Biochemistry and Physiology of Plant Hormones [2 ed.] 978-1-4612-8193-1, 978-1-4612-3654-2

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Thomas C. Moore

Biochemistry and Physiology of Plant Hormones Second Edition

With 177 Figures

Springer-Verlag New York Heidelberg Berlin London Paris Tokyo Hong Kong

Thomas C. Moore Professor of Botany Department of Botany and Plant Pathology Oregon State University Corvallis, Oregon 97331-2902 USA

Library of Congress Cataloging-in-Publication Data Moore, Thomas C. Biochemistry and physiology of plant hormones. Includes bibliographical references. 1. Plant hormones. I. Title. QK898.H67M66 1989 581.19'27 89-19737 ISBN-13: 978-1-4612-8193-1 DOT: 10.1007/978-1-4612-3654-2

e-ISBN-13: 978-1-4612-3654-2

Printed on acid-free paper

© 1979, 1989 Springer-Verlag New York Inc. Softcover reprint of the hardcover 2nd edition 1989 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer-Verlag New York Inc., 175 Fifth Avenue, New York, NY 10010, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use of general descriptive names, trade names, trademarks, etc., in this publication, even if the former are not especially identified, is not to be taken as a sign that such names, as understood by the Trade Marks and Merchandise Marks Act, may accordingly be used freely by anyone. Typeset by Technical Typesetting Inc., Baltimore, Maryland.

987654321

Biochemistry and Physiology of Plant Hormones

Second Edition

Preface to the First Edition Biochemistry and Physiology oj Plant Hormones is intended primarily as a textbook or major reference for a one-term intermediate-level or advanced course dealing with hormonal regulation of growth and development of seed plants for students majoring in biology, botany, and applied botany fields such as agronomy, forestry, and horticulture. Additionally, it should be useful to others who wish to become familiar with the topic in relation to their principal student or professional interests in related fields. It is assumed that readers will have a background in fundamental biology, plant physiology, and biochemistry. The dominant objective of Biochemistry and Physiology oj Plant Hormones is to summarize, in a reasonably balanced and comprehensive way, the current state of our fundamental knowledge regarding the major kinds of hormones and the phytochrome pigment system. Written primarily for students rather than researchers, the book is purposely brief. Biochemical aspects have been given priority intentionally, somewhat at the expense of physiological considerations. There are extensive citations of the literature-both old and recent-but, it is hoped, not so much documentation as to make the book difficult to read. The specific choices of publications to cite and illustrations to present were made for different reasons, often to illustrate historical development, sometimes to illustrate ideas that later proved invalid, occasionally to exemplify conflicting hypotheses, and most often to illustrate the current state of our knowledge about hormonal phenomena. The lists of references at the ends of the chapters, containing some references which are cited and others that are not, are not intended as comprehensive bibliographies of the most recent, or even exclusively the most important, publications on each subject. Each list is intended both to document the text and provide other examples of the extensive literature on each topic. An explanation should be given for inclusion of the subject matter comprising Chapter 1, since it is acknowledged that many readers will regard Chapter 1 as quite elementary information with which they already are familiar. That is fully to be expected. But for those readers whose background may be deficient, as has been found to be true of a fair percentage of students, Chapter 1 will provide a reasonable overall introduction to and perspective about growth and development of whole plants throughout ontogeny and set the stage for consideration of hormonal regulation. Books such as this invariably disappoint some readers, which is to say that they cannot be-perhaps should not even purport to be-all things that all readers might wish or expect. In my judgment, Biochemistry and Physiology oj Plant Hormones most likely might disappoint some readers in each of two ways. First, the book does not contain as lengthy and integrated a discussion either of the physiological roles of the different kinds of hormones or of hormonal

vi

Preface to the First Edition

interactions as some readers will wish, although, of course, these topics definitely are covered. To the extent that this is true it is by design. For in my ten years of experience teaching a graduate course in hormonal regulation of plant growth and development, I personally have found that it is more effective to guide students from an information base such as this book provides to a more integrated understanding of regulation of growth and development than to undertake the converse approach. Another way this book might disappoint some prospective users is that it lacks detailed and comprehensive coverage of practical uses of synthetic plant growth regulators, except for synthetic auxins and auxin-type herbicides. Such information is largely beyond the scope of this small volume. Moreover, practical uses of plant growth regulators are covered in many specialized books in agronomy, forestry, and horticulture. It seems to be a good time in some ways, and not so good a time in other ways, for a new book on the biochemistry and physiology of plant hormones. On the negative side, so far during the decade of the 1970s there seems to be a relative lull in the field as regards dramatic new developments-the "acid growth theory" and other important advancements notwithstanding-compared, let us say, to either of the previous two decades. In view of the relative scarcity of "big news," it could be argued that it is not a particularly good time. But, on the other hand, there is really good and highly significant research going on, and there is a steady output of important new knowledge. The literature-the state of the science-probably is in the best shape ever as far as unequivocal validation of facts and concepts is concerned. It is a time of separation of fact from fiction and devising new approaches to old problems, as well as asking new, important, exciting questions. For these reasons, it seems, therefore, timely for a new book to call attention to this healthy state of the science. In any case, it is an excellent time to be a student at any level of the fascinating subject discussed in

Biochemistry and Physiology of Plant Hormones.

Acknowledgments The real credit for Biochemistry and Physiology of Plant Hormones ultimately should go to the many Plant Physiologists whose research during the last half century disclosed information comprising the book. While too numerous to mention individually, the names of many of these scientists are contained in the literature lists at the ends of the chapters. Certain specitic contributions by particular authors, of course, are acknowledged also in the forms of citations in the text and notations in legends to figures and tables. The actual writing and production of the book naturally has involved several forms of assistance by many persons, to all of whom I express my sincere gratitude. For directly supplying or assisting to make available certain illustrations, I thank Douglas O. Adams, James D. Anderson, Gerard W. M. Barendse, Michael L. Evans, Peter Hedden, Hans Kende, Anton Lang, A. Carl Leopold, Morris Lieberman, Bernard O. Phinney, Folke Skoog, Nobutaka Takahashi and Jan A. D. Zeevaart. Donald J. Armstrong and Ralph S. Quatrano are thanked for the advice and technical assistance that they provided regarding various topics. I thank Ellen Witt and Leona Nicholson for typing and clerical assistance, and E. Kay Fernald for photographic service. Mark Licker, Science Editor, and Judi Allen, Production Editor, at Springer-Verlag Inc., New York, and their staff were very helpful throughout the review and production processes. Finally, I wish to acknowledge the financial support provided by the National Science Foundation for those of my own investigations during the past fifteen years which are cited in the book. Corvallis, Oregon January 1979

Thomas C. Moore

Preface to the Second Edition Ten years ago, when writing the Preface to the first edition of Biochemistry and Physiology of Plant Hormones, I observed that, "The literature (on plant hormones)-the state of the science-probably is in the best shape ever as far as unequivocal validation of facts and concepts is concerned," and further that, "It is a time of separation of fact from fiction and devising new approaches to old problems, as well as asking new, important, exciting questions." Assuredly, those observations are as valid in 1989 as they were in 1979. Progress during the last decade has not occurred by dramatic major discoveries, but rather by a careful, methodical, and definitive slow pace. The increasingly widespread application of a large number of techniques of plant molecular biology in hormone investigations has enabled many impressive advances, such as (1) elucidation of the nucleotide sequences of phytochrome cDNA clones and deduction of the amino acid sequences by Peter H. Quail and others; (2) isolation and partial characterization of hormone receptors by Michael A. Venis and others; and (3) identification of early gene products resulting from the action of hormones on chromatin by Gretchen Hagen, Tuanhua David Ho, and others. We seem to be very close to understanding in precise molecular terms the early events in the mechanisms of action of the auxins, gibberellins, and abscisic acid in particular. Because of the efforts of Anthony J. Trewavas and others, there is a growing appreciation of the concept that plant responses are not correlated only with the amounts or concentrations of endogenous growth substances but as well to the state of sensitivity of the tissues to those substances. The increasing use of mutants in hormone research by Bernard O. Phinney, James B. Reid, and others is enabling an ever closer connection between genes and the hormones they control. Through the efforts of N. Bhushan Mandava. Nobutaka Takahashi, Takao Yokota, and others we are gaining increased appreciation of the brassinosteroids, which, in the" opinion of many plant physiologists, deserve to be treated as a new class of plant growth substances. And problems of long standing continue to receive attention. Kenneth V. Thimann and an associate only recently reported new evidence concerning the role of ethylene in apical dominance, for example. As exciting and important as the emerging discoveries of plant molecular biology are, the challenge to understand hormonal regulation at the organismal level is ever present. We still seem a long way from understanding the role of hormones in phenomena such as photoperiodic induction of flowering, endogenous circadian and other rhythms, gravitropism, and senescence. The changes made in the second edition of Biochemistry and Physiology of Plant Hormones are substantial. Part of the former Chapter 1 has been deleted, as that subject matter has become common knowledge; a new chapter on

