CONTRIBUTORS NELSON J. LEONARD PETER M. RAY J. MACMILLAN J. E. VARNER B. V. MILBORROW S. F. YANG THE CHEMISTRY AND BIOCHEMISTRY OF PLANT HORMONES Recent Advances in Phytochemistry Volume 7 Edited by V. C. RUNECKLES Department of Plant Science University of British Columbia Vancouver, British Columbia, Canada E. SONDHEIMER College of Environmental Science and Forestry State University of New York Syracuse, New York and D. C. WALTON College of Environmental Science and Forestry State University of New York Syracuse, New York ACADEMIC PRESS New York and London 1974 A Subsidiary of Harcourt Brace Jovanovich, Publishers COPYRIGHT © 1974, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER. ACADEMIC PRESS, INC. Ill Fifth Avenue, New York, New York 10003 United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NW1 LIBRARY OF CONGRESS CATALOG CARD NUMBER: 77-182656 PRINTED IN THE UNITED STATES OF AMERICA LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin. NELSON J. LEONARD (21), School of Chemical Sciences, Department of Chemistry, University of Illinois, Urbana, Illinois J. MACMILLAN (1), School of Chemistry, The University, Bristol, England B. V. MILBORROW (57), Shell Research Limited, Milstead Laboratory of Chemical Enzymology, Sittingbourne Laboratories, Sittingbourne, Kent, England PETER M. RAY (93),* Department of Biological Sciences, Stanford Uni- versity, Stanford, California J. E. VARNER (123), MSU/AEC Plant Research Laboratory, East Lansing, Michigan S. F. YANG (131), Department of Vegetable Crops, University of Cali- fornia, Davis, California * Present address: Institut für Biologie III, Universität Freiburg, Freiburg, West Germany. vii PREFACE At first glance one might suspect that our understanding of the chemistry and biochemistry of plant hormones should be further advanced than is the case with animal regulator substances. There are only five generally recognized groups of higher plant hormones: the auxins, the gibberellins, the cytokinins, abscisic acid, and ethylene. On the other hand, animal hormones are more diverse and are often more complex in structure. For example, proteins, peptides, steroids, and phenolic amines, which have major roles as animal hormones, are not presently known to be represented among the regulators of higher plants. Structure proofs for the proteins, and for many of the peptides and steroids, certainly represent more of a challenge than those for the hormones thus far encountered in plants. With regard to the biochemistry of hormones, one might also expect greater progress with plants. The latter have neither a nervous nor a muscular system and therefore require fewer control mechanisms than do their animal counterparts. Yet it must be admitted at the outset that progress in the understanding of the chemistry, and particularly the biochemistry, of plant hormonal regulation lags behind the understanding of regulation in the animal king- dom. There is no counterpart in plant biochemistry to the elegant work that has been done on the influence of estrogens on uterine processes. We know nothing about carrier proteins involved in the transport of plant hormones, a subject in which considerable progress has been made with animal hormones. The role of cyclic adenosine monophosphate as a second messenger in animal metabolic regulation has been elucidated. Such a role for this compound in higher plants is still in doubt. We mention these contrasts not in any spirit of discouragement, but rather to point out what a truly challenging research area is represented by the chemical and biochemical aspects of plant hormones. Furthermore, it is our hope that the present volume will be of aid to continued research efforts in this field. ix X Preface The authors attempted to bring the reader up to date in an area in which the literature is often highly dispersed among physiologically and chemi- cally oriented journals; they have critically evaluated their respective fields and have identified those topics where application of the discussed techniques and experiments can be expected to lead to a deeper under- standing of the mode of action of plant hormones. While there is no complete separation in the treatment of the hormones between chemistry and metabolism, the first three chapters display an orientation toward the chemical approach. In the review on gibberellins, modern isolation and characterization techniques are stressed. After read- ing this contribution, one can readily appreciate the powers of combining gas-liquid chromatography with mass spectral analysis in the solution of problems that have proven refractory to the more classical approaches The present status of cytokinin chemistry is described with emphasis on methods of structure elucidation, synthesis, and structure-activity rela- tions. This chapter illustrates very well the synergistic