Advances in Botanical Research, Volume 9

Advances in

BOTANICAL RESEARCH VOLUME 9

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Advances in

BOTANICAL RESEARCH Edited by

H. W. WOOLHOUSE John Innes Institute, Norwich, England

VOLUME 9

1981

ACADEMIC PRESS A Subsidiary of Harcourt Brace Jovanovich, Publishers

London New York Toronto Sydney San Francisco

ACADEMIC PRESS INC. (LONDON) LTD. 24/28 Oval Road, London NW1 7DX

U.S. Edition published by ACADEMIC PRESS INC. 111 Fifth Avenue New York, New York 10003

Copyright

0 1981 by Academic Press Inc. (London) Ltd

AN Rights Reserved

No part of this book may be reproduced in any form by photostat, microfilm, or any other means, without written permission from the publishers

British Library Cataloguing in Publication Data

Advances in botanical research.-Vol. 9 1. Botany-Periodicals 581.05 QK45.2 ISBN C-12405909-6

Filmset in Great Britain by Latimer Trend & Company Ltd, Plymouth and printed by Thomson Litho Ltd, East Kilbride, Scotland

CONTRIBUTORS TO VOLUME 9 D. BOULTER, Department

of Botany, The University of Durham, Science Laboratories, South Road, Durham DH1 3LE, England A. CROZIER, Department of Botany, University of Glasgow, Glasgow GI2 8QQ, Scotland T. SACHS, Department of Botany, The Hebrew University, Jerusalem, Israel

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PREFACE The seed legumes comprise the most abundant source of vegetable protein for consumption by man or his livestock in many parts of the world. The breeding of legumes for improvement of the yield and quality of their seed proteins is a young science compared to the study of cereals but is now attracting a major investment of effort. In the first article in this volume Boulter describes the characterization of the storage proteins of legumes and their biochemical composition. The deposition of storage proteins is discussed in the context of the development of the seed. Boulter then turns to the challenging questions surrounding the isolation of the genes for the storage proteins and discusses possible approaches to the study of their control. It is clear that work in this rapidly advancing area of plant molecular biology will have enormous economic importance. The study of plant hormones may be regarded as the great challenge or perennial nightmare of the botanist’s world according to ones disposition. The subject is bewildering and in a sense disappointing in that after the heyday of “apply it and see” or “spray and pray” in which a wide range of effects of applying hormones were described, there has been only limited progress which bears no comparison with work on mechanisms of hormone action in animals. Even in well-defined systems such as amylase induction in the barley endosperm the role of receptors and second messengers remains obscure. It seems increasingly clear that hormone action in plants has many important features of difference from animal systems, and this is brought out by the emphasis on the chemistry of the gibberellins by Crozier. The opening section of this article emphasizes not only the vast complexity of gibberellin chemistry but also the need for the most rigorous chemical methods if progress is to be made in understanding the relationships and interconversions of the gibberellins, which are evidently of central importance to their mode of action in plants. Ultimately gibberellins affect plant growth and one has some sympathy with Crozier in his conclusion that further progress in understanding the mode of action of gibberellins may well rest on the development of a more definitive picture of the events involved in growth. In our previous volume Gross considered the biochemistry of lignification; it is a logical extension of his review to enquire concerning the control of the elaborate patterns which are observed in the disposition of lignified and other vascular tissues. Sachs takes up this subject of the control of vascular patterns

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in a deeply thought article which concludes the present volume. This article is built around the development of a hypothesis involving signals in the form of hormone fluxes and cellular responses which have been observed or inferred and suggests important general principles for the control of development. Sachs’ discussion should provide a stimulus to more students to take up this difficult but fascinating aspect of plant development. I thank the authors for their efforts in minimizing the editor’s task; my indexers for their patient endeavours, and Miss Justine Speed for invaluable secretarial assistance. Norwich 1981

H. W. Woolhouse

CONTENTS CONTRIBUTORS TO VOLUME 8 .

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Biochemistry of Storage Protein Synthesis and Deposition in the Developing Legume Seed D . BOULTER I . Introduction

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IV . Synthesis and Deposition . . . . . . . . . . . . A. Intracellular Sites of Synthesis and Deposition . . . . . B. Post-translational Modifications . . . . . . . . . C. Sites of Post-translational Modifications . . . . . . D . Protein Bodies: Origins and the Protein Transport Pathway . Biochemical Mechanism of Protein Synthesis and its Control E. F. Some Genetic Aspects . . . . . . . . . . . .

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111. Storage Proteins .

V . Conclusions . . Acknowledgements References . . .

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Aspects of the Metabolism and Physiology of Gibberellins ALAN CROZIER I . Introduction

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I11 . Gibberellin Biosynthesis . . . . . A. Mevalonic Acid to Enr-Kaurene . B. Em-Kaurene to G A , , aldehyde .

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Pathways beyond G A aldehyde . . . . . . . . Sites of Gibberellin Biosynthesis and Compartmentation . .

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The Control of the Patterned Differentiation of Vascular Tissues TSVI SACHS I . The Problems

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I1 . A Flux from the Leaves to the Roots Which Controls Differentiation 155 A. Introductory Summary . . . . . . . . . . . 155 B. The Induction of Differentiation by Leaves and by Auxin . 158 C. The Orienting Effect of Roots on the Flux of the Signals for Differentiation . . . . . . . . . . . . . . 170 D . A Relation of Vascular Differentiation to a Flux of Inductive Signals . . . . . . . . . . . . . . . . 172 E. Evidence for Additional Controls . . . . . . . . 176 111. Cell Polarization by a Flux of Signals . . . . . A. Introductory Summary . . . . . . . B. Facilitation of Signal Transport as a Basis Formation . . . . . . . . . . . C. The Stability of Polarity and its Possible Basis. D . The Formation of Vascular Networks . . .

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IV . Cellular Responses Involved in Oriented Differentiation . . . . 199 A. Introductory Summary . . . . . . . . . . . 199 B. Early Events Indicating Determination and Differentiation . 200 C. Is Cellular Differentiation Dependent on the Gradient or the Flux of Signals? . . . . . . . . . . . . . 205 V . Special Development Processes in the Cambium A. Introductory Summary . . . . . B. Quantitative Controls of Cambial Activity C. The Constant Changes in the Cambium Ray Formation and the Radial Polarity of D.

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VI . The Cellular Complexity of the Vascular System . . . . . . A. Introductory Summary . . . . . . . . . . . B. The Relation Between the Xylem and the Phloem . . . . C. The Controls of Fibre Differentiation . . . . . . . D. The Controls of Parenchyma Formation . . . . . .

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VII . The Relation of the Controls of Vascular Differentiation to other Aspects of Plant Morphogenesis . . . . . . . . . .

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VIII. The Major Characteristics of the Hypothesis IX.

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Summary . . . Acknowledgements References. . .

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Biochemistry of Storage Protein Synthesis and Deposition in the Developing Legume Seed

D. BOULTER

Department of Botany, University of Durham, Science Laboratories, South Road, Durham D H l 3LE, England

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I. INTRODUCTION The process of storage protein synthesis in developing legume seeds attracts investigators for several reasons : curiosity* and the accumulation of know* My own interest in legumes was first aroused as an Oxford undergraduate by seeing Luthyrus juponicus (Willd), the “Sea Pea”, flowering and setting pods in great profusion on otherwise bare shingle beaches.

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ledge for its own sake; as a model system in the study of differentiation; and, because legumes are a very important source of protein for man and animals (Boulter, 1977a; Boulter and Crocomo, 1979). It is for this latter reason that I have investigated Vicia fubu and Pisum sativum, the important agricultural legumes of the UK, and a sub-tropical legume, Vigna unguiculatu, which is of great importance to the Third World. Most other workers in the field have been similarly motivated so that it is exclusively crop plants which have been investigated. Foremost among these have been the temperate food legumes, Pisum sutivum (peas), Vicia jabu (broad and field beans), Phaseolus vuigaris (“dry” beans) and to a lesser extent Lupinus spp. (lupins) and the cash crops, Glycine max. (soya beans) and Arachis hypogaea (ground-nuts). More recently, increasing attention has been paid to food crops of the Third World, including Vigna unguiculata (cow peas), Vigna rudiata (mung beans), Cajanus cujun (pigeon peas) and to a lesser extent Cicer arietinum (chick peas) and Lens culinaris (lentils). However, the developing seed is also of academic interest. Most studies of differentiation, the process whereby cells become more specialized at some stage in their life, are based on the current paradigm that it is a consequence of differential gene expression in space and in time. This explains why systems such as the developing seed, where cells make one or a few organ-specific proteins in large amounts, are favourable material for study, since the protein(s) synthesized is a part of the differentiation process which can be related directly to genetic events. Proteins such as the seed storage proteins, which are only produced in significant amounts in cells at a specific period in the life cycle of an organism, can be contrasted with so-called “housekeeping enzymes”, which are needed throughout their life by most cells. The latter may be partly subjected to different and more complex homeostatic controls than the former. In many ways, the events occurring during storage protein synthesis in seed development can be compared to those in differentiating animal cells, e.g. those synthesizing haemoglobin or ovalbumin (see O’Malley et al., 1977). However, the interpolation of a phase of metabolic inactivity during drying out, dispersal and/or dormancy of seeds is a feature unique to this system. Determination is the process whereby the control mechanisms necessary to establish and stabilize differentiation are produced. We are beginning to piece together a good biochemical and fine-structure microscopic description of the sequence of events taking place during storage protein synthesis in developing legume seeds, but an understanding of determination at the level of the molecular mechanism, whilst now partially available for some prokaryotes and viruses, is not yet possible here. In these prokaryote and virus studies, a wide variety of methods were used including in vitro assays for protein and nucleic acid synthesis, protein and nucleic acid structure determinations, recombinant DNA techniques, E.M. methods and especially

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the availability of a range of mutants (Szybalski, 1977). Whilst the methods have been adapted for use with higher plants, suitable developmental mutants are lacking. Since the controls involved in differentiation are interrelated in a complex network with many possible control points, i.e. during transcription, post-transcription, translation, post-translation, transport and protein storage, each of which has many possible trigger mechanisms, a qualitative and quantitative biochemical analysis of seeds at different stages of development will only lead slowly to an understanding of the underlying molecular mechanisms. 11. BIOLOGY OF THE SEED

The seed is normally the sexually produced offspring of higher plants and the organ of dispersal. Seed development therefore can be viewed as a preparation for survival during dispersal and for subsequent successful germination. The seed is a more complex propagule than the functionally related spore of lower plants and considerable development of the fertilized ovule, nourished by the maternal plant, takes place before its separation from the latter. Functionally, three phases can be identified in seed development : initially, cell division gives rise to vegetative tissues, but then instead of continuing to seedling formation, development changes to a phase devoted to ensuring a successful future for the offspring as a separate entity. As a preparation for subsequent germination, since the nitrogen and mineral uptake and photosynthetic capacity of the mature seed is low, a food store of carbon, nitrogen and inorganic materials is laid down in the cotyledons, which in most legumes function primarily as the storage tissue. Then, in the third phase, there is a drastic reduction in metabolic activity, accompanied by the drying out of the seed and its protective seed coat. In the dispersal phase and that prior to germination, the metabolism must remain inactive and this inactivity is achieved by the low water content, the impermeable seed coat and the possible presence of inhibitors, although the extent to which these factors are involved, may vary in different legumes (see Taylorson and Hendricks, 1977). Much of the general metabolic machinery of the cells must survive the dehydration process since many active enzymes have been extracted from dry mature seeds, but the extent to which developed seeds contain the metabolic machinery required for the mobilization of reserves on germination is not clear (see Bewley and Black, 1978). Associated with each of these phases are morphological and biochemical changes, but how much they overlap is not known; Cullis (1976) has shown, for example, that it is not obligatory for cell division to cease throughout the cotyledon before the laying down of the reserves in Pisum, line JI 181. The mature seed consists of several tissues, some produced as a result of fertilization, others of maternal origin, but all with a diploid number of

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chromosomes except in the endosperm when it survives. The tissues develop at different stages and at different rates and some will be senescing whilst others are being formed. The fertilization of the ovule, the formation of the triploid endosperm (usually transient) and hormonal and nutritional “triggers” are undoubtedly part of the developmental process, but so far their precise influence is little understood and they will not be discussed in this chapter. This chapter deals with only one aspect of this whole process of seed development, namely the biochemistry of storage protein synthesis and deposition in the developing seed : since there are several recent reviews on this topic (Dure, 1975; Millerd, 1975; Boulter, 1977b, 1979), I shall not attempt to cover the extensive literature fully. The objective, rather, is critically to illustrate with results drawn largely from my own and my colleagues’ work,* the concepts, experimental approaches and present understanding of the field and to relate them briefly to other biological information. Although results from a variety of legume sources is referred to, the basic “picture” of the biochemistry, fine-structure and control of storage protein synthesis can be safely generalized to all large-seeded food legumes. Pisurn will continue to be a favourite material since the genetics is so well known. 111. STORAGE PROTEINS

A comprehensive review of the extensive literature on the purification and taxonomic distribution of storage protein types in legumes would be inappropriate here (see Derbyshire et a / . , 1976); however, some mention will be made of the present state of our knowledge in this field. It is sometimes not easy to decide whether or not a seed protein is a storage protein. Storage proteins senm stricto should be deposited in membrane-bound protein bodies and used subsequently after proteolytic breakdown as a nitrogen supply on germination. Occasionally a nonprotein-body protein, such as urease in Canavalia ensifomis appears to have been secondarily adapted to a storage role (Bailey and Boulter, 1971). Although some legumes occasionally store nitrogen in their seeds in the form of unusual amino acids, proteins are by far the most important storage component and so far as is known from amino acid analyses, these unusual amino acids are not incorporated into storage proteins. Storage proteins are large multimeric molecules of at least two main types, vicilin (7s) and legumin (1 IS), each of which consists o f a family of closely related molecules. They contain more amide and arginine and less sulphur amino acid residues (Boulter and Derbyshire, 1971) than the average metabolic protein (Smith, 1966), but have an acid P I ; vicilin proteins are usually glycosylated. *Some unpublished work from our laboratory is referred to without particular attribution as (U).

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Whilst it is possible that from an evolutionary point of view storage proteins need to be less well conserved than enzyme proteins, and yet still function, they are nevertheless under some structural constraints. There is a need for leader sequences for membrane attachment, glycosylation sites, and other processing sites (e.g. legumin 60,000+40 and 20,000 subunits (Croy et al., 1980a)), packaging and subunit interaction sites and their structure must allow the enzymic machinery of the germinating seed to make their constituent amino acids available during germination. Evidence for the conservative nature of their structure comes from results such as those of Jackson et al. (1967,1969), who showed that there was great similarity in the tryptic peptide maps of the storage proteins of the different genera of the legume tribe Vicieae. Gel electrophoresis of storage proteins of this tribe and another, the Trifolieae, also indicate considerable similarity, although less rigorously (Boulter et al., 1967). The protein-separation techniques now available, provided they are used in correct sequence, are powerful enough to separate the several different storage proteins which exist in any one tissue or cell. Ammonium sulphate precipitation, zonal isoelectric column chromatography, molecular sieving, sucrose gradient separations and hydroxylapatite chromatography are particularly useful. The major difficulty is that separatory methods for undissociated proteins are much less effective than those for the separation of constituent protein subunit polypeptide chains. The use of two-dimensional gels is particularly helpful ( & mercaptoethanol, & dissociating conditions, molecular weight and charge separation gels) for characterization, although more chemical data, especially of amino acid sequences, are required. The major storage proteins of Vicia faba and Pisum sativum have been well studied and these can serve as our models. The major legumin-type protein in Vicia faba consists of six acidic (MW about 40,000) and six basic (MW about 20,000) disulphide-bonded subunit pairs with a MW of about 350400,000. Minor legumin molecules also occur in which some acidic subunits are of higher MW (Matta et al., 1980). Considerable charge and size heterogeneity in the subunits of any one type has been demonstrated, probably leading to microheterogeneity in the assembled legumin molecules. Thus whilst legumin molecules have the same overall size, shape, charge, solubility and other characteristics, they exhibit microheterogeneity at other levels. The other major component of the storage proteins of Viciafaba is vicilin, a glycosylated protein of MW about 170,000, made up essentially of three or four subunits of approximately 50,000 MW with no disulphide bonding between subunits (U). Once again, considerable charge and size heterogeneity has been observed in these subunits. The situation in Pisum sativum is very similar, with the two major storage proteins being equivalent to the major legumin and vicilin of Vicia;however, the minor legumin subunits are normally absent or in extremely low concen-

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tration and another vicilin-type protein is usually present in relatively high concentration. This protein has been named convicilin, has a MW of between 250-300,000 and consists of 70,000 subunits which are not disulphide bonded or glycosylated (Croy et al., 1980b) ;tryptic peptide mapping (U) and serological studies (Croy et al., 1980b) show it to be structurally similar to vicilin. In addition, major subunits of 3 3 , 19, 16 and 12,000 MW are present although as yet unassigned to a protein; it is likely that these are posttranslational products of certain vicilin 50,000 subunits (U). A similar storage protein profile probably occurs in all five genera of the Vicieae, as suggested by the similarity of protein electrophoretic patterns and tryptic fingerprint maps (Jackson et a!., 1967, 1969). The proportion of the different storage proteins can vary considerably in different species and even in different varieties of the same species. Furthermore, variation in size and charge can occur in the subunits of homologous proteins in different species and in varieties of the same species (Casey, 1979a,b). Legumin-type proteins are widely distributed in other legumes, occurring in large amounts in Glycine mux (“glycinin”) where amino acid sequence data prove its homology to Vicia legumin (Gilroy et [ I / . , 1979), and in smaller amounts in Lupinus spp., Vignu unguiculata, Phaseolus vulgciris and Phaseolus uureus (now Vigna rudiatu) (Derbyshire et a/., 1976). However, in many important food legumes a major storage protein of the “vici1in”-type predominates, i.e. a protein of 140-180,000 MW (7S), three heterogeneous nondisulphide linked subunits of MW between 43-63,000 and containing small amounts of covalently linked carbohydrate. Examples of this type of protein are the glycoprotein I1 of Phaseolus vulgaris (Derbyshire et al., 1976) and the major proteins of Vigna unguiculatu (Khan et al., 1980) and Vignu radiatu (Ericson and Chrispeels, 1973, 1976). The homologous relationships, if any, of these different 7 s proteins have not been demonstrated and await the results of amino acid sequence studies ; serological cross-reactivity can be misleading as a method of establishing homologies since it is lacking between the legumin of Vicia and Glycine (Dudman and Millerd, 1975) despite amino acid sequence similarity suggesting homology (Gilroy et al., 1979). Many 7 s legume seed storage proteins are glycoproteins containing between about 2-6% sugar, usually mannose, N-acetylglucosamine and possibly glucose (Pusztai and Watt, 1970; Derbyshire et al., 1976; Thanh and Shibasaki, 1976; Ericson and Chrispeels, 1973; Basha and Beevers, 1976; Eaton-Mordas and Moore, 1978; Davey and Dudman, 1979).These workers have reported relatively low but variable amounts of carbohydrate in vicilin preparations. Certain of these preparations, however, usually contained another protein called convicilin, which contains no carbohydrate (Croy et al., 1980b). Furthermore, the work of Davey and Dudman (1979) and Gatehouse (unpublished observations) show that not all vicilin subunits are glycosylated and that most if not all of the carbohydrate is associated with the 12,000

STORAGE PROTEIN SYNTHESIS AND DEPOSITION

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subunit. Since the amounts of contaminating convicilin and the relative proportions of the known carbohydrate-containing vicilin subunits can vary in different varieties and probably in preparations made from material at different developmental stages, both the variation and the overall low levels of carbohydrate in vicilin can probably be explained. Legumin (1 lS), the other important storage protein of GIyciiw m i x , Arachis hypogaea, Viciu juhu, Pisum scitivum and Lupinus, contains little (usually < 1%) carbohydrate (Derbyshire et u/., 1976; Eaton-Mordas and Moore, 1978). Technically, it is difficult to remove all non-covalently bound carbohydrate and often phenol/borate partitioning and other necessary procedures have not been used, making interpretation of the results difficult. Thus, Koshiyama and Fukushima (1976) concluded that the small amounts of carbohydrate associated with the 11s protein of Gl-ycine may were not covalently bound. Casey (1979a) and Gatehouse et al. (1980a) have both concluded that Pisum legumin, as isolated, does not contain carbohydrate, whereas Basha and Beevers (1976) reported the presence of 1% neutral sugars (principally mannose with some glucose) and 0.176 N-acetylglucosamine. Davey and Dudman (1979) found small amounts of carbohydrate in their preparation of Pisum legumin and whilst calling legumin a glycoprotein, expressed some doubts as to whether carbohydrate was covalently bound. Bailey and Boulter (1970) determined the amount of carbohydrate in Vicia legumin to be less than O.lO,.;. The difference in the legumin results could be explained by : (i) Variable trimming of the carbohydrate content. In animals there is good evidence that oligosaccharides containing glucose and mannose may undergo trimming and the relevant enzymes such as glucosidases, mannosidases and N-acetylglucosamidase do occur in plants. (ii) Presence of contaminating vicilin. (iii) Presence of non-covalently linked carbohydrate. (iv) Differences in legumin from different sources. In summary, legumes normally contain two different types of storage protein, legumin and vicilin, one or other type usually predominating. Both types are multimeric proteins with considerable subunit heterogeneity in size and charge. IV. SYNTHESIS AND DEPOSITION A. INTRACELLULAR SITES OF SYNTHESIS AND DEPOSITION

The storage proteins are made on the rough endoplasmic reticulum (RER) (Bailey er al., 1970; Miintz, 1978; Bollini and Chrispeels, 1979) which is specially assembled for the purpose (Opik, 1968; Payne and Boulter, 1969), and eventually they are deposited in single membrane-bound protein bodies

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which do not themselves have protein synthesizing capacity (Wheeler and Boulter, 1966; Morris et al., 1970). Whether legumin and vicilin are transported as subunits and assembled into proteins later in the protein bodies is not known. This information should soon be available, as isolated subunits are in part antigenic, leaving open the possibility of using ferritin-labelled antibody EM studies to answer the question. That both vicilin and legumin-type proteins are deposited in the same protein bodies has been established by immunofluorescent light microscopy (Graham and Gunning, 1970; Craig ct L I I . , 1979b; Harris, unpublished observations). B . POST-TRANSLATIONAL MODIFICATIONS

Considerable post-translational modifications of some storable protein polypeptides takes place as is evident from a comparison of translation products of poly A-containing RNA and microsomes in in vitro protein synthesizing systems. Higgins and Spencer (1980) have shown that the 50,000 vicilin subunit is synthesized with an additional sequence of about 1000 MW which is subsequently removed by enzymes in the RER. Evidence from cyanogen bromide cleavage (Croy rt a/., 1980b) shows that this additional piece must be either N - or C-terminal. Leader sequences were first demonstrated in mammalian systems by Blobel and Sabatini (1971) and Blobel and Dobberstein (1975), who proposed the so-called “signal” hypothesis, and since then the concept has gained general acceptance. However, not all proteins which are synthesized and secreted from the RER carry a signal sequence which is subsequently removed (Palmiter et al., 1978) nor need the signal be at the N - or C-terminus (Di Rienzo er a [ . , 1978). Legumin is made as a subunit of about 60,000 MW which is subsequently cleaved to 40,000 and 20,000 subunits (Croy et al., 1980b), although the intracellular site of this cleavage has not yet been demonstrated. Other post-translational changes include the glycosylation of some of the storage protein subunits. C. SITES OF POST-TRANSLATIONAL MODIFICATIONS

In animals, proteins synthesized on the RER and destined for secretion from it after passing into the lumen are usually, although not always, glycosylated. The route of secretion, established primarily from specialized secretory cells but accepted as of general application, is RER+ SER (smooth endoplasmic reticulum)+golgi apparatus+secretory vesicles (Jamieson, 1978). The core sugars are attached in the RER with or without subsequent glycosylations taking place in the SER, the initial glycosylation (Molnar, 1975) occurring either just before or just after completion of the peptide chain. There is now

STORAGE PROTEIN SYNTHESIS AND DEPOSITION

9

some evidence that translational control of protein synthesis is exerted via the glycosylation reaction. Usually a core oligosaccharide consisting of N-acetylglucosamine (Glc NAc) and a variable number of mannose residues is joined to the protein. The first step of this process is the linking of N-acetylglucosamine to a polyisoprenoid lipid, dolichol phosphate, followed by another N-acetylglucosamine residue and usually two mannose residues by their respective sugar nucleotide glycosyl transferases. In the last phases of synthesis, mannose is transferred from GDP-mannose to dolichol phosphate to form mannosylphosphoryldolichol which donates mannosyl residues to the oligosaccharide, which is then transferred from the lipid carrier to the protein by formation of a N-glycosidic bond, probably with asparaginyl residue in the polypeptide chain (Waechter and Lennarz, 1976). This may not be the only mechanism and glycoproteins are synthesized elsewhere in the cell. Lipid oligosaccharides and associated enzyme systems have been found in subcellular sites other than the ER (Elbein, 1979). Furthermore, even in animal cells, a positive role for the oligosaccharide core in drainage of secretory proteins from the RER has not been firmly established (Jamieson, 1978). There is good evidence that plants contain enzyme systems which can form mannosyl-phosphoryldolichol from G D P mannose, incorporate Glc NAc into a lipid (possibly dolichol) intermediate (Elbein, 1979) and contain several lipid-linked oligosaccharides which probably consist of mannose and Glc N Ac residues in similar structures to those established in animals. Nagahashi and Beevers (1978) using M g 2 + shift and coincidence with antimycin-insensitive N ADH-cytochrome c reductase have shown that enzymes capable of transferring N-acetylglucosamine from UDP-N-acetylglucosamine to both lipid and protein acceptors were primarily located in the ER of developing pea cotyledons. Some activity was also detected in an IDPase-containing fraction (Nagahashi rt a/., 1978). In Vignu rudiatu (Phasrolus aureus), Lehle rt a / .(1978) provided less firm evidence of mannosyl transfer to dolichol phosphate in the ER, whilst enzymes which transfer mannose to protein were found in the golgi (Lehle rr ul., 1978), although unfortunately, the glycoprotein product was not identified. Davies and Delmer (1979) showed that a particulate membrane fraction, isolated from Phaseolus vulgaris and probably originating predominantly from the RER, incorporated labelled N-acetylglucosamine in vitro into glycoprotein 2 (vicilin) and also into phytohaemagglutinin. Little mannose was incorporated and the predominant glycosylation reaction was a transfer of N-acetylglucosamine (Glc N Ac), but not from a preformed oligosaccharide. Gardiner and Chrispeels (1975) have localized a glycoprotein glycosyl transferase for a cell wall protein in the golgi apparatus. However, in plants no one has yet demonstrated that lipid-linked oligosaccharides are attached to known glycoproteins by in vitro enzyme assays.

10

D. BOULTER

The presence, however, of the necessary enzymes and components, including oligosaccharide lipids and the fact that vicilins are known to contain mannose and Glc NAc would suggest that the reactions involved are similar to those established in animals. Furthermore, the structure of the carbohydrate chain of soya bean agglutinin has been shown to contain a mannose Glc NAccontaining core-oligosaccharide linked to asparagine of the protein (Elbein, 1979). In addition to mannose and Glc NAc, glucose has also been found as a component of some lipid-linked oligosaccharides in animals but not in plants. The suggestion that some plant glycoproteins may contain glucose therefore needs careful re-examination, especially as it is well known that non-covalently bound glucose, a substantial component of seed extracts and a contaminant from various separatory media, e.g. Sephadex, is normally found in purified protein preparations. D . PROTEIN BODIES: ORIGINS AND THE PROTEIN TRANSPORT PATHWAY

Proteins are deposited for storage within a cell probably exclusively in protein bodies (Craig et ul., 1979b), sometimes called aleurone grains or aleurone vacuoles. The statements which follow about protein body origins and homologies are restricted to legume cotyledonary protein bodies, since protein bodies occurring in the legume embryonic axis and those of cereal endosperm may differ in origin. Protein bodies are roughly spherical organelles, usually 1-10 pm in diameter, bounded by a single membrane and found principally in the cotyledons (Miege, 1975).They contain not only the protein reserves but also phytin, the calcium salt of inositol hexaphosphoric acid, which sometimes also exists as a magnesium salt, and some other minerals. Phytin occurs both in globoids and also in the proteinaceous matrix (Lott and Buttrose, 1978). Protein bodies may be unstable during conventional subcellular preparative procedures (Pusztai et a/., 1979)and since the purity of protein body preparations has normally been confined to qualitative microscopic examination, the suggested presence in them of other proteins with proteolytic and hydrolytic activities, although often reported, has to be viewed cautiously (Quail, 1979). During seed development, a phase of cell division is followed by a period when the cells enlarge and develop a peripheral cytoplasm containing the nucleus and one or a few large apparently empty vacuoles (Fig. 1). The formation of smaller vacuoles by division of large ones at about the time storage protein synthesis starts has been recorded for Viciufbba (Briarty er al., 1969), Vigna unguiculata (Harris and Boulter, 1976), Pisum sativurn (Bain and Mercer, 1966) and several other legumes. Protein material is subsequently seen to be deposited in these sub-vacuoles (Figs 2-5). However, some workers, e.g. Neumann and Weber (1978), using Viciafaba, do not accept a

STORAGE PROTEIN SYNTHESIS AND DEPOSITION

11

developmental continuity between vacuoles and protein bodies. At seed maturity large vacuoles are not present; instead the cell contains numerous (> 100,000) small protein bodies containing dense protein deposits (Fig. 4). This development could have taken place in several different ways which are not mutually exclusive : (i) vacuoles may have divided to give protein bodies which were filled by the deposition of protein contained in E R and/or golgi vesicles; (ii) vacuoles may have been dismantled and protein bodies formed by coalescence of E R and/or golgi vesicles; (iii) vacuoles may have been dismantled and protein bodies formed directly from the E R . In the latter two cases, since most legume protein body membranes do not have ribosomes on their outside surface, the R E R ribosomes must have been removed or smooth E R used in protein body formation. However, Craig rt ul. (1979a) have reported the presence of some protein bodies with ribosomes on their outside surface during the later stages of Pisurn seed development. Bain and Mercer (1960) suggested a dual origin of protein bodies in Piarn7. protein being laid down early on in development on the inner surface of cytoplasmic vacuoles which divided and subsequently in protein bodies originating from E R vesicles. Once the latter type of protein body started to accumulate, no further protein was laid down in cytoplasmic vacuoles. Harris and Boulter (1976) reported a similar dual origin in Vigncr unguiculattr, except that they identified the second type of protein body as probably originating from golgi vesicles (also Fig. 7). In contrast to Bain and Mercer (1960), these authors showed that in cow pea during the period of protein deposition, protein deposition continued in both types. This difference in relative importance of the two routes was tentatively correlated with the major protein types, legumin or vicilin being synthesized in the two legumes (Harris and Boulter, 1976). However, since the site of synthesis of the protein was in the R E R , they did not exclude the possibility of a direct route, i.e. from E R vesicles. Although many cytoplasmic vesicles can be observed in the electron microscope, these are thought to come from the golgi rather than the E R , since, by using thick sections, many apparent E R vesicles can be shown to be artefacts of thin sectioning (Harris, 1979) (Figs 5 and 6 ) . Others, however, have refuted the role of golgi (Bain and Mercer, 1966; Neumann and Weber, 1978; Craig et al., 1979a). The present balance of E.M. evidence suggests that the completed protein bodies in legumes are homologous with the vacuolar system of the cell (Matile, 1975; Bergfeld et a / . , 1980). Furthermore, there is some EM evidence to suggest that proteins (i.e. pronase-digestible material) are transported to the protein bodies by golgi vesicles (Dieckert and Dieckert, 1976; Harris, 1979, unpublished observations) and that in some legumes these may form a second type of protein body by coalescence (Harris and Boulter, 1976;

Fig . 1. Highly vacuolate cotyledon parenchyma cells of developing Pisum, 7 daysi after floweiring. Magnification x 2550. Fig . 2 . Storage protein deposited in both main vacuole and cytoplasmic vesicles in cot)rledon cells (,f developing Pisurn, 10 days after flowering. Cytoplasm contains rough cysternal endoplasmiic reticulum (ER) and numerous dicotyosomes (circled). S , starch. Magnification x: 3400; glutai.aldehyde and osmium tetroxide fixation.

Fig . 3. Membrane bound protein bodies in cotyledon cell of developing Pisurn, 15 daysi after flower,ing. Rough cysternal endoplasmic reticulum in shorter profiles than at 10 days after flower.ing. D, dicotyosomes. Magnification x 12,750. Fig. 4.Cotyledon cells of developing Pisum, 19 days after flowering, with numerous pi.otein bodiesi of approximately uniform size, none equivalent to original large vacuoles. CW, cell wall. Magn ification x 2550.

Fig. 5 . Cotyledon cell of developing Viciafaba at mid-protein deposition phase. Conspicuous rough cysternal endoplasmic reticulum and protein deposition in cytoplasmic vesicles. M, mitochondrion. Magnification x 10,200.

Fig. 6 . Thick (1 pm) section of similar tissue at same magnification as Fig. 5 after glutaraldehyde and zinc iodine-osmium tetroxide fixation. The grey endoplasmic reticulum cysternae are interconnected by tubular endoplasmic reticulum (TER). Numerous dictyosomes (D) are present adjacent to protein bodies. Magnification x 10,200. Fig. 7 . Dictyosomes in cotyledon cell of developing Vicia ,firha with electron dense vesicles; spin vesicles (SU). Magnification x 21,250.

16

D. BOULTER

Harris, 1979). There is little evidence for a direct connection of the ER lumen with protein bodies as is the case in many cereals (Harris and Juliano, 1977 ; Larkins and Hurkmann, 1978). The most important unresolved question is whether all the “apparent” ER vesicles seen in thin section are artefacts or whether, as suggested by Bain and Mercer (1966) and Pernollet (1978) they coalesce to form protein bodies. Briarty (1978) when reviewing the subject, discussed the difficulty of attempting to identify the transport system using only EM evidence and it is unlikely that EM studies alone will answer this question. Thus, though Opik’s work (1968) suggested formation of protein bodies only by division of the main vacuole, she was unable to find any physical connections between the ER and vacuoles or any vesicles which might transport protein. Similarly, although Bain and Mercer (1966) found isolated patches of granular ER they considered that these were not involved in protein synthesis because of their late appearance relative to maximum protein synthesis. Stereology can be used to provide information on the three-dimensional structure of the cell and EM autoradiography (Bailey et a / . , 1970) and the use of ferritin-labelled antibodies might in the future supply an answer. Perhaps the best indication could come from a biochemical study. There is now a convincing body of experimental evidence for the concept of organelle marker enzymes, i.e. enzymes whose presence identifies a subcellular organelle (De Duve, 1971; Quail, 1979). Rough ER, golgi and vacuoles have all “accepted” marker enzymes (Quail, 1979), i.e. antimycin A-insensitive NAD(P)H cytochrome c reductase, combined with Mg2+ shift for the RER, latent inosine diphosphatase for golgi and phosphodiesterase and RNAase for vacuoles. It should be possible, therefore, to establish the origin of protein bodies by this method. The activities of marker enzymes should be assayed quantitatively across complete sucrose gradients, with the provision of complete balance sheets. However, there are many technical and interpretative difficulties in the use of “markers”, as discussed by Quail (1979), and several problems remain. The fact that the endomembrane system involves membrane flow and differentiation necessarily complicates questions of origin (Morre, 1975). Matile (1968) suggested that the protein bodies not only contain the storage reserves but were also responsible, on germination, for their hydrolysis as well. He further postulated that non-storage macromolecules were engulfed and broken down by protein body acid hydrolases on germination. Briarty et d.(1970) showed that whilst the protein bodies swelled in Vicia faba during the first four days of germination, they appeared to retain their electron dense material and that subsequently this was lost just prior to the coalescence of protein bodies, to form large vacuoles. The cytological evidence suggested that proteolysis occurred uniformly throughout the protein bodies and indicated the presence of a latent protease in them. They also

STORAGE PROTEIN SYNTHESIS AND DEPOSITION

17

demonstrated that the development of RER in the cotyledon during the first few days of germination indicated that synthesis as well as degredation of protein was occurring. Subsequent work by Chrispeels and co-workers with germinating Vigna radiuta showed that the low level of acid protease activity detectable in protein bodies at the onset of germination did not initiate protein breakdown, but that the mobilization of these reserves depended on an endoprotease (Chrispeels and Boulter, 1975) which was synthesized de n o w and transported into the protein bodies (Baumgartner and Chrispeels, 1979). Acid phosphatases which could bring about the utilization of phytin have also been primarily localized in protein bodies (Quail, 1979). In Vigna radium, Van der Wilden et al. (1980) have presented good evidence that the cotyledon protein bodies contain a-mannosidase, carboxypeptidase, phosphatase, phosphodiesterase, phospholipase D and low levels of ribonuclease activity. This work suggests therefore that protein bodies act as lysosomal organelles. In contrast, Pusztai et al. (1979) showed that isolation of protein bodies from imbibed seeds of Phaseolus vulgaris contained no internal proteases. It was proposed that on germination a controlled release of protein from the protein bodies might occur without prior proteolytic breakdown inside the organelles, since in v i m they could show a substantial loss of matrix protein without disrupting the continuity of the limiting membrane. Payne and Boulter (1974) and Dyer and Payne (1974) showed that virtually all the rRNA isolated from mature cotyledons was undegraded, yet if these were homogenized, ribonuclease became active, probably due to the disruption of a membrane. They suggested therefore that in vivo, during germination, some ribosomes pass into lysozomes and/or vacuoles and are destroyed. Since protein bodies coalesce in later stages of germination to form one or a few large vacuoles (Briarty et ul., 1970), these vacuoles might be considered the site of ribosome degradation. In fact, as mentioned earlier, Matile (1968, 1975) and more recently, Van der Wilden et al. (1980), have suggested this to be the case. However, Leigh (1979) has argued strongly against the mature vacuole acting as a lysosome and the whole question of the presence, at this stage in development, of lysosomes and vacuoles and their relative origins and activities is still unresolved. E. BIOCHEMICAL MECHANISM OF PROTEIN SYNTHESIS AND ITS CONTROL

1. The Biochemical Machinery Protein synthesis is a complex, energy requiring multi-enzymic process which takes place on polysomes consisting of an RNA template (mRNA) and several associated ribosomes (“work-bench”) (see Boulter et al., 1972). The biochemical reactions involved were first elucidated for the 70s bacterial systems, but the steps and mechanisms are basically the same in both

18

D. BOULTER

the bacterial and eukaryotic systems (Boulter, 1976; Yarwood. 1977). However, during evolution, considerable changes have taken place in both ribosomal and soluble proteins, probably reflecting the increasing need for translational controls in eukaryotes. Thus the initiation factors for protein synthesis in the cytosol in eukaryotes are more numerous than in prokaryotes ; there are, for example, seven mammalian initiation factors (Revel and Groner, 1978). There is also a requirement for ATP hydrolysis and the initiator t-RNA is not formylated except in organelle protein synthesis, and the order of the steps in the formation of the initiation-ribosome complex differs from prokaryotes (Revel and Groner, 1978). Although not all of the constituents have been isolated and characterized, the steps and mechanisms of legume storage protein synthesis are essentially the same as in other 80s eukaryotic systems (Payne et ul., 1971a,b; Yarwood et ul., 1971). These studies elucidated the basic biochemical machinery of protein synthesis, but in vitro systems of the stability and fidelity of the reticulocyte lysate (Pelham and Jackson, 1976) and wheatgerm systems (Marcus et ul.. 1974) were not established at that time from the cotyledons of developing legume seeds. Recently, however, Peumans et ul. (1980) have isolated an in vitro system from the primary axes of dry pea seeds which is as active as the wheatgerm system; it should prove quite feasible to develop similar in vitro systems from developing legume seed cotyledons in spite of the presence in them of relatively high levels of proteases and RNAase. These systems are now needed. For example, the differences obtained by different laboratories about glycosylation reactions of membranes could be resolved by using suitable legume in virro systems. The criteria that in virro systems must fulfil to be satisfactory were discussed by Boulter (1976).

2. Description of the Changes in Protein and Nucleic Acids ( a ) Phase I . The results in Fig. 8 show the changes in nucleic acid and protein content during seed development in Pisum. During the first phase, cell division occurs, intermediates are built up (Boulter and Davis, 1968) and the rate of protein synthesis is low with very little storage protein being synthesized (see later). Ribosomes (polysomes) are free in the cytoplasm and they are presumed to be making the housekeeping enzymes, although very little work has been done on their levels during seed development. Boulter and Davis (1968) showed a changing pattern of the major albumin proteins present during seed development in Vicia faba by using non-dissociating acrylamide gel electrophoresis. Clearly, levels of required enzymes must change, but these are probably regulated by feed-back controls on enzyme activity rather than by transcriptional controls. ( b ) Phase ZZ.During this phase, days 7 to 22 in Fig. 8, the rate of protein synthesis increases greatly and the type of protein being synthesized changes and storage proteins are the main product. It can be postulated that ac-

140

-

28

120 -

100

-

-

OI

-a

X 80-

z

n ,--

-?

I

3

f

60-

0

40

-

20

-

Days after flowering

-.-

Fig. 8. Changes in dry weight, protein and nucleic acids during development of seeds of Pisuni sarivurn, var. Feltham First. Dry weight, --O--; Legumin, - - -:I- -; and Vicilin, . . .A.. ., determined by rocket immunoelectrophoresis. D N A (pg), -0determined by diphenylamine assay method (Burton, 1956). Polysomal RNA (pg), - - - determined by A260 of isolated polysomes. Left-hand scale refers to both dry weight (mg) and DNA (pg). Right-hand scale: inner scale refers to amounts of legumin and vicilin (mg) and outer scale refers to RNA big). All values per cotyledon. M. mature.

20

D. BOULTER

companying the massive increase in protein synthesizing work-bench (polysomes) visualized in the EM and measured by quantitative and qualitative RNA analysis (Payne and Boulter, 1969), that there was a concommitant increase in the cellular concentration of the many enzymes involved in protein synthesis (Boulter et ( I / . , 1972). It will be recalled that over this period the cell numbers in the cotyledon remain virtually constant. In bacteria where the controls involved in changing rates of protein synthesis have been investigated in much more detail, the whole protein synthesizing machinery is co-ordinately controlled, although transcriptional controls of the genes are at least in part independent (Nierlich, 1978). Legumin and vicihn can both be detected in very small amounts as early as four days after ovule fertilization, by using sensitive immunological methods ( U and Domoney et a/., 1980) and probably both vicilin and legumin are synthesized at least in very small amounts throughout seed development. However, the amount of storage protein being accumulated increases greatly about one-third of the way through seed development. The onset of the increased rates of deposition (synthesis, see later) of vicilin and legumin are sequential (Thompson er ul., 1979). Vicilin. legumin, convicilin, accumulate fastest in that order (U), although there is considerable overlap in the periods of their synthesis, the cessation of which is also probably sequential (see Fig. 8). This sequence of events was established by determining quantitatively the accumulation of these proteins using immunological methods and also demonstrating that there was very little turnover, i.e. the changing amounts were not due to the balance between the same rate of synthesis and different rates of degradation. Thus, pulse/chase radioactive tracer studies with isolated cotyledons which synthesize storage protein about as actively as when on the plant, showed that storage protein did not turn over significantly since the number of counts in storage protein did not diminish in the chase period (U). Immature pea embryos and cotyledons can be cultivated in vitro (Stafford and Davies, 1979) so that storage proteins are synthesized at about the same rate as when the seeds are on the plant and this provides a very useful tool for pulse-chase and inhibitor studies. A previous report (Millerd rt a / . , 1975) that legumin synthesis was not initiated in embryos cultured at an early stage irz vitm without pods, seems to have been a result caused by the low sucrose concentration used; if 18%) sucrose is used, legumin synthesis is obtained (Domoney et a/., 1980). More recently, it has been shown that by using polysomes extracted at different stages of development and comparing their translation products, that vicilin, but not legumin and convicilin, is synthesized at 8 days, vicilin, convicilin and legumin at 13 days and only legumin and convicilin at 19 days (U). (c) Phase / I / . During drying out, polysomes are dismantled (Payne and

STORAGE PROTEIN SYNTHESIS AND DEPOSITION

21

Boulter, 1969) but the ribosomes appear not to be degraded at this stage but later during germination (Payne and Boulter, 1974). Although some messenger RNAs seem to be stored in the dry seed (Payne, 1976), mRNA for storage proteins appears not to be among them (Gordon and Payne, 1976) suggesting that the polysomes break down during dehydration because the message is destroyed. After seeds have dried out, housekeeping enzymes are still present since many active enzymes have been extracted from dry seeds which on inbibition would be metabolically active. Although it is the embryo axis which is particularly active in germination, the cotyledons are also active anabolically after inbibition. Before, during and after the period of storage protein synthesis, there are polysomes free in the cytoplasm of cotyledon cells, although their ratio relative to membrane-bound polysomes is small during Phase 11 (Payne and Boulter, 1969). These free polysomes have been shown 1101to synthesize storage proteins (Bollini and Chrispeels, 1979). It is possible that the enzymes which mobilize storage proteins on germination are also synthesized but remain inactive during seed development. Thus some enzymes required for this purpose are not synthesized on germination, since inhibitors of protein synthesis do not suppress their appearance (Bewley and Black, 1978); some enzymes may be present in zymogen form, but at least some of the enzymes needed specifically to mobilize protein reserves are synthesized de novo during germination (Baumgartner and Chrispeels, 1979). Very little critical evidence, however, is available (see Bewley and Black, 1978). 3 . Control of’Storugr Protein Synthesis Control of the overall process of protein synthesis could occur at transcription, post-transcription, translation or post-translation or at several of these stages (Boulter, 1976). From the knowledge that protein is synthesized on polysomes containing specific mRNA templates, models of control can be proposed which can be experimentally tested. If transcription of mRNA is reset to a higher rate as a one step process at time 1 ( t l ) and then remains constant till time 2 ( t 2 ) ,and then falls rapidly, the amount of mRNA accumulated in the cells will depend on its stability; it is assumed that the reset rate is not “switched off’ before the new steady state is reached. Taking the two extremes of either very stable or very instable mRNA, the results will be as in Fig. 9a and Fig. 9b respectively. If the mRNA has a half-life comparable to the interval ( t l - f 2 ) / 2 then an intermediate situation would ensue (Fig. 9c). If (a) obtains, protein accumulation should be quadratic over the period fl-t2 (unless some other constraint, e.g. translation level control, comes into effect at higher rates of protein synthesis), and then continue linearly. If (b), protein accumulation would be virtually linear as the amount of mRNA at the steady

22

D. BOULTER

a z LL

E

c

E

a

I(c)

.-v1

fl [L

E

(d)

I-=

Fig. 9. Control model. t , , time of “switch on”; t 2 . time of “switch off”; t , 1 2 mRNA, mRNA.

life of

state would remain constant until t 2 . If (c), accumulation of protein would be between (a) and (b). The results in Fig. 8 show that the increase in the amount of storage protein is initially quadratic (up to 14 days), then approximately linear in rate till day 21, when it remains approximately constant. Following protein accumulation, however, does not decide whether increased transcription accounts for the results, since although storage protein synthesis apparently followed a pattern consistent with mRNA accumulation as (c), it is very difficult to distinguish this pattern from that due to (a) if the later stages of protein synthesis were also constrained by processes other than transcription. It is necessary, therefore, in order to see if there are transcription controls (or at least pre-translational controls, since it is possible that control of processing of the RNA could give the same results), to assay mRNA for individual storage proteins throughout seed development. Polysomes can be recovered more or less quantitatively from developing pea seeds (U) and since most of the mRNA they contain is storage protein message as indicated by translation assays (Evans et af., 1979 and U) the amount of RNA of polysomes will correlate with the amount of storage protein mRN A present at any one developmental stage, providing small corrections are made for the differences in size of individual polysomes. We have performed such a preliminary experiment. The results show a linear increase in mRNA till day 15, then a levelling off and a decrease about day 18. Thus the initial exponential increase in rate of protein synthesis is correlated with a linear increase in mRNA up to day 15, i.e. it follows the

STORAGE PROTEIN SYNTHESIS AND DEPOSITION

23

pattern of (c). This result is very tentative and clearly it is important to follow the amounts of individual mRNAs during seed development. Messenger RNA for storage proteins from Vicia (Puchel et al., 1979) and Pisum (Evans et ul., 1980) has been partially purified, but in both cases the different storage protein messengers were not separated from each other or completely from contaminating rRNA. Puchel et ul. (1979) identified their impure messenger fraction as a discrete peak in the 12-18s region on agarose gels, whereas the preparations of Evans et ul. (1980) gave a polydisperse peak with three maxima at 18S, 14s and 12s. Both vicilin and legumin subunits were translated in vitro from mRNA preparations separated on sucrose gradients starting with a minimum size of 14s (Evans et al., 1980). 14s and 18s poly A-containing RNA correspond to about 1400 and 2100 nucleotides respectively, so allowing for poly(A) sequences of up to 10% of the mRNA length (Puchel et al., 1979) these messages could code for subunits of 49,000 and 76,000. Thus whilst the 14s species could code for either vicilin (50,000)or legumin (60,000 subunits), the 18s is larger than expected and probably contains non-coding sequences (Evans e f al., 1980). Attempts to separate pure mRNA for individual storage protein messages in reasonable amounts have not yet been successful (U). In the model proposed earlier, it was postulated that transcriptional activity might increase rapidly to a faster rate at onset of increased storage protein synthesis ( t l ) and then continue at this rate till t 2 (Fig. 9d). In an early paper of considerable interest, Millerd and Spencer (1974) demonstrated a burst of transcriptional activity, starting at the onset of storage protein synthesis, increasing for two days and then rapidly falling. They used isolated pea nuclei and determined transcriptional activity by determining the number of counts incorporated into RNA. This probably largely represented transcription into r RNA. These authors concluded from experiments with added E. coli. polymerase that excess template capacity was available and that the whole process was constrained in vivo by RNA polymerase activity. However, different RNA polymerases are used by plants to transcribe rRNA and mRNA and these assays did not distinguish between them. Furthermore, no attempt was made to show whether or not transcription had been initiated and terminated correctly and that the observed activity was not due to nicked template, etc. In view of the technical difficulties involved, these experiments now need repeating using product characterization with mRN A probes. What is required are specific probes for individual storage protein messengers. The most feasible strategy to obtain these is to make ds-copy DNA against partial purified mRNA using AMV reverse transcriptase and DNA polymerase (Klenow’s enzyme A) and to clone this using microbiological vectors. Evans et al. (1980) have successfully made ds-cDNA to partially purified mRNA of Pisurn storage proteins and so have Hall and co-workers

24

D. BOULTER

for Phaseolus glycoprotein I1 (Hall et al., 1980) and Beachy et al. (1980) for soya bean storage proteins. Cloning of storage protein cDNA using standard recombinant DNA techniques is now in progress in several laboratories, e.g. Pisum (Chandler, Higgins and Spencer; and Croy, Evans and Boulter); Vicia (Muntz and collaborators); Glycine (Goldberg, Breidenbach and collaborators; and Beachy and collaborators); Phaseolus vulgaris (Hall and collaborators). This work has so far only been reported briefly, without details, in conference reports. The availability of mRNA probes cannot be far away, however, and these will be used for both in vitro transcription assays and for probing the amounts and distribution of specific mRNAs during seed development. During seed development, after cell division has stopped, the amounts of DNA per cell increases in both Pisum (Smith, 1973) and Vicia (Wheeler and Boulter, 1967; Cionini et a/., 1978) and possibly in all legumes. In pea, for example, DNA levels up to 64C are attained (Smith, 1973 ; Scharpe and Van Parijs, 1973; Millerd and Spencer, 1974; Davies and Brewster, 1976). This is not due to storage gene amplification but to both endomitosis and endoreduplication, i.e. both polyploid and polytene nuclei occur (Marks and Davies, 1979). These authors also showed that it was possible to induce both types of nuclei to divide in culture. The fact that DNA continues to increase after the amount of mRNA per cell (RNA of polysomes) has levelled off (see Fig. 8) suggests that it is not required in order to supply extra template for storage protein synthesis, a result in agreement with the conclusions of Millerd and Spencer (1974) and Cullis (1976). However, Cullis (1976) has shown that in two pea lines the optimum rate of RNA polymerase activity per pg DNA occurs well before “excess” replication of DNA, whereas the RNA polymerase activity per cell reaches a maximum after this DNA replication is well under way. Assuming that the efficiency of the polymerase does not change, he was able to conclude that some of the “excess” DNA is used as a template for rRNA. Another suggestion, namely that the “excess” DNA acts as a nitrogen store to be used during germination has little experimental support, although studies are few. These indicate that cotyledon tissues are unable to supply the nucleotides required by the growing axis, which come instead from nitrogen provided by storage protein hydrolysis (Bewley and Black, 1978). F. SOME GENETICAL ASPECTS

1. Storage Protein Genes The fact that storage proteins are multimeric probably means that several different genes are involved in the specification of each, otherwise different subunits (polypeptides) must have been generated post-translationally from a single gene product. This is not to imply, however, that considerable posttranslational activity does not go on.

STORAGE PROTEIN SYNTHESIS AND DEPOSITION

25

In order to study the genetic basis for the variable storage protein subunit patterns shown by different taxa in a genus, certain pre-requisites are necessary : (i) The subunit patterns for each storage protein must be established and where the constituent subunits are of approximately the same molecular weight, either charge or amino acid sequence differences must be shown to exist which are not post-translationally generated. (ii) If genetically, rather than environmentally or developmentally determined variants are available, it should be possible to establish the genetic basis by carrying out F1 and F2 crosses. Thus, if the F1 protein patterns are additive, and F2 give a 1 :2 :1 (parentall :additive : parental2) ratio, this would indicate alleles acting at a single locus. Casey (1979) has fulfilled criteria (i) and (ii) for a number of major 40,000 legumin subunits of two Pisum varieties. Since several subunits were involved, the genes responsible must be tightly linked so as to behave as a single focus, suggesting that gene duplication had taken place. The fact that 40,000 and 20,000 subunits are derived from a 60,000 precursor, and that amino sequenceheterogeneity has been established in one case by Gilroy et al. (1979) for the 20,000 subunits, supports this contention. However, the same results could arise from mRNA processing. In a broader study, Thompson and Schroeder (1978), showed that separate loci for three vicilin polypeptides and two legumin polypeptides behaved as single loci. However, the subunits investigated were not strictly identified with individual storage proteins and, for example, the suggestion that LD 20,000 MW bands are multigenetically controlled may not be correct if these bands are not exclusively legumin subunits (U). Davies (1980) has shown, using near isogenic lines of smooth and wrinkled peas, that the structural genes for the subunit polypeptides of Pisum legumin are on chromosome 7 and closely linked to the ra locus. Legumes are first limiting nutritionally, in their sulphur amino acid content. Since different protein fractions- albumin, legumin, vicilin, convicilin -differ in their sulphur amino acid content (Boulter and Derbyshire, 1971) and since the proportions of these fractions varies in different varieties (Gatehouseet al., 1980b),these differences may be reflected in varying amino acid profiles of the seed meal. Screening world collections, however, may not easily reveal a correlation between a protein fraction and better than average sulphur amino acid content, since the latter can arise in a variety of ways. In order to demonstrate a possible correlation, it will be necessary to follow the sulphur amino acid content and protein profile in the different offspring of a cross between high and low sulphur amino acid containing varieties. 2. Mutants Developmental mutants, which should be of great help in these investiga-

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tions, are unlikely to become readily available due to lack of powerful screening methods. Storage proteins normally do not have enzyme activity and so direct screening methods are excluded. In the case of urease, which does, Polacco (1977) has taken advantage of the enzyme activity of urease to screen for high urease-containing clones using cell cultures. Urease of soya bean is relatively rich in methionine, the nutritionally limiting amino acid of seeds, compared with the total protein of soya bean seeds (Bailey and Boulter, 1971) and higher levels of urease therefore should also result in improved nutritional quality. Polacco (1977) devised several selective systems to recover overproducing urease mutants : (i) Utilization of urea in the presence of urease inhibitors. (ii) Utilization of urea in the presence of known metabolizable repressors of urease production. (iii) Utilization of urea in the presence of high levels of nitrate. These selective procedures may also produce in the case of (i) mutants with altered urease, and in the case of (iii) nitrate reductase constitutive mutants, i.e. those that produce nitrate reductase without the prior metabolism of a reduced nitrogen source. Another possible approach to obtaining mutants might be to select temperature-conditioned mutants, the seeds of which were only viable at permissive temperatures. Lastly, it is possible to screen directly, at least on a smaller scale, using one or another form of gel electrophoresis or serology. Recently, Davies (1980) has identified smooth and wrinkled peas as genetic variants in which the relative proportion of the storage proteins has changed. Wrinkled peas contain less starch and more soluble sugars, so that their seeds do not fill, and wrinkle. These mutants are similar therefore to barley mutants such as Riser 1508. During seed development, carbohydrates, protein and lipid metabolism interact in complex ways. Thus storage proteins are synthesized on lipid “containing” membranes, which proliferate at this time. Vicilin is glycosylated, probably via lipid-oligosaccharide intermediates and there are increased energy requirements with increased rates of protein synthesis. Starch and protein metabolism also interact. Pleiotrophic mutants such as Riso 1508 and wrinkled peas therefore, although useful for gene mapping, may be less useful in studies on development. Fortunately, the lack of mutants is not quite so serious as might appear, since there are already many excellent examples of control mechanisms from viruses and prokaryotes and more recently from mammalian systems. Even in a relatively simple system, such as coliphage lambda, in which less than 50 genes occur, the control systems involve both transcription and translation, positive and negative controls, and attenuation. Control is affected by proteins and the control circuits interact in a complex network. In the T

STORAGE PROTEIN SYNTHESIS A N D DEPOSITION

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phage system, control additionally involves a change in function of the components of the basic biosynthetic machinery, namely DNA dependent RNA polymerase. In the more complex Escherichia coli, for example in the lac operon, small effector molecules (inducers and co-repressors) are also involved and in mammalian systems such as those which synthesize ovalbumin, hormones may act as effectors. These examples all supply possible models for consideration in eukaryotes. However, although our ideas on control will rely heavily on prokaryote and virus examples, eukaryote control mechanisms must differ to some extent. In eukaryotes, in contrast, to prokaryotes, transcription and translation are separated in time and space and genes are split so that the RNA processing of HnRN A to mRNA is an additional processing step. Eukaryote mRNA is more stable than prokaryote and hence translation level controls may play a greater part in the former. Lastly, the chromatin of eukaryotes is more complex than the DNA of prokaryotes (see Walker, 1977) and long range controls are apparent. V . CONCLUSIONS

Storage protein synthesis can be thought of as a short phase interpolated between an initial phase of development of the fertilized ovule, and its continued development on germination to the adult plant. A special “machinery” of protein synthesis is assembled and is used to produce specialized proteins which are sequestered from the cytoplasm by a transport and storage system. We can speculate therefore that the controls of storage protein synthesis are an independent sub-set of the cellular controls operating during seed development and can be effectively investigated as if isolated from accompanying metabolic changes (i.e. changes in the supply of energy, building blocks and protein synthesis machinery), which can be thought of as co-ordinated consequences of the developmental shifts. The developing legume seed will continue to be a source of important problems in cell biology, intra-cellular transport, protein structure-function relationships, molecular evolution, optimization of crop protein yields, as well as in developmental biology, for some time to come. ACKNOWLEDGEMENTS

I would like to thank my colleagues who have allowed me to use their unpublished results, and especially Drs R. R. D . Croy, J. A. Gatehouse and N. Harris, who additionally made many helpful comments on the text. Figures 1-7 were provided by Dr N. Harris and Fig. 8 by Dr J. A. Gatehouse. I also wish to thank the Agricultural Research Council, the Science Research Council and the Overseas Development Administration for financial support.

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REFERENCES Bailey, C . J. and Boulter, D. (1970). Eur. J . Biochem. 17,46@466. Bailey, C . J. and Boulter, D . (1971). In “Chemotaxonomy of the Leguminosae” (J. B. Harborne, D . Boulter and B. L. Turner, Eds), pp. 485-502. Academic Press, London and New York. Bailey, C. J., Cobb, A. and Boulter, D. (1970). Planta 95, 103-118. Bain, J. M . and Mercer, F. V. (1966). Aust. J . b i d . Sci. 19,49-67. Basha, S. M. M. and Beevers, L. (1976). Plant Physiol. 57,93-97. Baumgartner, B. and Chrispeels, M . J. (1979). In “Seed Proteins of Dicotyledonous Plants” (K. Muntz, Ed.), pp. 115-124. Academie Verlag, Berlin. Beachy, R . N., Barton, K. A,, Madison, J. T., Thompson, J. F. and Jarvis, N. (1980). In “Genome Organisation and Expression in Plants” (C. J. Leaver, Ed.), pp. 273-282. Plenum Press, New York and London. Bergfeld, R., Kiihne, T. and Schopfer, P . (1980). Planta 148, 146156. Bewley, J . D . and Black, M . (1978). “Physiology and Biochemistry of Seeds”, Vol. 1. Springer-Verlag, Berlin, Heidelberg and New York. Blobel, G. and Dobberstein, B. (1975). J . Cell. B i d . 67, 835-851. Blobel, G. and Sabatini, D. (1971). In “Biomembranes” (L. A. Manson, Ed.), Vol. 2, pp. 193-195. Plenum Press, New York. Bollini, R. and Chrispeels, M. J. (1979). Planta 146,487-501. Boulter, D. (1976). In “Genetic Improvement of Seed Proteins”. Proc. of a Workshop, pp. 231-250. National Acad. Sci., Washington, U.S.A. Boulter, D . (1977a). In “Protein Quality from Leguminous Crops”, pp. 1 1 4 7 . Commission of the European Communities, Brussels. Boulter, D . (1977b). In “Cellular and Molecular Plant Physiology” ( H . Smith, Ed.), pp. 256279. Blackwell Scientific Publications, Oxford. Boulter, D. (1979). In “Nitrogen Assimilation of Plants” (E. J . Hewitt and C . V . Cutting, Eds), pp. 359-368. Academic Press, London and New York. Boulter, D. and Crocomo, 0. J . (1979). In “Plant Cell and Tissue Culture. Principles and Applications” (W. R . Sharp, P. 0. Larsen, E. F. Paddock and V. Raghavan, Eds), pp. 615-631. Ohio State U . Press, Columbus, U.S.A. Boulter, D. and Davis, 0.J. (1968). New Phytol. 57,935-946. Boulter, D. and Derbyshire, E. (1971). In “Chemotaxonomy of the Leguminosae” (J. B. Harborne, D. Boulter and B. L. Turner, Eds), pp. 285-308. Academic Press, London and New York. Boulter, D., Thurman, D. A. and Derbyshire, E. (1967). New Phytol. 66,27-36. Boulter, D., Ellis, R. J . and Yarwood, A. (1972). Biol. Rev. 47, 113-175. Briarty, L. G. (1978). In “Plant Proteins” (G. Norton, Ed.), pp. 81-106. Butterworths, London. Briarty, L. G., Coult, D. A. and Boulter, D . (1969). J . exp. Bot. 20, 358-372. Briarty, L. G., Coult, D. A. and Boulter, D. (1970). J . exp. Bot. 21, 513-524. Burton, K . (1956). Biochem. J . 62,315-323. Casey, R . (1979a). Biochem. J . 177, 509-520. Casey, R. (1979b). Heredity 43,265-272. Chrispeels, M. J . and Boulter, D . (1975). Plant Physiol. 55, 1031-1037. Cionnini, P. G., Bennici, A. and D’Amato, F. (1978). Protoplasm 96, 101-1 12. Craig, S., Goodchild, D. J. and Hardham, A. R . (1979a). Ausr. 1.Plant Physiol. 6, 81-98. Craig, S . , Goodchild, D. J . and Millerd, A. (1979b). J . Histochem. and Cytochem. 27, 1312-1 3 16.

STORAGE PROTEIN SYNTHESIS AND DEPOSITION

29

Croy, R. R. D.. Gatehouse, J . A,, Evans, I. M. and Boulter, D. (1980a). Pluntu 148, 49-56. Croy, R. R. D.. Gatehouse, J. A,, Evans, I. M. and Boulter, D. (1980b). Plunru 148, 5743. Croy, R . R. D., Gatehouse, J. A,, Tyler, M. and Boulter, D. (1980~).Biochem. J . 191, 509-5 16. Cullis, C. A. (1976). Pluntu 131, 293-298. Davey, R. A. and Dudman, W. F. (1979). Aust. J . Plunt Physiol. 6,435437. Davies, D. R. (1980). Biochemicul Genetics 18, 167-179. Davies, D. R. and Brewster, V. (1975). Pluntu 124, 303-309. Davies, H. M. and Delmer, D. P. (1979). Pluntu 146,513-520. De Duve, C. (1971). J . Cell. Biol. 50, 20D-55D. Derbyshire, E., Wright, D. J. and Boulter, D. (1976). Phytochemistry 15, 3-24. Dieckert, J. W. and Dieckert, M. C. (1976). J . Food Sci.41,475482. Di Rienzo, J . M., Nakamura, K . and Inouye, M . (1978). Ann. Rev. Biochrm. 47, 48 1-532. Domoney, C., Davies, D. R . and Casey, R . (1980). Plunru 149,454460. Dudman, W. F. and Millerd, A . (1975). Biochem. Svst. Ecol. 2, 25-33. Dure, L. S . (1975). Ann. Rev. Plunr Physiol. 26, 259-278. Dyer, T. A. and Payne, P. I . (1974). Plnntu (Berl.) 117, 259-268. Eaton-Mordas, C. A. and Moore, R. G. (1978). Phytochrnristry 17, 619-621. Elbein, A. D. (1979). Ann. RKV.Plunt Physiol. 30, 239-272. Ericson, M. C. and Chrispeels, M. J . (1973). Plunt Physiol. 52, 98-104. Ericson, M. C. and Chrispeels, M. J. (1976). Aust. J . Plunt Physiiol. 3, 763-769. Evans, I. M., Croy, R . R . D., Hutchinson, P., Boulter, D., Payne, P. I . and Gordon, M. E. (1978). PIuntu 144, 455-462. Evans, I . M., Croy, R. R. D., Brown, P. and Boulter, D. (1980) Biochim. Biophys. Acru 610, 81-95. Gardiner, M. and Chrispeels, M. J. (1975). Plant Physiol. 55, 536-541. Gatehouse, J . A., Croy, R . R . D. and Boulter, D. (1980a). Biochrm. J . 185,497-503. Gatehouse, J. A., Croy, R . R. D., Mclntosh, R., Paul, C. and Boulter, D. (1980b). I n “Viciu fuhu. Feeding Value, Processing and Viruses” (D. A. Bond, Ed.), pp. 173-190. E.C.S.E., E.E.C., E.A.E.C. Brussels, Luxembourg. Gilroy, J., Wright, D . J. and Boulter, D. (1979). Phytochemistry 18, 315-316. Gordon, M. E. and Payne, P. I . (1976). Pluntu 130,269-273. Graham, T. A. and Gunning, B. E. S. (1970). Nuture (Lond.) 228,81-82. Hall, T. C., Sun, S. M., Buchbinder, B. U., Pyne, J. W., Bliss, F. A. and Kemp, J . D. (1980). I n “Genome Organisation and Expression in Plants” (C. J. Leaver, Ed.), pp. 259-272. Plenum Press, New York and London. Harris, N . (1979). Pluntu 146,63-69. Harris, N . and Boulter, D. (1976). Ann. Bot. 40,739-744. Harris, N . and Juliano, B. 0.(1977). Ann. Bot. 41, 1-5. Higgins, T. S. V. and Spencer, D. (1980). I n “Genome Organisation and Expression in Plants” (C. J. Leaver, Ed.), pp. 245-258. Plenum Press, New York and London. Jackson, P., Milton, J . M. and Boulter, D. (1967). New Phytol. 66, 47-56. Jackson, P., Boulter, D. and Thurman, D. A. (1969). New Phytol. 68,25533. Jamieson, J . D. (1978). I n “Transport of Macromolecules in Cellular Transport” (S. C. Silverstein, Ed.), pp. 273-287. Abakon Verlagsgesellschaft, Berlin. Khan, Md. R . I., Gatehouse, J. A. and Boulter, D. (1980). J . exp. Bot. 31, 1599-161 1. Koshiyama, I. and Fukushima, D. (1976). Cereal Chem. 53,768-769. Larkins, B. A. and Hurkmann, N. J. (1978). Plant Physiol. 62, 256-263.

30

D. BOULTER

Lehle, L., Bowles, D. J. and Tanner, W. (1978). Plant Sci. Lett. 11, 27-34. Leigh, B. A. (1979). Trends in Biochemical Sciences 4, N37-38. Lott, J. N. A. and Buttrose, M. S. (1978). Aust. J . Plant Physiol. 5 , 89-111. Marcus, A., Efron, D. and Weeks, D. P. (1974). Methods in Enzymology 30,749-754. Marks, G. E. and Davies, D. R. (1979). Protoplasma 101, 73-80. Matile, P., (1968). 2. Pfanzmphysiol. 58,365-368. Matile, P. (1975). “The Lytic Compartment of Plant Cells”. Cell Biology Monography, Vol. 1. Springer, Vienna and New York. Matta, N., Gatehouse, J . A. and Boulter, D. (1981). J . exp. Bot. 32, 183-197. Miege, M. (1975). In “Les Proteins des Graines” (J. Miege, Ed.), pp. 31-54, Geneve. Millerd, A. (1975). Ann. Rev. Plant Physiol. 26, 53-72. Millerd, A. and Spencer, D. (1974). Aust. J . Plant Physiol. 1, 331-341. Millerd, A,, Spencer, D., Dudman, W. F. and Stiller, M. (1975). Ausr. J . Plant Physiol. 2, 51-59. Molnar, J. (1975). Mol. Cell Biochem. 6, 3-20. Morre, D. J. (1975). Ann. Rev. Plant Physiol. 26,441481. Morris, G. F. I., Thurman, D. A. and Boulter, D. (1970). Phytochemistry 9, 1707-1714. Miintz, K. (1978). In “Regulation of Developmental Processes in Plants” (H. R. Shiilte and D. Gross, Eds), pp. 7G97. VEB Gustav Fischer, Jena. Nagahashi, J. and Beevers, L. (1978). Plant Physiol. 61,451459. Nagahashi, J., Mense, R . M. and Beevers, L. (1978). Plant Physiol. 62, 766772. Neumann, D. and Weber, E. (1978). Biochem. Physiol. Pflanzen. 173, 167-180. Nierlich, D. P. (1978). Ann. Rev. Microbiology 32, 393432. O’Malley, B. W., Towle, H. C. and Schwartz. R. J . (1977).Ann. Rev. Genet. 11.239-275. Opik, H. (1968). J . exp. Bot. 19,6476. Palmiter, R. D., Gagnon, J., Walsh, K . A . (1978). Proc. Natl. Acad. Sci. U.S.A. 75, 94-98. Payne. E. S., Brownrigg. A.. Yarwood. A . and Boulter, D. (1971a). Phytochem. 10, 2299-2303. Payne, E. S., Boulter, D., Brownrigg, A , , Lonsdale, D., Yarwood, A. and Yarwood, J . N. (197 1b). Phytochemistry 10,2293-2298. Payne, P. I. (1976). Biol. Rev. 51,329-363. Payne, P. I. and Boulter, D. (1969). Planta (Bed.) 84,263-271. Payne, P. I. and Boulter, D. (1974). Planta (Bed.) 117, 251-258. Pelham, H. R. B. and Jackson, R. J. (1976). Eur. J . Biochem. 67,247-256. Pernollet, J. C. (1978). Photochemistry 17, 1473-1480. Peumans, W. J., Carlier, A . R. and Schreurs, J. (1980). Planta 147, 302-306. Polacco, J. C. (1977). Plant Physiol. 54, 654655. Piichel, M., Miintz, K., Parthier, B., Aurich, O., Baussiimer, R., Manteuffel, R. and Schmidt, P. (1979). Eur. J . Biochem. 96, 321-329. Pusztai, A. and Watt, W. B. (1970). Biochim. Biophys. Acta. 207,413431. Pusztai, A,, Croy, R: R. D. and Grant, G. (1979). In “Seed Proteins of Dicotyledonous Plants” (K. Miintz, Ed.), pp. 133-144. Academie Verlag, Berlin. Quail, H. (1979). Ann. Rev. Plant Physiol. 30,425-484. Revel, M. and Groner, Y. (1978). Ann. Rev. Biochem. 47, 1079-1 126. Scharpe, A. and Van Parijs, R. (1973). J. exp. Bot. 24, 216222. Smith, D. L. (1973). Ann. Bot. 37,795-804. Smith, M. H. (1966). J . theoret. Biol. 13,261-282. Stafford, A. and Davies, D. R. (1979). Ann. Bor. 44,315-321. Szybalski, W. (1977). In “Cell Differentiation in Micro-organisms, Plants and Animals” (L. Nover and K. Mothes, Eds), pp. 262-316. VEB Gustav Fischer, Verlag, Jena.

STORAGE PROTEIN SYNTHESIS AND DEPOSITION

31

Taylorson, R. B. and Hendricks, S. B. (1977). Anti Rev. Planr Physiol. 28, 331-354. Thanh, V . H. and Shibasaki, K. (1976). Biochini.Biophys. Actu 439,326-338. Thompson, J. A. and Schroeder, H. E. (1978). Airst. J . Plunr Physiol. 5,281-294. Thompson, J. A,, Millerd, A. and Schroeder, H . E. (1979). “Seed protein improvement in cereals and grain legumes”. Proc. Symp. Neuherberg, Vol. 1, pp. 231-240. 1.A.E .A,, Vienna. Van der Wilden, W., Herman, F. M. and Chrispeels, M . J . (1980). Proc. Nor/. A c i d . Sci. U.S.A., 77,428432. Waechter, C. J. and Lennarz, W. J. (1976). Ann. Rev. Biochem. 45, 95-1 12. Walker,P. R. (1977). In “Essays in Biochemistry” (P. N . Campbell and W. N. Aldridge, Eds), Vol. 13, pp. 39-69. Academic Press, London and New York. Wheeler, C. T. and Boulter, D. (1966). Biochem. J . 100, 53-54. Yarwood, A. (1977). In “Plant Proteins” (G. Norton, Ed.), pp. 41-55. Butterworths, London. Yarwood, A,, Boulter, D. and Yarwood, J . N. (1971). Biocheni. Biophys. Res. Conzm. 44. 353-361.

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Aspects of the Metabolism and Physiology of Gibberellins

ALAN CROZIER Department of Botany. University of Glasgow. Glasgow GI2 8QQ. Scotland

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I1. Analytical Methods . . . . . . . . A . General Observations . . . . . B . Extraction and Partitioning Techniques C . Group Purification Procedures . . . D . Separatory Techniques . . . . . E . Identification Procedures . . . . . F. Verification of Accuracy . . . . .

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I . INTRODUCTION The gibberellins (GAS) are a group of diterpenoid acids which function as endogenous regulators of the growth and development of higher plants . General acceptance of their hormonal role is based on the observation that

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GAS are natural components of the vast majority of higher plants, and that exogenous application of pg quantities can induce a wide range of plant growth responses. They are very effective in promoting stem elongation in intact plants and the response is especially pronounced in dwarf varieties of pea (Pisum sativum), maize (Zeu mciys) and rice ( O r ~ m sativa). GAScan also overcome seed dormancy by substituting for prerequisite cold, light or dark treatments as well as promoting the growth of dormant buds of woody plants and tubers. De n o w synthesis of several hydrolysing enzymes, including a-amylase, can be induced by GA treatment of the aleurone layer of cereal grains. This finds practical application in the malting industry where GA is widely used to increase the rate of starch hydrolysis. GAS can promote stem elongation and the subsequent flowering of rosette plants grown in noninductive short day photoperiods and in a similar manner can circumvent the vernalization requirements of certain biennial species. Sex expression can be modified by GAS, particularly in the Cucurbitaceae where the production of staminate flowers is strongly enhanced. The effects of GAS on reproductive organs are not restricted to angiosperms as similar responses have been observed in some coniferous species. GA application results in prolific male strobilus production in 60-day-old seedlings of Arizona cypress (Cupressus nrizonica Greene), a species that does not usually produce strobili until it is 1G-15years of age. GAS will induce parthenocarpic fruit development in a number of plants including Lycopersicum esculentum Mill., Cucumis sativus L., Solanum melongena L. and Capsicum frutescans L. GAS are widely used in vineyards as they induce the growth of large, elongated berries in open clusters, thereby making the grapes more attractive for table use. It is also reassuring to know that Californian wines prepared from GA-treated grapes and tasted by a panel of experts scored just as highly as those made from untreated berries. A further effect of GA is to retard both leaf and fruit senescence. Treated citrus fruit attached to the tree remains green for six months or more. The senescence of detached fruit is slowed from three weeks to two months by G A application. Clearly GAS have far-reaching effects on many phases of growth and development. There have been many reviews on GAS,the most recent on the physiological role of GAS being that of Jones (1973). GA metabolism has been covered by Phinney (1979), Hedden et al. (1978) and Railton (1976). Hedden (1979) has reviewed selected aspects of GA chemistry while Graebe and Ropers (1978) have published a critical and very comprehensive review of GA chemistry, biochemistry, metabolism and physiology. Agricultural and horticultural uses of GAs have been outlined by Weaver (1972). The discovery of GAS originates from an investigation of the “foolish seedling” or “bakanae” disease of rice (Kurosawa, 1926). The disease had been observed in Japan for over 150 years and infected plants were characterized by both excessive shoot overgrowth of the seedlings and lowered seed

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production by mature plants. The first suggestion that a fungus might be involved was made in 1912 after hyphae were observed in infected rice plants (Sawada, 1912). The pathogen was subsequently identified as an ascomycete Gibberella ,fujikuroi (Saw) Wr. (called Fusarium nionilifornie Sheldon in the asexual stage) and it was shown that sterile culture filtrates of the bakanae fungus induced a marked growth stimulation in rice and maize (Kurosawa, 1926). In 1935 the active ingredient was isolated in a purified but noncrystalline form and given the name gibberellin (Yabuta, 1935). crystalline GA was obtained by Yabuta and Sumiki (1938) although this later proved to be a mixture of at least three compounds all of which promoted shoot growth when applied to rice seedlings (Takahashi et a / . , 1955). Many of these early reports were, contrary to popular belief, published in English, yet despite a great interest in hormonal regulation of growth by auxins, plant physiologists in the West did not become aware of the Japanese work on GA until the early 1950s when groups at the US Department of Agriculture and ICI Akers Laboratory in the UK initiated their own investigations. The British isolated “gibberellic acid” (Borrow et a / . , 1955) and the Americans “gibberellin X” (Stodola et ul., 1955). The compounds proved to be identical and the structure of gibberellic acid, or GA3 as it is now known, was fully elucidated by Cross et a / . (1959) (see Fig. I). The quantity of GA produced as a metabolic by-product by G. fujikuroi exceeds that found in higher plants by several orders of magnitude. Even so, growth promoting activity similar to that of GA was found to be present in higher plant tissues by Radley (1956) and Phinney et al. (1957) and the first characterization of a GA from a flowering plant was reported by MacMillan and Suter (1958) who isolated GA1 from immature seed of the scarlet runner bean, Phaseolus coccineus L. With the development of both improved purification procedures and analytical techniques, subsequent progress has been rapid and 62 GAS have now been characterized (Fig. 1). Nine GAShave been found only in Gibberella fujikuroi cultures, 38 are exclusive to higher plants while 15 are ubiquitous, having been detected in extracts from the fungus and higher plant tissues. Many more potential permutations of the GA structure exist and there will undoubtedly continue to be additions to this list for some time to come. So as to avoid confusion, the trivial nomenclature GA1-GA62 has been adopted and there will be a sequential allocation of numbers GA,,-GA, to any new naturally occurring, fully identified GA (MacMillan and Takahashi, 1968). Familiarity with the various GA structures is not such a daunting task as first impressions of Fig. 1 might suggest. All the GASpossess an ent-gibberellane skeleton and can be divided into two groups by virtue of the possession of either 19 or 20 carbon atoms (Fig. 2). The C19-GAs have lost carbon-20 and all but one possess a 19- 10 y-lactone bridge, the exception being GA, which has a 19+2 linkage. The C20-GAsare characterized by the presence of carbon-20 which can exist as either a CH3, C H 2 0 H , CHO or COOH

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(3

9 c,

c,

c

C

-:0

OJ

a

W

Q,

I

c

-a

(3

I

c

co

a W

I

I

p:

v

a

W

N t

c

z

a (3

c

I (I)

v

N

a

(3

Pz

x.0

-

u--

Q

0

z-

g 0-

x

O

0 rr,

I

c

-

U

W

OH -OH

I

co HO

CH3

HO

I

H

H

H COOH

CH3

COOH

CH,,'

rnnu

COOH

CHZ

"

H

HOW

OH

O

0-

H

a

HO

n

HO H CH,

H

H COW

CHZ

CH,

COOH

CHZ

CH3

H

co

O

-

-OH

m

-

CH2

.

.

HO

H CH3

COOH

co

HO

OH

r

co

co

co

OH

- -o-CH2

H COOH

CHZ

CH3

COOH

CH2

0

HO %O -H

co -OH

CH3

co

--OH H

H COOH

CH2

co

CHz

Fig. I . Structures of GA,-GA6,. F, endogenous component of Gihherellafuji~uroi;H, higher plant GA

CHZ

40

ALAN CROZIER

20

enl-gibberellane skeleton

CH3

u

COOH C - 2 0 methyl C ~ O - G A

O----CHOH

CHO

7

CH3

H

H

‘1 ‘\

,

CH3

COOH COOH

COOH

CH2

6-lactor CpO-GA

C-20aldehydic C ~ O - G A

m COOH

CH3

\

‘,

COOH

CH2

COOH C - 2 0 carboxylic C m G A

H CH3

COOH

C*Z

y - l o c t o n i c C19-GA

Fig. 2. Ent-gibberellane skeleton and basic structures of CI9-and C?o-GAs.

function. The 20-CH20H group forms a 19+20 d-lactone bridge and the 20-CHO function appears to exist, in solution, in equilibrium with a 19+20 Glactol ring (Harrison et al., 1968). The variations in the oxidation state at C-20 and the presence or absence of 38- and 13a-hydroxyl groups account for 20 of the GAS(Table I). The remaining GASare represented by additional

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

41

TABLE 1 GA structures bused on variations in the oxidation state at C-20 und the presence or absence of hydroxyl groups at C-3 and C-13

H ydroxylation Oxidation at C-20

None

38

13a

38, 13a

modifications to these basic configurations in the form of 2,3 and 1,lO epoxide groups, C-3 and C-12 keto groups, 8-hydroxylation at C-1, C-2, C-12 and C-15, a-hydroxylations at C-1 and C-2, C-12 and C-16, oxidation of the 18-methyl group to carbonyl and carboxyl functions and the introduction of 1,2 and 2,3 double bonds. GA-glucose conjugates have also been found in higher plants. The glucose is present in the pyranose form and the 0(3)B-~-glucosylether of G A I and GA3, the 0(2)~-D-glucosylether of GAB,GA26,GA2, and GA29the 0(11)/?-~glucosyl ether of GA35 and the 0(7)B-~-glucosylester of G A T ,GA4, GAS, GA9, GA3,, GA38and GA4, have all been characterized. The n-propyl ester of GA3 has been identified in cucumber seed extracts while O(3)P-acetyl GA, and O(3)B-acetylGA3 are the only conjugated GASto have been isolated from Gibberella fujikuroi cultures. In addition a sulphur containing derivative of GA3, called gibberethione, has been isolated from seed of Pharbitis nil (Yokota et al., 1974, 1976). The application of combined gas chromatography-mass spectrometry (GC-MS) to the analysis ofendogenous GAS(Binks et al., 1969; MacMillan, 1972) is the main reason why GAS have now been identified in more than 26 species of higher plants (see Graebe and Ropers, 1978). Nine GAShave been identified in extracts from immature seed of Calonyction gladiata and 13 in Phaseolus coccineus. Immature seed has proved to be a rich source of GAS and can contain up to 100 mg kg- fresh weight. It is therefore not surprising that the vast majority of GAs identified in higher plants have originated from seed material. The GASpresent in immature seed seemingly can become conjugated as the seed develops and seeds have also been the source of almost all the conjugated GAS that have been identified to date. It is debatable whether or not the high concentrations of GAS found in immature seed have a hormonal function. The little evidence that is available is equivocal and has been obtained from experiments utilizing either 2isopropyl -4 - (trimethylammonium chloride) - 5 - methylphenyl- 1- piperidine carboxylase methyl chloride (AMO-1618) or B-chloroethyltrimethylam-

'

42

ALAN CROZIER

monium chloride (CCC). Both these compounds inhibit GA biosynthesis in Gibberellu fujikuroi cultures and cell-free systems from higher plants by preventing the conversion of geranylgeranyl pyrophosphate to copalyl pyrophosphate (Robinson and West, 1970b; Shechter and West, 1969) but when applied to seedlings inhibition of growth is often mediated by other less specific effects (see Crozier et a/., 1973; Graebe and Ropers, 1978). Baldev rt ul. (1965) have shown that treatment of cultured immature Pisum sativum seed with 5 mg AMO-1618 1- results in a 60% fall in GA levels while the rate of growth is unchanged. This implies that at least a sizable portion of the GA pool is not involved in seed development. Application of CCC to immature seed of Phurbitis nil also produces depleted GA levels without adversely affecting seed growth (Zeevaart, 1966). When mature, the CCC-treated seeds were viable and germinated giving rise to dwarf seedlings with a reduced GA content. However, no conclusions can be drawn as to whether or not these symptoms are a consequence of lowered GA levels during seed maturation as the mature seed and germinating seedlings contained residual CCC which would inhibit the rate of stem elongation. It has been suggested that GAS produced during seed development are stored in the mature seed as GA conjugates and during the early stages of germination these biologically inactive conjugates are hydrolysed to release free GASwhich enhance the rate of stem elongation (see Lang, 1970). There is however no conclusive evidence for such a role and the available data imply that it is an unlikely proposition as hydrolysis of many GA conjugates yields 2P-hydroxy GAS which exhibit relatively little biological activity (see Section IV). Quantities of GA in tissues other than seed are rarely higher than 5@100 ,ug kg- fresh weight and as a consequence there are relatively few reports of GASbeing identified in extracts from such material (Table 11). Although they have been isolated from conifers (see Pharis and Kuo, 1977), there are only two examples of GAS being characterized in lower plants other than Gibberellufujikuroi. Yamane er nl. (1979) detected GA9 methyl ester in extracts from prothalli of the fern Lygodiumjaponicum while Rademacher and Graebe (1979) and Graebe et a / . (1980) have identified GA4 and small amounts of GAg, GA13,GA14 and GAZ4in culture media of Sphaceloma manihoticola, a pathogenic fungus that is a member of the Melanconiales and causes “superelongation disease” of cassava (Munihot esculentu). In contrast to this dearth of information there are many hundreds of reports of bioassays being used to detect GA-like activity in all types of higher plant tissues and organs as well as the occasional moss, fern, alga, fungus and bacterium. The data have been used to implicate endogenous GAS in many varied aspects of plant growth and development. While the use of bioassays in such circumstances is understandable, in view of their simplicity and the lack of readily available alternative methodology, it is none the less unfortunate as it is becoming increasingly apparent that bioassays are an unreliable analytical tool

TABLE I1 Identification of higher plant GAs from tissues other than seed niaterial

Gibberellin

Species

Tissue

Water sprouts Shoot apices and flower buds Shoot apices Phyllostachys edulis Althea rosea Shoot apices Seedlings Phaseolus roccineus Bryophyllum daigremontianum Shoots Sonneratia apetala Leaves Rhizophera mucranata Leaves Pinus attenuata Pollen Shoots Pinus attenuata Citrus reticulata Nicotiana tabacum

GA20>

GA29

Picea sitchensis Cupressus arizonica Juniperus scopulorum Pseudotsuga menziesii O r p a sativa Humulus lupulus Stevia rebaudiana Spinacea oleracea Ribes nigrum

Needles Shoots Shoots Shoots Seedlings Shoots Shoots Shoots Shoot apices

Pisum sativuni

Seedlings

Reference Kawarada and Sumiki (1959) Sembdner and Schrieber (1965) Murofushi er a / . (1966) Harada and Nitsch (1967) Bowen et a / . ( 1 973) Gaskin et al. (1973) Ganguly and Sircar (1974) Ganguly and Sircar (1974) Kamienska et a / . (1976) Crozier, Morris and Bell (unpublished data) Lorenzi et a / . (1976, 1977) See Pharis and Kuo (1977) See Pharis and Kuo (1977) See Pharis and Kuo (1977) Kurogochi et a / . (1978) Watanabe er a/. (1978) Alves and Ruddat (1979) Metzger and Zeevaart (1980) Crozier, MacMillan and Schwabe (unpublished data) Sponsel and Albone (unpublished data)

44

ALAN CROZIER

with which to monitor qualitative and quantitative changes in GA levels (Reeve and Crozier, 1975, 1980; Graebe and Ropers, 1978; Letham et d., 1978). Acceptance of this view implies that much of the available information on topics such as sites of GA biosynthesis and cellular compartmentation of GASin seedling tissues and the involvement of endogenous GAS in developmental processes such as dormancy, photoperiodism, leaf expansion, and dwarfism, may be based on something other than a firm experimental foundation, and so requires critical re-investigation using more definitive methods of analysis. Clearly, this is a contentious issue and the following sections will discuss the special problems associated with the analysis of trace quantities of endogenous GASand outline various approaches that can be used to overcome them. 11. ANALYTICAL METHODS A . GENERAL OBSERVATIONS

Theoretical concepts associated with the identification of hormones in plant extracts have been proposed by Reeve and Crozier (1980) who point out that to fully understand the nature of the problems encountered in practice it is necessary to take a general view of analytical theory. It is important to realize that the distinction made between “qualitative” and “quantitative” analysis is a semantic convenience rather than a logical reality. Because it is impossible to quantify an unknown in meaningful terms, quantitative analysis is in fact inherently qualitative. The converse also applies, since the statement that “X is C A I ” implies that ALL of sample X has ALL the properties associated with the chemical concept of C A I . Reeve and Crozier ( 1980) further argue that quantitative analysis displays all the enigmas of scientific induction. The identification and quantification of a substance can never be absolute, and thus must be considered in association with a complex probability term which defines the chances of making an error when concluding that Y = x p g of compound Z . Two types of error, namely precision and accuracy, independently contribute to the complex probability term. Precision is a measure of random errors that determine run-to-run variability. Thus when given a series of estimates of the same sample it is possible to use statistical methods to calculate the standard deviation (SD) of the data and, with a minimum of assumptions, state that the probability of the precision being no worse than k2SD is 0.95. An averaging process can be applied to enhance the precision of an analysis in proportion to the square root of the number of estimates averaged. Accuracy, however, refers to the non-random or systematic error of the analysis and its error and confidence limits are inordinately more difficult to ascertain. Understanding the distinction between accuracy and precision is critical as it is essential to realize that, regardless of the number of estimates con-

45

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

tributing to the final average, there is no guarantee that accurate results will be obtained as non-random error will apply the same bias to each estimate and so be present undiminished in the averaged result. A “target” analogy such as that illustrated in Fig. 3 is a useful means of demonstrating the total independence of the terms accuracy and precision. It is evident from Fig. 3 that a rifle must not only be aimed accurately but must also be designed so as to closely group its shots, i.e. it must be precise if there is to be a high probability of hitting the “bull’s eye”. In the case of plant hormones it is a common mistake to assume that an analysis is accurate because it offers adequate precision. It is apparent from Fig. 3 that the degree of repeatability provides no such assurance of accuracy. In practice, verification of accuracy is the single most crucial, yet neglected, factor in the analysis of plant hormones. Reeve and Crozier (1980) have proposed that in a general context verification of accuracy consists of (a) defining, in terms of probability limits, the complexity of the analytical problem, and (b) relating this to the amount of pertinent information obtained during an analysis. At present, practical solutions to this problem require making a number of less than ideal assumptions. Even so figures for accuracy obtained by such methods will be more reliable than those ascertained by conventional procedures where the criteria for verification can range from the whims of technical fashion to standards of intuition that vary enormously from one investigator to another. In order to put a complex situation into perspective it is necessary to review the practical procedures employed in GA analysis before discussing them in the context of the proposals of Reeve and Crozier (1980) for verification of accuracy. At a practical level the analysis of endogenous GAScan be divided into the

Inoccurote and imprecise

lnoccurote and precise

Accurate ond imprecise

Accurate and precise

Error

Error

Error

Error

Fig. 3. Target analogy demonstrating the independence of the error terms accuracy and precision.

46

ALAN CROZIER

following sequential steps : (i) extraction and partitioning, (ii) group purification procedures, (iii) separation and (iv) identification. GAS are comparatively major components in extracts from Gibberrllri ,fiijikur.ai so little purification is necessary before identification is attempted. In contrast, they are minor trace constituents in extracts from higher plants and substantial purification is essential before attempts are made at characterization. This problem is especially severe with vegetative tissues because the GA levels are several orders of magnitude lower than those encountered in developing seeds. The chances of inaccurate analysis are substantially enhanced in such circumstances as the possibility of mistaking an impurity for a G A are increased. Sample losses invariably occur during purification and this also adversely affects the accuracy of quantitative estimates. Such errors can be corrected through the use of an appropriate internal marker which is added to every sample at the extraction stage. The most suitable internal markers are isotopically labelled analogs of the particular GA under study. ['HI, [3H]and [I4C]GAs tend to behave in the same manner as their endogenous counterparts yet can be differentiated by mass spectrometry or radioassay. When the GA under investigation is not available in an appropriately labelled form the best alternative internal standard is a GA of similar structure. However, recourse to such a procedure increases the possibility of some degree of separation occurring between the standard and the endogenous GA before the ultimate analytical step, thereby degrading accuracy. B. EXTRACTION AND PARTITIONING TECHNIQUES

Over the years an array of GA extraction and partitioning procedures have been used and this must cause confusion to many budding gibberellinologists. Tissues are usually extracted with methanol or ethanol, although aqueous buffers have also been tried. In [3H]GA metabolism studies methanol removes >95% of the radioactivity in Phn.seo/u,sseedlings (Reeve and Crozier, 1978). It is, however, an open question as to whether or not endogenous GAS are removed from all cellular sites with equal efficiency. Browning and Saunders (1977) reported that extraction of isolated chloroplasts of Tviticurii ciestivum with the detergent Triton-X yielded far higher levels of GA4 and GAg than methanol extracts. Unfortunately, similar results have not been obtained when the experiment has been carried out in other laboratories (Railton and Rechov, 1979). Buffer extracts from pea seedlings contain fewer impurities and more GA-like activity than methanolic extracts (Jones, 1968). However, it does not necessarily follow that buffer is the more effective in removing GAS from plant tissues as the bioassay data could just as well reflect reduced inhibitor concentrations as increased GA levels. When methanol and buffer extracts are subjected to several purification steps prior to bioassay, the methanolic extract yields higher levels of G A-like activity (Reid and Crozier, 1970).

47

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

Macerate tissue and extract 3 times with an excess of cold methanol. Combine methanolic extracts and reduce to the aqueous phase in i~ucuo.Add at least an equivalent volume of pH 8.0,0.5 M phosphate buffer and if necessary adjust extract to pH 8.0 Partition at least five times against volumes of toluene

+

Toluene

Aqueous phase Slurry with PVP and polyamide (50 mg ml-') filter

I

I

Aqueous phase

PVP and polyamide

Adjust to pH 2.5 and partition against 5 x + volumes of ethyl acetate

I

1 Acidic, ethyl acetate-soluble fraction

Aqueous phase Partition against 3 x f volumes of n- butanol

(Free G A S , and unknown amounts of GA conjugutes)

I

I

Acidic butanol-soluble fraction

Aqueous phase

Fig. 4. Flow diagram of extraction and partitioning techniques.

The procedures that are currently in routine use in my own laboratory are shown in Fig. 4. Tissue is macerated and extracted three times with an excess of cold methanol. The combined methanolic extracts are reduced to the aqueous phase in vaciio and the aqueous residue diluted at least twofold with pH 8.0,0.5 M phosphate buffer.This stabilizes the pH and ensures a minimum ionic strength during the ensuing partition procedures. At pH 8.0 the

48

ALAN CROZIER

aqueous phase is sufficiently basic to retain even the less polar GAS when partitioning against toluene yet not so basic as to risk isomerization. Petroleum spirit could be used instead of toluene but our experience is that the aromatic solvent has a higher solubilizing power for compounds with a large number of conjugated double bonds (i.e. pigments) and it therefore removes more impurities. Furthermore, emulsion problems are less likely to arise when toluene is used. Many investigators partition the aqueous phase against ethyl acetate or diethyl ether although the GA partition coefficient data of Durley and Pharis (1972) clearly show that this results in the removal of significant quantities of non-polar GAS. After partitioning at pH 8.0 the buffer phase can be further purified by slurrying with insoluble polyvinylpyrrolidone (PVP) (Glenn et a / . , 1972) and polyamide before acidification to pH 2.5 and extraction with 5 x 2/5 volumes of ethyl acetate. At this pH the partition coefficients are such that the bulk of the free GAS are removed by the ethyl acetate. The tetrahydroxy compound GA32is the only known free GA that will be retained by the buffer to any extent (Yamaguchi et a/., 1970). Metabolism experiments with [3H]GAs indicate that certain GA conjugates also migrate into the ethyl acetate. It is difficult to assess what proportion of the conjugated GAS this represents as little is known at present about their partitioning behaviour . It is however possible to extract conjugates from the acidified aqueous phase with n-butanol. The GA moiety of GA conjugates is best released by enzymatic hydrolysis and the efficiencies of various enzymes has been investigated by Knofel et a / . (1974). C . GROUP PURIFICATION PROCEDURES

The concentration of GAS in the acidic, ethyl acetate-soluble fraction is usually very low so a multistep analytical procedure has to be employed in order to attain a degree of purity that facilitates an accurate determination of GA content. Both the GA levels and the nature and amounts of contaminants vary greatly from one tissue to another so that the exact combination of procedures to be used is best determined by an on the spot assessment rather than the application of “cookbook style recipes”. When deciding what particular techniques to utilize some general points should be borne in mind. In the initial stages of purification, the substantial sample weights encountered dictate the use of chromatographic techniques with a high sample capacity. It is also advisable at this stage to use procedures which separate the GAS as a group from other components in the extract, otherwise unwieldy numbers of sub-fractions are quickly generated and there will be a marked decrease in the overall speed of analysis. Finally, purification is most effectively achieved if the individual techniques display widely different separatory mechanisms. Gel permeation or steric exclusion chromatography (SEC) has proved useful as a preparative, group separatory procedure (Reeve and Crozier,

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

49

1976, 1978). The system consists of two 25 x 1000 mm columns connected in series, packed with Biobeads SX-4 and eluted with tetrahydrofuran (THF) at a flow rate of 2 ml min- '. This is the maximum flow rate the support can tolerate without excessive compression of the bed. The gel has an operating range of (k1500 M W and solutes elute in decreasing order of molecular size. The sample capacity is high and is readily realized because of the excellent solubilizing power of THF, 1.5 ml of which will readily dissolve up to 1.0 g of an acidic, ethyl acetate extract; recoveries, estimated with a range of [3H]GAs, are >90%. The absence of adsorption effects ensures that even with the most impure extracts all solutes will be eluted by a volume of solvent (630 ml) which corresponds to the total volume of the column (V,). This allows the system to be used repeatedly without fear of sample overlap. 13H]GA9 and [3H]GA43,which represent the extremes of the molecular weight range of the free GAS, have respective retention volumes (V,) of 550 ml and 470 ml in this system with peak widths (w) of 40 ml. Endogenous GAS in extracts can therefore be purified by collecting the 45Cb.570 ml zone. While this technique is well suited for large-scale extracts a high performance SEC procedure has recently become available which offers a very rapid speed of analysis for the purification of smaller sized samples (Crozier et a / . , 1980). It involves the use of a p-Spherogel support" with a nominal exclusion limit of < 2000 MW. p-Spherogel is a macroporous cross-linked polystyrene divinylbenzene copolymer support that has been specifically designed for high performance liquid chromatography (HPLC) (Krishen, 1977). An 8 x 300 mm column eluted with 0.5% acetic acid in T H F generates 9000 theoretical plates and has a sample capacity of > 100 mg. The exclusion or void volume (V,) is 5.5 ml and V , is 9.5 ml. The V R of [3H]GA43is 7.0 ml and that of [3H]GA9,7.6 ml. In both instances w=0.4 ml. Thus collection of the 6.8-7.8 ml zone provides a very simple means of separating the free GAS as a group from the many extraneous components typically present in plant extracts. The speed of analysis is greatly enhanced as at a flow rate of 1 ml min- samples can be analysed every 9.5 min. The salient features of the two SEC systems are summarized in Table 111. Grabner et a / .(1976) used DEAE Sephadex A25 anion exchange chromatography to separate abscisic acid (ABA), GA3, GA, and ~(3)/3-D-glucosyl ether of GA,. This procedure is readily adapted for use as a group separatory procedure for free GAS. A 25 x 150 mm column of DEAE Sephadex A25 charged in the acetate form is eluted with four void volumes (600 ml) of methanolic 0.1 M acetic acid to remove neutral and weakly acidic impurities. The GAS are then eluted with c. 90% efficiency with two void volumes of ~ acid. The technique has been used to reduce the methanolic 1 . 0 acetic weight of a Pinus attenuata shoot extract from 550 mg to 45 mg and similar

'

* Altex Scientific Inc., Berkeley, California.

50

ALAN CROZIER

TABLE 111

Perfbrniuiic~choructrristics

of' SEC using Bioheads SX-4 and p-Sphrrogd supporis" Biobeads SX-4

p-Spherogel

-.

Nominal exclusion limit Column dimensions Solvent Flow rate Sample capacity

v,,

v/. N N,, H Speed of analysis Elution zone of G A S Analysis time "

1500 amu Two 25 x I000 mm THF 2 mI min 1 gm 350 ml 630 ml 3600 650 0.55 mm 0. I9 plates s - I 450-570 ml 320 min

-'

< 2000 amu 8 x 300 mm 0.57" acetic acid in THF I ml mm > 100 mg 5.5 mi 9.5 ml 9000 1600 0.03 mm 15.8 plates s 6.8-7.8 ml 9.5 mill

N-efficiency in theoretical plates. NeJ, efficiency in effective plates, H-plate height.

reductions in sample size have been achieved with other tissues (Crozier and Bell. unpublished data). As long ago as 1939 purification of GAS was achieved by exploiting the unusual reverse phase effects of charcoal adsorption chromatography (Yabuta and Hayashi. 1939). As currently employed the sample is dissolved in 1-2 ml of 203:, aqueous acetone and applied to the top of a 20 x 120 mm charcoal-celite ( 1 :2) column. Weakly adsorbed impurities are eluted with 100 ml of 207; aqueous acetone which is equivalent to four column volumes. The GAS are then removed with 200 ml of acetone. The sample capacity of charcoal is high and a column of the dimensions described can accommodate extracts weighing up to 500 mg (Reeve and Crozier, 1978). The method is also readily adaptable for use as a simple slurry procedure in which case large numbers of extracts can be treated in a matter of minutes. The recovery of GAS from charcoal is usually 75-85"/,. However, inexplicably high losses do occur from time to time, even with the same batch of charcoal, and as a consequence the procedures should only be used when replicate samples are readily available. Other group separatory procedures have been used for the purification of GAS. Although PVP adsorption chromatography can significantly reduce the dry weight of an extract (Glenn et [)I., 1972), very low column efficiencies and long analysis periods are associated with this technique. Furthermore, it involves the use of aqueous solvents which is undesirable because of the risk of GA rearrangements (Pryce, 1973). It is therefore safer, easier and almost as effective to make use of the PVP slurry treatment at the partitioning stage,

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

51

as shown in Fig. 4. A Sephadex G-I0 column eluted with 0.1 M, pH 8.0 phosphate buffer will retain GAS by virtue of uncharacterized adsorption phenomena and can be of value as a purification tool (Crozier rf o/., 1969). However as this procedure also involves exposure of the extract to mildly alkaline conditions for several hours. with attendant risks of degradation, it should be avoided if the required degree of purity can be achieved without recourse to a hydrolytic environment. Counter-current distribution has been used as a preliminary purification procedure for GAS (Crozier rt ( I / . , 1969) but it is now somewhat outmoded and usually does little that cannot be more effectively achieved with other techniques. 1). SEPARATORY TECHNIQUES

In most instances extracts which have been subjected to a range of group purification procedures will still require further purification before successful attempts can be made at GA analysis. This is achieved through the use of analytical methods which separate the GAS to some degree. Originally paper chromatography (PC) was the method of choice (see Phinney and West, 1961) but it was superseded by thin layer chromatography (TLC) (MacMillan and Suter. 1963; Kagawa c’t ( I / . , 1963) which is still widely used especially in conjunction with bioassays. However liquid-liquid partition column chromatography systems offering a high peak capacity (Giddings, 1967) have the ability to simultaneously resolve a large number of components and as a consequence provide much better separations than TLC. Several such systems have been used with GAS although on occasions some extraordinary but unrepeatable separations have been claimed. In general, good separations have been obtained with techniques utilizing either silica gel or dextran gel supports. Adequate results can be achieved, without recourse to expensive instrumentation. with a silica gel partition column (Powell and Tautvydas, 1967), originally developed to analyse indole-3-acetic acid ( I A A ) and other indoles (Powell, 1963). The system involves partitioning a O-lOO~:( gradient of ethyl acetate in hexane against a 400,; ( v / w )0.5 M formic acid stationary phase on a Mallinckrodt CC-4 silica gel support. Separation is primarily determined by the degree of hydroxylation : G A S with no free hydroxyl groups elute at an early point in the gradient and in turn are followed by the mono-, di- and tri-hydroxy G A S as the solvent strength increases. The elution pattern of 12 GAS is presented in Table IV. Durley rt a / . (1972) have reported that the technique works well only with certain batches of silica gel as columns tend to temporarily “dry out” at an early point in the gradient. Because of this they developed an alternative procedure using a Woelm silica gel support with a 15% water stationary phase. While this technique provides better results than TLC, it does not perform as well as the Powell and Tautvydas column. This is probably due to the mixed nature of the separatory mechan-

52

ALAN CROZIER

TABLE IV Retention characteristics of GAS on a straight phase silica gel partition column (Durley et al., 1972)" Fraction number

Gibberellin

2 4 5 6 8 11 13-14 18 "Column: 13 x 200 mm Mallinckrodt CC-4 silica gel; stationary phase: 40%. 0-5 M formic acid; mobile phase: 160 min gradient, @loo% hexane in ethyl acetate; flow rate: c. 3 ml min -' ; sample: G A S as indicated; detector: 25 successive 20 ml fractions collected and G A content determined by gas chromatography.

ism which varies during the course of a gradient from silica gel adsorption to partitioning against a stationary phase that changes from water to 0.5 M formic acid. The "drying out" phenomenon experienced with the PowellTautvydas system is, in fact, due to out-gassing of the solvents and is easily overcome by degassing the hexane and ethyl acetate under reduced pressure, immediately prior to use. Re-absorption of atmospheric oxygen by the ethyl acetate can be suppressed by entraining a stream of nitrogen over the solvent reservoir (Reeve et al., 1976). When these precautions are taken the procedures of Powell and Tautvydas (1967) are reproducible and batch-tobatch variations in Mallinckrodt silica gel are not apparent. There is in fact nothing especially magical about Mallinckrodt CC-4 silica gel. Other silica gels work equally well and in certain instances their performance is far superior. In the early 1970s advances took place in liquid chromatography technology, especially the development of efficient microparticulate silica gel supports (see Majors and MacDonald, 1973). This facilitated vast improvements in the performance of the silica gel partition system, which is especially well suited for the separation of GAS in plant extracts, as a 10 x 450 mm column can accommodate multicomponent samples weighing up to 100 mg. The high sample capacity is a direct consequence of the high (40%)stationary phase loading. The relatively high miscibility of the ethyl acetate mobile phase and formic acid stationary phase does, however, present special problems that must be overcome if high column efficiency is to be maintained. This can be achieved through the use of a stationary phase trap in the solvent delivery line and a precolumn, which, along with the analytical column, is held at 30f0-05"C to ensure equilibration of the incoming mobile phase with the stationary phase (Reeve et al., 1976; Reeve and Crozier, 1978).

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

53

Reeve et a / . (1976) assessed the performance of various types of silica gel in this system by chromatographing UV absorbing phenol under various conditions and calculating column efficiencies by established procedures (see Karger, 1971). Three silica gel supports were used : (i) Mallinckrodt CC-4- irregularly shaped particles with a wide size range (approximately 60-250 pm). The support used by Powell and Tautvydas (1967). (ii) Merckogel SI 200-spherical, I 40-63 pm particles with a 200 A pore diameter. (iii) Partisil 20- irregularly shaped 20 pm particles of closely controlled size distribution with 55-60 8, pore diameters. The relationship between plate height, H , and linear solvent velocity, v, for columns packed with these gels is presented in Fig. 5. In each instance the concentration of ethyl acetate in the mobile phase was adjusted so that phenol eluted with a capacity factor, k', of 1.5. The performance of both the Mallinckrodt CC-4 and Merckogel SI 200 falls off at increased flow rates, the effect being much more marked with CC-4. Much better H values were obtained with Partisil 20 and no significant fall off was evident at higher solvent velocities. From the practical viewpoint Partisil 20 is clearly the superior support as it can generate high efficiencies without sacrificing the speed of analysis. Subsequently, 5 and 10 pm silica gel supports have become Mallinckrodt C C - 4

1.0

-2 E E

Merckogel

SI 200

0.5-

I-

Partisil 20

o.oJ,

0

1

2

I

1

1

3

4

5

v ( m m s-') Fig. 5. The relationship of plate height ( H )to linear solvent velocity (v). Column: 10 x 450 mm packed with Mallinckrodt CC-4 Silicar (triangles), Merckogel SI 200 (circles) and Partisil 20 (squares). Sample: phenol. Stationary phase: 40%, 0.5 M formic acid. Mobile phase: hexaneethylacetate, ratioadjusted togivea k'of 1.5 forphenol. Detector:absorbancemonitorat (Reeve et al., 1976.)

54

ALAN CROZIER

commercially available and they enhance performance even further although operating pressures are higher. A 10 x 450 mm column packed with Partisil 10 and eluted at a flow rate of 5 ml min- ',which corresponds to v = 1.5 ml s- ', generates up to 3800 theoretical plates for a solute with a k' of 1.2. Thus plate height and speed of analysis can be calculated at 0.12 mm and 1.1 effective plates per second. Depending upon solvent composition, a column inlet pressure of 14C200 p.s.i. is required. Recovery from the column is 95% for a wide range of compounds. These performance figures represent a considerable improvement in both efficiency and speed of analysis, when compared with classical liquid chromatography techniques used for the separation of GAS. The system is some ten times faster and twenty times more efficient than the silica gel partition column of Powell and Tautvydas (1967) from which it was derived. The transition from a silica gel partition to a high performance system necessitates the use of more elaborate instrumentation. The preparative HPLC that is used is illustrated in Fig. 6, along with an on-stream homogeneous radioactivity monitor which is employed in GA metabolism studies to detect [jH] and [14C] solutes eluting from the column. A dual pump gradient generator delivers mixtures of hexane and ethyl acetate saturated with 0.5 M formic acid to the analytical column via a pulse dampening network, a stationary phase trap and a precolumn. Samples are dissolved in the mobile phase and introduced into the analytical column via a six-port sample valve. Solvent emerging from the column is directed to a UV monitor before entering a stream splitter which subtracts a pre-set portion of the column eluant and restores the original flow rate with a make-up solvent of ethyl acetate :toluene (1 :1). After the addition of scintillation cocktail supplied from a metering pump the mixture is cooled to - 5°C and passed through a spiral glass flow cell positioned between the photomultiplier tubes of a manual scintillation counter. The output is processed by a spectrometer/ ratemeter and displayed along with the UV-absorbance trace on a dual channel recorder. The inclusion of a radioactivity monitor in the system requires a suitable compromise be made between chromatographic resolution and speed of analysis and detector sensitivity (Reeve and Crozier, 1977).This is achieved by selecting an appropriate scintillant :eluant ratio, matching the flow cell volume and geometry with the minimum chromatographic peak width and adjusting the overall flow rate to give an optimum value for flow cell transit time. When these parameters are optimized the monitor has a relative sensitivity of 3 x lo3 d.p.m. for and 1 x lo3 d.p.m. for I4C for a solute eluting with a k' of 1.7. The monitor does not contribute to the total bandspreading of the chromatograph for solutes where k' > 1.7. By manipulation of the hexane-ethyl acetate ratio a wide range of mobile phase solvent strengths can be used to provide rapid and effective separations. Figure 7 illustrates the use of a gradient designed for the analysis of samples

+ Stationary

i

Pulse dampener

Gradient generator and pumps

Splitter control unit Constant temperature circulotor (30°C)

73$

5

-

1

Make-up solvent reservoir

V 0

0

0

-

'u

Pump

c

a

0

) .

a

I

I

Solvent reservoirs

Sample volve

I I L - -- - - - -

Two-pen recorder

I I I

Hold-up coil

€3

Scintiliont reservoir

pump

Splitter r

UV cell

p P

Constant temperature

Q

Collect

Flow cell in scintillation Counter

UV monitor

-

.--U Spectrometer / ratemeter

____

Fig. 6. Preparative HPLC with UV and radioactivity monitors. (Reeve and Crozier, 1978.)

56

ALAN CROZIER E 0

h

r

0

~

1 1-

1

60

120

Retention time ( min)

Fig. 7. Preparative HPLC of radioactive GAS and GA precursors with UV-absorbing internal markers. Column: 10 x 450 mm Partisil 20. Stationary phase: 40%. 0.5 M formic acid. Mobile phase: 2 h gradient O-lOO% ethyl acetate in hexane. Flow rate: 5 ml min-'; sample: c. 24,000 d.p.m. ["C] ent-kaurene, 50,000 d.p.m. [14C]GA3, [3H]GA5, ['4C]GA12. [14C]GA15 and [3H]GAZo;100,000 d.p.m. [3H]enr-kaurenoic acid, [3H]GA1, [3H]GA4, [3H]GAs, ['H]GA9, [3H]GA1zaldehydeand t3H]GA14, and uncalibrated amounts of gibberic acid, allogibberic acid and gibberellenic acid. Detectors : radioactivity monitor I800 c.p.m. full-scale deflection, absorbance monitor at AZs4(Reeve et a / . , 1976).

whose components span a wide range of polarities. The GAS and GA precursors separate according to the degree of hydroxylation. Compounds with no hydroxyl groups such as ent-kaurene, ent-kaurenoic acid, GA, aldehyde, GA9, GA15and GA12elute first, followed by the monohydroxylated GAS (GAL,GAI4,GAS and GA,,), the dihydroxylated compounds GAl and GA3,and finally GA, which has three hydroxyl groups. When wide-range gradients of this type are employed UV-absorbing markers such as gibberic, allogibberic and gibberellic acid can be incorporated into the sample to allow precise determination of the relative retentions of radioactive peaks. In metabolic studies, metabolism of the applied G A often involves successive hydroxylations and the products are usually chromatographically distinct from each other and from the precursor molecule. In such cases a considerable saving can be made in the analysis time because good separations are obtainable without the need for high effective k' values. This is shown in

,

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

57

3'0

0 Retention time (rnin)

Fig. 8. Preparative HPLC of G A , , GA4 and GABusing a restricted solvent gradient designed for rapid analysis. Column: 10 x 450 mm Partisil 20. Stationary phase, 40%, 0.5 M formic acid. Mobile phase: 20 min gradient 80-100% ethyl acetate in hexane. Flow rate: 5 ml min-'; sample: c. 20,000 d.p m. [3H]GA,, ['HIGA, and [3H]GA8. Detector: radioactivity monitor, 600 c.p.m. full-scale deflection. (Reeve er at., 1976.)

Fig. 8, where the solvent programme has been adjusted to allow the repeated separation of GA4, GAI and GA8 at 30-min intervals. Silica gel supports with a chemically bonded stationary phase are now available (see Locke, 1973). Columns packed with this type of support are extremely stable and present fewer problems to the inexperienced chromatographer than the preparative HPLC system designed by Reeve et al. (1976). However, the stationary phase content of these supports is rarely more than 15% and is usually only 5% (Majors, 1975; Cooke and Olsen, 1979). As a consequence, the sample capacity is very limited when compared with a silica gel support carrying a 40% stationary phase loading. Reverse phase chromatography of free GAS using a chemically bonded octadecylsilane (ODS or C18) stationary phase has been reported by Jones et al. (1980). Four 6.5 x 600 mm columns connected in series, packed with Bondapak C18 (Waters Associates) and referred to as a preparative system, were eluted with a 25-min, 3&100% gradient of methanol in 1% aqueous acetic acid at a flow rate of 9.9 ml min-'. Thirty fractions were collected and the location of GA standards determined by gas chromatography (GC). The separations obtained are presented in Table V. An analytical system using a single 4 x 300 mm p-Bondapak c18 column (Waters Associates) gave similar results. The procedures were used to separate endogenous G A Sin an extract from immature seed of Pharbitis nil. Zones of biological activity from the

58

ALAN CROZIER

TABLE V Reverse phase chrornatogruphy of GAS (Jones et al., 1980)" Fraction number

Gibberellin

11 12 13 15 17 18 19 21 22 23 24 28 "Column: four 6 . 5 ~ 6 0 0mm Bondapak C,,/Porasil B, columns in series; mobile phase: 25 min gradient. 30-10070 methanol in 12, aqueous acetic acid; flow rate: 9.9 ml m i n - ' ; sample: GAS as indicated; detector: 30 successive 9.9 ml fractions collected and GA content determined by gas chromatography.

preparative column were rechromatographed on the analytical column, fractions from which were bioassayed and the active components subsequently identified as GA3, GAS,GA,,, GAI9, GA20, GA29and GA44 by GC-MS. Despite the successful identification of these GAS it would be unfortunate if other investigators were to use reverse phase chromatography procedures in the manner described by Jones et al. (1980). Although figures for column efficiency are not given, from the data presented, it would seem that the preparative columns, despite their length, generate fewer than 400 theoretical plates ( H = 6 mm), while N = 1600 and H=0.19 mm for the analytical column. A system comprising either a single Whatman Magnum 9 x 500 mm ODs-2 or Shandon 8 x 250 mm ODs-Hypersil column would be much cheaper yet would provide a higher sample capacity and far superior efficiency and peak capacity than the combined efforts of the five columns used by Jones et a l . (1980). In order to fully utilize the separatory capacity of such a system at least 150 fractions must be collected for analysis by bioassay. Jones et al. (1980) collected only 30 fractions and therefore the effective resolution of their reverse phase systems is, in practice, no better than that of a silica gel-formic acid partition column (Powell and Tautvydas, 1967) which requires neither an elaborate solvent programmer nor expensive pulse-free pumps. High column efficiencies and good G A separations have been achieved with dextran gels as a stationary phase support. The procedures of Pitel et a/.

59

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

(1971a) and Vining (1971) using Sephadex G-25, separate the double bond isomers G A , / G A 3 and G A 4 / G A 7 and some of their closely related derivatives. These columns are, however, of restricted general value as they do not provide adequate resolution of other groups of G A S(Durley rt ul., 1972). In contrast methods devised by MacMillan and Wels (1974) do separate a wide range of GAS and G A precursors. A biphasic solvent of light petroleumethyl acetate-acetic acid-methanol-water (100 :80 :5 :40 :7) was prepared and the aqueous phase used to swell the Sephadex LH-20 support and act as a stationary phase. The gel was packed into a column and eluted with the organic phase. Five thousand five hundred theoretical plates were generated on a 15 x 1450 mm column and excellent G A separations were obtained (Fig. 9). The sample capacity of this column is 100-200 mg so most plant extracts can be easily accommodated. The method has the advantage that it is relatively simple and does not require expensive, complex equipment. However, the 30-h analysis period is a major problem as far as routine analyses are concerned, as it severely limits sample throughput. The speed of analysis, calculated from GA3 in Fig. 9, is only 0.05 effective plates per second. Because of a lack of gel rigidity, it is unlikely that this situation could be improved by either increased solvent velocities or solvent programming (see Bombaugh, 1971). Despite this drawback the procedure is an attractive proposition for occasional use.

0)

C

P

?

D

LL

10

20

30

40

50

60

70 80 90 Fraction number

100

110

120

130

140

150

Fig. 9. Separation of GAs by liquid-liquid partition chromatography on a Sephadex LH-20 support. Column: 15 x 1450 mm Sephadex LH-20. Stationary phase: aqueous phase of light petroleum%thyI acetate-acetic acid-methanol-water (100:80:5:40:7) mixture. Mobile phase: organic phase of above. Flow rate: 50 ml h -l. Sample: ( I ) mi-kaurene, (2) ent-kaurenoic acid, (3) GAI2aldehyde,( 4 ) GA,,alcohol, ( 5 ) ent-7a-hydroxykaurenoic acid, GA9, (6) steviol, G A I 2 , (7) GA15, (8) GA~~aldehyde, (9) GA24, (10) GA4, (1 I ) GAT,(12) GA25. (13) GAT,GA3,. (14) GAI4,(15) GAS,( 16) mevalonic acid, (17) G A 3 6 . (1 8) C A I 6 , (19) GA2,(20) C A I ,G A l 3 , GA, ,, (21) GA3 and (22) CA,, GAZ8.Detector: 150-ml fractions collected and analysed by GC with a flame ionization detector (MacMillan and Wels, 1973).

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

61

Fig. 10. The response of (a) the Tanginbozu dwarf rice leaf sheath and (b) the cucumber hypocotyl bioassays to GA3.Controls on the left, plant on the right treated with 1 pg GA3. (c) The response of the lettuce hypocotyl bioassay to GA,. Controls on the left, seedlings on the right grown in 1 pg GA, ml-'.

Whatever method of chromatography is used to separate endogenous GAS,their subsequent detection can be a time-consuming process because of the lack of a specific label. [3H]and [14C]GAsare of course an exception as they can be readily detected with a radioactivity monitor. If a known GA is being analysed the appropriate zone of the chromatogram can be collected and subjected to additional analysis to facilitate identification and quantification. When the identity of the endogenous GAS are unknown, bioassays are commonly used to detect peaks of GA-like activity which are then subjected to physicochemical procedures such as GC-MS in order to characterize the active components. Very often, however, endogenous GASare not identified in this manner and analyses go no further than bioassay with the GA content of samples being compared on this basis.

62

ALAN CROZIER

E. IDENTIFICATION PROCEDURES

I . Bioussays und Rudioinznzunoussuys Historically bioassays have played an important role in the discovery of GAS. Many GAS were originally detected in plant extracts because of their biological activity, and without such an indicator it is unlikely that the extensive purification that must precede rigorous chemical analysis could have been achieved. Indeed it is interesting to speculate how much would currently be known about GAS if rice seedlings did not elongate so markedly when infected with G . fujikuroi. Over the years numerous bioassays have been devised. Bailiss and Hill (1971) listed 33 test systems based on processes such as coleoptile, leaf sheath, epicotyl, mesocotyl and radicle growth, bud dormancy and seed germination, a-amylase synthesis, leaf expansion and senescence and flower and cone induction. GA-induced elongation of the leaf sheath of Tanginbozu dwarf rice and hypocotyls of lettuce and cucumber seedlings is illustrated in Fig. 10. Typical dose-response curves for the barley aleurone, Tanginbozu dwarf rice microdrop, lettuce, cucumber and dwarf pea bioassays are shown in Fig. 11, while the essential features of these and other widely used GA assays are reviewed in Table VI. The relative activities of the individual GAS in some of these test systems are listed in Table VII. The data are compiled from Crozier et uI. (1970), Yokota ef a / . (1971), Fukui et u / . (1972), Yamane et a / . (1973), Reeve and Crozier (1975), Hoad et a / . (1976) and Sponsel et a / . (1977). When used to detect GAS in plant extracts, bioassays are moderately selective, especially when compared with many physicochemical detectors. However no known GA bioassay is entirely free from interaction with extract impurities. Thus, regardless of the repeatability of bioassay data, the accuracy is always open to question until such time as verification is achieved by reference to a more definitive technique. The following example involves the estimation of GA levels in Phuseolus cocciiwus seedlings and illustrates the problems that severely restrict the interpretation of bioassay data. The acidic, ethyl acetate-soluble fraction obtained from a methanolic extract of light grown Phuseolus seedlings was partially purified by Sephadex G- 10and charcoal-celite column chromatography and then divided into two. One portion was subjected to TLC and the other to liquid chromatography (LC) on a silica gel partition column (Powell and Tautvydas, 1967). When a 1/60 aliquot of each chromatographic fraction was tested in the Tanginbozu dwarf rice bioassay the LC fractions induced a greater overall response and revealed more zones of biological activity than did the TLC fractions (Fig. 12). Seemingly the higher peak capacity of LC resulted in a better separation of the GAS from one another as well as from impurities. Support for this view was obtained when a 1/120 aliquot of each chromatographicfraction was assayed. The LC fractions showed the expected

u n

I”

20

Cucumber bioassay 10

o

lo+

10-l

loo

,-.

E

E I

f av) l r

n

c 0

W

100

_I

50

o

m3

lo-’

loo

I

E E

o

10-l

pg GA3 ml-‘

o

I O - ~ 10-1

loo

pg G A T mL-’

Fig. 1 1 . Dose-response curves of the cucumber hypocotyl, Tanginbozu dwarf rice leaf sheath. Progress No. 9 dwarf pea epicotyl. barley aleurone and lettuce hypocotyl bioassays.

TABLE VI Gibberellin bioussuys Method Tanginbozu dwarf rice leaf sheath bioassay Progress No. 9 dwarf pea epicotyl bioassay Dwarf maize leaf sheath bioassay Cucumber hypocotyl bioassay Lettuce hypocotyl bioassay Barley aleurone bioassay Rumex leaf senescence bioassay

Reference Murakami (1968) Kohler and Lang (1963) Phinney (1956) Brian, Hemming and Lowe (1964) Frankland and Wareing (1960) Nichols and Paleg (1963) Jones and Varner (1967) Whyte and Luckwill (1966)

"Test compound GA4.hTest compound GA,.

Minimum detectable level of GA,

Range of linear response to GA,

TABLE VII Relative activities ofgibberellins injive bioassay systems Gibberellin

Barley aleurone

Dwarf Pea

Lettuce hypocotyl

Dwarf rice

++++ ++++ ++++ +++ ++

+++ ++ ++++ +++ +++ ++ +++ + ++

+++ ++ +++ ++ ++ ++ ++++ + +++

+++ +++ ++++ ++ +++ +++ +++

++ +++ + + + + 0 + 0 0

+

0 0 0

+ 0 +++ ++

0 0 0 0 0

+ +++ + +++ + 0 +++ ++ ++ + -

Relative activities: inactive.

0 0 0

0 0

+ ++ 0 ++

0

+ +++ ++ +

0

0 0

+

0 0

+++ ++ +++ + + ++ ++ ++ +++

0 0 0

++

0

0 0

0

+ ++ + 0 + + +++ + ++ +

0

0 0 0

0 0 0

+ ++ + 0 + + ++ 0 + 0

+ ++ +++ + + + + ++ + + +++ +++ +++ 0 +++ +++ +++

0 0

+ + + ++ ++ ++++ + +

++ +++ +++ +

+

Cucumber hypocotyl

++

++ ++ +++ + + ++++ 0 +++ ++ + +

0 0

++ +

0 0 0 0 0 0 0

+++

+

0

0 0 0

+ +++

0

0 0

+++ +++ +++ + ++

0

0 0

0 0

0

0

0

0

+

+

++

+ + + +, very high; + + + , high; + +, moderate; +, low; 0, very low to

66

ALAN CROZIER

TLC

LC

G A S standards

+10-3/1g

26

r

301

(b’

t

18 14

pg

+0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1

5

10

15

Fraction number

20

I I T 1

25 Rf

Fig. 12. Tanginbozu dwarf rice bioassays of eluates from a silica gel partition column (LC) and a silica gel G thin layer chromatogram (TLC) developed with ethyl acetate-chloroformformic acid (50: 50 : 1) of a semi-purified extract from red light-grown seedlings of Phaseolus coccineus cv. Prizewinner. Eluates were tested at (a) 60- and (b) 120-fold dilutions.

reduction in biological activity, whereas the TLC fractions actually displayed enhanced activity at the lower dose (Fig. 12). Such anomalous dose-response behaviour clearly indicates that the selectivity of the bioassay is insufficient to cope with the level of interfering substances. Even if it were possible to establish that the growth promotion in Fig. 12 was exclusively due to the action of G A S ,it would still be difficult to obtain a meaningful measure of the actual amount of G A present. To express the data as pg of G A 3 equivalents is misleading because the relative activities of individual G A Svary greatly (Table VII). The threshold doses differ by several orders of magnitude; there is often no parallelism between the slopes of the response curves, and, as a further complication, the size of the dose required to saturate the response is far from uniform (Reeve and Crozier, 1974). These factors must exclude the use of a biological response as the basis for the quantification of an unknown G A . Quantitative estimates of G A levels based on biological activity may however be of more value if they can be related to a specific G A . For instance, in the case of the Phaseolus extract,

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

67

illustrated in Fig. 12, it has been established by GC-MS that the material contains GAl which elutes from the LC column in fraction 14 (Bowen et al., 1973). It can therefore be argued that there are grounds for expressing the biological activity in fraction 14 as ng of GA1 by reference to the regression of the response on log-dose GA1. As this involves a log-normal distribution the estimate will be a median rather than a mean value and will have asymmetric confidence limits. Table VIII contains estimates of the GA1 content of the Pkaseolus extract based on the growth response induced by 1/60 and 1/120 aliquots of LC fraction 14 in the dwarf rice bioassay. The accuracy of estimates is dependent upon the validity of the assumption that the doseresponse curve of fraction 14 exactly mirrors that of the GA1 dose-response curve. As halving the dose size had no significant effect on the GA1 estimates it would seem that the assumption is at least partially valid and that LC fraction 14 is acceptably pure. However, interference from extraneous material need not necessarily be revealed by assaying at more than one dose level. It was therefore of interest to analyse the extract in other bioassays which offered different selectivities. When LC fraction 14 was tested in the lettuce hypocotyl and barley aleurone a-amylase bioassays, the GA1 estimates obtained were much higher than those based on the dwarf rice bioassay (Table VIII). Without further investigation it is impossible to establish which figure is the more accurate, so under the circumstances the best estimate of the GAl content of the Phuseolus extract is 4-1400 ng. It should be noted that at no stage has rigorous proof of accuracy been obtained and thus there is no guarantee that the actual GA1 content of the extract lies within even this broad range. The Pkaseolus analysis cited above is by no means a “worst case” example as the extract underwent a two-step purification and LC fractions were tested in three bioassays at various dilutions. In many published instances, purification is almost non-existent and estimates of GA content are based on TLC of crude, acidic ethyl acetate-soluble extracts and a single bioassay at TABLE VIII Estimated GA1 content of an extract from 60 light-grown Phaseolus coccineus seedlings Estimated G A , levels (ng) Bioassay Dwarf rice Lettuce hypocotyl Barley aleurone

’

Median value

Upper and lower 95% confidence limits

18“ 20b 600’ 700‘

5-60 448 13C-1400 3W1200

“1/60 aliquot assays; ljl20 aliquot assays; 1/6 aliquot assays.

68

ALAN CROZIER

one dilution. The relationship between estimates based on such data and the actual GA content of a sample is likely to range from minimal to nonexistent. Although immunological assays are extensively employed in the fields of mammalian endocrinology details of their application to GAS are limited to one report by Fuchs and Fuchs (1969). This should not be taken to indicate their general unsuitability in this role as the selectivity, limits of detection and simplicity of a well designed radioimmunoassay at least rival, and often exceed, those of GA bioassays. Fuchs and Fuchs (1969) showed that antibody raised against GA3 extensively cross-reacted with GA4, GA,, GA9 and to a lesser extent G A I 3 . While this lack of specificity is detrimental to radioimmunoassay of specific GAS it is a distinct advantage in developing a general assay to monitor overall GA levels. The limited ability of the GA3antibody to distinguish between individual GAS also suggests that it would be worthwhile investigating the possible use of affinity chromatography as a GA group separatory purification procedure. 2. Physicochemical Methods ( a ) Gas chromatography and combined gas chromatography-mass spectrometry. GC of the methyl esters of GAI-GA9 using a flame ionization detector (FID) was first reported by Ikegawa et al. (1963). Subsequently Cave11 et al. (1967) separated the methyl esters and trimethylsilyl ethers of the methyl esters of GA1-GAI5, GA18and GAI9 on 2% QF-1 and 2% SE-33 columns. The application of these procedures to the analysis of endogenous GAShas however not been a great success because the purity of most extracts is such that an FID, which is a non-specific mass detector, has to cope with high background levels and numerous extraneous peaks. As a consequence the great advantage of GC, its high peak capacity, is lost as there is no guarantee that the mass peaks being measured are in fact attributable to GAS.The analytical situation is greatly simplified when G C is used in metabolism studies as radioactive precursors and metabolites can be selectively monitored with a flow-through proportional gas radioactivity monitor (Simpson, 1968). In the light of more definitive analyses, identifications based on the retention times of [3H]GAmetabolites on 2% QF-l,2% SE-30 and 1% XE-60 columns have proved very reliable (see Durley and Pharis, 1973; Durley et al., 1974a; Railton et al., 1974a). The problems encountered with an FID can be overcome when a mass spectrometer is used as the G C detector, as all the GAS can then be distinguished from each other and from extract impurities on the basis of their mass spectra. Effluent from the GC column passes through a separator, which removes most of the carrier gas, and enters the ion source of the mass spectrometer where, a t c. Torr, it is ionized by bombardment with high energy electrons. This process is known as electron impact ionization. It

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

69

should be noted that instruments offering chemical ionization in a suitable reagent gas, such as methane or ammonia, are increasing in popularity although as yet they have not been widely used with GAS. Regardless of the method of ionization, a signal relative to the total ion current (TIC) is derived either by summing the ion current values using a data system or by intercepting a portion of the unresolved ion beam. The TIC gives an indication of the amount of material in the source and is analogous to a FID trace. However, the real analytical power of mass spectrometry lies in the fact that fragment ions can be resolved according to their mass to charge ratio (m/e) by means of a magnetic sector or quadrupole mass analyser to give spectra such as that illustrated in Fig. 13. The range, pattern and variety of fragments is so vast that each compound yields a characteristic spectrum. Identifications are based on the matching of spectra with those of compounds of known structure. If reference spectra are available they can be used for comparative purposes so it is unnecessary to have standards on hand. This is an important consideration as the great majority of the GAS are not readily available. GC-MS was first introduced to plant hormone analysis by Binks et al. (1969) who published reference electron impact spectra of the methyl esters and trimethylsilyl ether of the methyl esters of GA1-GAz4. One hundred nanograms or less of GA are required to obtain a mass spectrum, and provided adequate separation is achieved by GC, acceptable GA spectra can be obtained from relatively impure plant extracts. Primarily because of these attributes, GC-MS has rapidly become a technique of great importance to GA analysis and it is widely believed that mass spectral data are essential if a “conclusive, definitive or unequivocal” characterization is to be achieved. Identifications made in the absence of such data are invariably viewed with suspicion. GC-MS can also be used for selected ion current monitoring (SICM) and the technique is of particular value in the quantitative analysis of trace quantities of endogenous GAS.Instead of scanning the entire mass range the mass spectrometer determines the intensities at one or more selected m/e values that are prominent in the spectrum of the GA under investigation. In this role the mass spectrometer acts as a selective G C detector and, as it monitors only a few ions, detection limits can be as low as one picogram. However SICM does require that the identities of GASlikely to be present in a sample be known and that reference compounds are available to quantify the detector response and determine GC retention characteristics. Mass spectrometry reveals the relative isotope content of fragment ions provided the isotope content is sufficiently high. Although the level of 3H in [3H] labelled compounds is usually too low to measure, the [14C/’2C]and [’H/‘H] ratios can often be determined at the same time as a G A is identified from its mass spectrum (Bowen et al., 1972; Bearder et al., 1974). [14C] and [’HI

M+ 504

m 0

100

200

300

400

m /e

Fig. 13. Mass spectrum of the trimethylsilyl ether of the methyl ester ofGA,.

500

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

71

GAS can therefore be used as internal standards in quantitative analysis as they behave in a similar if not identical manner as their endogenous ["C/'H] counterparts yet can be distinguished from them by mass spectrometry. The ability of mass spectrometry to determine relative isotope content is also of value in metabolic studies as it provides a means of determining whether a GA mass spectrum is that of either a ["C/'H] endogenous component of indeterminate ancestry or a metabolite originating from a ['HI or ['"C] labelled precursor (see Sponsel and MacMillan, 1979). The flexibility and potential of GC-MS is greatly enhanced when it is coupled with an on-line computer (MacMillan, 1972). Copious details of the various modes in which such instrumentation can be operated, along with examples of the types of data obtained in assorted GA analysis, are contained in two practically orientated articles by Gaskin and MacMillan (1978) and Hedden (1978). ( h i High performance liquid chromatography. As commonly practised HPLC and G C are broadly equivalent in that they display similar efficiencies and speeds of analysis with the sample capacity of HPLC being about ten times that of GC. The major difference between the two techniques lies in the thermodynamics of the partitioning process. In liquid-solid and liquidliquid processes the differences in the free energies of distribution of the solutes d(dG') are usually far greater than for gas-solid or gas-liquid systems. Thus all other factors being equal HPLC will always give a superior separation to GC. In addition d(dG') is much more dependent upon the properties of the mobile and stationary phase in HPLC than it is in GC, thus HPLC is able to offer a much wider variety of column selectivities. HPLC has been applied to numerous diverse analytical problems (see Pryde and Gilbert, 1979). High column efficiencies and peak capacities, rapid speeds of analysis, the availability of many supports each offering markedly different separatory mechanisms, operation at ambient temperatures and ease of sample recovery all contribute to the overall effectiveness of the technique. It should however be noted that the high efficiencies ( > 40,000 theoretical plates m-') are achieved on columns with a 2-5 mm bore. The sample capacity of such columns rarely exceeds 500 pg. Thus, as far as the analysis of endogenous GASare concerned, the potential of HPLC, like that of GC-MS, can only be fully exploited when applied to extracts of relatively high purity. The application of HPLC to G A analysis is still in its infancy and the first problem confronting potential users of the technique is detection of the GAS. Although refractive index and far-UV monitors are often referred to as universal detectors they are not as useful as implied by the manufacturers' advertising literature which almost invariably fails to point out that they function in only a very restricted range of solvent conditions. Faced with this situation one answer is to use bioassays to detect G A S in HPLC eluates although this is not a particularly satisfactory solution, as it is time consuming

72

ALAN CROZIER

and much of the practicality of HPLC is lost. An alternative approach taken by Reeve and Crozier (1978) is to convert GAS to derivatives which absorb in an accessible region of the UV spectrum. GA benzyl esters (GABEs), synthesized by esterification with N ,N’ dimethylformamide dibenzylacetal, have a A,,,, of 256 nm and can be readily detected in a wide range of solvents with a standard UV monitor operating at 254 nm. The GABEs can be analysed on a silica gel adsorption column which generates up to 8000 theoretical plates (H=0.06 mm) and provides good separations of isomers because of its ability to distinguish subtle differences in the spatial relationships of the polar groupings of structurally similar molecules. An added advantage is that the selectivity of the silica gel can be substantially altered simply by changing the reagent used to modify the mobile phase. This point is illustrated in Fig. 14, which shows the separation of isomeric GABEs in hexane-dichloromethane based solvents modified with dimethylsulphoxide (DMSO) and THF. In the DMSO system the elution order is GA,,BE>GA,BE> GA,BE>GA,BE and GA,BE>GA,BE. The 13a-hydroxy GABEs (GA20BEand GA,BE) elute before their 3b-hydroxy equivalents (GA4BE and GA,BE) while the A ’ . and d 2 %isomers (GA3BE, GA7BE and GA,BE)

’

Mobile phase Hexane-dichloromethane - DMSO

Mobile phase Hexane-dichloromethane - DMSO (25 75 1)

GA.BE

0

8 12 16 20 Retention time (min )

4

Retention time (min)

Mobile Dhase Dichloromethane-THF

Mobile phase Dichlorornethane -THF

(97 31,

(92 81

I

0

. 4

.

8

I

.

12

16

Retention time (min)

20

24

0

. 4

. 8

. 12

.

16 20 Retention time (min)

24

.

. 24

Fig. 14. The influence of DMSO and T H F modifiers o n the HPLC retention characteristics of GABEs. Column : 4.6 x 500 m m Partisil 10. Mobile phase: as indicated on figure. Flow rate: 1.61111 m i n - ’ . Sample: GA,BE, GA,BE, GA,BE and GA,,BE or G A , B E and GASBE. Detector: absorbance monitor at AZS4.(Reeve and Crozier, 1978.)

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

73

are more retained than their saturated analogs ( G A I B E , G A 4 B E and GA2,BE). When T H F is substituted for DMSO there IS a complete reversal of the elution order with respect to both the location of the hydroxyl group and the degree of unsaturation. Such marked changes in column selectivity demonstrate the flexibility of silica gel adsorption HPLC and can be of value in the purification and isolation of G A S . A comprehensive discussion of silica gel adsorption HPLC of G A B E s has been published by Reeve and Crozier (1978). The procedures have been used in conjunction with an onstream HPLC radioactivity monitor and direct probe mass spectrometry to purify and identify [3H]GA metabolites from PhLisrolus c*occineusseedlings (Crozier and Reeve, 1977; Reeve and Crozier, 1978; Nash rt al., 1982) and lettuce hypocotyl sections (Nash " t al., 1978). Although they have proved useful in metabolism studies it should be noted that the c,,~,, of mono GABEs is 205 1 mol-'cm-' and the limit of detection at A254 is only c. 300 ng. This lack of sensitivity represents a serious constraint when it comes to fully utilizing the high resolving power of HPLC to analyse trace quantities of endogenous G A S . Other G A derivatives do however offer much greater potential in this regard. Heftmann r t u l . (1978) prepared p-nitrobenzyl GA esters (h,,, = 265 nm, c,, > 6000) using 0 pnitrobenzyl-N,N'-diisopropylisourea (Knapp and Krueger, 1975). Unfortunately, when the esters were chromatographed on a preparative silver nitrate impregnated silica gel column, the performance was very poor ( N = 1500, H = 3.25 mm) and peaks up to 200 ml in volume were obtained. As a consequence, the limit of detection achieved at A265 was 100 ng rather than the 10 ng that might have been expected if conventional HPLC procedures had been used. Morris and Zaerr (1978) used 18-Crown-6, according to the procedures of Durst et d.(1975), to catalyse the synthesis of pbromophenacyl G A esters (;.,,l,x=256 nm, F,,,,= 19,100).The p-biomophenacyl esters of G A 3 , G A 4 , G A , , GA,, G A 9 and G A I 3 were analysed on HPLC systems utilizing silica gel supports with a bonded C, or cyanopropyl stationary phase. The separations obtained with the reverse phase C, column are illustrated in Fig. 15. The limit of detection for nzoizo G A esters at was, as anticipated from their c,,,,, < 5 ng. Recently a marked increase in sensitivity has been obtained by using GA methoxycoumaryl esters (GACEs) for HPLC (Crozier et d., 1982). These derivatives are synthesized from 4-bromomethyl-7-methoxycoumarinin a Crown ether catalysed reaction (Fig. 16) that was originally used by Diinges (1977) to produce methoxycoumaryl esters of fatty acids. The GACEs are nm) (Fig. 17) and can be strongly fluorescent (A=;'= 320 nm, A5!?=400 detected at the low picogram level with a spectrophotofluorimeter after reverse phase HPLC. This is shown in Fig. 18 in which a log-log plot of relative response against sample size gives a line with a slope of 1 .O, indicating a linear response extending over almost four orders of magnitude. The limit

G

3

A254

c

0

5

15

10

Retention time ( rnin )

Fig. 15. Reverse phase HPLC of p-bromophenacyl GA esters. Column: 4 x 300 mm bBondapak,'CI8. Mobile phase: 15 min gradient. 5C-100; ethanol in 20 mmol pH 3.5 ammonium acetate buffer. Flow rate: 2 mi min - I . Sample: 1)-bromophenacyl esters of GA,, GA,, GAS, GAT.GA, and GAI3. Detector: absorbance monitor at (Morris and Zaerr. 1978.)

CH2Br 4 - bromornethyl-7- rnethoxycoumarin ( BMC 1

0 I1

R-C-OH

BMC

18- C r o w n - 6

Crystal K,CO, acetonitrile 60' for 2 h

R-C-O-CH2

Fig. 16. Crown ether catalysed synthesis of methoxycoumaryl esters.

Wavelength ( n m ) Fig. 17. Fluorescence spectra of GA,CE.

4

I

/i Fluorescence Excitation 320 nm

aJ

Emission 4 0 0 n m

'3x bockground noise 0

I

I

I

1

1 P9

l0pg

1oopg

1ng

Gibberellin A 3 Fig. 18. HPLC analysis of GA3CE. Column: 5 x 250 mm ODs-Hypersil. Sample: GA,CE, dose as indicated. Mobile phase: 45% ethanol in 20 mmol pH 3.5 ammonium acetate buffer (GA,CE k' = 2.3). Flow rate: 1 ml min -' , Detector: Perkin-Elmer 650-1OLC spectrophotoffuorimeter, excitation 320 nm, emission 400 nm, 10 nm slits.

76

ALAN CROZIER

of detection for niono esters is c. 1 pg as determined by the point at which the curve intersects the ordinate equivalent to three times the level of background noise. Good recoveries (>90:/,) and efficiencies ( N = 10,000, H=0.025 mm) were obtained when the GACEs were chromatographed on an ODSHypersil column. This HPLC system has the ability to distinguish between closely related GAS as the double bond isomers GA1/GA3,GA4/GA7 and GA5/GAzo,all separated with baseline resolution. It is also of interest to note the effect of solvents on column selectivity. When a methanol-buffer gradient was used GAI3CE and GA14CE co-chromatographed as did GA9CE and GA36CE (Fig. 19a). However the compounds are well resolved when ethanol is substituted for methanol (Fig. 19b). In general increasing the number of hydroxyl groups decreases retentions, 13a-hydroxylation to a greater extent than 3&hydroxylation which, in turn, is more effective than hydroxylation at either the la- or 2&positions. Similarly, A ' . 2 and GAS elute earlier than their saturated analogs. Methoxycoumaryl functions increase V R as the elution order is mono > bis> tris esters. The GACEs have been investigated by direct probe mass spectrometry. Electron impact and chemical ionization positive ion spectra were of no value as in all instances the dominant fragment was rn/e 191 with no other ions of significant intensity being present. However, chemical ionization negative ion spectra proved to be more diagnostic (Table IX). A strong molecular ion was GAS,GA3CE and GA,CE. M-189, arising from the obtained with the d loss of the ester moiety, was the main fragment in the spectra of the other C19-GACEs. Some C19-GAs (i.e. GA4CE and GA2,CE) have identical spectra but they can be readily distinguished on the basis of their HPLC retention characteristics (Fig. 19). Three CZO-GAswere analysed and M-189 was the strongest ion produced by GAI4CEwhile M-395 was the base peak in the spectra of both GA13CEand GAS6CE. As far as GAS are concerned HPLC and GC are mutually incompatible techniques. While G C derivatives such as GA methyl esters can be chromatographed on an HPLC column they are not readily detected with conventional on-line HPLC monitoring systems. Conversely GACEs and other derivatives that are suitable for HPLC are far from ideal candidates for GC as they lack the necessary volatility. This means that it is difficult to obtain mass spectra of GACEs by GC-MS. There are three ways around this problem. The first, and the one employed in obtaining the mass spectra presented in Table IX, is to use direct probe mass spectrometry. This practice has limitations with plant extracts as relatively large samples of high purity are required if acceptable spectra are to be obtained. The second approach would be to develop an effective transesterification process to convert fluorescent or UV-absorbing GA esters to a methyl ester that could be silylated and analysed by GC-MS. One potential problem with this procedure is that GA derivatives amenable to transesterification may well be somewhat unstable

',

GA13 GA14

5 16 GA9 GA36

G

GA25

i

L I

0

1

5

I

10

1

1

15

20

I

I

25

30

I

35

GA13

I

0

I

5

10

,

15

r

I

I

I

20

25

30

35

Retention time (min)

Fig. 19. Reverse phase HPLC of GACEs. Column : 5 x 250 mm ODS-Hypersil. Mobile phase: 30 min gradient (a) 6&100% methanol in 20 mmol, pH 3.5 ammonium acetate buffer, (b) 4&80% ethanol in 20 mmol, pH 3.5 ammonium acetate buffer. Flow rate: 1 ml min - I . Sample: methoxycoumaryl esters of G A 1 , G A 3 , C A I , G A S , GA,, G A a , GA9, GA13, GAL,, GA16, G A Z o ,G A Z 5 ,GA36, c. 9 ng mono, 4.5 ng bis and 3.0 ng tris esters. Detector: Perkin-Elmer 650-1OLC spectrophotofluorimeter, excitation 320 nm, emission 40 nm, 10 nm slits.

78

ALAN CROZIER

TABLE IX Methane chemical ionization negative ion mass spectra of gibbrrellin methoxycoumaryl esters Compound

Mol. wt.

GA,CE GA,CE

536 534

GA,CE GAJE GA,CE

520 518 518

GAsCE GAgCE GA 13CE

552 504 942

GA14CE GA 16CE GAZoCE GA36CE

724 536 520 738

347-100% (M-189) 534100% (M-), 34543% (M-189), 301-7% (M-233), 2837% (M-251) 331-100% (M-189) 329-100% (M-189) 518-100% (M-), 329-34% (M-189). 285-5% (M-233), 26715% (M-2-51), 2655% (M-253) 363-100% (M-189) 315-100% (M-189) 565-8% (M-377 [-189-1881), 5477100% (M-395 [-189-2061), 359-54% (M-583 [-189-188-206 and/or -189-206188]), 3155% (M-627), 3166% (M-628) 535-100% (M-189), 347718% (M-377 [-189-1881) 347-100% (M-189), 329-6% (M-207), 303-9% (M-233) 331-100% (M-189) 549-10% (M-189,343-100% (M-395 [-I89-206])

and some degree of breakdown could occur during HPLC. The long-term solution is the use of an HPLC directly coupled to a mass spectrometer. Two interfaces are currently being marketed although the technology is still in its infancy and performance, convenience and reliability have yet to be proven (see McFadden et al., 1977; McFadden, 1979; Karger et al., 1979; Arpino and Guiochon, 1979). When it becomes a practical proposition HPLC-MS will greatly increase the flexibility of HPLC. It will be possible to obtain spectra with smaller sized samples and, equally important, as far as GACEs and the analysis of endogenous GA is concerned, is that components eluting from HPLC columns could be analysed by SICM. The HPLC-fluorescence procedures can be used for GA analysis when the identity of individual GA(s) likely to be present in the sample is known or suspected and when reference compounds are available to determine HPLC retention characteristics and quantify the response of the fluorimeter. In view of the low picogram limits of detection of the GACEs it is evident that the amount of plant material that must be extracted can be reduced to gram quantities. This will make it possible to experiment with small tissues such as root caps and apical buds and individual plant parts that are either difficult to obtain and/or contain only small quantities of GA. While HPLC data may be compiled in these circumstances the amount of GA present will rarely be sufficient to enable a full scan mass spectrum to be obtained. This raises a question of great importance, namely, is it possible to identify a G A

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

79

solely on the basis of chromatographic retention indices? Although to date only about 20 of the 62 known GAShave been subjected to HPLC it is quite apparent that the separatory power of the technique is more than adequate to distinguish all known GAS. Contrary to popular belief this is not the problem. The real nub of contention, when dealing with trace components from natural sources is, do the chromatographic procedures employed have the capacity to separate the GA under study from all other components potentially present in the sample to which the detector will respond? Furthermore, and equally important, how can this capacity be demonstrated so that the accuracy of the analysis can be verified? F. VERIFICATION OF ACCURACY

Much confusion and dogma surround the entire process of verification of accuracy. Although it is widely accepted that only mass spectrometric evidence is acceptable this approach is not without its problems even in apparently favourable circumstances. The points of contention can be illustrated by a hypothetical conversation between a logician, (L), playing devil’s advocate, and a plant physiologist (PP) who has methylated and trimethylsilylated a purified plant extract prior to analysis by GC-MS, and on the basis of SICM at m/e 506 and a full scan mass spectrum has concluded that his sample contains 1 p g of GA1.Most of us will have some sympathy for the plant physiologist and consider that he has more than enough evidence to verify the accuracy of his analysis and that his data would certainly be published by even the most critical of journals. Nevertheless the points raised by the logician do demonstrate that the plant physiologist’s conclusions are based on very subjective criteria. The discussion runs as follows : PP “I estimate that the extract contains 1 pg of C A I.” L “You surely can’t mean 1~oooOOOO. . . . pg of GA,? There must be some uncertainty in the estimate.” PP “Of course, by analysing the sample five times I calculated that the 95% confidence limits are 0.1 pg.” L “Yes, that estimates the random error associated with the measurement process but is it the only source of uncertainty? Can you rule out the possibility that, say, 0.1% of the quantified response was due to compounds other than GAI?“ PP “No, I can’t be absolutely certain.” L “Perhaps then as much as 1% of the response was due to impurities.” PP “That is possible.” L “Then why not lo%, 50% or even loo%?’’ PP “Oh no. I don’t think that is at all likely.” L “Why not?” PP “Because the SICM data I used to quantify the GA, content were obtained by monitoring at m/e 506 which is the molecular ion of the trimethylsilyl ether of GA, methyl ester. This is a very selective procedure.”

80

ALAN CROZIER

L PP

L PP

L PP L

PP L PP L

“Do you mean to say that at m/e 506 the mass spectrometer responds only to the molecular ion of the G A , derivative? Surely fragment ions from other compounds at or near this nominal mass would also evoke a response.” “Yes but it seems improbable that an impurity giving rise to such a fragment would have the same GC retention time as the trimethylsilyl ether of G A , methyl ester.” “Perhaps, but how improbable is improbable? You must quantify that statement before accuracy can be defined.” “You forget that I obtained a full scan mass spectrum of the SICM peak that was used to determine the amount of G A L present in the sample.” “In that case you are transferring the source of the uncertainty of the estimate to the full scan mass spectrum.” “But this enables me to be far more certain of the accuracy of my analysis, as chemists tell me that mass spectra provide unique fingerprints of organic compounds.” “It now seems that verification of accuracy hangs on the word ‘unique’. This implies that mass spectra can distingukh between an infinite number of compounds.” “Of course not, that is impossible; but it is well known that the discriminating power of mass spectrometry is very high indeed and far exceeds that of other procedures.” “I agree, but just how high is very high indeed? It must be able to distinguish more compounds than the number that are present in your sample.” “No problem. It can certainly do that. The purity of the sample was more than adequate. It was extensively purified prior to GC-MS and was very clean indeed.” “The uncertainty in accuracy we were originally discussing has now manifested itself in the uncertainty associated with the purity of the extract. Thus you are no further forward as you must quantify this new uncertainty by deriving the numerical probability associated with your statement ‘The purity of the sample was more than adequate’. Until this is achieved the accuracy of your estimate will remain undefined and in doubt.”

At the start of the discussion between the plant physiologist and the logician the uncertainties associated with the analysis of GA, centred around whether SICM was sufficiently selective for the problem in hand. When the full scan mass spectrum was used as a basis for accuracy the uncertainty factor changed and became associated with the discriminatory power of a mass spectrometer relative to the complexity or purity of the sample. Reeve and Crozier (1980) have suggested a simple practical test, called a “Successive Approximation”, for detecting situations in which the selectivity of an analysis is inadequate. It relies on the fact that, as the purity of a sample is increased, estimates of GA concentration must show an improvement in accuracy, since even a totally non-selective method will provide accurate results with a perfectly pure sample. The successive approximation works in the following manner. When given a sample purporting to contain a given quantity of GA, ( E , ) the test for accuracy simply consists of purifying the sample by a factor of at least two and re-estimating the GA, content (E2).If

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

81

El is accurate, E 2 , taking into account the precision of the method, should not be significantly different. If a difference is found, El must be rejected as inaccurate and E2 retested by further purification and analysis. This process is continued for as long as is necessary to obtain an estimate that does not change on purification. At this point it is possible to conclude that on the basis of the available evidence, there are no grounds for believing the estimate is inaccurate. Reeve and Crozier (1980) have discussed several statistical methods that can be used to derive the probability of errors arising when making such an assumption. An alternative way to approach the verification of accuracy is through the uncertainty associated with sample purity. The nature of the problem is best grasped by viewing extracts under analysis as open-ended systems in which an infinite array of organic compounds are potentially present. For a variety of reasons only a limited number of these possible components are likely to occur in amounts that are significant in relation to the quantity of GA present. Reeve and Crozier (1980) used typical molecular weight distributions of plant extracts to draw probability limits on the number of compounds likely to be encountered. Information theory was then invoked to determine what type of mass spectrum, chromatogram or other analytical information was necessary to ensure that the discriminating power of the method was suficient to cope with the number of compounds likely to occur at a given probability level. Mass spectrometric data comprise the authoritative subjective standard that is widely used as a basis for the verification of accuracy, because of the enormous discriminating power of the technique. For instance, the mass spectrum of authentic GAzo trimethylsilyl ether methyl ester in Fig. 20a yields 436 binary digits (bits) of information according to the proposals of Reeve and Crozier (1980). This means that it can distinguish different compounds. However all this bewildering power is not transferred to the spectrum in Fig. 20b which has been used to identify GAzo in Steviu rebuudiunu extracts (Alves and Ruddat, 1979). Although Fig. 20b contains many of the features of the authentic GAzotrimethylsilyl ether methyl ester spectrum, the chances of a mistaken identification would be reduced if the sample had been purer and a more exact match had been obtained. In fact only 198 bits of information in Fig. 20b correlate with the spectrum of the standard in Fig. 20a. In accepting this less-than-perfect match the power of the technique has been reduced to such an extent that it can distinguish only one compound in lo6'. This represents an infinitesimal fraction of its potential discriminating power. Reeve and Crozier (1980) calculated that, at a probability level of 0.9, the number of different compounds potentially present in a typical plant extract is lo4' and thus c. 140 bits of information are required to ensure accuracy. The spectrum in Fig. 20b more than meets this standard so, provided the basis of the calculations is valid, it facilitates

( a ) Authentic GA2oTM Si M e

c

L

z

( b

.

Putative GA20TM Si M e

80-

6040

20 10

0

50

100

150

250

200

300

350

400

450

m/e

Fig. 20. Electron impact mass spectra of (a) authentic trimethylsilyl ether of the methyl ester of GAzoand (b) putative trimethylsilyl ether of the methyl ester of G A z ofrom a purified extract of Stevia rebaudiana shoots (Alves and Ruddat, 1979).

METABCLISM AND PHYSIOLOGY OF GIBBERELLINS

83

the accurate identification of GAZ0in S. rebuudiunu. It is evident that 140 bits of information can be furnished by something less than a full scan mass spectrum although there are limits, and if too few ions are monitored and/or too many spurious fragments are present, mass spectrometric evidence will almost certainly fail to provide the necessary verification of accuracy. When the upper limit of the molecular weight range of components in an extract is limited by SEC the potential complexity of the sample is greatly simplified. If, for instance, 90% of the mass of a fraction collected from an SEC column is comprised of compounds with a molecular weight of less than 400, only 36 bits of information are required to guarantee accuracy with probability of 0.9. SEC systems that can achieve this type of fractionation were described in Section IIC. When they are incorporated into purification procedures it becomes possible to use less powerful analytical techniques than mass spectrometry to yield accurate results provided the principles and attendant assumptions laid down by Reeve and Crozier (1980) are followed. For instance, any chromatogram can be treated in an analogous manner to the mass spectra in Fig. 20. The potential information yield in bits is related, on a one-to-one basis, to the peak capacity of the chromatographic system. Authentic GAx

I

Sample A

I 0

I

I

1

5

10

15

1

20

25

Retention time (min )

Fig. 21. Hypothetical analysis of GA, on a chromatogram with a peak capacity of c. 100.

84

ALAN CROZIER

Thus capillary G C has a potential of c . 300 bits, modern HPLC c. 100 hits, while classical procedures such as TLC and PC produce no more than 5 hits. Figure 21 illustrates a hypothetical chromatogram of an authentic sample of GA, in which the potential information by virtue of the peak capacity is 100 birs. All of this information is available for the verification of accuracy in the case of sample A, which produces a trace that is a perfect match with that of the GA, standard. Sample B, however, contains a large number of impurities and the correlation is far from perfect. Although GA, can be quantified on the basis of the appropriate peak area, the amount of information that can be used to verify the accuracy of the estimate is limited to only one bit as so few parts of the chromatogram match the authentic GA, trace. Clearly sample purity is an important consideration and cannot be ignored, as it is a major factor in determining whether or not sufficient evidence is accrued for verification of accuracy. When analysing impure samples the availability of a selective detector is advantageous as traces will yield more information than equivalent chromatograms obtained with a non-specific method. This is where the strength of SICM lies, why G C data obtained with a radioactivity monitor have proved reliable in identifying [3H]GA metabolites and why, in contrast, GC-FID analysis of endogenous GAS has produced many erroneous identifications. In this context the bioassays in Table VI can be looked upon as selective detectors for free GAS.Unforunately they are very labour-intensive and the problem is compounded as large numbers of fractions must be collected and analysed if the peak capacity of the chromatogram is to be maintained. In addition the response time can be anything from two to seven days and the precision is poor because of the log-linear doseresponse curve and the inherent variability of plant material. One appealing feature of all chromatographic methods is that by running a sample in a number of different solvent systems information can be accumulated. HPLC is particularly amenable to this approach because of the ease and efficiency of sample recovery and the variety of separatory mechanisms that are available. Indeed certain combinations of HPLC techniques can easily challenge the informing power of mass spectrometry. The analysis of GACEs is a case in point as in circumstances where the limited availability of sample prevents a mass spectrum being obtained, information can be readily accumulated by HPLC because of the low limits of detection of the spectrophotofluorimetric monitor. However, the interested reader is urged to consult the rules used to calculate the information yield of a combination of chromatographic techniques as in some situations the total information obtained is not additive (Reeve and Crozier, 1980). While the procedures of Reeve and Crozier (1980) provide a means of verifying accuracy it must be emphasized that they involve a number of less than perfect assumptions and it would be unwise, without detailed investigation, to pedantically adopt such criteria as universal standards for analysis.

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

85

However, it does appear that even rough objective standards provide an intuitively acceptable assessment of many of the imponderables usually left to subjective judgement. 111. GIBBERELLIN BIOSYNTHESIS A. MEVALONIC ACID TO ENT-KAURENE

All GAS originate from a common pathway leading to GAlz aldehyde; thereafter, depending upon the plant material, the pathway can branch in an assortment of directions. Primarily as a result of investigations with cellfree systems from Gibberella fujikuroi (Shechter and West, 1969; Evans and Hanson, 1972), Marah macrocarpus* (Graebe et al., 1965; Upper and West, 1967; Oster and West, 1968), Circurbira maxima? (Graebe, 1969, 1972), Pisutn sativum (Anderson and Moore, 1967; Coolbaugh and Moore, 1971; Coolbaugh et a / . , 1973; Graebe, 1968) and Ricinus communis (Robinson and West, 1970a) it has been established that the early stages of GA biosynthesis follow the normal isoprenoid pathway. The activation of mevalonic acid to the pyrophosphate form is followed by conversion to dimethylallyl pyrophosphate via isopentenyl pyrophosphate. Three condensation steps then occur, each involving isopentenyl pyrophosphate, in which the conversion of dimethylallyl pyrophosphate-geranyl pyrophosphate-tfarnesyl pyrophosphate+geranylgeranyl pyrophosphate takes place. Geranylgeranyl pyrophosphate is a precursor of ent-kaurene which is synthesized via copalyl pyrophosphate. The enzymes involved in the synthesis of ent-kaurene from mevalonic acid are soluble, remaining in the high speed supernatant when tissue homogenates are ultra-centrifuged, and require ATP, Mg’ ’ and Mn’ ’as co-factors. Liquid endosperm preparations from seed of Cucurbita maxima at the appropriate stage of development can convert mevalonic acid to ent-kaurene with an efficiency of c . 40% (Graebe, 1969). However, in other systems, especially seedling material, the ent-kaurene yield is much lower, as mevalonic acid is preferentially converted into products such as squalene via farnesyl pyrophosphate ; phytoene and casbene via geranylgeranyl pyrophosphate ; and ( + )-stachene, ( )-sandarocopimaradiene and trachylobane via copalyl pyrophosphate (Graebe, 1968 ; Robinson and West, 1970b; Hedden and Phinney, 1979). Certain synthetic growth retardants can inhibit the conversion of geranylgeranyl pyrophosphate to ent-kaurene via copalyl pyrophosphate in cell-free systems. The first step in the sequence is affected by N,N,N-trimethyl-1methyl (2’,6’,6’-trirnethylcyclohe~-2’-en1’-yl prop-2-enylammonium iodide (Hedden et al., 1977)), AMO-1618 and its isomer Carvadan, phosphon-D,

+

*Originally referred to as Echinocystis macrocarpa. toriginally referred to as Cucurbita pepo.

86

ALAN CROZIER

phosphon-S, 4-53, 4-58 and 4-64, inhibitors of sterol biosynthesis such as S K F 3301A and SKF 525A and high doses of CCC. The conversion of copalyl pyrophosphate to ent-kaurene is more resistant and is sensitive only to 4-53, 4-58, 4-64 and the steroid inhibitors (Fall and West, 1971; West, 1973; Frost and West, 1977). In the late 1960s and early 1970s some of these retardants, in particular AMO-1618 and CCC, were widely used in physiological experiments and it was often assumed, without appropriate experimentation, that their inhibitory effects on growth were a direct consequence of reduced ent-kaurene synthesis and the resultant depletion of endogenous GA levels. Anyone questioning the simplicity of these views was likely to become embroiled in a colourful debate (see Lang, 1970). There was, none the less, much data indicating that the mode of action of the retardants was considerably more complex in vivo than in vitro. Their inhibitory effects on plant growth are not universal (Cathey and Stuart, 1961) and when dwarfism is induced it can rarely be completely counteracted by exogenous GA treatment (see Lockhart, 1962; Crozier et al., 1973). In certain circumstances low doses of CCC and AMO-1618 can actually enhance growth and/or increase levels of endogenous GA-like activity (Mishra and Pradham, 1968; Van Bragt, 1969; Wunsche, 1969; Reid and Crozier, 1970, 1972; Hdevy and Shilo, 1970). To compound the situation still further, Douglas and Paleg (1974) have shown that phosphon-D, AMO-1618 and CCC inhibit growth and sterol biosynthesis in Nicotiana tabacum and the retardation of growth can be prevented by application of either sterol or GA. Unless the role of GA is to activate sterol biosynthesis, these data prove that the retardants have more than one potential site of action in higher plants. Indeed it is now generally accepted that they are not the elegant physiological tool once envisaged and that their effects on plant growth are unlikely to be exclusively due to an inhibition of ent-kaurene synthetase. Claims to the contrary are now expected to be accompanied by unequivocal evidence rather than assumptions. Seedlings of the d 5 single gene recessive mutant of Zea mays are characterized by shortened stems and leaves and, unlike normal seedlings, they contain little or no GA-like activity (Phinney, 1961). The growth rate of d5 mutants is enhanced by treatment with GAS and some GA precursors such as entkaurene, and it has been suggested that dwarfism is a consequence of a block in the GA biosynthesis pathway prior to ent-kaurene formation (Katsumi et al., 1964). Hedden and Phinney (1979) have investigated this possibility by using a cell-free system to study the production of ent-kaurene by shoots of etiolated normal and d 5 seedlings. In preparations from both tissues, most of the radioactivity from a [14C]mevalonic acid precursor became associated with phytoene and squalene. Although only relatively minor components overall, the main diterpene hydrocarbons produced were ent-kaurene (entkaur-16-ene) and its isomer, which is not on the pathway leading to GAS,

87

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

ent-isokaurene (ent-kaur-15-ene). Normal seedlings synthesized higher levels of ent-kaurene while ent-isokaurene was the major diterpene in the d5 incubations. Similar ent-kaurenelent-isokaurene ratios were obtained when [' 4C]geranylgeranyl pyrophosphate and [3H]copalyl pyrophosphate were used as substrates. This point is demonstrated in Table X which also shows that the total incorporation of radioactivity into diterpenes was higher in cell-free preparations from normal seedlings than it was from d 5 . TABLE X Incorporation of radioactivity into ent-kaurene and ent-isokaurenefrom [2-'4C]mevalonic acid ( M V A ) , ['4C]geranylgeranylpyrophosphate ( G G P P ) and [3H]copalylpyrophosphate ( C P P ) incubated in cell-free extracts from etiolated shoots of normal and ds seedlings of Zea mays. Data expressed as c.p.m. g-' fresh weight. (After Hedden and Phinney, 1979) -~~~~~ ~

~~

c.p.m. g Substrate

Tissue

-' fresh weight -

ent-kaurene ent-isokaurene MVA

GGPP

Normal ds Normal

CPP

Normal

d5 d5

673 16 103 10 766 153

84

127 26 37 81 608

ent-kaurene

ratio ent-isokaurene 8.0 0.: 4.0 0.3 9.5 0.3

The data of Hedden and Phinney (1979) indicate that the normal allele of the d 5 gene controls the conversion of copalyl pyrophosphate to ent-kaurene since mutation results in a marked reduction in ent-kaurene biosynthesis. Apparently the mutated gene codes for an altered enzyme which catalyses the production of ent-isokaurene at the expense of ent-kaurene. Hedden and Phinney (1979) have suggested a possible mechanism for the enzymic formation of ent-kaurene and ent-isokaurene from copalyl pyrophosphate. It involves ent-kaurene being synthesized from copalyl pyrophosphate by the loss of a proton from the C-17 carbofiinm ion (I) while loss of a proton from C-15 gives rise to ent-isokaurene (Fig. 22). Thus any alteration of the enzyme, such as a shift in the position of the proton-accepting group bringing it closer to C-15 than to C-17, would result in increased production of entisokaurene in preference to ent-kaurene as seemingly occurs in the d5 mutant of Zea mays.

88

ALAN CROZIER

Copalylpyrophosphate

J

@ \

enl- kaurene

enf - isokaurene

Fig. 22. Proposed scheme for the synthesis of rnt-kaurene and ent-isokaurene from copalyl pyrophosphate in maize seedlings (Hedden and Phinney, 1979).

B.

ENT-KAURENE TO C A I 2ALDEHYDE

Further conversion of ent-kaurene involves sequential oxidation at C-19 to produce em-kaurenol, enr-kaurenal, em-kaurenoic acid and em-7a-hydroxykaurenoic acid (Fig. 23). All the steps have been shown to occur in cell-free us et al., systems from liquid endosperm of both Marah ~ ~ ~ a c r o c a r p(Graebe 1965; Lew and West, 1971) and Cucurbita maxirna (Graebe and Hedden, 1974) and immature seed of Pisurii sativurii (Ropers et al., 1978). Murphy and Briggs (1975) have demonstrated the sequence from ent-kaurenol in cell-free preparations from embryos and young leaves of Hordeum distichon. In Gibberella jujikuroi the conversion of ent-kaurenoic acid to ent-7ahydroxykaurenoic acid has been established (West, 1973; Bearder et al., 1975a). The enzymes involved in this section of the pathway in Marah are particulate and located in the 105,000 x g pellet (Dennis and West, 1967). Activity is dependent upon the presence of O2 and NADPH2. The oxidation of ertt-kaurene to eut-kaurenol, and that of ent-kaurenal to ent-kaurenoic acid, is inhibited by carbon monoxide and in both instances the inhibition is

18-

1 9 ~

ent - kaurane skeleton

L

ent- kaurene

enf- kaurenol

ent - kaurenal

ent- kaurenoic acid

enf - 7a - hydroxykaurenoic acid

Fig. 23. Ent-kaurane skeleton and the conversion of ent-kaurene to ~nt-7a-hydroxykaurenoicacid

90

ALAN CROZIER

counteracted by light which exerts maximal effect at 450 nm. This suggests that the conversions are catalysed by mixed function oxidases and implies an involvement of cytochrome P450(Murphy and West, 1969). The growth retardant ancymidol (a-cyclopropyl-a-[p-methoxyphenyl]-5-pyrimidine methyl alcohol) which induces a GA-reversible inhibition of root and shoot growth (Leopold, 197l), blocks the oxidation of ent-kaurene, ent-kaurenol, ent-kaurenal but not ent-kaurenoic acid in cell-free preparations of Marah macrocarpus, and it has been suggested that it interacts with cytochrome P450 in the oxidase-catalysed steps between ent-kaurene to ent-kaurenoic acid (Coolbaugh et al., 1978). In cell-free systems from Cucurbita maxima and Marah macrocarpus, ent7a-hydroxykaurenoic acid undergoes either oxidative B-ring contraction to produce the GA precursor, GA1,aldehyde, or ent-6a-hydroxylation to form ent-6a,7a-dihydroxykaurenoicacid (Graebe et al., 1972, 1974~;West, 1973; Graebe and Hedden, 1974). To date only GAlzaldehydeformation has been reported in preparations from immature Pisum sativum seed (Ropers et al., 1978). In Gibberella fujikuroi, ent-7a-hydroxykaurenoic acid gives rise to GAlzaldehyde (Hanson et al., 1972) and in addition is seemingly the intermediate in the synthesis of both ent-6a,7a-dihydroxykaurenoicacid and 7pdihydroxykaurenolide from ent-kaurenoic acid (West, 1973). The ent-6a,7adihydroxykaurenoic acid does not accumulate to any extent, being converted to fujenal (Cross et al., 1970)while 7,LLhydroxykaurenolideacts as a precursor of 7/?,18-dihydroxykaurenolide(Cross et al., 1968a). All these steps are illustrated in Fig. 24. The conversion of ent-7a-hydroxykaurenoic acid to G Al ,aldehyde requires contraction of ring B from a six to a five carbon structure with the extrusion of C-7. Evans et al. (1970) proposed that ring contraction was initiated by abstraction of the ent-6a-hydrogen as feeding experiments with Gibberella fujikuroi using stereospecifically labelled [3H]mevalonic acids showed that the ent-6a-hydrogen was lost in the conversion of ent-7ahydroxykaurenoic acid to GA3 while hydrogen at the ent-6j?-position was retained. Experiments by Graebe et at. (1975) indicated a similar process may occur in Cucurbita maxima preparations as metabolism of ent-7a-hydroxy ['4C,6-3Hz]kaurenoic acid produced GA1,aldehyde and ent-6a,7a-dihydroxykaurenoic acid with half the 3H/14Cratio of the substrate. Timecourse studies on the synthesis of these two metabolites in preparations from the 200,000 x g microsomal pellet of Cucurbita endosperm revealed that both compounds were formed simultaneously at equivalent rates. LineweaverBurk and Hill plots of the rates of synthesis were linear and the Hill plot had a slope of almost 1.0 indicating first order kinetics. Co-factor, pH and temperature requirements for both reactions were similar implying that both metabolites were being formed from the same high energy intermediate whose rate of synthesis determined the overall rate of production (Graebe

// /

@-gQ \

oc-d \ H ',""

OH COOH

ent - 6a,7a - di hydroxy kau renoic acid

Fujenal

@- m COOH

COOH CHO

GA12 aldehyde

enl- 7a - hydroxykaurenoic acid

7p- hydroxykaurenolide

7&18 -dihydroxykaurenolide

Fig. 24. Metabolism of ent-7a-hydroxykaurenoic acid.

'R ent -70- hydroxykaurenoic acid

ent-6a,7a- dihydraxykaurenoic acld

t

QQ-(&Q R'

R

(0

c+

H/ '3 \

& c' / \

H O GA 12aldehyde

H

Fig. 25. Proposed mechanism for the conversion of ent-7a-hydroxykaurenoic acid to GA12aldehyde and enr-6a,7a-dihydroxykaurenoicacid. R = COOH (Evans et al., 1970; Hedden et al., 1978).

92

ALAN CROZIER

and Hedden, 1974). Graebe et d.(unpublished data quoted by Hedden et d., 1978) fed ent-7a-hydro~y[6a-~H,I 7-3Hz]kaurenoicacid containing 62 atoms :{ [’HI to the Cucurbitu cell-free system. The resultant GA1zaldehyde and enr-6a,7a-dihydroxykaurenoicacid had the same specific radioactivity as the substrate but contained only zero and 4 atoms :{ [’HI respectively thereby proving that they had both lost the ent-6a-hydrogen atom. The simplest mechanism commensurate with the experimental data obtained with Gibberellu and Circitr.bitcr was originally proposed by Evans et a / . (1970) and is illustrated in Fig. 25. Abstraction of the ent-6a-hydride from errt-7a-hydroxykaurenoic acid produces a putative carbonium ion intermediate which can either be hydroxylated at the ent-6a-position to give ent-6a,7a-dihydroxykaurenoic acid, or alternatively undergo B-ring contraction to form GA12aldehydevia migration of the 7,8 bond to the 6,8 position and the loss of a proton from the hydroxyl function of the extruded C-7. C. PATHWAYS BEYOND GA1 ,ALDEHYDE

I . Gibberella fujikuroi GA biosynthesis pathways have been thoroughly investigated in Gibherellu ,fi~jikur*oi. The GAS are metabolic bi-products and do not appear to be involved in mycelial growth in any way. Early studies by Cross et a / . (1968b) were suggestive of a branch at an early point in the pathway as although [ 17-’4C]GA1,aldehyde and [ I 7-14C]GA14 were effectively converted to [17-14C]GA3,[17-14C]GA12produced three unidentified acids and only relatively small amounts of GA3. Details of developments from a chronological point of view can be gauged from reviews by Cross (1968), Hanson (1971), MacMillan and Pryce (1973), MacMillan (1974), Graebe and Ropers (1978) and Hedden et N / . (1978) while an outline of the current status of knowledge is presented in Fig. 26. Much of the information in Fig. 26 was obtained from experiments using mutant strains of Gibbere//nfujikuroi.Originally genetic studies were virtually impossible because of an inability to consistently obtain the sexual stage of the fungus in the laboratory. However, Spector (1964) succeeded in routinely inducing perithecial production by growing Gibberella strains of opposite mating types on a Cirrus stem medium. Asci were removed from mature perithecia and ascospores from individual asci separated with a micromanipulator and transferred individually to potato-dextrose agar for culturing. With this technique Spector and Phinney (1966, 1968) provided direct evidence of genetic control of GA production in the fungus and demonstrated the presence of two non-allelic genes blocking different points Fig. 26. G A biosynthesis pathways beyond GA,2aldehyde in Gibberella fujikuroi. Thick arrows represent steps connecting major metabolites.

GA40

94

ALAN CROZIER

on the synthesis pathway. The first gene blocked an early stage in the metabolic sequence and controlled all GA production. The second blocked a later step as the production of only GA1 and GA3 was adversely affected. Further developments occurred because Phinney (unpublished data) was able to modify the barley aleurone a-amylase bioassay (Jones and Varner, 1967) to provide a simple non-labour intensive procedure to monitor the presence or absence of GA in Gibberellafujikuroi cultures. This facilitated a rapid screening of the GA production capacity of many thousands of strains of Gibberelln from both natural sources and mutants arising from UVirradiation of a wild type parent strain GF-la. The pathways illustrated in Fig. 26 were compiled from data obtained with the GA synthesizing strains ACC 917, GF-la and M-119 and two mutants, B1-41a and R9. R9 arose spontaneously from a wild type strain isolated from a paddyfield in Japan and is blocked for 13a-hydroxylation, producing neither GA, nor GA3 (Bearder et al., 1973a). B1-41a is a UV-induced mutant blocked between ent-kaurenal and ent-kaurenoic acid with a leakage of <3:4 for the conversion of [2-'4C]mevalonic acid to [I4C]GA3 (Bearder et al., 1974). This mutant has proved especially useful, as the absence of endogenous GASenables metabolism studies to be carried out with unlabelled precursors. The conversion of GA,,aldehyde to either G A 1 2 via C-7 oxidation or GA,, aldehyde via 3P-hydroxylation represents the major branch in the GA biosynthesis pathway (Bearder et a/., 1973b, 1975a; Evans and Hanson, 1975). GAI4aldehydeis probably the immediate precursor of GAI4as both compounds are converted via GA4 and GA? to GA3,which is the major GA in G A-producing strains of Gibberella. In addition to this main pathway, trace quantities of G A I 3 ,GA36and GA4, are synthesized from both GA14 aldehyde and GA14,while GA4 is the substrate for small amounts of GA1, GA2, GAI6 and GA4? (Bearder et al., 1975a; McInnes et ul., 1977). GAl, GA3, GA16 and GA36 are not further metabolized, nor is GA3,, which although not detected as a metabolite in these studies is an endogenous component of Cibberellu (MacMillan and Wels, 1974; Bearder et ul., 1975a). Some seemingly contradictory data have been obtained with G A I 4 feeding. Evans and Hanson (1975) have reported that it is essentially unchanged after a 24-h incubation while Cross et al. (1968a) observed a 4.7% incorporation into GA,. Both investigations used the wild type strain ACC-917. Studies by Hedden et al. (1974) with B1-41a showed that although GA14 and GA14aldehyde produced the same spectrum of metabolites, all the aldehyde was converted over a five-day period while 40% of the acid remained unmetabolized. This could be a consequence of poorer penetration of GA14 to the enzymic site in the fungal hyphae but the possibility that GA14 may not be on the direct pathway between GA,,aldehyde and GA4 cannot be discounted. The alternative pathway, from GAI2aldehyde via GA12, gives rise to

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

95

predominantly non-hydroxylated GAS (Fig. 26). [17-14C]GAI fed to strain ACC-917 yielded labelled GA9 (23%), GA15 (973, GA24 (25%) and GA25 (18”,J within six hours (Evans and Hanson, 1975). Similar results were obtained with the B1-41a mutant except that trace amounts of GA3, G A I 3and GA14 were also formed (Bearder et a / . , 1975a). Presumably this could be due to low level conversions of G A I Zto G A I 4 .GA9 was synthesized only from GA12,GA24 was converted to GA2s,which along with G A l S ,was not further metabolized. GA9 is not converted to GA4, instead it is slowly metabolized to small amounts ofthe hydroxylated G A S ,G A l o ,GA, 1 , GA20, GA4o and 16,17dihydro, 16,17dihydroxyGA9 (11) as well as the A1.’o,19,2 lactone (111) and possibly d ’GA9 (IV) as illustrated in Fig. 26 (Cross et a / . , 1964, 1968b; Bearder et a / . , 1976a). The and A ’ * ’ ’ compounds are potential precursors of GA, although this has not been experimentally tested. It is evident from the GA biosynthesis pathways illustrated in Fig. 26 that only C19-GAs undergo 13a-hydroxylation and that 3b-hydroxylation occurs only at the CIO-GA stage. Enzyme-substrate specificity, however, is low as some structural analogs of endogenous intermediates are metabolized to produce an array of natural GAS and GA analogs (see MacMillan, 1974; Hedden et al., 1978). While such studies provide information of intrinsic value they also result in the production of sizable quantities of many GAS that would otherwise be very difficult, if not impossible, to obtain. The GAdeficient mutant B1-41a is especially useful in this regard as the metabolites are not contaminated with endogenous GAS. This strain has been used to synthesize 15&hydroxy derivatives of GA1, GA3, GA4, GA7, GA9 (i.e. GA45), GA12. GA13 and GA15 from ent-l5a-hydroxykaurenoicacid (Bearder and Kybird, unpublished data). Metabolism of steviol (ent-13hydroxykaurenoic acid) by B1-41a produces ent-7a,l3-dihydroxykaurenoic acid, ent-6a,7a, 13-trihydroxykaurenoic acid, 7jl,13-dihydroxykaurenolide, 13-hydroxyfujenal, G A I , G A I 7 , G A l s , GAI9, GA20 and GA53 (Bearder et al., 1975b). In the absence of a GA deficient mutant, a similar spectrum of metabolites can be obtained, undiluted by endogenous GAS, by feeding steviol to normal Gibberella strains and inhibiting ent-kaurene synthetase with either AMO-1618, CCC or N,N,N-trimethyl-l-methyl-3-(3’,3’,5’trimethylcyclohexyl)-2-propenylammonium iodide (Murofushi et a/., 1979). There is a further facet to the interest in steviol since it occurs naturally in high concentrations as a glucoside in the leaves of Stevia rebaudiuna which is a member of the Compositae, native to Paraguay. Unlike Gibberellafujikuroi, many higher plants contain 13a-monohydroxylated C2,,-GAs and it has been hypothesized that steviol may be a key precursor in the synthesis of these compounds (Ruddat et al., 1963, 1965). It is envisaged that the pathway would run steviol+ ent-7a, 13-dih ydroxykaurenoic acid -,G A ,aldehyde+ GA53.Steviol is active in GA bioassays (Katsumi et a/., 1964) and as noted ‘ 3

96

ALAN CROZIER

above it is converted to 13a-hydroxy CZO-GAsby Gibberella fujikuroi. Similar conversions have not, however, been demonstrated in higher plant tissues and Stevia rebaudiana is the only plant in which steviol has been detected although the literature offers no indications of serious attempts being made to locate it in other species. The steviol hypothesis is interesting conjecture but no more than that as critical evidence is clearly lacking and the possibility that 13a-hydroxylation takes place after, rather than before, B-ring-contraction is equally feasible. The metabolic profile can be modified to some degree by varying the pH of the medium in which Gibberella is cultured. For instance, GAI4accumulates in 20-h feeds of ent-kaurenoic acid, ent-7a-hydroxykaurenoic acid, GA12 aldehyde and GA14aldehydeto B1-41a at pH 7.0, yet at pH 3.5 its appearance is transient, presumably because further conversion is no longer a rate limiting step (Bearder et al., 1975a). When the GA-producing strain ACC-917 is grown on synthetic medium the ratio of the GA3/GAl content is >20 and [14C]GA1is not converted to [14C]GA3.However when the fungus is grown in a glucose-soybean meal medium the GA3/GA1 ratio is < 1.5 and there is a 0.6% conversion of [1,2,3H2]GA1to [3H]GA3(Pitel et a/., 1971b; McInnes et al., 1977). Under the circumstances it seems plausible that some of the seemingly contradictory results that have been obtained in G A metabolism studies (see Hedden et al., 1978) could be due to non-uniform culturing techniques compounded by the use of different strains of Gibberellafujikuroi. 2. Cucurbita maxima When MnC12 is included in the incubating medium, the 20,000 x g supernatant from liquid endosperm of Cucurbita maxima seed converts mevalonic acid through to GA12aldehyde and GA12(Graebe et al., 1972). Oxidation of GA12aldehyde to GA12 is catalysed by both microsomal and soluble enzymes although further conversion is exclusively associated with the 200,OOOxg supernatant and requires NADPHz and Fei+ or F e + + + cofactors. The action of the soluble enzymes is inhibited by M n + + which explains why metabolism does not proceed beyond GA12in the presence of MnC12 (Graebe and Hedden, 1974). The G A biosynthesis pathways that operate in the Cucurbita maxima cellfree preparations are illustrated in Fig. 27. GA12aldehyde is primarily converted to GA,, which is metabolized to produce GA4, GA13,GA, 1 5 , GA24, GA25, GA36, GA37and GA43. Re-incubation of these compounds indicates that the main pathway from GA12 runs GA12-+GA24+GA36+GA13+ GA43 (Graebe et al., 1974a,b,c). The terminal product, GA43, is an endogenous constituent of Cucurbita maxima endosperm (Beeley et al., 1975) implying that the pathway operates in vivo as well as in vitro. GA24 feeds yield some GA25, which, on re-incubation, is converted to GA13, showing that an alternative route exists from GA24 to GA13, proceeding via GAzs

’ COOH

I

HO

H

o

COOH W C

H

z

GA43

Fig. 27. G A biosynthesis pathways beyond GA,,aldehyde in cell-free preparations from liquid endosperm of Cucurbita maxima seed. Thick arrows represent the preferred pathway based on rates of conversion and levels of metabolites.

98

ALAN CROZIER

rather than (3,436. However, this is probably a minor pathway as GA25 is converted to GA13far less efficiently than GA36.The formation of GA24 from GAlz represents two oxidation steps at C-20, and the S-lactone GA15 would seem to be the logical intermediate. In short-term incubations with [ 14C]GAI2 , a radioactive component with the G C retention characteristics of GA15accumulates and then disappears as GA43starts to build up. However, GA15is not the intermediate, as when it is added to cell-free preparations the only metabolite is GA37.Graebe et al. (1974~)postulate that the true intermediate between GA12 and GA24 may be the C-20 alcohol (V), which they suggest Iactonizes to form GAl 5 . The only CI9-GAmetabolite to be detected is GA4 which is produced in 5-10% yields from GA12aldehyde (Graebe et al., 1974b,c). It has recently been determined that GA36 is the immediate C20-GAprecursor of GA4 (Graebe et al., 1980). Although GA9 is also metabolized to GA4 the significance of the conversion is unclear because GA9 has never been detected as a metabolite in Cucurbita cell-free preparations (Graebe et al., 1974b,c). The conversion may therefore reflect a lack of specificity of the 3j?-hydroxylase involved in the production O f GA36 from GA24, although the possibility that GA9 is a natural substrate that goes undetected because it is rapidly metabolized to GA4,cannot be ruled out. When GA12aldehydeis incubated at the usual concentration of 0.1 pg 100 pl-I the main product is GA43. However when fed at 36 pg 100 plthere is a low yield of GA43rand the major metabolite is GA14aldehyde which is not detected at the lower precursor dose. GA14aldehyde feeds produce GA14, and both compounds are incorporated into GA37as well as into GA36, GA13 and GA43. On the basis of this evidence Graebe and Hedden (1974) have concluded that GA,,aldehyde is not a natural intermediate in the synthesis of GA43and that its production is induced by high concentrations of GA12aldehyde. This may be a somewhat simplified view of the. situation as it is unclear from the data provided whether there is indeed a genuine decrease in the amount of GA43 synthesized at the higher substrate concentration, or merely a reduction in relative terms compared to the increased levels of GA14aldehyde and unmetabolized GAl zaldehyde. If it is the latter, GA14aldehyde may well be a natural metabolite which under normal circumstances does not accumulate, as its rate of synthesis from GA12aldehyde does not exceed its rate of metabolism to GA14. When the substrate concentration is increased conversion to GA, becomes the rate limiting step in the pathway and as a consequence GA14aldehyde builds up and is readily detected. The data produced in biosynthetic studies can be less than straightforward even when closed in vitro systems are used. It is important to appreciate that the accumulation of metabolites merely indicates rate limiting steps in the pathway and that pool size does not necessarily reflect the importance of a compound as an intermediate in the biosynthetic sequence. It is quite poss-

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

99

ible for key intermediates in a pathway to be overlooked because they are rapidly metabolized and do not accumulate to any extent. Incubation of steviol in Cucurbita maxima preparations yields ent7a, 13-dihydroxykaurenoic acid and ent-6a,7a, 13-trihydroxykaurenoic acid (Graebe, 1974~).Surprisingly, in view of the data obtained by Bearder et a / . (1975b) with Gibberella fujikuroi there is no evidence of B-ring contraction to form 13a-hydroxy GA12aldehyde, GA53and other 13a-hydroxy CZO-GAs. Clearly there is a marked difference in the substrate specificity of GAl2 aldehyde synthetase in the two systems. 3 . Pisum sativum ( u ) Seeds. The 200,000 x g supernatant fraction from immature pea seed cv. Schnabel converts GAI2aldehydeto unknown metabolites and GA12alcohol, while the 2000 x g supernatant yields several products, two of which have been identified as the 13a-hydroxy GAS,GA44 and GAS3.Exactly how these GASare biosynthetically related has not yet been reported although the most likely sequences would seem to be GAlzaldehyde+GA12+GA53+ GA44 or alternatively GAIzaldehyde+ GA, 3aldehyde+ GAS +GA4, with GA,, alcohol being formed independently from GAl ,aldehyde in both instances. The activity of the enzymes involved in the production of these GAS is dependent upon the presence of Fe' i.and ATP in the incubating medium (Ropers et al., 1978). Although the pathways have not been fully elucidated in Pisum sativum it is evident that they are quite distinct from those operating in Cucurbira maxima and that 13a-hydroxylation occurs at a much earlier point than it does in Gibberellafujikuroi. The data provide no solace for proponents of the steviol hypothesis as 13a-hydroxylation takes place after rather than before B-ring contraction. The metabolism observed in vitro is probably closely related to that occurring in vivo as GA44 is an endogenous constituent of immature seed of Pisum sativum cv. Progress No. 9. The seeds contain relatively large amounts of GA20 and GA 29 and smaller quantities of GA9, GA17,GA3*,* GA44 and GA51 (Frydman et al., 1974; Sponsel and MacMillan, 1977). Frydman et at. (1974) used GC-SICM to quantitatively analyse GA9, GA1,, GAZ0and GA29 during seed maturation. They found that as the seed matures there is a sequential increase and subsequent decline in the levels of the individual GAS. The GA9 maxima occurs 20 days after anthesis, followed by the GA17and GAzopeaks two days later and GAZ9 at day 27 (Fig. 28). Against this background of changing endogenous GA levels, which presumably reflects changes in enzyme activity associated with *This identification has been withdrawn while further investigations are carried out as the mass spectrum upon which the characterization was based may have been that of the isomeric compound 2B-OH GAd4 (Sponsel era/., 1979).

100

ALAN CROZIER

0 15r

Days after anthesis

Fig. 28. Estimated endogenous levels of G A 9 , CAI,, G A Z Oand G A Z 9during maturation of seed of Pisum sativum cv. Progress No. 9 (Frydman et a / . , 1974).

seed development, GA metabolism studies have produced some intriguing data. The results of Frydman and MacMillan (1975), Sponsel and MacMillan (1977, 1978), Ropers et al. (1978) and Durley et al. (1979) have been used to compile the pathways illustrated in Fig. 29. At an early stage of seed development [15,17-3H]GA9undergoes 13a-hydroxylation and gives rise to [3H]GA20.However, the yields are < 10% and Sponsel and MacMillan (1977) point out that GA9 is unlikely to be the natural precursor of GAzoas at the time of the feed neither GA9 nor GAZ0are endogenous components of the immature seed. The conversion may well be a consequence of the lack of substrate specificity of the 13a-hydroxylase involved in the metabolism of GA12aldehydeto GA53.At a later stage of seed maturation, 13a-hydroxylation is suppressed and [15,17-3H]GA9acts as the precursor of [3H]GA51. This is believed to be an endogenous pathway as both substrate and product are now native constituents of the seed. [3H]12a-hydroxyGA9and a [3H]12a-

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

101

hydroxyGA, conjugate are also produced from [15,17-3H]GA9 at both stages of seed development. However, these are thought to be artifacts as neither compound has been isolated from Pisum sativum (Sponsel and MacMillan, 1977). If the biosynthesis of these compounds is due to low enzyme-substrate specificity, it seems possible that Pisum may naturally produce endogenous 12a-hydroxy GASwhich to date have not been detected. Although the CZo-GAprecursors of GAzohave not so far been determined, the conversion of GA44, or alternatively its hypothetical C-20 alcohol equivalent, to GA20via G A I 9is one option in view of the detection of GA44in Pisum seed extracts and its in vitro production from GA12aldehyde. GA19, which has recently been shown to be an endogenous Pisum GA (Ingram and Browning, 1979), may act as the precursor of G A L 7as well as GAzo(Fig. 29). The predominance of 13a-hydroxylation during the early stages of seed development, the high levels of endogenous GAZo,and the low GA, content imply that GAzo is on the main GA biosynthesis pathway and that GA9 originates via a minor route. No obvious intermediates between GA12aldehyde and GA9 have either been isolated from Pisum sativum or detected in metabolic studies. Metabolism of [ 14,15,17-3H]GA20during the later stages of seed development results in a 5&90% yield of [3H]GA29.Highest conversions are found when the time of the feed and the extraction of the tissue coincide with the respective maxima of native GAZ0and GA29. This suggests that the 2phydroxylation step is a normal endogenous process. Small quantities of conjugates of [3H]GAzo and [3H]GA29 also originate from [14,15,173H]GA20although neither conjugate has been found to occur naturally in Pisum. [3H]GA29fed to seed 27-28 days after anthesis is not metabolized to any extent over an eight day period. [3H]GA29produced in situ by feeding [14,15,17-3H]GAzois also metabolized at a very slow rate despite the fact that the endogenous GA29 pool rapidly declines (Sponsel and MacMillan, 1977). The identification of [3H]GA metabolites from Pisum sativum seed by Sponsel and MacMillan (1977) was based on GC-radioactivity retention times. Although mass spectra were obtained from mass peaks co-chromatographing with the radioactivity, this did not distinguish between ['HI endogenous and [3H] metabolite GA because the specific activity of the [3H]GAs was much too low for [3H] fragments to show up in the spectra. To overcome this problem Sponsel and MacMillan (1978) used precursor GAS labelled with [3H] and a high level of ['HI. The [3H] served as an indicator of metabolic processes while deuterated and non-deuterated species in the molecular ion cluster of mass spectra were used to estimate the ['HI metabolite and ['HI endogenous GA content. As would be anticipated from the results of previous experiments, differential rates of metabolism of exogenous and endogenous GA29 were observed

-OH

GA,,

aldehyde

CH2

CH3':

-OH+

GA44

-OH

+[Hypothetical]

COOH

0 -OH

G A 2 9 conjugate+

129-OH GAS

Compound B (tentative)

1

120-OH G A g conjugate

m,P -unsaturated ketone Fig. 29. GA metabolism pathways in developing seed of Pisum sativum. Thick arrows represent established conversions and thin arrows indicate hypothetical steps.

103

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

TABLE XI Levels of endogenous and exogenous GAZ9 in developing Pisum sativum seed following ] (Ajtey Sponsel and MacMillan, 1978) the application of’ [ Z U - ~ H [2a-3H]GA29. Age of seed (days after anthesis)

Metabolism period (days)

21 30 33 36 40

0 3 6 9 13

Recoverable GAz9(pg seed -’) Endogenous Exogenous

7.7 3.6 1.9

5.1 4.5 4.1

1.5 1.2

3.6 2.8

2, recovery of exogenous GA29 100 79

12 63 49

when [2a-2H][2a-3H]GA29was fed to Pisum sativum seed 27 days after anthesis (Sponsel and MacMillan, 1978). At the start of the experiment each seed contained 5.7 p g of the deuterated substrate and 2.8 p g remained after 13 days, indicating a 49% recovery of the exogenous GA29. Over the same period the endogenous GA29 pool fell from 7.7 pg to 1.2 pg seed-’ which is 160,; of the original level (Table XI). The metabolism 0 f G A 2 9 resulted in a loss of label as, except for a minor accumulation of a putative GAZ9conjugate, no metabolites were detected. Presumably this was due to oxidation at C-2 and Sponsel and MacMillan (1978) have proposed that the data are consistent with the conversion of [2u-’H] [ ~ U - ~ H I to G the A ~a,B-unsaturated ~ ketone shown in Fig. 29 which is an endogenous component of both seeds and seedlings of Pisum sativurn. The data of Durley et a / . (1979) support this view, and indicate that [3H]GA29,formed from [2,3-3H]GA20,is converted to the [3H]ketonevia [3H]compound B which has been tentatively assigned the structure illustrated in Fig. 29. The [3H]ketone was the major labelled product in mature seed although moderate amounts of [3H] compound B were also present. Both compounds seem to be metabolized only slowly and Durley et ul. (1979) suggest that they serve as biologically inactive catabolite sinks for GAS produced during the development of Pisum seed. In other species this role is thought to be played by GA conjugates which are only minor constituents in mature pea seed. The [3H]a,/3-unsaturated ketone was not identified by Sponsel and MacMillan (1977) in their experiments with [ 14,15,17-3H]GA20 and [14,15,17-3H]GA29 because the relevant radioactive G C peak was attributed to the partially derivatized ether, mono trimethylsilyl ether G A 2 9 methyl ester. Sponsel and MacMillan (1978) have postulated that the slow rate of conversion of exogenous [ ~ u - ~[H~ ]u - ~ H I G compared A ~ ~ , to that of the endogenous species, is due to the cleavage of the 2a-H bond being subject to a primary isotope effect. In such circumstances, breakage of a C-2H bond

104

ALAN CROZIER

would be expected to be about eight times slower and that of a C-3H bond up to sixteen times slower than breakage of a C-’H bond. In support of this proposal Sponsel and MacMillan (1978) contend that endogenous GA29 and [lj?,3a-2H][lj?,3a-3H]GA29, produced in situ from [lj?,3~-~H][lp,3a-~H] CiAzo,are metabolized at equivalent rates by maturing Pisum seed. However the experimental data that are produced are not convincing. [1/3,3~-~H] [ ~ ~ ? , ~ U - ~ was H ] injected G A ~ ~into 23-day-old seeds and samples for analysis were harvested immediately and at two to four day intervals for a period of 13 days. The levels of recoverable endogenous and exogenous GAzo and Ga29 that were obtained are presented in Table XII. Estimates of the endogenous GA content show that the GAzo pool was depleted by day 9. Endogenous G A 2 9 was not detected in 23-day-old seed at day 0 although by day 9 it was present at a level of 10.3 bg seed-’. At the termination of the experiment four days later this had fallen to 7.7 pg seed- The estimated level of recoverable exogenous GAzo at day 0 was 5.4 pug seed-’. Thirteen days later this had all been metabolized and each seed contained 2.6 pg of GAZ9that had been formed by 2p-hydroxylation of the exogenous deuterated GAZO.This represents a 48% recovery of the applied label which is almost identical to the 49% obtained with exogenous [ ~ u - ~[H~ ]U - ~ H I G A ~ ~ . ]G metabolized A~~ at an equivalThe contention that [ lP,3a-’H] [ I / ~ , ~ U - ~ H is ent rate to endogenous GA29 while [2a-’H] [ ~ U - ~ H ] G is Ametabolized ~~ more slowly is, therefore, not based on differences in the rates of metabolism of the applied labels but on differential rates of disappearance of the endogenous GA29 pool in the two experiments. The reasons for this can be seen in Tables XI and XI1 and Fig. 28. When [2a-2H][2a-3H]GA29was applied to seed 27 days after anthesis, the endogenous GA29 pool was at its highest measured value containing 7.7 p g seed- and during the course of the 13 day

’.

’

TABLE XI1 Levels of exogenous and endogenous GAZoand GA29 in developing Pisum sativum seed following the application of [ I @ ,3a-’W [ I P , ~ U - ~ H I G(After A ~ ~Sponsel . and MacMillan, 1978)

Age of seed (days after anthesis)

23 25 28 32 36

Metabolism period (days)

0 2

Recoverable GA (pg seed-’) Endogenous Exogenous

% recovery of

exogenous GAZOGA19 GAzo GAZ9 GA,,andGA,,

5

2.4 3.2 2.3

9 13

-

-

-

1.3 5.3 10.3 7.7

5.4 2.9 0.9 -

-

100

1.6 2.4 3.2 2.6

83 61 59 48

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

! I

105

metabolism period it fell to 1.2 pg seed-’. When [ 1/3,3a-2H][l/3,3a-3H]GA20 was used as a substrate for [l/3,3a-2H][l/3,3a-3H]GA29the endogenous GA29 level peaked 32 days after anthesis, four days later than anticipated, and the experiment was terminated when the seeds were 36 days old. Hence, metabolism of the applied label was monitored for only four days beyond the endogenous GA29 maximum, whereas in the experiment with [2/3-’H][2/3 3H]GA29it was followed for 13 days. In view of this lack of synchrony and variance in metabolism periods it is difficult to draw any firm conclusions as [I/3,3a-3H]GA29 to the relative rates of metabolism of exogenous [ l/3,3~-~H] and [2/3-’H] [2/3-3H]GA29with respect to that of endogenous GAZ9.The dynamics of GA metabolism in developing Pisum seeds are inherently complex and experimental data are exceedingly difficult to interpret because (i) the kinetics of the system are only partially characterized, (ii) the label is applied as a pulse and not at a steady dose rate and (iii) the changing endogenous GA pool sizes clearly indicate that the system is not operating under steady state conditions. There is therefore a lack of persuasive evidence to support the suggestion that a primary isotope effect at C-2 is responsible for the slow rate of conversion of [2a-2H][2a-3H]GA29.It is, in fact, difficult to envisage how this model could account for the similarly slow metabolism of [ 14,15,17-3H]GA29.Other explanations that warrant consideration include the slow transport of exogenous GA29 to the metabolic site, although this is unlikely to be a limiting factor when labelled GA29 is produced in situ from exogenous GA20.However, differential rates of metabolism could occur if there were two pools of endogenous GA29, one arising from GAzoand the other from a different precursor. In this context it would be interesting to learn how GA51 and its precursor GA9 are metabolized by Pisum sarivum seed during the later stages of maturation. Although neither GA would be present in the seed in detectable quantities this does not necessarily eliminate them as potential intermediates in the synthesis of GA29 as endogenous pool sizes need not reflect rates of turnover. More informed discussion on this topic will only be possible when GA feeds are made throughout seed maturation instead of being restricted to the developmental stage at which the endogenous pool is maximal. ( b ) Seedlings. GA metabolism has also been studied in etiolated seedlings of Meteor dwarf pea. [3H] metabolites were identified on the basis of their GC retention characteristics using three different stationary phases, although in some instances mass spectrometric evidence was also obtained. The results are summarized in Table XIII. The fates of [3H]GA9 and [3H]GA20are similar to those observed in developing seed, while [3H]GA3accumulates after the application of 13H]GA5.The metabolism of [3H]GA14is of special interest as it involves the conversion of a Cz0-GAprecursor to two C19-GAs, GAI and its metabolite GAB.On the basis of metabolite pool sizes in a time course study, and the structural relationships of the GAS involved, Durley

106

ALAN CROZIER

TABLE XI11 Metabolism oj [ 3 H ] G A sby dark-grown Pisum sativum seedlings Precursor [17-3H]GA9

Metabolities 12a-hydroxyGA9,GAlo",GA20GA29, GA51

[2,3-3H]GA20 [l-3H]GA5 [17-3HlGAi4 [1,2-3H]GA,

Reference

GAZ9 GA3 GA,, ' 3 . 4 8 , GAi,, GA23, (3.438 GAS

Railton et 01. (1974a,b) Railton et ul. (1974a,c) Durley et al. (1973) Durley et cd. (1974a,b) Durley et a / . (1974b)

This compound may be an artifact resulting from the sample being in prolonged contact with active sites on silica gel particles during either T L C or Woelm silica gel partition colum chromatography as described by Durley et a / . (1972) and discussed in Section 1I.D. Also see Grove (1961) and Hanson (1966).

rt 01. (1974a, 1974b) proposed a GAI4+GAl 8+GA38 +GA23 + G A +GAB

pathway with G A 2 8 being formed as a side branch from G A 2 3 (Fig. 30).The evidence for such a pathway is circumstantial as the metabolism of the potential CZO-GAintermediates in the sequence leading to GAl has not been investigated. It is impossible to assess the degree to which the conversions listed in Table XI11 and illustrated in Fig. 30 reflect native GA biosynthetic pathways operating in Pisum sativunz seedlings, because the low GA levels have so far precluded a detailed investigation of endogenous GAS. This is a major stumbling block and is reflected in the slow progress made in attempts to elucidate the regulatory role of GAS in stem elongation of pea seedlings. Much of the interest in this subject originates from the elegant studies of Lockhart (1956,1959) with the dwarf and tall Pisum cultivars Progress No. 9 and Alaska. These investigations showed that GA3 treatment promoted the growth rate of light-grown tall seedlings to that of etiolated plants. There was no observable effect when GA3 was applied to tall seedlings grown in darkness although when dwarf plants were used, GA3 promoted the growth of both light- and dark-grown material and the final heights were similar. Lockhart (1959) reasoned that light inhibited stem growth either by (i) inhibiting GA biosynthesis, (ii) enhancing the destruction of endogenous GA or (iii) decreasing the sensitivity of the seedling to GA. These mechanisms could also account for the reduced rate of stem elongation in dwarf varieties. Subsequent attempts to relate endogenous GA-like activity to rates of growth of pea seedlings have been based almost exclusively on bioassay data, and there are several seemingly contradictory reports in the literature. Jones and Lang (1968) estimated the GA content of light- and dark-grown Alaska and Progress No. 9 pea seedlings using agar diffusion and solvent extraction procedures. GAl-like and GA5-like compounds were detected

H CH3

‘’\

O coon

a CH2

-OH H CH3

CHZ

COOH GA8

Fig. 30. Proposed metabolism of [17-’H]GAI4by light-grown seedlings of Pisum sarivum cv. Meteor (Durley et al., 1974a,b).

108

ALAN CROZIER

when tissues were extracted, but only the GA -like component diffused from excised shoot apices into agar blocks. There were no significant differences in either diffusible or extractable GA-like activity obtained from light- and dark-grown dwarf and tall plants. Kende and Lang (1964) had previously obtained similar results to Jones and Lang (1968), but in addition demonstrated that while light- and dark-grown Pisum scrtivurn seedlings responded equally well to the CAI-like compound, the growth response of light-grown plants to the GA,-like substance was considerably less than that of darkgrown tissues. GA1 and GAS promoted growth in the same way as the endogenous GA,-like and CA,-like components. These observations support Lockhart’s third hypothesis which suggested that the inhibitory effects of light on stem growth are due to changes in the sensitivity of the seedling to GAS rather than to qualitative or quantitative changes in the GAS themselves. The physiological relevance of these experiments has, however, been questioned by Frydman and MacMillan (1973) who, after identifying GAzo and GA29 in immature pea seed suggested that these GAS and not GAS and GAl were responsible for the two GA-like compounds present in seedling extracts. Although Sponsel and Kirkwood (unpublished data quoted by Hedden et a / . , 1978) have since characterized GAzo and GA29 in Pisz.int seedlings it should be noted that GA29 induces only a relatively small response in GA bioassays (Reeve and Crozier, 1975; see Table VII). Thus, unless G A 2 9 is present in very high amounts, it is unlikely to account for the CAI-like zone of biological activity in Pisum seedling extracts. Kohler (1970) investigated GA levels in a tall variety of Pisunt (Schnabel) and a dwarf cultivar (Kleine Rheinlanderin) and estimated that light-grown plants contained more GA-like activity than dark-grown seedlings, and dwarf varieties more than tall. Thus, there was an inverse correlation between GA content and the rate of stem elongation. The projected GA levels are quite different from those determined by Kende and Lang (1964) and Jones and Lang (1968) and are not in full agreement with earlier data obtained by Kohler (1965). It is difficult to assess whether this is due to the use of different varieties of pea or merely a consequence of the use of different analytical procedures. Kende (1967) estimated that light- and dark-grown Progress No. 9 dwarf pea seedlings metabolize [l ,2-’H]GAI at a similar rate although [1-’H]GA5 is metabolized faster by dark-grown plants (Musgrave and Kende, 1970). Railton (1974a) reported that etiolated seedlings of dwarf peas cv. Meteor convert [l 7-’H]GA9 into [3H]GAzo-like and [3H]12a-hydroxyGA9-like compounds, more readily than light-grown plants, although metabolism into a polar metabolite is unaffected by light. The conversion of [2,3-3H]GA20 into a [’H]GAz9-like component appeared to be reduced in light-grown tissues. The loss of the applied label was not determined in any of these investigations ; instead the size of metabolite pools was monitored. In practice

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

109

this is of limited value as the various products are being further metabolized so that their rate of biosynthesis cannot be ascertained from pool sizes. The relative rates of metabolism in the light- and dark-grown seedlings are more readily assessed by estimating the amount of unmetabolized precursor GA remaining at set intervals after the application of a standard dose. When this is done, there is no significant difference in the rate of metabolism of [2,33H]GA9 applied to light- and dark-grown Progress No. 9 and Alaska pea seedlings (Dunbar and Crozier, unpublished data). There is, however, strong evidence to suggest that the rate of GA biosynthesis varies in these tissues. i n vitro studies by Ecklund and Moore (1974) have shown that the 78,000 x g supernatant from the shoot tips of light-grown Alaska pea seedlings has a greater capacity for synthesizing rnr-kaurene from mevalonic acid than similar preparations from Progress No. 9 seedlings. In addition, cell-free preparations from light-grown shoot tips of both varieties exhibited higher rnt-kaurene synthetase activity than equivalent extracts from their etiolated counterparts. Railton and Reid (l974a) detected six zones of GA-like activity in lightgrown Alaska pea seedlings although the total number of endogenous GAS is likely to be much higher than this figure. Other than GA2,, and GA29 their identity is at present a matter of conjecture, and the manner in which the levels of individual GAS vary in light- and dark-grown dwarf and tall seedlings, is a topic well into the realms of unproductive speculation. Investigations into endogenous GA levels in Pisum seedlings by Kende and Lang (1964), Jones and Lang (1968) and Kohler (1970) utilized what were, at the time, “state of the art” analytical procedures. However it is now evident that the experimental system is so complex and the shortcomings of bioassays, used in conjunction with chromatographic techniques of low peak capacity, so severe, that any similarity between estimates of GA-like activity and the true GA status of the samples will be extremely fortuitous. There have been considerable advances in the analytical and separatory sciences in recent years and from the discussion in Section 11, it is clear that procedures are becoming available which, if correctly used, can provide accurate analysis of trace components in highly impure samples. It will be of interest to see what progress is made when these methods are applied to the quantitative analysis of endogenous GAS from Pisum sativum seedlings. Despite the shortcomings of the estimates of GA-like activity in Pisum seedlings it is evident that slow growing tissues do contain GAS. Thus reduced growth rates are unlikely to be a direct consequence of a blockage at an early point in the GA biosynthesis pathway as it is in the d 5 mutant of Zea mays. It is therefore doubtful that comprehensive information on the levels of individual GAS in Pisum seedlings will, on its own, provide a clear view of the role played by GAS in regulating the rate of stem elongation. The dynamics of the systems will have to be carefully investigated and attempts

110

ALAN CROZIER

made to determine the rate of turnover of endogenous GAS as it is this, rather than pool sizes, which may determine the quantity of GA that is available for growth. Rates of turnover are notoriously difficult to estimate (see Brown and Wetter, 1972) and at the very least it is necessary to establish rates of synthesis, investigate the compartmentation of site of synthesis and catabolism and determine the rates of transport between them, before even rudimentary conclusions can be drawn. At the present time, the information available on G A biosynthesis pathways in seedling material in general is very limited indeed and even less is known of the kinetics involved. Railton (1974b) has reported that the rate of turnover of GA20in dwarf pea seedlings is enhanced by cytokinin treatment. Unfortunately, the data presented are insufficient to even start to meet the criteria listed above, and the conclusion must therefore be seriously questioned until a much more thorough study is undertaken. Other pieces of fragmentary evidence do, however, suggest that further patient experimentation will provide insights into the complexities of GA metabolism at the cellular and sub-cellular level. For instance, studies on the early stages of G A biosynthesis have shown that etioplasts isolated from dark-grown Pisum shoot tips, but not mitochondria, can convert [ 1-3H]copalyl pyrophosphate to [3H]ent-kaurene. In contrast [ 1-14C], geranylgeranyl pyrophosphate does not serve as an effective substrate for ent-kaurene production in the in vitro etioloplast preparations (Simcox et al., 1975). It is not known if this is due to the conversion of geranylgeranyl pyrophosphate acting as a regulatory point in the GA biosynthesis pathway, or a consequence of the instability of copalyl pyrophosphate synthetase. The biosynthesis of [ 14C]ent-kaurene from [ 14C]geranylgeranyl pyrophosphate has however been observed in sonicated chloroplast preparations from Pisum seedlings (Moore and Coolbaugh, 1976). Direct proof that mevalonic acid is converted to ent-kaurene is lacking, as isolation of the organelle in aqueous media results in the loss of mevalonic acid-kinase activity, which is required for activation of the initial steps in the terpenoid pathway. Chloroplasts isolated in a non-aqueous medium retain mevalonic acid-kinase activity (Buggy et a]., 1974), implying that they do have the capacity to synthesize ent-kaurene from mevalonic acid. Indirect evidence to support this view was obtained when Moore and Coolbaugh (1976) showed that the in vitro production of ent-kaurene from [2-'4C]mevalonic acid by 100,000x g supernatant preparations from Alaska pea shoot tips was enhanced by the addition of chloroplast enzymes. The synthesis of GAS from ent-kaurene has not been demonstrated in chloroplast preparations from peas or any other species for that matter. However, chloroplasts from Hordeum distichon incorporate ent-kaurenol into ent-kaurenal, and ent-kaurenoic acid into ent-7a-hydroxykaurenoic acid (Murphy and Briggs, 1975), while Hordeum vulgare chloroplasts have been shown to convert ['4C]ent-kaurenoic acid into an unidentified GA-like sub-

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

111

stance (Stoddart, 1969). Chloroplast-rich preparations from Pisum sutivum seedlings contain at least five GA-like substances (Railton and Reid, 1974a) and can also metabolize [3H]GA substrates (Railton and Reid, 1974b; Railton, 1977). The only report in which [3H]GA metabolites from chloroplasts have been convincingly identified involves the formation of 16,17 dihydro,l6,17dihydroxy GA9 from [17-3H]GA9,with GAlo,which may be an artifact for the reasons outlined in Table XIII, acting as an intermediary (Railton, 1977). On balance it seems that Pisum chloroplasts may well support the entire GA biosynthetic sequence. In quantitative terms it is difficult to gauge their contribution to the overall GA complement of the plant and a considerable amount of critical investigation will be necessary if detailed information on the involvement of chloroplasts in GA biosynthesis is to be forthcoming. 4. Phaseolus coccineus and Phaseolus vulgaris ( a ) Seeds. Large numbers of GAs have been found in seeds of the broad bean Phaseolus vulgaris and the scarlet runner bean Phuseolus coccineus. GAl,GA4,GA5,GA6, G A S , G A l 7 GA20, , GA29r GA37,GA38 and GA44r as well as GASglucosyl ether and the glucosyl esters of GA1,GA,, GA37and GA,,, have all been shown to be present in extracts from developing or mature seed of Phaseolus iiulgaris (West and Phinney, 1959; Durley er a/., 1971 ; Hiraga et a/., 1972, 1974a,b; Yamane et ul., 1977). The spectrum in Phaseolus coccineus seed is very similar as G A , , GA3,GA,, GAS,GA6,GAs, GA17, GA19, GA20,GA28, GA3,, GA38and GA,, have all been identified along with the glucosyl ether of GAS and undetermined conjugates of GA17, GAZoand GA28 (Jones, 1964; Sembdner et a/., 1968; Schreiber et al., 1970; Durley et al., 1971; Gaskin and MacMillan, 1975; Sponsel and Albone, unpublished data). Studies by Durley et al. (1971) with Phaseolus coccineus seeds of increasing size, indicate that the GA levels vary as the seeds mature (Fig. 31). The pattern is similar, although not identical, to that observed in Pisum sativum seed with the main GA present in the later stages of development being GASrather than GA29. Consideration of the structures of endogenous C19- and C 2 0 - G Ain ~ Phaseolus coccineus and Phaseolus vulgaris, with regard to hydroxylation at the 3P- and 13a-positions (see Table XIV) suggests that two biosynthetic pathways may be operating as the result of a branch at an early point in the sequence. One branch would involve 3P-hydroxylation and lead to GA37 and GA,, while the alternative route would result in 13ahydroxylation of a C20-GAand give rise to G A , 7 , GA19 and GA,, as well as GAS,GA6 and GA20.Convergence of the pathways would facilitate the production of 3P,13a-dihydroxy GAS, such as G A , , GA3, GAS,GA28 and GA38.More precise details of the operation of such pathways are not available as there are no reports on the metabolism of potential C20-GAinter-

TABLE XIV

3fi-hydroxy, 13a-hydroxy und 3fi.13a-dihydroxy

c,9-and CzO-und C 2 0 - G Afrom ~ seed of Phaseolus vulgaris and Phaseolus coccineus. (After Sponsel et al., 1979)

Species

__-

3fi-OH

Phaseolus vulgtrris

~~

I3a-OH

~

~~~

3fi.13a-diOH

3B-OH

13a-OH

3/?,13a-diOH

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

4-

0 -

-

c

.

-

-

--

113

GA5/20

Seed length (rnm)

Fig. 31. Estimated changes in the endogenous levels of GA,, GASIZO, GA6 and GABduring maturation of seed of Phuseo/us coccineus (Durley ei d., 1971).

mediates in seed of either Phaseolus vulgaris or Phuseolus coccineus. However [3H] labelled endogenous C19-GAs have been fed to developing seed of Phaseolus vulgaris c. 10 days and 18 days after anthesis (Yamane et al., 1975, 1977). The conversions that were observed are summarized in Fig. 32. [1,2-3H]GA1applied to 10-day-old seed yielded [3H]GA8, [1,2-3H]GA4 was metabolized to [3H]GA8via [3H]GA1,while [2,3-3H]GAzowas converted to [3H]GAz9as well as [3H]GA1and [3H]GA8.[l-3H]GA5 did not yield any identifiable metabolites. Evidence of the existence of similar pathways was obtained with feeds to 18-day-old Phaseolus vulgaris seed. However, the kinetics of the system were different primarily because of a marked enhancement of the activity of glycosylating enzymes with [3H]GA8glucosyl ether being the major metabolite obtained from [l ,2-3H]GA1, [l ,2-3H]GA4, [1-3H]GA8and [2,3-3H]GA20.In addition, as shown in Fig. 32, [’H]GA1 glucosyl ester, [3H]GA4glucosyl ester and putative glucosyl ethers of both [3H]GA1and [3H]GA20were also formed. [1-3H]GA5 applied to the older

GA4glucosyl ester

GAI glucosyl ester

0

3 p - hydroxylation pathway

+-b

GAB glucosyl ether

HO CH3

COOH

CHZ

GA4

130- hydroxy lat ion pathway

-b+

G A ~ oglucosyl ester (tentative) GA20

GAS glucosyl ester (tentative ) GAS

t t

13a- hydroxylation pathway

GA29

Fig. 32. G A metabolism pathways in developing seed of Phaseolus vulgaris (Yamane et a!., 1975, 1977).

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

115

seeds yielded a putative glucosyl ether of t3H]GAS,['HIGAS glucosyl ether and [3H]GAB.['H]GA6 is the expected intermediate in the conversion of [1-3H]GA5to ['HIGAB. However, it was not detected although GAGis a naturally occurring constituent of the seed and it has been shown to be converted to GAB by Phaseolus coccineus (Sembdner et a/., 1968). The origins of GASare unknown and there were no indications of it being synthesized from either [1,2-3H]GA1or [2,3-3H]GA2~. The data suggest that the main GA biosynthesis pathway in Phuseolus vulgaris seed leads to GABand that its 3-deoxy analog, GA29ris on a minor route while in Pisum sativum the major pathway appears to proceed via GA29. The subsequent fate of the main 2P-hydroxy GAS in Phaseolus vulgaris and Pisum sativum is also different. In Pisum, catabolism of GA29 gives rise to an @-unsaturated ketone and compound B, with only small quantities of a putative conjugate of GA29 being detected (see Fig. 29). Glycoxylation is the dominant mechanism in Phuseolus vulgaris with GAB being readily converted to GAB glucosyl ether. However, the 3P-hydroxy derivative of the a,Punsaturated ketone has been detected in Phaseolus vulgaris seed (Sponsel et a/., 1979) implying that a portion of the GABpool may undergo catabolism by a route analogous to that operating in Pisum sativum. The 2P-hydroxylation of GAl to form GABhas been studied using a cellfree preparation from the cotyledons of germinating Phaseolus vulgaris seed (Nadeau and Rappaport, 1972; Patterson and Kappaport, 1974; Patterson et al., 1975). Enzyme activity located in the 95,000 x g supernatant, requires F e + + or F e + + + ,NADPH and O2 co-factors and is insensitive to CO. The conversion of [1,2-'H]GA1 to ['HIGAS involves the loss of [3H] from the 2-position which results in the stoichiometric formation of [3H]H20. The cell-free system exhibits high stereospecificity as it does not 28-hydroxylate ~ ~16-keto ) GA1. either pseudo GAl [ 3 ~ - h y d r o x y G A or ( b ) Seedlings. GC-MS has been used to identify GAl, GA4, GA5 and GAZ0in extracts of Phaseolus coccineus seedlings although bioassays indicate the presence of many more as yet unidentified GAS (Crozier et ai., 1971; Bowen et a/., 1973). The metabolism of a number of [3H]GAs has been investigated using seven-day-old light-grown seedlings (Nash and Crozier, 1975; Nash, 1976; Nash et al., 1982). [3H]GAs injected into the apical bud were retained, with little metabolism, in the apical region, whereas injection into the hypocotyl, 5 mm below the cotyledonary node, resulted in redistribution of label throughout the seedling and a more extensive conversion of the applied [3H]GA to other products. These observations are illustrated in Table XV with typical data obtained using [l ,2-3H]GA1. Basal injection of [3H]GAs was also associated with a more substantial accumulation of metabolites. This method of application was therefore used when investigating GA metabolism pathways in Phaseolus coccineus seedlings. Although bioassays indicate that light-grown Phaseolus coccineus seed-

116

ALAN CROZIER

TABLE XV Distrihutioir qf radioartivity in Phaseolus coccineus srrdlings 24 h nfter injecting [I,23H]GA1 inro either the upicnl bud (A) or the hypocotyl (B) (Nash and Crozier, 1975) distribution of [3H]" Tissue Apical bud Stem Cotyledons H ypocotyl Roots

A

B

95.8 0.5 2.5 0.3 0.9

12.1 55.4 11.7

18.2 2.6

"Overall recovery of applied label 66.37, (A). 24.6';b (B) ~

lings contain only nanogram quantities of endogenous GAS (Fig. 12, Table VIII), preliminary feeds with [3H]GAsrevealed that there were no qualitative changes in the [3H]metabolite profile when the substrate levels were increased from c. 50 ng to 10 pg seedling- '. The investigation into the effects of precursor dose was combined with an evaluation of the metabolism periods required to produce an accumulation of optimal levels of conversion products. Experiments using purification and analytical procedures outlined in Section I1 were then carried out to identify the various [3H]GA metabolites. After extraction of the plant tissues, acidic ethyl acetate-soluble and acidic n-butanol-soluble fractions were obtained. Ethyl acetate extracts were purified by SEC (Reeve and Crozier, 1976) which provided a convenient means of separating low molecular weight (LMW) free GAS from high molecular weight (HMW) GA conjugates. The putative conjugates in the HMW ethyl acetate fraction and the butanol extract were hydrolysed with cellulase to release free GAS. The extracts were then further purified on charcoal-celite before being subjected to preparative HPLC (Reeve et al., 1976). [3H]GA metabolite peaks were benzyl esterified and re-examined by preparative HPLC prior to being analysed by silica gel adsorption HPLC (Crozier and Reeve, 1977; Reeve and Crozier, 1978). Identification of the [3H]GAs was based on HPLC retention indices obtained with an on-line radioactivity monitor (Reeve and Crozier, 1977). When sufficient sample was available, characterizations were confirmed with mass spectra obtained by direct probe mass spectrometry. In view of the low levels of native GAS in Phaseolus coccineus seedlings, it is valid to conclude that the GA benzyl ester mass spectra were derived from metabolite GAS and not endogenous species. [1,2-3H]GA1, [1,2-3H]GA4 and [1-3H]GA8 feeds indicated a GA,+ GA1-+GA8 pathway with all three GAS yielding HMW conjugates. [2,3-3H]GAzo gave rise to a [3H]GAz0 conjugate and trace amounts of

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

1 I7

[3H]GA3.Although the seedlings metabolized [1-3H]GA5,no metabolites 3GA9was converted to [3H]GA5and [2,3-3H]GA9 were detected. [1-3H]d2~ to [3H]GA20and [3H]GA3.However, as neither precursor has been isolated from Phuseolus coccineus, it is unlikely that native GAS and GA20are synthesized by these routes. Their appearance as metabolites is probably the result of the low substrate specificity of the enzymes involved in the 13ahydroxylation of endogenous CZO-GAs.The results are summarized in Fig. 33. It is interesting to note that [1-’H]GA8 was metabolized far more rapidly than any of the other [3H]GAs,with only 7.9% of the applied radioactivity being recovered after a 2 h metabolism period. Much of the radioactivity became associated with a volatile component, presumably [3H]Hz0,which was lost via transpiration and during preliminary partitioning of extracts. The only [1-3H]GA8 metabolite to be detected was a butanolsoluble conjugate with properties similar to those of GAS glucosyl ether. This compound was present at low levels indicating that it was either a minor catabolite or that it was being converted to other products. The detection of potential catabolites of [1-3H]GA8 and the [3H]GA8conjugate may well have been obscured by rapid rates of turnover, as well as low specific activities resulting from partial or complete expulsion of [3H] from the 1 position on the ent-gibberellane skeleton. In an effort to obtain information on CZO-GAprecursors of GA4, GAS and GA20in Phuseolus coccineus seedlings, the metabolism of GA1zaldehyde and GA14 was investigated. The seedlings rapidly metabolized [17-3H]GA14. After 8 h, only 52% of the applied radioactivity remained, distributed between [17-3H]GA14(35.5%) and a [3H]GA14conjugate (16.5%). No free [3H]GAmetabolites were detected in the LMW ethyl acetate extract, perhaps because of an absence of rate limiting steps in the ensuing metabolic sequence or conversions associated with a loss of [3H] from C-17. O n occasions, preparative HPLC traces indicated the presence of very small amounts of LMW [3H]GAmetabolites but their appearance was transitory and attempts to induce their accumulation with GA3and GA4cold traps were unsuccessful. In contrast to [17-3H]GA14, [17-3H]GA12aldehyde gave rise to several metabolites. This is illustrated in Fig. 34 which shows preparative HPLC traces of the LMW and hydrolysed HMW ethyl acetate extracts and of the hydrolysed butanol fraction. Most of the metabolite peaks yielded two to three products on benzyl esterification. Despite their number, none of the metabolites had HPLC retention characteristics corresponding to those of either G A l , GA4,GA5or GAzo.Because the applied label became associated with so many products, none of the metabolites was present in sufficient quantity to permit identification by mass spectrometry. The experiments therefore provide no positive information on the endogenous C20-GAprecursors of C1,-GAS in Phaseolus coccineus seedlings. Crozier and Reid (1971, 1972) investigated the involvement of roots in

GA4 conjugate

A2v3 GA,

GA, conjugate

GA5

Fig. 33. G A metabolism pathways in light-grown Phaseolus roccineus seedlings (Nash, 1976; Nash et al., 1982).

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

119

Retention time (min 1

Fig. 34. Preparative HPLC traces of LMW and hydrolysed H M W ethyl acetate extracts and the hydrolysed butanol-soluble fraction obtained from light-grown Phaseolus coccineus seedlings eight hours after applying [l 7-’H]GAI2aldehyde. Column, stationary phase, mobile phase and flow rate: as in Fig. 7. Detector: radioactivity monitor 1800 c.p.m. full-scale deflection (Nash, 1976; Nash et al., 1982).

GA biosynthesis in light-grown Phaseolus coccineus seedlings. Extracts from

control plants and seedlings from which root apices had been removed, were chromatographed on a Powell and Tautvydas (1967) silica gel partition column. Twenty-five fractions were collected and analysed with the Tanginbozu dwarf rice (Murakami, 1968) and barley aleurone bioassays (Jones and Varner, 1967). Several zones of GA-like activity were detected with major

120

ALAN CROZIER

peaks being located in fractions 7-9 and 12-15. A comparison of the biological activity in fractions 12-15 with the data of Crozier et al. (1971) suggested that GAl was present. On similar grounds, it was thought likely that fractions 7-9 contained GA19.However, GAS and GAZ0also elute in this zone and the identification of these GAS in extracts from Phaseolus coccineus seedlings by Bowen et al. (1973) implied that they, rather than GA19,were responsible for the GA-like activity in fractions 7-9. Removal of the root apices from seedlings resulted in the disappearance of the GA,-like activity, which was the major peak in control plants, and a concomitant increase in activity in the GAS and/or GAZ0zone. The data therefore suggest that shoot-synthesized GAS and/or GAZ0may be transported to the root apices where conversion to GA1 occurs. Subsequent studies have, however, failed to produce any evidence to support this hypothesis. [1-3H]GASand [2,3-3H]GAz0applied to the shoot apex of Phaseolus coccineus seedlings are not transported to the roots in substantial quantities ; neither precursor appears to be conkerted to GA1 and there are no marked differences in the [1-3H]GASand [2,3-3H]GAzometabolite profile obtained from intact seedlings, excised shoots and excised roots (Nash and Crozier, 1975; Nash, 1976; Nash et al., 1982). Railton (1979) has also reported that the transport of [2,3-3H]GAZ0is seemingly not compatible with the proposals of Crozier and Reid (1971, 1972). Further detailed experimentation will therefore be necessary if the significance of the changes in GA-like activity in Phaseolus coccineus seedlings following root surgery is to be determined. The effects of light on the GA metabolism and growth of Phaseolus coccineus seedlings have been investigated by Bown et a / . (1975) and the results are somewhat different to the data obtained with Pisum sativum discussed in Section III.C.3b. Light inhibits the rate of stem elongation in Phaseolus coccineus seedlings and GA promotes growth in light but not in darkness. This indicates that the availability of GA is a limiting factor only in the growth of light-grown plants. However, the effect of light is not solely to reduce the amount of GA available for growth as its inhibitory effects on stem elongation are not completely counteracted by application of exogenous GA (Fig. 35). The rate of metabolism of applied [3H]GAsis much higher in light-grown plants. In addition, light lowers the estimated level of endogenous GA-like activity c. 20-fold, although the means by which this reduction occurs is by no means clear as pool sizes merely represent the balance between rates of GA formation and utilization. There is a lack of information on rates of GA synthesis so it would be premature to assume that the increased capacity for GA metabolism is the major causal factor in the reduction of GA levels in light-grown plants. This is an important consideration as ultimately the amount of GA available for growth is more likely to be related to the rate of GA synthesis than be regulated by GA pool sizes. While the relevance of the metabolism of [3H]GAs to the growth process is un-

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

121

'"1

--g 120h

c

g

w

100-

80-

60-

,

40)

0

01

1

10

70

100

700

Gibberellin A, ( p g )

Fig. 35. Effect ofexogenous GA4 on stem growth of light- and dark-grown Phaseolus coccineus seedlings. Data expressed as mean stem length i standard error (Bown et al., 1975).

determined, the possibility of secondary control of GA availability through the operation of a competitive utilization pathway cannot be excluded. For instance, although there is a reduced capacity to metabolize GAS in darkgrown Phaseolus coccineus seedlings a greater proportion may be involved in growth than in light-grown plants in which metabolism could be primarily associated with de-activation or diversionary pathways. 5 . Conversion of CIO-GAs to CI9-GAs The studies by Graebe et al. (1974a,b,c, 1980) with Cucurbita maxima cell-

free preparations, indicate that the oxidative sequence at C-20 prior to the formation of C19-GAs,follows the general scheme outlined in Fig. 36, with the C-20 aldehyde acting as the immediate precursor of both y-lactonic CI9-GAsand C-20 carboxylic CIo-GAs. The main point of uncertainty in the pathway is the identity of the intermediate between the C-20 methyl and C-20 aldehydic CIo-GAs. d-lactonic CIo-GAs do not appear to fulfil this role as GA15undergoes 3P-hydroxylation to form GA37which is not further metabolized. Although critical evidence is lacking, it may be that the hypothetical C-20 alcohol shown in Fig. 36 is the intermediate in the conversion. On extraction, C-20 alcohol CIo-GAs would almost certainly lactonize to

122

R

ALAN CROZIER

Q-1-

CH3

RJ-J]-RG}-R CH,OH H

CH \H 3 COOH C - 2 0 methyl Cz0-GA

CH3 \COOH C-20olcohol CZaGA [ hypothetical I

H

CH \COOH C-20aldehydic C2cGA

CH3 7 - lactonic C,,-GA

‘a} Jj

0 ---- CH, R

CH3 8-lactonic C G -,A

\H CH3 COOH C - 2 0 carboxylic C2G ;P

Fig. 36 Oxidation sequence at C-20 prior to the conversion of C,,-GAs to CI9-GAs by Cucurbita maxima cell free preparations. R-H or OH

produce S-lactonic C20-GAswhich would explain why they have never been isolated as endogenous constituents. This suggestion also implies that 6lactonic GAS such as GA15,GA37rGA38 and GA44 are artifacts, although the possibility that they are also natural products of GA metabolism cannot be ruled out. Gibberella fujikuroi has been widely used to investigate the conversion of Czo-GASto C19-GAs although the results are less straightforward than those obtained with Cucurbita maxima. Fungal cultures convert GA12 to GA9 and GA14to C A I but the point in the pathway at which the C-20 group is expelled has not been demonstrated (Fig. 26). The Czo-GASoriginating from GA12 and GAI4, represent successive stages in the oxidation of the C-20 methyl group and it was originally thought that a C-20 carboxyl function might be removed by oxidative decarboxylation. This now seems unlikely as neither GA13nor GAZ5are converted to CI9-GAsby Gibberella jujikuroi (Cross e t a / . , 1968b; Bearder e f al., 1975a). Dockerill et al. (1977) observed that GAl 3aldehyde is incorporated into GA4,7;(0.9%)and GA3 (12.9%). However, Bearder and Phinney (1979) have shown that the B1-41a mutant converts GA13aldehydeanhydride to GA13 aldehyde (80%) and GAI3(2073, while GA13 alcohol anhydride is metabolized to GA13alcohol(98%) and trace quantities of GA13and GA,,aldehyde. As no C19-GA metabolites were detected it was concluded that GA13 aldehyde, GAl 3alcohol and their anhydrides (Fig. 37) do not act as precursors of CI9-GAs in Gibberella. Although Hanson and Hawker (1972) have reported the conversion of GA13anhydride (Fig. 37) to GA3 by Gibberellu cultures the data are equivocal as only 0.015% of the applied label was incorporated into GA,. Evidence obtained by Bearder et al. (1976b) con-

123

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

GA12 alcohol

HO CH3

CH20H

CHZ

COOH GA13alcohol anhydride

GA13 alcohol

COOH

GA13 aldehyde anhydride

GA13aldehyde

0----co

GA,3 anhydride

Fig. 37. Structures of GAI,alcohol, GA,,alcohol. GA,,alcohol anhydride, GA,,aldehyde, GA,,aldehyde anhydride and G A , ,anhydride.

vincingly demonstrates that the h-lactone G A , 5 is not an intermediate in the conversion of G A 1 2to GA9, and by analogy, the involvement of GA3, in the biosynthesis of GA4 from GA14 is similarly excluded. However, the data do not rule out the possible involvement of the corresponding C-20 alcohol CIO-GAs in the production of C19-GAs. Unexpectedly, in view of the synthesis of GA, from GA36by Cucurbitu maxima, Gibberella fujikuroi does not convert the C-20 aldehydes, GA24 and GA36rto CI9-GAs (Bearder et ul., 1975a). This could, however, be due to poor substrate penetration of the fungal hyphae. Cross and Norton (1966) speculated that C-20 was lost by decarboxylation of a B,y-unsaturated acid. This theory was disproved when Hanson and

124

ALAN CROZIER

peracid oxidation

Fig. 38. A Baeyer-Villiger oxidation.

0

CH3

\ / 0

CH3

\ /

C

0

C I

'80

@-D

Fig. 39. A biological Baeyer-Villiger reaction : the metabolism of pregn-4-ene-3,20-dione by CIadesporrum resinae (Nakano et a / . , 1968).

White (1969) demonstrated that all the hydrogen atoms at the C-1, C-5 and C-9 positions were retained in the transformation of C2,-GAs to C ,-GAS by Gibberella jiijikuroi. It was postulated by Hanson and White (1969) that the C-20 group might be lost at the aldehyde stage by a Baeyer-Villiger (1 899) type oxidation. Baeyer-Villiger oxidations are unusual in that they involve the insertion of an oxygen atom a to a carbonyl function to form an ester (Fig. 38). Traditionally, the reaction is associated with the action of a peracid. Although this is difficult to envisage in a biological system, the existence of analogous enzymic processes is a distinct possibility as BaeyerVilliger type oxidations are quite common in the microbial degradation of steroid ketones (Fig. 39) (Smith, 1974; see Bearder and Sponsel. 1977). In

TABLE XVI Metabolites produced from [19-"0]GA12 alcohol, containing 55 atom % ["O], by Gibberella fujikuroi mutant Bl-4Ia (Bearder et al., 1976bj Metabolite

"801%

125

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

the case of the C-20 aldehydic C20-GAstructure (VI) illustrated in Fig. 40, a Baeyer-Villiger type oxidation would produce an intermediate (VII) which could undergo acyl-oxygen fission, hydrolysis and lactonization to form a CIg-GA (VIII)

a 1 a] H

\/

O

/O

H-C?

' 0

Baeyer - Villiger

Alkyl- oxygen

____t

R

oxidation

R

CH3 "0

2,

"OH

(E)

F Lfission yI-oxygen

R

7-

} J ? H' $ 80R

H

CH3

(mn)

Half label retained

k H +

H

] J - J R

H+

H3

H CH3

(X) All label retained

Fig. 40. Proposed mechanisms for the conversion of C2,-GAs to CI9-GAs by Cihberelln fujikuroi. R = H or OH (after Bearder and Sponsel, 1977).

Bearder et al. (1976b) obtained an insight into the mechanism involved in the loss of the C-20 group during the conversion of C 2 0 - G Ato ~ C19-GAsby Gibberellafujikuroi. [19-'80]GA12alcohol (Fig. 37) containing 55 atom % of ['*O] was fed to cultures of the GA deficient mutant B1-41a and the metabolites analysed after periods of two and five days. In the virtual absence of native GASany fall in the ["O] content of the metabolites, as determined by mass spectrometry, is due to loss of label rather than dilution by endogenous ['60]GAs. The results presented in Table XVI show that both oxygen atoms are incorporated into the y-lactone in the C-19 carboxyl group of C20-GA~ ring ofC19-GAs.Bearder et al. (1976b) concluded that GA,,alcohol and its metabolites are not covalently bound through the C-19 carboxyl group to enzymes catalysing the formation of C19-GAs, although this view is debatable since knowledge of the enzymes involved and their surrounding

126

ALAN CROZIER

microenvironment is lacking. However, the data do show that the conversion of CZ0-GAs to C19-GAs involves an intermediate with an electrophilic centre at C-10 which undergoes nucleophilic attack by the C-19 carboxyl group to form the 1'-lactone. Bearder and Sponsel (1977) point out that this rules out the lactonization mechanism of VII discussed above, as this would involve loss of half the ['*O]label (Fig. 40). They propose an alternative mechanism in which the intermediate ester (VII) is eliminated by alkyl-oxygen fission rather than acyl-oxygen fission. The carbonium ion (IX) could then cyclize by attack of the C-19 carboxyl group to generate the 7-lactone C19-GA (X) which would contain both oxygen atoms from the C-19 carboxyl group (Fig. 40). A pragmatic touch, in view of the uncertainty concerning the immediate CzO-GAprecursor of CI9-GAsin Gibberella, is that the model is flexible as in addition to the C-20 aldehyde (VI), the carbonium ion (IX) could be generated from Czo-GAs with either a C-20 carboxyl or alcohol group. Dockerill and Hanson (1978) have reported that the C-20 carbon of CZO-GAsformed from ['4C]ent-kaurene is released as ['4C]COz during the production of C I9-GAs by Gibberella fujikuroi. Dilution analysis failed to find any radioactivity associated with either formaldehyde or formic acid. Dockerill and Hanson (1978) postulated that the entire sequence from a putative peracid intermediate (XI) to the formation of a y-lactone CI9-GA (XII) may occur via a single concerted process (Fig. 41). This proposal, along with that of Bearder and Sponsel(l977) illustrated in Fig. 40, is highly speculative. The data presented by Dockerill and Hanson (1978) should not be regarded as irrefutable proof of the immediacy of C 0 2 to the expulsion of the C-20 carbon from CZo-GAs.It is possible, for instance, that the ['4C]C02 arose from small ['4C]labelled pools of either formaldehyde or formic acid which were not detected because of the inherent lack of sensitivity of dilution analysis. At present the only firm conclusion that can be made is that the conversion of CZO-GAsto C19-GAs by Gibberella fujikuroi must in effect follow the general scheme in Fig. 42. Far too little is known to permit meaningful speculations about the oxidative level at C-20 prior to its expulsion. The fact that Czo-GAs representing all stages of C-20 oxidation are produced by the fungus is of little value since ifa Baeyer-Villiger type mechanism is operating, and this is by no means certain, the insertion of oxygen between C-10 and C-20 could occur at any stage from the alcohol to acid. Alternatively, the entire sequence from a C-20 methyl Czo-GA onwards could take place without the intermediates involved leaving the enzyme complex. It seems likely that progress will be enhanced by the development of a cell-free system from Gibberella fujikuroi which can efficiently convert CZo-GAsto C19-GAS. In view of the in vitro metabolism of GA36 to GA4 by Cucurbita maxima (Graebe et al., 1980), it will be of interest to learn if the inability of Gibberella

127

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

(XII) Fig. 41. Concerted decarboxylation and lactonization of a hypothetical C,&A intermediate. R = H or O H (Dockerill and Hanson, 1978).

peracid

Fig. 42. General schematic for C,,-GA formation in Gibberellafujikuroi.

fujikuroi to carry out similar conversions in vivo is due to either (i) a major difference in the mechanism of C19-GA biosynthesis or (ii) a limited availability of substrate at the enzymic site in the fungal hyphae. Exact details of the conversion mechanism will, however, probably remain a mystery until such time as a purified, characterized enzyme is isolated that can synthesize a CI9-GAfrom a CZO-GAsubstrate. D. SITES OF GIBBERELLIN BIOSYNTHESIS AND COMPARTMENTATION

The sequential changes in endogenous G A levels associated with maturation of Pisum sativum seed occur in material grown in vitro as well as in vivo (Sponsel and MacMillan, 1977). It is also known that the endogenous GA pools in detached cultured Pisum fruits are depleted by AMO-1618 treatment (Baldev et a / . , 1965). Thus, if Pisum sativum is typical, it appears that GASare synthesized in the developing seeds rather than being imported from the parent plant. The incorporation of [2-14C] mevalonic acid into GAS by liquid endosperm preparations from Marah macrocarpus (West, 1973) and Cucurbita maxima (Graebe et al., 1972) provides direct evidence of GA biosynthesis in seeds as does the conversion of ['4C]ent-kaurene to1 GA aldehyde by cell-free preparations from Pisum sativum seed (Ropers et a/., 1978). Coolbaugh and Moore (1972) have shown that ent-kaurene synthetase is principally located in the cotyledons of immature Pisum seed and is not found in either the embryo or the seed coat.

128

ALAN CROZIER

The embryo of germinating barley grain may be a site of GA biosynthesis although the evidence is circumstantial and based on reports that (i) the production of GA-like activity in excised embryos is inhibited by CCC and phosphon D, and (ii) exogenous GA can substitute for the embryo in the induction of a-amylase synthesis in the aleurone layer of germinating seed (Paleg, 1960; Yomo, 1960; Yomo and Iinuma, 1966; Radley, 1967). Despite careful experimentation, Murphy and Briggs (1973) were unable to obtain any metabolic conversion of ['4C]ent-kaurene by cell-free preparations from germinating barley seeds. They did, however, show that the endogenous entkaurene content of the grain falls during the first twelve hours of germination. While this implies that in vivo metabolism of ent-kaurene takes place, it does not necessarily mean that GASare among the products. There is a paucity of critical evidence on sites of GA biosynthesis in seedling systems. Most, ifnot all, of the available evidence is physiological rather than biochemical in nature and is based on the detection of GA-like activity in xylem sap (Carr et al., 1964; Phillips and Jones, 1964) and the subsequent experiments of Jones and Phillips (1966, 1967) which indicated that the young leaves of the apical bud and the root apices of Helianthus annus seedlings act as sites of GA biosynthesis. Jones and Phillips (1966,1967) showed that when excised apical buds from Helianthus seedlings were incubated on agar for twenty hours, more GA, as determined by PC and bioassay, diffused into the agar than was extracted from the tissue. As no reduction in the extractable GASwas observed during the diffusion period, it was concluded that the apical buds had synthesized GAS in amounts corresponding to the level of diffusible GA-like activity. Similar results were obtained with root apices, although stem internodes and sub-apical root sections appeared not to act as sites of GA biosynthesis. Other publications followed with GA-like activity being detected in phloem exudate (Hoad and Bowen, 1968) and in the xylem sap of a range of species (Reid and Carr, 1967; Skene, 1967; Jones and Lacey, 1968; Luckwill and Whyte, 1968; Reid and Burrows, 1968) and the levels were shown to be affected by CCC (Reid and Carr, 1967), excision of various organs (Sitton et al., 1967; Crozier and Reid, 1971, 1972) and flooding of the root system (Reid et al., 1969; Reid and Crozier, 1971). In strictly biochemical terms, these reports demonstrated very little regarding sites of GA production and as a consequence, the widely accepted view that GAS are synthesized in root and shoot apices is, in essence, solely based on the results of Jones and Phillips (1966, 1967). This is unfortunate as these data provide only indirect physiological evidence, and with the passage of time they have begun to look somewhat dated and less convincing. This is because the supposition that root and shoot apices synthesize GA is dependent upon the PC-bioassay data of Jones and Phillips (1966,1967) providing a relatively accurate quantitative measure of the GA content of the tissues and agar diffusates. In view of the

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

129

shortcomings of bioassay-based estimates discussed in Section I1 .E.1, it is difficult to accept such data at face value especially as comparisons were made between GA-like activity in highly impure plant extracts and diffusates containing considerably less inhibitory material. There is therefore, despite much interest in the subject, an absence of compelling evidence on potential sites of GA biosynthesis in seedlings. Although in the 1960s there were no viable alternatives to the analysis of GASin shoot and root apices and diffusates by bioassay, this situation is now changing. If the main endogenous GAS in Heliunthus unnus seedlings could be identified, it should be feasible to employ the highly sensitive GC-SICM or HPLC procedures reviewed in Section II.E.2. to monitor diffusible and extractable GA levels. The Helianthus system appears to be particularly suitable for a detailed re-investigation and it is perhaps surprising that it has not already taken place, as Phillips (1971) reported that addition of mevalonic acid to the agar on which excised tissues are incubated, increased the levels of GA-like activity diffusing from shoot apices to such an extent, that microgram quantities of GA3 equivalents were produced by only fifty apical buds. This holds out the prospect of a considerable easing of the analytical situation. What is more, if the increased GA levels are due to enhanced biosynthesis, it may be possible to demonstrate an incorporation of radioactive mevalonic acid into GAS and this would certainly constitute unequivocal evidence of shoot apices acting as a site of GA biosynthesis. Evidence for the compartmentation of GAS in chloroplasts has already been outlined in Section III.C.3b. Other cellular components that might be involved in GA biosynthesis and metabolism include etioplasts, proplastids and vacuoles. Both vacuole and proplastid fractions from Hordeum vulgare convert [I ,2-’H]GA1 to [3H]GA8 and [3H]GA8 glucosyl ether (Rappaport and Adams, 1978). It is also known that proplastid extracts from Marah macrocarpus endosperm catalyse the synthesis of ent-kaurene from geranylgeranyl pyrophosphate and copalyl pyrophosphate although similar preparations from etiolated Pisum sutivum shoot tips and Ricinus communis endosperm convert only copalyl pyrophosphate to ent-kaurene. There is little or no enf-kaurene synthetase activity in the mitochondria1 fraction from these tissues (Simcox et a / . , 1975). Chloroplasts are impermeable to mevalonic acid so the precursor must be produced in situ (Rogers et ul., 1966). In contrast mevalonic acid can be transported across etioplast membranes although the capacity is lost following illumination (Cockburn and Wellburn, 1974; Wellburn and Hampp, 1976). There is currently no biochemical evidence to implicate etioplasts in the later stages of GA biosynthesis although bioassay data has been used to establish a link between GA-like activity and phytochrome in etioplasts of Hordeum vulgare and Triticum aestivum. The subject has attracted much attention and there is a general belief that compartmentation of endogenous

130

ALAN CROZIER

growth regulators coupled with environmentally-mediated release mechanisms could offer a ready solution to many problems of plant development including the interchangeability of red light and exogenous GA in the control of leaf unrolling in etiolated cereals (see Loveys and Wareing, 1971b; Stoddart, 1976). Reid et al. (1968) were the first to show a substantial transient rise in endogenous GA-like activity 15 min after treating etiolated barley leaf segments with red light (660 nm). This was thought to be the result of an enhanced rate of GA biosynthesis as no increase in activity occurred when the leaf sections were pretreated with either AMO-1618 or CCC. Evans and Smith (1976a) obtained similar data with authenticated barley etioplast preparations and also showed that far-red light (730 nm) reverses the red lightinduced increase in GA-like activity (Fig. 43). Thus, the effects of light on G A-like substances are mediated via phytochrome which appears to be located in the etioplast envelope (Evans and Smith, 1976b). It is also known that the GA-like activity that accumulates after red light treatment becomes associated with the incubating medium and is not retained by the etioplasts to any extent and that sonication of non-irradiated etioplast preparations results in a four-fold increase in extractable GA (Table XVII) (Evans and Smith, 1976a). Evans and Smith (1976a) propose that red light induces the photoconversion of phytochrome from the P, ,to the P,, form so facilitating changes in membrane permeability which enable GAS in the etioplast to be

Fig. 43. The effect of red and far-red light on the level of GA-like activity extracted from intact Hordeum vulgare etioplasts. GA-like activity is measured by a modification of the barley aleurone bioassay of Jones and Varner (1967). LSD-least significant difference when P=0.05 (Evans and Smith, 1976a).

131

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

TABLE XVll GA-like uctivity extracted f r o m intuct Hordeum vulgare rtioplust prrpurutions und rtioplasr prrpurutions ultrusonicutrd prior to rxtruction. ( A f t e r Evuns und Smith, 1 9 7 6 ~ ) “

Etioplasts

Dark

5 min red light

T

T

T

P

S

4.0 17.5

10.8 17.9

22.0 24.9

3.0

24, I

5 min red light+5 min dark

_________~~ ~

Intact Sonicdted

~

-

GA-like activity determined by a modified barley aleurone bioassay (Jones and Varner, 1967) and data expressed as pmol maltcse rnin mg I protein. Extracts made from either the total suspension (T) or the pellet (P) and supernatant (S) obtained by centrifugation o f T for 1 min at 6000 x g.

-’

released into the surrounding medium. They further suggest that the depletion in GA levels resulting from this efflux leads to increased production of active GASpossibly by release of feed back control of late steps in the biosynthetic pathway. