o g i c a l l y t o what a r e c a l l e d p r o m i t o c h o n d r i a ( 1 4 ) , and the c e l l s become a u x o t r o p h i c f o r s t e r o l (11) and u n s a t u r a t e d f a t t y a c i d such as o l e a t e ( 1 5 ) . Aerob i c a l l y y e a s t d e r i v e s i t s s t e r o l and u n s a t u r a t e s by b i o s y n t h e s i s , and f o r b o t h types o f compound t h e r e a r e b i o s y n t h e t i c s t e p s i n v o l v i n g mixed f u n c t i o n o x i d a s e s (1,2). T h i s means t h a t a v a i l a b i l i t y o f oxygen i s d e n i e d , i t i s o b v i o u s t h a t n e i t h e r s t e r o l n o r u n s a t u r a t e s can be formed. By o p e r a t i n g a n a e r o b i c a l l y we can t h e r e f o r e d i c t a t e what s t e r o l i s i n the c e l l s s i m p l y by a d d i n g the s t e r o l o f our c h o i c e to the medium. Andreasen and S t i e r (11) were the f i r s t to demonstrate t h a t t h i s c o u l d be done and they were the f i r s t a l s o to show t h a t w i t h o u t s t e r o l (11) and u n s a t u r a t e d f a t t y a c i d (15) y e a s t w i l l not grow. T h i s was an e l e g a n t d e m o n s t r a t i o n that these l i p i d s p l a y a v i t a l r o l e . The

F u n c t i o n of

Sterols

There i s e x t e n s i v e e v i d e n c e , which I have reviewed e a r l i e r (1,16)» to i n d i c a t e t h a t the p r i n c i p a l r o l e o f s t e r o l s throughout n a t u r e i s t o a c t as a r c h i t e c t u r a l components o f some though n o t n e c e s s a r i l y a l l membranes. The plasma membrane i s f r e q u e n t l y the p r i n c i p a l r e s i d e n c e of the s t e r o l ( 12,17., 1 8 ) . E u k a r y o t i c as w e l l as most p r o k a r y o t i c membranes a r e b i l a y e r s o f p h o s p h o l i p i d and p r o t e i n . Each monolayer, which i s about 2.1 nm t h i c k (19,20), i s b e l i e v e d to c o n t a i n the s t e r o l i n a nonhomogeneous d i s t r i b u t i o n , and a t l e a s t i n some c a s e s s t e r o l can move between the monolayers. This process i s c a l l e d " f l i p - f l o p " . S t e r o l has been found b o t h i n t h e m i t o c h o n d r i a (21) and the plasma membrane o f . c e r e v i s i a e (13,18), and the a b i l i t y t o s u p p o r t the growth o f a n a e r o b i c y e a s t presumably i s a s s o c i a t e d w i t h i t s membranous function. I t i s n o t f u l l y c l e a r how s t e r o l i s i m p o r t a n t a r c h i t e c t u r a l l y , but much e v i d e n c e p o i n t s t o a m o d u l a t i o n o f the p h y s i c a l p r o p e r t i e s of the membrane, e s p e c i a l l y o f the t r a n s i t i o n from the g e l t o the l i q u i d c r y s t a l s t a t e of the p h o s p h o l i p i d t h e r e b y c o n t r o l l i n g the r i g i d i t y , u s u a l l y e x p r e s s e d i n the r e v e r s e as f l u i d i t y , o f the membrane. T h i s has been c a l l e d the " b u l k membrane f u n c t i o n " (22) o f s t e r o l to d i s t i n g u i s h i t from a n o t h e r f u n c t i o n o r f u n c t i o n s which require(s) less sterol. The n a t u r e o f the l a t t e r f u n c t i o n ( s ) i s more e l u s i v e , a l t h o u g h we (.23,24) and o t h e r s (2*18,25.) have p r e s e n t e d good e v i d e n c e f o r i t s e x i s t e n c e i n y e a s t , and a s i m i l a r phenomenon e x i s t s a l s o i n p r o k a r y o t i c mycoplasmas (22,26). We have c a l l e d the f u n c t i o n

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

16.

NES

Structure-Function Relationships for Sterols

257

requiring less s t e r o l "regulatory" (23), while Parks and h i s group use the word "sparking" (25), and Bloch and h i s associates apply the term " s y n e r g i s t i c " (18). The Experimental System We discovered very early that the growing of yeast anaerobically, i.e_., r e a l l y anaerobically, i s quite d i f f i c u l t . Or, to put i t another way, i t i s f a r from easy to remove enough oxygen to prevent at l e a s t some growth from occurring on a synthetic medium containing no s t e r o l . We achieved success by c a r e f u l l y sealing i n l e t s into our growth chamber with p a r a f f i n wax and p r i o r to i n o c u l a t i o n by using a prolonged f l u s h of e l e c t r o n i c grade nitrogen (< 0.2 ppm of oxygen) which had been passed through a s o l u t i o n of chromous c h l o r i d e to reduce r e s i d u a l oxygen to the water stage. Under these conditions i t became possible to take a log phase inoculum of yeast from a continu­ ous c u l t u r e and f i n d i t presence of Tween-80 (a same time, growth ensued w e l l (10 c e l l s / m l from 10 c e l l s / m l i n 3 days) when ergosterol (5 mg/liter of medium) had been emulsified i n the Tween-80 and then dispersed i n the medium. Our i n i t i a l plan was to make a systematic i n v e s t i g a t i o n of the s t r u c t u r a l features of ergosterol by replacing the ergosterol with other s t e r o l s which either lacked a p a r t i c u l a r feature, e_.£., the Δ -unsaturation, or had some other i n t e r e s t i n g aspect to them, e.^., a l t e r e d stereochemistry. The c o n t r i b u t i o n of the feature was then to be assessed by evaluation of the number of c e l l s a f t e r three days. The c e l l population could be measured both v i s u a l l y and by means of a 16-channel Coulter counter which also permits determina­ t i o n of the d i s t r i b u t i o n of c e l l sizes and volumes. In a d d i t i o n , e x t r a c t i o n of the c e l l s , or s u b c e l l u l a r f r a c t i o n s , would permit us to examine the amount, d i s t r i b u t i o n , and metabolism of the added s t e r o l . This experimental design (27,28) proved adequate to show a great deal about what i s and what i s not important about the s t e r o l mole­ cule. However, some growth and i t should be added v a r i a b l e growth i n the presence of c h o l e s t e r o l occurred. By examining the s t e r o l d i s ­ t r i b u t i o n i n the n e u t r a l l i p i d f r a c t i o n of the yeast grown i n the manner described we found that there were s t i l l s u f f i c i e n t traces of oxygen l e f t i n the medium to allow very small but s i g n i f i c a n t amounts of endogenous s t e r o l synthesis to occur. We could i d e n t i f y (28) not only ergosterol but also l a n o s t e r o l , zymosterol, 24-methylene-7-dehydrocholesterol, and 22-dihydroergosterol a l l of which are i n t e r ­ mediates i n the ergosterol pathway (1). Such biosynthesis presumably accounts f o r growth on c h o l e s t e r o l i n the experiments of Hossack and Rose (13) who reported that ergosterol was also present. Ergosterol produced b i o s y n t h e t i c a l l y of course complicates the i n t e r p r e t a t i o n of the r e s u l t s obtained with other s t e r o l s . In order to reduce biosynthesis to an i n s i g n i f i c a n t l e v e l i n the oxygen-deprived yeast we added 2,3-iminosqualene i n our more recent experiments (29). This compound i s a competitive i n h i b i t o r of 2,3-oxidosqualene cyclase (30), and when i t was incorporated into the medium along with c h o l e s t e r o l no growth then occurred although on replacement of c h o l e s t e r o l with ergosterol growth proceeded w e l l . Thus, stepwise we were able to achieve a no-growth condition 7

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

258

ECOLOGY AND METABOLISM OF PLANT LIPIDS

without s t e r o l by using prolonged flushing with highly p u r i f i e d nitrogen and then by adding 2,3-iminosqualene we also achieved a nogrowth condition i n the presence of c h o l e s t e r o l . One f i n a l experi­ mental thing must be mentioned. If we want to have both ergosterol and some other s t e r o l present, i t i s obvious we could add both, and we have done j u s t that. Such a method has the advantage of allowing various r e l a t i v e amounts to be added, but we also found f o r some pur­ poses generation of ergosterol i n s i t u by the addition of a i r was a simple a l t e r n a t i v e . With 10 ml of a i r added to the headspace (ca. 100 ml) of our growth chamber i n the presence of the iminosqualene but with no s t e r o l we (23) obtained 24 χ 10 c e l l s / m l which i s a quarter or a f i f t h of what we obtained with added ergosterol. The e f f e c t of added s t e r o l could then be determined by measuring the increase i n c e l l count. 6

The P o s i t i o n i n g of C-21 and C-22 i n Space I t has been possible to the configuration at C-20 of s t e r o l s . The configuration at C-20 of cholesterol (Figure 1) i s known to be R from degradation of the analog and c o r r e l a t i o n of the product containing C-20 with D-(+)glyceraldehyde (30,31). Cholesterol i s therefore a primary standard. We prepared 20-epicholesterol and both for i t and f o r c h o l e s t e r o l we determined H- and C-NMR spectra, the melting points, the o p t i c a l r o t a t i o n s , and the chromatographic rates of movement (32-34). In some cases t h i s was also done f o r a series of other s t e r o l s (33-35) such as 20R- and 20S-halosterol (35) which i s the C 6-analog of cholesterol which has one less CH2~group i n the side chain and occurs i n the marine environment (1) . There were several consistent s h i f t s . The 20R-isomers moved slower i n both adsorption and g a s - l i q u i d chromatography, showed a downfield s h i f t i n the C-21 proton NMRs i g n a l , a greater separation of the terminal gem-dimethyl proton s i g n a l s , and a more p o s i t i v e (actually less negative), o p t i c a l r o t a ­ t i o n at the D-line of sodium. There was also a tendency for the S-isomer to have a lower melting point, although t h i s was not so f o r the epimeric c h o l e s t e r o l p a i r . This information along with x-ray d i f f r a c t i o n permitted a survey of the C-20 configuration of natural sterols, , r e f . 32_. With one w e l l documented but curious excep­ t i o n (36) along with one dubious exception lacking strong p h y s i c a l evidence (37), a l l s t e r o l s i n both the marine and t e r r e s t r i a l environments have the 20R-configuration. Why should t h i s be so? I t i s not necessary from a biosynthetic point of view, since reasonable mechanisms are possible to give both the R- and the S-epimers (32). I believe the answer to why the R-configuration at C-20 i s chosen n a t u r a l l y l i e s i n the mating of the shape and s i z e of the s t e r o l to the dimensions of the monolayer i n the l i p i d l e a f l e t of membranes. In order to keep the smallest group on C-20 adjacent to C-18 which l i e s on the front or 3-face of the nucleus, r o t a t i o n of C-20 about the 17(20)-bond should occur preferably so that the 20-H-atom i s i n front. This means that i n the R-series C-22 i s on the r i g h t ("right-handed") and C-21 on the l e f t ("left-handed") when the s t e r o l i s viewed i n the usual way, I.e., with C-3 to the l e f t and C-18 and C-19 toward the observer. X-ray data f o r many natural s t e r o l s corroborates not only the R-configuration but the existence 1

13

2

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

16. NES

Structure-Function Relationships for Sterols

20R

Ε

20S

_ Ι7(20) Δ

Ζ-Δ

, 7 ( 2 0 )

Figure 1 . Stereochemistry of sterols at C - 2 0 . Structures i l l u s t r a t e ring-D and the cholesterol side chain. The carbon bearing the Η-atom shown i s C - 2 0 . In halosterol the isohexyl moiety [(CH )3-CH-(CH ) ] on C - 2 0 i s replaced by an isopentyl group [ (CH )2 -CH-(CH3)2 ] . Other replacements were made with n-alkyl groups. 2

3

2

2

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

259

260

ECOLOGY AND METABOLISM OF PLANT LIPIDS

of the side chain i n the right-handed conformation. We have also been able to show from x-ray d i f f r a c t i o n with the t e t r a c y c l i c t r i t e r penoids euphol and t i r u c a l l o l that the o r i e n t a t i o n of C-22 r e a l l y i s dependent on the configuration of C-20 and the presence of C-18 (38). The right-handed conformation of s t e r o l s i s probably chosen i n an evolutionary sense because with C-22 on the r i g h t and the side chain i n the staggered conformation the long dimension of c h o l e s t e r o l (and other s t e r o l s with the same chain length) coincides almost exactly with that of the thickness of the monolayer. However, the s t e r o l length i s much shorter than the l a t t e r when C-22 i s on the l e f t as we think i t would be i n the 20S-epimer. Direct evidence f o r the above i n t e r p r e t a t i o n was obtained as follows. When incubated with our oxygen-deprived yeast i n the absence of 2,3-iminosqualene, c h o l e s t e r o l , i t w i l l be r e c a l l e d , permitted some growth. However, when c h o l e s t e r o l was replaced by 20e p i c h o l e s t e r o l no growth at a l l occurred (28) S i m i l a r l y while cholesterol or h a l o s t e r o pyriformis, the 20-epi-analog not i t s epimer at C-20 induced oospores i n the fungus Phytophthora cactorum (41). In order to show that t h i s negative e f f e c t of inversion at C-20 correlates with the p o s i t i o n i n g of C-22 on the l e f t , we synthesized E- and Z-17(20)-dehydrocholesterol (42). In the E-isomer C-22 i s r i g i d l y oriented to the r i g h t , and to the l e f t i n the Z-isomer. The Ε-isomer permitted some growth of yeast, but as expected the Z-isomer did not (28). Again i n agreement with r e s u l t s with the yeast system, only the Ε-isomer was metabolized by T. pyriformis (39), the Z-isomer being recovered unchanged from the c e l l s (39). S i m i l a r l y , only the Ε-isomer induced formation of oospores i n P_. cactorum (43). The Length of the Side Chain A d i r e c t test of the importance of the long dimension of the s t e r o l molecule was obtained by our synthesizing androst-5-en-33-ol, which i s a Δ - C i 9 - s t e r o l with no side chain at a l l , and by also making a v a r i e t y of A - s t e r o l s i n which the isohexyl group (C-22 to C-27) on C-20 of c h o l e s t e r o l was replaced e i t h e r with hydrogen (pregn-5-en33-ol) or with longer or shorter arrays of carbon atoms than occur i n c h o l e s t e r o l , ±.e., a series of 20R-n-alkylpregn-5-en-33-ols (34). The epimeric 20S-sterols were also prepared and as already mentioned 20R- and 20S-halosterol were made (35). We also prepared 21-isopentylcholesterol (44) which we c a l l e d "wingsterol , since i t has an isohexyl group on both sides of C-20. Neither the C i g - s t e r o l nor wingsterol permitted growth of yeast (44). The C i g - s t e r o l also pre­ vented growth of T. pyriformis (44) as w e l l as of the larvae of the insect H e l i o t h i s zea (44), although H. zea larvae grow w e l l on c h o l e s t e r o l . In work which has only been p a r t l y published (45) we also found that as the length of the s t e r o l side chain i s lengthened growth of yeast r i s e s to a maximum at f i v e C-atoms on C-20, , with 20R-n-pentylpregn-5-en-33-ol and f e l l v i r t u a l l y to zero with more than seven C-atoms on C-20. The 20S-analogs were a l l i n a c t i v e . The influence of length of the side chain was also assessed i n oospore induction with cactorum (46), metabolism i n T_. pyriformis (47), e s t e r i f i c a t i o n i n T. pyriformis (44), i n h i b i t i o n of t e t r a 5

5

11

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

16.

NES

Structure-Function Relationships for Sterols

261

hymanol production by T. pyriformis (44), and e s t e r i f i c a t i o n with mammalian ACAT (44). In a l l cases maximal a c t i v i t y occurred either with the 20R-n-pentyl or 20R-n-hexyl derivative of pregn-5-en-33-ol agreeing w e l l with the yeast r e s u l t s . The observed length for maximal a c t i v i t y (5 or 6 C-atoms on C-20) correlates w e l l with the length of natural s t e r o l s (8). Importance of the 243-Methyl Group i n Yeast While the stereochemistry at C-20 and the length of the side chain should and a c t u a l l y do appear to have general s i g n i f i c a n c e i n terms of the s i z e of the l i p i d l e a f l e t i n membranes, i t i s not as easy a p r i o r i to assign a r o l e to the 243-methyl group. This i s e s p e c i a l l y d i f f i c u l t , since from data on occurrence (1) the methyl group c e r t a i n l y does not seem to have general importance. Geometrically i t should thicken the side chain and probably make i t more r i g i d . This sort of phenomenon might protect a membrane agains tures. However, yeast, with an optimum temperature of 28-36 and a maximum temperature of 40-42°, does not grow very w e l l at temperatures much above those which are normal f o r mammals which have c h o l e s t e r o l , and there i s no c o r r e l a t i o n between the optimum temperature f o r a fungus and the s t e r o l i t makes. Nevertheless, the early work of ourselves (27,28) and of Andreasen and S t i e r (11) showed that ergosterol i s better f o r J5. c e r e v i s i a e than i s c h o l e s t e r o l . More recently Ramgopal and Bloch (18) concurred i n t h i s using the GL7 mutant which i s auxotrophic f o r s t e r o l a e r o b i c a l l y having l o s t the oxidosqualene cyclase. We went on to show that t h i s difference i n effectiveness i s not due to the A -bond, since neither l a t h o s t e r o l nor 7-dehydrocholesterol were any better than cholesterol i n supporting the growth of our semi-anaerobic w i l d type yeast (28). On the other hand, 243-methyl-22E-dehydrocholesterol (brassicasterol) was v i r t u a l l y as e f f e c t i v e as was e r g o s t e r o l , and when the double bond was removed to give 243-methylcholesterol the compound was s t i l l 75% as a c t i v e as ergosterol (28), although under the same conditions cholesterol was only 23% as a c t i v e . The mystery deepened when we showed recently (48) that growth on cholesterol or on other s t e r o l s lacking a 243-methyl group (Table I I I ) can be brought to zero by using 2,3-iminosqualene even though the yeast retains i t s a b i l i t y to grow w e l l on ergosterol, b r a s s i c a s t e r o l , 243-methylcholesterol, and desmosterol which we were able to show was converted i n high y i e l d i n the yeast to 243-methylcholest e r o l (48). I n t e r e s t i n g l y , neither 24a-methylcholesterol nor 243e t h y l c h o l e s t e r o l supported growth i n d i c a t i n g that both the exact s i z e and configuration of the substituent at C-24 are important. Thus, the 243-methyl group i t s e l f i s e s s e n t i a l and the idea that ergosterol i s j u s t better than cholesterol i s not quite r i g h t . In some v i t a l way cholesterol can not replace ergosterol at a l l . Yet, i t appeared that i n some way i t could, because we (27,_28) and others (11,13) had gotten yeast to grow i n the absence of 2,3-iminosqualene to v a r i a b l e degrees on c h o l e s t e r o l . Since i n our system without the i n h i b i t o r there was some ergosterol present, though not enough by i t s e l f to l e t growth occur (28), i t appeared that i t must be this small amount of endogenous s t e r o l which was responsible f o r the a b i l i t y of choies7

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

24a-Alkylsterols 24a-Methylcholesterol 24a-Ethylcholesterol

243-Alkylsterols 243-Methylcholesterol 243-Methyl-22E-dehydrocholesterol 243-Methyl-7,22E-bisdehydrocholesterol (ergosterol) 24 3-Ethylcholes t e r o l

C.

D.

b.

0.2 0.2

0.1 0.2 0.5 99 0.1 0.2 0.1

0.1 0.2

air

95 108 114 0.2

No

a

103

107 89

80 16

-85

112 97 100

21 61

Added a i r

b

C e l l Count ( M i l l i o n s of Cells/ml)

After 3 days, average of 3 experiments. C e l l s adapted to oxygen-deprivation were used. 10 ml of a i r added to cultures which had not grown a f t e r i n i t i a l three days and c e l l count obtained a f t e r another 3 days. Without s t e r o l , the c e l l count was 24 m i l l i o n . Data are averages f o r 3 experiments.

24-Desalkylsterols Cholesterol Lathosterol 7-Dehydrocholes t e r o l 24-Dehydrocholesterol (desmosterol) 5a-Cholestanol E-17(20)-Dehydrocholesterol Z-17(20)-Dehydrocholesterol

B.

a.

Sterols lacking an H0-group Choiest-5-ene 5a-choles tan-3-one

Sterol (5 mg/liter)

Growth Response of Oxygen-Deprived S_. cerevisiae to Sterols with 2,3-Iminosqualene (50 μΜ) Present (From r e f s . 23, 2A> and 48)

A.

Table I I I .

16.

NES

Structure-Function Relationships for Sterob

263

t e r o l to be a c t i v e . This i n turn suggested the existence of a dual (or perhaps multiple) r o l e f o r s t e r o l i n yeast. In one r o l e choles­ t e r o l could replace ergosterol, but i n the other, r e q u i r i n g less s t e r o l , c h o l e s t e r o l must be i n e f f e c t u a l . Evidence f o r a Dual Role of S t e r o l The supposition discussed above that 243-methylsterol i n an amount too small to support growth by i t s e l f would permit s t e r o l s lacking the methyl group to induce growth was demonstrated d i r e c t l y i n two ways. In the f i r s t place, i n the presence of 2,3-iminosqualene a f t e r three days following i n o c u l a t i o n , cultures containing the s t e r o l s l i s t e d i n Table I I I which had not grown were oxygenated with 1 0 ml of a i r to generate ergosterol i n s i t u 0 2 3 , 2 4 ) . A f t e r an a d d i t i o n a l three days the only cultures which had not now grown more than the control (lacking s t e r o l ) were the ones which contained cholest-5-ene and Z - 1 7 ( 2 0 ) - d e h y d r o c h o l e s t e r o one which was reduced t was metabolized, or more p r e c i s e l y a l l except the ketone were recovered unchanged i n s u b s t a n t i a l quantity, and no metabolites were observed ( 2 3 ) . The growth data a f t e r a d d i t i o n of a i r (Table I I I ) show that quite a v a r i e t y of s t e r o l s w i l l support growth i f a small amount of 2 4 3 - m e t h y l s t e r o l i s present. This kind of e f f e c t was f i r s t found i n insects by Clark and Bloch ( 5 0 ) f o r c h o l e s t e r o l and choles­ tanol, and cholestanol was said to "spare (partly replace) the c h o l e s t e r o l . We ( 2 3 ) have adopted t h i s term, 1.e., cholesterol, for instance, w i l l spare ergosterol. Table I I I reveals that sparing a c t i v i t y occurs with s t e r o l s not only lacking a substituent at C - 2 4 but also with those having 24a-methyl, 2 4 a - e t h y l , or 2 4 3 - e t h y l groups. Unsaturation i n r i n g Β i s unnecessary, as i s also the case i n insects ( 5 0 ) , e i t h e r Δ -, Δ -, or Δ > -unsaturation can be present and C - 2 2 must be oriented to the r i g h t i n the usual view of the molecule. We have also constructed growth curves using mixtures of choles­ t e r o l and ergosterol i n various r a t i o s from 0 - 1 0 0 % of each ( 2 3 ) instead of generating ergosterol with a i r . The r e s u l t s are shown i n Table IV i n an abbreviated form. I t w i l l be seen that neither a l o t (5 mg/1) of c h o l e s t e r o l alone nor a l i t t l e (< 0 . 5 mg/1) ergosterol alone was supportive of growth, yet maximal growth could be obtained when the s t e r o l s were combined at these l e v e l s , e^g.. » 4 . 5 mg/1 of c h o l e s t e r o l and 0 . 5 0 mg/1 of ergosterol. Since i t takes 1 . 5 0 mg/1 of ergosterol alone f o r the yeast to grow maximally, c h o l e s t e r o l i s c l e a r l y sparing most (about two-thirds) but not a l l of the ergos­ t e r o l . Under these circumstances, the c h o l e s t e r o l i s presumably f u l f i l l i n g the bulk membrane function. Since the ergosterol (or other 243-methylsterol) i s required i n only a small amount, so long as another s t e r o l i s present i n large amount, we think 243-methylsterols play some regulatory function. This may have something to do with c o n t r o l l i n g r a t i o s of saturated and unsaturated f a t t y acid ( 5 0 , 5 1 ) , although the exact nature of the function needs more exploration. 11

5

7

5

7

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

264

ECOLOGY AND METABOLISM OF PLANT LIPIDS Table IV. Sparing A c t i v i t y of Cholesterol for Ergosterol i n _S. cerevisiae with 50 μΜ 2,3-Iminosqualene Present (From r e f . 23)

S t e r o l Cone, (mg/1) Ergosterol Cholesterol o.oo

a

5.00

0.05

a

4.95

o.io

a

i.oo

0.2

4.90

12

a

4.80

48

4.70

71

C

4.50

107

4.00

108

0.50 a) b) c) d)

0.2

b

0.20 0.30

C e l l Count ( M i l l i o n s of Cells/ml)

d

This This This This

cone. cone. cone. cone.

without without cholesterol gave 0.3 χ 10 c e l l s / m l . without cholesterol gave 0.8 χ 10 c e l l s / m l . without cholesterol gave 54.( 3 χ 10 c e l l s / m l . 6

6

Use of mutants. Ramgopal and Bloch (18) have come to conclusions s i m i l a r to ours using the yeast mutant GL7, but for reasons not r e a l l y clear neither they nor others (25,53) have been able to obtain a no-growth-condition with GL7 on c h o l e s t e r o l . However, Ramgopal and Bloch (18) f i n d that the mutant grows better on ergos­ t e r o l than on c h o l e s t e r o l , and a small amount of ergosterol substan­ t i a l l y enhances growth on cholesterol which these authors refer to as a s y n e r g i s t i c e f f e c t . Bloch and hie associates have also found a related synergism f o r cholesterol and lanosterol i n mycoplasmas (26). S i m i l a r l y , with GL7 and a mutant (FY3) with much the same defects Parks and h i s associates (25) f i n d ergosterol i s necessary i n small amount i n order for 5a-cholestan-33-ol to support growth. However, Parks and h i s group (25,54) interpret the r e s u l t s only to mean a A -grouping i s being supplied by ergosterol for what they c a l l the sparking function, and they f e e l a 243-methyl group i s not necessary (54) i n view of the a b i l i t y of cholesterol alone to permit growth of GL7 and FY3. The reason for t h i s growth remains unclear. Since the mutants are grown a e r o b i c a l l y and require much less s t e r o l than our w i l d type, e_.£., 0.3 vs. 1.5 mg/1 of ergosterol to give maximum growth for GL7 (18) and w i l d type (23), respectively, s l i g h t l e a k i ness through the block of s t e r o l biosynthesis i n the mutants might account for the apparent lack of an absolute requirement for a 243methylsterol. That i s , a minute amount of regulatory s t e r o l may acually be biosynthesized. I t of course i s also possible that the mutants a c t u a l l y do have a less stringent requirement for 243-methylsterol. 5

S t r u c t u r a l Effects on Feedback I n h i b i t i o n of Sterol

Biosynthesis

Another way to gain evidence on whether the structure of ergosterol i s e s p e c i a l l y important to yeast, would be to measure the e f f e c t of ergosterol and other s t e r o l s on the a b i l i t y of the organism to

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

16.

NES

Structure-Function Relationships for Sterols

265

biosynthesize s t e r o l s . Under oxygen-deprived conditions squalene accumulates i n yeast, because i t can not proceed on to squalene oxide. The squalene l e v e l , therefore, should be a measure of the flow of carbon into the s t e r o l pathway. Ergosterol i s already known by other parameters, to depress s t e r o l biosynthesis i n yeast, and i n agreement we found squalene accumulation was increasingly depressed as we raised the concentration of ergosterol i n the medium (55). However, c h o l e s t e r o l was much less e f f e c t i v e (55). Since c h o l e s t e r o l might have been less e f f e c t i v e l y absorbed, we also measured uptake (and e s t e r i f i c a t i o n ) . Based on the amount of free s t e r o l i n the c e l l s , ergosterol was about four times as e f f e c t i v e as c h o l e s t e r o l i n depressing squalene accumulation (55) . This agrees with the supposit i o n that the choices made i n the biosynthetic process are determined by a mating of the 243-methyl structure with function (23,48). Sterol Uptake Since the concentration an important to an understanding of function, we have been measuring among other things the r e l a t i o n s h i p of structure to uptake from the medium. Many d i f f e r e n t steroids were absorbed (56). They included not only the 33-hydroxysterols i n Table I I I which support growth with added a i r but also steroids such as 5a-cholestan-3-one which do not have an HO-group at C-3 (49). E s p e c i a l l y i n t e r e s t i n g was the observation that when a small amount of growth was induced by addit i o n of a i r (10 ml), the s t e r o l s with the wrong, left-handed o r i e n t a t i o n of C-22 (20-epicholesterol and Z-17(20)-dehydrocholesterol) were not absorbed a t a l l (56) even though s t e r o l s with the natural r i g h t handed o r i e n t a t i o n (cholesterol and E-17(20)-dehydrocholesterol were absorbed w e l l under the same conditions (23,56)). This means that the reason why the left-handed s t e r o l s f a i l e d to support growth (23, 28) was that they a c t u a l l y never even entered the c e l l s . This i n turn implies the existence of a s t r u c t u r a l l y s p e c i f i c gate f o r absorption, and t h i s gate seems to be s t e r e o c h e m i c a l ^ r e l a t e d to function, since we can r e l a t e r i g h t - and left-handedness to membrane geometry (see e a r l i e r ) . The existence of a gate i s also manifested i n the f a c t that yeast c e l l s can turn absorption on and o f f . For instance, while ergosterol i s r e a d i l y absorbed anaerobically, i t i s not absorbed a e r o b i c a l l y (56,57). This means that absorption can simply not be a passive entry of s t e r o l into the plasma membrane (56). A c a r r i e r protein may conceivably be involved (56). Such a protein i s w e l l known to e x i s t i n mammals (58) and evidence also e x i s t s i n yeast (58). The t r i g g e r i n g of the entry mechanism by oxygen-deprivation i s believed to occur v i a e l i m i n a t i o n of an oxygenrequiring step i n heme biosynthesis (59). The loss of heme then sets i n motion events leading to s t e r o l auxotrophy (59). Acknowledgmen t s I should l i k e to thank the National I n s t i t u t e s of Health f o r support of t h i s work through Research Grant AM-12172.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

266

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

Nes, W. R., McKean, M. L. "The Biochemistry of Steroids and Other Isopentenoids"; University Park Press: Baltimore, 1977. Nes, W. R. Adv. Lipid Res. 1977, 15, 233. Goad, L. J.; Goodwin, T. W. In "Progress in Phytochemistry"; Reinhold, L.; Liwschitz, Y., Eds.; Interscience: New York, 1972; Vol. III, p. 113. Garg, V. K.; Nes, W. R. Phytochemistry 1984, 23, 2919. Garg, V. K.; Nes, W. R. Phytochemistry 1984, 23, 2925. Djerassi, C. Pure and Appl. Chem. 1981, 53, 873. Barbier, M. In "Marine Natural Products"; Scheuer, P. J., Ed.; Academic Press: New York, 1981; Vol. IV, p. 147. Nes, W. R. In "Isopentenoids in Plants: Biochemistry and Function"; Nes, W. D.; Fuller, G.; Tsai, L-S., Eds.; Marcel Dekker, Inc.: New York, 1984; p. 325. Adler, J. H.; Young Lederer, E. Quart Rev Andreasen, Α. Α.; Stier, T. J. B. J. Cell Comp. Physiol. 1953, 41, 23. Proudlock, J. W.; Wheeldon, L. W.; Jollow, D. J.; Linnane, A. W. Biochim. Biophys. Acta 1968, 152, 434. Hossack, J. Α.; Rose, A. J. J. Bacteriol. 1976, 127, 67. Morpurgo, G.; Serlupi-Crescenzi, G.; Tecce, G.; Valente, F.; Venettacci, D. Nature (London) 1964, 201, 897. Andreasen, Α. Α.; Stier, T. J. B. J. Cell. Comp. Physiol. 1954, 43, 271. Nes, W. R. Lipids 1974, 9, 596. Lange, Y.; Ramos, Β. V. J. Biol. Chem. 1983, 258, 15130. Ramgopal, M.; Bloch, K. Proc. Nat'l Acad. Sci., U.S.A. 1983, 80, 712. Huang, C.; Mason, J. T. Proc. Nat'l Acad. Sci., U.S.A. 1978, 75, 308. Dalton, A. J.; Haguenau, F. "The Membranes"; Academic Press: New York, 1968. Bottema, C. K.; Parks, L. W. Lipids 1980, 15, 987. Dahl, J. S.; Dahl, C. E.; Bloch, K. J. Biol. Chem. 1981, 256, 87. Pinto, W. J.; Lozano, R.; Sekula, B. C.; Nes, W. R. Biochem. Biophys. Res. Commun. 1983, 112, 47. Pinto, W. J. Ph.D. Dissertation, Drexel University, Philadel­ phia, 1982. Rodriquez, R. J.; Taylor, F. R.; Parks, L. W. Biochem. Biophys. Res. Commun. 1982, 106, 435. Dahl, J. S.; Dahl, C. E.; Bloch, K. Biochemistry 1980, 19, 1467. Nes, W. R.; Adler, J. H.; Sekula, B. C.; Krevitz, K. Biochem. Biophys. Res. Commun. 1976, 71, 1296. Nes. W. R.; Sekula, B. C.; Nes, W. D.; Adler, J. H. J. Biol. Chem. 1978, 253, 6218. Corey, E. J.; de Montellano, P. R. O.; Lin, K.; Dean, P. D. G. J. Am. Chem. Soc. 1967, 89, 2797. Riniker, B.; Arigoni, D.; Jeger, O. Helv. Chim. Acta 1954, 37, 546.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

16.

31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59.

NES

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267

Conforth, J. W.; Youhotsky, I.; Popjak, G. Nature (London) 1954, 173, 536. Nes, W. R.; Varkey, T. E.; Krevitz, K. J. Am. Chem. Soc. 1977, 99, 260. Zarembo, J. E. Ph.D. Dissertation, Drexel University, Philadel­ phia, 1980. Joseph, J. M. Ph.D. Dissertation, Drexel University, Philadel­ phia, 1980. Joseph, J. M; Nes, W. R. J. Chem. Soc. Chem. Commun. 1981, 367. Vanderah, D. J.; Djerassi, C. Tetrahedron Letters 1977, 693. Ikekawa, N.; Tsuda, K.; Morisaki, N. Chemistry and Industry 1966, 1179. Nes, W. D.; Wong, R. Y.; Benson, M.; Landrey, J. R.; Nes, W. R. Proc. Nat'l Acad. Sci., U.S.A. 1984, 81, 5896. Nes, W. R.; Joseph, J. M.; Landrey, J. R.; Conner, R. L. J. Biol. Chem. 1978, 253, 2361. Nes, W. R.; Joseph J. Lipid Res. 1981, 22, 770. Nes, W. D.; Patterson, G. W.; Bean, G. A. Plant Physiol. 1980, 66, 1008. Nes, W. R.; Varkey, T. E.; Grump, D. R.; Gut, M. J. Org. Chem. 1975, 41, 3429. Nes, W. D.; Stafford, A. E. Lipids 1984, 19, 544. Nes, W. R.; Adler, J. H.; Billheimer, J. T.; Erickson, Κ. Α.; Joseph, J. M.; Landrey, J. R.; Marcaccio-Joseph, R.; Ritter, K. S.; Conner, R. L. Lipids 1982, 17, 257. Nes, W. R.; Joseph, J. M. Fed. Proc. 1981, 40, 1561. Nes, W. D.; Nes, W. R. Experientia 1983, 39, 276. Nes, W. R.; Joseph, J. M.; Landrey, J. R.; Conner, R. L. J. Biol. Chem. 1980, 255, 11815. Pinto, W. J.; Nes, W. R. J. Biol. Chem. 1983, 258, 4472. Pinto, W. J.; Nes, W. R. Abstracts of the Ann. Meet. of the Am. Soc. Microbiol., Las Vegas 1985, p. 247. Clark, A. J.; Bloch, K. J. Biol. Chem. 1959, 234, 2583. Buttke, T. M.; Jones, S. D.; Bloch, K. J. Bacteriol. 1980, 144, 124. Buttke, T. M.; Reynolds, R.; Pyle, A. L. Lipids 1982, 17, 361. Kumari, S. N.; Ranadive, G. N.; Lala, A. L. Biochim. Biophys. Acta 1982, 692, 441. Rodriquez, R. J.; Parks, L. W. Arch. Biochem. Biophys. 1983, 225, 861. Pinto, W. J.; Lozano, R.; Nes, W. R. Biochim. Biophys. Acta 1985, 836, 89. Nes, W. R.; Dhanuka, I. C.; Pinto, W. J. Lipids 1985, in press. Trocha, P. J.; Sprinson, D. B. Arch. Biochem. Biophys. 1976, 174, 45. Dempsey, M. E. Current Topics in Cellular Regulation 1984, 24, 63. Lewis, Τ. Α.; Taylor, F. R.; Parks, L. W. J. Bacteriol. 1985, 163, 199.

Received September 10, 1986

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 17

Mechanism of Fungal Growth Suppression by Inhibitors of Ergosterol Biosynthesis John D. Weete Department of Botany, Plant Pathology, and Microbiology, Alabama Agricultural Experiment Station, Auburn University, A L 36849

There are a large number of chemically diverse nitrogenous substances that block the C-14 demethylation of lanosterol (or 24-methylene dihydrolanosterol), and have proven to be effective antifunga site of action is well-defined formation of ergosterol or functionally equivalent sterol results in growth inhibition is not known. It appears from the available evidence that growth inhibition may be the result of a deterioration in membrane function, mainly the plasma membrane, due to an alteration in physical properties caused by changes in sterol content directly, or other modifications in membrane lipid composition brought about by changes in sterol content. I n h i b i t o r s o f v a r i o u s r e a c t i o n s i n t h e pathway of s t e r o l b i o s y n t h e s i s have been known f o r many y e a r s , but more r e c e n t l y t h e r e has been c o n s i d e r a b l e i n t e r e s t i n a l a r g e and w i d e l y d i v e r s e group of such c h e m i c a l s s y n t h e s i z e d d u r i n g t h e past 15 y e a r s t h a t b l o c k s e v e r a l r e a c t i o n s i n t h e l a t e r stages of t h i s pathway. Although not t e c h n i c a l l y c o r r e c t i n a l l c a s e s , t h e term " s t e r o l i n h i b i t o r " (SI) i s o f t e n used t o r e f e r c o l l e c t i v e l y t o t h e s e s u b s t a n c e s . The reason f o r t h e h i g h l e v e l of i n t e r e s t i n t h e s e substances i s t h a t most of them a r e potent growth i n h i b i t o r s of a broad spectrum o f a g r i c u l t u r a l l y and m e d i c a l l y important f u n g i , most of which have e r g o s t e r o l as t h e p r i n c i p a l s t e r o l ; t h u s , they a r e a l s o c a l l e d " e r g o s t e r o l b i o s y n t h e s i s i n h i b i t o r s " ( E B I ) . Most of t h e s e i n h i b i t o r s have a common b i o c h e m i c a l s i t e of a c t i o n , which seems remarka b l e i n view of t h e wide d i v e r s i t y of chemical s t r u c t u r e s . At t h e i r lowest b i o l o g i c a l l y a c t i v e c o n c e n t r a t i o n s , they i n h i b i t t h e d e m e t h y l a t i o n of l a n o s t e r o l (or 24-methylene d i h y d r o l a n o s t e r o l ) a t C - 1 4 , which i s t h e f i r s t step i n t h e metabolism o f t h i s s t e r o l t o ergosterol or other f u n c t i o n a l l y equivalent s t e r o l s i n f u n g i . I n h i b i t o r s w i t h t h i s mode of a c t i o n i n c l u d e c e r t a i n a z o l e , p y r i d i n e , p y r i m i d i n e , and p i p e r a z i n e d e r i v a t i v e s . Azoles are 5-membered c y c l i c molecules w i t h one o r more heteroatoms i n t h e r i n g , a t l e a s t one of which must be n i t r o g e n , and t h e maximum number of noncumulated double bonds (I). They a r e t h e l a r g e s t

0097-6156/87/0325-0268$06.00/0 © 1987 American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

17.

WEETE

Ergosterol Biosynthesis

269

group of such i n h i b i t o r s , and i n c l u d e compounds w i t h i m i d a z o l e and triazole rings. In a d d i t i o n t o t h e i n h i b i t o r s t h a t block C-14 d e m e t h y l a t i o n , c e r t a i n morpholine d e r i v a t i v e s i n h i b i t t h e r e d u c t i o n a t C-14( 15) i n an i n t e r m e d i a t e formed by d e m e t h y l a t i o n at C-14 of l a n o s t e r o l (2^,2)» o r Δ8 -> Δ7 i s o m e r i z a t i o n ( 4 ) . A z a s t e r o l s a r e a n o t h e r group of a n t i f u n g a l substances t h a t i n t e r f e r e w i t h s t e r o l metabo­ l i s m . The s i t e of a c t i o n of t h e s e s u b s t a n c e s depends on t h e l o c a ­ t i o n of the n i t r o g e n atom i n t h e s t e r o i d m o l e c u l e ; f o r example, 1 5 a z a s t e r o l b l o c k s C-14(15) r e d u c t i o n . U n l i k e t h e above s u b s t a n c e s which are a l l x e n o b i o t i c s , t h e a z a s t e r o l s are p r o d u c t s of t h e fungus Geotrichum flavo-brunneum (j>). A group of x e n o b i o t i c s t h a t t r u l y i n h i b i t s t e r o l b i o s y n t h e s i s are t h e a l l y l a m i n e s which block squalene e p o x i d a t i o n and hence the c o n v e r s i o n of squalene t o l a n o ­ s t e r o l (6). The names and s t r u c t u r e s of some of t h e most common s t e r o l i n h i b i t o r s of c u r r e n t i n t e r e s t are given i n T a b l e 1 and Figure 1, respectively. It i s w e l l known t h a s t e r o l , i s r e q u i r e d f o r t h e optimum growth of most f u n g i . T h i s i s based on t h e f o l l o w i n g e v i d e n c e : 1) a s t e r o l i s r e q u i r e d f o r t h e a n a e r o b i c growth of Saccharomyces c e r e v i s i a e (7_), 2) a v a r i e t y of c h e m i c a l s t h a t b l o c k v a r i o u s s t e p s i n t h e pathway of s t e r o l b i o s y n ­ t h e s i s a l s o i n h i b i t fungal growth (see b e l o w ) , and 3) t h e growth r a t e s of mutants d e f e c t i v e i n v a r i o u s a s p e c t s of e r g o s t e r o l b i o s y n ­ t h e s i s are lower than those of c o r r e s p o n d i n g w i l d - t y p e s t r a i n s (see below). T h i s requirement accounts f o r t h e a n t i f u n g a l p r o p e r t i e s of the sterol i n h i b i t o r s . S i n c e r e s e a r c h on t h e growth and b i o c h e m i c a l r e s p o n s e s , as w e l l as r e s i s t a n c e , t o s t e r o l i n h i b i t o r s has been r e c e n t l y reviewed (8-14) t h i s i n f o r m a t i o n w i l l be covered here o n l y i n s o f a r as n e c e s ­ sary to e s t a b l i s h the background f o r a d i s c u s s i o n on t h e molecular b a s i s f o r fungal growth i n h i b i t i o n by t h e s e s u b s t a n c e s . S i n c e most of t h e work has been conducted w i t h C-14 d e m e t h y l a t i o n i n h i b i t o r s , emphasis w i l l be given t o them, p a r t i c u l a r l y the a z o l e s . I n h i b i t i o n of Demethylation of L a n o s t e r o l at C-14 and Other E f f e c t s on L i p i d M e t a b o l i s m The c y c l i z a t i o n of 2 , 3 - e p o x i d o s q u a l e n e , which i s formed from meva­ l o n i c a c i d v i a t h e i s o p r e n o i d pathway, t o l a n o s t e r o l i s t h e process that constitutes sterol synthesis. The l a n o s t e r o l molecule t h e n undergoes c o n s i d e r a b l e m o d i f i c a t i o n t o form a f u n c t i o n a l l y compe­ t e n t s t e r o l , such as c h o l e s t e r o l i n animals and e r g o s t e r o l i n most f u n g i , as reviewed by Mercer ( 1 5 ) . The f i r s t of t h e s e m o d i f i c a ­ t i o n s i n c e r t a i n y e a s t s (_S. c e r e v i s i a e and T o r u l o p s i s g l a b r a t a ) i n v o l v e s the o x i d a t i v e removal of methyl groups a t t h e C - 4 and C-14 p o s i t i o n s , whereas i n most f u n g i a l k y l a t i o n at C - 2 4 o c c u r s p r i o r t o demethylation. R e g a r d l e s s of when a l k y l a t i o n o c c u r s , t h e C-14 methyl group i s removed p r i o r t o those a t C - 4 . Although some of t h e d e t a i l s of C-14 d e m e t h y l a t i o n are not w e l l e s t a b l i s h e d , t h e o v e r a l l r e a c t i o n i n v o l v e s a P-450 c y t o c h r o m e - c a t a l y z e d o x i d a t i o n of t h e methyl group t o t h e c o r r e s p o n d i n g hydroxymethyl which i s subsequently o x i d i z e d t o a formyl group and removed from t h e s t e r o l molecule as f o r m i c a c i d . The product of the d e m e t h y l a t i o n would be

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

C.

B.

A.

Enilconazole (Imazalil )

Miconazole

Clotrimazole

2.

3.

4.

Propiconazole

Triadimefonb

2.

3.

1.

Buthiolate

PYRIDINE

Diclobutrazol

1.

TRIAZOLE

Bifonazole

1.

IMIDAZOLE

a

inhibitors

methane

1

S-n-butyl-S -p-tert-butyl benzyl-N-3py r i dy 1 di t h i o c a r b o n i mi date

l-(4-chlorophenoxy ) - 3 , 3 - d i m e t h y l - l - ( 1 , 2 , 4 - t r i a z o l - 1 - y l ) butanone

l-[2-(2,4-dichl orophenyl)-4-propyl-1,3-dioxolan-2-ylmethyl]lH-l,2,4-triazole

[(2RS,3RS)-l-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1H-1,2,4tri azol-1-yl)pentan-3-ol]

bis-phenyl[1,2-chlorophenyl]-l-imidazolyl

Denmert

Bayleton

Tilt

Vigil

Canesten

Fungaflor/ Fecundal

l-[2-(2,4-dichlorophenyl)-2-(2-propenyloxy)]-lH-imidazole

l-{2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy] e t h y l } - I H - i m i d a z o l e mononitrate

Mycospor

TRADE NAME

l-[(4-biphenylyl)-phenyl methyl]-lH-imidazole

SYSTEMATIC NAME

R e p r e s e n t a t i v e a n t i f u n g a l a z o l e s and o t h e r s t e r o l

TRIVIAL NAME

TABLE I.

6

5

r

Η

> ζ

r

"Ό

τι

Ο

Ο r

Η > DO

m

>

ο

r Ο

ο

m π

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Fenarimol

Fenpropimorph

2.

(E)-N-methyl-N-(l-naphthylmethyl)-3-phenyl2-propen-l-amine hydrochloride

= t h e reduced form o f t r i a d i m e f o n , B a y t a n .

Vangard.

ne-methanol

p i p e r a z i ne

15-aza-24-methylene-cholesta-8,14-dien-3 -ol

1,4-di-(2,2,2-trichloro-l-formamidoethyl)

{±-cis-4-[3-(4-tert-butylphenyl)-2-methylpropyl]2 , 6 - d i m e t h y l m o r p h o l i ne}

N - t r i d e c y l - 2 , 6 - d i m e t h y l m o r p h o l i ne

a

a-(2-chlorophenyl)- -(4-chlorophenyl)-5-pyrimidi

e t a c o n a z o l e = t h e e t h y l homologue of p r o p i c o n a z o l e ,

Naftifine

ktriadimenol

a

1.

ALLYLAMINE

H.

T r i f o r i ne

AZASTEROL

1.

PIPERAZINE

Tridemorph

1.

MORPHOLINE

1.

PYRIMIDINE

G.

F.

Ε.

D.

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In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

273

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17. WEETE

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In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

274

ECOLOGY AND METABOLISM OF PLANT LIPIDS

e i t h e r 4 , 4 - d i m e t h y l - e r g o s t a - 8 , 1 4 , 2 4 ( 2 8 ) - t r i enol o r 4 , 4 - d i m e t h y l c h o l e s t a - 8 , 1 4 , 2 4 - t r i e n o l depending on whether a l k y l a t i o n a t C-24 o c c u r s b e f o r e or a f t e r d e m e t h y l a t i o n at C - 1 4 . The double bond at C-14(15) i n t h e s e i n t e r m e d i a t e s i s s u b s e q u e n t l y reduced i n a r e a c t i o n r e q u i r i n g NADPH. The C-14 demethylase i s a monooxygenase c o n s i s t i n g of c y t o ­ chrome P-450 and a cytochrome P-450 r e d u c t a s e t h a t r e q u i r e s m o l e ­ c u l a r oxygen and NADPH f o r a c t i v i t y (16). The s t e r o l i n h i b i t o r s block the f i r s t r e a c t i o n c a t a l y z e d by t h i s enzyme by b i n d i n g t h e heme i r o n of cytochrome P-450 ( 1 7 - 2 2 ) . No oxygenated i n t e r m e d i a t e s i n t h e d e m e t h y l a t i o n p r o c e s s a r e known t o accumulate i n i n h i b i t o r t r e a t e d t i s s u e s . An unhindered n i t r o g e n atom at p o s i t i o n 3 t o t h e main backbone of the i n h i b i t o r molecule i s f a v o r a b l e f o r b i n d i n g . M i c o n a z o l e and k e t o c o n a z o l e are b e l i e v e d t o show s e l e c ­ t i v i t y f o r t h e pathogen i n c l i n i c a l s i t u a t i o n s because they have a h i g h e r a f f i n i t y f o r t h e cytochrome P-450 of t h e y e a s t enzyme t h a n f o r t h a t of t h e host ( 2 1 ) Fungi t r e a t e d w i t t h e C-14 demethylase accumulate C-14 methyl s t e r o l s , which a r e u s u a l l y only f a i n t l y d e t e c t a b l e i n most n o n - t r e a t e d c e l l s . With C-14 d e m e t h y l a t i o n b l o c k e d , t h e methyl groups at C-4 a r e s e q u e n ­ t i a l l y removed as would n o r m a l l y o c c u r i n t h e absence of t h e i n h i b ­ itor. Thus, 2 4 - m e t h y l e n e - d i h y d r o l a n o s t e r o l [ 2 4 - m e t h y l - 1 a n o s t a 8,24(28)-dienol], o b t u s i f o l i o l [4a,14a-dimethyl-ergosta-8,24(28)d i e n o l ] , and 14a- m e t h y l - f e cos t e r o l [ 1 4 a - m e t h y l - e r g o s t a - 8 , 2 4 ( 2 8 ) d i e n o l ] accumulate i n most fungi t r e a t e d w i t h s t e r o l inhibitors (Figure 2). I t appears t h a t t h e double bond t r a n f o r m a t i o n s of the s t e r o l molecule t h a t t y p i c a l l y o c c u r i n t h e l a t e r s t a g e s of e r g o s t e r o l b i o s y n t h e s i s cannot proceed i n t h e presence of t h e C-14 methyl group. S i m i l a r r e s u l t s have been o b t a i n e d f o r t h e v a r i o u s s t e r o l i n h i b i t o r s and s e n s i t i v e f u n g i t e s t e d ( 2 3 - 4 3 ) . Sterols that accumulate i n S . c e r e v i s i a e and T. g l a b r a t a t r e a t e d w i t h t h e C-14 demethylase i n h i b i t o r s a r e t h e c o r r e s p o n d i n g C-24 d e m e t h y l , Δ24(25) d e r i v a t i v e s ( F i g u r e 2 ) . Some f u n g i t r e a t e d w i t h s t e r o l i n h i b i t o r s produce a p p r o x i ­ mately t w i c e t h e amount of s t e r o l of n o n - t r e a t e d c e l l s ( 3 8 , 4 2 ) . For example, t h e t o t a l s t e r o l content of mycelium o f the peanut l e a f s p o t pathogen C e r c o s p o r a a r a c h i d i c o l a t r e a t e d w i t h 0.1 ppm o f p r o p i c o n a z o l e i s 1.5 ug/mg dry w e i g h t , 80% of which i s C-14 methyl s t e r o l s , compared t o 0.7 ug/mg i n n o n - t r e a t e d mycelium, ( 4 2 ) . The e r g o s t e r o l content i s reduced 50% on a dry weight b a s i s by t r e a t ­ ment w i t h t h e i n h i b i t o r . T h i s i n c r e a s e i n t o t a l s t e r o l may be due t o a r e d u c t i o n i n feedback i n h i b i t i o n of s t e r o l b i o s y n t h e s i s by e r g o s t e r o l or one of i t s m e t a b o l i t e s . However, t h e i n c r e a s e i n t o t a l s t e r o l does not occur i n some f u n g i ( e . g . _S. c e r e v i s i a e ) . T h i s may be accounted f o r by t h e f a c t t h a t t h e a c t i v i t y of the key r e g u l a t o r y enzyme of s t e r o l b i o s y n t h e s i s , 3 - h y d r o x y - 3 - m e t h y l g l u t a r y l - C o A r e d u c t a s e , i s reduced i n t h i s y e a s t by C-14 methyl s t e r o l s such as l a n o s t e r o l t h a t accumulate i n response t o treatment with these i n h i b i t o r s (44,45). Since other v i t a l a c t i v i t i e s tested (protein s y n t h e s i s , n u c l e i c a c i d s y n t h e s i s , and r e s p i r a t i o n ) are not a f f e c t e d t o t h e same e x t e n t , o r as soon a f t e r t r e a t m e n t , by sub-MIC (minimum i n h i b ­ i t o r c o n c e n t r a t i o n ) l e v e l s of t h e a n t i f u n g a l a z o l e s and r e l a t e d compounds ( 2 5 , 2 6 , 2 8 , 3 2 , 3 5 , 3 9 ) , i t i s g e n e r a l l y agreed t h a t t h e

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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276

ECOLOGY AND METABOLISM OF PLANT LIPIDS

primary s i t e of t h e i r a c t i o n i s the C-14 demethylase. This i s f u r t h e r supported by r e s u l t s o b t a i n e d by Walsh and S i s l e r (46) who showed t h a t a mutant of U s t i l a g o maydis d e f i c i e n t i n C-14 d e m e t h y l a t i o n i s not s e n s i t i v e t o r e p r e s e n t a t i v e s of s e v e r a l c l a s s e s of s t e r o l i n h i b i t o r s ( m i c o n a z o l e , e t a c o n a z o l e , and f e n a r i m o l ) . This mutant accumulates t h e same s t e r o l s as f u n g i t r e a t e d w i t h s t e r o l i n h i b i t o r s , and t h e d o u b l i n g t i m e i s o n e - t h i r d t h a t of the w i l d t y p e . However, a s t e r o l auxotroph of cerevisiae supplied with exogenous e r g o s t e r o l i s reported t o be as s e n s i t i v e t o miconazole and c l o t r i m a z o l e as t h e w i l d - t y p e ( 4 7 ) . In a d d i t i o n t o b l o c k i n g t h e d e m e t h y l a t i o n of 24-methylene d i h y d r o l a n o s t e r o l a t C - 1 4 , t h e r e are s e v e r a l o t h e r a l t e r a t i o n s of l i p i d metabolism i n f u n g i t r e a t e d w i t h C-14 demethylase i n h i b i t o r s . These i n c l u d e : 1) an i n c r e a s e i n f r e e f a t t y a c i d s , l i n o l e i c a c i d , and i n some cases major s a t u r a t e d f a t t y a c i d s , and 2) a decrease i n o l e i c a c i d ( 8 , ^ 2 , 3 8 , 3 9 , 4 2 , 4 7 , 4 8 ) . The i n c r e a s e i n f r e e f a t t y a c i d s has been a t t r i b u t e d t o a d e g r a d a t i o n of a c y l l i p i d changes i n s t e r o l content r a t h e r than an a l t e r n a t e a c t i o n of the i n h i b i t o r s s i n c e i t a l s o o c c u r s i n a mutant of U. maydis d e f i c i e n t i n C-14 demethylase ( 4 6 ) . T h i s i n c r e a s e seems to occur subsequent t o d e t e c t a b l e changes i n s t e r o l metabolism and i s not c o n s i d e r e d t o be a major i n i t i a l f a c t o r i n t h e growth i n h i b i t o r y a c t i v i t y of the inhibitors. The i n c r e a s e i n f r e e f a t t y a c i d s does not o c c u r i n a l l cases ( 4 2 , 5 0 ) . The decrease i n o l e i c a c i d , which i s accompanied by an i n c r e a s e i n l i n o l e i c and i n some cases l i n o l e n i c a c i d , i s p a r t i c u l a r l y pronounced i n , but not r e s t r i c t e d t o , t h e p o l a r l i p i d (21,38,42,48,49). These a l t e r a t i o n s i n t h e r e l a t i v e p r o p o r t i o n s of f a t t y a c i d s have been a t t r i b u t e d t o a d a p t i v e - t y p e responses t o changes i n t h e f l u i d p r o p e r t i e s of membranes brought about by s t e r o l i n h i b i t o r - i n d u c e d m o d i f i c a t i o n of t h e s t e r o l content (21,38,42,48,49). In some but a p p a r e n t l y not a l l f u n g i , p a l m i t i c and i n some cases s t e a r i c a c i d accumulate w i t h treatment by a n t i f u n g a l a z o l e s (see r e f e r e n c e s c i t e d a b o v e ) , van den Bossche et a l . (21) have a t t r i b u t e d t h i s t o a decrease i n t h e a c t i v i t y of a membrane-bound d e s a t u r a s e . Fungal Growth I n h i b i t i o n and

Tolerance

A l a r g e number of f u n g i have been screened i n v i t r o f o r t h e i r s e n s i t i v i t y t o t h e C-14 d e m e t h y l a t i o n i n h i b i t i n g s u b s t a n c e s . Although t h e r e are e x c e p t i o n s , f u n g i show growth r e d u c t i o n (about 50% i n h i b i t i o n ) w i t h i n one to s e v e r a l hours of treatment w i t h 0 . 1 t o 2 . 0 ppm of a s t e r o l i n h i b i t o r ( 3 , 2 6 , 3 1 , 3 3 , 3 9 , 4 1 , 4 2 , 5 1 - 5 3 ) . The human pathogen Candida a l b i c a n s and t h e p l a n t pathogenic U s t i l a g o m a y d i s , U_. avenae, and Taphrina deformans, a l l of which grow as y e a s t - l i k e or s p o r i d i a l forms i n v i t r o , and t h e f i l a m e n t o u s M o n i l i n i a f r u c t i g e n a , P é n i c i l l i u m i t a l i c u m and A s p e r g i l l u s n i d u l a n s a r e some of t h e s p e c i e s used f o r more i n depth mode of a c t i o n s t u d i e s on t h e s t e r o l i n h i b i t o r s . A few of t h e most a c t i v e a z o l e s , w i t h t h e i r ED50 c o n c e n t r a t i o n s , and f u n g i most s e n s i t i v e t o them are g i v e n i n Table I I .

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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TABLE I I . A z o l e s t e r o l i n h i b i t o r s and t h e i r E D ^ Q c o n c e n t r a t i o n s for selected s e n s i t i v e fungi Fungal SPECIES Candida a l b i c a n s Pénicillium italicum T a p h r i n a deformans Torulopsis glabrata a

INHIBITOR miconazole imazalil propiconazole bifonazole

APPROXIMATE E D CONCENTRATION 10-8 t o 1 0 - / M 0 . 0 1 ppm 0.073 ppm 0.125 ppm

ED5Q = c o n c e n t r a t i o n g i v i n g 50% growth

5 0

a

REFERENCE (37) (35) (51) (52)

inhibition.

C e l l d i v i s i o n i n y e a s t i s more s e n s i t i v e t o s t e r o l i n h i b i t o r s t h a n dry matter a c c u m u l a t i o n ( 3 2 , 5 1 ) . Spore g e r m i n a t i o n i n most s p e c i e s i s not i n h i b i t e d at c o n c e n t r a t i o n s of t h e i n h i b i t o r s t h a t prevent hyphal e l o n g a t i o n ( 2 6 , 3 4 ) . A l s o , growth i n h i b i t i o n by t h e s e substances i s e i t h e ergosterol supplied i n th v a r i e t y of u n s a t u r a t e d l i p o p h i l i c s u b s t a n c e s , such as Tween 20 and 4 0 , o l e i c a c i d , t o c o p h e r o l , 3 - c a r o t e n e , f a r n e s o l , t r i l i n o l e n i n and s e v e r a l o t h e r s can a l l e v i a t e t h e growth i n h i b i t o r y e f f e c t s of a z o l e s ( 3 4 , 5 4 - 5 6 ) as w e l l as morpholines ( 5 8 ) . The s t e r o l i n h i b i t o r s a r e g e n e r a l l y c o n s i d e r e d t o be f u n g i s t a t i c r a t h e r t h a n f u n g i c i d a l (51). The C - l T ~ d e m e t h y l a t i o n i n h i b i t o r s a r e g e n e r a l l y c o n s i d e r e d t o be s i n g l e - s i t e i n h i b i t o r s , and t h e r e has been concern about t h e development of f i e l d r e s i s t a n c e or t o l e r a n c e t o them. (The terms r e s i s t a n c e and t o l e r a n c e a r e used i n t e r c h a n g e a b l y here t o conform t o the t e r m i n o l o g y used by authors of the work b e i n g c i t e d . To my knowledge, t r u e r e s i s t a n c e t o a s t e r o l i n h i b i t o r has not been reported.) Indeed, fungal i s o l a t e s w i t h a high degree of t o l e r a n c e t o t h e s e substances can be r e a d i l y s e l e c t e d f o r i n l a b o r a t o r y c u l t u r e ( 3 3 , 3 4 , 5 9 ) , o r induced w i t h mutagenic agents ( 6 0 , 6 1 ) . There had been no r e p o r t s of f i e l d r e s i s t a n c e t o t h e s e i n h i b i t o r s up t o 1982 ( 6 2 ) , but more r e c e n t l y t h e r e have been r e p o r t s i n Europe (63). The absence of c o n f i r m e d r e s i s t a n c e i n t h e f i e l d has been a t t r i b u t e d t o reduced f i t n e s s o r p a t h o g e n i c i t y of r e s i s t a n t s t r a i n s r e l a t i v e to the w i l d - t y p e s t r a i n s (11). G e n e r a l l y , mutants t o one C-14 demethylase i n h i b i t o r show c r o s s - r e s i s t a n c e t o o t h e r s ( 6 2 ) . S t u d i e s on t h e mechanism of r e s i s t a n c e have been conducted w i t h a s t r a i n of A s p e r g i l l u s n i d u l a n s t h a t c a r r i e s genes f o r r e s i s t a n c e t o t h e p y r i m i d i n e i n h i b i t o r f e n a r i m o l (60) and t h e i m i d a z o l e i m a z a l i l (64). R e s i s t a n c e to f e n a r i m o l has been a t t r i b u t e d t o reduced uptake of t h e i n h i b i t o r ( 6 5 ) . Both t h e w i l d - t y p e and r e s i s t a n t s t r a i n s possess energy-dependent e f f l u x mechanisms w i t h d i f f e r e n t e f f i c i e n c i e s t o e x c r e t e f e n a r i m o l , t h e r a t e of e x c r e t i o n being higher i n the r e s i s t a n t s t r a i n (66). The mechanism f o r r e s i s t a n c e t o i m a z a l i l i s , at l e a s t i n p a r t , s i m i l a r t o t h a t f o r fenarimol (59). C e r t a i n Mucorales examined have shown a r e l a t i v e l y h i g h degree of t o l e r a n c e t o the s t e r o l i n h i b i t o r s . F o r example, 80 ppm of p r o p i c o n a z o l e i s r e q u i r e d f o r 50% growth i n h i b i t i o n of Mucor r o u x i i i n l i q u i d shake c u l t u r e ( 5 7 ) . Growth i n h i b i t i o n by t h i s r e l a t i v e l y h i g h c o n c e n t r a t i o n of t h e i n h i b i t o r i s probably due t o d i r e c t i n t e r a c t i o n of the t r i a z o l e w i t h the plasma membrane, and not t o

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

278

i n h i b i t i o n of the C-14 d e m e t h y l a s e , as has been shown f o r m i c o n a ­ z o l e i n (^. a l b i c a n s ( 2 1 ) . In f a c t , C-14 d e m e t h y l a t i o n i n Mucor i s i n h i b i t e d by 2 ug/ml p r o p i c o n a z o l e . The b a s i s f o r the observed t o l e r a n c e t o p r o p i c o n a z o l e i s not known, but p r e l i m i n a r y r e s u l t s suggest t h a t i t i s not due t o metabolism o r reduced uptake of the i n h i b i t o r (57). A p p a r e n t l y not a l l of t h e Mucorales possess t h e same degree of t o l e r a n c e to t h e s e i n h i b i t o r s s i n c e t h e growth of Rhizopus s t o l o n i f e r i s i n h i b i t e d 50% by 5 t o 8 ug/ml p r o p i c o n a z o l e (51). A l s o , o t h e r ρ hy corny c e t es (Oomycetes) a r e s e n s i t i v e t o f e n a r i m o l (_4 and c i t e d i n 6 5 ) . L i k e w i s e , s p e c i e s of the p y t h i a c e o u s genera Pythium and Phyt o p h t h o r a show a h i g h degree of t o l e r a n c e t o t h e s t e r o l i n h i b i t o r s . F o r example, 50 ppm of p r o p i c o n a z o l e i s r e q u i r e d f o r 50% growth i n h i b i t i o n of Phytophthora cinnamomi ( 5 7 ) . The r e l a t i v e l y h i g h t o l e r a n c e to s t e r o l i n h i b i t o r s by s p e c i e s of these genera can be accounted f o r by t h e f a c t t h a t they n e i t h e r produce s t e r o l s nor r e q u i r e them f o r v e g e t a t i v r e l a t i v e l y high c o n c e n t r a t i o n i n t e r a c t i o n w i t h membranes. Morphology,

U l t r a s t r u c t u r e , and P e r m e a b i l i t y

There are s e v e r a l common m o r p h o l o g i c a l responses by v a r i o u s f u n g i and y e a s t s t o treatment w i t h s t e r o l i n h i b i t o r s (^ and r e f e r e n c e s cited therein). While spore g e r m i n a t i o n i s g e n e r a l l y not i n h i b i t e d , germ tube and hyphal e x t e n s i o n i s c u r t a i l e d , f r e q u e n t l y w i t h s w e l l i n g and b u r s t i n g of the hyphal t i p s . Yeast c e l l s may s w e l l , become v a c u o l a t e d , and form c h a i n s or aggregates of 2 t o 6 c e l l s r e s u l t i n g from f a i l u r e of buds t o s e p a r a t e from mother c e l l s . Yeast-hypha c o n v e r s i o n i s p r e f e r e n t i a l l y i n h i b i t e d i n £ . a l b i c a n s by b i f o n a z o l e ( 6 7 ) . These r e s u l t s are s u g g e s t i v e of abnormal c e l l w a l l f o r m a t i o n , but t h e r e i s l i t t l e d i f f e r e n c e i n t h e c h i t i n content of the c e l l w a l l s of Cercospora and C e r c o s p o r i d i u m ( 4 2 ) , o r s e v e r a l o t h e r s p e c i e s (57) from p r o p i c o n a z o l e - t r e a t e d and n o n t r e a t e d c u l t u r e s ; i n f a c t , c e l l w a l l s from t r e a t e d c u l t u r e s u s u a l l y c o n t a i n s l i g h t l y more c h i t i n / c h i t o s a n t h a n t h o s e from n o n - t r e a t e d c u l t u r e s . F u r t h e r m o r e , b u t h i o l a t e (S-1358) has l i t t l e e f f e c t on t h e i n c o r p o r a t i o n of [ 1 4 c ] l a b e l e d g l u c o s e and glucosamine i n t o t h e c e l l w a l l of M o n i l i n i a f r u c t i g e n a ( 2 6 ) . Y e t , a l t e r a t i o n of c e l l w a l l s t r u c t u r e has been d e t e c t e d w i t h s c a n n i n g and t r a n s m i s s i o n e l e c t r o n microscopy ( 6 8 , 6 9 ) . Using a s p e c i f i c f l u o r e s c e n t marker, i t has been shown t h a t b i f o n a z o l e - and i m a z a l i l - t r e a t e d c e l l s of C_. a l b i c a n s and T o r u l o p s i s g l a b r a t a l a c k s e p t a and have i r r e g u l a r d e p o s i t i o n s of c h i t i n i n t h e i r w a l l s ( 6 9 , 7 0 ) . The most obvious u l t r a s t r u c t u r a l e f f e c t s of the s t e r o l i n h i b i ­ t o r s have been found i n c e l l w a l l s and membrane s y s t e m s . Except f o r m i t o c h o n d r i a l s w e l l i n g , f u n g i s t a t i c l e v e l s of c l o t r i m a z o l e have l i t t l e e f f e c t on t h e c y t o l o g y of C_. a l b i c a n s c e l l s , whereas f u n g i ­ c i d a l l e v e l s ( 1 . 5 x 1 0 - 4 M) r e s u l t i n a p r o l i f e r a t i o n of membranes by i n v a g i n a t i o n of t h e plasma membrane and t h e development of mem­ brane p a r t i c l e s between t h e r e t r a c t e d c e l l membrane and c e l l w a l l (71). D e t e r i o r a t i o n of t h e i n t e r n a l s t r u c t u r e of m i t o c h o n d r i a and n u c l e i a l s o has been o b s e r v e d . In a d d i t i o n , c e l l w a l l and septum t h i c k e n i n g , a c c u m u l a t i o n of l i p i d b o d i e s , and v a r i o u s changes i n membrane o r g a n i z a t i o n w i t h i n t h e c e l l s have been observed i n

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s p o r i d i a of U. avenae (72) and o t h e r f u n g i (71a,71b) t r e a t e d w i t h s e v e r a l C-14 demethylase i n h i b i t o r s . As might be expected f o r c e l l s i n which the plasma membrane has been a l t e r e d , leakage of c e l l u l a r c o n s t i t u e n t s ( 5 , 7 3 , 7 4 ) and a l t e r e d s u b s t r a t e uptake ( 3 5 , 5 1 , and c i t e d i η 37,75) i n c l u d i n g an i n c r e a s e d p e r m e a b i l i t y t o protons (_75), have been observed i n sterol inhibitor-treated c e l l s . Mechanism of Growth

Inhibition

The mechanism of growth i n h i b i t i o n by s t e r o l i n h i b i t o r s must be e x p l a i n e d i n t h e c o n t e x t of s p e c i f i c s t e r o l f u n c t i o n s . Unfortu­ n a t e l y , our knowledge of t h i s area i s somewhat l i m i t e d . In f u n g i , s t e r o l s such as e r g o s t e r o l e x e r c i s e t h e i r p r i n c i p a l b i o l o g i c a l a c t i v i t y i n membranes (76) where they are b e l i e v e d t o p a r t i c i p a t e i n t h e modulation of f l u i d p r o p e r t i e s (77). In a d d i t i o n t o s a t i s ­ fying t h i s "bulk" function t i o n a l , perhaps m e t a b o l i c These r o l e s have been d e s c r i b e d i n d e p e n d e n t l y f o r e r g o s t e r o l i n y e a s t s t e r o l auxotrophs and mutants and r e f e r r e d to as " s p a r k i n g " (78,79) and " s t e r o l s y n e r g i s m " ( 8 0 ) , both of which probably r e f l e c t t h e same f u n c t i o n s . The d i f f e r e n t r o l e s f o r s t e r o l s have c e r t a i n s t r u c t u r a l requirements (79) and d i s t i n c t s t e r e o s p e c i f i c i t i e s ( 8 1 ) . The s y n e r g i s t i c r o l e of s t e r o l s can be s a t i s f i e d by c o n s i d e r a b l y lower amounts of e r g o s t e r o l t h a n are r e q u i r e d t o a f f e c t the bulk f l u i d p r o p e r t i e s of membranes ( 8 0 ) . I t i s wel1-documented t h a t C-14 methyl s t e r o l s ( i . e . l a n o s t e r o l ) w i l l not f u l l y s u b s t i t u t e f o r e r g o s t e r o l i n s u p p o r t i n g t h e growth of _S. c e r e v i s i a e ( 8 2 ) . M o r e o v e r , endogenous l a n o s t e r o l i n a heme d e f i c i e n t mutant of _S. c e r e v i s i a e i s d e t r i m e n t a l t o c e l l g r o w t h , and a d a p t a t i o n f o r growth on media supplemented w i t h c h o l e s t e r o l i s c o r r e l a t e d w i t h the absence of l a n o s t e r o l ( 8 3 ) . B l o c h (84) p o s t u l a t e d t h a t the reason l a n o s t e r o l cannot f u l l y s a t i s f y the s t e r o l requirement f o r growth i s t h a t the a x i a l C-14 methyl group i n t e r f e r e s w i t h van der Waals i n t e r a c t i o n s of t h e s t e r o l w i t h f a t t y a c y l chains of membrane lipid. F u r t h e r m o r e , t h e Δ8(9) double bond present i n C-14 methyl s t e r o l s i s r e s p o n s i b l e f o r a bend i n the molecule t h a t i s a p p a r e n t ­ l y u n f a v o r a b l e f o r p a c k i n g i n the l i p i d b i l a y e r of t h e membrane. N e v e r t h e l e s s , C-14 methyl Λ^(9) s t e r o l s a r e g e n e r a l l y not c o n ­ s i d e r e d t o be l e t h a l because s e v e r a l C-14 demethylase mutants a r e a b l e t o grow w i t h o u t exogenous s t e r o l ( 4 6 , 8 5 , 8 6 ) . C-14 methyl s t e r o l s are b e l i e v e d t o s a t i s f y o n l y t h e bulk requirement and not t h e more s p e c i f i c r o l e f o r s t e r o l ( 7 8 , 7 8 a ) . With t h i s l i m i t e d background, some of the b i o l o g i c a l responses of f u n g i t o t r e a t m e n t w i t h s t e r o l i n h i b i t o r s , p a r t i c u l a r l y the C - 1 4 demethylase i n h i b i ­ t o r s , might be e x p l a i n e d , and a h y p o t h e s i s f o r t h e m o l e c u l a r b a s i s f o r growth i n h i b i t i o n by these compounds p r o p o s e d . The s e p a r a t i o n i n time of up t o s e v e r a l hours between t h e i n h i b i t i o n of C-14 d e m e t h y l a t i o n , o r o t h e r r e a c t i o n s r e l a t e d t o s t e r o l b i o s y n t h e s i s , and t h e onset of growth r e d u c t i o n ( i . e . 10 hours f o r P_. i t a l i cum t r e a t e d w i t h f e n p r o p i m o r p h , (3)) suggests t h a t e i t h e r b l o c k i n g e r g o s t e r o l b i o s y n t h e s i s i s not t h e growth i n h i b i t i n g mode of a c t i o n of these substances o r , perhaps more l i k e l y i n t h i s c a s e , the a c t u a l growth l i m i t i n g a c t i v i t y ( i e s ) i s s e v e r a l s t e p s removed from t h e primary b i o c h e m i c a l s i t e of a c t i o n .

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T h i s delay might be e x p l a i n e d i n p a r t by the time r e q u i r e d f o r e r g o s t e r o l - d e f i c i e n t membranes t o become s u f f i c i e n t l y i n t e g r a t e d i n t o t h e plasma membrane t o have an impact on growth e s s e n t i a l a c t i v i t i e s a s s o c i a t e d w i t h t h i s membrane. It i s d i f f i c u l t t o determine t h e n a t u r e , i f any, o r e x t e n t of the p o s s i b l e e f f e c t s of C-14 methyl s t e r o l s on t h e apparent growth l i m i t i n g e f f e c t s of having l i t t l e or no e r g o s t e r o l i n t h e membranes. They may tend t o compound the problems a s s o c i a t e d w i t h e r g o s t e r o l - d e f i c i e n t membranes, a c o n t e n t i o n supported by s e v e r a l authors ( 1 3 , 2 1 ) . The p e r p l e x i n g i n a b i l i t y t o be a b l e t o r e v e r s e growth i n h i b i ­ t i o n w i t h e r g o s t e r o l might be e x p l a i n e d by the f a c t t h a t exogenous s t e r o l i s not t a k e n up by t h e c e l l and i n c o r p o r a t e d i n t o t h e mem­ brane. T h i s i s c o n s i s t e n t w i t h the f a c t t h a t _S. c e r e v i s i a e c e l l s grown a n a e r o b i c a l l y r e a d i l y t a k e up s t e r o l s from t h e medium, and indeed r e q u i r e an exogenous source under t h i s c o n d i t i o n , but c e l l s do not t a k e up s t e r o l s from t h e medium when grown under a e r o b i c c o n d i t i o n s which are f a v o r a b l i n h i b i t i o n by s t e r o l i n h i b i t o r d e m e t h y l a t i o n a l s o cannot be reversed by s t e r o l s ; f o r example, growth i n h i b i t i o n of S o r d a r i a f i mi c o l a by AY 9944 and SKF 3301-A cannot be reversed by s t e r o l s ( 8 7 ) , and n e i t h e r can t h e i n h i b i t i o n of U. maydis by 1 5 - a z a s t e r o l be r e v e r s e d by e r g o s t e r o l ( 8 8 ) . Although i t has not been shown u n e q u i v o c a l l y , t h e s p e c i f i c changes i n f a t t y a c i d c o m p o s i t i o n ( i . e . r e d u c t i o n i n C i 8 : i d increase i n 0χ8:2) probably not caused d i r e c t l y by the C - 1 4 d e m e t h y l a t i o n i n h i b i t o r s , but i n s t e a d they may be a d a p t i v e responses t o p e r t u r b a t i o n of t h e membrane by changes i n s t e r o l com­ p o s i t i o n r e s u l t i n g from treatment w i t h t h e i n h i b i t o r s . In P h y t o p h t h o r a cinnamomi, which does not produce s t e r o l s , t h e s h i f t t o h i g h e r u n s a t u r a t i o n was not observed when t h e fungus was t r e a t e d w i t h t h e ED5Q c o n c e n t r a t i o n of p r o p i c o n a z o l e ( 5 7 ) . It i s w e l l known t h a t such changes o c c u r i n p o i k i l o t h e r m i c organisms u n d e r ­ going a d a p t a t i o n t o a temperature below t h e optimum f o r growth (89). It i s not c e r t a i n i f t h e C i s i + C i 8 : 2 sterol i " n h ~ i b i t o r - t r e a t e d f u n g i i s due t o t h e i n c r e a s e i n C-14 methyl s t e r o l s , decrease i n e r g o s t e r o l , or a c o m b i n a t i o n of b o t h . This s h i f t has been observed i n T. deformans grown i n a medium c o n ­ t a i n i n g very h i g h l e v e l s (250 ug/ml) of l a n o s t e r o l ( 3 8 ) ; however, s i m i l a r changes i n f a t t y a c i d c o m p o s i t i o n accompany r e d u c t i o n s i n b r a s s i c a s t e r o l caused by n a f t i f i n e which does not cause C-14 methyl s t e r o l s t o accumulate ( 9 0 ) . C o n t r i b u t i o n s of t h e observed changes i n f a t t y a c i d s t o t h e growth i n h i b i t o r y process brought about by C-14 demethylase i n h i b i ­ t o r s are d i f f i c u l t t o a s s e s s . The a b i l i t y of a wide v a r i e t y of u n ­ s a t u r a t e d , l i p o p h i l i c substances at r e l a t i v e l y high c o n c e n t r a t i o n s t o r e v e r s e growth i n h i b i t i o n by s t e r o l i n h i b i t o r s suggests t h a t t h e s e l i p i d s may s u b s t i t u t e f o r t h e bulk r o l e of s t e r o l s i n mem­ branes d e f i c i e n t i n e r g o s t e r o l . van den Bossche et a l . (21) s u g ­ g e s t t h a t h i g h e r l e v e l s of s a t u r a t e d f a t t y a c i d s , a l o n g w i t h C - 1 4 methyl s t e r o l s , i n a z o l e t r e a t e d f u n g i d i s t u r b membrane p e r m e a b i l ­ i t y and t h e a c t i v i t y of membrane-bound enzymes. On t h e o t h e r hand, i t appears t h a t t h e s t e r o l c o m p o s i t i o n of t h e membranes may, a t l e a s t i n p a r t , determine t h e optimum f a t t y a c i d c o m p o s i t i o n f a v o r a b l e f o r growth. For example, c h o l e s t e r o l a l l o w s t h e s t e r o l r e q u i r i n g c e l l s of M^. c a p r i c o l u m t o grow w i t h a wide range of f a t t y a n

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a c i d supplements t o t h e medium, but l a n o s t e r o l s u p p o r t s growth o n l y when supplemented w i t h c e r t a i n f a t t y a c i d c o m b i n a t i o n s (91). It i s a l s o c l e a r t h a t t h e nature of t h e s t e r o l supplement f o r t h e y e a s t mutant GL 7 ( d e f i c i e n t i n squalene epoxide c y c l a s e and heme s y n t h e s i s ) determines t h e f a t t y a c i d t h a t b e s t s a t i s f i e s t h e growth requirement ( 8 0 ) . I t i s tempting t o s p e c u l a t e t h a t t h e d e c r e a s e i n o l e i c a c i d i n the l i p i d , p a r t i c u l a r l y i n phospholipid, that occurs u n i v e r s a l l y i n f u n g i t r e a t e d w i t h C-14 demethylase i n h i b i t o r s i s r e l e v a n t t o t h e growth i n h i b i t o r y p r o c e s s . There i s e x p e r i m e n t a l e v i d e n c e t h a t s t e r o l s a r e s p e c i f i c a l l y i n v o l v e d i n o l e i c a c i d metabolism ( 8 0 , 9 2 ) . For example, t h e GL 7 mutant (see above) grown i n a medium c o n t a i n i n g a s y n e r g i s t i c m i x t u r e of e r g o s t e r o l and c h o l e s t e r o l ( 1 : 3 ) i n c o r p o r a t e s more [14C] o l e i c a c i d i n t o p h o s p h o l i p i d s t h a n when grown on c h o l e s t e r o l a l o n e ( 8 0 ) . Based on o l e i c a c i d d e p r i v a t i o n s t u d i e s u s i n g t h e GL 7 mutant, i t appears t h a t t h i s a c i d i s r e q u i r e d f o r normal l i p i o l e i c a c i d has been foun e n r i c h e d f r a c t i o n from 1_. deformans t r e a t e d w i t h 0 . 0 7 3 ug/ml p r o p i conazole (49,94). It i s not known whether t h e decrease i n membrane o l e i c a c i d i s s u f f i c i e n t t o have harmful consequences t o t h e growth of f u n g a l c e l l s , o r even i f t h e a l t e r a t i o n i n f a t t y a c i d c o m p o s i t i o n i n general r e s u l t i n g from t r e a t m e n t w i t h a C-14 d e m e t h y l a s e - i n h i b i t o r c o n t r i b u t e s t o t h e growth i n h i b i t o r y mechanism o f t h e s e s u b s t a n c e s . There are no plasma membrane-bound enzymes whose a c t i v i t i e s a r e known t o be s p e c i f i c a l l y and s u f f i c i e n t l y a l t e r e d t o account f o r growth i n h i b i t i o n by t h e s t e r o l i n h i b i t o r s at sub-MIC d o s e s . A l i k e l y c a n d i d a t e i s c h i t i n s y n t h e t a s e , but the a c t i v i t y of t h i s enzyme i s not r e d u c e d ; i n f a c t , s t e r o l i n h i b i t o r - t r e a t e d f u n g i c o n t a i n more glucosamine polymers t h a n c o n t r o l s . However, t h e a l t e r e d d e p o s i t i o n of c h i t i n (see above r e f e r e n c e s ) may r e f l e c t a d i s c o n t i n u i t y between c y t o s k e l e t a l elements which are b e l i e v e d t o be i n v o l v e d i n c e l l w a l l f o r m a t i o n and t h e plasma membrane, but i t i s u n l i k e l y t h a t t h i s i s at t h e root of growth i n h i b i t i o n s i n c e a w a l l - l e s s s l i m e mutant of Neurospora c r a s s a i s as s e n s i t i v e as t h e w i l d - t y p e s t r a i n to propiconazole (57). Cytochrome o x i d a s e and microsomal ATPase a r e i n h i b i t e d by h i g h c o n c e n t r a t i o n s (10*5, 10-4 M) o f m i c o n a z o l e and k e t o c o n a z o l e , but t h i s has been a t t r i b uted t o t h e i r d i r e c t d e s t r u c t i v e a c t i o n on t h e membrane systems r e q u i r e d f o r t h e a c t i v i t y of t h e s e enzymes ( 5 6 ) . The w e i g h t of e v i d e n c e l e a n s toward a l t e r e d p e r m e a b i l i t y of t h e plasma membrane as t h e g r o w t h - l i m i t i n g f a c t o r i n f u n g i t r e a t e d w i t h s t e r o l i n h i b i tors. Perhaps t h e most important element i n t h i s regard i s t h e increased p e r m e a b i l i t y to protons. B a l d w i n (13) has shown t h a t t h e h a l f - t i m e f o r t h e e n t r y of protons i n t o _S. c e r e v i s i a e c e l l s t r e a t e d f o r 18 hours w i t h d i c l o b u t r a z o l i s o v e r t h r e e times h i g h e r t h a n corresponding c o n t r o l s . T h u s , he a t t r i b u t e s t h e i n a b i l i t y of i n h i b i t o r - t r e a t e d c e l l s t o t a k e up n u t r i e n t s ( e . g . amino a c i d s ) a g a i n s t t h e c o n c e n t r a t i o n g r a d i e n t t o t h e absence of s u f f i c i e n t e l e c t r o c h e m i c a l g r a d i e n t a c r o s s t h e plasma membrane, and t h e r e f o r e t h e c o n t i n u e d e x p u l s i o n of protons from t h e c e l l by t h e ATPase i s a w a s t e f u l use of energy l e a d i n g t o c e l l d e a t h .

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Summary The s t e r o l i n h i b i t o r s of c u r r e n t i n t e r e s t a r e a l a r g e and d i v e r s e group of substances t h a t b l o c k v a r i o u s r e a c t i o n s i n t h e l a t e r stages of s t e r o l b i o s y n t h e s i s , and have potent a n t i f u n g a l p r o p e r t i e s a g a i n s t a wide v a r i e t y of a g r i c u l t u r a l l y and m e d i c a l l y important s p e c i e s . W i t h i n t h e l i m i t s of our knowledge, t h e major b i o c h e m i c a l responses by f u n g i t o s u b l e t h a l doses of t h e s e i n h i b i ­ t o r s , p a r t i c u l a r l y t h e C - 1 4 demethylase i n h i b i t o r s , appear t o be r e s t r i c t e d t o l i p i d m e t a b o l i s m . The m o l e c u l a r b a s i s f o r t h e a n t i ­ fungal p r o p e r t i e s appears t o r e l a t e t o changes i n t h e p h y s i c a l p r o p e r t i e s o f membranes, p a r t i c u l a r l y t h e plasma membrane, brought about i n i t i a l l y by a decrease i n e r g o s t e r o l o r f u n c t i o n a l l y e q u i v a ­ l e n t s t e r o l and perhaps an i n c r e a s e i n C-14 Δ 8 s t e r o l s which i n t u r n lead t o subsequent o t h e r changes i n l i p i d c o n t e n t . This r e s u l t s i n i m p a i r e d membrane f u n c t i o n s , t h e most important of which may be p e r m e a b i l i t y . Th with s t e r o l i n h i b i t o r s ar w i t h t h e i n d i v i d u a l f u n g u s , and most l i k e l y c o l l e c t i v e l y c o n t r i b u t e t o a r e d u c t i o n i n growth p o t e n t i a l of t h e t r e a t e d organism ( 4 9 ) . Acknowledgments The work presented from t h i s l a b o r a t o r y was supported by funds from t h e Alabama A g r i c u l t u r a l Experiment S t a t i o n ( P r o j e c t A L A - 5 - 8 7 7 ) , t h e Herman F r a s c h Foundation and t h e C i b a - G e i g y C o r p o r a t i o n . C o n t r i b u t o r s t o t h i s work a r e D r . M i c h e l S a n c h o l l e , H. Gary Hancock, Dory K i r b y , M i t c h W i s e , V i v i K a r a t h a n a s i s , S a b r i n a B a r b e r - A z z o u z , Mi eke Van Den Reek, and S c o t t McCraney.

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Kerkenaar, Α.; Barug, D. Pestic. Sci. 1984, 15, 199-205. Iwata, K.; Yamaguchi, H.; Hiratani, T. Sabouraudia 1973, 11, 158-166. Stiers, D. L.; Fellman, J. K.; LeTourneau, D. Environ. Exper. Botany 1980, 20, 181-189. Sancholle, M. Docteur D'Etat. Thesis, Universite Paul Sabatier, Toulouse, 1984. Hippe, S. Pestic. Biochem. Physiol. 1984, 21, 170-183. Iwata, K.; Kanda, Y.; Yamaguchi, H.; Osumi, M. Sabouraudia 1973, 11, 205-209. Swamy, K. H. S.; Sirsi, M.; Rao, G. R. Antimicrob. Agents Chemother. 1974, 5, 420-425. Thomas, P. G.; Haslam, J. M.; Baldwin, B. C. Brit. Biochem. Soc. Trans. 1983, 11, 713. Nes, W. R. Lipids 1974, 9, 596-612. Demel, R. Α.; de Kruiff, Β. Biochim. Biophys. Acta 1976, 457, 109-132. Rodriguez, R. J.; Taylor Biophys. Res. Commun. 1982, 106, 435-441. Taylor, F. R.; Rodriguez, R. J.; Parks, L. W. J. Bacteriol. 1983, 155, 64-68. Rodriguez, R. J.; Parks, L. W. Arch. Biochem. Biophys. 1983, 225, 861-871. Ramgopal, M.; Bloch, K. Proc. Natl. Acad. Sci. USA 1983, 80, 712-715. Pinto, W. J.; Lozano, R.; Sekula, B. C.; Nes, W. R. Biochem. Biophys. Res. Commun. 1983, 112, 47-54. Nes, W. R.; Sekula, B. C.; Nes, W. D.; Adler, J. H. J. Biol. Chem. 1978, 253, 6218-6225. Taylor, F. R.; Parks, L. W. Biochem. Biophys. Res. Commun. 1980, 95, 1437-1445. Bloch, K. E. CRC Crit. Rev. Biochem. 1979, 7, 1-5. Trocha, P. J.; Jasne, S. J.; Sprinson, D. B. Biochemistry 1977, 16, 4721-4726. Pierce, A. M.; Mueller, R. B.; Unrau, A. M.; Oehleshlager, A. C. Can. J. Biochem. 1978, 56, 794-800. Elliott, C. G. J. Gen. Microbiol. 1969, 56, 331-343. Woloshuk, C. P.; Sisler, H. D.; Dutky, S. R. Antimicrob. Agents Chemother. 1979, 16, 98-103. Martin, C. E.; Hiramitsu, K.; Kitajima, Y.; Nozawa, Y.; Skriver, L.; Thompson, G. A. Biochemistry 1976, 15, 5218-5227. Weete, J. D.; van den Reek, M., unpublished data. Dahl, J. S.; Dahl, C. E.; Bloch, K. Biochemistry 1980, 19, 1467-1472. Dahl, J. S.; Dahl, C. E.; Bloch, K. J. Biol. Chem. 1981, 256, 87-91. Buttke, T. M.; Pyle, A. Z. J. Bacteriol. 1982, 152, 747-756. Sancholle, M.; Weete, J. D.; Touze-Soulet, J. M.; Dargent, R. In "Structure, Function, and Metabolism of Plant Lipids"; Siegenthaler, P. Α.; Eichenberger, W., Eds.; Elsevier Science Publ., B.V., 1984; pp. 347-352.

Received January 10, 1986

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 18

Antifungal Activity of Plant Steroids James G. Roddick Department of Biological Sciences, University of Exeter, Exeter, United Kingdom

Antifungal steroids in plants are represented mainly by the glycoalkaloids (especially the Solanum type) and the saponins. Both types of compound are strongly fungistatic/fungicida their ability to comple disrupt membrane integrity. The suggestion that the aglycone (rather than the glycoside) is the active moiety is questioned partly on the basis of data from studies on synthetic lipid membranes. Available evidence suggests that glycoalkaloids and saponins are not key factors in the resistance of vegetative organs to fungal infections although they may be of greater significance in reproductive structures and also contribute to the general defences of the plant. Plants produce a wide range of steroids (Table I) and accumulate some i n considerable quantities but our knowledge of the functions of most of these compounds i s meagre (1). Probably the major exception i s the s t e r o l s which are known to be important membrane components {2,3) as well as precursors of other steroids (4). Estrogens, androgens, corticosteroids {5_,6) and brassinosteroids Ç7) may have growth regulatory a c t i v i t y although t h i s i s s t i l l not c e r t a i n . For the remaining groups, no d e f i n i t e role has been established within the plant but the toxic nature of many of these steroids suggests they could have an ecological rather than metabolic function cont r i b u t i n g to plant resistance t o pathogens (especially fungi) and predators (especially insects) (8). This a r t i c l e w i l l review the antifungal a c t i v i t y of plant steroids but i t should be noted that not a l l steroids act i n an i n h i b i t o r y capacity towards fungi. Certain s t e r o l s have a stimulatory e f f e c t on fungal development promoting growth, the d i f f e r e n t i a t i o n of reproductive structures, and f e r t i l i s a t i o n i n species of Pythium and Phytophthora which lack the a b i l i t y to synthesize s t e r o l s (9). This subject i s considered elsewhere i n t h i s volume. Antifungal a c t i v i t y has been demonstrated mainly i n two groups of plant steroids, glycoalkaloids of the Solanum type and saponins. Members of both groups are comprised of a s t e r o i d a l aglycone

0097-6156/87/0325-0286$06.00/0 © 1987 American Chemical Society

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attached to one or more carbohydrate moieties. In addition, alka­ l o i d s contain a basic nitrogen group which renders them much more t o x i c , e s p e c i a l l y to homoiothermic organisms (10). Steroidal sapogenins are usually based on a spirostane skeleton (Figure 1). Various types of Solanum glycoalkaloids e x i s t but those so f a r shown to be fungitoxic i n v a r i a b l y possess a spirosolane- or solanidane-type aglycone (Figure 1). These glycoalkaloids and the monodesmosidic saponins are b i o l o g i c a l l y - a c t i v e amphipathic molecules with an o l i g o ­ saccharide comprising up to f i v e monosaccharides attached at C - 3 ; b i desmosidic saponins have an additional sugar moiety (usually one glucose) at C-26 and are b i o l o g i c a l l y i n a c t i v e . The chemistry of the Solanum alkaloids and saponins has been reviewed by various workers (11-14). Of the two groups, the glycoalkaloids have received more research attention undoubtedly because they are present i n edible parts of the important food plants, potato and tomato, and have caused i l l n e s s and death of humans and livestock on a number of occasions (15). Steroida parts of some less prominen caused serious incidences of poisoning. The b i o l o g i c a l properties of these compounds are d e t a i l e d i n a number of reviews ( 1 3 , 1 4 , 1 6 - 2 0 ) . Table I.

Major Groups of Steroids found i n Plants

Group Sterol Estrogen Androgen Corticosteroid Brassinosteroid Progestogen Withanolide Ecdysteroid Cardiac glycoside Saponin Glycoalkaloid

Example S i t o s t e r o l , stigmasterol E s t r a d i o l , estrone Testosterone, androstenedione 11-deoxycorticosterone Brassinolide Progesterone, pregnenolone Withaferin, nicandrenone Ecdysone, ecdysterone Calotropin Digitonin, avenacosides A and Β Solanine, j ervine

Reference 3, 96 5, 6 5, 98 5, 102 7 97, 98 99 100 101 13, 14

11/ 11

In the following section some of the more important i n v i t r o studies which established the f u n g i t o x i c i t y of glycoalkaloids and saponins are described leading, i n the two subsequent sections, to a c r i t i c a l assessment of how these compounds a f f e c t fungi at the bio­ chemical l e v e l and what contributions they make to the resistance of plants to fungal pathogens. Fungitoxicity of Glycoalkaloids and Saponins i n v i t r o A large number of studies have been made i n t h i s f i e l d with reports dating from a t least 1933 when the potato glycoalkaloid 'solanine' (Figure 2) was reported to i n h i b i t growth of Cladosporium fulvum (21 ,£2) . (At t h i s time 'solanine' preparations probably contained both potato a l k a l o i d s , α-solanine and α-chaconine, the l a t t e r not being discovered u n t i l 1954). The f i n d i n g around the same time that expressed juice from tomato plants i n h i b i t e d Fusarium oxysporum f.sp. l y c o p e r s i c i (23) subsequently l e d to the discovery of the tomato glycoalkaloid, α-tomatine (Figure 2), the active ingredient of the extracts. Further work (24,25) demonstrated the general t o x i c i t y of

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Figure 1. Aglycone skeletons of fungitoxic s t e r o i d a l saponins and Solanum glycoalkaloids.

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Figure 2 .

Solanum s t e r o i d a l glycoalkaloids.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

tomatine towards fungi, with F. oxysporum and various human dermatophytic fungi proving p a r t i c u l a r l y s e n s i t i v e . The d i f f e r e n t i a l s u s c e p t i b i l i t y of fungi to tomatine was l a t e r confirmed i n a more comprehensive investigation involving 3 0 species from 1 9 genera ( 2 6 ) . The maximum and minimum concentrations of tomatine to completely i n h i b i t mycelial growth ranged from 8 5 0 mM to 1 3 0 uM, a factor of 6 5 0 0 . An important feature of glycoalkaloid t o x i c i t y was highlighted by McKee ( 2 7 ) when he demonstrated the pH dependence of solanine-, tomatine- and demissine- (Figure 2 ) induced disruption of spores of a number of fungi, including Fusarium caeruleum. Disruption was greatest i n a l k a l i n e conditions, the L D of solanine at pH 5 . 6 being lOOx greater than at pH 7 . 6 . The saponin d i g i t o n i n (Figure 3 ) , although more toxic than these a l k a l o i d s , was r e l a t i v e l y unaffected by pH (Table I I ) . The pH dependence of glycoalkaloid action has since been shown i n other fungi (e.g. 2 8 - 3 0 ) and to be a general e f f e c t . Although d e t a i l s remain uncertain, protonation of the a l k a l o i d produces a form with less b i o l o g i c a alkaline conditions y i e l d 5Q

Table I I . 5 0 ( g/D of Glycoalkaloids and Saponins against Spores of Fusarium caeruleum i n r e l a t i o n to pH. After McKee (_27) . L

D

m

PH Steroid Tomatine Solanine Demissine Digitonin

5.0

27

5.6

7.0 32

7.6

8.0

460

220

6.5 64

2000

1100

260

85

20

11

260

72

22

11

-

8

-

18

-

-

14

-

-

5.9

13

-

8.3 7 8

In the study by McKee ( 2 7 ) , chaconine (Figure 2 ) proved more toxic than solanine with t h e i r common aglycone, solanidine (Figure 2 ) , much less so. S i m i l a r l y , tomatine (a tetraoside) was more e f f e c t i v e than i t s trisaccharide hydrolysis products against Helminthosporium turcicum, Septoria l i n i c o l a and Colletotrichum orbiculare with the aglycone, tomatidine (Figure 2 ) , being l e a s t e f f e c t i v e ( 2 8 ) . The e f f e c t of tomatidine varied considerably with the t e s t organism. The greater t o x i c i t y of glycosides compared with aglycones has also been shown with B o t r y t i s cinerea ( 3 1 ) and Phytophthora cactorum ( 3 2 ) , the l a t t e r authors also demonstrating t h i s feature f o r solasonine/solasodine (Figure 2 ) . Nevertheless, a few reports also e x i s t of tomatidine and solanidine being more fungitoxic than t h e i r respective glycosides ( 3 3 , 3 4 ) . V i r t u a l l y a l l the reports on fungal development i n the presence of glycoalkaloids or saponins describe i n h i b i t o r y e f f e c t s , but recently tomatine was found to stimulate sporulation i n F. oxysporum f.sp. l y c o p e r s i c i even though i t depressed colony growth, spore germination and germ tube growth (_35) . Promotion of reproductive development, however, was not observed i n P. cactorum; on the contrary, various s t e r o i d a l alkaloids of both the Solanum (solanine, tomatine) and Veratrum (jervine, muldamine, Figure 4 ) types, as well as tomatidine, solanidine and solasodine, a l l i n h i b i t e d s i t o s t e r o l induced spore production ( 3 2 ) . However, i n h i b i t i o n of vegetative hyphae by these compounds was not so marked or consistent, and varied with the s i t o s t e r o l content of the medium. McKee ( 2 7 ) also found

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solanine less damaging towards hyphae than towards spores but a t t r i buted t h i s to the a b i l i t y of hyphae to degrade the a l k a l o i d . The d i f f e r e n t response of vegetative hyphae and reproductive structures towards such compounds obviously has important implications for the experimental assessment of f u n g i t o x i c i t y . Probably the most widelyused method has been measurement of colony diameter but the v a l i d i t y of t h i s parameter has recently been questioned (36). Few i n v i t r o studies with fungi make reference to the c e l l u l a r nature of growth impairment. However, the disintegration of Phytophthora infestans zoospores (which lack a c e l l wall) by solanine (27) and the release of amino acids from digitonin-treated Pythium ultimim hyphae (37) , point to damage to l i m i t i n g (and possibly other) membranes. Mode of Fungitoxic Action Almost 60 years ago Fischer (38) and Boas (39) proposed that 'solanine' and d i g i t o n i n cause the erythrocyte membrane gotten for the next 30 years , propertie of glycoalkaloids and saponins were elucidated and figured prominentl y i n explanations of membrane l y s i s by these compounds. Consistent with t h i s thinking was the observation that aglycones had lower surface a c t i v i t y and were less disruptive than glycosides. Resurrection of the steroid-binding hypothesis probably dates from 1957 when Schulz and Sander (40) demons rated the formation of 1 : 1 molecular complex i n v i t r o between tomatine and 33-hydroxy steroids such as cholesterol. Since then, a large number of glycoalkaloids and saponins have been shown capable of complexing with various sterols (including the fungal s t e r o l ergosterol) i n v i t r o (41,42). Evidence was presented (28) that the reduced f u n g i t o x i c i t y of hydrolysis products of tomatine (including tomatidine) could not be explained solely on the basis of surfactant properties but was more l i k e l y related to t h e i r i n a b i l i t y to complex with s t e r o l s . I t was further reported (_28,43) that binding to sterols i n v i t r o only occurs to a s i g n i f i c a n t degree with the unprotonated a l k a l o i d i n alkaline conditions (Figure 5). Such a mode of action i s reminiscent of the sterolbinding polyene a n t i b i o t i c s (44), and species of Pythium and Phytophthora which are unaffected by polyenes because t h e i r membranes lack sterols are also r e l a t i v e l y insensitive to tomatine (26) and d i g i t o n i n (31_,45). However, when grown i n the presence of s t e r o l s , these fungi incorporate them into t h e i r membranes, a process which sensitizes them to polyenes, saponins, etc. (37,45). Work by t h i s author (46) indicated that tomatine disrupts isolated organelles i n a similar manner to polyenes such as f i l i p i n and nystatin, causing loss of lysosomal contents and i n h i b i t i o n of chloroplast PS II a c t i v i t y , but having no e f f e c t on the respiratory a c t i v i t y of mitochondria. Nystatin-resistant mutants of Fusarium solani with lower s t e r o l levels were also less sensitive to tomatine (47,48). These mutants were unaffected by 800 ppm tomatine whereas wild-type strains succumbed to 100 ppm tomatine. Crossing experiments between mutants and wildtypes showed that low s t e r o l content and i n s e n s i t i v i t y to tomatine or d i g i t o n i n were always inherited together (49). As with polyenes, the complexing of glycoalkaloids and saponins to membrane sterols i s thought to lead to the formation of pores i n membranes (50^,5^) -

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Ν)

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

glucosidase

R*

R l I x

I

Plant

glucosidase

R"

R'

S t e r o i d a l saponins.

= H

2 6 - D e s g l u c o a v e n a c o s i d e B, R' (active) _„

glycosidase

;glu-

;glu

A v e n a c o s i d e B, (inactive)

N u a t i g e n i n , R» = R" (inactive)

fungal

rham"

= glu

= H

rham^

= glu

= glu

Figure 3, Continued.

2 6 - D e s g l u c o a v e n a c o s i d e A, R' (active)

Plant

A v e n a c o s i d e A, (inactive)

rham

glu-glu-

H

rham'

glu-glu^

glu

;glu'

-glu'

to

I

Co

t

294

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Figure 5. E f f e c t of pH on binding of tomatine to cholesterol i n vitro.

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Freeze-fracture E.M. studies of natural and synthetic membranes treated with f i l i p i n , d i g i t o n i n and tomatine revealed c h a r a c t e r i s t i c protuberances on the surfaces of membranes (52,_53) but how these structures r e l a t e to the chemical complexes or to the formation of pores i s not understood. Despite the evidence supporting a sterol-binding mechanism, a number of doubts s t i l l remain. For example, no quantitative r e l a t i o n ship could be established between the i n v i t r o sterol-binding capacity of glycoalkaloids and saponins and their haemolytic action (41). Nor has the greater antifungal a c t i v i t y of aglycones than glycosides observed by Wolters (33) and Sinden et a l . (34) yet been s a t i s f a c t o r i l y explained. Also, although less potent than tomatine, tomatidine was s t i l l capable of i n t e r f e r i n g with mycelial growth of H. turcicum, S. l i n i c o l a and (particularly) C. orbiculare (28). The minimum concentrations of the aglycone required to cause complete growth i n h i b i t i o n were respectively 400x, 2000x and only 3x the minimum concentration o also been shown to reduc proposed weakness of the g hypothesi d i r e c t evidence of incorporation of glycoalkaloids or saponins into the fungal mycelium or membrane (36). In evidence, solanine did not influence sporulation i n P. cactorum and, i n t r i t i a t e d form, was not incorporated into mycelium whereas PH]-solanidine was both deleterious to sporulation and incorporated into mycelium. The a b i l i t y of t h i s fungus to hydrolyse solanine to solanidine suggested to the authors that t h e i r findings might be explicable on the basis of a quite d i f f e r e n t hypothesis for mechanism of action which l a i d emphasis on the aglycone rather than the glycoside. This hypothesis has i t s origins i n haemolysis experiments c a r r i e d out by Segal et a l . (54). Following treatment of erythrocytes with d i g i t o n i n , solanine or tomatine, aglycones but not glycosides could be detected i n haemolysed ghosts whereas both aglycones and glycosides were associated with non-haemolysed c e l l s . Aglycones alone were also haemolytic. I t was proposed that the aglycone was the active moiety being released from the glycoside by a membrane glycosidase. Consistent with t h i s claim was the subsequent finding (55) that the addition of gluconolactone or galactonolactone (reputedly s p e c i f i c i n h i b i t o r s of glycosidases) i n h i b i t e d tomatineand (to a lesser extent) digitonin-induced haemolysis. When these experiments were repeated on B. cinerea and Rhizoctonia s o l a n i , e s s e n t i a l l y s i m i l a r r e s u l t s were obtained (56). Thus, i n addition to the requirement for membrane s t e r o l to bind the glycoside, t h i s hypothesis necessitates a second prerequisite i n the form of a membrane glycosidase. How the aglycone brings about l y s i s was not made c l e a r . The glycoside was thus considered to be simply the "water-soluble transport form" (56), but such a r o l e has recently been disputed (57,58). Although there are good grounds for accepting that inactive, non-sterol-binding bidesmosidic saponins (e.g. avenacosides A and B, Figure 3) are enzymically hydrolysed i n damaged c e l l s to active, monodesmosidic saponins (59), a number of problems arise when attempting to explain the t o x i c i t y of monodesmosidic saponins and glycoalkaloids on the basis of a similar hydrolytic a c t i v a t i o n process. The p r i n c i p a l objections come from work on synthetic l i p i d membranes which lack glycosidases. E l f e r i n k (60), for

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ECOLOGY AND METABOLISM OF PLANT LIPIDS +

example, found that d i g i t o n i n caused s i g n i f i c a n t leakage of K from phosphatidylcholine liposomes only when cholesterol was also present. A similar observation was reported recently by t h i s author (43) using tomatine (Table I I I ) . The extent of liposome disruption was directly related to the concentration of both the s t e r o l and the a l k a l o i d (with a strong interaction between the two) as well as to pH. The aglycone was not active i n t h i s system. In experiments on planar l i p i d b i l a y e r s and monolayers (61), d i g i t o n i n was p a r t i c u l a r l y e f f e c tive i n causing channel-like conductance changes i n membranes containing sterols; i n sterol-free membranes, e f f e c t s could only be achieved with s i g n i f i c a n t l y higher concentrations. Obviously, caution i s essential when attempting to extrapolate from a r t i f i c i a l membranes to c e l l membranes but the s i m i l a r i t i e s i n responses/suscept i b i l i t y of the two systems to glycoalkaloids and saponins i n relation to pH, s t e r o l content, etc. suggest that such comparisons have some validity. Table I I I . E f f e c t of Tomatin Liposomes containing d i f f e r e n t Phospholipids and Sterols Sterol ErgoNo CholeStigmaPhospholipid Sterol sterol Treatment sterol sterol 19.2 19.6 Control 19.5 26.0 Phosphatidylcholine 51.4 69.2 15.4 Tomatine 52.0 Sphingomye1in 7.4 48.4 Control 10.6 6.3 50.0 24.9 31.8 Tomatine 29.8 Values are % of liposome peroxidase a c t i v i t y released into supernatant. Liposomes were treated with 150 uM a l k a l o i d at pH 7.2 for 1 hour. Adapted from Roddick and Drysdale (43). Some aspects of the experimentation which support the aglycone hypothesis are also open to doubt. For instance, aglycones were apparently dissolved i n 20-25% DMSO which i s i t s e l f strongly haemol y t i c , and no control data were presented. With tomatidine i n 1% (or less) DMSO and proper controls, no haemolysis could be attributed to either of these components (Roddick, unpublished). High concentrations of sugar lactones can depress pH i n weakly-buffered solutions and thus possibly also the action of pH-dependent glycoalkaloids. The i n a b i l i t y of sugar lactones to i n h i b i t aglycone-induced haemol y s i s could be explained by l y s i s being caused by DMSO, as indicated above. The f a c t that haemolysed erythrocyte ghosts were washed whereas non-haemolysed c e l l s were not could account for the f a i l u r e to detect glycosides i n the former but t h e i r presence i n the l a t t e r . The s i t u a t i o n i s further confused by claims that a fungal membrane glycosidase activates tomatine (56) whereas a fungal wall glycosidase, e f f e c t i n g an i d e n t i c a l hydrolysis, i n h i b i t s tomatine action (62). In neither case was the proposed location of these enzymes established using unequivocal (e.g. fractionation) techniques. However, t h i s i s not to refute the existence or a c t i v i t y o f such glycosidases. Numerous reports exist of the hydrolysis of glycoalkaloids by fungal glycosidases (e.g. 27, 3^, 63-65) but i n virtually every case hydrolysis was viewed as an i n a c t i v a t i o n of a toxic glycoside. Nor do the doubts attaching the aglycone hypothesis necessarily

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mean that membrane d e s t a b i l i z a t i o n by glycoalkaloids and saponins can be explained i n a l l cases by sterol-binding. Many anomalies s t i l l e x i s t i n t h i s area e.g. the lack of a quantitative relationship between s t e r o l binding i n v i t r o and haemolysis (41); the greater d i s ­ ruption of s t e r o l - f r e e than sterol-containing liposomes by the saponin p a r i l l i n (Figure 3) (60). I t may be that d i f f e r e n t membrane-active s t e r o i d a l glycosides operate i n s l i g h t l y , or even markedly, d i f f e r e n t ways. Even so, the weight of evidence points to s t e r o l binding as being an important factor, perhaps q u a l i t a t i v e l y rather than quanti­ t a t i v e l y , i n the d e s t a b i l i z a t i o n of membranes by the compounds i n question. Interactions, of either a d i r e c t or i n d i r e c t nature, between glycoalkaloids/saponins and membrane phospholipids and/or proteins may also be involved. Opinions have been voiced both i n favour (36,j52) and against (37) such p o s s i b i l i t i e s . Obviously more work i s required i n t h i s f i e l d . Glycoalkaloids and Saponin The t o x i c i t y of s t e r o i d a l glycoalkaloids and saponins to various p a r a s i t i c fungi i n v i t r o has naturally led to suggestions that these compounds might also operate i n a similar capacity i n the i n t a c t , i n ­ fected plant. Early studies (24,33,66) gave some support to t h i s idea with tomato pathogenic fungi apparently less susceptible to tomatine than non-pathogens. From a more critical investigation (26) under standard conditions with 30 species of fungi, including tomato pathogens, non-pathogens and general saprophytes, a ranking order of increasing s u s c e p t i b i l i t y to tomatine was produced i n which tomato pathogens occupied 14 of the f i r s t 16 places. The p r o b a b i l i t y of such a ranking occurring by chance was calculated as 1 i n 10^. How­ ever, demonstration of a c o r r e l a t i o n between s u s c e p t i b i l i t y i n v i t r o and pathogenicity i s not i n i t s e l f s u f f i c i e n t evidence that compounds l i k e tomatine play a major role i n resistance to fungal pathogens. Although only one of many factors, d i f f e r e n t i a l s u s c e p t i b i l i t y of fungi to toxic plant metabolites i s undoubtedly not without s i g ­ n i f i c a n c e . A probable explanation for t h i s i n Pythium and Phytophthora spp. i s the lack of membrane s t e r o l s , a supposition which finds support i n the f a c t that growth i n a sterol-containing medium sensi­ t i z e s these fungi to glycoalkaloids and saponins (37,45). An alterna­ t i v e explanation i s that these fungi may not assimilate the glycosides (36) although the significance of assimilation has yet to be ascer­ tained. Detoxification of glycoalkaloids or saponins by extracellular glycosidases which hydrolyse glycosides to aglycones may also con­ tribute to the r e l a t i v e i n s e n s i t i v i t y of these species (32,67) a l ­ though t h i s mechanism may be more important i n other species of fungi. The leaf-spot fungus Septoria l y c o p e r s i c i , for example, inactivates tomatine by removing one glucose to produce 3 2 ~ (63,64) whereas F. oxysporum f.sp. l y c o p e r s i c i (65) and B. cinerea (31) both remove the whole tetrasaccharide moiety. In A l t e r n a r i a solani (62) and F. caeruleum (27) there i s evidence of a stepwise removal of a l l the monosaccharide units of glycoalkaloids. In damaged oat leaves, the inactive, bidesmosidic saponins avenacosides A and Β are hydrolysed at C-26 by leaf enzymes to y i e l d the active, monodesmosidic derivatives. However on i n f e c t i o n by Helminthosporium avenae, the monodesmosidic saponins are further degraded to the inactive agly­ cone, nuatigenin, by a fungal glycosidase which removes the carbot o m a t : i

n e

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hydrate moiety at C - 3 (Figure 3 ) ( 5 9 ) . In potato tubers, the conversion of solanine to solanidine observed i n tissue damaged by I>. infestans or the bacterium Erwinia atroseptica was reported to be due to the release of h o s t - c e l l hydrolases ( 6 8 ) . In infected, but nondamaged, r e s i s t a n t v a r i e t i e s the aglycone was not detectable. The proposal that tomatine might be a factor i n the resistance of tomato v a r i e t i e s to F. oxysporum f.sp. l y c o p e r s i c i ( 2 3 , 6 9 ) was not confirmed by Kern ( 6 6 ) who considered the a l k a l o i d neither sufficientl y i n h i b i t o r y to the fungus nor s u f f i c i e n t l y abundant i n lower stems and roots. Drysdale and coworkers have argued that the tomatine concentration i n these organs i s adequate to i n h i b i t growth and spore germination of F. oxysporum ( 7 0 ) but rule out a primary resistance role for t h i s a l k a l o i d because i t was not detectable i n f l u i d from xylem (where t h i s w i l t fungus i s located) and because i t s l e v e l s i n creased to the same extent i n both r e s i s t a n t and susceptible cultivars following i n f e c t i o n Ç71_) . Others ( 3 5 ) have detected tomatine i n tracheal f l u i d but agai centration i n r e s i s t a n t f e c t i o n . Further evidenc agains primary that the a l k a l o i d stimulated sporulation of F. oxysporum f.sp. l y c o p e r s i c i ( 3 5 ) , a process which the authors point out i s more important i n invasion by t h i s fungus than mycelial growth. On the other hand, some workers have arrived at the opposite conclusion regarding the importance of tomatine i n fusarium w i l t of tomato. Sarhan and Kirâly (12) concluded that i n f e c t i o n was related to s o i l nutrients and fungicide treatments and that resistance was mediated v i a elevated tomatine l e v e l s . A s i m i l a r claim was made from a study of the fungit o x i c i t y of extracts from infected and non-infected tomato plants of r e s i s t a n t and susceptible c u l t i v a r s (13); differences from the conclusions of other workers (e.g. 7 0 ) were explained by v a r i e t a l differences. The above information together with the f a c t that F. oxysporum f.sp. l y c o p e r s i c i i s a fungus of low (or zero) tomatine regions capable of detoxifying t h i s a l k a l o i d ( 6 5 ) suggest that tomatine i s probably not a major factor i n resistance to fusarium w i l t . Claims that tomatine may contribute to resistance to b a c t e r i a l w i l t (Pseudomonas solanacearum) i n roots of Lycopersicon pimpineHifolium ( 7 4 ) require substantiation. Glycoalkaloid l e v e l s i n whole potato tubers tend to be similar to the low l e v e l s found i n roots and stems although the skin and peel of tubers have much higher concentrations Ç 7 5 ) . Nevertheless, glycoalkaloids are not thought to be important i n combatting tuber i n f e c tions by F. caeruleum ( 7 6 ) or R. solani ( 7 7 ) . I t may be s i g n i f i c a n t that F. caeruleum i s able to degrade potato glycoalkaloids ( 2 7 ) . S i m i l a r l y , no correlations were observed between a l k a l o i d l e v e l s i n potato roots/stems and V e r t i c i l l i u m w i l t (V. albo-atro), or between tuber a l k a l o i d s and the b a c t e r i a l disease, common scab (Streptomyces scabies) ( 7 8 ) . Nor are glycoalkaloids apparently involved i n r e s i s tance of potatoes to b a c t e r i a l r i n g rot caused by Corynebacterium sepedonicum ( 7 9 ) . Much work into the role of glycoalkaloids i n potato tuber resistance to the l a t e - b l i g h t fungus (P. infestans) has been done by Kuc and coworkers and has recently been reviewed by Kuc ( 8 0 ) . Early investigations ( 8 1 _ , 8 2 ) pointed to glycoalkaloids as being a possible factor i n tuber resistance but the subsequent demons t r a t i o n that a l k a l o i d accumulation (which increases i n damaged

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tubers) i s suppressed by compatible and incompatible races of l>. infestans (83) questioned the involvement of these compounds i n R gene and hypersensitive resistance. A study of t h i s disease i n 15 potato clones (84) also produced no evidence of a glycoalkaloid cont r i b u t i o n to multigenic (field) resistance. Despite these conclusions, Kuc (80) i s of the opinion that tuber glycoalkaloids may s t i l l play a role i n general resistance to disease. In view of t h e i r higher concentrations i n leaves and reproductive structures, i t has been proposed (63) that glycoalkaloids might be more important against l e a f - or f r u i t - i n f e c t i n g fungi. The leaf pathogen C. fulvum was adversely affected by tomatine but i t was not clear whether t h i s fungus, which grows i n i n t e r c e l l u l a r spaces, could cause tomatine leakage from c e l l s (30). In potato leaves, elevated resistance to the early b l i g h t fungus A. solani observed i n continuous l i g h t was not attributable to glycoalkaloids (85). Sinden et a l . (34) concluded likewise for older (120 day) leaves which had a lower alkal o i d content (260 ppm) bu higher a l k a l o i d levels (157 s o l a n i . In a study of lea i n f e c t i o by . solan and . infestans in 10 potato c u l t i v a r s , no correlations were apparent between glycoalkaloids and resistance to disease (78) although t h i s might be explained by the age of the plants (approx. 8 weeks) i n the case of A. solani. Tomato f r u i t s are interesting organs for phytopathological studies being s i t e s of tomatine synthesis and high tomatine accumulations (in small, green f r u i t s ) as well as tomatine degradation and low to zero a l k a l o i d levels (in large green to ripe f r u i t s ) (58,86). Of i n t e r e s t i n t h i s respect i s the finding that B. cinerea germ tubes penetrated epidermal c e l l s of green f r u i t s but no further development of the fungus occurred (31). However, the authors expressed doubt that t h i s was due to tomatine as the fungus i s able to degrade t h i s a l k a l o i d and also d i d not resume growth i n ripe f r u i t s , though s t i l l a l i v e . More clear-cut r e s u l t s have been reported for F. solani, low s t e r o l mutants of which caused severe r o t of green tomato f r u i t s whereas wild type strains d i d not (48). Both types were equally aggressive on ripe tomatoes. The authors suggested that tomatine could be a major resistance factor i n f r u i t s (at least to t h i s fungus). Further evidence i n support of t h i s claim was that i n crossing experiments the a b i l i t y to r o t green f r u i t s was inherited along with i n s e n s i t i v i t y to tomatine (49). Ripening tomato f r u i t s show a decline in their resistance to Colletotrichum phomoides (87) but i t i s not known i f t h i s i s related to the concomitant decrease i n f r u i t txmatine. A study of colonisation of tomato f r u i t s (of d i f f e r e n t developmental stages) by the f r u i t r o t pathogens Corticium r o l f s i i , B. cinerea, Monilia fructigena and Gloeosporium fructigenum revealed an order of pathogenicity (as shown) which corresponded to that of i n s e n s i t i v i t y to tomatine i n v i t r o (29). Colonization was explained, not by secret i o n of hydrolytic enzymes, but by a l t e r a t i o n to the pH at the inocul a t i o n s i t e s . The most successful pathogen (C. r o l f s i i ) lowered the pH from 5.7 to 3.6, the least successful (G. fructigenum) increased the pH to 6.4, and the two intermediate pathogens gave intermediate pH changes (to 4.4). The relationship between pH and resistance was explained on the basis of the pH dependence of tomatine t o x i c i t y . In another report, the same author (62) showed that invasion of tomato f r u i t s by various species and races of A l t e r n a r i a , pathogenic and non-

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pathogenic to tomato, was related, not to pH changes ( a l l test fungi increased t h i s equally), but to t h e i r a b i l i t y to secrete tomatinehydrolysing enzymes. The species/strains which did not produce such enzymes were incapable of successful colonization. The major factors which influence the t o x i c i t y of glycoalkaloids and saponins to fungi i n vivo can thus be i d e n t i f i e d as the l e v e l of glycoside, fungal s t e r o l content, secretion of fungal hydrolases, production of suppressors of glycoside synthesis and a l t e r a t i o n of pH. To t h i s l i s t could be added a number of other factors which may be important i n moderating the above or having a bearing i n t h e i r own r i g h t , but which have been l i t t l e researched. For example, glyco­ a l k a l o i d l e v e l can be influenced by age (34) and/or developmental stage (86) as well as by v a r i e t y , environmental factors and c u l t u r a l practices (88,89). Consideration should also be given to the i d e n t i t y (and ratios) of compounds present as some are more toxic than others (27,34). In view of the d i f f e r e n t i a l s e n s i t i v i t y of vegetative hyphae and reproductive structure aspects of the fungal pathoge pounds of host o r i g i n liberated along with glycoalkaloids or saponins following damage to t h e i r main storage s i t e , the vacuole (90,91), or to other c e l l compartments are known to enhance, ameliorate or even n u l l i f y t o x i c i t y of these compounds. These include sugars (27), ions such as Na , K and C a (27), s t e r o l s (32) and glycosidases (92-94). +

+

2 +

Conclusions Despite t h e i r potent antifungal action i n v i t r o , s t e r o i d a l glyco­ alkaloids and saponins do not appear to be key factors i n the r e s i s ­ tance of roots, stems and leaves to fungal disease, although they may play a greater role i n resistance to f r u i t i n f e c t i o n s . Neverthe­ less, many authors s t i l l believe these compounds play some role i n the general defences of the plant against fungi. The f a c t that many successful fungal parasites have means of degrading or i n a c t i v a t i n g glycoalkaloids or saponins or of suppressing t h e i r synthesis, or do not synthesize t h e i r target molecules, etc. suggests that i n evolu­ tionary, i f not e c o l o g i c a l , terms these compounds do have a poten­ t i a l l y protective function. Many p a r a s i t i c fungi are not pathogenic to plants which elaborate glycoalkaloids or saponins (26), a s i t u a t i o n which could be due to the presence of these compounds. In the coevolution of plants and fungal parasites, any equilibrium i s usually a dynamic one and the r e l a t i v e importance of a p a r t i c u l a r protective device i n a plant undoubtedly changes with the evolution of i t s parasites. Such f l e x i b i l i t y necessitates the maintenance of a variety of defence mechanisms only one, or a few, of which may oper­ ate as the ' f r o n t - l i n e ' system at any one time and/or against any one pathogen, with the others acting i n an a u x i l i a r y or 'back-up' capacity. Thus, although i t may not be possible to demonstrate a primary role for a p a r t i c u l a r compound, i t i s not unreasonable to assume that i t s synthesis and accumulation s t i l l represent an impor­ tant, and possibly v i t a l , investment i n evolutionary terms. F i n a l l y , i t must be borne i n mind that chemical defences i n plants have evolved i n response to a number of b i o t i c pressures, not least those from herbivores, and p a r t i c u l a r l y insects. The r a p i d i t y with which damage i s caused by feeding i s such that, to be e f f e c t i v e ,

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counter measures must usually be equally rapid and the release o f preformed i n h i b i t o r s , toxins or repellants i s one way of achieving t h i s . The p o s s i b i l i t y therefore that s t e r o i d a l glycoalkaloids and saponins evolved primarily i n response to prédation pressures mainly from insects i s a very r e a l one (95). Even so, the general t o x i c i t y of glycoalkaloids and saponins renders i t u n l i k e l y that any deterrent role played by these compounds would be a highly s p e c i f i c one.

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11. 12. 13. 14. 15. 16. 17. 18.

19. 20. 21. 22. 23.

Heftmann, E. Phytochemistry 1975, 14, 891. Demel, R.A.; de Kruyff, B. Biochim. Biophys. Acta 1976, 457, 109. Nes, W.D.; Heftmann, E. J. Nat. Prod. 1981, 44, 377. Heftmann, E. Lipids 1971, 6, 128. Geuns, J.M.C. Phytochemistry 1978, 17, 1. Hewitt, S.; Hillman, J.R.; Knights, Β.A. New Phytol. 1980, 85, 329. Mandava, N.G.; Thompson, M.J. In "Isopentenoids in Plants"; Nes, W.D.; Fuller, G.; Tsai, L.-S., Eds.; Marcel Dekker: New York, 1984; p. 401. Swain, T. Ann. Rev. Plant Physiol. 1977, 28, 479. Elliott, C.G. Adv. Microbiol. Physiol. 1977, 15, 121. Roddick, J.G. In "Secondary Plant Products"; Bell, E.A.; Charlwood, B.V., Eds.; ENCYCLOPAEDIA OF PLANT PHYSIOLOGY, NEW SERIES, Springer-Verlag: Berlin, Heidelberg, 1980; Vol. 8, p. 167. Schreiber, K. In "The Alkaloids. Chemistry and Physiology"; Manske, R.H.F., Ed.; Academic: New York, 1978; Vol. X, p. 1. Schreiber, K.; Ripperger, H. In "The Alkaloids. Chemistry and Physiology"; Manske, R.H.F.; Rodrigo, R.G.A., Eds.; Academic: New York, 1981; Vol. XIX, p. 81. Tschesche, R.; Wulff, G. Fortschr. Chem. Org. Naturstoffe 1973, 30, 462. Mahato, S.B.; Ganguly, A.N.; Sahu, N.P. Phytochemistry 1982, 21, 959. Morris, S.C.; Lee, T.H. Food Technol. (Australia) 1984, 36, 118. Roddick, J.G. Phytochemistry 1974, 13, 9. Defago, G. Ber. Schweiz. Bot. Ges. 1977, 87, 79. Schonbeck, F.; Schlosser, E. In "Physiological Plant Pathology"; Heitefuss, R.; Williams, P.H., Eds.; ENCYCLOPAEDIA OF PLANT PHYSIOLOGY, NEW SERIES, Springer-Verlag: Berlin, Heidelberg, 1976; Vol. 4, p. 653. Jadhav, S.J.; Sharma, R.P.; Salunkhe, D.K. Crit. Rev. Toxicol. 1981, 9, 21. Roddick, J.G. In "The Biology and Systematics of the Solanaceae"; D'Arcy, W.G.; Hawkes, J.G., Eds.; Columbia Univer­ sity: New York, In press. Agerburg, L.S.; Schick, R.; Schmidt, M.; Sengbusch, R.V. Züchter 1933, 5, 172. Schmidt, M. Planta 1933, 20, 407. Fisher, P.L. Maryland Agr. Expt. Sta. Bull. 1935, 374.

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Chapter 19

Biosynthesis and Requirement for Sterols in the Growth and Reproduction of Oomycetes W. David Nes Plant and Fungal Lipid Group, Plant Development and Productivity Research Unit, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Albany, C A 94710

All Oomycetes fungi possess an obligatory requirement for sterols to complet cycle. Growth is mediate synthesizing Oomycetes additionally by "sterol-like" pen­ tacyclic and tetracyclic triterpenoids. The structural features and levels which are determinant for bioregulator activity differ amongst the fungi. Herein the bio­ synthesis and sterol function(s) which operate to control growth are examined and compared with the involvement of sterol in reproduction. Oomycetes are less-advanced fungi Which are grouped apart from the lineage to the more-advanced fungi, eg. Basidiomycetes ( F i g . 1 ) , based on t h e i r having coenocytic mycelia and double-walled oospores. The Oomycetes may be divided into 2 groups - those which synthesize s t e r o l s and those which do not (1-10). While s t e r o l s are not o b l i gatory f o r the growth of a l l the Oomycetes, both groups require s t e r o l s f o r sexual reproduction (oospore production). As discussed i n what follows, the s t e r o l u t i l i z e d to mediate growth may be, a t a l a t e r period i n the vegetative cycle, responsible f o r regulating oosporogenesis without necessarily having undergone metabolism. With c e r t a i n Oomycetes, ••sterol-like** molecules, eg. t e t r a c y c l i c and pentacyclic triterpenoids ( F i g . 2), may produce the same physiolog i c a l end response as s t e r o l s (2). The growth response to these p o l y c y c l i c isopentenoids i s intimately associated with t h e i r 3-dimens i o n a l geometry, molecular features and s u b c e l l u l a r quantity. I n order to discuss s t e r o l function f o r What we assume i s an evolutionary determinant of developmental change, a b r i e f overview of s t e r o l occurrence and biosynthesis i s given f i r s t . S t e r o l occurrence. McCorKindale, et a l . , were the f i r s t to s t r u c t u r a l l y and q u a n t i t a t i v e l y examine the s t e r o l composition of a variety of Oomycetes (8). From t h e i r study i t was evident that: the Oomycetes produced a s t e r o l p r o f i l e d i s s i m i l a r from the more-advanced fungi, This chapter not subject to U.S. copyright. Published 1987, American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Sterols in the Growth and Reproduction of Oomycetes

19. NES

BASIDIOMYCETES

DEUTEROMYCETES ASCOMYCETES

ZYGOMYCETES .TRICHOMYCETES HYPHOCHYTRIDIOMYCETES -

OOMYCETES

CHYTRIDIOMYETES

SLIME MOLDS MYXOMYCETES: ACRAISIOMYCETES: LABYRINTHULOMYCETES

ALGAE

ALGAE

(CHRYSOPHYCOPHYTA)

/ RHODOPHYCOPHYTA \ V PHAEOPHYCOPHYTA I

ALGAE (DINOPHYCOPHYTA)

Figure 1. Phylogenetic r e l a t i o n s h i p s of fungi (based on c l a s s i c considerations of Morphology, L i f e Cycle, C e l l Wall Chemistry, and Amino Acid Pathways).

TETRAHYMANOL

β-AMYRIN Figure 2.

tt-AMYRIN

As shown.

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eg. the ergosterol-producing Ascornycetes; some members f a i l e d to synthesize detectable levels of s t e r o l s ; and the predominant end products characterized by GC-MS, included both 24-desalkyl and 24alkylidene s t e r o l s . In the intervening 15 years a d d i t i o n a l Oomycetes have been examined f o r t h e i r s t e r o l content (11-21. Table I ) , and i n select fungi the s t e r o l i d e n t i f i c a t i o n has been confirmed by proton magnetic resonance (PMR) spectroscopy (4,11). The s t e r o l content ranged from .01 to .25% of the mycelial dry weight. The lower value may i n some cases represent differences i n the d e t a i l s of extraction and i s o l a t i o n . For instance, as we have found, mycelia extracted as fresh and freeze dried versus oven dried material generate about 5 to 10 times as much s t e r o l unless water i s added back i n the extraction of the l a t t e r material. I f digitonides are made then long chain f a t t y alcohols would not contaminate the TLC p u r i f i e d samples and hence would not i n t e r f e r e with GLC q u a n t i f i c a t i o n . From these studies, c f . Table I , three findings are s i g n i f i c a n t . F i r s t l y , th Peronosporales. Secondly accompaniment of cycloartenol ( a l b e i t , see Sterol Biosynthesis and Metabolism Section). Thirdly, some investigators have found ergosterol i n numerous Oomycetes (12,13,21), although t h i s observation has not been confirmed by others i n which the same fungus, precursor material, eg. cycloartenol and lanosterol fed to Phytophthora and Lagenidum. and culture conditions were used (1.18.20). The occurrence of ergosterol, however, can be explained. I t has been our experience that Phytophthora i s r e a d i l y contaminated by other fungi (spores) during poor c u l t u r i n g p r a c t i c e s . Consequently, we routinely monitor the s t e r o l content of our fungal c o l l e c t i o n by GLC and HPLC. When ergosterol i s detected, e s p e c i a l l y i n the Oomycetes s t e r o l p r o f i l e , the culture i s destroyed and the parent s t r a i n regenerated, from material maintained under an envelope of l i q u i d nitrogen. Another p o s s i b i l i t y i s that ergosterol was derived from the media, eg. yeast extract. As shown i n Table I I , s t e r o l s and p a r t i c u l a r l y - c h o l e s t e r o l , are widely d i s t r i b u t e d i n commercial sources. Cholesterol, l i k e ergosterol, has been i s o l a t e d i n various Oomycetes, although, l i k e ergosterol, i t may not be an endogenous component depending on the composition of the ' synthetically-compounded media. When P. cactorum ( a s t e r o l - l e s s fungus, c f . Table I ) was 14 cultured with [2- C]mevalonic acid (MVA) on a synthetic media and inoculated with agar plugs, s u f f i c i e n t non-radioactive cholesterol was absorbed by the mycelia to be chemically i d e n t i f i e d , although 14 cholesterol f a i l e d to possess C-label (20). Fryberg e t a l . . s i m i l a r i l y found an unusual s t e r o l i n yeast i e . , s i t o s t e r o l (22), and concluded f o r biosynthetic reasons that the s t e r o l must have been dietary (present i n the o i l supplement) rather than endogenous. We (1) and others (20) have attributed the trace l e v e l s of cholesterol present i n P. cactorum cultured on agar-supplemented media to be derived from the agar plug - agar i s an i n d u s t r i a l by-product of red algae; the dominant s t e r o l of red algae i s cholesterol. The s o l u b i l i t y c h a r a c t e r i s t i c s of s t e r o l s i n the various dispersing agents also have an influence on the amount of dietary s t e r o l made a v a i l a b l e to the fungus, thereby a f f e c t i n g quantitative

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differences i n content and development. For instance, a 1.0 ppm l e v e l of c h o l e s t e r o l dispersed i n ether, ethanol (1.5) or no dispersing agent may not be as r e a d i l y accumulated as s t e r o l dispersed i n the same concentration i n l e c i t h i n . Thus, differences i n growth-response and oospore numbers may vary although the amount of the s t e r o l supplement i s s i m i l a r i n the separate treatments. I t follows that growth stimulation of pythiaceous fungi (24-28) and production of oospores (23.29.30) by Tweens, l e c i t h i n s and vegetable o i l s , without (additional) s t e r o l supplementation, can be r a t i o n a l i z e d not i n terms of an obligatory requirement f o r a f a t t y acid but due to the contaminant s t e r o l i n the media preparation. This trace s t e r o l may also permit other compounds, eg. cycloartenol (6.31), to act i n a s y n e r g i s t i c or sparking (32) capacity allowing for growth stimulation and oogonia induction ( c f . S t e r o l Function Section). Concerning the cholesterol i d e n t i f i e d i n other Oomycetes 14 (Table 1), such as S. fera 14 S. ferax. [ CIcholesterol i s i s o l a t e d i n radiochemically pure form, thus demonstrating i t s biosynthesis de novo (11). Apparently, those fungi which synthesize cholesterol f a i l to a c t i v e l y accumulate the dietary s t e r o l precluding a s i g n i f i c a n t contamination e f f e c t i n s i t u , u n l i k e P. cactorum. which r e a d i l y absorbs exogenous cholesterol. The s t e r o i d biosynthetic pathway of Oomycetes may be operationally divided into 4 stages: (1) acetate conversion to squalene-oxide; (2) p o l y c y c l i z a t i o n of squalene-oxide to a t e t r a c y c l i c product; (3) metabolism of the t e t r a c y c l e to a series of amphipathic neutral s t e r o l s having a free 3S-0H, planar nucleus and i n t a c t side chain of 8 to 10 C-atoms; and (4) side chain hydroxylation and reduction followed by a d d i t i o n a l nuclear metabolism of s p e c i f i c a l l y the 24-aIkylidenesterol-fucosterol to polar steroids (Fig.3). Phytophthora and related " s t e r o l - l e s s Oomycetes possess an enzymic defect i n Stage I . They synthesize squalene (9.19) but f a i l to produce squalene- oxide (9.10.18). While some fungi c y c l i z e squalene-oxide to pentacyclic triterpenoids i t remains unknown whether the pythiaceous fungi s i m i l a r i l y perform t h i s kind of cyclization. S. ferax and related sterol-producing Oomycetes synthesize lanosterol rather than cycloartenol (10) i n Stage I I . The mechanism of squalene-oxide c y c l i z a t i o n to lanosterol involves a nonconcerted process i n which a terminal intermediate, chair-boat-chair-boat-"left-handed" unfolded (33.34) side chain, proceeds through stereoelectronic r i n g and side chain inversion (Fig. 4) to y i e l d a product with an a l l - c h a i r nucleus and "right-handed" unfolded side chain (34, and r e f . c i t e d t h e r e i n ) . There are suggestions i n the l i t e r a t u r e , however, that Oomycetes (13.36) may synthesize cycloartenol, and metabolize i t to a 4-desmethylsterol, such as f u c o s t e r o l ; i f the fungus cannot synthesize or metabolize the cyclopropyl s t e r o l , then i t may use the compound unaltered as a unique s t r u c t u r a l membrane i n s e r t (31) due to the b u t t e r f l y or bent shape of the cyclopropyl s t e r o l (32). Our o r i g i n a l i n t e r e s t i n cycloartenol u t i l i z a t i o n by Oomycetes was phylogenetic rather than functional. The u t i l i z a t i o n of a cycloartenol-versus a lanosterol-based pathway i s known to 1

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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308

T a b l e I.

S t e r o l c o m p o s i t i o n of

Oomycetes*

Group

Saproleqniales Saprolegnia ferax S . Ferax S . Ferax Achlya C a r o l i n a A. hypogyna A. b i s e x u a l i s A. americana P y t h i o p s i s cymosa P. i n t e r m e d i a D i c t y u c h u s monosporus Leptomitales Apodachlya mimima A. brachynema A p o d a c h y l e l l a compléta A. compléta Peronosporales Phytophthora cactorum Phytophthora i n f e s t a n s P. cinnanomi Pythium ultimum P. g r a m i n i c o l a P. debaryanum Zoophagus i n s i d i a n s Lagenidiales A t k i n s i e l l a dubia Lagenidium c a l l i n e c t e s Lagenidium giganteum Haliphthoros milfordensis s t r a i n , HAL - 223

Sterol 1

2

3

4

5

4 1 -

13 37 -

68 43

15 12 -

18

2 -

17 22 73

70 67 4

4

-

14

39

85 68 3 tr

1 2 3

6 8 41 6

1 22 56 88

tr. 11

18 -

69 67

13 13

3 4

-

-

Content* 6

7

55

26

* As per cent of t o t a l s t e r o l : 1, c h o l e s t e r o l ; 2, desmosterol; 3, 24-methylene c h o l e s t e r o l ; 4, f u c o s t e r o l ; 5, zymosterol; 6, f e c o s t e r o l ; 7, stigmasta-8, E-24(28)-dienol; 8, l a t h o s t e r o l ; 9, ergosta-7, 24(28)d i e n o l ; 10, stigmasta-7, E-24(28)-dienol; 11, 7-dehydrofucosterol, 12, lanosterol. kit

These cultures may have been contaminated y i e l d i n g ergosterol i n otherwise a non-sterol synthesizing fungus. See text for further querries.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

19.

NES

Sterols in the Growth and Reproduction of Oomycetes

10

11

12

Sterol Absent

1 15 11,15

7 1 10 4 Yes 11

-

7

25

Ref.

1

17 15 8 13 15 8 8 8 15_

Yes Yes Yes Yes Yes Yes ? **

1,20 8 9,18 8,19 19 19 21

Yes

16 14,15 10,14

Yes

16

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

309

310

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Table I I .

S t e r o l s i s o l a t e d from commercial sources

Sterol Content

Cholesterol

Tween 80

Soybean Lecithin (ICN)

37'

Soybean Lecithin (Sigma)

1

Noble Agar

1

99

Campesterol

16

22

15

tr.

Sitosterol

31

52

68

tr.

Stigmasterol

16

25

16

tr.

Ergosterol Unknown TotaV

13.4ug/100g

150ug/100g

212ug/100g

1 As per cent t o t a l s t e r o l ; t r - t r a c e ; dash i n d i c a t e s not u n p u b l i s h e d o b s e r v a t i o n from t h i s l a b o r a t o r y .

5ug/63g

detectable;

^ T o t a l r e p r e s e n t s the amount o f f r e e s t e r o l q u a n t i f i e d per u n i t m a t e r i a l e x t r a c t e d w i t h 5% a q . a c e t o n e . Q u a n t i f i c a t i o n was a s s e s s e d u s i n g GLC (3? SE and 3% 0V-17 packed columns) and RP-HPLC ( d e t e c t o r s e t a t 205 nm o r 282 nm). Tween 80 and y e a s t e x t r a c t were s a p o n i f i e d w i t h 10% m e t h a n o l i c KOH w h i l e V-8 j u i c e was c e n t r i f u g e d and the c l a r i ­ f i e d V - 8 j u i c e e x t r a c t e d w i t h d i e t h y l e t h e r ; V - 8 j u i c e and the o t h e r samples were chromatographed w i t h o u t p r i o r s a p o n i f i c a t i o n . 3 Confirmation of s t e r o l structure

was by GC-MS.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

19. NES

Difco Agar

311

Sterob in the Growth and Reproduction of Oomycetes

Yeast E x t r a c t (Difco)

99

Corn meal Agar (Difco)

P o t a t o Dextrose Agar (Difco)

V-8 Juice (Campbell)

54

71

1

7

10

13

—

21

12

41

—

17

7

45

tr.

—

tr. tr.

88 12 16ug/70g

100ïig/100g

22ug/90g

14ug/108g

664ug/143mL(4.6ug/ml)

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

FUCOSTEROL 24 · METHYLENE CHOLESTEROL DESMOSTEROL CHOLESTEROL DEHYDROPOLLINASTANOL

2,3 - OXIDOSQUALENE

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Sterols in the Growth and Reproduction of Oomycetes

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

314

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Figure 4. Two possible conformations of cycloartenol, (A) and (B); established conformation of l a n o s t e r o l , (C).

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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315

disassociate organisms with a photosynthetic (cycloartenol) and a non-photosynthetic (lanosterol) ancestry. We have recently determined by feeding and trapping experiments (Table I I I ) that cycloartenol i s not formed i n s i t u by the Oomycetes, although i n S. ferax (and presumbly related s t e r o l synthesizers) i t may be metabolized to the cyclopropyl 4-desmethyl sterol-dehydropollinastanol (8). We have interpreted the r e s u l t s to imply that Oomycetes are not of a l g a l o r i g i n . In l i g h t of the f u n c t i o n a l implications, we continued our conformational analysis of cyclo­ propyl s t e r o l s . We have deduced by PHR ( i n p a r t i c u l a r with nuclear Overhauser e f f e c t s ) that the conformational preference of cyclo­ artenol and other cyclopropyl s t e r o l s i n s o l u t i o n (35) i s c h a i r boat-boat-chair "right-handed" unfolded (conformer Β i n F i g . 4) which confirms the s o l i d state X-ray data (37.). This new information has permitted us to reassess the sequence of events i n the terminal stages of squalene-oxide c y c l i z a t i o n to cycloartenol (38-40) and the contributio function. With regards suggested that the bent conformer e x i s t s i n membrane systems. However, i t seems u n l i k e l y that a membrane which has physiochemical properties intermediate between pure s o l u t i o n and s o l i d state would induce cyclopropyl s t e r o l s to adopt a d i f f e r e n t conformer than chair-boat-boat (A,B,C). A l t e r n a t i v e l y , i t seems that the cyclopropyl group i t s e l f , without concurrently having the c y c l o s t e r o l s adopt an e n e r g e t i c a l l y unfavorable bent conformer, can perturb the membrane b i l a y e r i n a fashion s i m i l a r to that shown f o r the cyclopropyl group introduced into f a t t y acids (41). We (11) and Berg, et a l . (42), have studied the sequencing i n stage I I I . A d d i t i o n a l l y , we (unpublished observation) have deter­ mined the mechanism of C-24 t r a n s a l k y l a t i o n (Fig 5). The r e s u l t s are as follows: the substrate i n s i t u f o r C-24 a l k y l a t i o n i s zymosterol, rather than l a n o s t e r o l , analogous to some higher-fungi, eg., yeast (43), but not others, eg., Gibberella f u j i k u r o i (44,45) ; by incubation with i n h i b i t o r s and labeled substrates we have shown that reduction of the (zymosterol) C-24 bond, leading to c h o l e s t e r o l 8 7 formation, was found to preceed the Δ -» Δ isomerization step. 8(9) Feeding and trapping experiments have confirmed the steps: Δ -> Δ^ -» Δ ^ ^ -» Δ^. S i m i l a r s t e r o l i c enzyme systems have been shown f o r P. cactorum (Nes, unpublished and (46)) i n d i c a t i n g that P. cactorum presumably possessed a complete s t e r o l pathway at one point i n i t s evolutionay h i s t o r y , then l o s t i t (2). As shown i n F i g . 5, through incubations with 2 4 - t r i t i o l a n o s t e r o l , the mechanism by which S. ferax a l k y l a t e s the A - b o n d i s by a 1, 2-hydride s h i f t analogous to yeast (47) and G. fu.iikuroi (Le and Nes unpublished observations). Since the second a l k y l a t i o n can be completely blocked by a carbonium ion high energy intermediate enzyme blocker (48), i . e . , 25-azacholesterol, i n addition to the loss of t r i t i u m at C-25 following iodine isomerization, i t i s assumed the f i r s t and second a l k y l a t i o n s proceed through a s i m i l a r mechanism. Presumably the other Oomycetes which synthesize these same s t e r o l s biosynthesize them i n an analogous manner. Stage IV has been studied p r i m a r i l y by McMorris. His group has over a period of 20 years i d e n t i f i e d the various male and female 2 4 ( 2 5 )

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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ECOLOGY AND METABOLISM OF PLANT LIPIDS

Table I I I . R a d i o l a b e l l i n g feeds t o Saprolegnia ferax

Substrates

Starting specific activity (DPMAiG/50 mL Medium)

4

3

Incorporation 1 C o r H I n t o S t e r o l M i x t u r e (DPM)

[°HlCholesterol 3

2.1 χ 10 /500uG

1,.0 χ Ι Ο

3

1.0 χ 1 0 / 4 0 0 μ 6

4..0 χ ί ο

5

1 4

2.0 χ 1 0 / 2 5 0 μ 6

1..0 χ ί ο

5

1 4

3.1 χ 10 /205^G

1..3 χ Ί 0

8.0 χ 1 0 / 5 0 0 μ 6

1..1 χ Ί Ο

2.5 χ 1 0 / 5 0 0 μ β

8..5 χ ί ο

[ H]Desmosterol

6

[ H124-Methylene

6

6

cholesterol [ C]Zymosterol

5

[ ClFecosterol 3

[ H]Lanosterol

5

2

6

3

[ HlCycloartenol

2

6

3

[ HlDihydro-

5

3

3

6

3..9 χ Ί Ο

3

1.0 χ 10 /500uG

7

3,.5 χ Ί Ο

6

8.3 χ 1 0 / 4 5 0 μ ΰ 5

7..1 χ Ί Ο

3

7

1..5 χ Ι Ο

5

4.2 χ 1 0 / 6 0 0 μ 6

lanosterol 3

[ HlDehydropollinastanol 1 4

[ C]S0 + 3

Tridemorph 1 4

[ ClAcetate

2

1.4 χ

10

1. The l a b e l was introduced i n t o the s t e r o l by base-catalyzed exchange with H 0, or by incubating the GL-7 yeast s t e r o l mutant or S. ferax with [ acetate i n the presence and absence of tridemorph. 2. R e c r y s t a l l i z e d products to constant s p e c i f i c a c t i v i t y . 3· Accumulated A - s t e r o l s ; s t r u c t u r e s confirmed by MS and PMR SO = squalene oxide. 3

2

8

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

19.

Sterob in the Growth and

NES

Reproduction ofOomycetes

R a t i o of a c t i v i t y i n HPLC p u r i f i e d end Cholesterol

1.0

X

10

4

Desmosterol

8.0

0 0.1

X

X

10

3

1.1

3.8

5

X

10

3

0 10

3

3.2

0 0.9

10

0

0 2.0

X

X

10

3

24-Methylene cholesterol

317

products

Fucosterol

2.5

X

10

3

1.5

X

10

2

2.6

X

10

5

0.6

X

10

3

0.8

X

10

3

0.9

X

10

3

1.8

X

10

4

0.8

X

10

3

4.1

X

10

3

1.6

X

10

4

Dehydropollinastanol

— — — — —

11 11 11 11 11

0

0

0

05. χ 1 0

0

0

0

—

0

0

0

0

2.8 χ 1 0

0

0

0

0

X

X

10

10

3

3

1.0

X

10

4

1.2

X

10

4

3.4

X

3

10 10

6

— !0

Ref.

4

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

11 10 10,

318

ECOLOGY AND METABOLISM OF PLANT LIPIDS

gametangia inducing factors (GIF) from Achlya (4). GIF are s t e r o i d hormones b i o s y n t h e t i c a l l y derived from fucosterol (4). Our a b i l i t y to block fucosterol metabolism i n S. ferax with 25-azacholesterol, thereby i n h i b i t i n g sporulation (unpublised data), leads us t o conclude S. ferax synthesizes s i m i l a r s t e r o i d (GIF) hormones. S t e r o l Functions. The c l a s s i c a l view, reviewed i n r e f . 32,49. f o r how s t e r o l s regulate m i c r o b i a l growth envisages an asymmetric s u b c e l l u l a r d i s t r i b u t i o n of s t e r o l aligned with f a t t y acyl chains i n d i s c r e t e domains along the plane of the monolayer such that changes i n s t e r o l l e v e l s w i t h i n l o c a l i z e d areas i n each of the b i l a y e r halves should s u f f i c i e n t l y perturb the physicochemical properties ( g e l ^ l i q u i d phase t r a n s i t i o n s ) of the membrane l i p i d core to a l t e r i t s permea­ b i l i t y , thereby stimulating, as a cascading response to the i n f l u x of n u t r i e n t s , the growth dynamics. Bloch (32) has referred to the accumulation of s t e r o l i support of a bulk r o l e i the plasmalemma (32.49) and the s t e r o l concentration dependence, ca 22-23 mol % r e l a t i v e to f a t t y acids, noted i n studies designed to measure sterol-induced physicochemical and permeability properties of n a t u r a l and a r t i f i c i a l membrane systems (32.49). The question which we have posed f o r Oomycetes physiology i s what i s the developmental s i g n i f i c a n c e of the bulk r o l e and what s t r u c t u r a l features of the s t e r o l are e s s e n t i a l f o r bulk a c t i v i t y . As we examined t h i s problem, we found that s t e r o l s possessed a d d i t i o n a l non-metabolic roles each of which expressed i t s e l f with development (Fig. 6). Our i n i t i a l s t r u c t u r e - a c t i v i t y studies were not designed to reveal bulk importance but to discriminate the s p e c i f i c t y f o r molecular groups (3B -OH, length of side chain, etc) i n s t e r o l controlled growth and reproduction . We found ( F i g . 7_) that a v a r i e t y of s t e r o l s and " s t e r o l - l i k e " compounds, i e . , t r i t e r p e n o i d s , added to the cultures a t lOppm stimulate growth (1.6.7.24). although only s p e c i f i c s t e r o l s induced oospore production (1.50-53). The lack of a c t i v i t y was not due to uptake per se (2,6). The s p e c i f i c i t y f o r Δ - s t e r o l s i n reproduction coupled with the well-described s t e r o i d hormones of Achlya, derived b i o s y n t h e t i ­ c a l l y from a Δ - s t e r o l , seemingly indicated that P. cactorum metabolized the dietary s t e r o l s to analogous GIF. However, our recent s t e r o l structure-oospore production studies (1,50,53) with P. cactorum f a i l to show s t r u c t u r a l homology f o r GIF of Achlya with those produced by Phytophtora. Had the hormones been s i m i l a r then ( i n d i c a t i n g sine qua non, s i m i l a r metabolites), f o r example, 20-epicholesterol should have f a i l e d to induce Phytophthora oogonia which i t did not. While the sexual hormones f o r Achlya and Phytophthora appear now to be d i f f e r e n t , i t remains unknown whether there i s any s i m i l a r i t y i n s t e r o l s t r u c t u r a l features governing growth. To answer t h i s question we have recently repeated a series of experiments designed to study the influence of s t e r o l s on growth of P. cactorum cultured on a s t e r o l - f r e e media. The amount of s t e r o l required to "spark" (a euphemism coined by Parks, 55) and stimulate maximal growth was found to be very small - 0.5 ppm, analogous to the s t e r o l auxotrophs of yeast (54,55). This l e v e l , we 5

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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BLOCK

319

ι -

BIOSYNTHESIS CHEMICAL SYNTHESIS

Figure 5. Mechanism o interference by 25-azacholesterol was induced i n the presence of iodine to confirm biochemical 1,2-hydride transfer. STATIONARY

VEGETATIVE GROWTH

TIME DECREASE DUE TO FERTILIZATION, MATURATION

SEXUAL STRUCTURE INDUCTION

SEXUAL SPORE FORMATION

SPORE GERMINATION

s

3

TIME

Figure 6. Sequence of developmental events i n the fungal l i f e cycle which may be c o n t r o l l e d by s t e r o l s . In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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4) STEREOCHEMISTRY AND EXTENT OF ALKYLATION AT C - 2 4

3) CONFORMATION OF SIDE CHAIN 1) STEREOCHEMISTRY AT C-20

5) BRANCH AT C-26(27) 2) BRANCH AT C-21 R= CH C H 3

™ D R Y

m

HYPHAL EXENSION

2

OOGONIA INDUCTION

5

OOSPORE PRODUCTION

1)

++

++

Ο

++

2) 3)

Ο ++

Ο ++

Ο Ο

Ο + +

4) 5)

Ο Ο

++ Ο

Ο Ο

+ + Ο

6)

+-»-

++

Ο

++

+ + VERY SIGNIFICANT; Ο, NOT SIGNIFICANT Figure 7. Effect of s t e r o l s t r u c t u r a l features on b i o l o g i c a l properties o f P. caetorum.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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and others had previously found, f a i l s to produce oogonia or oospores when the media i s supplemented with agar (2,3,5 and r e f . c i t e d therein). In the more recent studies, we found that the fungus w i l l respond to the sterol-mediated "spark" p r i m a r i l y at an early stage-specific phase i n the growth cycle i e . , the lag to log t r a n s i t i o n period (Fig. 8), a c e l l cycle mediated process; addition of s t e r o l p r i o r or post to t h i s developmentally predisposed event (note the growth c h a r a c t e r i s t i c s of control) produced s i g n i f i c a n t l y 3 less of a stimulatory influence, even though s t e r o l ( Η-labelled) i s a c t i v e l y accumulated by the mycelia throughout logarithmic growth. That trace levels of s t e r o l s ( i n some organisms a s t r u c t u r a l l y s i m i l a r s t e r o l may be f u n c t i o n a l l y equivalent although the amount of the same s t e r o l f o r induction of a c t i v i t y may be d i f f e r e n t at a l a t e r point i n the growth cycle) and s t e r o l - l i k e compounds may regulate temporal changes i n growth rate indicates gene regulation of developmental protein by something other than sterol-induce physicochemical ( f l u i d i t y ) properties. Perhaps the m i t o t i c index and growth-stimulation (a manifestation of increased nucleation, c e l l d i v i s i o n , hyphal elongation and branching) i s associated with s t e r o l (and " s t e r o l - l i k e " compounds)-protein interactions (the so-called bridge model, proposed by Nes and Heftman 49, F i g . 9). The association of some bridged s t e r o l s might act to control vegetative development by signaling to the fungus that large masses of s t e r o l s w i l l subsequently be made a v a i l a b l e f o r i n s e r t i o n into the membrane to play the bulk r o l e . Other bridged s t e r o l s may be an e s s e n t i a l binding factor which affects the topology of the protein strands and without i t or a suitable replacement molecule produces a diminished response u l t i m a t e l y impacting on l i f e cycle events. The p h y s i o l o g i c a l importance of the bulk r o l e appears to be linked to the development of the hyphal growth u n i t (56). More s p e c i f i c a l l y , the bulk r o l e appears to be a component of reproductive f i t n e s s which q u a n t i t a t i v e l y controls gametangial induction. In support of t h i s view, we have observed that, i n contrast to the b e n e f i c i a l a f f e c t s of cholesterol on the morphogenesis of Phytophthora mycelia (Fig. 10), 20-epicholesterol induced aberrant mycelia. The deleterious morphogenesis was evidenced i n an altered volumetric and s p a t i a l d i s t r i b u t i o n of the hyphal branches (Fig. 11), which presumably produced the hyphal t i p fragmentation and aborted oospores observed i n Fig. 11 (1.50.53). When the hyphal growth u n i t f a i l s to assume the appropriate s i z e f o r gametangial i n i t i a t i o n , which has also been observed i n the t r i t e r p e n o i d treatments, the mycelia remain i n the vegetative mode. There are reasons to believe (57.58) that calcium (5.7), c y c l i c nucleotide metabolism (58) and perhaps f a t t y acid levels (58), i n addition to s t e r o l s , a f f e c t the hyphal growth u n i t and reproduction i n d i c a t i n g the p o t e n t i a l f o r a p h y s i o l o g i c a l interdependency with minimally these four biochemical components. Achlya and Saprolegnia may, l i k e P. cactorum. u t i l i z e s t e r o l s i n a sparking and bulk r o l e during vegetative growth. As a r e s u l t , each of the 4 end products may e x h i b i t nonequivalent functions. When 25-azacholesterol i s fed to S. ferax. (^-transferase a c t i v i t y i s blocked, desmosterol accumulates and oosporogenesis i s prevented. Presumably, cholesterol i s a sparking agent,

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CONTROL

Dry Weight ^

4 6 8 10 1214 16 1820 22 24 26 |

Time (days)

Days — · > 7)4.05

11)103 96 14)-

18)— 21)—

248 85

278 158 297 310

25) 350 371 381 353 343 F i g u r e 8. Growth c u r v e ( l i q u i d media; 50 ml i n 250 ml f l a s k s ) o f P h y t o p h t h o r a cactorum supplemented w i t h 0.5 ppm o f c h o l e s t e r o l a t d i f f e r e n t times f o l l o w i n g i n o c u l a t i o n and a c o n t r o l experiment s e t - u p t o g e t h e r w i t h t h e c h o l e s t e r o l t r e a t m e n t . C u l t u r e s were i n c u b a t e d a t 20°C i n t h e d a r k and seeded w i t h a m y c e l i a l s u s p e n s i o n p r e v i o u s l y grown i n a s t e r o l - f r e e l i q u i d media. The d r y w e i g h t s ranged by as much as 20 mg i n mid t o l a t e l o g phase growth and 10 mg i n e a r l y l o g and s t a t i o n a r y phase growth.

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Figure 10. Micrographs of cultures of P^ cactorum supplemented with 10 ppm c h o l e s t e r o l were obtained with a scanning electron (top; also note lower left-hand corner with a d i f f e r e n t magnification) and l i g h t (bottom and top r i g h t i n s e r t ) microscope.

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Figure 11. Micrographs of cultures of cactorum supplemented with 10 ppm 20-epicholesterol were obtained with a scanning electron (top) and l i g h t (bottom and top r i g h t i n s e r t ) microscope.

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24~methylenecholesterol and desmosterol play the bulk r o l e and f u c o s t e r o l has no apparent vegetative r o l e , although i t i s synthesized to play another r o l e i e . , as the metabolic precursor of the s t e r o i d hormones Which regulate gametangia induction. In conclusion, the hyphal growth u n i t and other vegetative c h a r a c t e r i s t i c s i e . , growth rate and stationary phase population density, are influenced by s t e r o l s and " s t e r o l - l i k e " molecules. The s t r u c t u r a l features and levels of s t e r o l s f o r sparking may be more s p e c i f i c from those used f o r bulk a c t i v i t y . Apparently, f o r a l l the Oomycetes a c r i t i c a l mass of non-select s t e r o l s (bulk r o l e ) must accumulate p r i o r to growth arrest i n order that the hyphal growth u n i t assume a vegetative habit capable of producing and responding to GIF hormones. Cholesterol and f u c o s t e r o l accumulated by P. cactorum appear to t r i g g e r sexual reproduction without a d d i t i o n a l metabolism. The s e l e c t i v e advantage, however, f o r some but not a i l Oomycetes e.g. Saprolegnia and Achlya to synthesize and then metabolize f u c o s t e r o l t i s not c l e a r . Nevertheless s t e r o l structure-function changes during the vegetative phase, thereby influencing reproduction. This implies that a p h y s i o l o g i c a l dependence e x i s t s f o r s t e r o l - c o n t r o l l e d developmental regulation. In terms of fungal evolution, the change i n s t e r o l p r o f i l e induced by e c o l o g i c a l , environmental or fungal host-mediated events may have been one of many i n t e r r e l a t e d forces which have acted v i a informational molecules i n the vegetative phase to d i r e c t the gene(s) governing sporulation. I f the l a t t e r i s so, then p h y s i o l o g i c a l adapation or the lack thereof to developmental modulation of s t e r o l biosynthesis and function may be a s i g n i f i c a n t contributing f a c t o r to evolutionary change and perhaps speciation. Literature Cited 1. Nes, W.D.; Stafford, A.E. Proc. Natl. Acad. Sci. 1983, 80, 3227. 2. Nes, W.D. in "Isopentenoids in Plants: Biochemistry and Function"; Eds. Nes, W.D.; Fuller, G.; Tsai, L . ; Marcel Dekker, 1984; pp. 267. 3. Elliott, C.G. Adv. Microbiol Physiol. 1977, 15, 121. 4. McMorris, T.C. Lipids 1978, 13, 716. 5. Hendrix, J.W. Ann. Rev. Phytopathol. 1970, 8, 111. 6. Nes, W.D.; Saunders, G.A.; Heftmann, E. Lipids 1982, 17, 178. 7. Nes, W.D.; Patterson, G.W. J. Nat. Prod. 1981, 44, 215. 8. McCorkindale, N.J.; Hutchinson, S.A.; Pursey, B.A.; Scott, W.T.; Wheeler, R. Phytochemistry 1969, 8, 861. 9. Gottlieb, D.; Knaus, R.J.; Wood, S.G. Phytopathology 1978, 68, 1168. 10. Nes, W.D.; Le, P.H.; Berg, L . ; Patterson, G.W.; Kerwin, J.K. Experientia 1985, in press. 11. Le, P.H.; Nes, W.D.; Parish, E.J. J . Amer. Oil Chem. Soc. 1984, 19, 544 (A). 12. Warner, S.; Sovocool, G.; Domnas, A. Mycologia 1983, 75, 285. 13. Warner, S.; Eirman, D.; Sovocool, G.; Domnas, A. Proc. Natl. Acad. Sci. 1982, 79, 3769. 14. Warner, S.; Domnas, A. Experimentl. Mycol. 1981, 5, 184. 15. Berg, L.R. Ph.D. Thesis 1982, Univ. Md.

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Ludwig-Kohn, H.; Jahnke, K.D.; Bahnweg, G. Biochim. Biophys. Acta. 1982, 713, 463. Popplestone, C.R.; Unrau, A.M. Phytochemistry 1973, 12, 1131. Wood, S.G.; Gottlieb, D. Biochem. J . 1978, 170. 355. Schlosser, E . ; Shaw, P.D.; Gottlieb, D. Arch. Mikrobiol. 1969, 66, 147. Richards, J.B. and Hemming, F.W. Biochem. J . 1972, 128, 1345. Warner, S.A.; Sovocool, G.W.; Domnas, A. Phytochemistry 1982, 21, 2135. Fryberg, M.; Oehlschlager, A.C.; Unrau, A.M. Arch. Biochem. Biophys. 1975, 173,171. Ko, W.H. J . Gen. Microbiol. 1985, 131, 2591. Nes, W.D.; Patterson, G.W.; Bean, G.A. Lipids 1979. 14, 458. Hendrix, J.W.; Apple, J.L. 1964 Phytopathology 54. 987. Hendrix, J.W.; Norman, C.; Apple, J.L. Physiol. Plant 1966, 19, 159. Hohl, H.R. Phytopathog Bahnweg, G. Botanic Ko, W.; Ho, W. Ann Phytopathol. Soc. Japan 1983, 49, 316. Zakai, A.I.; Zentmeyer, G.A.; Sims, J . J . ; Keen, N.T. Phyto­ pathology 1983, 73, 199. Ricci, P.; Benveniste, P.; Bladocha, M. C.R. Acad. Sc. Paris 1985, 300, 119. Bloch, K.E.; CRC Crit. Rev. Biochem. 1983. 14, 47. Nes, W.R.; Varkey. T.E.; Krevitz, K. J . Amer. Chem Soc. 1977, 99, 260. van Tamelen, E.E. J . Amer. Chem. Soc. 1982, 104, 6480. Nes, W.D.; Benson, M. Le, P.H. J . Amer. Chem. Soc. Manuscript submitted. Bu'Lock, J.D.; Osagie, A.U. Phytochemistry 1976, 15, 1249. Ducruix, Α.; Pasaard-Billy, C.; Devys, M.; Barbier, M.; Lederer, Ε.; J . Chem. Soc. Chem. Commun. 1973, 929. Heintz, R.; Benveniste, P. J . Biol. Chem. 1974, 249, 4267. Goad, L . J . ; in "Lipids and Lipid Polymers in higher Plants"; Eds., Tevini, M.; Lichtenthaler, H.K.; Springer-Verlag, 1976, 146. Rees, H.H.; Goad, L . J . ; Goodwin, T.W. Biochem. J . 1968, 107, 417. Silbert, D.F.; Ruch, F.; Vagelos, P.R. J . Bacteriol. 1968, 95, 1658. Berg, L.R.; Patterson, G.W.; Lusby, W.R. Lipids 1983, 18, 448. Fryberg, M.; Oehlschlager, A.C.; Unrau, A.M. J. Amer. Chem. Soc. 1873, 95, 5747. Nes, W.D.; Heupel, R.C. Arch. Biochem. Biophys. 1986, 244, 211. Nes, W.R.; Heupel, R.C.; Le, P.H. J . Chem. Soc. Chem. Commun. 1985, 1431. Knights, B.A.; Elliott, C.G. Biochim. Biophys. Acta 1976, 441, 341. Akhtar, M.; Hunt, P.F.; Parvez, M.A. Biochem. J . 1968, 103, 616. Rahier, Α.; Genot, J.; Schuber, F . ; Benveniste, P.; Narula, A.S. J. Biol. Chem. 1984, 259, 15215. Nes, W.D.; Stafford, A.E. Lipids 1984, 544. Nes, W.D.; Heftmann, E. J . Nat. Prod. 1981, 44, 377. Nes, W.D.; Patterson, G.W.; Bean, G.A. Lipids 1979, 14, 458.

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52. Nes, W.D.; Hanners, P.K.; Bean, G.A.; Patterson, G.W. Phytopathology 1981, 72, 447. 53. Nes, W.D.; Nes, W.R. Experientia 1983, 39, 276. 54. Pinto, W.J.; Luzano, R.; Sekula, B.C.; Nes, W.R. Biochem. Biophys. Res. Commun. 1983, 112, 47. 55. Rodriguez, R.J.; Low, C.; Bottema, D.K.; Parks, L.W. Biochim. Biophys. Acta 1985, 837, 336. 56. Trinci, A.P.J. J. Gen. Microbiol. 1974, 81, 225. 57. Kerwin, J.L.; Washino, R.K. Experimentl Mycol. 1984, 8, 215. 58. Kerwin, J.L.; Washino, R.K. Can. J. Microbiol, in Press. Received August 20, 1986

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Chapter 20

Fatty Acids and Fungal Development: Structure-Activity Relationships James L. Kerwin Department of Entomology, University of California, Davis, CA 95616

Free and esterified fatty acids and their metabolites regulate fungal morphogenesis in diverse and poorly understood ways. Through use of structure-function relationships, the physiological bases for morphological developmen directly investigated classification, e.g. saturated vs. unsaturated or preferential partitioning in fluid or gel phases, can be misleading due to the presence of segregated lipid domains in cell membranes or to selective metabolism. Fatty acid composition has been shown to regulate a variety of membrane transport processes, oxidative phosphorylation, and a number of transferases and other enzymes. Fungal growth requirements for fatty acids can vary among genera, species or even isolates of the same species. Sporulation and spore germination are also affected by fatty acid composition as are the interactions between fungal parasites and their plant or insect hosts. Preliminary investigations support a role for oxygenated fatty acid derivatives in fungal morphogenesis. Fatty acids, which are i n t e g r a l components of c e l l u l a r and organelle membranes, primary storage products and substrates f o r secondary metabolism, are involved i n the regulation of fungal morphogenesis. Changes i n f a t t y acid composition and metabolism associated with fungal growth and d i f f e r e n t i a t i o n (reviewed i n 1-6) have been extens i v e l y documented; although a l t e r a t i o n s i n f a t t y acid components have been associated with the development of fungi, s p e c i f i c structurefunction relationships have r a r e l y been established. Secondary metabolism of these compounds has also l a r g e l y been ignored, espec i a l l y concerning possible regulatory roles of e.g. oxidized f a t t y acids i n fungi. The following review focuses on studies which attempt to more p r e c i s e l y r e l a t e f a t t y acid structure to p h y s i o l o g i c a l and morphological development i n fungi, often i n terms of t h e i r physical properties. I n i t i a l observations are also presented concerning potent i a l regulatory roles f o r f a t t y acid cyclooxygenase and lipoxygenase products i n fungal systems. 0097-6156/87/0325-0329$06.00/0 © 1987 American Chemical Society

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Fatty Acid Structure Reviews of fungal f a t t y acid composition (5>7_>8) reveal that t h e i r primary constituents are 12- to 20-carbon chain length unbranched compounds, with even-numbered chains predominant. Both saturated and unsaturated compounds occur, with p a l m i t o l e i c (C-16:l), o l e i c (C-18.-1), l i n o l e i c (C-18:2) and l i n o l e n i c (C-18:3) acids the most common unsaturated moieties C5). As with most n a t u r a l l y occurring f a t t y acids (9), monounsaturated compounds usually contain a c i s o l e f i n i c bond and polyunsaturated acids have methylene-interrupted c i s double bonds. Although rare i n occurrence and subjected to l i m i t e d study, branched chains, hydroxy, oxo and epoxy acids are also synthesized (5). L i s t s of the structures of unusual f a t t y acids which can be employed i n structure a c t i v i t y studies are presented i n d e t a i l elsewhere (9-14). When discussing diverse f a t t y acid structures, i t i s convenient to group related compound t i o n of structure-functio i s to group compounds as being saturated or (poly) unsaturated; however, problems a r i s e i n instances where e.g. short chain saturated compounds and longer chain unsaturated acids have s i m i l a r e f f e c t s on membrane f l u i d i t y . An alternate approach involves grouping according to p r e f e r e n t i a l p a r t i t i o n i n g i n t o f l u i d or g e l phases (15,16), which separates cis-unsaturated f a t t y acids from saturated and trans-unsaturated compounds. In some developmental systems categorization may not be possible due to p r e f e r e n t i a l secondary metabolism of s p e c i f i c compounds, e.g. arachidonic acid, which subsequently regulates pivot a l events i n fungal morphogenesis. Quantitative s t r u c t u r e - a c t i v i t y studies using l i n e a r free energy-related parameters such as p a r t i t i o n c o e f f i c i e n t s , e l e c t r o n i c substituent constants and s t e r i c properties (17,18) would p r e c i s e l y define r e l a t i o n s h i p s among diverse f a t t y acids, but this approach has not been exploited i n fungal research. Physical Properties and Concepts A c y l carbon chains i n f u l l y saturated phospholipids occur i n extended a l l - t r a n s conformations below the phase t r a n s i t i o n temperature (19, 20). The presence of a s i n g l e c i s double bond disrupts the close packing of the a11-trans conformation and can profoundly a f f e c t physi c a l properties. The p o s i t i o n and configuration of double bonds a f f e c t basic c h a r a c t e r i s t i c s such as melting point, with lower melting points found f o r compounds i n c i s rather than trans configurations and i n f a t t y acids with double bonds near the center of the fatty acid chain (21-23). Carbon chain length, degree and p o s i t i o n of unsaturation and the presence of c y c l i c or oxygenated moieties a l l a f f e c t membrane f l u i d i t y which i n turn regulates a v a r i e t y of c e l l u l a r functions (24-26). Their e f f e c t s are mediated i n part by influencing the membrane l i p i d phase t r a n s i t i o n temperature (27-29) and related physical properties such as range and rate of a c y l chain motion; however, extreme changes i n l e v e l s of unsaturation often r e s u l t i n i n s i g n i f i c a n t changes i n f a t t y a c y l motional parameters i n both b i o l o g i c a l and model membrane systems (30). Recent observations on the properties of cyclopropane-containing membranes revealed that although the c y c l i c moiety perturbs membrane l i p i d packing, i t a c t u a l l y s t a b i l i z e s membrane structure by acting as a b a r r i e r to the

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propagation of perturbations (31). Unexpected r e s u l t s such as these emphasize the complex nature of lipid-mediated events, and care should be taken i n extrapolating properties exhibited by free f a t t y acids to e f f e c t s on b i o l o g i c a l systems. An added complication when attempting to i n t e r p r e t membrane-mediated phenomena i n terms of f a t t y a c y l composition i s the possible existence of l a t e r a l l y segregated l i p i d domains (32-35) which have been proposed to have both s t r u c t u r a l and f u n c t i o n a l s i g n i f i c a n c e . Previous discussion has emphasized membrane-mediated (phosphol i p i d ) phenomena; free f a t t y acids, which can act on membranes or have completely d i f f e r e n t modes of action, have also been used to perturb fungal morphogenesis. Although the same physical properties outlined above may regulate t h e i r e f f e c t s , s e l e c t i v e metabolism of a l i m i t e d number of s p e c i f i c compounds, e.g. by lipoxygenase enzymes, can often be of greater regulatory s i g n i f i c a n c e . This aspect of f a t t y acid metabolism i s presented i n greater d e t a i l i n appropriate sections. Membrane Transport Fungi have f a t t y acid c a r r i e r systems, often d i s p l a y i n g some degree of s p e c i f i c i t y e.g. f o r 12- and 14-C compounds vs. 16- and 18-C saturated or unsaturated f a t t y acids i n Saccharomyces uvarum and Saccharomyces l i p o l y t i c a (36). Exogenous f a t t y acids can be i n c o r porated or p a r t i t i o n e d i n t o membrane phospholipids and subsequently modify c a r r i e r systems f o r other classes of compounds. Short chain compounds, e.g. formate, acetate, butyrate, c a p r y l i c and caproic acids i n h i b i t phosphate (37) and thiamine (38) uptake by Saccharomyces cerevisiae, perhaps by blocking the use of energy from adenosine triphosphate. The k i n e t i c s of arginine uptake by anaerobically grown S* cerevisiae i s considerably a l t e r e d following incorporation of l i n o l e i c acid i n t o c e l l s when compared to o l e i c acid-enriched c e l l s (39). On the basis of comparative i n h i b i t i o n studies, i t was concluded that the a c t i v i t y of membrane-bound transport proteins was probably altered by the nature of surrounding f a t t y acids. Several related studies exploited d i f f e r i n g f a t t y acid compositions of thermophilic, mesophilic and psychrophilic species of Torul o p s i s (40) and several other species of yeast (41). Glucose and leucine uptake occurred at measurable rates only at temperatures at which the various fungi grew. The f a t t y acid unsaturation index was high for psychrophilic species, intermediate for mesophiles and low f o r thermo-tolerant yeasts, and t h i s was related to possible mediation of transport processes by membrane f l u i d i t y . Enzymic Regulation Fatty acid auxotrophs of j>. cerevisiae have been extensively u t i l i z e d to probe r e l a t i o n s h i p s between f a t t y a c i d unsaturation and enzymic a c t i v i t y . A major area of i n v e s t i g a t i o n has been e l u c i d a t i o n of the bases f o r loss of oxidative phosphorylation by yeast cultures characterized by less than ca. 20-30% unsaturated f a t t y acid (42-44). Much reduced or no a c t i v i t y i n mitochondrial membranes with highly saturated f a t t y a c y l compositions has been documented f o r cytochromes a, a3, b and c, mitochondrial ATPase, succinate oxidase and NADH oxidase (45-47). Fatty acids apparently mediate many mitochondrial functions

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including the rate of p r o t e o l y s i s of mitochondrial t r a n s l a t i o n prod­ ucts (48), dihydrolipoate-induced ATP synthesis i n promitochondria (49), mitochondrial p r o l i f e r a t i o n (50) and release from glucose r e ­ pression (51). The Δ-9 isomer was the most e f f i c i e n t of the c i s octadecenoic acid compounds evaluated i n t h i s l a t t e r study, with eicosaenoic acid supporting 15% of the release induced by o l e i c a c i d . A l l of the l i s t e d processes were i n h i b i t e d by saturated f a t t y acids. L i n o l e n i c acid generally accelerates the a c t i v i t y of a number of enzymes i n anaerobic yeast cultures, including i s o c i t r a t e lyase, malate synthase and malate dehydrogenase (52), and s t e r o l synthesis (53). Linoleate and oleate had progressively less e f f e c t on synthesis of the three enzymes i n the former study (52). Oleic and l i n o l e i c acid supplementation at 0.01% i n anaerobic yeast cultures induced a 5- to 10-fold increase i n s t e r o l formation while e l a i d i c acid i n h i ­ bited induction (54). Stearic and p a l m i t i c acids suppressed anaerobic growth while l a u r i c acid had no e f f e c t on e i t h e r parameter. In a related i n v e s t i g a t i o n o abolished growth i n h i b i t i o saturated acids having no e f f e c t (55). In other instances unsaturated f a t t y acids can strongly repress S^. cerevisiae enzymic a c t i v i t y e.g. alcohol acetyltransferase (56), acyltransferases involved i n g l y c e r o l i p i d synthesis (57), f a t t y acid biosynthesis ( i n both cerevisiae and Candida l i p o l y t i c a , 58) and a c e t y l CoA carboxylase (59). Some enzymes are repressed by long chain f a t t y acids (57-59), while unsaturated compounds have s p e c i f i c e f f e c t s i n others (56). Defined mechanisms f o r these diverse respon­ ses are not c l e a r , although nonspecific surfactant action i s probably rare (57) and f a t t y acid metabolites mediate some repressive f a t t y acid e f f e c t s (59). Temperature Adaptation and Fatty Acid Composition The f a t t y acid composition of microorganism membranes can be a l t e r e d by varying temperature during growth, with the usual pattern being an increase i n short chain and/or unsaturated compounds with decreasing growth temperature (60-64). Membrane f l u i d i t y appears to be under regulatory c o n t r o l (65) since i t must be maintained at a l e v e l com­ p a t i b l e with c e l l growth and function (60,66,67). A number of fungi produce more highly unsaturated f a t t y acids when grown or adapted to grow at low temperatures (68); however, tem­ perature adaptation can involve increased synthesis of d i s t i n c t 18-C monounsaturated or diunsaturated compounds by Mucor mucedo and Asper­ g i l l u s ochraceous, respectively (69); s i g n i f i c a n t s h i f t s from s t e a r i c to p a l m i t i c acid by IS. cerevisiae (70); increasing amounts of palmit­ o l e i c a c i d s p e c i f i c a l l y without changing the t o t a l quantity of unsat­ urated f a t t y acids by the psychrophilic yeast Candida u t i l i s (71); enhancement of l i n o l e n i c acid at the expense of mono- and diunsat­ urated 18-C compounds by three species of Leucosporidium (72); or the synthesis of rare highly unsaturated compounds, e.g. Δ-6,9,12,15octadecetetraenoic acid by the Zygomycete Thamnidium elegans (73). A related study documented greater amounts of unsaturated f a t t y acids i n spores and mycelium of several species of thermophilic and thermotolerant mucoralean fungi when grown at 25°C rather than 48°C (74). These authors suggested that perhaps low dissolved oxygen i n growth media at high temperatures would support only reduced l e v e l s

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of f a t t y acid desaturase a c t i v i t y as had been proposed previously for other species of fungi (75,76). This may have occurred i n t h e i r ex­ perimental system; however, considering the wide range of fungal species examined under diverse c u l t u r a l conditions, i t i s u n l i k e l y that t h i s hypothesis can serve generally to explain r e l a t i o n s h i p s between unsaturation and temperature acclimation. Exceptions to cold temperature adaptation being accompanied by shorter chain or more unsaturated f a t t y acids include m y c e l i a l phos­ pholipids with greater unsaturation when grown at 36°C vs 20°C (77), and the lack of c o r r e l a t i o n of cold temperature acclimation with increased l i p i d unsaturation or membrane f l u i d i t y by four fungi representative of four d i f f e r e n t fungal classes (78). Many of these changes i n membrane f a t t y acid composition proba­ bly act by modulating enzymic a c t i v i t y by regulating l o c a l i z e d or bulk membrane f l u i d i t y ; however, since most studies have not measured rates or ranges of l i p i d or protein motion as a function of changes i n f a t t y acid composition and phospholipid composition hypothesis. An a d d i t i o n a l complication i s the possible presence of l a t e r a l l y segregated l i p i d domains which could have profound e f f e c t s on protein assembly or enzyme a c t i v i t y with minimal changes i n bulk membrane f l u i d i t y . Fatty Acids and Fungal Growth There has been extensive use of anaerobic cultures or desaturase mu­ tants of S_. cerevisiae to probe the unsaturated f a t t y a c i d growth requirements of t h i s yeast (79). A number of i n v e s t i g a t i o n s have attempted to formulate empirical rules r e l a t i n g f a t t y a c i d structure to t h e i r a b i l i t y to support growth. Using a desaturase mutant of IS. cerevisiae, 23 f a t t y acids were evaluated and only those possessing a c i s double bond at C-9 supported growth (80). This claim was d i s ­ puted using a d i f f e r e n t desaturase mutant of t h i s yeast i n which 16C to 22C f a t t y acids with double bonds i n positions M through Δ19 a l l supported growth (81). Arachidonic, eicosapentaenoic and docosahexaenoic acids had the highest e f f i c i e n c i e s ; i t was concluded that there was a d i r e c t c o r r e l a t i o n between greater number of double bonds and increased growth. A t h i r d study using a d i f f e r e n t desaturase mutant examined a number of cis-octadecenoate isomers and found a l l isomers between Δ7 and Δ12 able to support extensive growth (82). The Δ6 isomer was also e f f e c t i v e , which i s i n contrast to r e s u l t s from other studies (79,80) which showed no a c t i v i t y using t h i s compound. A more recent study using anaerobic cultures of J>. cerevisiae (79) concluded that those f a t t y acids which supported growth a l l had a maximum of 9 saturated C atoms before the occurrence of a c i s double bond or a hydroxyl group, or 7 saturated C atoms before the occurrence of a trans ethylenic bond. These rules are i n apparent general agreement with most published work although there are excep­ tions, e.g. the i n h i b i t o r y action of cis-All-octadecenoate re­ ported f o r one desaturase mutant (80) while supporting growth by S^. cerevisiae under anaerobic conditions (75,79). Requirements f o r s p e c i f i c f a t t y acids f o r S. cerevisiae growth can vary with s t e r o l structure (83) and i t i s probable that d i f f e r e n t desaturase mutants and d i f f e r e n t s t r a i n s of t h i s yeast grown anaerobically under d i f f e r ­ ent culture conditions could also have varying f a t t y a c i d require-

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merits. There i s no compelling reason to assume d i f f e r e n t i s o l a t e s should have i d e n t i c a l metabolic requirements; t h i s alone i s s u f f i cient to explain occasional discrepancies i n the l i t e r a t u r e concerning unsaturated f a t t y acid needs. Saturated f a t t y acids are also required f o r growth by :S. cerev i s i a e (84,85), with a negative c o r r e l a t i o n of growth demonstrated for high l e v e l s of dodecenoic and tridecenoic acids and a p o s i t i v e c o r r e l a t i o n with the presence of m y r i s t i c , pentadecenoic and p a l m i t i c acids (86). Fatty acid supplementation a f f e c t s the growth of a number of fungi. Among the documented e f f e c t s are the promotion of growth of the crustacean parasite Haliphthoros milfordensis by o l e i c a c i d , t r i o l e i n and t r i p a l m i t o l e i n . Saturated f a t t y acids were not e f f e c t i v e while polyunsaturated f a t t y acids were i n h i b i t o r y (87). When s i x thermophilic fungi were grown at 45°C on various f a t t y acids, optimum y i e l d s were obtained for a l l species on m y r i s t i c , p a l m i t i c , s t e a r i c and o l e i c acids except fo grow on m y r i s t i c a c i d (88) arachidic acids were not u t i l i z e d by any of the fungi. Comparative studies of several i s o l a t e s of saprobic s k i n fungi provided d i r e c t evidence that d i f f e r e n t s t r a i n s of the same species can have diverse growth requirements as speculated previously concerning S_. c e r e v i s i a e . A l l three s t r a i n s of Pityrosporum ovale u t i l i z e d o l e i c acid, but i n addition one used m y r i s t i c acid only, one used l a u r i c and m y r i s t i c acids, and a t h i r d could use those two acids plus p a l m i t i c and s t e a r i c (89,90). A related species, Pityrosporum orbiculare, u t i l i z e d only m y r i s t o l e i c and p a l m i t o l e i c acids (90). It i s apparent from t h i s b r i e f o u t l i n e that fungi have diverse f a t t y acid requirements to sustain growth, r e f l e c t i n g t h e i r adaptation to a v a r i e t y of saprobic and p a r a s i t i c habits. E l u c i d a t i o n of the precise r o l e of s p e c i f i c f a t t y acids w i l l probably r e f l e c t some of the enzymic a c t i v i t i e s outlined above, and requires research directed towards s p e c i f i c morphogenetic controls before more than the simplest of generalizations can be made. Sporulation Changes i n d i s t r i b u t i o n and abundance of l i p i d s have been documented i n fungi undergoing asexual or sexual reproduction (91-93); however, r a r e l y have s p e c i f i c structure-function r e l a t i o n s h i p s been establ i s h e d . Recently an exogenous requirement f o r s p e c i f i c f a t t y acids has been documented f o r the induction and maturation of the sexual oospore stage of Lagenidium giganteum, a f a c u l t a t i v e parasite of mosquito larvae (94,95). Free f a t t y acids and t r i a c y l g l y c e r o l s cont a i n i n g saturated f a t t y acids from 14C to 20C i n length induce l i m i ted numbers of v i a b l e oospores i n l i q u i d c u l t u r e . P a l m i t o l e i c acid added to basal growth media containing appropriate s t e r o l s induced s i g n i f i c a n t oosporogenesis, while o l e i c acid was less e f f e c t i v e and l i n o l e i c and l i n o l e n i c acids were t o x i c at r e l a t i v e l y low concentrations; however, o l e i c and l i n o l e i c acids added as mono-, d i - or t r i a c y l g l y c e r o l s produced the maximum number of oospores. T r i l i n o l e n i n , arachidonic and 11,14,17-eicosatrienoic acids induced no or few oopores (95). Analysis of whole c e l l f a t t y acids confirmed that a l l exogenous f a t t y acids were taken up from the media, although saturated f a t t y acids were incorporated more slowly. Metabolism of the

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exogenous f a t t y acids occurred but to a l i m i t e d extent. Delayed addition of t r i o l e i n to developmentally synchronized cultures r e s u l ­ ted i n a gradual loss of oospore induction and maturation, which varied according to the concentration of added c h o l e s t e r o l (95). Exogenous f a t t y acids are incorporated by L. giganteum i n t o a l l major classes of l i p i d s including phospholipids (unpubl. observ.). Membrane-mediated metabolism may p a r t l y explain the p o s i t i v e e f f e c t of c e r t a i n unsaturated f a t t y acids on oosporogenesis, which would increase b i l a y e r f l u i d i t y since the major f a t t y acid synthesized by L^. giganteum on unsupplemented media i s p a l m i t i c acid (95). A regu­ l a t o r y r o l e i n oospore induction has been documented f o r c y c l i c nucleotides (96) and b i l a y e r f l u i d i t y i s known to modify the a c t i v i t y of adenylate cyclase (30). Neurospora crassa mutants characterized by an aberrant colony morphology and a corresponding c y c l i c AMP de­ f i c i e n c y reverted to normal c y c l i c nucleotide l e v e l s and colony morphology when l i n o l e i c , o r l i n o l e n i c or arachidonic acid was added to basal growth media (97) ated compounds had no e f f e c t cess i s operating i n the induction of L^. giganteum oosporogenesis. I t i s i n t e r e s t i n g to note that the Myxomycete Dictyostelium d i s c o i deum, whose morphogenesis i s also regulated by c y c l i c AMP, dramati­ c a l l y increases i t s c e l l u l a r unsaturated f a t t y acids, e s p e c i a l l y octadeca-5,11-dienoic a c i d , during i t s development from yeast to mature sporocarp (98). Arachidonic acid and i t s methyl, e t h y l and propyl esters a f f e c t the movements of the m u l t i c e l l u l a r migratory slug stage of t h i s organism at micromolar concentrations (99). Methyl esters of o l e i c , l i n o l e i c and l i n o l e n i c acids also disoriented slug phototaxis at 100-200 μΜ concentrations. An unspecified prostaglandin at 10 μΜ and 0.1 μΜ leukotriene B4 had no e f f e c t . Two other i s o l a t e s of L. giganteum (ATCC nos. 48336 and 48337) do not produce oospores i n v i t r o using the protocol b r i e f l y described above for the C a l i f o r n i a s t r a i n (ATCC no. 52675) of the fungus. The two former i s o l a t e s also r a r e l y produce oospores following i n f e c t i o n of laboratory-reared Culex t a r s a l i s mosquito larvae; however, large numbers of oospores are induced following i n f e c t i o n of an alternate l a r v a l host, Chaoborus astictopus, the Clear Lake gnat. They also produce oospores i n media supplemented with cod l i v e r o i l (unpubl. observ.). Preliminary i n v e s t i g a t i o n of t h i s phenomenon has implicated 5,8,11,14,17-eicosapentaenoic acid (EPA) as the s p e c i f i c compound responsible f o r oospore induction. EPA i s present i n low q u a n t i t i e s i n laboratory-reared 0· t a r s a l i s larvae, which are incapable of syn­ thesizing t h i s f a t t y acid and must obtain i t from t h e i r d i e t (R. Dadd, unpubl. observ.); however, i t i s present i n r e l a t i v e l y high concentrations i n cod l i v e r o i l and fourth i n s t a r diapausing C_. astictopus larvae (unpubl. observ.). This phenomenon i s discussed i n more d e t a i l below i n r e l a t i o n to lipoxygenase mediation of fungal morphogenesis. Spore Germination Regulation of spore germination by s p e c i f i c f a t t y acids has been documented f o r several fungi. Macroconidial germination by Microsporum gypseum i s stimulated by o l e i c , l i n o l e i c and arachidonic acids, while short chain saturated compounds were i n h i b i t o r y and longer chain f a t t y acids had no e f f e c t (100). An even more s p e c i f i c

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response has been demonstrated f o r asexual conidia produced by Ernyia v a r i a b i l i s , a parasite of small dipteran insects (101). Conidia (asexual spores) are f o r c i b l y discharged by the fungus and subse­ quently develop i n one of four ways: d i r e c t vegetative germination which leads to i n f e c t i o n of a p o t e n t i a l insect host; formation and discharge of a smaller secondary conidium with the same developmental p o s s i b i l i t i e s as primary conidia; mycelial growth dependent s o l e l y on endogenous reserves; or death. In the presence of a basal agar medium containing yeast extract, c h i t i n or chitosan, vegetative c o n i d i a l germination i s induced by o l e i c a c i d . Oleic acid on water agar alone resulted i n secondary sporulation or death of discharged c o n i d i a . P a l m i t o l e i c , l i n o l e i c and l i n o l e n i c acids were t o x i c to conidia over a wide range of concentrations, and an excess of o l e i c acid could mitigate these t o x i c e f f e c t s . P a l m i t o l e i c acid was unique among the f a t t y acids which k i l l e d conidia i n i t s promotion of mycelial growth subsequent to spore germination. Saturated f a t t y acids from 14C to 20C induced p r i m a r i l y secondar In a related study i minate vegetatively on the c u t i c u l a r surface of adults of the lesser housefly, Fannia c a n i c u l a r i s , and on basal media containing yeast extract plus l i p i d extracts of adult f l y c u t i c l e s . In contrast, conidia developed p r i m a r i l y i n t o secondary spores when discharged onto puparia of Έ_· c a n i c u l a r i s or basal media supplemented with puparial c u t i c u l a r l i p i d s . Although the r e l a t i v e free f a t t y acid compositions of the two d i f f e r e n t stages of housefly were nearly i d e n t i c a l , adult c u t i c l e s contained nearly f i v e times as much free f a t t y acid as puparial c u t i c u l a r surfaces. I t was concluded that the l i m i t a t i o n of host range of E_. v a r i a b i l i s to adult dipterans was due i n part to c h a r a c t e r i s t i c s of the f a t t y acids of t h i s order of i n ­ sects, i . e . s u f f i c i e n t o l e i c acid to induce spore germination; high l e v e l s of p a l m i t o l e i c acid to enhance mycelial growth; and r e l a t i v e l y low l e v e l s of i n h i b i t o r y polyunsaturated 18C acids (102). Subsequent research has shown that of several s t r a i n s of a re­ lated entomophthoralean fungus, Erynia radicans, i s o l a t e d from a diverse group of insects, only c e r t a i n s t r a i n s respond to o l e i c a c i d by vegetative c o n i d i a l germination, and t h i s can be related to host range (A. U z i e l , R. Kenneth and I . Ben-Ze'ev, pers. communication). I t appears, therefore, that c u t i c u l a r f a t t y acid composition may have a d e f i n i t i v e r o l e i n regulating host range by a number of entomopathogenic fungi. Host-pathogen i n t e r a c t i o n s In addition to the entomophthoralean fungi discussed above, other laboratories have documented a r o l e for f a t t y acids i n the regulation of host-parasite i n t e r a c t i o n s (103). Research on a number of physio­ l o g i c a l races of Phytophthora infestans, some of which i n f e c t c u l t i ­ vars of potato, Solanum tuberosum, has shown that both v i r u l e n t and a v i r u l e n t s t r a i n s of the fungus contain chemical e l i c i t o r s of hyper­ s e n s i t i v e resistance, including induction of sesquiterpenoid phytoa l e x i n accumulation (104). A f t e r a number of inconclusive reports, the e l i c i t o r s were i d e n t i f i e d as arachidonic (AA) and eicosapentaenoic (EPA) acids (105-107). Methyl esters of AA and EPA also e l i c t e d sesquiterpene accumulation (106) a f t e r a s l i g h t delay, while the propyl ester was much less active (103). A l l saturated f a t t y

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acids, 18C compounds and arachidonyl cyanide f a i l e d to induce hyper­ s e n s i t i v e responses (103,108). Among the e i c o s a t r i e n o i c compounds tested, the Δ11,14 and Δ11,14,17 isomers were weakly active while the Δ5,8,11 and Δ5,11,14 compounds were highly active (103,108). The Δ5 bond appears to be of importance f o r a c t i v i t y , perhaps due to enzymic substrate s p e c i f i c i t y , leading to a more metabolically active compound. Such enzymic modification could be due to l i p o x y ­ genase or cyclooxygenase a c t i v i t y , which i s discussed i n the f i n a l section of t h i s review. Regulation of fungal morphogenesis by lipoxygenase and cyclooxygenase products Using the P^. infestans-potato tuber system outlined above, e l i c i t o r induced accumulation of phytoalexins has been i n h i b i t e d by s a l i c y l hydroxaraic acid and d i s u l f i r a m (103,109,110); these compounds i n h i b i t both cyanide-resistant r e s p i r a t i o l a t t e r acting s p e c i f i c a l l 1,4-pentadiene system to form hydroperoxides (111). I t has been speculated without further data that lipoxygenase a c t i v i t y may be involved i n e l i c i t i n g the hypersensitive response i n potato tubers (103). Direct i m p l i c a t i o n of cyclooxygenase enzymes i n fungal morpho­ genesis has been obtained using the Oomycetes Achlya caroliniana, Achlya ambisexual!s and Saprolegnia p a r a s i t i c a . A s p i r i n and indomethacin, both cyclooxygenase i n h i b i t o r s , reduced or eliminated mycelial growth by these fungi, often inducing abnormal colony mor­ phology i n l i q u i d culture (112). Inhibited colonies i n the presence of 0.1 mM indomethacin, when allowed to grow f o r more than 10 days, assume normal colony morphology, but do not undergo sexual reproduc­ t i o n (oosporogenesis). Addition of prostaglandin F i ( P G F i ) at 2 μg/ml p a r t l y overcame indomethacin induced growth i n h i b i t i o n , while PGF2 and PGE had no e f f e c t . The authors suggested growth and oosporogenesis may be regulated by the i n t e r a c t i o n of a prosta­ glandin or PG-like substance produced v i a cyclooxygenase f a t t y acid metabolism with s t e r o i d or other hormones (112). Using a developmentally synchronized L. giganteum culture system, our laboratory has documented that stage-specific addition of l i p o x y ­ genase i n h i b i t o r s (nordihydroguaiaretic acid, esculetin, ornaphthol, propyl g a l l a t e , salicylhydroxamic acid), at concentrations which do not a f f e c t asexual reproduction, blocks the induction and maturation (gametangial fusion, meiosis, spore c e l l w a l l formation, v e s i c u l a r fusion w i t h i n the immature spore) of oospores by t h i s fungus. Cyclo­ oxygenase i n h i b i t i o n using ibuprofen and to a lesser extent indo­ methacin had comparable s p e c i f i c effects on oosporogenesis. The cyclooxygenase i n h i b i t o r s a s p i r i n , s a l i c y l i c acid and phenylbutazone, however, had minimal effect on L. giganteum oosporogenesis at concen­ trations less than those which had a deleterious effect on mycelium (unpubl. observ.). This does not preclude a r o l e f o r cyclooxygenase metabolism i n oosporogenesis by t h i s fungus since the L. giganteum enzymes may be r e l a t i v e l y i n s e n s i t i v e to i n h i b i t i o n by these com­ pounds. Also, the e f f e c t s of these i n h i b i t o r s on L. giganteum oxygenases, which may be d i f f e r e n t from those documented i n mammal­ ian systems, have not been investigated. Tentative confirmation by t h i n layer chromatography (114) has a

a

a

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been made for the production of both cyclooxygenase and lipoxygenase products by L . giganteum, with more of both types of fatty acid metabolites i s o l a t e d from the l i q u i d culture medium than from the my­ celium (unpubl. observ.). This implicates these compounds i n some type of i n t e r c e l l u l a r communication r o l e . An i n t r a c e l l u l a r regulatory r o l e i s also suggested for the lipoxygenase metabolites by the i n h i ­ b i t o r y effects on oospore maturation. The i n h i b i t o r y effects of nordihydroguaiaretic acid on oospore induction could be reversed using p a r t l y p u r i f i e d eicosanoid extracts from growth media (113). Further e l u c i d a t i o n of the nature and stage-specific synthesis of these prod­ ucts i s i n progress. Arachidonic a c i d , which i s synthesized by L . giganteum (95), and eicosapentaenoic acid, which i s not, are suitable substrates for cy­ clooxygenase and lipoxygenase. Since two strains of t h i s f a c u l t a t i v e parasite appear to i n i t i a t e oosporogenesis only i n the presence of exogenous EPA (unpubl. observ.), t h i s compound i s h i g h l y suspect as a p o t e n t i a l regulatory molecule ted metabolism to a more i s present, often as the p r i n c i p a l fatty acid, i n a number of Oomy cetes (103,107,112,114). Further evaluation of the r o l e of EPA i n sexual reproduction by t h i s p r i m i t i v e class of fungi i s warranted. Leukotrienes are a recently discovered class of lipoxygenase metabo­ l i t e s with highly potent b i o l o g i c a l effects (116-118). I t i s possible that these or related metabolites play a central regulatory role i n fungal morphogenesis. Lipoxygenase regulation of morphogenesis may not be l i m i t e d to the oomycetous fungi described above. As an extension of a previous hypothesis regarding possible fatty acid hydroperoxide involvement i n c o n i d i a l germination by the Zygomycete Erynia v a r i a b i l i s (101), a s t r a i n of Erynia delphacis, a related parasite of small adult d i p teran insects, was investigated. Conidia discharged by 15. delphacis, which germinate vegetatively on yeast extract supplemented with t r i ­ o l e i n , undergo nearly 100% secondary sporulation when 150 μΜ n o r d i hydroguaiaretic acid i s added to the germination medium (unpubl. observ.) These i n i t i a l observations suggest that lipoxygenase-mediated morphogenesis may be widespread among fungi. Research on fungal eicosanoids w i l l provide insights into t h e i r basic developmental biology and serve as useful models for the role of these metabolites i n mammalian systems. Acknowledgments Writing of t h i s review was supported i n part by USDA research grant CR 806771-02, RF-4148A, R. K. Washino, P r i n c i p a l Investigator. I thank Dr. R. Herman for the preprint of h i s cyclooxygenase a r t i c l e .

Literature Cited 1. 2. 3.

Madelin, M. F., Ed. "The Fungus Spore"; Colston Papers: Butterworths, London, 1966; Vol. 18, 338 pp. Hess, W. M.; Weber, D. J. In "Fungal Lipid Biochemistry"; Weete, J. D., Ed.; Plenum: New York, 1974; p. 358. Reisener, H. J. In "The Fungal Spore: Form and Function"; Weber, D. J.; Hess, W. M., Eds.; Wiley: New York, 1976; p. 165.

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103. Kúc, J . ; Preisig, C. Mycologia 1984, 76, 767. 104. Varns, J. L.; Currier, W. W.; Kúc, J. Phytopathology 1971, 61, 968. 105. Bloch, C. B.; Kúc, J. Phytopathology 1983, 73, 828. 106. Bostock, R. M.; Kúc, J. Α.; Laine, R. A. Science 1981, 212, 67. 107. Bostock, R. M.; Laine, R. Α.; Kúc, J. A. Pl. Physiol. (Lancaster) 1982, 70, 1417. 108. Preisig, C. L.; Kúc, J. Phytopathology 1983, 73, 831. 109. Theologis, A. J . ; Laties, G. G. Pl. Physiol. (Lancaster) 1978, 62, 232. 110. Stelzig, D. Α.; Allen, R. O.; Bhatia, K. Pl. Physiol. (Lancaster) 1983, 72, 746. 111. Vliegenthart, J. F. G.; Veldink, G. Α.; Verhagen, J . ; Slappendel, S.; Vernooy-Gerritsen. J. In "Biochemistry and Metabolism of Plant Lipids"; Wintermans, J. F. G. M.; Kuiper, P. J. C., Eds.; Elsevier: Amsterdam, 1982; p. 265. 112. Herman, R. P.; Herman 113. Kerwin, J. L.; Simmons Leukotrienes and Med. (in press). 114. Salmon, J. Α.; Flower, R. J. Meth. Enz. 1982, 86, 477. 115. Gellerman, J. L.; Schlenk, H. Biochim. Biophys. Acta 1979, 573, 23. 116. Chakrin, L. W.; Bailey, D. M., Eds.; "The Leukotrienes ­ -Chemistry and Biology"; Academic: New York, 1984. 117. Piper, P. J . , Ed.; "Leukotrienes and other Lipoxygenase Prod­ ucts"; Research Studies Press (John Wiley): New York, 1983. 118. Piper, P. J. Physiol. Rev. 1984, 64, 744. Received May 1, 1986

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 21

The Involvement of Membrane-Degrading Enzymes During Infection of Potato Leaves by Phytophthora infestans Robert A. Moreau Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Philadelphia, PA 19118

During the infectio thora infestan membrane components (chlorophyll, galactolipids, and phospholipids). We have identified an inducible phospholipase Β activity which is secreted by the fungus in culture and also occurs during the infection of potato leaves. Under conditions which result in maximal in­ duction of phospholipase activity in culture filtrates, comparable levels of galactolipase and triacylglycerol lipase are also observed. During the infection of potato leaves there was a large (>l4-fold) increase in total phospholipase activity. Since potato leaves also contain very high levels of phospholipase and galactolipase activities, experiments were conducted to elucidate the possible involvement of the lipolytic enzymes of the pathogen and the host during infection and resistance. Phytophthora infestans i s the fungal pathogen that causes late b l i g h t of potatoes. The I r i s h "potato famine" of the 1840's was caused by P. infestans. Even today, l a t e b l i g h t i s s t i l l the single most important disease of potatoes worldwide (1). With modern farming techniques approximately 10% of the world potato crop and 4% of the U.S. potato crop are l o s t i n the f i e l d to t h i s disease each year (2,3). In addition to causing these large losses i n the f i e l d , l a t e b l i g h t and the secondary b a c t e r i a l infections which often accompany i t cause comparable losses during the storage of tubers. Despite many years of intensive breeding research, no e x i s t i n g c u l t i v a r s of European or North American potatoes allow commercial c u l t i v a t i o n i n humid regions without fungicide p r o t e c t i o n (_1). At best, farmers can choose c u l t i v a r s with a moderate l e v e l of general resistance ( i . e . , Sebago) which are protected by fewer applications This chapter not subject to U.S. copyright. Published 1987, American Chemical Society

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of fungicide than are required by other c u l t i v a r s . Our laboratory i s interested i n studying disease resistance mechanisms at the subcellular l e v e l , e s p e c i a l l y at the membrane l e v e l . For our preliminary studies we have chosen to study the changes i n membrane l i p i d composition that occur during a susceptible host-pathogen i n t e r a c t i o n . We have attempted to correlate changes i n l i p i d composition with changes i n the l e v e l s of l i p o l y t i c enzymes of the host and pathogen separately and during i n f e c t i o n . This report w i l l summarize our recent studies (4-7) of the P. infestans-potato l e a f i n t e r a c t i o n and compare them with comparable studies of other host-pathogen i n t e r a c t i o n s . Changes i n Host U l t r a s t r u c t u r e and L i p i d Composition during Infection In the f i e l d , i n f e c t i o n of potato plants by P. infestans i s usually l o c a l i z e d i n the leaves surface, form an appressorium of the leaf. The recent work of Wilson and Coffey (8) indicates that d i r e c t penetration of epidermal c e l l s adjacent to stomatal c e l l s i s the most common mode of entry. Hyphae spread both i n t r a and i n t e r c e l l u l a r l y forming haustoria when host c e l l s are penetrated (9,10). In susceptible i n t e r a c t i o n s , host c e l l s contain "organelles the structure of which i s disorganized" (10) or "very disintegrated organelles" (9). In contrast, fungal organelles remain i n t a c t long a f t e r the surrounding host t i s s u e has been disrupted (10). In a r e s i s t a n t i n t e r a c t i o n the fungus a c t i v e l y i n f e c t s the leaves f o r the f i r s t 9-12 h but i s then subsequently k i l l e d by 24 h (9). Unfortunately, no histochemical or cytochemical studies of the involvement of l i p o l y t i c enzymes during the i n f e c t i o n of potato leaves by P. infestans have yet been reported. Histochemical and biochemical observations of potato tubers infected by P. infestans revealed elevated l e v e l s of esterase (measured with a-naphthol acetate) i n u n i d e n t i f i e d host organelles during i n f e c t i o n (11). Cytochemical techniques were used to i d e n t i f y l i p o l y t i c a c t i v i t y i n fungal and host c e l l walls during the i n f e c t i o n of lettuce (Lactuca sativa L.) cotyledons by Bremia lactucae (12). Very l i t t l e i s known about the changes that occur i n the composition of membrane l i p i d s during the i n f e c t i o n of plants by fungal pathogens. Three such studies have been reported (13-15) and deal with the i n f e c t i o n of susceptible leaves by rust species. In each case there was a rapid disappearance of chloroplast glycol i p i d s and phosphatidyl g l y c e r o l , and evidence f o r the presence of fungal phospholipids during l a t t e r stages of i n f e c t i o n . During the i n f e c t i o n of bean plants (Phaseolus v u l g a r i s ) by Uromyces phaseoli, there was an increase i n the proportion of unsaturated f a t t y acids and t h i s change was a t t r i b u t e d to fungal growth (16). In our preliminary studies of the i n f e c t i o n of susceptible potato leaves by P. infestans we also observed a s i m i l a r rapid degradation of g l y c o l i p i d s and phosphatidyl g l y c e r o l . However, upon further i n v e s t i g a t i o n we r e a l i z e d that the unique composition of membrane l i p i d s i n P. infestans may provide a useful marker during i n f e c t i o n . The polar l i p i d composition of healthy potato leaves and cultured P. infestans i s shown i n Table I . The l i p i d

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Infection of Pota to Leaves by Phytophthora infestans

MORE AU

Table I . Polar L i p i d Composition of Potato Leaves and P. infestans. Fungal cultures were grown i n l i q u i d French bean media (4) f o r 14 days at 14° without shaking. L i p i d s were extracted (42) separated by 2-dimensional TLC (18). I n d i v i d u a l spots were i d e n t i f i e d by comparison with standards, scraped from TLC p l a t e s , and analyzed f o r phosphorous (6) and hexose (43). mole % Lipid Class

3

Healthy Potato Leaf

MGDG DGDG SQD PC PG PE PI CAEP

P. infestans

53 20

0 0

3 6 1 0

0 39 2 13

Abbreviations: MGDG = monogalactosyldiglyceride, DGDG = d i g a l a c t o s y l d i g l y c e r i d e , SQD = sulfoquinovasyldiglyceride, PC = phosphatidylcholine, PG = phosphatidylglycerol, PE = phosphatidylethanolamine, PI = p h o s p h a t i d y l i n o s i t o l , CAEP = ceramide aminoethylphosphonate. composition of potato leaves i s comparable to that of the leaves of other species of angiosperme (17). P. infestans contains high l e v e l s of phosphatidylcholine and phosphatidylethanolamine, as i s common f o r fungi (13), but i t also contains an unusual sphingol i p i d , ceramide aminoethylphosphonate (CAEP) (Figure 1). This sphingolipid was previously i d e n t i f i e d i n two c l o s e l y related fungi Pythium prolatum and Phytophthora p a r a s i t i c a var. nicotianae (18). We have recently observed that a f t e r 6 days of i n f e c t i o n CAEP i s detectable i n t h i n layer chromatograms of l i p i d s from infected leaves. Since the fungal ceramide lacks carboxyl ester bonds i t i s r e s i s t a n t to hydrolysis by phospholipase Β and i t s presence may render c e r t a i n fungal membranes less vulnerable to degradation during i n f e c t i o n . P. infestans also contains high l e v e l s of two unusual f a t t y acids, arachidonic acid (20:4) and eicosapentaenoic acid (20:5) (19) which are absent i n the potato plant and could also be used as markers of fungal l i p i d s during i n f e c t i o n . We are currently studying the quantitative changes i n these membrane l i p i d components during i n f e c t i o n . Properties of Phospholipases

from Phytopathogens

Phospholipases are enzymes that catalyze the hydrolysis of membrane phospholipids. There are s i x types of phospholipases (A A , B, C, D, and lysophospholipase). Each hydrolyses a d i f f e r e n t part of the phospholipid molecule and results i n the formation of d i f f e r e n t lt

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products (Figure 2). Although some assay techniques are capable of i d e n t i f y i n g the type of phospholipase a c t i v i t y , several of the common techniques only measure t o t a l breakdown of phospholipid. Phospholipase a c t i v i t y has been reported to occur i n eleven phytopathogens (ten fungi and one bacteria) (4,16,20-27) (Table I I ) . In s i x of these species, phospholipase Β (which hydrolyzes two f a t t y acids per phospholipid molecule) was detected. Only three of these studies (20,21,22) have reported the e f f e c t of fungal phospholi­ pases on plant t i s s u e . In Table I I , i t i s noted whether these enzyme a c t i v i t i e s were detected i n fungal cultures ( i n t r a c e l l u l a r or e x t r a c e l l u l a r ) , or i n infected plant t i s s u e . The pH optimum for each enzyme i s also l i s t e d . Table I I . Occurrence of Phospholipase A c t i v i t y i n Phytopathogens

Species B o t r y t i s cinerea Erwinia carotovora

Β C

Erysiphe p i s i

A

Fusarium solani Phoma medicaginis Phytophthora infestans Rhizoctoni solani S c l e r o t i a sclerotiorum Sclerotium r o l f s i i Thielaviopsis basicola Uromyces phaseoli

2

?

Β Β ?

Β Β Β A or Β or C

5.0 Assayed only at 8.0 Assayed only at 8.9 4.0 ?

9.0 7.5 - 8.5 4.0 4.5 4.5 and 8.5 4.0 - 5.0

M Ε

21 22

I

20

Ε M Ε,I,Ρ Ε Ε,Ρ Ε,Ρ Μ,Ρ Ρ

23 24 4 23 25 26 27 16

Abbreviations: Ε = from e x t r a c e l l u l a r fungal culture; I = from i n t r a c e l l u l a r fungal culture; M = from mixture of i n t r a c e l l u l a r and e x t r a c e l l u l a r ; Ρ = from infected plant t i s s u e . The properties of the phospholipase a c t i v i t i e s from cultures of P. infestans (4) are summarized i n Table I I I . Of the f i v e types of l i q u i d media tested, the greatest phospholipase a c t i v i t y was obtained when P. infestans was grown i n rye steep media. The phospholipase a c t i v i t y i n rye culture f i l t r a t e s was stimulated 15-fold by omitting glucose from the medium. The addition of 100 mg/liter of phosphatidylcholine (PC) to the rye medium caused an a d d i t i o n a l 35-fold stimulation i n the a c t i v i t y of phospholipase. These experiments suggest that t h i s enzyme a c t i v i t y i s inducible. The addition of other l i p i d s (sunflower o i l , wax ester, and cholesterol oleate) to rye media also caused an apparent induction of phospholipase a c t i v i t y . We also detected e x t r a c e l l u l a r phospho­ lipase a c t i v i t y i n lima bean agar cultures of P. infestans as shown i n the second column of Table I I I . When the fungus was collected

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Infection of Potato Leaves by Phytophthora infestans

OH

Η

Η

I

I

I

c

H

S

NH

H

C—0

I

(ÇH ), 2

CH

2

(ÇH ) 2

CH

3

0 H H I

I

I

c—o—P—c—c—Nu

c

î

347

I

I

Η

0 H H

I

I

3

(-)

1 8

3

Figure 1. Ceramide aminoethylphosphonat from P. infestans the and the most common long chain base i s sphingosine.

PHOSPHOLIPASE A, PHOSPHOLIPASE Β PHOSPHOLIPASE A ->y

\

2

^

II

0 CH -0-C-R R-C-O-CH 0 " C H - 0 - Ρ - 0 - CH CH N ( CH ) 2

1

2

PHOSPHOLIPASE C

2

2

3

3

PHOSPHOLIPASE D

Figure 2. S i t e s of a c t i o n of various types of phospholipases on phosphatidylcholine. Lysophospholipase hydrolyzes the carboxyl ester bond of a lysophospholipid ( i . e . , lysophosphatidylcholine).

American Chemical Society Library 1155 16th St., N.W. Washington, D.C. 20036

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

348

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Table I I I . A Summary of the Properties of the Phospholipase A c t i v i t y from Cultures of P. infestans (4,7) Liquid Culture Media Regulation Localization pH optimum E f f e c t of 5 mM DTT E f f e c t of 5 mM EDTA E f f e c t of 5 mM CaCl Km f o r PC Substrate s p e c i f i c i t y

Agar Culture

Rye steep Repressed by glucose induced by PC Mostly e x t r a c e l l u l a r 9.0 96% I n h i b i t i o n None

Lima bean Not tested

TG - PC - MGDG

PC > TG

Extracellular 9.0 94% I n h i b i t i o n 55% I n h i b i t i o n

2

Abbreviations: PC = phosphatidylcholine, TG = t r i a c y l g l y c e r o l , MGDG = monogalactosyldiglyceride, DTT = d i t h i o t h r e i t o l , EDTA = ethylenediaminetetraacetic acid. from l i q u i d rye cultures (with added PC to induce phospholipase a c t i v i t y ) much more phospholipase a c t i v i t y (30-fold) was found e x t r a c e l l u l a r l y than i n t r a c e l l u l a r l y . The pH optimum of the enzyme a c t i v i t y from both sources was 9.0. D i t h i o t h r e i t o l severely i n h i b i t e d both enzyme a c t i v i t i e s . EDTA (5 mM) had no e f f e c t on the enzyme from l i q u i d rye c u l t u r e , but i n h i b i t e d the enzyme from agar cultures. Both enzymes were s l i g h t l y i n h i b i t e d by 5 mM CaCl . A very low Km (2.86 μΜ) f o r phosphatidylcholine was measured using induced rye culture f i l t r a t e as a source of enzyme. When l i p i d substrates other than PC were tested the enzymes i n the induced l i q u i d culture were able to hydrolyze t r i a c y l g l y c e r o l (TG) and g a l a c t o l i p i d s (GL) at rates very s i m i l a r to that f o r PC. In contrast, the enzymes from agar culture hydrolyzed PC at a rate about s i x times higher than f o r TG. We have recently completed a study of the production of extra­ c e l l u l a r enzymes by germinating cysts of P. infestans (7). Although we were able to i d e n t i f y an esterase a c t i v i t y (p-nitrophenyl butyrate hydrolase) that appeared to be secreted during germination (0 to 20 h ) , phospholipase and lipase a c t i v i t i e s were apparently not secreted (Table IV). We are currently studying whether t h i s esterase can hydrolyze any p h y s i o l o g i c a l substrates. This i s an example of a case where the use of a nonphysiological substrate (PNP-butyrate) resulted i n new and i n t e r e s t i n g information. However, extreme care must be exercised when t r y i n g to draw physio­ l o g i c a l conclusions from studies using nonphysiological substrates ( i . e . , p-nitrophenyl esters or 4-methylumbelliferyl e s t e r s ) . We are currently i n v e s t i g a t i n g a new phospholipase assay (28), which employs a fluorescent phospholipid substrate, l-acyl-2-[6-[(7nitro-2,1,3 benzoxadiazol-4*yl)amino]-caproyl] phosphatidyl­ choline (C -NBD-PC) . Our preliminary studies (Table IV) indicate 2

6

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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349

Infection of Potato Leaves by Phytophthora infestans

Table IV. Changes i n the l e v e l s of e x t r a c e l l u l a r enzyme a c t i v i t i e s during germination (0 to 20 h) of cysts of P. infestans. The l e v e l s of the f i r s t three enzymes were pre­ v i o u s l y reported (7). C,-NBD-PC hydrolysis was measured as described (28). Enzyme A c t i v i t y η mol/min/10 Spores 6

Enzyme

0 h

5 h

10 h

20 h

p-nitrophenyl butyrate hydrolase Lipase Phospholipase ( C-PC assay)

.590

.599

.604

.610

Phospholipase (C -NBD-PC assay)

.738

.735

.733

.732

14

6

that very s i m i l a r values are obtained when^phospholipase a c t i v i t y i s measured with authentic phospholipid ( C-PC) or the fluorescent phospholipid (C,-NBD-PC). I f i t proves r e l i a b l e , t h i s new fluorometric assay w i l l be much more convenient (30 assays per hour verses 15 assays per day using C-PC). 14

Properties of the Phospholipase A c t i v i t y i n Healthy Potato Leaves Many types of plant tissue contain high l e v e l s of l i p o l y t i c acyl hydrolase (LAH) a c t i v i t y (29). These enzymes are capable of hydrolyzing phospholipids (phospholipase Β), g a l a c t o l i p i d s (galact o l i p a s e ) , and acyl glycerols. The LAH s i n potato tubers and bean leaves have been p u r i f i e d and extensively studied (29). In 1979 Matsuda and Hirayama (30) reported that the t o t a l a c t i v i t y of phos­ pholipase i n potato leaves was about 400-fold lower than i n potato tubers. They subsequently p u r i f i e d a l i p o l y t i c acyl hydrolase from potato leaves (31). The enzyme had a molecular weight of about 110,000 and a pH optimum of 5.0. The rate of hydrolysis of galac­ t o l i p i d s was 7-fold higher than f o r phospholipids. The following experiments i l l u s t r a t e that when studying the involvement of phospholipase i n the host-pathogen i n t e r a c t i o n , the t o t a l contribution of enzyme of host o r i g i n may be considerably higher than previously r e a l i z e d . Rodionov and Zakharova (32) recently reported very high rates of a u t o l y t i c hydrolysis of mem­ brane l i p i d s i n homogenates of potato leaves (26-37% of the phospholipids were hydrolyzed a f t e r 2 h at 0-1°). Our laboratory recently confirmed t h i s observation and proceeded to study some of the properties of the l i p o l y t i c acyl hydrolase a c t i v i t y i n potato leaves (6). L i p o l y t i c acyl hydrolase a c t i v i t y i s apparently inactivated by polyphenol oxidase or i t s t o x i c quinone products. 1

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

350

ECOLOGY AND METABOLISM OF PLANT LIPIDS

When polyphenol oxidase a c t i v i t y was c o n t r o l l e d , phospholipase a c t i v i t i e s ranged from 1.04 to 11.60 μ mol/min/gfw i n the leaves of 41 North American c u l t i v a r s (6). These values are much higher than those previously reported f o r potato leaves (.009 pmol/min/gfw) (30) and nearly as high as i n potato tubers (2 to 30 μπιοΐ/min/gfw) (5,30). Change i n the Levels of L i p o l y t i c Enzymes during I n f e c t i o n Hoppe and Heitefuss (16) reported a 2-3-fold increase i n phospho­ lipase a c t i v i t y during the i n f e c t i o n of susceptible bean leaves by U. phaseoli. During the i n f e c t i o n of potato leaves by P. infestans we observed a greater than 14-fold increase i n t o t a l phospholipase a c t i v i t y (Figure 3) (4). The increase i n phospholipase a c t i v i t y was roughly proportional to the amount of leaf surface area that was covered by the fungus. When phospholipase a c t i v i t y was measured with the fluorescen described (28), i t c l o s e l C-PC. However, when esterase a c t i v i t y was measured with another fluorometric substrate, 4-methylumbelliferyl laurate, i t was highest at day 0 and decreased during i n f e c t i o n . This indicates that 4-methylumbelliferyl laurate appears to be hydrolyzed by a l i p o l y t i c enzyme of the host. To determine whether the increase i n phospholipase a c t i v i t y during i n f e c t i o n (Figure 3) was due to enzymes of fungal or host o r i g i n , the following experiments were performed. We observed that the pH optima of the phospholipase a c t i v i t y i n fungal cultures was about 9.0 (Figure 4A,B) (4). Phospholipase a c t i v i t y was present i n the uninfected leaves (Figure 4C), but i t was much lower and ex­ h i b i t e d optimum phospholipase a c t i v i t y at pH 6.0 as reported elsewhere (6). The infected l e a f had a large peak of phospholipase a c t i v i t y at pH 8.0 to 9.0 and a smaller peak a t pH 6.0. The two peaks of phospholipase a c t i v i t y i n the infected leaves were further resolved by assaying i n the presence of 5 mM DTT (Figure 4D) which i s a potent i n h i b i t o r of fungal phospholipase. The peak of phos­ pholipase a c t i v i t y a t pH 9.0 was DTT-sensitive and l i k e l y to be of fungal o r i g i n . The peak of phospholipase a c t i v i t y at pH 6.0 was DTT-insensitive and i s probably of host o r i g i n although further work i s required to prove t h i s beyond doubt. 14

Conclusions and Perspectives These results i n d i c a t e that during the i n f e c t i o n of potato leaves by P. infestans, there i s a rapid degradation of the host*s membrane l i p i d s ( e s p e c i a l l y g a l a c t o l i p i d s ) and a gradual increase i n l i p i d s of fungal o r i g i n (ceramide aminoethylphosphonate being the most unique). An analysis of phospholipase a c t i v i t y revealed that a DTT-sensitive phospholipase with an a l k a l i n e pH optima accumulated i n infected leaves and was probably of fungal o r i g i n . Although these preliminary studies i n d i c a t e that l i p o l y t i c enzymes are produced by the fungus during i n f e c t i o n , much more work i s required to determine t h e i r actual role i n i n f e c t i o n and patho­ genesis. Because the symptomology and biochemistry of i n f e c t i o n are very s i m i l a r to those events that occur during normal l e a f

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Infection of Potato Leaves by Phytophthora infestans

4 r

0

3 DAYS

AFTER

5

7

INOCULATION

Figure 3. Time-course study of t o t a l phospholipase a c t i v i t y during i n f e c t i o n of potato leaves by P. infestans. Phospholipase values were previously reported (4). C -NBD-PC hydrolysis assays were performed at pH 9.0 as described (28). Hydrolysis of 4-methylumbelliferyl laurate was measured by a fluorometric technique (44). 6

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

352

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Figure 4. E f f e c t of pH on t o t a l phospholipase a c t i v i t y i n : A. culture f i l t r a t e s of fungus grown on rye steep media + 10 mg PC for 7 days. B. Culture wash of fungus grown on lima bean agar for 14 days. C. Potato l e a f , uninfected or infected f o r 7 days. D. Infected potato leaf (7 days) assayed i n the presence of 5 mM d i t h i o r e i t o l (DTT-insensitive a c t i v i t y ) . DDT-sensitive a c t i v i t y was calculated by subtracting the phospholipase a c t i v i t y i n the presence of DDT from that measured i n the absence of DDT. Reproduced with permission from Ref. 4. Copyright 1984, Academic Press.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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senescence ( i . e . , breakdown of c h l o r o p h y l l , g a l a c t o l i p i d s , and p r o t e i n ) , the two processes need to be very c a r e f u l l y compared (33). Both disease and senescence cause increases i n membrane permeability (34,35). Recent work has shown that s i m i l a r increases i n the levels of free r a d i c a l s , lipoxygenase a c t i v i t y , and l i p i d peroxidation occur during i n f e c t i o n and senescence (36-38). Other studies indicate that under c e r t a i n conditions phospholipid d e e s t e r i f i c a t i o n can be mediated by superoxide ( 0 ) i n the absence of phospholipases (39). The most recent studies irom our laboratory suggest that some of the l i p o l y t i c enzymes i n potato leaves are regulated by calmodulin (40). Even i f produced, free f a t t y acids may not accumulate i n healthy or infected leaves because peroxisomes are capable of metabolizing f a t t y acids v i a β-oxidation (41). These factors a l l indicate that membrane l i p i d metabolism i n healthy and diseased leaves i s a dynamic process and any change i n membrane composition needs to be interpreted very carefully. 2

Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Thurston, H. D.; Schultz, O. In "Compendium of Potato Diseases"; Hooker, W. J., Ed.; American Phytopathological Society: St. Paul, 1981; p. 40. Currier W. W. Trends in Biochemical Science 1981, 6, 191. Bills, D. D. In "Host Plant Resistance to Pests"; Hedin, P. Α., Ed.; ACS SYMPOSIUM SERIES No. 62, American Chemical Society: Washington, DC, 1977, p. 47. Moreau, R. Α.; Rawa, D. Physiol. Plant Path. 1984, 24, 187. Moreau, R. A. J. Ag. Fd. Chem. 1985, 33, 36. Moreau, R. A. Phytochemistry 1985, 24, 411. Moreau, R. Α.; Seibles, T. S. Can. J. Bot. (in press). Wilson, U. E . ; Coffey, M. D. Ann. Bot. 1980, 45, 81. Shimony, C.; Friend, J. New Phytol. 1975, 74, 59. Coffey, M. D.; Wilson, U. A. Can. J. Bot. 1983, 61, 2669. Pitt, D.; Coombes, C. J. Gen. Microbiol. 1969, 56, 321. Duddridge, J . Α.; Sargent, J. A. Physiol. Plant Path. 1978, 12, 289. Hoppe, H. H.; Heitefuss, R. Physiol. Plant Path. 1974, 4, 11. Lösel, D. M. New Phytol. 1978, 80, 167. Lösel, D. Μ.,; Lewis, D. H. New Phytol. 1974, 73, 1157. Hoppe, Η. H.; Heitefuss, R. Physiol. Plant Path. 1974, 4, 25. Harwood, J. L. In "The Biochemistry of Plants: A Comprehen­ sive Treatise"; Stumpf, P. K.; Conn, Ε. E . , Eds.; Academic Press: New York, 1980; Vol. 4, p. 1. Wassef, M. K.; Hendrix, J. W. Biochem. Biophys. Acta 1977, 486, 172. Bostock, R. M.; Kuc, J . Α.; Laine, R. A. Science 1982, 22, 67. Faull, J. L . ; Gay, J. L. Physiol. Plant Path. 1983, 22, 55. Shepard, D. V.; Pitt, D. Phytochemistry 1976, 15, 1465. Tseng, T. C.; Mount, M. S. Phytopathology 1974, 64, 229. Tseng, T. C.; Bateman, D. F. Phytopathology 1968, 58, 1437.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38.

39. 40. 41. 42. 43. 44.

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Plumbley, R. Α.; Pitt, D. Physiol. Plant Path. 1979, 14, 313. Lumbsden, R. D. Phytopathology 1970, 60, 1106. Tseng, T. C.; Bateman, D. F. Phytopathology 1969, 59, 359. Lumbsden, R. D.; Bateman, D. F. Phytopathology 1968, 58, 219. Wittenaur, L. Α.; Shirai, K.; Jackson, R. L.; Johnson, J. D.; Biochem. Biophys. Res. Commun. 1984, 118, 894. Galliard, T. In "The Biochemistry of Plants: A Comprehensive Treatise"; Stumpf, P. K.; Conn, Ε. E., Eds.; Academic Press: New York, 1980; Vol. 4, p. 85. Matsuda, H.; Hirayama, O. Bull. Fac. Agric. Shimane Univ. 1979, 13, 105. Matsuda, H.; Hirayama, O. Biochim. Biophys. Acta 1979, 573, 155. Rodionov, V. S.; Zakharova, L. S. Soviet Plant Physiol. 1980, 27, 298. Novacky, A. In "Biochemical Plant Pathology"; Callow J. Α. Ed.; John Wiley an Wheeler, H. In "Plan Horsfall, J. G.; Cowling, Ε. B., Eds.; Academic Press: New York, 1978; Vol. 3, p. 327. Barber, R. F.; Thompson, J. E. J. Exp. Bot. 1980, 31, 1305. Lupu, R.; Grossman, S.; Cohen, Y. Physiol. Plant Pathol. 1980, 16, 241. Dhindsa, R. J.; Plumb-Dhindsa, P.; Thorpe, T. A. J. Exp. Bot. 1981, 32, 126. Thompson, J. E.; Pauls, K. P.; Chia, L. S.; Sridhara, S. In "Biosynthesis and Function of Plant Lipids"; Thomson, W. W.; Mudd, J. B.; Gibbs, Μ., Eds. American Society of Plant Physiologists: Rockville, MD, 1983; p. 173. Niehaus, W. J., Jr. Bioorg. Chem. 1978, 7, 77. Moreau, R. Α.; Isett, T. Plant Science (in press). Gerhardt, B. FEBS Letts. 1981, 126, 71. Hara, Α.; Radin, N. S. Anal. Biochem. 1978, 90, 420. Christie, W. W. "Lipid Analysis"; Pergamon Press: Oxford, U.K., 1982. Hasson, E.P.; Laties, G. G. Plant Physiol. 1976, 57, 142.

RECEIVED May 1, 1986

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Chapter 22

Use of Cerulenin and Butyrate in the Study of Candida albicans Germination 1

Ronald L. Cihlar and Kathryn A. Hoberg

Department of Microbiology, Schools of Medicine and Dentistry, Georgetown University, Washington, DC 20007

The antilipogenic agent, cerulenin, has been shown to be an effective inhibitor of fatty acid biosyn­ thesis in a variet property has le lenin to assess the role of phospholipids in the morphogenesis of these organisms. We have examined the utility of cerulenin and the fatty acid salt, sodium butyrate, in studies concerning vegetative germination and pathogenesis of Candida albicans. Both agents are effective as inhibitors of C. albicans germination at concentrations that do not significantly affect cell viability during the time course of experiments. As expected, cerulenin prevented germination by the inhibition of lipid biosynthesis, and such inhibition could be overcome by supplementation of cultures with palmitate. Buty­ rate had no effect on lipid biosynthesis, and presum­ ably inhibits germination by an alternate mechanism. Since both agents inhibited germination by different routes, their effectiveness in identifying biochemical correlates of germination was assessed. The increase in chitin biosynthesis that normally accompanies C. albicans germination was inhibited by cerulenin and butyrate. Cerulenin-resistant mutants of C. albicans have also been isolated and partially characterized. Resistance was verified by unaltered germination capacities, growth kinetics, ultrastructural organi­ zation and continued lipid biosynthesis in the presence of the drug. In addition, the mutants have surface compositions which differ from the parental strain and from each other. These differences have been correlated with diminished capacity of the mutant strains to adhere in vitro to human buccal epithelial cells or to fibrin-platelet matrices. 1

Current address: Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205 0097-6156/87/0325-0355$06.00/0 © 1987 American Chemical Society

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

356

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Dimorphism i s a c h a r a c t e r i s t i c of a number of filamentous fungi, and may be defined as t h e i r a b i l i t y to grow as e i t h e r a y e a s t - l i k e or m y c e l i a l morphology. Generally, the t r a n s i t i o n from one morphological form to the other occurs i n response to environmental factors such as growth medium, incubation temperature or atmospheric composition (1,2). Since such conditions can e a s i l y be controlled and adjusted i n the laboratory, several dimorphic fungi have been exploited experimentally to examine questions r e l a t e d to developmental processes (2,3). In a d d i t i o n , since many fungal pathogens of man are dimorphic, an array of investigations have attempted to assess the s i g n i f i c a n c e of dimorphism i n r e l a t i o n to the invasiveness of these organisms. Studies concerning morphogenesis and pathogenesis of Candida albicans i n p a r t i c u l a r , have appeared with increasing frequency. C. albicans i s an indigenous member of the human m i c r o b i a l f l o r a , and i s a major contributor to the development of opportunistic disease (4). Investigations directed toward understandin r e l a t i o n , i f any, to i t i n c e l l w a l l structure and composition (5,6), as w e l l as concom i t a n t adjustments i n the a c t i v i t y of enzymes involved i n c e l l w a l l biosynthesis that accompany vegetative germination (yeast to hyphal t r a n s i t i o n ; referred to hereafter as germination) (7,8). In contrast, l i t t l e information i s a v a i l a b l e concerning the funct i o n of the plasma membrane i n the dimorphic response and i n w a l l assembly; however, the association of a number of c e l l w a l l b i o synthetic enzymes with the membrane, and the p a r t i c i p a t i o n of membrane components i n transport and secretion processes, implicate the membrane i n morphogenesis and c e l l w a l l assembly. We have approached t h i s issue by employing agents that i n h i b i t germination of C. albicans, with the aim of i d e n t i f y i n g key biochemical and molecular events that may be membrane-associated, and that serve to regulate the yeast to hyphal t r a n s i t i o n . S p e c i f i c a l l y , we have investigated the s u i t a b i l i t y of the a n t i b i o t i c , cerulenin, and the f a t t y acid s a l t , sodium butyrate, f o r use i n these types of i n v e s t i g a t i o n s . Cerulenin, an a n t i l i p o g e n i c agent produced by Cephalosporium caerulens, s p e c i f i c a l l y i n h i b i t s f a t t y acid biosynthesis i n a v a r i e t y of b a c t e r i a , yeast and fungi (9). I t has been u t i l i z e d to study the r o l e of membrane b i o genesis and f a t t y acid synthesis i n d i f f e r e n t i a t i o n of several fungal systems including Rhizopus s t o l o n i f e r (10), Ceratocystis ulmi (11), and B o t r y o d i p l o i d i a theobromae (12). Sodium butyrate, on the other hand, has been found to influence diverse processes, including c e l l u l a r d i f f e r e n t i a t i o n (13), regulation of enzyme a c t i v i t y (14), modification of histones (15) and c e l l cycle events (16), and has been u t i l i z e d l a r g e l y i n studies with higher eukary o t i c c e l l s . The d i r e c t mechanism whereby butyrate potentiates such e f f e c t s has not been defined; however, the agent has not been reported to d i r e c t l y i n h i b i t l i p i d synthesis. The r e s u l t s of our investigations u t i l i z i n g cerulenin and butyrate are discussed below.

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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Materials and Methods. Culture conditions. C. albicans, s t r a i n 4918 (17), was the w i l d type s t r a i n employed i n a l l experiments. Unless otherwise noted, a l l cultures were grown for 18 hr at 28°C i n Phytone-peptone broth supplemented with 0.1% glucose. C e l l s were then c o l l e c t e d by c e n t r i f u g a t i o n , washed and resuspended i n PBS (0.12M sodium chlor i d e ^ 0.037M sodium phosphate, pH 7.2), to f i n a l concentration of 5x10 c e l l s / m l . At the onset of experiments, appropriate amounts of a standardized culture were resuspended i n Phytone-peptone broth. Induction of germination was mediated by s h i f t i n g cultures to an incubation temperature of 37°C. Germination was monitored by l i g h t microscopy. The agents cerulenin and butyrate were u t i l i z e d at f i n a l concentrations of 1 ug/ml and 20 mM, respectively. Biochemical Determinations. Protein biosynthesis, l i p i d b i o synthesis and c h i t i n biosynthesi e a r l i e r (18). U l t r a s t r u c t u r a l Analysis. Samples to be examined by electron microscopy were prepared as described by P e r s i and Burnham (19). A l l samples were f i x e d using the following schedule: g l u t a r a l dehyde (4% (w/v), 6h), osmium tetroxide (20% (w/v), 6h) tannic acid (10% (w/v), 3h), and osmium tetroxide (20% (w/v), 2h). Each f i x a t i v e was prepared i n 0.2M cacodylate b u f f e r , pH 7.2, and samples were washed between f i x a t i o n s with cacodylate buffer. Subsequently, a l l c e l l s were treated with uranyl acetate, washed, dehydrated through a graded ethanol s e r i e s , and f i n a l l y embedded i n Maraglas (Polysciences, Inc.). U l t r a t h i n sections were cut, post-stained with lead c i t r a t e and examined i n a P h i l l i p s 300 transmission electron microscope operating at 60 Kv. Results and

Discussion.

E f f e c t of cerulenin and butyrate on germination of C. albicans. We have previously demonstrated that cerulenin and butyrate e f f e c t i v e l y i n h i b i t germination of C. albicans, s t r a i n 4918 (18). At cerulenin concentrations between 1.0 and 5.0 ug/ml, germinat i o n i s i n h i b i t e d 90 to 95%. In c e l l s i n which germination does occur, germ tubes are s i g n i f i c a n t l y stunted when compared to untreated controls (Figure l a and b). A small percentage of c e l l s also germinate (1-3%) i n the presence of 20 mM butyrate and these l e v e l s can be reduced to v i r t u a l l y zero at concentrat i o n of 50 mM or greater. Butyrate-treated c e l l s remained i n chains and obtained a larger s i z e then those i n untreated or cerulenin-treated c u l t u r e s . In a l l other experiments cerulenin and butyrate were used at concentrations of 1 ug/ml and 20 mM, respectively. The e f f e c t of cerulenin and butyrate upon c e l l architecture was examined further by electron microscopy. No differences were apparent between untreated controls (Figure 2) and butyratetreated c e l l s (data not shown). In both cases membrane bound organelles (e.g., mitochondria, n u c l e i etc.) appeared normal,

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Figure 1. Photomicrographs of C. albicans c e l l s a f t e r 3.5 h of incubation at 37°C. (a) Untreated c o n t r o l . (b) Cultures supplemented with 1 pg/ml of cerulenin 30 min before incubat i o n , (c) Cultures supplemented with 20 mM butyrate 30 min before incubation. Magnification = 360X. (Reprinted with permission of the authors (18), and the American Society f o r Microbiology).

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Figure 2. U l t r a s t r u c t u r e of cerulenin-treated and untreated C. albicans c e l l s . Electron micrographs of albicans, 4918 grown i n phytone peptone broth f o r 3 hr at 28 °C. Panel a: Untreated c e l l s ; Magnification = 28,400X. Panel b: Cerulenin treated c e l l s ; Magnification = 22,135X; m designates mitochondria.

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and numerous invaginations were observed i n the plasma membrane. In contrast, several u l t r a s t r u c t u r a l changes were noted i n cerulenin-treated c e l l s . These include an apparent disorganizat i o n of mitochondria and an absence of plasmalemma invaginations. These observations are consistent with the idea that cerulenin i n t e r f e r e s with the biogenesis of the plasma membrane and membrane organelles i n C. albicans, by v i r t u e of i n h i b i t i o n of l i p i d synthesis. On the other hand, butyrate treatment does not r e s u l t i n u l t r a s t r u c t u r a l changes i n membraneous c e l l u l a r structures, suggesting that the agent may not d i r e c t l y a f f e c t l i p i d synthesis. The e f f e c t of cerulenin and butyrate on l i p i d synthesis was examined ^ i r e c t l y by determining t h e i r influence on i n c o r poration of [ H]acetate into hot methanol/chloroform-extractable material a f t e r induction of C. albicans germination. The k i n e t i c s of [ H]acetate incorporation into l i p i d containing material were essentially identical i for a period of at l e a s cultures with ceruleni approximately i n such synthesis. Both butyrate and cerulenin i n h i b i t e d germinat i o n during these experiments, while untreated c e l l s undergo germination. Experiments were performed to determine whether the addition of exogenous f a t t y acids might overcome the e f f e c t s of e i t h e r cerulenin or butyrate on germination. The addition of as l i t t l e as .01% palmitate to cerulenin-treated cultures restores germinat i v e capacity "Table I . " Supplementation with oleate increased germination of s i m i l a r l y treated cultures by only 5%. Addition of either palmitate or oleate to butyrate treated cultures or to cultures containing both cerulenin and butyrate was not e f f e c t i v e i n overcoming the i n h i b i t o r y action of the agents. The r e s u l t s of t h i s group of experiments suggest that: (1) Cerulenin and butyrate i n h i b i t C. albicans germination. (2) Cerulenin most l i k e l y i n t e r f e r e s with morphogenesis by i n h i b i t i n g l i p i d synthesis. The mechanism whereby butyrate blocks morphogenesis i s unknown; however the data suggest that i n h i b i t i o n i s not d i r e c t l y r e l a t e d to reduced l i p i d synthesis. This might be expected since ACP-butyrate i s an intermediate i n long chain f a t t y acid synthesis, and i s u n l i k e l y to i n h i b i t synthesis, although a u t o - i n h i b i t i o n by free butyrate cannot be excluded. In any event i t i s most l i k e l y that cerulenin and butyrate i n h i b i t C. albicans germination by d i f f e r e n t mechanisms. (3) The fact that l i p i d synthesis continues i n butyrate-treated c e l l s despite the fact that germination i s i n h i b i t e d , implies that although such synthesis may be required during germination, t h i s i s not s u f f i c i e n t to insure that morphogenesis w i l l occur. Cerulenin and butyrate i n i n v e s t i g a t i o n s concerning biochemi c a l correlates of germination. Since butyrate does not i n h i b i t germination by i n t e r f e r i n g d i r e c t l y with l i p i d biosynthesis, i t can be used to help d i s t i n g u i s h between biochemical events i n h i bited by cerulenin that are correlates of morphogenesis from

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those events merely coupled to i n h i b i t i o n of l i p i d biosynthesis. In t h i s regard, we have examined the e f f e c t s of the agents on c h i t i n synthase a c t i v i t y . The enzyme was chosen f o r analysis since i t i s known to be d i f f e r e n t i a l l y expressed during C. albicans germination (8,20), i s associated with the plasma membrane (21), and has been reported to require phosphatidyl serine for maximum a c t i v i t y . In these experiments cultures containing cerulenin or butyrate were s h i f t e d to 37°C, aliquots were removed at 20 min i n t e r v a l s (e.g., +20 min, +40 min^ +60 min), and were then pulsed f o r an a d d i t i o n a l 20 min with [ H]N-acetyl-glucosamine. C h i t i n was extracted and quantiated as described previously. The r e s u l t s indicated that c h i t i n deposition was i n h i b i t e d by both Table I.

Fatty Acid Supplementation of Cerulenin-and/or Butyrate Treated Cultures

Cerulenin

3

Butyrat

++++ + + + + + + +

+ + + + + +

.02 .02 .02 .01

.02 .01 .01

.01 + + + + + + + + + + + + +

+ ++++ ++++ -H-++

+ ++++

.02 .02 .02 .01

.02 .01 .01

.01 .02 .02 .01

.02 .02 .01 .01

.01

^Cerulenin was added to a f i n a l concentration of 1 ug/ml. Sodium butyrate was employed at a f i n a l concentration of 20 mM. Palmitate and oleate were maintained as 1% stock solutions i n 10% BRIJ 35. BRIJ 35 controls were negative. Germination was determined microscopically, 3 hr following a s h i f t to an incubation temperature of 37°C. (++++) = >90% of germination, (+) = 5-10%, (-) = no germination. Overnight cultures of C. albicans s t r a i n 4918 were resuspended i n phytone-peptone broth (5x10 /ml) under the conditions indicated above. A l l cultures were pre-incubated for 30 min at 28°C p r i o r to the s h i f t up i n temperature (37°C). d

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agents. During each successive pulse cerulenin i n h i b i t e d c h i t i n synthesis 46%, 73% and 80%, r e s p e c t i v e l y , while butyrate i n h i b i t e d c h i t i n synthesis 46%, 73% and 80%, r e s p e c t i v e l y , when compared to untreated c o n t r o l s . Overall protein synthesis was not i n h i b i t e d during these time i n t e r v a l s as judged by incorporation of [ H]leucine i n t o TCA p r e c i p i t a b l e counts. These r e s u l t s suggest that the apparent increase i n c h i t i n synthesis that accompanies C. albicans germination (8,20), i s a v a l i d biochemical correlate of development. In addition the combined use of cerulenin and sodium butyrate may provide a general approach to assess the importance of membrane biogenesis, as w e l l as adjustments i n the a c t i v i t y of membrane associated enzymes, to morphogenesis. Cerulenin r e s i s t a n t mutants of C. albicans. In order to extend our investigations concerning the molecular regulation of dimorphism of C. albicans, cerulenin-resistant mutants have been i s o l a t e d and p a r t i a l l y characterized. The mutants, designated 4918-2 and 4918-10, hav surface structure that i n s i g h t s concerning the virulence of C. albicans. Spontaneous cerulenin-resistant mutants were i s o l a t e d as described elsewhere (22). The mutants were conclusively i d e n t i f i e d as C. albicans s t r a i n s on the basis of sugar assim­ i l a t i o n patterns, and by t h e i r a b i l i t y to form germ tubes and chlamydospores. The only consistent difference noted among s t r a i n s 4918, 4918-2 and 4918-10 was i n the a b i l i t y of s t r a i n 4918-10 to u t i l i z e g l y c e r o l as i t s sole carbon source; the other two s t r a i n s were unable to grow with g l y c e r o l as the carbon source. V a r i a b i l i t y was also observed i n the a s s i m i l a t i o n by the mutant s t r a i n s of the sugar alcohols, a d o n i t o l , x y l i t o l and sorbitol. The nature of the cerulenin resistance of the mutant s t r a i n s was f i r s t investigated by constructing growth curves of both s t r a i n s i n the presence of cerulenin. In these experiments samples were removed hourly from newly i n i t i a t e d cultures i n YEPD (2.0% yeast e x t r a c t , 1% peptone, 1% glucose) incubated at 37°C, and the o p t i c a l density at 540 nm f o r each aliquot was determined. The growth k i n e t i c s of untreated cultures of s t r a i n s 4918, 4918-2 and 4918-10 and cerulenin-treated cultures of 4918-2 and 4918-10 were i d e n t i c a l . In each case logarthmic growth was entered at approximately 4 hr a f t e r the onset of the experiments, while the stationary growth phase was reached at 10-12 hr time points. The f i n a l c e l l density ( o p t i c a l density of 1.6) was the same i n a l l cases. In contrast, wild-type cultures treated with cerulenin reached a c e l l density of only 12-15% of the other cultures. The resistance of 4918-2 and 4918-10 to the i n h i b i t o r y e f f e c t s of cerulenin was substantiated by determining whether the agent affected the rate of l i p i d synthesis i n each s t r a i n . As before, the rate οξ l i p i d synthesis was quantitated by determining incorporation of [ H]acetate into hot methanol/chloroform extractable-material. The r e s u l t s demonstrated that the k i n e t i c s of l i p i d synthesis was not s i g n i f i c a n t l y affected by cerulenin.

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Whether or not q u a l i t a t i v e differences i n l i p i d composition e x i s t between w i l d type and mutant s t r a i n s remains under i n v e s t i g a t i o n . I t was reasoned that cerulenin r e s i s t a n t s t r a i n s of C. albicans might a r i s e i n at l e a s t two ways: 1) A mutation i n the gene sequences encoding 3-Ketoacyl-ACP synthase, the enzyme i n h i b i t e d by the action of cerulenin (9), or 2) Mutation(s) r e s u l t i n g i n c e l l surface changes rendering the organism impermeable to the agent. Several observations suggest that cerulenin resistance of 4918-2 and 4918-10 may f a l l into t h i s l a t t e r category. U l t r a s t r u c t u r a l analysis was f i r s t performed to determine whether obvious surface differences existed between wild-type and mutant s t r a i n s . The notion that such differences might e x i s t stemmed from observations that growth of the mutant s t r a i n s i n l i q u i d media resulted i n f l o c c u l a n t appearing cultures i n comparison to the w i l d type, and that such c e l l s were d i f f i c u l t to resuspend a f t e r c o l l e c t i o However, no difference surface architecture of mutant s t r a i n s concerning the number or thickness of the layers of the c e l l w a l l . Although c e l l surface u l t r a s t r u c t u r e was i d e n t i c a l i n mutant and w i l d type s t r a i n s , differences have been observed i n the composition of c e l l w a l l material of the mutant s t r a i n s i n comparison to w i l d type. One dimensional polyacrylamide gel analysis of proteins extracted from c e l l walls of each s t r a i n showed that s t r a i n 4918-2 c e l l walls contained a protein of an apparent molecular of 26,000 daltons that was absent i n w a l l preparations of 4918 and 4918-10. In a d d i t i o n , a quantitative difference i n a protein with a molecular weight of approximately 46,000 daltons was detected. This protein was present i n lower amounts i n s t r a i n 4918-2 than i n the other s t r a i n s . A s i g n i f i c a n t difference was also observed i n c e l l w a l l polysaccharide composition. S p e c i f i c a l l y , s t r a i n 4918-10 and 4918-2 had 15.8% and 18.4% more reducing sugar, r e s p e c t i v e l y , associated with t h e i r walls when compared with the parental s t r a i n . Direct quantitation of glucose, however, revealed that the amount of glucose present i n the walls of mutant s t r a i n s was nearly 11% less than that present i n wild-type c o n t r o l s . I t appears that the difference i n t o t a l reducing sugar content was due to s i g n i f i c a n t l y higher mannose content i n c e l l walls i s o l a t e d from mutant s t r a i n s . The w a l l of each mutant s t r a i n contains at l e a s t 27% more mannose than does that of the wild-type s t r a i n (23). The differences observed prompted experiments to determine i f functions associated with the c e l l surface were affected by such a l t e r a t i o n s . In p a r t i c u l a r , the adherence c a p a b i l i t i e s of the s t r a i n s was assessed by measuring adherence to buccal e p i t h e l i a l c e l l s and to f i b r i n - p l a t e l e t matrices (24,25). Adherence of s t r a i n s 4918-2 and 4918-10 was greatly reduced i n both systems when compared to c o n t r o l values. Adherence of the mutant s t r a i n s was reduced to approximately 60% and 85% i n comparison to r e s u l t s obtained with s t r a i n 4918 i n the buccal c e l l and f i b r i n - p l a t e l e t systems, r e s p e c t i v e l y .

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Since the a b i l i t y of C. albicans to adhere to c e l l s i n vivo may be a factor contributing to virulence of the organism, inves­ t i g a t i o n s with the mutant s t r a i n s have turned toward examining t h e i r r e l a t i v e virulence and t h e i r antigenic composition as opposed to the w i l d type. In t h i s respect recent i n v e s t i g a t i o n s have shown that the mutant s t r a i n s 4918-2 and 4918-10 are s i g n i ­ f i c a n t l y less v i r u l e n t i n the rabbit model of Candida albicans (26). Work i s i n progress to a s c e r t a i n the molecular mechanisms for the reduced v i r u l e n c e . Acknowledgments This work was supported i n part by Public Health Service Grants DE07168 and HL21370 from the National I n s t i t u t e s of Health. We thank Dr. Richard Calderone f o r h i s help and advice, and Adriana Cabrales f o r manuscript preparation.

Literature Cited 1. Stewart, P.R., and P.J. Rogers. 1978. In: "The Filamentous Fungi." (J.E. Smith and D.R. Berry, eds.) pp. 164-196. Wiley and Sons, New York, New York. 2. Szanizlo, P.J., C.W. Jacobs, and P.A. Geis. 1983. In: Fungi Pathogenic for Humans and Animals." (D.H. Howard, ed.) pp.323-436. Marcel Dekker Inc., New York, New York. 3. Sypherd, P.S., P.T. Borgia, and J. Paznokas. 1978. Adv. Microb. Physiol. 18:67-104. 4. Odds, F.C. 1979. "Candida and Candidosis." University Park Press. Baltimore, Maryland. 5. Schewitz, C.R., R. Martin, and H. Verberberg. 1978. Sabouraudia. 16:115-124. 6. Cassone, Α., Ν. Simonetti, and V. Strippoli. 1973. J. Gen. Microbiol. 77:417-526. 7. Chattaway, F.W., R. Bishop, M.R. Holmes, F.C. Odds, and A.J.E. Barlow. 1973. J. Genl. Microbiol. 75:97-109. 8. Braun, P.C., and R.A. Calderone. 1979. J. Bacterol. 140:666-670. 9. Omura, S. 1981. Meth. Enzymol. 72:520-532. 10. Nickerson, K.W., and E. Leastman. 1978. Exp. Mycol. 2:26-31. 11. Brambl, R., M. Wenzler, and M. Josephson. 1978. J. Bacteriol. 128:21-27. 12. Nickerson, K.W., D.J. McNeel, and R.K. Kulkarni. 1982. FEMS Microbiol. Lett. 13:21-25. 13. Leder, Α., and P. Leder. 1975. Cell 5:319-322. 14. Littlefield, B.A., N.B. Cidlowski, and J.A. Sidlowski. Arch. Biochem. Biophys. 201:174-184. 15. Kruh, J. 1982. Mol. Cell. Biochem. 42:65-82. 16. van Wijk, R., L. Tichonicky and J. Kruh. 1983. Eur. J. Biochem. 129:456-460. 17. Manning, M. and T.G. Mitchell. 1980. J. Bacteriol. 142:714-719.

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18. Hoberg, K.A., R.L. Cihlar, and R.A. Calderone. 1983. Anti­ microb. Agents and Chemother. 24;401-408. 19. Persi, M.A., and J.C. Burnham. 1981. Sabouraudia. 19:1-8. 20. Chiew, Y.Y., M.G. Shepherd, and P.A. Sullivan. 1980. Arch. Microbiol. 125:97-104. 21. Duran, Α., Β. Bowers, and E. Cabib. 1975. Proc. Nat. Acad. Sci. U.S.A. 72:3952-3955. 22. Cihlar, R.L., K.A. Hoberg, and R.A. Calderone. 1984. In: "Microbiology, 1984". (L. Leive and D. Schlessinger, eds.) pp. 148-149. American Society of Microbiology, Washington, D.C. 23. Hoberg, K.A., R.L. Cihlar, and R.A. Calderone. 1986. Inf. Immun. 51:102-109. 24. King, R.D., J.C. Lee, and A.L. Morris. 1980. Infec. Immun. 27:667-674. 25. Maisch, P.Α., and R.A. Calderone. 1981. Infec. Immun. 32:92-97. 26. Calderone, R.A., R.L W.M. Scheld. 1985. J. Inf. Dis. 152:710-715. Received May 1, 1986

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365

Author Index Lichtenthaler, Hartmut Κ., 109 Lozano, Ruben, 187, 200 Lusby, William R., 200 Meudt, Werner J., 53 Moreau, Robert Α., 343 Mudd, J. B., 10 Nes, W. David, 2, 140, 304 Nes, William R., 252 Parish, Edward J., 140 Phinney, B. 0., 25

Andrews, J., 10 Bach, Thomas J., 109 Bishop, D. G., 10 Bradford, Susan, 140 Chitwood, David J., 200 Cihlar, Ronald L., 355 Crawford, Mark S., 152 Croteau, Rodney, 76 Elakovich, S t e l l a D., 93 Ettinger, William F., 152 Feldlaufer, Mark F. F u l l e r , Glenn, 2, 44 Geisler, V i c t o r i a J. Hanners, Patrick Κ., 140 Heupel, Rick C , 140 Hoberg, Kathryn Α., 355 Johnson, Mark Α., 76 Kabara, Jon J., 220 Kannenberg, Elmar, 239 Kerwin, James L., 329 Kleppinger-Sparace, 10 Kolattukudy, P. E., 152 Le, Phu H., 140

, , Soliday, Charles L., 152 Sparace, S. Α., 10 Spray, C. R., 25 Stumpf, P. Κ., 44 Svoboda, James Α., 176, 187, 200 Thomas, S., 10 Thompson, Malcolm J., 176, 187, 200 Weete, John D., 268 Weirich, Gunter F., 187 Woloshuk, Charles P., 152

Subject Index A

Abies grandis, interactions with the f i r engraver beetle, 79 Absidia coerulea, phosphorylation of compactin, 115 Absidia cylindrospora, phosphorylation of compactin, 115 Absidia glauca, phosphorylation of compactin, 115 Acetyl coenzyme A, formation, 45 Achlya, s t e r o l u t i l i z a t i o n , 318,321 Achlya ambisexualis, growth i n h i b i t i o n , 337 Achlya c a r o l i n i a n a growth i n h i b i t i o n , 337 Acyl c a r r i e r protein, fatty acid synthase sequence, 45-46 Acyl c a r r i e r protein derivatives, desaturation, 13,l4f Acyl l i p i d s , formation, 47-48 Aglycone skeletons saponins, 288f Solanum glycoalkaloids, 288f t

Allelopathic agents, structure, 98f Alternaria solani, removal of mono­ saccharide units of glycoalkaloids, 297,299 Ambrosia psilostachya, production of sesquiterpene lactones, 104 Antifungal a c t i v i t y , plant steroids, 286-301 Antifungal azoles, names, 270-271t Aphelenchus avenae, ecdysteroids, 214 Apis mellifera, effect of sterols on brood production, 183 Artemisia absinthium, sesquiterpene inhibitor production, 104 Ascaris, s t e r o l biosynthesis, 202-203 Ascaris suum, ecdysteroids, 214 Aspergillus nidulans, studies of s t e r o l i n h i b i t o r s , 276-277 Aspergillus niger, studies of fatty acids, 222,223 Aspergillus ochraceous, temperature adaptation, 332 Aspergillus terreus, production of mevinolin, 110

366 In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

Author Index Lichtenthaler, Hartmut Κ., 109 Lozano, Ruben, 187, 200 Lusby, William R., 200 Meudt, Werner J., 53 Moreau, Robert Α., 343 Mudd, J. B., 10 Nes, W. David, 2, 140, 304 Nes, William R., 252 Parish, Edward J., 140 Phinney, B. 0., 25

Andrews, J., 10 Bach, Thomas J., 109 Bishop, D. G., 10 Bradford, Susan, 140 Chitwood, David J., 200 Cihlar, Ronald L., 355 Crawford, Mark S., 152 Croteau, Rodney, 76 Elakovich, S t e l l a D., 93 Ettinger, William F., 152 Feldlaufer, Mark F. F u l l e r , Glenn, 2, 44 Geisler, V i c t o r i a J. Hanners, Patrick Κ., 140 Heupel, Rick C , 140 Hoberg, Kathryn Α., 355 Johnson, Mark Α., 76 Kabara, Jon J., 220 Kannenberg, Elmar, 239 Kerwin, James L., 329 Kleppinger-Sparace, 10 Kolattukudy, P. E., 152 Le, Phu H., 140

, , Soliday, Charles L., 152 Sparace, S. Α., 10 Spray, C. R., 25 Stumpf, P. Κ., 44 Svoboda, James Α., 176, 187, 200 Thomas, S., 10 Thompson, Malcolm J., 176, 187, 200 Weete, John D., 268 Weirich, Gunter F., 187 Woloshuk, Charles P., 152

Subject Index A

Abies grandis, interactions with the f i r engraver beetle, 79 Absidia coerulea, phosphorylation of compactin, 115 Absidia cylindrospora, phosphorylation of compactin, 115 Absidia glauca, phosphorylation of compactin, 115 Acetyl coenzyme A, formation, 45 Achlya, s t e r o l u t i l i z a t i o n , 318,321 Achlya ambisexualis, growth i n h i b i t i o n , 337 Achlya c a r o l i n i a n a growth i n h i b i t i o n , 337 Acyl c a r r i e r protein, fatty acid synthase sequence, 45-46 Acyl c a r r i e r protein derivatives, desaturation, 13,l4f Acyl l i p i d s , formation, 47-48 Aglycone skeletons saponins, 288f Solanum glycoalkaloids, 288f t

Allelopathic agents, structure, 98f Alternaria solani, removal of mono­ saccharide units of glycoalkaloids, 297,299 Ambrosia psilostachya, production of sesquiterpene lactones, 104 Antifungal a c t i v i t y , plant steroids, 286-301 Antifungal azoles, names, 270-271t Aphelenchus avenae, ecdysteroids, 214 Apis mellifera, effect of sterols on brood production, 183 Artemisia absinthium, sesquiterpene inhibitor production, 104 Ascaris, s t e r o l biosynthesis, 202-203 Ascaris suum, ecdysteroids, 214 Aspergillus nidulans, studies of s t e r o l i n h i b i t o r s , 276-277 Aspergillus niger, studies of fatty acids, 222,223 Aspergillus ochraceous, temperature adaptation, 332 Aspergillus terreus, production of mevinolin, 110

366 In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

367

INDEX

Aubergenone, structure, 95 f Auxin-brassinolide interaction, 64-67 Azole s t e r o l i n h i b i t o r s , 276,277t

B Bacillu s acidocaldarius, hopanoid and l i p i d content, 244-247 Bacillus s u b t i l i s , reproduction of Turbatrix a c e t i , 201 Bark beetle attack, biochemistry of conifer resistance, 78-79 Bioassays, quantitative, brassinosteroids, 61-64 Bisabolene, structure, 98f Bombyx mori, metabolism of sterols, 177 Botryodiploidia theobromae, use of cerulenin to distinguish, 356 Botrytis cinerea, t o x i c i t y of glycosides, 290,295-299 Brassica compestris, presence of 28-norbrassinone, 59 Brassica napus, b i o l o g i c a l l y active agent, 53 Brassinolide b i o l o g i c a l a c t i v i t y , 59 chemical and b i o l o g i c a l aspects, 53-73 effect on crop production, 70-73 effect on geotropism, 70,71f effect on growth of p u l v i n i , 70,72f effect on proton secretion, 67 structure, 55,56f Brassinolide-auxin interaction, 64-67 Brassinolide-ethylene interaction, 67-70,71f Brassinolide-gibberellic acid interaction, 67-69 Brassinosteroids bean f i r s t internode bioassay, 62-64 bean second internode bioassay, 61-62 natural occurrence i n plants, 55 quantitative bioassays, 61-64 rice-lamina i n c l i n a t i o n test, 62 structure, 55,58f Bremia lactucae, i n f e c t i o n of lettuce, 344 Brugia pahangi, s t e r o l biosynthesis, 202 Bursaphlenchus xylophilus, amine t o x i c i t y , 205 Butyrate, investigations concerning biochemical correlates of germination, 360-362

C Caenorhabditis, n u t r i t i o n a l requirement for sterols, 201-203 Caenorhabditis elegans effects of i n h i b i t o r s on s t e r o l metabolic pathways, 208-213 general discussion, 202-205,208 Calliphora v i c i n a , ecdysone conversions, 189 Campesterol, structure, 179f Candida albicans cerulenin-resistant mutants, 362-364 effect of cerulenin and butyrate on germination, 357-360 growth i n h i b i t i o n , 276,278 morphogenesis and pathogenesis, 356 photomicrographs, 358f

Candida u t i l i s , temperature adaptation, 332 Capsenone, structure, 95f Capsicum annuum, as a source of sesquiterpenoid phytoalexin, 100 Capsicum frutescens, use i n hostparasite studies, 101 Capsidiol, structure, 95f Caryophyllene, structure, 98f Castanea crenata, as a source of 6-deoxocastasterone, 59-60 Castasterone b i o l o g i c a l a c t i v i t y , 60 IUPAC equivalent name, 57 Cephalosporum caerulens growth regulation, 115 i n h i b i t i o n of fatty acid synthesis, 356 Ceramide aminoethylphosphonate, 347f Ceratocystis, growth i n h i b i t i o n , 81 Ceratocystis fimbriata, infection of sweet potatoes, 102 Ceratocystis ulmi infection of Wych elm, 103 use of cerulenin to distinguish, 356 Cercospora arachidicola, s t e r o l content, 274 Cerulenin, investigations concerning biochemical correlates of germination, 356,360-362 Chaetomium, growth of isolated compounds on acids, 334 Chaoborus astictopus, infection, 335 Chloroplast g l y c e r o l i p i d s , biosynthesis, 10-23 5,22,24-Cholestatrienol, structure, 179f Cholesterol, structure, 179f Chondrostereum purpureum, i n f e c t i o n of elm, 103 C i r c i n e l l a muscae, phosphorylation of compactin, 115

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

368

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Cladosporium fulvum, growth i n h i b i t i o n , 287 Coleoptera phytophagous insects that convert phytosterols to cholesterol, 180-182 phytophagous insects unable to convert phytosterols to cholesterol, 183 Colletotrichum, c a t a l y s i s of ester hydrolysis, 157 Colletotrichum c a p s i c i , spore suspensions, 163 Colletotrichum gloeosporiodes, cutinase secretion, 163,166 Colletotrichum graminicola, spore suspensions, 163 Colletotrichum orbiculare, growth i n h i b i t i o n , 290,295 Colletotrichum phomoides, resistanc of tomato f r u i t s , 299 Compactin, structure, 112 Conifer resistance to bark beetles, biochemistry, 76-89 Conifer resistance to t h e i r fungal symbionts, biochemistry, 76-89 Coriolus versicolor, infection of elm, 103 Corticium r o l f s i i , f r u i t rot, 299 Corynebacterium sepedonicum, bacterial ring rot of potatoes, 298 Cotton—See Gossypium species Cucurbita maxima, 4-desmethylsterols, 253,254t Culex t a r s a l i s , infection, 335 Cutin, fungal interaction with plants, 152-173 Cutinase comparison of homologous peptides, 159,l62f i s o l a t i o n and characterization, 154-158 mechanism of c a t a l y s i s , 157,158f mRNA content, 170,171f nucleotide sequence, 159-161 role i n pathogenesis, 163-170 structure, 159 Cutinase a c t i v i t y e x t r a c e l l u l a r f l u i d of Fusarium solani p i s i , 170,171Γ released into the e x t r a c e l l u l a r f l u i d during spore germination, 166,168t Cyclooxygenase, regulation of fungal morphogenesis, 337-338 Cyperus serotinus, isolated inhibitory f r a c t i o n , 104 D Dacrydium cupressinum, i s o l a t i o n of podocarpic acid, 140

Datura stramonium, production of antifungal compounds, 102 Dehydroipomeamarone, structure, 96f Dendroctonus brevicomis, aggregation pheromone precursor, 79-80 Dendroctonus ponderosae, resistance of pine, 80 6-Deoxocastasterone b i o l o g i c a l a c t i v i t y , 59 IUPAC equivalent name, 57 Deoxodolichlosterone b i o l o g i c a l a c t i v i t y , 59 IUPAC equivalent name, 57 4-Desmethylsterols, Cucurbita maxima, 253,254t Diacylglycerol, biosynthesis, 18 Diacylglycerol moiety of chloroplast glycerolipids, 15-16

biosynthesis, 18 4a,5-Dihydrocompactin, structure, 112 4a,5-Dihydromevinolin, structure, 112 Diplodea pinea, i n h i b i t i o n of spore germination, 81 D i r o f i l a r i a immitis, s t e r o l biosynthesis, 202,214 Dolicholide b i o l o g i c a l a c t i v i t y , 60 IUPAC equivalent name, 57 Dolichos lablab, i s o l a t i o n of brassinosteroid, 59 Dolichosterone b i o l o g i c a l a c t i v i t y , 59-60 IUPAC equivalent name, 57 Dolico lablab, i s o l a t i o n of dolicholide, 60 Dysdercus fasciatus, s t e r o l metabolism, 183

Ε

Ecdysone IUPAC equivalent name, 57t structure, 178f Ecdysteroid(s) content i n meconium f l u i d , 191-194 embryonated eggs of Manduca sexta, 194-198 i s o l a t i o n from Manduca pupae, 192f i s o l a t i o n from Manduca sexta, 190f structure, 178f Eggplant—See Solanum melongena Electron micrographs, pea-stem c u t i c l e , I64f Elm—See Ulmus glabra 3-Epiecdysome, metabolic scheme, l89,190f Epilachna v a r i v e s t i s , s t e r o l content, 180

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

369

INDEX

Ergosterol biosynthesis, inhibitors, 268-282 Erynia delphacis, c o n i d i a l germination, 338 Erynia radicans, response to o l e i c acid, 336 Erynia v a r i a b i l i s , c o n i d i a l germination, 336,338 Escherichia c o l i acyl c a r r i e r protein, 45-46 effect on Caenorhabditis, 201 Esterase a c t i v i t y , released into the e x t r a c e l l u l a r f l u i d during spore germination, 166,168t Esters, antimicrobial i n s e c t i c i d a l agents, 220-236 Ethylene-brassinolide interaction, 67-70,71f Europhium clavigerum, resistanc f conifers, 80,82

Fungitoxicity—Continued saponins, 287-291 Furanosesquiterpenoid stress metabolites from sweet potato, structure, 96f Fusarium caeruleum, disruption of spores, 290,298 Fusarium oxysporum, i n h i b i t i o n , 287,290,297-298 Fusarium solani p i s i bioassays on pea e p i c o t y l , 163-167 cutinase mRNA content, 170,171f degradation by fungal pathogens, 154 isolated enzyme, 159 nystatin-resistant mutants, 291 pectinase production, 172 penetration of pea-stem c u t i c l e , I63,l64f

G F Fannia c a n i c u l a r i s , germination on the cuticular surface, 336 Farnesol, structure, 96f,98f Farnesyl acetate, structure, 98f Fatty acids and fungal developments, 329-338 antimicrobial a c t i v i t y , 221-227 antimicrobial i n s e c t i c i d a l agents, 220-236 composition, temperature adaptation, 332-333 enzymic regulation, 331-332 e s t e r i f i c a t i o n , 224 fungi spore germination, 334-336 general discussion, 4-5 host-pathogen interactions, 336-337 membrane transport, 331 physical properties, 330-331 plants, a model system, 44-51 structure, discussion, 330 synthase sequence, 45-47 synthesis i n plants, 11-13,44-45 Foeniculum vulgare, monoterpene c y c l i z a t i o n , 87 Fomes annosus, growth i n h i b i t i o n , 81 Fucosterol, structure, 179f Fungal e l i c i t o r s and conifer resistance, 81-82 Fungal growth fatty acids, 333-334 i n h i b i t i o n by i n h i b i t o r s of ergosterol biosynthesis, 268-282 Fungal l i f e cycle, sequence of development controlled by sterols, 319f Fungitoxicity glycoalkaloids, 287-291

Galactolipids biosynthesis, 18-19 general discussion, 5,6t Geotrichum flavobrunneum a n t i b i o t i c i s o l a t i o n , 129 azasterol production, 269 Geotropism, effect of brassinolide, 70,71f Gibberella f u j i k u r o i early 3-hydroxylation pathway, 32,34f i s o l a t i o n of g i b b e r e l l i n s , 25 Gibberellic acid-brassinolide interaction, 67-69 Gibberellins biosynthesis, 29-41 p u r i f i c a t i o n steps, 26,28f Gloeosporium fructigenum, f r u i t rot, 299 Glutinosone, structure, 95f Glycerol-3-phosphate, acylation, 16-17 Glycoalkaloids as resistance factors, 297-300 fungitoxicity, 287-291 Gossypium species, as a source of sesquiterpenoid phytoalexins, 103 Gossypol, structure, 97f Growth i n h i b i t o r s , i n radish seedlings, 131,132f

H Haemonchus contortus, ecdysteroids, 214 Haliphthoros milfordensis, growth promotion by o l e i c acid, 334 Heliothis zea growth prevention of larvae, 260 metabolism of phytosterols, 177

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

370

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Helminthosporium avenae, i n f e c t i o n of saponins, 297 Helminthosporium turcicum, growth i n h i b i t i o n , 290,295 Hemiptera, phytophagous insects unable to convert phytosterols to cholesterol, 180-181 28-Homobrassinolide b i o l o g i c a l a c t i v i t y , 60 IUPAC equivalent name, 57 28-Homodolicholide b i o l o g i c a l a c t i v i t y , 60 IUPAC equivalent name, 57 28-Homodoli chosterone b i o l o g i c a l a c t i v i t y , 60 IUPAC equivalent name, 57 Hopanoids B a c i l l u s acidocaldarius content, 244-245,247 function i n model membran systems, 246-249 occurrence, 242-244,245f s t e r o l equivalents i n bacteria, 239-249 structures, 242,243f Zymomonas mobilis content, 246,247f 7-Hydroxycostal, structure, 96f 7-Hydroxycostoi, structure, 96f 11-Hydroxy-9,10-dehydronerolidol, structure, 95f 20-Hydroxyecdysone, structure, 178f 9-Hydroxynerolidol, structure, 95f Hymenoptera, phytophagous insects unable to convert phytosterols to cholesterol, 183-184

I Ipomeabisfuran, structure, 96f Ipomeamarone, structure, 96f Ipomoea batatus, as a source of sesquiterpenoid phytoalexins, 102 Ips typographis, production of aggregation pheromone, 80 ent-Isokaurene, 37,40f Isopentenoid compounds, 7

J Jimsonweed—See Datura stramonium

Κ ent-Kaurene, 37,40f

L Lactuca sativa, changes during infection, 344

Lagenidium giganteum, sporulation, 334-335,337-338 Lanosterol, i n h i b i t i o n of demethylation, 269-276 Laurie acid, structure, 4t Lemna minor as a plant model system, 48-50 d i s t r i b u t i o n of f a t t y acids, 49t labeled acetate incorporation, 49-51 Lepidoptera, phytophagous insects that convert phytosterols, 177,179f Leucosporidium, enhancement of l i n o l e i c acid, 332 L i n o l e i c acid, structure, 4t Lipids biosynthesis i n chloroplasts, 10-23 classes, 4-7 derived from f a t t y acids, 5

overview, 2-9 polar, 5 L i p o l y t i c enzymes change i n l e v e l s , 350 during i n f e c t i o n , 350 L i p o p h i l i c antimicrobial agents, 221-227 Lipoxygenase, regulation of fungal morphogenesis, 337-338 Lippia n o d i f l o r i a , as a source of sesquiterpenoid phytoalexins, 104 Locusta migratoria, ecdysteroids, 189 Lycopersicon esculentum, as a source of sesquiterpenoid phytoalexins, 102 Lycopersicon pimpinellifolium, resistance to bacterial w i l t , 298

M Makisterone A, structure, 178f Malonyl coenzyme A, formation, 45 Manduca sexta free and conjugated ecdysteroids, 187-198 metabolism of s t e r o l s , 177 t o x i c i t y of 29-fluorostigmasterol, 202 Meconium f l u i d , ecdysteroid content, 191-194 Megachile rotundata, use of dietary phytosterols, 183 Meloidogyne incognita, i n h i b i t i o n of sterol metabolism, 205 Meterodera zeae, s t e r o l content, 204 24-Methylene-cholesterol, structure, Methylococcus capsulatus synthesis of hopanoids, 249 synthesis of s t e r o l s , 240,249

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

179f

371

INDEX

Mevalonic acid chemical structure, 7 early pathway to gibberellins-12-aldehyde, 29,30f Mevinolin effect on growth and chemical composition of c e l l suspension cultures of Silybum marianum, 126,127t effect on isoprenoid accumulation i n radish seedlings, 118-124 effect on isoprenoid synthesis i n primary leaves of wheat, 124,125t effect on p l a s t i d i c p r e n y l l i p i d s and prenylquinones, 122,123t plant-growth regulation, 109-134 secondary physiological responses, 126-129 structure, 112f Mevinolin-type metabolites of i n h i b i t i o n constants, 110,113-114t Microsporum gypseum, macroconidial germination, 335 Monacolin, structure, 112f Monascus ruber, mevinolin production, 110 Monilinia f r u c t i c o l a , inoculation of peppers, 101-102 Monilinia fructigena f r u i t rot, 299 sterol i n h i b i t o r studies, 276,278 Monogalactosyldiacylglyceride, substrate, 47 Monogalactosyldiacylglycerol biosynthesis, 18 positional d i s t r i b u t i o n i n l e a f g l y c e r o l i p i d s , 15-16,17t Monoterpene i n conifers alterations, 76 biosynthesis, 82-84 components, 77f quantitative analysis, 8 3 Monoterpene c y c l i z a t i o n , mechanism, 84-88 Mucor hiemalis, compactin hydroxylation, 110 Mucor mucedo, temperature adaptation, 332 Mycoplasma, dependence on s t e r o l s , 240 Mycosphaerella, i n f e c t i o n of papaya f r u i t s , 166 Myristic acid, structure, 4t Hyrothecium cerrucaria, studies of f a t t y acids, 223

Ν

Nannocystis exedens, s t e r o l content, 240

Nematodes function of s t e r o l s , 213-215 i n h i b i t i o n of s t e r o l metabolism, 205-208 lack of de novo s t e r o l biosynthesis, 202-203 p a r a s i t i c , s t e r o l composition, 203 Nematospiroides dubius, growth i n h i b i t i o n , 208 Neurospora crassa mutants, reversion to normal c y c l i c nucleotide l e v e l s , 335 slime mutant, s e n s i t i v i t y to propiconazole, 281 Nicotiana species, as a source of sesquiterpenoid phytoalexins, 101 Nippostronglylus b r a s i l i e n s i s , s t e r o l requirements, 202,208

sativum, 31-33f 2 8 -Nor bras s i no 1 i de b i o l o g i c a l a c t i v i t y , 60 IUPAC equivalent name, 57 28-Norbrassinone b i o l o g i c a l a c t i v i t y , 59 IUPAC equivalent name, 57

0 Octahydrophenanthrene lactones, structure, 141f Oleic acid, structure, 4t Oncopeltus fasciatus, s t e r o l conversions, 180 Oomycetes s t e r o l biosynthesis, 304-326 sterol composition, 308-309t s t e r o l occurrence, 304-318 s t e r o l requirements, 304-326 Oryza sativa, as a source of dolichosterone, 60,62 Ostertagia ostertagi, t o x i c i t y of alkylamines and amides, 205 9-0xonerolidol, structure, 95f 6-0xydendrolasin, structure, 96f

Ρ

Palmitic acid, structure, 4t Panagrellus redivivus, 203-205 ecdysteroids, 214 s t e r o l biosynthesis, 203 Pectinaceous barrier, enzymatic penetration, 172,173f Pénicillium, compactin production, Pénicillium italicum, growth i n h i b i t i o n , 279 Pepper—See Capsicum annuum

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

110

372

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Phaseolus vulgaris castasterone content, 60,62 changes during i n f e c t i o n , 344 presence of 6-deoxocastasterone, 59-61 Phocanema decipiens, ecdysis, 214 Phosphatidylcholine, p o s i t i o n a l distribution i n leaf g l y c e r o l i p i d s , I6,17t Phosphatidylglycerol in chloroplasts, biosynthesis, 19,21t positional d i s t r i b u t i o n i n l e a f glycerolipids, I6,17t Phospholipase a c t i v i t y during i n f e c t i o n of potato leaves, 351f effect of pH, 352f from phytopathogens, 345-349 in healthy potato leaves on phosphatidylcholine, 347 Phytoalexins, discussion, 99-100 Phytopathogens, phospholipase a c t i v i t y , 346 Phytophagous insects, s t e r o i d metabolism, 176-185 Phytophthora cactorum growth curve, 322f metabolism of s t e r o l s , 260 micrographs, 324-325f sporulation, 295 s t e r o l occurrence, 306-309,315-321 t o x i c i t y of glycosides, 290 Phytophthora cinnamomi, growth i n h i b i t i o n , 278,280 Phytophthora infestans changes i n host ultrastructure during i n f e c t i o n , 344-345 changes i n levels of e x t r a c e l l u l a r enzyme a c t i v i t i e s during germination, 349t changes i n l i p i d composition during infection, 344-345 disintegration of zoospores, 291 host-pathogen interactions, 336-337 membrane-degrading enzymes during infection of potato leaves, 343-353 polar l i p i d composition, 345t potato resistance, 100,299 Phytophthora p a r a s i t i c a , sphingolipid content, 345 Phytosterol synthesis, e f f e c t of i n h i b i t o r s , 129-133 P i e r i s brassicae, ecdysteroids, 189 Pinus pinaster, formation of a c y c l i c hydrocarbons, 84 Pinus radiata monoterpenes, 81 seedlings, 84 Pinus virginiana, r e s i n flow from wounds, 81

Pisum sativum nonhydroxylation pathway, 31-33 pathogen, 154 Pityrosporum orbiculare, u t i l i z a t i o n of acids, 334 Pityrosporum ovale, u t i l i z a t i o n of acids, 334 Plant growth regulation by mevinolin, 109-134 by s t e r o l biosynthesis i n h i b i t o r s , 109-134 Plant l i p i d s — S e e Lipids Plant steroids antifungal a c t i v i t y , 286-301 See also Steroids Plodia interpunctella, s t e r o l metabolism, 177 Podisus maculiventris, ecdysteroid

synthesi scheme, 142-144,I45f structure, I 4 l f synthesis and f u n g i s t a t i c a c t i v i t y , 140-147 Podocarpic acid derivatives chemical synthesis scheme, 142-144,I45f structure, I 4 l f Podocarpus cupressins, i s o l a t i o n of podocarpic acid, 140 Podocarpus dacrydioides, i s o l a t i o n of podocarpic acid, 140 Potato—See Solanum tuberosum Potato leaves, polar l i p i d composition, 345t Pseudomonas lachrymans, i n f e c t i o n of tobacco, 101 Pseudomonas solanacearum, resistance in plant roots, 298 Pythium prolatum, sphingolipid content, 345 Pythium ultimum, release of amino acids, 291

R Raphanus sativus, c u l t i v a t i o n of seedlings, 116 Resin composition and conifer resistance, 79-81 Rhabditis maupasi, n u t r i t i o n a l requirement for s t e r o l s , 201 Rhizoctonia s o l a n i , i n f e c t i o n of tubers, 295,298 Rhizopus nigricans, inoculation of cotton tissues, 103 Rhizopus s t o l o n i f e r growth i n h i b i t i o n , 278 use of cerulenin to distinguish, 356

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

INDEX

373

Rhodospirillaceae, content, 244

hopanoid

S Saccharomyces cerevisiae anaerobic growth, 269 entry of protons into c e l l s , 281 growth i n h i b i t i o n , 279-280 growth response to sterols, 262t phosphate and thiamine uptake, 331-334 primary s t e r o l , 255-256 sparing a c t i v i t y of cholesterol for ergosterol, 264t s t e r o l content, 274,276 structure-function relationship of sterols, 252-265 Saccharomyces l i p o l y t i c a , membrane transport, 33 Saccharomyces uvarum, f a t t y acid membrane transport, 331 Salvia o f f i c i n a l i s , use i n synthesis of monoterpene o l e f i n s , 86-87 Saponins as resistance factors, 297-300 fungitoxicity, 287-291 steroids, 292-293f Saprolegnia ferax mechanism of C-24 a l k y l a t i o n , 319f radiolabeling feeds, 316-317t Saprolegnia p a r a s i t i c a , growth i n h i b i t i o n , 337 Schizophyllum commune, transformations of compactin, 115 Scolytus v e n t r a l i s , interactions with grand f i r , 79 Septoria l i n i c o l a , growth i n h i b i t i o n , 290,295 Septoria l y c o p e r s i c i , tomatine inactivation, 297 Sesquiterpenes a l l e l o p a t h i c agents, 93-104 as phytoalexins, 93 structures, 93-98f Sesquiterpenoid stress metabolites from cotton, structure, 97f from eggplant, structure, 95f from elm, structure, 97f from potato, structure, 94f from sweet pepper, structure, 95f from sweet potato, structure, 96f from tobacco, structure, 95f Silybum marianum, c e l l suspension cultures, 1l6,126,127t S i t o s t e r o l , structure, 179f Solanum melongena, as a source of sesquiterpenoid phytoalexins, 101 Solanum s t e r o i d a l glycoalkaloids, 289f Solanum tuberosum infection by Phytophthora infestans, 336

Solanum tuberosum—Continued phytoalexin research, 100 Sordaria fimicola, growth i n h i b i t i o n , 280 Spirostane skeleton, 288f Spodoptera frugiperda, s t e r o l metabolism, 177 Staphylococcus aureus, s t e r o l synthesis, 240 Stearic acid, structure, 4t Steinernema f e l t i a e , n u t r i t i o n a l requirement f o r s t e r o l s , 201 Steroids found i n plants, major groups, 287t v a r i a b i l i t y of metabolism among phytophagous insects, 176-185 Sterols biosynthesis, 240,241f,264-265

general discussion, 7 in membranes, function, 240-242 in nematodes metabolism and function, 200-215 n u t r i t i o n a l requirement, 201-202 i n Saccharomyces cerevisiae, structure-function relationships, 252-265 inhibitors names, 270-271t structures, 272-273f isolated from commercial sources, 310-311t metabolism i n Caenorhabditis elegans, 208-213 metabolism i n f r e e - l i v i n g nematodes, 203-207 occurrence, 239-240 structural v a r i a b i l i t y , 252-255 synthesis i n h i b i t o r s , 129,130f Stigmasterol, structure, 179f Streptomyces scabies, e f f e c t of tuber alkyloids, 298 Sulfoquinovosyldiacylglycerol biosynthesis, 20-22 positional d i s t r i b u t i o n i n l e a f glycerolipids, 15-16,17t Sweet potato—See Ipomoea batatus Synecephalastrum nigricans, hydroxylation of compactin, 110

Τ

Tanacetum vulgare, c y c l i z a t i o n s of geranyl pyrophosphate, 87 Taphrina deformans growth i n h i b i t i o n , 280,281 s t e r o l i n h i b i t o r studies, 276 Teasterone, IUPAC equivalent name, 57 Tenebrio molitor, s t e r o l l e v e l s , 180 Tetrahymena, s t e r o l metabolism, 249

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

374

ECOLOGY AND METABOLISM OF PLANT LIPIDS

Tetrahymena pyriformis, s t e r o l metabolism, 260 Thamnidium elegans, synthesis of unsaturated compounds, 332 Thea sinensis, presence of 28-norbrassinone, 59 Tobacco—See Nicotiana species Tobacco hornworm—See Manduca sexta Tomatine effect of pH on binding to cholesterol, 294f effect on release of peroxidase from liposomes, 296t Tomato—See Lycopersicon esculentum Torulopsis glabrata depositions of c h i t i n , 278 oxidative removal of methyl groups, 269 sterol accumulation, 27 s t e r o l i n h i b i t i o n , 277t Tribolium castaneum, s t e r o metabolism, 180 Tribolium confusum, s t e r o l metabolism, 180 Trichoderma v i r i d e , studies of fatty acids, 223 Trichophyton i n t e r d i g i t a l e , studies of fatty acids, 222 Trichophyton mentagrophytes, studies of fatty acids, 223 Trichophyton purpureum, studies of fatty acids, 222 Trichosporium, resin production i n conifers, 81 Trichosporium symbioticum, interactions with grand f i r , 80 Trichostrongylus colubriformis, i s o l a t i o n of hormones, 214 Triticum aestivum, seedling c u l t i v a t i o n , 116 Trogoderma granarium, s t e r o l metabolism, 183-184 Turbatrix a c e t i , n u t r i t i o n a l studies, 201,203-205

Typhasterol b i o l o g i c a l a c t i v i t y , 60 IUPAC equivalent name, 57

U Ulmus glabra, i s o l a t i o n of sesqui­ terpenes, 103 Uridinediphosphate-galactose, 18 Uromyces phaseoli, infection of bean plants, 344 Ustilago maydis s t e r o l content, 280 sterol i n h i b i t o r studies, 276 V

V e r t i c i l l i u m albo-atrum, inoculation of cotton tissues, 103 Vertrum steroidal alkaloids, 294f Vibrio parahaemolyticus, f a t t y acid studies, 226 W Waxes, as l i p i d s i n organisms, 7 Y Yeast general discussion, 255-256 importance of methyl groups, 261-263 Ζ

Zea mays, dwarf mutants, 35-39 Zymomonas mobilis, hopanoid content, 246,247f

Production by Cara Aldridge Young Jacket design by Pamela Lewis Elements typeset by Hot Type Ltd., Washington, DC Pnnted and bound by Maple Press Co., York, PA

In Ecology and Metabolism of Plant Lipids; Fuller, G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.