effects possible when workers from different areas are able to collaborate. The review of the chemistry of abscisic acid, the most recently discovered hormone dis- cussed in this volume, indicates that with modern techniques, progress in isolation, quantitative determination, characterization, synthesis, etc., can be extremely rapid. It is most remarkable that our knowledge concerning the chemical aspects of this compound is more or less at the same level as that of the other hormones, even though some of these were described almost 40 years ago. Elucidation of the role of auxins at the molecular level has proven a tremendous challenge to the plant biochemist, and perusal of the chapter by Ray shows that even sophisticated experiments have not yet provided any generally accepted explanations concerning the mode of action of indoleacetic acid. Here, clearly, is an area that will be a challenge to any investigator who wants to take up the gauntlet. In the fifth chapter, evi- dence is presented that gibberellic acid has an accelerating effect on the synthesis of rough endoplasmic reticulum in barley half-seeds. Since gib- berellic acid is also known to enhance the synthesis of various hydrolases in this system, it is of special significance that the membrane effects are observed before de novo enzyme synthesis is detectable. The suggested paths for the biosynthesis of ethylene are critically reviewed in the final chapter. At present, the best evidence indicates that methionine is the pre- cursor in higher plants, and glutamic acid in fungi. This work demonstrates the difficulties in determining the biogenesis of simple molecules which arise from common intermediary metabolites. This knowledge of the bio- synthesis should facilitate work on the regulation of ethylene biosynthesis. Preface xi The work summarized and discussed in the present volume shows the vast scope and multi faceted aspects of modern chemical and biochemical investigations of plant hormones. While progress may be a little slower in some areas than one had anticipated in the first flushes of discovery, the overall outlook is encouraging and will, we hope, lead additional investi- gators to pursue research in this fascinating and significant field. We wish to thank the University-Wide Programs, State University of New York, Albany, New York, for its financial support through the Con- versations in the Disciplines program. Acknowledgment is also made of the help provided by the SUNY College of Environmental Science and Forestry for hosting the Twelfth Annual Symposium of the Phytochemical Society of North America, under whose auspices this meeting was held. The Editor- in-Chief particularly wishes to thank the contributors to the volume for their cooperation, and also Ms. Diane Green, who achieved miracles in interpreting editorial changes and preparing the final manuscript copy. V. C. RUNECKLES D. C. WALTON Ernest Sondheimer 1923-1973 ERNEST SONDHEIMER The organizer of the symposium on which this book is based, Dr. Ernest Sondheimer, died during the preparation of this volume. Ernest Sondheimer was a long-time member of PSNA. Trained as an organic chemist, he became deeply interested in the biochemical and physiological aspects of seed dormancy. During the past nine years, he had been using his training and considerable talents in this area, bringing to the work a fresh viewpoint. His untimely death occurred when his studies on seed dormancy were making rapid progress. Ernest was the rare combination of dedicated researcher and highly effective teacher, enjoying both activities. Those of us who were his col- leagues, students, and friends will miss his interest in and enthusiasm about both his work and ours. Phytochemistry has lost one of its most able and ardent practitioners. xiii RECENT ASPECTS OF THE CHEMISTRY AND BIOSYNTHESIS OF THE GIBBERELLINS J. MacMILLAN School of Chemistry, The University, Bristol, England Introduction 1 Chemical Structures 3 Structure Determination 5 Identification in Plants 8 Biosynthetic Metabolism 10 Catabolic Metabolism 16 Concluding Remarks 17 References 18 Introduction The gibberellins comprise a large family of diterpenoid acids which were initially discovered as metabolites of Fusarium moniliforme (Gibberella fujikuroi), the causative fungus of the Bakanae disease of rice. The story of their discovery and emergence as an important group of ubiquitous plant hormones is a fascinating one which has been described in several reviews (e.g., Stodola, 1958; Stowe and Tamaki, 1957; Brian et dl., 1960). The gibberellins fulfill the following reasonable criteria for endogenous plant hormones: (a) when applied exogenously in extremely small amounts they induce a wide range of plant growth responses; (b) they are present in most, and probably all, higher plants; and (c) there is circumstantial 1