x

Preface to the Second Edition

brassinosteroids has been added, and every other chapter has been revised and updated. The use of references continues to be as it was described in the Preface to the first edition, in that I have not attempted to provide comprehensive lists of references on each subject. The chapters are not comprehensive reviews but rather guides for students to what I believe to be the most relevant and important subject matter on each topic.

Acknowledgments Many persons have contributed in many ways to the production of the second edition of Biochemistry and Physiology of Plant Hormones, and my sincere gratitude is extended to them all. In particular I wish to thank Donald J. Armstrong, Derek J. Baisted, Ronald C. Coolbaugh, Tuan-hua David Ho, Terri L. Lomax, N. Bhushan Mandava, Noboru Murofushi, B. W. Poovaiah, Peter H. Quail, Nobutaka Takahashi, and Takao Yokota. I thank Ellen Witt for her typing of the manuscript and clerical assistance. Corvallis, Oregon January 1989

Thomas C. Moore

Contents CHAPTER 1

INTRODUCTION

Fundamental Terms and Concepts Patterns and Kinetics of Growth in Cells, Tissues, Organs, and Whole Plants Discontinuities in Growth, Growth Periodicities, and Problems of Relative Growth Rate Mechanisms Controlling Cellular Differentiation Introduction to Plant Hormones References CHAPTER

2

AUXINS

Brief History of Discovery Went's A vena Coleoptile Curvature Test Early Isolations of IAA Synthetic Auxins Controversy Surrounding the Use of Certain Chlorophenoxy Acids as Herbicides and Defoliants Natural Occurrence of Auxins Auxin Biosynthesis "Free" and "Bound" Auxin Destruction of lAA Auxin Transport Relationships between Auxin Content and Growth Correlative Differences in Auxin Relations between Etiolated and Light-Grown and Dwarf and Normal Plants Mechanism of Auxin Action References CHAPTER

3

GIBBERELLlNS

Brief History of Discovery Chemical Characterization of GAs Natural Occurrence of GAs GA Biosynthesis in Seeds GA Biosynthesis in Systems Other Than Seeds Effects of Light on GA Biosynthesis Role of GAs in Dwarfism Other Aspects of GA Metabolism

1

3 12 14 23 25 28 28 31 32 33 34 38 39 42 44 47 54 58 61 85

94 94 96 104 104 108 113 114 121

Contents

xiv

Quantitative Changes in GA Content during Development Sites of GA Biosynthesis in Seed Plants Transport of GA Anatomical and Biophysical Basis of GA-Induced Growth Mechanism of Action of GA References CHAPTER

4

CYTOKININS

History of Discovery Terminology Isolation of Kinetin and the Search for Other Naturally Occurring Cytokinins Discovery of Natural Cytokinins Effects of Cytokinins and Other Hormones in Organisms Other Than Seed Plants Structure/Activity Relationships of the Cytokinins Biosynthesis and Metabolism Auxin and Cytokinin Biosynthesis in Crown Gall Tumors Mechanisms of Origin in tRNA Metabolic Consequences of the Presence of Cytokinins in tRNA Hormonal Activity of Free Cytokinins Cytokinin Binding Protein Effects on Moss Protonemata Some Physiological Effects on Seed Plants Translocation References CHAPTER

5

ABSCISIC ACID AND RELATED COMPOUNDS

Introduction History of Discovery Chemical Characterization Biosynthesis and Other Features of Metabolism Natural Occurrence of ABA Physiological Effects State of Chromatin in Dormant Tissues and the Mechanism of Action of ABA References CHAPTER

6

ETHYLENE

Historical Background Ethylene and Fruit Ripening Interaction between Auxin and Ethylene

123 134 136 136 139 150 158 158 159 159 160 166 167 170 174 174 178 179 180 181 184 188 189 196 196 197 201 202 207 208 220 221 228 228 229 235

Contents

Ethylene and Apical Dominance Inhibition of Root Growth and Role in Root Gravitropism Role in Emergence of Dicot Seedlings Role in Abscission Effects of Ethylene on Planes of Cell Expansion Other Effects of Ethylene Ethylene Biosynthesis and Mechanism of Action Ethylene Receptor References CHAPTER

7

BRASSINOSTEROlDS

Introduction Brief History of Discovery Chemistry of BRs Natural Occurrence of BRs Biological Effects Effects on Nucleic Acid and Protein Metabolism Practical Applications in Agriculture Some Problems and Prospects References CHAPTER

8

PHYTOCHROME

xv

238 239 240 242 244 244

248 249 250 255 255 255 256 256 260 263 263 264 264

267

Introduction History of Discovery and Modern Description Sequence Analysis of Phytochrome Occurrence, Distribution, and Intracellular Localization Induction-Reversion versus High Irradiance Responses Non-Phytochrome-Mediated Photoresponses to Blue Light Introduction to Mechanism of Phytochrome Action Phytochrome Action in Nonphotoperiodic Photoresponses Phytochrome and Photoperiodic (Flowering) Responses Calcium Messenger System in Plants References

267 267 275 278 280 281 283 284 293 308 312

Index

321

CHAPTER

1

Introduction Fundamental Terms and Concepts The term "development," as it applies to whole seed plants arising by sexual reproduction, denotes the gradual and progressive changes in size, structure, and function that collectively comprise the transformation of a zygote into a mature, reproductive plant. It is also a correct and common practice to speak of the development of particular organs from initials or primordia, and to refer to development of a whole plant from any single cell. Whatever the specific case, development is a gradual process that takes time to be fully realized, generally is accompanied by increases in size and weight, involves the appearance of new structures and functions and the loss of former ones, is characterized by temporal and spatial discontinuities and changes in rate, and eventually slows down or ceases when mature dimensions are reached. Unfortunately, but perhaps not surprisingly, there is no rigorously standardized terminology applied to the phenomena of plant growth and development. Some physiologists, the author included, consider that there are three interrelated processes that together comprise development, namely, "growth," "cellular differentiation," and "morphogenesis." "Growth" is defined as an irreversible increase in size that is commonly, but not necessarily (e.g., the growth of an etiolated seedling), accompanied by an increase in dry weight and in the amount of protoplasm. Alternatively, it may be viewed as an increase in volume or in length of a plant or plant part. In any case, it must be emphasized that growth can occur only by an increase in volume of the individual cells. Some authors consider cell division as a separate process that accompanies growth in meristems, but a more generally held view is that growth includes cell division as well as cell enlargement. "Cellular differentiation" is the transformation of apparently genetically identical cells of common derivation from a zygote or other single cell into diversified cells with various biochemical, physiological, and structural specializations. It is the sum of the processes by which specific metabolic competences are acquired or lost and distinguish daughter cells from each other or from the progenitor cell. "Morphogenesis" is the integration and coordination of growth and differentiative events occurring at the cellular level and is the process which accounts for the origin of morphological characters and gross form. Other authors have used the terminology somewhat differently. For example, E. W. Sinnot (1960) in a book entitled Plant Morphogenesis wrote, "The process of organic development, in which are posed the chief problems for the

