Plant Growth Substances 9780841205185, 9780841206762, 0-8412-0518-3

Table of contents :
Title Page......Page 1
Half Title Page......Page 3
Copyright......Page 4
ACS Symposium Series......Page 5
FOREWORD......Page 6
PdftkEmptyString......Page 0
PREFACE......Page 7
1 Chemistry and Physiology of Conjugates of Indole-3-Acetic Acid......Page 9
The Structure and Concentrations of Indoles of Zea mays......Page 10
Methods of Assay......Page 11
Metabolic "Turnover" of Plant Indoles......Page 15
The "Seed Auxin Precursor"......Page 17
The Equilibrium Between IAA and IAA-myo-inositol in vivo......Page 18
Is the Equilibrium Between IAA and its Conjugates Perturable by an Environmental Input......Page 19
An Hypothesis Concerning Hormonal Homeostasis......Page 20
Abstract......Page 22
Literature Cited......Page 23
2 Aspects of Gibberellin Chemistry......Page 26
Stability......Page 27
Qualitative and Quantitative Analysis......Page 28
The Preparation of Less-readily Available GAs......Page 45
The Preparation of Labeled GAs......Page 53
Structure-Activity Relationships......Page 57
Conclusions......Page 58
Literature Cited......Page 59
3 Gibberellin Biosynthesis in the Fungus Gibberella fujikuroi and in Higher Plants......Page 64
From MVA to GA12-aldehyde in the Fungus and in Higher Plants (Figure 2)......Page 66
GA12-Aldehyde and the Gibberellins......Page 68
GAs and Shoot Elongation......Page 77
Literature Cited......Page 80
4 Anticytokinins as Probes of Cytokinin Utilization......Page 86
Tobacco Explants are Used for the Bioassay of Cytokinins......Page 87
A Rationale for the Preparation of Specific Anticytokinins......Page 89
Substituted Pyrazolo [4,3-d]pyrimidines as Potential Anticytokinins......Page 90
Substituted Pyrrolo[2,3-d]pyrimidines as Potential Anticytokinins......Page 93
Anticytokinins Elicit Responses in Several Plant Bioassays......Page 101
Literature Cited......Page 102
Historical Background......Page 106
Methods for Detection and Measurement of Abscisic Acid......Page 107
Occurrence......Page 109
Metabolism of Abscisic Acid......Page 110
Physiological Roles of Abscisic Acid......Page 114
Concluding Remarks......Page 118
Literature Cited......Page 119
Ethylene Biosynthesis......Page 122
Ethylene Action......Page 123
Conclusions......Page 139
Literature Cited......Page 140
7 Natural Products in Plant Growth Regulation......Page 142
Aliphatic Compounds.......Page 144
Unsaturated Lactones.......Page 146
Fatty Acids and Other Lipids.......Page 151
Aromatic Compounds.......Page 153
Terpenoids.......Page 158
Steroids.......Page 167
Alkaloids and N-Heterocycles.......Page 173
Purines and Nucleosides.......Page 174
Miscellaneous Natural Products.......Page 184
New Plant Growth Regulators From USDA Laboratories......Page 190
Growth Substances Involved in Biochemical Interactions between Plants in Natural Habitat - Allelopathy......Page 201
Mechanism of Action......Page 203
2) Effect on Gibberellin or Auxin-Induced Growth.......Page 204
3) Effect on Sulfhydryl Enzymes.......Page 205
4) Effect on Ethylene Production.......Page 206
Problems and Prospects......Page 207
Acknowledgments......Page 209
Literature Cited......Page 210
Qualitative vs. Quantitative Analysis.......Page 221
Use of HPLC for PGS Analysis.......Page 228
Other Selective Detection Procedures for PGS.......Page 240
Futher Refinement of PGS Analysis.......Page 241
Literature Cited.......Page 247
9 Hormonal Regulation of Genome Activity in Higher Plants......Page 251
Hormone Receptors......Page 252
Regulation of Nuclear RNA Polymerase Activity......Page 253
Regulation of the Formation of Messenger RNAs for Specific Enzymes......Page 256
Do Hormones Regulate Genome Activity?......Page 258
LITERATURE CITED......Page 261
Uses of Growth Regulants......Page 268
Pineapple.......Page 269
Sugarcane.......Page 270
Citrus.......Page 271
Cotton......Page 272
Fruit.......Page 273
Gibberellins......Page 274
Control of Plant Size......Page 275
Biochemistry and Cell Biology......Page 276
Soybeans......Page 279
Temperate Fruits.......Page 280
Sugarcane......Page 281
Discussion and Remarks......Page 282
Literature Cited......Page 283
11 Factors Affecting Commercialization of Specialty-Use Plant Growth Regulating Chemicals......Page 285
Scientific Factors......Page 286
Economic Factors......Page 290
Future Of Specialty-Use Compounds......Page 295
REFERENCES......Page 296
A......Page 298
C......Page 299
E......Page 301
G......Page 302
I......Page 304
M......Page 305
P......Page 306
R......Page 308
S......Page 309
X......Page 310
Z......Page 311

Citation preview

PLANT GROWTH SUBSTANCES

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Plant Growth Substances Ν.

Bhushan Mandava,

EDITOR

Beltsville Agricultural Research Center

Based on a symposium presented at the 13th Middle Atlantic Regional Meeting of the American Chemical Society at West Long Branch, New Jersey, March 19-23, 1979.

ACS

SYMPOSIUM AMERICAN

CHEMICAL

WASHINGTON, D. C.

SERIES SOCIETY 1979

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

111

Library of Congress CIP Data Plant growth substances. (ACS symposium series; 111 ISSN 0097-6156) Based on a symposium presented at the 13th Middle Atlantic Regional Meeting of the American Chemical Society at West Long Branch, New Jersey, March 1923, 1979. Includes bibliographies and index. 1. Plant regulators—Congresses. I. Mandava, N. Bhushan, 1934. II. American Chemical Society. III. Series: American Chemical Society. ACS symposium series; 111. SB128.P57 ISBN 0-8412-0518-3

581.3'1 79-18933 ACSMC8 111 1-310 1979

Copyright © 1979 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article 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. 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 new collective works, for resale, or for information storage and retrieval systems. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval byACSof 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. P R I N T E D IN THE U N I T E D

STATES

OF

AMERICA

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

ACS Symposium Series M . Joan Comstock, Series Editor

Advisory Board K e n n e t h B . Bischoff

James P. Lodge

D o n a l d G . Crosby

J o h n L . Margrave

Robert E . Feeney

Leon

Jeremiah P. Freeman

F. Sherwood

E. Desmond Goddard

A l a n C. Sartorelli

Jack H a l p e r n

R a y m o n d B . Seymour

Robert A . Hofstader

Aaron W o l d

James D . Idol, J r .

Gunter

Petrakis Rowland

Zweig

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

FOREWORD T h e A C S SYMPOSIUM SERIES was founded in 1974

to provide

a medium for publishing symposia quickly in book form. T h e format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that in order to save time the papers are not typese

bu

reproduced

the

sub

mitted by the author viewed 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 since symposia may embrace both types of presentation.

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PREFACE T ) l a n t growth substances occupy an important place in the growth and -*· developmental processes of all plant species. Although Charles Darwin, Boycen-Jensen, and many others were credited for their pioneering work i n recognizing that the plant growth phenomenon was under the control of some chemical substances produced by the plants, an actual beginning for the hormone concept was made in 1928 when F . W . Went successfully demonstrated the existence of growth-regulating substances in plants. In fact, two landmark events took place simultaneously in this field. Went and his co-workers in the West discovered auxins from oat seedlings and Kurosawa Gibberella jujikuroi, the later were identified as a group belonging to indoles derived from tryptophan; all gibberellins share a common basic enf-gibberellane (gibbane) skeleton. Recent additions to these two groups of natural plant growth substances are cytokinins (1964) with a common 6-aminopurine ring system, abscisic acid (1967), a growth inhibitor, and a fruit-ripening agent, ethylene (1962). Thus a correlation was made between the chemical structures and the physiological responses of these compounds. It now is believed that they are present in all plant species, particularly in higher plants, and that plant growth is regulated by these compounds, commonly known as plant hormones. Several secondary plant products such as phenolics, lipids, steroids, and terpenoids also were shown to be responsible for growth and development and some of them elicit growth responses in conjunction with these endogenous growth hormones. Several synthetic compounds, although different from the natural growth substances, also induce similar biological responses. There was considerable interest in synthetic growth substance research mainly in view of practical applications and some have found limited agricultural uses. Because of the presence of natural plant growth substances in very minute amounts, the progress i n this field, particularly in the characterization, was really slow until the recent introduction of new analytical methods. Food production i n the last few decades has been improved greatly by applying chemical fertilizers, irrigation methods, rotation of crops, and plant-breeding practices. Despite these new technologies for increased food production, the crop losses caused by insect pests, pathogens, ix

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

and weeds are so great that i n the United States alone the resulting damage amounts to several billion dollars. It appears likely that future research (e.g., allelopathic compounds) w i l l provide a solution to this problem, and proper pest management by natural methods might further increase agricultural production. In view of the increasing worldwide demand for food, feed, energy, and safe environment, several new approaches are being sought now for increasing agricultural productivity, particularly the crop and biomass production, by modifying such plant control mechanisms as photosynthetic efficiency, light- and stress-related phenomena, and nitrogen fixation of the plants. Undoubtedly, plant growth substances play a key role in understanding these physiological processes. It was considered timely for the American Chemical Society to take an inventory by reviewin chemistry, and physiology plan growt symposiu at the Middle Atlantic Regional Meeting ( M A R M ) i n March, 1979. The titles and subjects for this symposium were selected to provide broad coverage in both synthetic and natural growth substances including the analytical methods for their detection and characterization. The partici­ pants, experts in their respective fields, were chosen to provide a wellbalanced program covering auxins, gibberellins, cytokinins, abscisic acid, ethylene and other natural products, and synthetic growth substances. Nine chapters on natural plant growth substances are followed by two chapters on synthetic growth regulators. It is hoped that this volume w i l l initiate and stimulate work by chemists, biochemists, plant physiologists, and other related scientists. This multidisciplinary approach provides a better understanding of plant internal control mechanisms via growth substances and results i n finding practical applications of these compounds for increasing agricultural productivity. U.S. Department of Agriculture

N . BHUSHAN MANDAVA

Beltsville, MD June 19, 1979

χ In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1 Chemistry and Physiology of Conjugates of Indole-3-Acetic

Acid

ROBERT S. BANDURSKI Department of Botany and Plant Pathology, Michigan State University, East Lansing, MI 48824 Auxins are hormone t h a t promot p l a n t growth The n a t u r a l l y and, i n s t r u c t u r e r i n g , an u n s u b s t i t u t e d , electronegatively positio ortho to a side chain o f , at l e a s t , two carbons, and w i t h a c a r boxyl group on the s i d e chain 2_). Examples of auxins are i n d o l e - 3 - a c e t i c a c i d ( 3 J , p h e n y l a c e t i c a c i d ( 4 J , and 4 - c h l o r o i n d o l e - 3 - a c e t i c a c i d (_5, 6). Knowledge o f the s t r u c t u r e o f i n d o l e - 3 - a c e t i c acid--IAA--(7_) l e d to the chemical p l a n t growth r e g u l a t o r i n d u s t r y w i t h annual a g r i c u l t u r a l savings i n Michigan alone equal to the cost o f a l l U . S . financed plant hormone research. U t i l i z a t i o n of auxins date to a n t i q u i t y , as f o r example, the use of germinating seeds to promote r o o t i n g o f c u t t i n g s ( 8 ) . The d i s c o v e r y of hormones i n roots by the P o l i s h h o r t i c u l t u r a l i s t , T e o f i l C i e s i e l s k i {9) and i n shoots by the B r i t i s h n a t u r a l i s t , Charles Darwin (lOJ was made one century ago. They observed t h a t the t i p o f the root or shoot c o n t r o l l e d growth of the t i s s u e some distance from the t i p . Thus, an " i n f l u e n c e " must have d i f f u s e d from t i p to growing r e g i o n . S i x t y years l a t e r , F.W. Went developed the "Avena curvature t e s t " f o r the " i n f l u e n c e " (3)* and Bonner developed the " s t r a i g h t growth assay" (11,). These t e c h niques plus knowledge that tryptophan could be converted to auxin i n fungal c u l t u r e s (12) and t h a t "precursors" i n seeds could be converted to auxins by a l k a l i n e h y d r o l y s i s (13^, 14_, 15_, 16_) l e d to knowledge o f the s t r u c t u r e o f IAA (7_). We s t r e s s these assays because, w h i l e l e a d i n g to the d i s covery o f IAA, they imposed s t r u c t u r e - a c t i v i t y requirements prec l u d i n g study of the IAA conjugates—and p o s s i b l y the d i s c o v e r y o f other a u x i n s . For a substance to be a c t i v e i n the assays r e q u i r e d t h a t they: 1) permeate membranes i n a cut t i s s u e s u r f a c e , 2) be transported to the growing zone, and 3) promote growth i n t h a t zone. Hopefully these three requirements may someday be s t u d i e d independently. The b i o l o g i c a l e f f e c t s o f the auxins are d i v e r s e and range from r a p i d e f f e c t s , u s u a l l y growth promotion and o c c u r r i n g w i t h i n 0-8412-0518-3/79/47-lll-001$05.00/0 © 1979 American Chemical Society In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2

PLANT GROWTH SUBSTANCES

minutes, to growth d i f f e r e n t i a t i o n , obvious o n l y i n days ( 1 7 ) . How IAA can e l i c i t profound changes i n the s i z e and form o f a p l a n t i s t o t a l l y unknown. At the c e l l u l a r l e v e l i t i s known t h a t the p l a n t c e l l w a l l s must be "softened" f o r growth promotion to occur ( c f . 1 8 ) . Such e f f e c t s , however, may be concomitants o f growth and not the "primary" e f f e c t s of auxin ( 1 9 J . We suggest t h a t research d i r e c t e d towards c o r r e l a t i n g and developing b i o l o g i c a l and physiochemical assays o f the a u x i n s , s t r u c t u r a l c h a r a c t e r i z a t i o n o f auxins and auxin conjugates and s t u d i e s of what permits auxins to move to d i f f e r e n t parts o f the p l a n t and, perhaps s e l e c t i v e l y i n t o d i f f e r e n t o r g a n e l l e s , w i l l be p r o d u c t i v e approaches. Thus, I s h a l l confine my remarks t o , 1) the chemistry o f IAA conjugates, 2) the q u a n t i t a t i v e assay o f IAA and i t s conjugates, 3) the "turnover" o f the i n d o l y l i c compounds of the p l a n t , 4) an i n d i c a t i o n of how knowledge of pool s i z e and turnover permitted i d e n t i f i c a t i o a demonstration o f the e q u i l i b r i u 6) a demonstration o f the p e r t u r b a b i l i t y o f the e q u i l i b r i u m and, l a s t l y , 7) a working hypothesis concerning how a hormonal homeos t a t i c system can be attuned to the environment. The

S t r u c t u r e and Concentrations o f Indoles o f Zea mays

Figure 1 summarizes the s t r u c t u r e s and c o n c e n t r a t i o n s o f the IAA conjugates o f the kernels o f corn {Zea mays), the only p l a n t to have been s t u d i e d i n d e t a i l . This work was done by my c o l leagues Drs. Labarca, N i c h o l l s , Ueda, P i s k o r n i k and Ehmann (2025). We have not detected a p p r e c i a b l e amide l i n k e d IAA i n Zea but there are three major c l a s s e s o f e s t e r s : the IAA-zm/tf-inositols, c o n s t i t u t i n g about 15%; the I A A - r a / o - i n o s i t o i g l y c o s i d e s , about 25%; and the high molecular weight IAA 31+4 g l u c a n , about 50% o f the t o t a l IAA. Free IAA, the 2 - 0 , 4-0 and 6-0 IAA glucose e s t e r s and the ( I A A ) i n o s i t o l s comprise the remainder. The v e g e t a t i v e t i s s u e of corn contains 300 yg/kg fresh weight o f e s t e r IAA and 30 yg/kg o f free IAA (26.). A major p o r t i o n o f the e s t e r s o f the shoot i s IAA-tfz?/o-inositol ( c f . 27 and Nowacki, u n p u b l i s h e d ) . The seeds o f oats {Avena sativa) have been s t u d i e d by Dr. P e r c i v a l and shown to have 85% of t h e i r IAA e s t e r i f i e d to a g l u c o p r o t e i n ( 2 8 ) . The glucan i s o f the l i c h e n a n type having both 31+3 and 31+4 l i n k a g e s . R e c e n t l y , Ms. P. Hall i n our l a b o r a t o r y (personal communication) has i s o l a t e d I AA-mz/c-inositol from r i c e (Oryza sativa) thus showing the compound o r i g i n a t e d e a r l y i n cereal e v o l u t i o n . This completes our knowledge o f the chemistry o f the n a t u r a l l y o c c u r r i n g IAA conjugates. IAA-aspartate i s known to be formed f o l l o w i n g exogenous a p p l i c a t i o n o f IAA to p l a n t s o f Pisum sativum and was the f i r s t IAA conjugate to be s t r u c t u r a l l y chara c t e r i z e d ( 2 9 J . There are some data i n d i c a t i n g t h a t IAA-aspartate occurs n a t u r a l l y ( 3 0 ) . In a d d i t i o n , 1-0 g l u c o s y l IAA has been reported to be formed f o l l o w i n g a p p l i c a t i o n o f IAA to p l a n t s (31). n

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

BANDURSKi

3

Indole-3-Acetic Acid Conjugates

D e f i n i t i v e c h a r a c t e r i z a t i o n o f the b i o s y n t h e s i z e d 1-0 glucose e s t e r has not been published although the compound has been s y n ­ t h e s i z e d c h e m i c a l l y (32). Q u a n t i t a t i v e data on the amounts o f free IAA, e s t e r IAA and amide l i n k e d IAA have been s u p p l i e d by Ms. A. Schulze (33). Her d a t a , shown i n Table I , permit several c o n c l u s i o n s : f i r s t , a l l plants examined c o n t a i n g r e a t e r amounts o f IAA conjugates than free IAA; secondly, the c e r e a l s c o n t a i n mainly e s t e r IAA; and, t h i r d l y , legumes c o n t a i n mainly amide l i n k e d IAA. The d i s c o v e r y by Ms. Schulze o f the u b i q u i t y o f IAA-conjugates convinced us that c o v a l e n t l y bonded hormone conjugates were of metabolic importance. E s p e c i a l l y s i g n i f i c a n t was the occur­ rence o f IAA conjugates i n seedlings where growth r a t e i s a func­ t i o n o f IAA c o n c e n t r a t i o n . We concluded t h a t , i f an e q u i l i b r i u m e x i s t s between IAA and i t s conjugates, then anything t h a t s h i f t s t h a t e q u i l i b r i u m would a f f e c system f o r hormonal homeostasi r e q u i r e d , one, a more convenient and s e n s i t i v e assay f o r IAA and, two, knowledge of pool s i z e and turnover rates o f the i n d o l y l i c components o f the p l a n t . Methods o f Assay B i o - a s s a y s , i n the hands o f c a r e f u l workers ( c f . 3)> have provided almost a l l o f our knowledge of p l a n t hormonal metabolism. Such assays, however, measure a c t i v i t i e s o f e x t r a c t s and are not equatable to amounts o f a chemical e n t i t y . Thus, one must r e l y upon both chemical and b i o a s s a y s . With regard to chemical assays, i t i s our contention t h a t the planar i n d o l e s t r u c t u r e and the number o f π bonding e l e c t r o n s renders IAA so unstable t h a t r e c o v e r i e s w i l l be both low and v a r i a b l e thus making i n t e r n a l standards o b l i g a t o r y ( c f . 33, 3 4 ) . We f i r s t used C - I A A - i s o t o p e d i l u t i o n assays i n 1961 ( 3 5 j and r e f i n e d the assays i n 1974 ( 3 3 ) . The o r i g i n a l assays r e q u i r e d kilogram amounts o f t i s s u e and a week to o b t a i n a s i n g l e v a l u e . More r e c e n t l y we developed two new assay procedures which permit one assay i n a day or two o f work and r e q u i r e only 10 to 50 grams o f t i s s u e (36, 3 7 ) . Our assays are l a b o r i o u s but, i n t h i s e a r l y stage o f chemical assays o f IAA, such l a b o r may be d e s i r a b l e . One method i n v o l v e s the use of 4 , 5 , 6 , 7 t e t r a d e u t e r o - I A A , r e c e n t l y synthesized by Dr. V . Magnus ( 3 6 ) . There are two advan­ tages to use o f t h i s as an i n t e r n a l standard: f i r s t , the deu­ terium i n these p o s i t i o n s i s s t a b l e to the a l k a l i n e h y d r o l y s i s we use to assay the IAA i n the conjugates, and s e c o n d l y , the pre­ sence o f four deuterium i n the standard moves the ions o f the standard away from the isotope c l u s t e r normally observed i n mass spectrometry owing to the n a t u r a l l y o c c u r r i n g heavy i s o t o p e s . We add d^-IAA, plus a t r a c e o f C - I A A , to the acetone i n which we homogenize the p l a n t m a t e r i a l . Then, wit h or without a l k a l i n e h y d r o l y s i s , depending upon whether we wish to measure free or 14

1I+

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

acid

)-myo-\inositol

5.6%

15.4

23.2%

15.2% 10.1

4.7%

0.8%

10.5%

PERCENT OF TOTAL

0.5

IN DRY SEED MG/KG

5-£-6-L-arabinopyranosyl-l-DL( i n d o l e - 3 - a c e t y l ) - m y o - \ nos i t o i

AMOUNT

17.6%

STRUCTURE

5-0-3-L-arabinopyranosyl-2-0( i n d o l e - 3 - a c e t y l ) - m y ο-Λnosi t o i

Indoleacetylinositoi-arabinosides

l-DL-(indole-3-acetyl

2-C-(indole-3-acetyl)-mz/o-inositoi

Indoleacetylinositols

Indole-3-acetic

COMPOUND

Ο Μ

•

Η

S d w

H

I

S

H

>

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

compounds

toi

itoi

Figure 1.

(indole-3-acetyl)-glucan

LOW M . W . COMPOUNDS — TOTAL 3 1 4 c e l l u l o s i c glucan with 7 t o 50 g l u c o s e u n i t s p e r IAA

2

CHOH

0.05

47.6%

52.5% 35.0

0.3%

0.2

31.2

.1%

5.4

The structure and concentration of indolylic compounds in kernels of Zea mays

6-0-(indole-3-acetyl)-D-glucopyranose

4-0-(indole-3-acetyl)-0-glucopyranose

2-0-(indole-3-acetyl)-D-glucopyranose

Tri-Ο-(i ndole-3-acetyl)-myo-inosi

Di-0-(i ndole-3-acetyl)-myo-\nos

Trace

5 - o - a - L - g a l a c t o p y r a n o s y l -2-0(indole-3-acetyl )-ra/oinositoi

n

Ci

sCιιO

§•

>

6

PLANT GROWTH SUBSTANCES

TABLE I Concentrations o f Free and Bound IAA i n Various P l a n t Tissues Species

IAA content

Tissue Free IAA

1

E s t e r IAA

2

Peptidyl IAA 3

yg/kg CEREALS Avena

sativa

Avena sativa Hordeum vulgare

vegetative

seed 40 (milled) 1703 seed 366 seed 123 seed vegetative 24 tissue 500 to seed 1000

329 2739 3198 511

-

328 71600 to 78500

60

5

Ovyza sativa Panicum miliaceum Triticum aestivum Zea mays Zea mays

-

LEGUMES Glycine max Phaseolus vulgaris Pisum sativum Pisum

sativum

seed seed vegetative tissue seed

4 20

50 30

5

5 5

35 93

5 n.d..

0 40 30 trace

905 127 no trace

524 136 43 202

OTHERS liquid endosperm Fagopyrum esculentum seed seed Helianthus annus Lycopersicum esculentum f r u i t Saccharomyces cevevisea packed cells Cocas

nucifera

5

290

5

n.d.

-

25

-

*No a l k a l i n e h y d r o l y s i s . I A A a f t e r h y d r o l y s i s with 1 Ν a l k a l i minus the free IAA. I A A a f t e r h y d r o l y s i s wit h 7 Ν a l k a l i minus the free and e s t e r IAA. ^Seedlings and f r u i t s are fresh weight, seeds are a i r dry and yeast c e l l s c o n t a i n 30% dry m a t t e r . A v i s u a l estimate o f IAA on a TLC p l a t e as c o l o r i m e t r y was pre­ cluded by contaminants. 2

3

5

Reprinted by permission o f P l a n t Physiology ( 3 3 ) .

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

BANDURSKi

7

Indole-3-Acetic Acid Conjugates

t o t a l IAA, we r e - i s o l a t e the IAA by p a r t i t i o n i n g , DEAE-sephadex, and high pressure l i q u i d chromatography. The r e s u l t a n t mixture of d^-IAA and the p l a n t d e r i v e d IAA i s methylated and then the i n d o l e n i t r o g e n i s a c y l a t e d w i t h h e p t a f l u o r o b u t y r i c anhydride. The methylheptafluorobutyryl IAA d e r i v a t i v e i s used f o r gas chromatography-selected i o n mass spectrometry (gc-sim-ms). As shown i n Figure 2 , we monitor four masses, 385 and 389, the molecular i o n f o r methylheptafluorobutyryl IAA (and i t s d a n a l o g ) , and 326 and 330, the base peaks f o r IAA and the d s t a n dard. The agreement between the r a t i o s o f d - I A A to IAA at the molecular i o n and at base peak g i v e assurance o f the v a l i ' i t y o f the assay. Is t h i s f i n a l l y an absolute assay, g i v i n g data such t h a t e r r o r i s impossible? We t h i n k i t i s c l o s e i n t h a t f o r an e r r o r to occur a compound would have t o c o f r a c t i o n a t e w i t h IAA on a DEAE and HPLC column and coemerge from the gc column and then y i e l d the same percentage S t i l l , we do o c c a s i o n a l l warn other workers t h a t d e a l i n g w i t h nanogram amounts o f i n d o l e s is d i f f i c u l t . A second method o f assay o f IAA has been developed by Mr. J . Cohen and i n v o l v e s a "double i n t e r n a l standard" u s u a l l y C - I A A and C - i n d o l e - 3 - b u t y r i c a c i d ( 3 7 ) . I w i l l not discuss t h i s method o f assay except to i n d i c a t e t h a t i t i s p o s s i b l e to develop assays not i n v o l v i n g mass spectrometry but w i t h comparable s e n s i t i v i t y and good s e l e c t i v i t y . k

4

4

U

11+

M e t a b o l i c "Turnover" o f P l a n t

Indoles

Ms. Pat H a l l and D r s . J . Nowacki and E . E p s t e i n have provided our knowledge o f the amounts and r a t e o f metabolic turnover of the i n d o l y l i c components o f the kernels o f Zea mays ( c f . 27, 28; Nowacki, unpublished; E p s t e i n , unpublished). This knowledge ïïâs enabled u s , 1) to i d e n t i f y the "seed auxin p r e c u r s o r " - - t h a t i s the compound which i s transported from the seed to the growing shoot ( 3 9 ) , and 2) has provided a p o r t i o n o f the proof t h a t IAAra/o-inositol and IAA are i n r e v e r s i b l e e q u i l i b r i u m i n the shoot tissue. Proving t h a t IAA and IAA e s t e r s are i n r e v e r s i b l e e q u i l i b r i u m i n the t i s s u e i s e s s e n t i a l i f we wish to p o s t u l a t e hormonal homeostasis. These experiments r e q u i r e d l a b e l e d IAA and tryptophan, which are a v a i l a b l e commercially, and C - l a b e l e d I A A - z m / o - i n o s i t o l . This compound was synthesized by D r . Nowacki by r e a c t i n g C-IAAimidazole w i t h m ^ - i n o s i t o l ( 4 0 ) . A p p l i c a t i o n o f these l a b e l e d compounds to corn k e r n e l s , followed immediately by homogenization of the t i s s u e i n acetone permitted us to determine the amounts o f each constiuent i n the kernel by the isotope d i l u t i o n method o f R i t t e n b e r g and Foster ( 4 1 0 · An extension o f t h i s method, whereby the kernels are incubated f o r v a r y i n g periods o f time a f t e r a p p l i c a t i o n o f the i s o t o p i c a l l y l a b e l e d compound permits determination o f the "turnover" o f the p o o l . Such data are shown i n 1I+

1 4

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

8

PLANT GROWTH SUBSTANCES

HYDROLYZED CORN

A-3717 389.2

A

A-97II

^—

J I\

385.2 Av. 2 8 . 1

% d

4

A* 9 0 2 4

/Λ 330.2 Λ

|1

28.4%

/ ;\

1 7

4

Α · 31826

/!V

326.2 -

d

β

-— 9

TIME (MIN.)

Figure 2. Selected ion chromât ο gram of a mixture of the methyl esters of tetrafluorobutyryl IAA and d IAA. The IAA was from an extract of corn seedlings and the d -IAA added during homogenization. Retention time is in minutes and the masses monitored are 326.2 and 385.2 for IAA and 330.2 and 309.2 for d -IAA. The percent d -IAA has been computed by the area of the peaks at 330.2/326.2 + 330.2 (base peak) and 389.2/385.2 + 389.2 (molecular ion). r

h

k

h

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

BANDURSKi

9

Indole-3-Acetic Acid Conjugates

Table I I . They showed t h a t tryptophan, IAA and I A A - T m / s - i n o s i t o l are t u r n i n g o v e r - - t h a t i s made and then d e s t r o y e d , or used, at rates such t h a t t^ was 5, 3.2 and 12 h r s , r e s p e c t i v e l y . Such data permitted several important conclusions concerning the metabolism o f these compounds, f o r example, t h a t i t i s the IAA e s t e r s , and not tryptophan, which serve as a source o f IAA f o r the germinating seed and secondly t h a t the I A A - i n o s i t o i s are t u r n i n g over at such a r a p i d r a t e t h a t they must be i n e q u i l i brium w i t h the I A A - r a / 2 - i n o s i t o l g l y c o s i d e pool perhaps a c t i n g as g l y c o s y l a t i o n reagents ( E p s t e i n , u n p u b l i s h e d ) . TABLE II Concentration and M e t a b o l i c Turnover of Some I n d o l y l i c Compounds i n Zea Kernels

Compound

Incubatio Time hrs

dpm/yg

0

31,000

4

8,900

8

5,400

0

15,200

8

5,000

0

935

8

590

IAA

Tryptophan

IAA-mz/o-inositol

The "Seed Auxin

Sp. A c t .

k hrs

- 1

hrs

0.22

3.2

0.14

5.0

0.06

12.0

Precursor"

Of great importance, f o r these s t u d i e s , was knowledge o f both pool s i z e and t u r n o v e r . T h i s knowledge permitted c a l c u l a t i o n of the s p e c i f i c a c t i v i t y o f the a p p l i e d i s o t o p i c a l l y l a b e l e d compound at any d e s i r e d t i m e . Thus, r a d i o a c t i v i t y , from a l a b e l e d compound a p p l i e d to the endosperm and appearing i n the shoot could be t r a n s l a t e d i n amounts o f compound moved from seed to shoot. For these experiments, minute amounts o f l a b e l e d IAA, t r y p t o phan, or I A A - r a / o - i n o s i t o l were a p p l i e d to an i n c i s i o n i n the semil i q u i d endosperm o f 4 day germinated Zea s e e d l i n g s . A f t e r 8 hrs o f i n c u b a t i o n , the shoots were harvested and the IAA, tryptophan or IAA-777z/0-inositol i s o l a t e d using r i g o r o u s p u r i f i c a t i o n t e c h niques. Now, knowing the s p e c i f i c a c t i v i t y o f the a p p l i e d compound at the m i d - p o i n t of the experiment we could c a l c u l a t e the

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

10

PLANT GROWTH

SUBSTANCES

average r a t e of t r a n s p o r t o f t h a t compound from endosperm to shoot. A summary o f a p o r t i o n o f these data ( c f . 27/, Nowacki, unpublished; E p s t e i n , unpublished) i s shown i n Table I I I . As can be seen IAA-Tm/s-inositol moves from endosperm to shoot at a r a t e of 6 p m o l s - s h o o f ^ h r . We had p r e v i o u s l y estimated t h a t about 9 p m o l S ' S h o o t - ^ h r " of IAA compound must be moving from endosperm to shoot to s u s t a i n i n d o l e concentrations i n the shoot (38) and G i l l e s p i e and Thimann (42)measured 5 p m o l s - s h o o t ^ - h r - of IAA d i f f u s i n g down from e x c i s e d Zea t i p s . On the assumption t h a t what goes down must come up, at l e a s t 5 p m o l s - s h o o f ^ h r " must be going up. By c o n t r a s t free IAA moves from endosperm to shoot at a r a t e of 0.015 p m o l - s h o o t " ^ h r - and tryptophan i n the endosperm appears as IAA i n the shoot at l e s s than 0.2 p m o l - s h o o t " · h r * . This l a s t f i g u r e i s high and c o u l d , i n f a c t , be zero s i n c e non-enzymatic conversion o f tryptophan to IAA occurs so r e a d i l y . Thus these data e s t a b l i s h t h a IAA-mz/s-inositol i th "seed auxin precursor" f o r Zea - 1

1

1

1

1

1

1

TABLE I I I Rate o f Transport o f I n d o l y l i c Components From Endosperm to Shoot Compound a p p l i e d to endosperm

Compound i s o l a t e d from shoot

Rate pmols.shoot-^hr0.015

3

H-IAA

IAA + e s t e r IAA

3

H-tryptophan

IAA + e s t e r IAA

0.15

IAA + e s t e r IAA

6.2

lif

C-IAA-ra/0-inositol

1

The E q u i l i b r i u m Between IAA and IAA-myo-inosito! in vivo Dr. Hamilton e a r l i e r observed t h a t ether e x t r a c t i o n of t i s s u e induced a u t o l y s i s , l i b e r a t i n g a c t i v e esterases and g l y c o s i dases, and thus l e a d i n g to more free IAA than e x t r a c t i o n of t i s s u e by p o l a r , and t h u s , enzyme-denaturing s o l v e n t s ( 3 5 ) . Thus, we knew then t h a t there were enzymes i n the t i s s u e capabTe o f h y d r o l y z i n g IAA e s t e r s . Much l a t e r , Kopcewicz demonstrated the presence o f an enzyme system which could s y n t h e s i z e Ihh-myoi n o s i t o l from IAA, ATP, M g and CoASH (43). More r e c e n t l y , Mr. Lech Michalczuk (unpublished) has shown t h a t IAA-CoA w i l l a c y l a t e i n o s i t o l only i n the presence of other n u c l e o t i d e s . Thus, the r e a c t i o n i s complex, but there i s no doubt t h a t enzymes to make and hydrolyze the IAA e s t e r s are present i n c o r n . Now, are the enzymes a c t i v e in vivo! The data of Ms. Schulze and H a l l and Nowacki, E p s t e i n and Cohen (27_, 38; Nowacki, unpubl i s h e d ; E p s t e i n , unpublished) demonstrate t h a t they a r e . We + +

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

1.

BANDURSKi

Indole-3-Acetic Acid Conjugates

11

showed i n 1972 t h a t there i s 93% e s t e r i f i e d IAA and 7% free IAA i n the shoots o f Zea s e e d l i n g s (26). The d e s i r e d experiment then was to apply l a b e l e d Ihh-myo-inositol o r l a b e l e d IAA to the endosperm of Zea k e r n e l s and determine whether we approach the same value--93% e s t e r , 7% f r e e - - s t a r t i n g e i t h e r from e s t e r o r free l a b e l e d IAA. H y d r o l y s i s or s y n t h e s i s o f e s t e r i n the endosperm becomes unimportant s i n c e the pools are so l a r g e t h a t we would not see a p p r e c i a b l e r a d i o a c t i v i t y i n the shoot i f h y d r o l y s i s or e s t e r i f i c a t i o n occurred i n the endosperm. Thus, we can determine whether e q u i l i b r i u m i s a t t a i n e d a f t e r the l a b e l e d compound enters the shoot. Mr. Nowacki a p p l i e d l a b e l e d I A A - r a ^ - i n o s i t o l to the endosperm and found 94% e s t e r and 6% free IAA i n the shoot (unp u b l i s h e d ) . Ms. H a l l a p p l i e d l a b e l e d IAA to the endosperm and found 70% e s t e r and 30% free IAA i n the shoot ( 3 8 ) . These values approximate those found f o r n a t u r a l in vivo c o n c e n t r a t i o n s by Ms. S c h u l z e . The conversio H a l l , are low but thes were aware o f the ease o f h y d r o l y s i s o f the e s t e r s . The r e s u l t s demonstrate t h a t one approaches the same e q u i l i b r i u m amounts o f e s t e r and free IAA s t a r t i n g from e i t h e r compound. We use the word e q u i l i b r i u m to denote t h a t e s t e r IAA can be hydrolyzed to free IAA and free IAA can be converted t o e s t e r IAA. We do not imply t h a t t h i s i s a r e v e r s i b l e r e a c t i o n c a t a l y z e d by a s i n g l e enzyme ( 4 3 ) . This c o n s t i t u t e s the f i r s t demonstration i n b i o l o g y of an in vivo e q u i l i b r i u m between a hormone and i t s c o v a l e n t l y l i n k e d conjugates. Is the E q u i l i b r i u m Between IAA and i t s Conjugates P e r t u r a b l e by an Environmental Input An attempt to answer the question o f whether the environment c o n t r o l s p l a n t growth by p e r t u r b i n g the e q u i l i b r i u m between free and c o v a l e n t l y conjugated hormone i s the major e f f o r t o f our l a b o r a t o r y . To date only one environmental input has been t e s t e d and t h a t i s p h o t o i n h i b i t i o n o f growth. I t has been known f o r many years t h a t a b r i e f f l a s h o f l i g h t w i l l i n h i b i t the e x t e n s i o n growth o f an e t i o l a t e d s e e d l i n g p l a n t ( c f . 4 £ ) . The question then becomes, when p h o t o i n h i b i t i o n o f growth o c c u r s , w i l l there be a concomitant decrease of free IAA and a commensurate i n c r e a s e i n e s t e r IAA? The r e s u l t s o f t h i s experiment are shown i n Table IV. A 20 second l i g h t f l a s h r e s u l t e d i n a 43% i n h i b i t i o n o f growth as measured 90 minutes a f t e r the l i g h t f l a s h . The free IAA decreased by 35% and e s t e r IAA increased by a commensurate amount (45). Thus, our working hypothesis t h a t growth i s c o n t r o l l e d by the r e l a t i v e amounts o f free and conjugated hormone and t h a t i t i s t h i s r a t i o which r e f l e c t s the environment i s , i n t h i s case, confirmed.

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

12

PLANT GROWTH

SUBSTANCES

TABLE IV Photo-Induced Change i n Growth and i n Free and Free Plus E s t e r IAA Dark

Light

Δ

%

-1.1

-34

mm/90 mi η Growth

3.6

2.6 yg/kg

Free IAA

23

13

-10

-42

Free plus e s t e r IAA

68

77

+9

+11

An Hypothesis Concerning Hormonal Homeostasis The "take home l e s s o n " I wish to leave wit h you i s t h a t the hormones, IAA, the g i b b e r e l l i n s , c y t o k i n i n s , and a b s c i s i c a c i d , a l l occur i n free and conjugated form ( 4 £ , 47, 48, 49) and t h a t anything t h a t a f f e c t s the r e l a t i v e amounts of free and conjugated hormone w i l l c o n t r o l growth. A s i m i l a r h y p o t h e s i s , concerning mainly the g i b b e r e l l i n s has been made ( 5 0 ) . In the s p e c i a l case of IAA we have demonstrated t h a t IAA i s i n e q u i l i b r i u m w i t h i t s conjugates and t h a t t h i s e q u i l i b r i u m can be s h i f t e d by l i g h t . Thus, from these l i m i t e d d a t a , we propose, as a working hypothe­ s i s , t h a t the environment a f f e c t s the r a t e of p l a n t growth by causing changes i n the r e l a t i v e amounts o f free and conjugated hormone. This concept i s i l l u s t r a t e d d i a g r a m a t i c a l l y i n Figure 3. We envisage, Figure 3, t h a t environmental s t i m u l i , as f o r example l i g h t , heat, g r a v i t y , water s t r e s s , e t c . , impact upon one or more sensory apparatuses. In the case of l i g h t t h i s would be a pigment, whereas other s t i m u l i would impact upon the p l a n t counterpart of a " s o l i o n " ( 5 1 ) . A " s o l i o n " senses changes i n heat, sound, p r e s s u r e , g r a v i t y , e t c . , using a r e v e r s i b l e redox system and a minute a p p l i e d p o t e n t i a l . Since p l a n t c e l l s have a s u i t a b l e b i o - e l e c t r i c p o t e n t i a l (52) and redox systems i n t h e i r cytoplasm, they can be, i n a very r e a l sense, " s o l i o n s " . The sensor then t r a n s f e r s i t s s i g n a l , perhaps a hydride ion from a f l a v i n , to one o f the transducer enzymes. Chemically t h i s could mean using the hydride i o n to reduce a d i s u l f i d e bond i n an en­ zyme t h a t synthesizes or hydrolyzes hormone conjugates--thus changing the a c t i v i t y o f the enzyme. I f the h y d r o l y z i n g t r a n s ­ ducer i s a c t i v a t e d then more a c t i v e hormone r e s u l t s . I f the s y n t h e s i z i n g transducer i s a c t i v a t e d then there i s l e s s free hormone and l e s s hormone e f f e c t o c c u r s . We do not know what hor­ mones do to c o n t r o l growth nor how many processes must occur f o r

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

BANDURSKi

lndole-3-Acetic Acid Conjugates

ENVIRONMENTAL STIMULUS

TRANSDUCER HORMONE

CONJUGATE

HYDROLASE FREE HORMONE + X

[HORMONE-X] HORMONE

CONJUGATE

SYNTHETASE

I GROWTH SYSTEM

Figure 3.

Diagram of a system for control of plant growth by varying the relative amounts of free and conjugated hormone

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

14

PLANT GROWTH

SUBSTANCES

growth to r e s u l t so we show a dotted arrow l e a d i n g to growth. My personal f e e l i n g i s t h a t what the hormone does cannot be d i s covered u n t i l in vitro hormone-responsive systems are a v a i l a b l e (19). Here we encounter the Heisenberg u n c e r t a i n t y p r i n c i p l e as a p p l i e d to b i o l o g y . Conclusion I wish to c l o s e now upon an o p t i m i s t i c but cautious note. U l t r a - m i c r o s c a l e chemical assays o f l a b i l e p l a n t hormones w i l l never be easy and r o u t i n e . Nonetheless, use o f i n t e r n a l standards and the detectors now a v a i l a b l e f o r gc and h p l c , or the s e n s i t i v e and s e l e c t i v e mass spectrometer, permits assays of the hormones i n 1 to 10 gm amounts o f t i s s u e thus p e r m i t t i n g p h y s i o l o g i c a l experiments. We must someday return to b i o - a s s a y s , using a d e f i n i t i v e chemical assa assay s i m u l t a n e o u s l y . Thi both hormones and high amounts o f , u s u a l l y i n h i b i t o r y , phenylpropanes, as w i l l be discussed l a t e r i n t h i s symposium. A l s o , as Professor J . van Overbeek has i n d i c a t e d (personal communication), we w i l l not understand the physiology o f the organism u n t i l we simultaneously know what happens to a l l the hormones and t h e i r conjugates during complex developmental phenomenon. D i f f i c u l t as t h i s sounds, i t w i l l be p o s s i b l e , provided we confine ourselves to a few p l a n t s and s e r i o u s l y t r y to understand the hormonal system. L a s t l y , I b e l i e v e our working hypothesis o f hormones and t h e i r conjugates as homeostatic hormone systems w i l l lead to new and answerable q u e s t i o n s . I emphasize, however, our data apply to one hormone, and to one environmental input and only to seedl i n g Zea p l a n t s . W i l l there be a g e n e r a l i t y to our working hypothesis t h a t the environment c o n t r o l s the r a t i o of free to conjugated growth hormone and thus c o n t r o l s growth? We f e e l t h a t the answer to t h i s question has great i m p l i c a t i o n s f o r the cont r o l o f food and f i b e r production by a p p l i e d growth r e g u l a t o r s . I t h i n k we should work hard to answer t h i s q u e s t i o n . Abstract Most of the indole-3-acetic acid (IAA) in plants occurs as ester or amide-linked conjugates. This preponderance and apparent ubiquity led to a study of the functions of the conjugates. Evidence for four physiological roles has been found: 1) Conjugation i s reversible and provides the plant with a way to regulate its IAA l e v e l s , and thus i t s growth rate, in accordance with the environment; 2) one of the conjugates (IAA-myo-inositol) i s the chemical form in which IAA is transported from the seed of corn to the shoot, suggesting that conjugation provides the plant with information concerning where the hormone-precursor should be del i v e r e d ; 3) IAA conjugates serve as a source of IAA for the seed

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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BANDURSKi

Indole-3-Acetic Acid Conjugates

15

and seedling; and 4) conjugation of IAA protects it against peroxidative attack. To our knowledge, this i s the first case in biology where hormone levels are controlled by the formation and hydrolysis of a covalent bond. Acknowledgements The support o f the M e t a b o l i c B i o l o g y S e c t i o n o f the National Science Foundation PCM 76-12356 i s acknowledged. Without sust a i n e d support t h i s work could not have been accomplished. This i s j o u r n a l a r t i c l e 8963 from the Michigan A g r i c u l t u r a l Experiment Station. I am indebted to my colleagues D r s . Cohen, E p s t e i n , and Nowacki and to Mr. M i c h a l c z u k , Ms. S c h u l z e , and Ms. Hall f o r permission to use t h e i r unpublished d a t a ; to Ms. Joanne Di Lucca Schlub f o r valuable help i n manuscript p r e p a r a t i o n ; and to Dr. R. Chapman and a grant to Professo the use o f the MSU-NIH

Literature Cited 1.

Wain, R . L . ; Fawcett, C.H. In F . C . Steward, Ed. "Plant Physiology VA"; Academic Press: New York, 1966, pp. 231-296.

2.

Katekar, G.F. Phytochem., 1979, 18, 223.

3.

Went, F.W.; Thimann, K.V. "Phytohormones"; Macmillan: New York, 1937.

4.

Haagen-Smit, A.J.; Went, F.W. Konikl. Ned. Akad. Wetenschap., Proc., 1935, 38, 852.

5.

Gandar, J.C.; Nitsch, J . P . In J . P . Nitsch, Ed. "Regulateurs Naturels de l a Croissance Végétale"; Centre Nat. de l a Rec. S c i . Quai-Anatole: Paris, 1964, pp. 169-178.

6.

Marumo, S.; Hattori, H . ; Abe, H . ; Manakata, K. Nature, 1968, 219, 959.

7.

Haagen-Smit, A.J.; Leech, W.D.; Bergen, W.R. Amer. J. Bot., 1942, 29, 500.

8.

Weaver, R . J . "Plant Growth Substances in Agriculture"; Freeman: San Francisco, 1972, p. 120.

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C i e s i e l s k i , T. B e i t r . Bio. Pflanzen, 1872, 1, 1.

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Darwin, C . ; Darwin, F. "The Power of Movement in Plants"; D. Appleton: London, 1880.

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Bonner, J. J. Gen. P h y s i o l . , 1933, 17, 63.

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Thimann, K.V. Ann. Rev. Biochem., 1935, 4, 545.

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Avery, G.S.; Creighton, H.B.; Shalucha, B. Amer. J . Bot., 1940, 27, 289.

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Avery, G.S.; Berger, J.; Shalucha, B. Amer. J . Bot., 1941, 28, 596.

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Haagen-Smit, A . J . ; Leech, W.D.; Bergren, W.R. Science, 1941, 93, 624.

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van Overbeek, J . Amer. J . Bot., 1941, 27, 1.

17.

Evans, M.L. Ann. Rev. Plant Physiol., 1974, 25, 195.

18.

Cleland, R. Ann. Rev Plan Physiol.

19.

Bandurski, R.S.; Piskornik, Z. In F. Loewus, Ed. "Biogenesis of Plant Cell Wall Polysaccharides"; Academic Press: New York, 1973, pp. 297-314

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Labarca, C.; Nicholls, P.B.; Bandurski, R.S. Biochem. Biophys. Res. Commun., 1966, 20, 641.

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Ueda, M.; Bandurski, R.S. Phytochemistry, 1974, 13, 243.

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Piskornik, Z . ; Bandurski, R.S. Plant Physiol., 1972, 50, 176.

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Ueda, M.; Bandurski, R.S. Plant Physiol., 1969, 44, 1175.

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Ehmann, A. Carbohy. Res., 1974, 34, 99.

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Ehmann, Α.; Bandurski, R.S. Carbohy. Res., 1974, 36, 1.

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Bandurski, R.S.; Schulze, A. Plant Physiol., 1974, 54, 257.

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Bandurski, R.S. In W.W. Wells and F. Eisenberg, Eds. "Cyclitols and Phosphoinositides"; Academic Press: New York, 1978, pp. 35-54.

28.

Percival, F . ; Bandurski, R.S. Plant Physiol., 1970, 58, 60.

29.

Andreae, W.A.; Good, N.E. Plant Physiol., 1955, 30, 380.

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Tillberg, E. Physiol. Plant, 1974, 31, 271.

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Zenk, M.H. Nature, 1961, 191, 493.

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Keglevic, D. Carbohy. Res., 1971, 20, 293.

1971

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Bandurski, R.S.; Schulze, A. Plant Physiol., 1977, 60, 211.

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Little, C.H.H.; Heald, J . K . ; Browning, G. Planta, 1978, 139, 133.

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Hamilton, R.H.; Bandurski, R.S.; Grigsby, B.H. Plant Physiol., 1961, 36, 354.

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Magnus, V.; Bandurski, R.S. Plant Physiol., 1978, 61(S), 63.

37. 38.

Cohen, J.D.; Schulze, Α.; Bandurski, R.S. Plant Physiol., 1978, 61(S), 63. Hall, P.L.; Bandurski, R.S. Plant Physiol., 1978, 61, 425.

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Skoog, F. J . Gen.

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Nowacki, J.; Cohen, J.D.; Bandurski, R.S. J . Labelled Comp., 1978, 15, 325.

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Rittenberg, D.; Foster, G.L. J. Biol. Chem., 1940, 133, 737.

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Gillespie, B.; Thimann, K.V. Plant Physiol., 1963, 38, 214.

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Kopcewicz, J.; Ehmann, Α.; Bandurski, R.S. Plant Physiol., 1974, 54, 346.

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Elliott, W.M.; Shen-Miller, J . Photochem. and Photobiol., 1976, 23, 195.

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Bandurski, R.S.; Schulze, Α.; Cohen, J.D. Biochem. Biophys. Res. Commun. 1977, 79, 1219.

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Yogota, T.; Hiraga, K.; Hisakazu, Y. Phytochem., 1975, 14, 1569.

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Milborrow, B.V. Ann. Rev. Plant Physiol., 1974, 25, 259.

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Peterson, J . B . ; Miller, C.O. Plant Physiol., 1977, 59, 1026. Morris, R.O. Plant Physiol., 1977, 59, 1029.

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Sembdner, G. In K. Schreiber, H.R. Schütte, and G. Sembdner, Eds. "Biochemistry and Chemistry of Plant Growth Regulators"; Inst. of Plant Biochem.: Halle, GDR, 1974, pp. 283-302.

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2 Aspects of G i b b e r e l l i n Chemistry

PETER HEDDEN Department of Biology, University of California, Los Angeles, CA 90024

Since their discovery as secondary metabolites of the phytopathogenic fungus have been identified fro some gymnosperms. In addition there are many more reports of GA-like substances (detected by biological assays) occurring in species of both groups. It seems probable that GAs are ubiquitous in seed plants. There are also reports of GA-like substances occurring in lower organisms, including fungi other than G. fujikuroi, algae and bacteria, but none of these have been conclusively identified as GAs. Gibberellins elicit a variety of physiological responses in seed plants and are well established as hormones controlling plant growth and development. Gibberellic acid (GA3) is used extensively in agriculture, and is produced commercially from large scale cultures of G. fujikuroi. Other GAs have been found to have specific agricultural applications where they are more effective than GA3. There is therefore interest in methods for producing GAs, other than GA3, in commercially useful quantities. GAs are also required for research purposes, both for testing their biological activity and as standards for GA identification and quantitation. In most cases it is impractical to extract sufficient quantities of GAs from their plant sources and they must be prepared chemically or microbiologically from more-accessible compounds. T h i s review discusses aspects of GA chemistry which may be u s e f u l to p l a n t p h y s i o l o g i s t s or b i o c h e m i s t s . I t covers GA i d e n t i f i c a t i o n and q u a n t i t a t i o n , p a r t i c u l a r l y d i f f i c u l t tasks c o n s i d e r i n g the low l e v e l s of GA i n p l a n t t i s s u e s . However the problems o f GA a n a l y s i s have been c o n s i d e r a b l y a l l e v i a t e d by recent technology, p a r t i c u l a r l y combined gas chromatography-mass spectrometry. A l s o d e s c r i b e d are methods f o r p r e p a r i n g l e s s a c c e s s i b l e GAs and f o r i s o t o p i c a l l y l a b e l i n g GAs f o r metabolism s t u d i e s . F i n a l l y some c o n s i d e r a t i o n i s given to s t r u c t u r e a c t i v i t y r e l a t i o n s h i p s . Such c o r r e l a t i o n s may shed l i g h t on the mechanism o f a c t i o n of GAs at the molecular l e v e l and

0-8412-0518-3/79/47-lll-019$09.50/0 © 1979 American Chemical Society

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suggest how the GA s t r u c t u r e might be m o d i f i e d t o produce com­ pounds w i t h enhanced b i o - a c t i v i t y or s p e c i a l i z e d a p p l i c a t i o n . Structure The g i b b e r e l l i n s (GAs) are a c l a s s o f t e t r a c y c l i c d i t e r penoid a c i d s o f which 53 members (Figure 1) have been i d e n t i f i e d from higher p l a n t s or the fungus, G i b b e r e l l a f u j i k u r o i . For con­ venience, each f u l l y - c h a r a c t e r i z e d , n a t u r a l l y - o c c u r r i n g GA i s a l l o c a t e d a number ( 1 ) , thus the GAs are r e f e r r e d to as GAi GA53. S t r u c t u r a l l y , the GAs can be subdivided i n t o two groups, the C20 Ο19 GAs. The C20 GAs c o n t a i n the e n t - g i b b e r e l l a n e s k e l e t o n as i s t y p i f i e d by the simplest member o f t h i s group, GA12 (Figure 2 ) . The C19 GAs have an ent-20-norgibberellane s k e l e t o n i n which carbon-20 has been r e p l a c e d by a hydroxyl group. With one exception, the C19 GAs c o n t a i n a 19*10 γ-lactone as f o r example, i n GAo (Figur d i f f e r mainly i n the degre s k e l e t o n . The C19 GAs are d e r i v e d b i o s y n t h e t i c a l l y from the C20 GAs by an as y e t unknown mechanism. C20 GAs are found n a t u r a l l y having carbon-20 at each p o s s i b l e o x i d a t i o n l e v e l and C^o, GA b i o ­ s y n t h e s i s may i n v o l v e successive o x i d a t i o n o f t h i s carbon atom. GAs w i t h an a l c o h o l f u n c t i o n at carbon-20 are i s o l a t e d as the 19,20 δ-lactones i n which carbon-20 i s prevented from f u r t h e r o x i d a t i o n (2). I t i s l i k e l y that t h i s l a c t o n e i s formed during i s o l a t i o n and t h a t these GAs occur as f r e e a l c o h o l s i n v i v o . Those GAs w i t h C-20 at the aldehyde o x i d a t i o n l e v e l appear t o e x i s t as the 19>20 l a c t o l s i n the s o l i d s t a t e and as an e q u i l i ­ brium mixture o f l a c t o l and aldehyde i n s o l u t i o n ( 3 ) . The C20 and C19 GA r i n g systems can be hydroxylated at a number of p o s i t i o n s , 2β, 33 and 13 hydroxyled GAs being encountered most f r e q u e n t l y . Hydration o f the 16,17 double bond i s observed i n G. f u j i k u r o i t o g i v e the s a t u r a t e d C-l6 a l c o h o l . Other l e s s frequent f u n c t i o n a l i t i e s i n c l u d e a double bond at e i t h e r the 1,2 or 2,3 p o s i t i o n s i n C]_a, GAs; ketone and epoxide f u n c t i o n s . a n ( i

Stability The g e n e r a l chemistry of the GAs has been reviewed (h). Many o f the GAs c o n t a i n a h i g h c o n c e n t r a t i o n o f f u n c t i o n a l groups rendering them s u s c e p t i b l e t o rearrangement and degradation. Therefore, as a g e n e r a l r u l e high temperatures and extremes o f pH should be avoided when working w i t h them. In m i n e r a l a c i d 13-hydroxy GAs undergo a Wagner-Merwein rearrangement o f the c/D r i n g system (Figure 3) . When the 13-hydroxyl group i s absent the 16,17 double bond may be isomerized by a c i d t o the e n d o c y c l i c 15,16 p o s i t i o n or may be hydrated to g i v e the s a t u r a t e d l 6 a l c o h o l . Four GAs w i t h the l 6 - h y d r o x y l group (GA2, GA^o, GAuJL, GA^) have been i d e n t i f i e d from G• f u j i k u r o i (6,7,8). Since the fungus i s u s u a l l y grown at a c i d i c pH, these GAs c o u l d be

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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the r e s u l t o f non-enzymatic h y d r a t i o n . GAs such as GA^ o r GA^ w i t h o n l y a 33-hydroxyl group i n t h e A r i n g a r e s e n s i t i v e t o d i l u t e aqueous a l k a l i , underoing epimeri z a t i o n a t thfe 3 p o s i t i o n t o g i v e a mixture o f epimers. A r e t r o a l d o l mechanism has been proposed f o r t h i s e p i m e r i z a t i o n ( F i g ure k) ( 9 ) , a mechanism supported b y the f i n d i n g t h a t t h e 3orhydrogen i s r e t a i n e d i n t h e rearrangement (10). The epimerizat i o n does not occur i f there i s a l s o a 2-hydroxyl group o r a 1,2 double bond i n the A r i n g . In t h e l a t t e r case there i s a s h i f t o f t h e 1,2 double bond t o the 1,10 p o s i t i o n and t h e formation o f a 19,2 l a c t o n e . T h i s i s o m e r i z a t i o n i s r a t h e r f a c i l e and can occur during gas chromatography o f GAs, such as GA3 or GAy, r e s u l t i n g i n broad double peaks. Many GAs i n aqueous s o l u t i o n a r e s l o w l y degraded, the process b e i n g a c c e l e r a t e d a t h i g h e r temperatures a s , f o r i n s t a n c e , during a u t o c l a v i n g . A f t e an autoclave a t 120° f o The chemical processes i n v o l v e d i n t h i s degradation o f GA3 have been s t u d i e d i n some d e t a i l (12). The proposed pathway o f decomposition i s shown i n F i g u r e 5. The major products a r e i s o g i b b e r e l l i c a c i d , g i b b e r e l l e n i c a c i d , a l l o g i b b e r i c a c i d , 9-epia l l o g i b b e r i c a c i d and 9,11-didehydroallogibberic a c i d . The l a s t compound i s formed from g i b b e r e l l e n i c a c i d v i a a proposed t r i e n e intermediate by an o x i d a t i o n which appears t o i n v o l v e hydroperoxide i n t e r m e d i a t e s . Q u a l i t a t i v e and Q u a n t i t a t i v e A n a l y s i s E x t r a c t i o n and P u r i f i c a t i o n . Many methods have been used t o e x t r a c t and p u r i f y GAs from p l a n t m a t e r i a l , the procedure o f t e n depending on the t i s s u e s b e i n g e x t r a c t e d . Graebe and Ropers (13) i n t h e i r review on GAs have c r i t i c a l l y d i s c u s s e d e x t r a c t i o n and p u r i f i c a t i o n techniques. The c o n c e n t r a t i o n o f GAs i n h i g h e r p l a n t t i s s u e s v a r i e s from about 10 p,g per g f r e s h weight i n seeds o f c e r t a i n species t o l e s s than 1 ng p e r g f r e s h weight i n v e g e t a t i v e t i s s u e s . The extent o f p u r i f i c a t i o n r e q u i r e d w i l l depend on t h e p a r t i c u l a r p l a n t t i s s u e under i n v e s t i g a t i o n . T y p i c a l l y the m a t e r i a l i s homogenized i n a watermethanol mixture (about 75$ methanol) a t low temperature. Acetone has been used as the organic solvent but can cause problems due t o t h e formation o f acetonides w i t h v i c i n a l d i o l s i n s l i g h t l y a c i d c o n d i t i o n s (1^*15) · A f t e r t h e aqueous methanol e x t r a c t i o n , t h e homogenate i s f i l t e r e d and the methanol removed from the f i l t r a t e under reduced pressure a t h0° o r below. A t t h i s stage i t i s common t o b u f f e r t h e aqueous r e s i d u e , u s u a l l y w i t h potassium phosphate. With some t i s s u e s , f o r example l i q u i d endosperm, i t i s convenient t o e x t r a c t d i r e c t l y with a b u f f e r s o l u t i o n a t about pH 8 r e s u l t i n g i n a c l e a n e r e x t r a c t ( l 6 ) . The b u f f e r e d aqueous e x t r a c t i s a d j u s t e d t o pH 8 and n e u t r a l and b a s i c compounds a r e e x t r a c t e d w i t h an organic s o l v e n t ,

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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PLANT GROWTH

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1 CO

"So CO

1 en

3.

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Gibbereïlin Chemistry

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-s S S Ο

v.

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PLANT GROWTH

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In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Gibberellin Chemistry

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•S ο Ο

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PLANT GROWTH

SUBSTANCES

ο

fcJD

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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.g Ο

Ο

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

P L A N T G R O W T H SUBSTANCES

GA

1 2

(C

2 0

-GA)

GA

9

(C

1 9

-GA)

Figure 2. Structures of GA (a C o GA) and GA (a C GA) possessing the ent-gibberellane and ent-20-norgibberellane skeletons, respectively. The numbering system and ring designations are shown also. 12

2

9

19

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Gibberellin Chemistry

Figure 3. Acid-catalyzed Wagner-Merwein rearrangement of the C/D ring 13-hydroxy GAs (5)

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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PLANT GROWTH

Figure 4.

SUBSTANCES

Proposed retro-aldol mechanism for the hase-catalyzed epimerization of ^-hydroxy GA's (9)

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Gibberellin Chemistry

9,11-didehydroollogibberic acid

Figure 5.

9 / ? - H ; allogibberic acid 9 a - H ; 9-£/?/-allogibberic acid

Proposed pathway for the decomposition of GA in aqueous solution (12) 3

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u s u a l l y e t h y l a c e t a t e . Some o f the l e a s t p o l a r GAs, p a r t i c u l a r l y GA9 and GAi2> and GA-glucosyl e s t e r s are a l s o e x t r a c t e d i n t h i s f r a c t i o n . Most GAs a r e e x t r a c t e d i n t o e t h y l a c e t a t e a f t e r adjustment o f the pH t o 3.0 w i t h h y d r o c h l o r i c a c i d * Very p o l a r GAs and GA-glucosides can be e x t r a c t e d w i t h n-butanol. It i s important t h a t the a c i d i c e t h y l a c e t a t e and b u t a n o l e x t r a c t s be washed w i t h water before being concentrated t o dryness. Otherwise t r a c e s o f a c i d (phosphoric a c i d i f phosphate b u f f e r was used) w i l l be concentrated l e a d i n g t o rearrangement o r h y d r a t i o n o f any GAs present. Many methods have been used f o r a d d i t i o n a l p u r i f i c a t i o n o f the a c i d i c e x t r a c t s . I f the weight o f the e x t r a c t i s not too l a r g e then t h i n - l a y e r chromatography, e i t h e r a d s o r p t i o n chromatography on s i l i c a g e l o r p a r t i t i o n chromatography on k i e s e l g u h r , i s most convenient, although the s e p a r a t i o n obtained by t h i s method i s poor. B e t t e r e s o l u t i o ha bee obtained u s i n t i t i o n chromatography o sephadex (17,20). R e c e n t l y hig performanc l i q u i chromatog raphy has been used w i t h GAs and gives e x c e l l e n t s e p a r a t i o n (21). The disadvantage o f chromatographic methods i s t h a t they separate the GAs from each other as w e l l as from other components i n the e x t r a c t . Thus numerous f r a c t i o n s are generated, each o f which has t o be analyzed s e p a r a t e l y f o r GAs. The most s a t i s f a c t o r y method a v a i l a b l e f o r i d e n t i f y i n g microgram o r l e s s q u a n t i t i e s o f GAs i n a complex mixture i s combined gas chromâtographymass spectrometry (GC-MS), a technique which i t s e l f contains a s e p a r a t i o n s t e p . T h e r e f o r e , i d e a l l y , p u r i f i c a t i o n methods p r i o r to GC-MS a n a l y s i s should separate the GAs as a group from other components* Short columns o f c h a r e o a l - c e l i t e (22), g e l f i l t r a t i o n chromatography on sephadex (23), and anion-exchange chromatography (2*+,25) have been used f o r t h i s purpose. I n s o l u b l e PVP i s o f t e n used t o remove phenolic compounds (26) and can be added t o the aqueous e x t r a c t b e f o r e p a r t i t i o n a g a i n s t organic s o l v e n t . The use o f a f f i n i t y chromatography i n which a n t i bodies s p e c i f i c t o GAo were bound t o sepharose has been i n v e s t i g a t e d (27) and c o u l d provide a r a p i d method f o r p u r i f y i n g GAs i f a n t i b o d i e s t o a l l GAs c o u l d be developed. G i b b e r e l l i n g l u c o s y l ethers and e s t e r s are d i f f i c u l t t o analyze by GC-MS although they can be gas chromatographed as t r l m e t h y l s i l y l (TMS ) ethers (28,29). The conjugates a r e g e n e r a l l y hydrolyzed e n z y m a t i c a l l y i n the crude e x t r a c t and the f r e e GAs subsequently p u r i f i e d and analyzed* Commercial c e l l u l a s e (30) o r p e c t i n a s e (31) have been used f o r the enzyme h y d r o l y s i s w i t h v a r y i n g success. A c i d o r base h y d r o l y s i s i s a l s o p o s s i b l e but may l e a d t o rearrangement o f the GAs. This complicates t h e i d e n t i f i c a t i o n u n l e s s the rearranged products f o r each GA are a v a i l a b l e f o r comparison (Ik). I d e n t i f i c a t i o n . Combined GC-MS has advanced p l a n t hormone r e s e a r c h g r e a t l y i n recent years and w i t h the i n t r o d u c t i o n o f

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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33

Gibberellin Chemistry

computerized systems (32) the s e n s i t i v i t y and v e r s a t i l i t y o f t h i s technique have been i n c r e a s e d s t i l l f u r t h e r . The g e n e r a l methodology f o r GA a n a l y s i s by GC-MS has been reviewed r e c e n t l y (33, 3*0 « The v o l a t i l i t y o f GAs i s i n c r e a s e d p r i o r t o GC by forming the methyl e s t e r s w i t h diazomethane. Hydroxylated GAs are o f t e n converted t o t r i m e t h y l s i l y l (TMS) ethers a f t e r m e t h y l a t i o n . The mass s p e c t r a o f GA methyl e s t e r s TMS ethers f r e q u e n t l y c o n t a i n i n t e n s e molecular ions and c h a r a c t e r i s t i c fragmentation p a t t e r n s , which are e a s i e r t o i n t e r p r e t than those o f the f r e e hydroxy compounds. When recovery o f GAs i s r e q u i r e d a f t e r GC, GA TMS ether e s t e r s are a convenient d e r i v a t i v e s i n c e the f r e e GA can e a s i l y be recovered a f t e r h y d r o l y s i s i n water. Combined GC-MS-computer systems w i t h r e p e t i t i v e scanning can l e a d t o the i d e n t i f i c a t i o n o f GAs as minor components o f complex e x t r a c t s at l e v e l s down t o ÎCT- - g. In such cases mass fragmentograms can be c o n s t r u c t e ions o f p a r t i c u l a r m/e value Thus i f the presence o f a p a r t i c u l a r GA i s suspected, charact e r i s t i c ions i n the mass spectrum o f the d e r i v a t i z e d GA are p l o t t e d . An i d e n t i f i c a t i o n can be made i f the ions peak at the same r e t e n t i o n time as the GA and have the same r e l a t i v e i n t e n s i t y as i n the mass spectrum o f the a u t h e n t i c compound (see F i g ure 6 ) . In t h i s way GAs can be detected which are masked i n the GC t r a c e by other compounds o f s i m i l a r r e t e n t i o n time ( c f . 33). In order f o r an i d e n t i f i c a t i o n t o be made from mass fragmentograms s u f f i c i e n t ions (at l e a s t s i x ) must be scanned. I t i s sometimes p o s s i b l e t o o b t a i n a f u l l , interprétable mass spectrum i n such cases by background s u b t r a c t i o n . The scan (spectrum) i n which the ions due t o the compound o f i n t e r e s t are at a maximum i s determined from the mass fragmentograms. T h i s spectrum i s then "cleaned up" by s u b t r a c t i o n o f those ions c o n t r i b u t e d by the contaminant. I t i s a l s o p o s s i b l e t o compare the backgrounds u b t r a c t e d spectrum d i r e c t l y w i t h a u t h e n t i c s p e c t r a s t o r e d i n the instrument. The s e n s i t i v i t y o f GC-MS can be i n c r e a s e d s t i l l f u r t h e r by s e l e c t i v e i o n c u r r e n t m o n i t o r i n g (SICM) whereby o n l y a l i m i t e d number o f c h a r a c t e r i s t i c ions i n the mass spectrum o f the compound are monitored. Therefore the time f o r which each i o n i s monitored, and hence the s e n s i t i v i t y , i s i n c r e a s e d so that the amount o f an i d e n t i f i a b l e GA i s reduced t o Î C T ^ g. As i n mass fragmentometry, s u f f i c i e n t ions must be monitored f o r an i d e n t i f i c a t i o n t o be made. 1

1

1

Q u a n t i t a t i o n . Combined GC-SICM has been used mainly f o r q u a n t i t a t i o n . For a p a r t i c u l a r GA the absolute i n t e n s i t y o f a c h a r a c t e r i s t i c i o n i n i t s mass spectrum i s r e l a t e d t o the amount o f GA p r e s e n t , u s i n g standards t o c a l i b r a t e the instrument. Frydman et a l . (35) used t h i s " e x t e r n a l standard method" t o measure the l e v e l s o f a number o f GAs throughout the development o f pea seeds. An a l t e r n a t i v e and p r e f e r a b l e approach employs

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

34

PLANT GROWTH

SUBSTANCES

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

HEDDEN

Gibberellin Chemistry

DK1:G05JNZ RUNS 4 0 1 - 5 9 0 MRRflH E N D O S P E R M R C I D S M E T M S , I

410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 56 0 570 580 590 ΙΙΙΙΙΙΙΙΐ1ΐΙΙΙΙίΙ|ΙΐΙ)ΙΙΙΙΙΐΙΐίΙΙΙΙΙΐΙΐΙΙΙΙΙΙΙΐΙΐΙΙ|ΙΙΙΙΐ1ΐΙΙΙΙΙΙΙΐΙΐΙΙΙΙΙΙΙΐ1ΐΙ1ΙΙΙΙΙΐ|ΐΗ lllllllllllllHllllllllllllllllll Illlllllllllll lllllllllllllllllllllll I

20.00

8.00

32.00

36. 00

2 8.00

32.00

36. 00

2 8. 00

32. 00

24.00

MRSS 418

20.00

24.

MASS 22

^8.00

24. 00

12.00

INT >69

-I-

^.00

12.00

16.00

20.00

24.00

2 8.00

32.

36.00

410, 420 430 440, 450 400, 470 480 490 500 510 520 530 540 550 56 0 570, 580 590

8.00

iiiiHiiliiiiiiiilliiiiHiiilmiiiiiliiiiiiiiilu^ 12.00

. 00

TIME

20. 00

24.

2liHiiiilimiiiiiliiimiHl— 8.

36.00

(MINUTES)

Figure 6. Mass fragmentograms of the acidic fraction from an extract of Marah macrocarpus endosperm. Ions in the mass spectrum of GA Me TMS (shown above) were examined. The extract was run as the Me TMS derivative on a MS 902 spectrometer coupled to a Varian 2700 GC via a membrane separator. GCMS conditions—3% OV-17 on 100-120-mesh Gas Chrom Q in a 2 m X 0.2 cm i.d. column. Temperature—200°C for 5 min; then programmed at 4°C/min. Heliumflow—16.5cm /min. Electron energy—70 eV; accelerator potential— 2.9 KV; separator temperature—226°C; source temperature—250°C. 4

3

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

36

PLANT GROWTH

SUBSTANCES

the use of an internal standard such as an isotopicallylabeled analog of the GA being quantitated. A known amount of the standard i s added to the plant extract at any early stage of the purification procedure so that account i s taken for losses, which can be quite considerable. The most conveniently prepared standards are deuterated GAs (see l a t e r ) . The natural and deuterated GA have almost the same GLC retention time and the group of ions i n the region of the molecular ion (M") of the natural GA are monitored throughout the mass peak (see Figure 7 ) . The relative intensities of the ions at m/e M and M + X, where X i s the number of deuterium atoms i n the standard, are calculated and, after correction for natural heavy isotopes i n the M + X ions, the relative amounts of the natural and deuterated GAs are determined. Sponsel and MacMillan (36,25) have i l l u s t r a t e d the use of GAs labeled with deuteriu also to follow their metabolism injected a measured amount of [ H][3H]GA29 (both species labeled at the same position) into immature pea seeds. Some seeds were extracted immediately and others at regular time intervals thereafter so that the metabolism of GA29 could be studied. The [3H] label was present to determine now much of the added GA29 remained unmetabolized after a particular time. Then by comparison of the relative amounts of the natural and deuterated GA29 mass spectrometry, the amount of endogenous GA29 was calculated. Using this method they also compared the rates of metabolism of exogenous and endogenous GA29. 4

Structure determination for new GAs. Mass spectrometry i s a useful tool for identifying GAs whose structures have been previously determined, i n which case comparison of mass spectra is s u f f i c i e n t . In contrast, the characterization of GAs of unknown structure is a much more d i f f i c u l t and time-consuming task. In these cases mass spectrometry can give information such as molecular weight and some indications of structure. For instance i n the mass spectra of the methyl esters TMS ethers a strong ion at m/e 129 indicates that the A ring contains a single hydroxyl group at the 1 or 3 positions. 13-Hydroxy GAs produce ions at m/e 207/208 and v i c i n a l alcohol functions, such as i n GA8, result i n a strong ion at m/e lk7 (37). If the structure of a GA can be inferred from i t s mass spectrum the suspected compound may be synthesized and i t s mass spectrum compared with that of the unknown. Thus the structures of GAJ4.6 and G A I ^ Y were confirmed by the p a r t i a l synthesis of their methyl esters from GA1+ (38). This synthesis w i l l be discussed i n detail l a t e r . GAl^ was identified i n immature seeds of Pyrus communis (pear) i n an analogous manner by comparison of i t s mass spectrum with that of a product obtained from incubating ent-15or-hydroxykaurenoic acid with a mutant of the fungus G. fujikuroi (29).

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

HEDDEN

Gibberellin Chemistry

6A

2 0

PEA SEED E X T WITH H - G A METMS

METMS H

M/E 418 419 420 421 422

37

INTY 100,0 31.5 8.4 1.7 0.2

G A

2" 20

2

M E T M S

2

2 0

M/

420 421 422

100.0 30.8 8.2

420 421 422

100.0 31.3 10.1

Plant Growth Regulator Working Group Figure 7. An example of the use of deuterated GA's as internal standards for the quantitation of GAs in plant extracts. [2a- H ]GA was added to an extract of young pea seeds in order to quantitate GA (cf. 25). Ions in the region of the molecular ion were scanned. The acidic fraction from the pea seed extract was run as the Me TMS derivative on a MS 30 coupled to a Pye 104 GC via a singlestage silicone membrane separator (34). 2

1

29

29

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PLANT GROWTH

38

SUBSTANCES

Where i t has been possible to obtain sufficient material, GAs of previously unknown structure have been f u l l y characterized and their structure determined by a combination of chemical and spectroscopic methods. Proton Nuclear Magnetic Resonance (NMR) spectroscopy provides a great deal of structural information (k). 1 3 NMR promises to be a very powerful technique for both structure determination and metabolism studies of GAs . Yamaguchi et a l . (h2) used a combination of proton and 1 3 c NMR to determine the structure of G A ^ Q (2 by formation of the toluene-psulfonate and treatment of this with b o i l i n g collidine ÇJ+2). Thus the structures of G A q , whose structure wa (tf) and absolute stereochemistry has been confirmed by X-ray diffraction ( ^ > ^ , ^ U 8 ) . Recently the t o t a l synthesis of GA3 has been completed by Corey and co-workers (kg). The structures of C20 GAs were related ultimately to entkaurene v i a 7P-hydroxykaurenolide ( 5 0 ) . However, the two classes of GAs have now been related directly by the oxidative decarboxylation of GA^ to give GA^ ( 5 1 * 5 2 ) · Bearder and MacMillan ( 5 1 ) treated GA^^ with lead tetra-acetate to obtain a mixture of GAlj. and the isomeric 2 0 , 4 lactone (Figure 8 ) . Murofushi et a l . ( 5 2 ) employing a more lengthy procedure, decarboxylated the dimethyl ester of GAv* with lead tetra-acetate and lactonized the resulting o l e f i n with iodine (Figure 8 ) . The t o t a l syntheses of GA15 ( 5 3 ) and GAj_2 ( 5 ^ > 5 5 ) have also been reported. C

The Preparation of Less-readily Available

GAs

The isolation of significant quantities of many of the less-accessible GAs from plant tissues i s usually impractical. GAs are required as standards, both for qualitative and quantitative analysis, as substrates for metabolism studies (often isotopically labeled) and for b i o l o g i c a l assays. The most practical methods for preparing these compounds are the chemic a l or b i o l o g i c a l conversion to the more available GAs or entkaurenoids. Gibberella fujikuroi produces a number of GAs ( 5 6 ) , of which GA3, GAip GA7, GA13 and GA^ can be obtained i n r e l a t i v e l y large amounts. These fungal GAs are the starting point for the p a r t i a l synthesis of less-accessible GAs by r e l a t i v e l y simple chemical procedures. Also microbiological methods have been developed to convert GAs and GA-precursors (or analogs of these) of both fungal and higher plant origin to useful products.

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

HEDDEN

GA

13

Gibberellin Chemistry

dimethyl ester

Figure 8.

Methods for the oxidative decarboxylation of GA

13

to GA (51,52)

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

h

PLANT GROWTH

SUBSTANCES

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

Figure 9. 3h

h0

7

51

Chemical methods for the partial syntheses of the methyl esters of GA GA , GAj , and GA from GA 4 (38,42)

42

PLANT GROWTH

SUBSTANCES

Chemical Methods. As an example, Figure 9 shows a scheme for the preparation of four 2-hydroxy GAs from GA^. Two of the products are less abundant fungal GAs (GA^Q and GAI4.7) and the others (GAo^ and G A 5 1 ) occur i n higher plants. The preparation of the methyl esters of GAI±Q GA^y and GAolj. was described by Beeley and MacMillan ( 3 8 ) and the conversion of G A ^ Q to GAi^ by Yamaguchi et a l . (k2). The starting point was a mixture of GAJ4. and G A y , GAs which are not easily separated. This mixture was treated with OsOlj. and NalO^ to convert GA^ to the 1 7 - n o r - l 6 ketone which is easily separated from the G A y product i n which the 1,2-double bond i s also oxidized. The exocyclic double bond is thus protected as the norketone and can be restored later by the Wittig reaction. This reaction also gives an opportunity to introduce a label at the 17 position (see l a t e r ) . To prevent complications due to the presence of a free carboxylic acid group, the GA^ norketone was methylated and subsequent reactions were carried out on th for the 3-deoxy product for the 3-hydroxy GAs using the method described by Nagata et a l . (53)· The reduction of GA^Q-ketone with NaBHlj. gives a mixture of the 2or (GA^Q) and 20 ( G A c ^ ) alcohols which can be separated by preparative TLC (k2)• A similar series of reactions has been used ( 3 8 ) to convert the C 2 0 GA, GA^o, to GAJ4.3 and GAj^g, both of which occur i n the Cucurbitaceae ( 5 8 ) . 9

The synthesis of GA-60-aldehydes i s of particular interest since GA^-aldehyde i s the immédiate product of ring Β contrac­ tion i n GA biosynthesis ( 5 9 ) . GA]_2-aldehyde also appears to be a substrate for hydroxylation i n G . fujikuroi and at least one higher plant ( 6 θ ) . GA^-aldehyde was f i r s t synthesized from 70-hydroxykaurenolide, a metabolite of G . f u j i k u r o i , by treating the 7ff-toluene-p-sulfonate with KOH i n methanol ( 6 1 ) . The y i e l d was poor and the methyl ester was obtained as product. The y i e l d has been improved using t-butanol as solvent (59) with a small amount of water ( 6 2 ) • S t i l l higher yields were obtained with p-bromobenzenesulfonate as the leaving group ( 6 3 ) . The aldehydes of GA]_lj. and GA53 have also been prepared from 3$ 7B- and 7 3 , 1 3 dihydroxykaurenolides, respectively, using the same basic method ( 6 4 , 6 5 ) . The proposed mechanism for ring contraction of the kaurenolide i s shown i n Figure 1 0 ; the reaction requires an antiperiplanar relationship for the migrating 5>6-bond and the leaving group. Thus the 73-alcohol of the kaurenolide must be epimerized to the or position before formation of the sulfonate ester. The i n i t i a l product i s the 6cy-aldehyde but epimerization occurs under the basic conditions of the reaction to give the thermodynamically-favored 6g-aldehyde. The γ-lactone i s not essential for the reaction and the opened hydroxy acid w i l l also undergo ring contraction on treatment with base (collidine or sodium hydride). The reaction involves abstraction of the 6-hydroxy proton but requires the free 19-oic acid. It was proposed that the proton was abstracted from the s t e r i c a l l y 9

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

HEDDEN

Gibberellin Chemistry

Figure 10. The conversion of 7β-hydroxykaurenolide to GA -aldehyde showing the proposed mechanism of the reaction (59,). Τs = toluene-p-sulfonyl. 12

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PLANT

44

GROWTH

SUBSTANCES

hindered 6OR-hydroxy group by internal attack of the carboxyl anion ( 6 2 ). Node et a l . ( 6 6 ) i n a detailed investigation of the mechanism of ring contraction concluded that opening of the lactone precedes bond migration so the reaction i s not concerted. Microbiological Methods. The low substrate s p e c i f i c i t y of many of the enzymes involved in GA biosynthesis i n Gibberella fu j ikuroi has been u t i l i z e d for the preparation of higher plant GAs. Suitable analogs of the natural GA-precursors are converted by the fungus to the corresponding GA analogs. It i s usual to prevent the synthesis of the natural GAs i n order to f a c i l i t a t e purification of the unnatural products. A mutant strain, B l - 4 L A , in which GA biosynthesis i s blocked early i n the pathway ( 6 7 ) (between ent-kaurenal and ent-kaurenoic acid) has been used. It i s also possible to block GA synthesis chemically using i n hibitors such as AMO-L6L8 ( 6 8 ) dide compound i n Figur formation. Steviol, the 13-hydroxy analog of ent-kaurenoic acid, occurs naturally as the glucoside, stevioside, i n leaves of the shrub, Stevia rebaudiana. Steviol i s converted by B l - 4 L A to a number of 13-hydroxylated GAs (71). Although 13-hydroxylation i s a normal process i n fungal GA biosynthesis, i t i s the f i n a l step i n the pathway so that the end product, G A 3 , i s normally the only 13-hydroxy GA formed i n large amounts. When steviol i s incubated with B l - U l a the major products are GA]_ (equivalent to the natural fungal metabolite, GA^) and GAj£ (equivalent to GAi^). Other GAs such as G A 5 3 , GA^o,, and G A 2 0 are also produced. There i s no GA3 because the presence of a 13-hydroxy group i n hibits formation of the 1 , 2 double bond. When steviol acetate was fed the major GA-products were the acetates of G A 2 0 i G A 1 7 (72) since the presence of a 13-acetoxyl group prevents 33-hydroxylation. Thus i t was possible to predetermine which products were obtained. Relative yields of products could also be manipulated by changing the concentration of the substrate. The B l - Û l a mutant was found also to metabolize ent-ljpor-hydroxykaurenoic acid to a number of 150-hydroxylated GAs (J.R. Bearder & K. Kybird, unpublished information), one of which, 5B-hydroxy GA9, was subsequently identified as a new GA, GAj^.5, i n seeds of Pyrus communis (39)• Other fungi have also proved useful for preparing GAs. GAo, methyl ester was hydroxylated by Rhizopus nigricans to a number of products among which the methyl esters of G A ^ Q (2orhydroxylation), GA o (13-hydroxylation), GA45 (158-hydroxylation) and a 12-hydroxy G A n of undetermined stereochemistry were identified (73). Interestingly the free acid was metabolized only to GA "by hydration of the 1 6 , 1 7 double bond. While the individual yields i n the above conversions were not high, another species, R. Arrhizus, 13-hydroxylates GAs and GAprecursors i n very high y i e l d ( 6 5 ) . a n (

2

1Q

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

HEDDEN

Gibberellin Chemistry

Figure 11. Inhibitors of GA biosynthesis: AMO-1618 (2'-isopropyl-4'-(trimethylammonium chloride)-5'-methylphenylpiperidine-l-carboxylate) and Ν, N, N-irimethyl-l-methyl-(2ifi\6'-trimethylcyclohex-2'-en^'-^^^ iodide.

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

P L A N T G R O W T H SUBSTANCES

46 The Preparation of Labeled GAs

Is otopically-labeled GAs and GA-precursors are required for metabolic studies and as internal standards for quantitation by mass spectrometry. Numerous chemical methods have been used for the synthesis of labeled GAs from readily-available GAs. Labeled GAs have also been prepared biochemically using either fungal cultures or cell-free preparations from higher plants. Chemical Methods. has been introduced by chemical means exclusively at carbon-17 by the Wittig reaction. The compound to be labeled i s oxidized to the 17-nor-L6-ketone with osmium tetroxide and sodium metaperiodate. The ketone i s then reacted with the labeled y l i d , [l^C-methylene]triphenylphosphorane. There are many examples of the use of this method i n which the y l i d was generated usin [^"C-methyl]triphenylphos phonium iodide and η-buty tive methods which give higher yields have been published re cently by Bearder et a l . ( 7 2 , 7 3 ) . They used potassium t-butoxide as base or salt-free y l i d prepared from methyltriphenylphosphonium bromide and excess sodium hydride i n tetrahydrofuran. The Witting reaction has been used also to introduce into GAs by reaction of the norketones with [3IÎ2-methylene] triphenylphosphorane. has an advantage over ^J+C i n being less expensive and obtainable with a higher specific radioactivity. Bearder et a l . ( 7 2 ) labeled the methylphosphonium bromide by exchange with 3 H 0 i n tetrahydrofuran containing triethylamine. In the f i n a l product there i s scrambling of label between the 1 7 and 1 5 positions. The strongly basic conditions required i n the Wittig reaction necessitate the protection of base-labile groups. Thus the 3 - and 13-hydroxy groups can be converted to t r i m e t h y l s i l y l ethers ( 3 8 ) or tetrahydropyranyl ethers which are then easily removed by mild acid treatment. T r i t i a t e d GAs of very high specific radioactivity have been prepared by catalytic reduction. The 1 , 2 double bond of GAo can be selectively reduced, using a p a r t i a l l y poisoned palladium catalyst, to give [l,2- H2]GA ( 7 4 , 7 5 * 7 6 ) > although some reduction of the 1 6 , 1 7 double bond and the lactone also occurs ( 7 6 ) . Introduction of % at sites other than carbon atoms 1 and 2 has also been found ( 7 6 ) . [^HlGA^ has been prepared from GAy by a similar method T 7 7 ) . [ 3 H ] G A I was converted to [ H]GA by eldjriination of the 3-toluene-p-sulfonate ( 7 8 , 7 9 ) . Murofushi et a l . ( 8 ) protected the 1 6 , 1 7 double bond of GA5 methyl ester by forming the epoxide with metachloroperbenzoic acid. After catalytic reduction of the 2 , 3 double bond they restored the exomethylene group by treatment with a mixture of sodium iodide, sodium acetate and zinc and hydrolyzed the methyl ester to obtain [ 2 , 3 - 3 H 2 ] G A O * Yakota et a l . ( 8 1 ) prepared [ 2 , 3 - 3 H 2 ] G A Q , from GA^ by an analogous method v i a 2 , 3 dehydro G A 9 . Selective catalytic reduction of the 3-methane2

3

1

3

5

2

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

HEDDEN

47

Gibberellin Chemistry

sulfonate of GA3 methyl ester was used by Murofushl et a l . to prepare [ I - S H J G A C , from which [1-3H]GA8 was obtained by treatment with osmium tetroxide ( 8 0 ) . A convenient method for the specific introduction of H or 3H (or both) into a molecule i s by ketone reduction with labeled metal hydride. Beale and MacMillan (10) have u t i l i z e d this method for the preparation of GAs labeled at the 1, 2 or 3 positions from GA3 or GAy (Figure 1 2 ) . One point of interest i s the lithium borohydride reduction of the enone formed by manganese dioxide oxidation of GA3 or GAy. When the reaction i s carried out i n anhydrous tetrahydrofuran i t proceeds i n two steps. I n i t i a l l y the lithium enolate i s formed which incorporates a proton at carbon-2 from the acid used i n the work-up, forming the 3-ketone. This ketone i s reduced to the 3o?-alcohol by the borohydride which i s decomposed more slowly than i s the lithium enolate. Thus labels i n a single reaction Acid or base exchangeable protons can be easily labeled with 3H or H. Bearder et a l . (67) labeled the 15 and 17 positions of ent-kaurene by treatment with CF3C003H( H). A mixture of the 16,17 and 15,16 double bond isomers i s obtained and they are separated by AgN03 TLC. This method could be used with some 13-deoxy GAs although separation of the resulting isomers would be more d i f f i c u l t than for ent-kaurene. The 6-hydrogen i n GAi2~ lûehyde and GA^k-aldehyde has been labeled by treatment with Me0 H( H) or 3 H ( H ) 0 and sodium methoxide (62,64). 2

2

2

a

3

2

2

2

2

Biological Methods. Microbiological methods have been used i n conjunction with chemical synthesis to convert chemicallylabeled precursors to labeled GAs. Hanson and Hawker (82) prepared [17-^0] GAQ by incubating chemically-synthesized TTf-l^C] GA^p-7-alcohol with G. fujikuroi cultures. In this case the product was diluted by endogenous GA3. The method could be improved by using cultures i n which the endogenous GA levels are reduced, either by mutation (Bl-4la) or with inhibitors of GA biosynthesis (70). Bearder et a l . (72) used the G. fujikuroi mutant, Bl-4la, to prepare [17-^H2TgA o with high specific radioactivity by feeding [17-^H ] steviol acetate. This method has the potential for the preparation of a number of labeled 13-hydroxy GAs (see 71,72) · Cell-free systems provide a rapid and convenient method for preparing GA and GA-precursors with high specific radioa c t i v i t y , although on a r e l a t i v e l y small scale. [l^C]-labeled ent-kaurenoid precursors of GAs have been obtained from [2-l^C] mevalonic acid by incubating with c e l l - f r e e systems from endosperm of Marah macrocarpus (83) or Cucurbit a maxima (84,85). The £. maxima system can be used also to prepare labeled C o GAs from L^C]mevalonic acid (86). 2

2

2

American Chemical Society Library 1155 16th St. N. W. In Plant Growth Substances; Washington, D. C.Mandava, 20036N.;

ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

PLANT GROWTH

SUBSTANCES

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

2.

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Gibberellin Chemistry

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

49

50

PLANT

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SUBSTANCES

Structure-Activity Relationships Gibberellins vary greatly i n the degree of response they e l i c i t i n biological assays, and indeed, of the 53 naturally occurring GAs, less than half have appreciable bio-activity i n the standard assays. The relationship between the structure of a GA and i t s bio-activity has attracted considerable attention and the available information has been extensively reviewed (13,87,88).

Two explanations for the different responses of plants to a particular GA have been suggested (87). The f i r s t assumes that the GA must bind to a specific receptor s i t e to give a response, the degree of response being related to the binding efficiency. Variations i n the structure of the receptor from one plant species to another would then explain the differences in the bio-activity of p o s s i b i l i t y i s that th metabolism to active products by the assay plants. The presence or absence of bio-activity i n an assay would then reflect the a b i l i t y of the assay to metabolize the applied GA. The struc­ ture of the GA-receptor and the a b i l i t y of the assay plant to metabolize the applied GA probably both influence the result of a bioassay, and i t i s i n fact d i f f i c u l t to distinguish between these p o s s i b i l i t i e s . Thus those C Q GAs which show bio-activity may do so because they are converted to C^y GAs. However, C Q GAs with the 19,20 δ-lactone are probably active per se since they are protected from farther oxidation at carbon-20 and therefore probably from conversion to C 1 9 GAs. It has been pointed out by several workers that i t i s often misleading to compare bioassay data from different laboratories since the s e n s i t i v i t y of a response can be very dependent on the bioassay technique used (13,88)· Therefore the publications of Brian et a l . (89), who compared the bio-activities of 134 GArelated compounds i n four bioassays under the same conditions, and of Crozier et a l . (90), who compared the bio-activities of 26 GAs i n nine bioassays, are very useful. Thus i t i s possible to make some general observations about the structural features of the GA molecule which are necessary for high biological a c t i v i t y . A free carboxyl group on the B-ring appears to be essential. The γ-lactone characteristic of C]_a, GAs i s required for high bio-activity but substantial, although reduced, a c t i v i t y is exhibited by C Q GAS with an aldehyde at carbon-20 or with the δ-lactone. GAs with a methyl or carboxyl group at carbon20 have l i t t l e a c t i v i t y . Reeve and Crozier (87) suggested that the δ-lactone and δ-lactol formed from the C - 2 0 aldehyde might mimic the γ-lactone of the C19 GAs. The isomeric 20,4-lactone of GAlj. was found to have a c t i v i t y equal to GkU i n some assays and only s l i g h t l y reduced a c t i v i t y i n others (57). It was con­ cluded that the lactone was necessary only because of the shape i t conferred on the molecule. The slight change i n the position 2

2

2

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of the lactone i n GAo from 19,10 to 19,2 has no effect on bioa c t i v i t y (89,91). In most bioassays a 30-hydroxyl group increases bio-activity as does a 13-hydroxyl group. An exception i s the cucumber hypocotyl assay i n which 13-hydroxy GAs have lower a c t i v i t y than the equivalent 13-deoxy compounds. In general the most active GAs have both 30- and 13-hydroxyl groups and a 1,2 double bond or some combination of these. Interestingly, when the 30-hydroxyl group i s epimerized to the 3or position bio-activity i s v i r t u a l l y eliminated (89). The effect of hydroxylation at positions other than 30 or 13, with the exception of the 20-position, i s d i f f i cult to assess because of insufficient examples. 20-Hydroxylation causes loss of bio-activity and i s quite possibly a deactivating process i n higher plants (60). The deactivation i s f a i r l y stereospecific since 2a-hydroxylation, while reducing bio-activity idea that higher plant tion mechanism has led to methods for producing GA-derivative s with very high biological a c t i v i t y . 20-Methyl GAlj. has been synthesized i n an attempt to prevent 20-hydroxylation from taking place (M. Beale and J . MacMillan, personal communication). This compound was found to have higher bio-activity than GA^, especially when the duration of the bioassay was increased (J. MacMillan et a l . , unpublished information). A more dramatic effect was seen with 2,2-dimethyl GA^ which, i n the dwarf-5 maize assay, i s a hundred times more active than G A 3 . The enhanced a c t i v i t y i s much less marked i n short-duration bioassays. The unexpected result that dimethyl GA\± i s more active than the monomethyl compound complicates the interpretation and more derivatives need to be tested. However the preliminary results indicate that blocking the 2 position leads to higher (or prolonged) bio-activity. 20-Methoxylation, as i n 20-methoxy GA9, reduces a c t i v i t y as effectively as hydroxylation ( j . MacMillan et a l . , unpublished information). Conclusions Since the f i r s t attempts to determine the structure of GA3, the chemistry of GAs has been the subject of a large number of publications. Chemically, the GAs have proved to be d i f f i c u l t compounds to work with, a consequence of the high number and arrangement of functional groups i n the molecule. G A 3 i s particularly l a b i l e and i t i s only recently that i t s t o t a l synthesis has been completed (j+9), more than twenty years after i t s structure was established. Total chemical synthesis i s not a feasible method for preparing useful quantities of GAs; G A 3 and some of the other GAs produced by Gibberella fuj ikuroi are more practically obtained from cultures of this fungus. However, preparatively useful chemical methods have been developed for the p a r t i a l synthesis

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of some less-accessible GAs from more abundant precursors, such as the fungal GAs. Microbiological conversion, using G. f u j i ­ kuroi and other fungi, i s also a promising method for obtaining higher-plant GAs from readily-available substrates. The identification and quantitation of GAs i n plant extracts are particularly d i f f i c u l t problems due both to the very low amounts of GAs present i n plant tissues and to the large number of different GA structures that can be encountered. When only a limited number of GAs were known, identification was often based on co-chromatography of the unknown with standards on thin-layer plates. It i s now realized that comparison of chro­ matographic behaviour with that of standards i n any system i s not sufficient basis for identification. Furthermore bioassays have proved to be very unreliable methods for GA-quant i t at ion. Combined gas chromatography-mass spectrometry has the advantage of giving conclusive identification on ver lo amounts of com ponents in complex mixtures creasingly for the detectio plan hormones. It also provides an accurate means for quantitation. The mechanism of action of GAs at the molecular l e v e l s t i l l eludes plant physiologists. There have been reports of stereospecific binding of GAs to protein ( 9 2 , 9 3 ) and other c e l l frac­ tions (9k) but i t has not been demonstrated that the binding i s associated with a physiological response. However, correlations of the biological a c t i v i t i e s of GAs with their structures are one possible method for obtaining information on the site of action. Furthermore, a possibly valuable "spin-off" from structure-activity studies i s the design of GA-like molecules which, because of increased bio-activity or specific physio­ l o g i c a l properties, may have important agricultural applications. Acknowledgment s The preparation of this a r t i c l e was supported by a grant from the N.S.F. to B. 0. Phinney. The author wishes to thank Professor B. 0. Phinney for helpful comments on the manuscript. Literature Cited 1. 2.

3.

MacMillan, J . , Takahashi, N. Nature, 1968, 217, 170-171. Graebe, J.E., Hedden, P. In "Biochemistry and Chemistry of Plant Growth Regulators"; Schreiber, Κ., Schütte, H.R., Sembdner, G., Ed. Institute of Plant Biochemistry: Halle, G.D.R. 1974, pp. 1-16. Harrison, D.M., MacMillan, J . J . Chem. Soc. (C), 1971, 631-636.

4.

MacMillan, J . , Pryce, R.J. In "Phytοchemistry"; M i l l e r , L.P., Ed. Van Nostrand-Reinhold: New York, 1973, pp. 283326.

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6. 7. 8.

Grove, J.F., MacMillan, J . , Mulholland, T.P.C., Turner, W.B. J . Chem. Soc. 1960, 3049-3057. Grove, J.F. J . Chem. Soc. 1961, 3545-3547. Hanson, J.R. Tetrahedron. 1966, 22, 701-703. Bearder, J.R., MacMillan, J . J.C.S. Perkin Trans. 1. 1973,

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MacMillan, J . , Pryce, R.J.

2824-2830.

J . Chem. Soc. (C), 1967,

740-742.

10. 11. 12. 13.

MacMillan, J. Pure & Appl. Chem. 1978, 50, 995-1004. Pryce, R.J. Phytochemistry. 1973, 12, 507-514. Pryce, R.J. J.C.S. Perkin Trans. 1. 1974, 1179-1184. Graebe, J.E., Ropers, H.-J. In "Phytohormones and Related Compounds: A Comprehensive Treatise. Volume 1." Lethem, D.S., Goodwin, P.B., Higgins, T.J.V., Ed. Elsevier/ North Holland Biomedical Press: Amsterdam, Oxford, New York. 1978,

14.

pp. 107-203

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15. 16. 17. 18. 19. 20. 21.

Yamaguchi, T., Yokota, T., Murofushi, N., Takahashi, N., Ogawa, V. Agric. B i o l . Chem. 1975, 39, 2399-2403. McComb, A.J. Nature. 1961, 192,575-576. P i t e l , D.W., Vining, L.C., Arsenault, G.P. Can. J . Biochem. 1971, 49, 185-193. Durley, R.C., Crozier, Α., Pharis, R.P., McLaughlin, G.E. Phytochemistry. 1972, 11, 3029-3033. Rao, T.P., Nagor, P.K. J . Expt. Botany. 1973, 24, 412-417. MacMillan, J., Wels, C.M. J . Chromatog. 1973, 87, 271-276. Reeve, D.R., Crozier, A. In "Isolation of Plant Growth Substances." Hillman, J.R., Ed. Cambridge Univ. Press, 1978,

22.

pp. 41-77.

Durley, R.C., MacMillan, J . , Pryce, R.J.

Phytochemistry.

1971, 10, 1891-1908.

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Crozier, Α., Aoki, Η., Pharis, R.P. J . Exp. Botany. 1969,

24. 25. 26.

Browning, G., Saunders, P.F. Nature, 1977, 265, 375-377. Sponsel, V.M., MacMillan, J . Planta. 1978, 144, 69-78. Glenn, J.L., Kuo, C.C., Durley, R.C., Pharis, R.P. Phytochemistry. 1972, 11, 345-351. Fuchs, Y., Gertman, E. Plant C e l l Physiol. 1974, 15, 629-

20, 786-795.

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633.

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Schneider, G., Jänicke, S., Sembdner, G. J . Chromatog. 1975,

109, 409-412.

29. Yokota, T. Hiraga, K., Yamane, Η., Takahashi, N. Phytochemistry. 1975, 14, 1569-1574. 30. Knöfel, H.-D., Müller, P., Sembdner, G. In "Biochemistry and Chemistry of Plant Growth Regulators." Schreiber, Κ., Schütte, H.R., Sembdner, G., Ed. Institute of Plant Biochemistry: Halle, G.D.R. 1974, pp. 121-124. 31. Frydman, V.M., MacMillan, J . Planta. 1975, 14, 1575-1578.

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35. 36. 37. 38. 39. 40. 41. 42.

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MacMillan, J . In "Plant Growth Substances 1970." Carr, D.J., Ed. Springer: Berlin, Heidelberg, New York. 1972, pp. 790-797. Gaskin, P., MacMillan, J . In "Isolation of Plant Growth Substances." Hillman, J.R., Ed. Cambridge Univ. Press. 1978, pp. 79-95. Hedden, P. In "Proceedings of the Plant Growth Regulator Working Group." Abdel-Rahman, Μ., Ed. Plant Growth Regulator Working Group: Syracuse, New York. 1978, pp. 33-44. Frydman, V.M., Gaskin, P., MacMillan, J . Planta. 1974, 118, 123-132. Sponsel, V.M., MacMillan, J . Planta. 1977, 135, 129-136. Binks, R., MacMillan, J . , Pryce, R.J. Phytochemistry. 1969, 8, 271-284. Beeley, L.J., MacMillan 1022-1028. Bearder, J.R., Dennis, F.G., MacMillan, J., Martin, G.C., Phinney, B . O . Tet. Letters. 1975, 669-670. Yamaguchi, I., Takahashi, N., Fujita, K. J.C.S. Perkin Trans. 1. 1975, 992-996. Evans, R., Hanson, J.R., Siverns, M. J.C.S. Perkin Trans. 1. 1975, 1514-1517. Yamaguchi, I., Mujamoto, M., Yamane, H., Murofushi, N., Takahashi, N., Fujita, K. J.C.S. Perkin Trans. 1. 1975, 996-999. Murofushi, Ν., Yokota, T., Watanabe, Α., Takahashi, N. Agric. B i o l . Chem. 1973, 37, 1101-1113. Grove, J.F. Quart. Rev. (Chem. Soc. London) 1961, 15, 56-70. Hartsuck, J.A., Lipscomb, W.N. J . Am. Chem. Soc. 1963, 85, 3414-3419. McCapra, F., McPhail, A.T., Scott, A.I., Sim, G.A., Young, D.W. J . Chem. Soc. (C). 1966, 1577-1585. Kutschabsky, L., Reck, G., Adam, G. Tetrahedron, 1975, 31, 3065-3068. Höhne, E., Schneider, G., Schreiber, K. J . Prakt. Chem., 1970, 312, 816-822. Corey, E.J., Danheiser, R.L., Chandrasekaran, S., Keck, G.E., Gopalan, B., Larsen, S.D., Siret, P., Gras, J.-L. J . Am. Chem. Soc. 1978, 100, 8034-8036. Gross, B.E., Galt, R.H.B., Hanson, J.R. J . Chem. Soc. 1963, 2944-2961. Bearder, J.R., MacMillan, J . J.C.S. Chem. Comm. 1976, 421. Murofushi, N., Yamaguchi, I., Ishigooka, Η., Takahashi, N. Agric. B i o l . Chem. 1976, 40, 2471-2474. Nagata, W., Wakabayashi, T., Hayase, Y., Narisada, M., Kamata, S. J . Am. Chem. Soc. 1970, 92, 3202-3203. Mori, K., Takemoto, I., Matsui, M. Tetrahedron. 1976, 32, 1497-1502.

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55. 56.

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Nakata, T . , Tahara, A. Tet. Letters. 1976, 1515-1518. MacMillan, J., Wels, C.M. Phytochemistry 1974. 13, 14131417. 57. Sponsel, V.M., Hoad, G.V., Beeley, L . J . Planta. 1977, 135, 143-147. 58. Beeley, L.J., Gaskin, P., MacMillan, J. Phytochemistry. 1975, 14, 779-783. 59. Gross, B . E . , Norton, Κ., Stewart, J . C . J . Chem. Soc. (C), 1968, 1054-1063. 60. Hedden, P., MacMillan, J., Phinney, B.O. Ann. Rev. Plant Physiol. 1978, 29, 149-192. 61. Galt, R.H.B., Hanson, J.R. J . Chem. Soc. 1965, 1565-1570. 62. Bearder, J.R., MacMillan, J., Phinney, B.O. Phytochemistry. 1973, 12,, 2173-2179. 63. Hanson, J.R., Hawker, J. Tet. Letters 1972, 4299-4302. 64. Hedden, P., MacMillan Trans. 1. 1974, 587-592 65. Down, G. Ph.D. Thesis. 1978, University of Bristol, England. 66. Node, M., Hori, Η., Fujita, E. J.C.S. Perkin Trans. 1. 1976, 2144-2149. 67. Bearder, J.R., MacMillan, J., Wels, C.M., Chaffey, M.B., Phinney, B.O. Phytochemistry. 1974, 13, 911-917. 68. Dennis, D.T., Upper, C.D., West, C.A. Plant Physiol. 1965, 40, 948-952. 69. Haruta, H . , Yagi, H . , Iwata, T . , Tamura, S. Agric. Biol. Chem. 1974, 38, 417-422. 70. Hedden, P., Phinney, B.O., MacMillan, J., Sponsel, V.M. Phytochemistry. 1977, 16, 1913-1917. 71. Bearder, J.R., MacMillan, J., Wels, C.M., Phinney, B.O. Phytochemistry. 1975, 14, 1741-1748. 72. Bearder, J.R., Frydman, V.M., Gaskin, P., MacMillan, J., Wels, C.M., Phinney, B.O. J.C.S. Perkin Trans. 1. 1976, 173-178. 73. Bearder, J.R., Frydman, V.M., Gaskin, P., Hatton, I.K., Harvey, W.E., MacMillan, J., Phinney, B.O. J.C.S. Perkin Trans. 1, 1976, 178-183. 74. Kende, H. Plant Physiol. 1967, 42, 1612-1618. 75. Pitel, D.W., Vining, L . C . Can. J. Biochem. 1970, 48, 259-263. 76. Nadeau, R., Rappaport, L. Phytochemistry. 1974, 13, 1537-1545. 77. Durley, R.C., Pharis, R.P. Planta. 1973, 109, 357-361. 78. Musgrave, Α . , Kende, H. Plant Physiol. 1970, 45, 56-61. 79. Durley, R.C., Railton, I.D., Pharis, R.P. Phytochemistry. 1973, 12, 1609-1612. 80. Murofushi, N . , Durley, R.C., Pharis, R.P. Agric. Biol. Chem. 1974, 38, 475-476. 81. Yokota, T . , Reeve, D.R., Crozier, A. Agric. Biol. Chem. 1976, 40, 2091-2094.

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82.

Hanson, J.R., Hawker, J. Phytochemistry. 1973, 12, 1073-

83.

Dennis, D.T., West, C.A. J . B i o l . Chem. 1967, 242, 3293-

1075. 3300.

84. 85. 86. 87. 88

Graebe, J.E. Planta. 1969, 8 5 , 171-174. Graebe, J.E., Hedden, P., MacMillan, J. In "Plant Growth Substances 1973," Hirokawa: Tokyo. 1974, pp. 260-266. Graebe, J.E., Hedden, P., Gaskin, P., MacMillan, J . Phytochemistry. 1974, 13, l433-1440. Reeve, D.R., Crozier, A. J . Exp. Botany. 1974, 25, 431-445. Reeve, D.R., Crozier, A. In "Gibberellins and Plant Growth." Krishnamoorthy, H.N., Ed. Wiley Eastern: New Delhi, 1975,

pp. 35-64. 89. 90.

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Can. J . Bot., 1970 91. 92. 93. 94.

Hoad, G.V., Pharis, R.P., Railton, I.D., Durley, R.C. Planta. 1976, 130, 113-120. Stoddart, J . , Breidenbach, W., Nadeau, R., Rappaport, L. Proc. Nat. Acad. S c i . U.S.A. 1974, 71, 3255-3259. Konjevic, R., Grubisic, D., Morkovic, R., Petrovic, J . Planta. 1976, 131, 125-128. Jelsema, C.L., Ruddat, M., Norre, D.J., Williamson, F.A. Plant C e l l Physiol. 1977, 18, 1009-1019.

RECEIVED

June 19, 1979.

In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

3 Gibberellin Biosynthesis in the Fungus Gibberella fujikuroi and in Higher Plants

BERNARD O. PHINNEY Department of Biology, University of California, Los Angeles, CA 90024

The gibberellins (GAs)1 o r i g i n a l l identified secondary metabolites o (Fusarium moniliforme Sheld.) compound apparent r o l e i n the fungus, they have been found to elicit a variety of responses i n higher plants (seed plants) including shoot elongation, sex expression, f r u i t growth, and seed germination (1,2). The early descriptions of GA-induced elongation, especially those associated with genetic dwarfism ( 3 , 4 ) , led to the idea that GAs might be naturally-occurring i n normal, nondwarf strains of higher plants. The f i r s t evidence for the presence of gibberellin-like substances i n higher plants came from the fact that semipurified extracts from such material would mimic a GA-induced growth response when applied to genetic dwarfs ( 5 , 6 , 7 ) . This evidence was soon followed by the isolation and chemical identification of GAs from higher plants (8,9,10). Since then 53 GAs have been identified as naturally occurring, 22 of them being found i n the fungus G. fujikuroi, and40of them i n higher plants including members of the Gymnospermae (e.g. pines) and the Angiospermae (flowering plants) (1,11). Although gibberellin-like substances have also been obtained from other groups of plants such as algae, other fungi, bacteria, mosses and ferns, these substances have yet to be i d e n t i f i e d chemically as GAs. It seems l i k e l y that GAs w i l l be found to occur universally i n the plant kingdom. A l l GAs are tetracyclic diterpene acids; they can be d i v i ded into two types, the C20~GAs and the C ] q - G A s (Figure l ) . The unraveling of the details of the biosynthetic origin of the GAs has proven to be less d i f f i c u l t than the diversity of structures would suggest. Some of the simplifying factors are (l) that the origin of the GAs i n both the fungus and higher plants i s through a single terpenoid pathway leading to the common GA-precursor, GAi ~ aldehyde ; (2) that 20 of the 22 fungal GAs have now been biosynthetically related to each other v i a two pathways; and (3) that, although kO GAs have been identified from higher plants, less than 15 have been found i n any one 2

2

0-8412-0518-3/79/47-lll-057$05.50/0 © 1979 American Chemical Society In Plant Growth Substances; Mandava, N.; ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

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Figure 1.

SUBSTANCES

Numbering system for ent-kaurene and the GA's (GA ) and structures of ent-kaurene, GA , GA , and GA 12

12

9

3

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the

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Gibberellin Biosynthesis

species of plant. This review w i l l present an overall picture of GA biosyn­ thesis i n plants. In addition the role of GA biosynthesis i n relation to GA-induced shoot elongation w i l l be b r i e f l y dis­ cussed, as w i l l the correlation of levels of endogenous GA-like substances with elongation growth i n higher plants. A l l biosynthetic steps relating GAs to each other and to GA-precursors presented here are based on feeds of radiolabeled GAs and their precursors to plant preparations, including intact plants, tissue sections, and c e l l free homogenates The evidence for the specific steps described here has recently been reviewed i n de­ t a i l by Hedden et a l . (11) and by Graebe and Ropers (1). The latter also includes a c r i t i c a l evaluation of information on the physiology of the GAs. The details of the biochemistry of polyisoprenoid biosynthesis has recently been reviewed by Beytia and Porter (12) and Goodwi (13) From MVA to GA^-aldehyde i n the Fungus and i n Higher Plants (Figure 2) The earliest steps (MVA to GGPP) for polyisoprenoid biosyn­ thesis are identical for a l l plants and animals (12,13). They involve the well-known diterpene pathway, MVA — M V A P — M V A P P — I P P + DMAPP — G P P —»> FPP — G G P P . The enzymes catalyz­ ing these steps have been studied extensively, especially from animals (liver) and yeast, and to a more limited extent from higher plants. In some cases the enzymes have been purified to homogeneity; most have been only p a r t i a l l y purified. In both plants and animals a major branch at FPP leads to the production of squalene and the steroids. In plants, three major branches occur at GGPP, of which one leads to the carotenoids v i a phytoene, a second to the phytyl group of chlorophyll, and a t h i r d to the GAs. The steps i n the pathway from GGPP to GAi2~aldehyde are unique to plants and involve the reactions GGPP — e n t - k a u r e n e — ^ ent-kaurenol — e n t - k a u r e n a l — ^ ent-kaurenoic acid — ^ ent-7cy-hydroxy kaurenoic acid—GA]_2~aldehyde. The two step reaction, GGPP — ^ CPP — ^ ent-kaurene, i s catalyzed by entkaurene synthetase (15). This enzyme or enzyme complex i s responsible for (1) the proton-initiated cyclization to form the A and Β rings of the b i c y c l i c intermediate CPP (A a c t i v i t y ) , and (2) the loss of pyrophosphate, cyclization and rearrangement of the resulting carbonium ion, and loss of H from carbon-17 to produce ent-kaurene (B a c t i v i t y ) . ent-Kaurene i s the f i r s t com­ mitted intermediate i n the biosynthetic pathway leading to the GAs, and i t has been suggested that the A a c t i v i t y may be a limiting step i n GA biosynthesis ( l 6 ) . The enzymes catalyzing the steps from MVA to ent-kaurene are soluble. After production of ent-kaurene, carbon-19 i s sequentially oxidized to give ent-kaurenol, ent-kaurenal, and ent-kaurenoic +

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CPP

é?/7/-KAURENE

Figure 2. The GA hiosynthetic pathway from MVA to GA -aldehyde. This pathway is found in the fungus Gibberella fujikuroi and higher plants 12

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acid. This i s followed by a hydroxylation on carbon-7 to give ent-7a-hydroxykaurenoic acid. There i s some evidence that the enzymes for the pathway from MVA to ent-7a-hydroxykaurenoic acid are present i n subcellular organelles - proplastids and chloroplasts (ώ,Ιδ,ΐΧίΙ^) . characteristics of the enzymes cataly­ zing the steps between ent-kaurene and ent-7