2

1. Introduction

science of morphogenesis, occurs in the great maJonty of cases as an accompaniment of the process of growth. The association between these two activities (growth and development) is not an invariable one, for there are a few organisms in which growth is completed before development and differentiation are finished, but far more commonly the fonn and structure of a living thing change while it grows." One example of an exception is the development of the female gametophyte from an 8-nucleate stage in embryo sac development in angiospenns. Some have employed the tenn "morphogenesis" in a strictly descriptive sense, essentially as synonymous with classical developmental morphology. More generally and properly, however, it includes, besides descriptive facts as to the origin of form, a study of the results of experimentally controlled development and an analysis of the effects of factors, external and internal, that determine how the development of form proceeds. In other words, it attempts to get at the underlying formativeness in the development of organisms and especially to reach an understanding of the basic fact of which form is the most obvious manifestation, namely, biological organization itself. According to morphogeneticists like Sinnott, "The organism may thus be said to make the cells rather than the cells to make the organism." F. B. Salisbury and Cleon Ross (1985) used the tenn "development" (or "morphogenesis") as an inclusive tenn and regard the phenomenon as consisting of two primary functions: growth and differentiation. They consider growth primarily as an increase in size, and differentiation as the process by which cells become specialized. P. F. Wareing and 1. D. J. Phillips (1981) likewise adopted the view that development should be applied in its broadest sense to the whole series of changes that an organism goes through during its life cycle, while noting that it may also be applied to individual organs, to tissues, or even to cells. According to these authors, "plant development" involves both "growth" and "differentiation." "Growth" is used to denote quantitative changes occurring during development and is defined as an irreversible change in the size of a cell, organ, or whole plant. "Differentiation" is applied to qualitative changes. Thus, in their view growth and differentiation are the two major developmental processes. They reserve the term "morphogenesis" as one used by experimental morphologists (morphogeneticists) to denote origin of form. F. C. Steward (1968) employed a quite inclusive connotation of growth. Essentially he used the term "growth" in a very general way to include what others consider more explicitly to be growth, differentiation, and morphogenesis. With the variable usage of terminology, as has been illustrated, perhaps it is understandable why the coupled tenns "growth and development" are used so prevalently. This couplet connotes the kind of concept that James Bonner and A. W. Galston (1952) had when they wrote. "The changing shape, form, degree of differentiation, and state of complexity of the organization constitute the process of development." They viewed "growth" as a quantitative matter concerned with the increasing amount of the organism. On the other hand, "development,"

Patterns and Kinetics of Growth in Celis, Tissues, Organs, and Whole Plants

3

in their view, refers to changes in the nature of the growth made by the organism. Many biologists have emphasized the biological importance of organization and emphasize that the characteristics of life itself are characteristics of a system arising from, and associated with, the organization of materials and processes. It is of utmost importance to keep in mind the fact that there are unique emergent qualities associated with each successively higher level of biological organization-from molecular and subcellular to the levels of cells, tissues, organs, whole organisms, and beyond. In no instance is the cliche that the whole is more than the sum of its parts more vividly exemplified than when we observe the complicated changes that a seed plant manifests during the repeating cycle of development. We can arbitrarily conceive of this cycle as starting with the germination of a seed and continuing with the passage of a juvenile phase of growth and the graduation into maturity. With maturity the organism is capable of shifting from vegetative to reproductive development, with the development of flowers, the development of fruits, and the production again of a new generation of seed. mtimately, the development of the individual plant ends with senescence and death. This book is concerned with the processes involved in and the mechanisms which control the growth and development of seed plants.

Patterns and Kinetics of Growth in Cells, Tissues, Organs, and Whole Plants The curve which typically describes the changing size of a growing organism, organ, tissue, cell population, or individual cell is sigmoid in shape (Fig. 1.1a). The sigmoid growth curve can, for convenience, be considered in three parts. First, there is an accelerating phase in which growth starts slowly and gathers momentum. During this period of constantly accelerating growth rate, increase in size is exponential. When a plant is growing exponentially, it is said to be in the logarithmic phase of its growth. Second, there is either a point of inflection or a phase, more or less protracted, in which the course of growth is approximately linear with time. That is, equal increments of growth tend to occur in equal intervals of time. This is often termed the linear phase of growth. In some cases, however, there is no linear phase but merely a point of inflection in the curve, when the first rising rate of growth gives way to a decreasing rate of growth. Third, there is a phase of declining growth rate until, in fact, growth subsides and the organism may maintain only the size it has already achieved. In annual plants this is followed by senescence and death. In the case of woody perennials, each period of shoot growth is approximately sigmoidal and is followed by dormancy. Shoots of biennials, of course, exhibit two seasons of sigmoidal growth. Instead of plotting merely size versus time (Fig. 1.1a), it is often useful to plot the loge of size versus time (Fig. Lib) or growth rate versus time (Fig. 1.1c) when analyzing growth. Examples of various specific growth curves and a brief discussion of each follow.

4

1. Introduction FIGURE 1.1. Generalized growth curves. a Size-versus-time sigmoid growth curve; b logarithmic growth curve; and c growth rate curve. (Based on data for corn, Zea mays, in Whaley, 1961.)

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Even the growth of single cells has been demonstrated to be sigmoidal (Fig. 1.2). Organs such as leaves follow a sigmoidal pattern also on the basis of several parameters, including fresh weight, area oflamina, length oflamina, and cell number (Fig. 1.3). Many fruits exhibit common sigmoid growth curves-e.g., apple (Fig. 1.4), pineapple, strawberry, pea, and tomato. However, fruits of many species exhibit more complicated growth curves, which are essentially double sigmoid growth curves. This type of curve is common to probably all the stone fruits such as cherry (Fig. 1.5), apricot, plum, and peach. Such curves are also typical of some nonstone fruits such as fig, grape, and currant. However, neither type of growth curve seems to be distinctive for a particular morphological type of fruit because there are berries, pomes, and simple and accessory fruits that manifest each.

Patterns and Kinetics of Growth in Cells, Tissues, Organs, and Whole Plants FIGURE 1.2. Growth curves of single cells in the com (Zea mays) root tip. a Fresh weight; b dry weight. (Redrawn, with permission, from Boss, 1964.)

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The double sigmoid growth curve can be explained on the basis of asynchrony of growth of the different parts of the fruit. Two general types of growth centers occur in fleshy fruits, the pericarp and ovule(s). The growth of the pericarp commonly accounts for the initial enlargement. Growth in later stages generally is associated with seed development. There is wide variation in the extent to which cell division participates in the growth of fruits, ranging from cases in which cell division (except in the developing seeds) has been completed at the time of pollination (Ribes, Rubus), to cases in which there is a brief period of cell division just following pollination (tomato, Citrus, cucurbits, apples, Prunus), or rather extended periods of cell division (strawberry). The curve describing the kinetics of growth of whole plants also is typically sigmoidal. Herbaceous annual plants, such as garden pea (Pisum sativum L.), exhibit a single sigmoid growth curve throughout ontogeny from germination to senescence and death (Fig. 1.6). Size versus time plots of the shoot growth of woody perennials are sigmoidal throughout ontogeny (Fig. 1.7), although there is a gradual progression toward the asymptote. Notably, however, the size versus time plot for shoot growth of woody plants is composed of increments of growth, each sigmodal, which alternate with the periods of dormancy. This is true of both primary shoot growth (Fig. 1.8) and secondary shoot growth (Fig. 1.9). Growth and rate curves for different parameters in woody plants reveal other

1. Introduction

6

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Extract activity evaluated by tbe Xanthium biossay. One hundred plants were used in each of tbe six treatments. Ten plants were used to evaluate each oftbe 20 extracts from flowering or vegetative plants. The figures represent tbe total number of plants in each stage of flowering . Plants in stage 2 or above are considered to be flowering. GA, gibberellic acid. (Witb permission from Hodson, H . K. and K. C. Hamner. 1970. Floral inducing extract from Xanthium. Science 167: 384-385, 23 January 1970. Copyright 1970 by the American Association for tbe Advancement of Science.)

a

304

8. Phytochrome

TABLE 8.5. Effects of an extract from Xanthium strumarium on flowering of Lemna perpusilla. a

Treatment Flowering plants Flowering plants + GA Vegetative plants Vegetative plants + GA Gibberellic acid Water

Plants flowering (%)

45

31

54

37

58

46

45

51

39

0 0 0 0 0

0 0 0 0 0

0

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7

Extract activity evaluated by the Lemna biossay. A total of30,000 plants were used to evaluate the activity of the 20 extracts. The extracts were tested with and without the addition of gibberellic acid (GA). Each figure represents the average response of five lots of 100 plants each. (With permission from Hodson, H. K. and K. C. Hamner, 1970. Floral inducing extract from Xanthium. Science 167: 384-385,23 January 1970. Copyright 1970 by the American Association for the Advancement of Science.) a

Anton Lang flrst reported in 1957 that exogenous GA could cause flowering in numerous species of long-day and vernalization-requiring plants under noninductive environmental conditions (Fig. 8.24), but GA is not the long sought after florigen. One reason is that GAs do not cause flowering of short-day plants under noninductive conditions, or even in all long-day plants. Moreover, there is now good evidence that flowering and flower-bearing stem elongation (bolting) are separate processes in plants such as Silene armeria (Cleland and Zeevaart, 1970), with GA promoting stem elongation only. The latter conclusion was based on the flndings that (1) treatment of Silene armeria with Amo-1618, an inhibitor of GA biosysthesis, inhibited stem elongation but not flowering under long-day conditions; and (2) with GA treatment on short days, stem elongation sometimes occurred in the absence of flowering. Thus it seems clear that although flowering and stem elongation are closely related in rosettetype long-day plants under normal conditions, the two processes can be experimentally separated and thus represent separate developmental processes. While GA is not"florigen," it conceivably might be "vernalin." Jan A. D. Zeevaart of the MSU-DOE Plant Research Laboratory at East Lansing, Michigan and his associates have elucidated in remarkable detail the GA metabolism that evidently is causally involved in the photoperiodic control of stem growth in spinach (Spinacia o!eracea), a long-day rosette plant. Very early in their research it was determined that photoperiodic control of stem growth in spinach, as in other long-day, rosette plants like Silene armeria and Agrostemma githago, is mediated by GA (Zeevaart, 1971). They identifled six endogenous C-13 hydroxylated GAs in spinach shoots: GA 17 , GAI9, GA20, GA29 , ｇｾＬ＠ and GAS3 ' Five of these (except GA 17) were shown to be metabolically related as illustrated in Fig. 8.25 (Metzger and Zeevaart, 1980a; Gilmour et al., 1986, 1987). In spinach, long-day treatment of plants did not increase the total GA level, but levels of individual GAs did vary (Fig. 8.26), indicating a profound change in GA metabolism. Thus GAI9 showed a 5-fold decline, while the levels of GA20 and GA29 increased dramatically during the same period. The levels of GAI7 and ｇｾ＠ did not change signiflcantly with long-day treatment. GAS3 occurred in quantities too small to be measured.

Phytochrome and Photoperiodic (Flowering) Responses

305

FIGURE 8.24. Carrot (Daucus carota) plants showing the application of GA as a substitute for a cold requirement for flowering. Treatments: Left, no GA, no cold treatment. Center, 10 p.g GA per day, no cold treatment. Right, no GA but 8 weeks cold treatment. The plants were grown in long days. (From Lang, 1957, with permission; photo courtesy of A. Lang, 1978.)

306

8. Phytochrome

GaI2 13-

hydroxylase

GaS3 oxidase

*GAJ2

GA44

oxidase

HO GA 20 Ｒｾﾭ

GA 19

hydroxylase

oxidase

*GA20 FIGURE 8.25. GA conversions in cell-free systems from spinach leaves grown in long days, except for the step GAzo -> GAZ9 that was not observed. GAs marked with an asterisk are endogenous to spinach. GA12 has not been shown to be endogenous to spinach. (Redrawn in slightly modified form, with permission, from Gilmour et al., 1987).

Gilmour et al. (1986) investigated further the metabolic relations among the endogenous GAs by using cell-free enzyme extracts prepared from spinach leaves and by identifying the products by gas chromatography-mass spectrometry. The pathway shown in Fig. 8.25 was found to operate in the cell-free enzyme extracts, except that the conversion of GA20 to GA29 was not observed. Extracts from plants given long days (LD) or short days (SD) were examined, and enzymic activities were measured as a function of exposure to LD, as well as to darkness following 8 LD. The results indicate that the activities of the enzymes oxidizing GA53 and GA 19 are increased in LD and decreased in SD or darkness, but that the enzyme activity oxidizing ｇｾ＠ remained high irrespective of light or dark treatment. This photoperiodic control of enzyme activity was shown not to be due to the presence of an inhibitor in plants grown in SD. These observations provided an explanation for the higher GA20 content of spinach plants in LD than in SD. It is thought to be the increased endogenous GA20 that brings about stem growth in LD (Metzger and Zeevaart, 1980b). More recently Gilmour et al. (1987) reported on the extraction and partial purification of four enzyme activities catalyzing the following oxidative steps in the GA pathway in spinach: GA I2 -> GA53 -> ｇｾ＠ -> GAI9 -> GA2o . During the early investigations of the effects of exogenous ABA on plants, it

Phytochrome and Photoperiodic (Flowering) Responses GA20

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.-

X""""- /X-X - 100 /

/1,

l.!:! 60

.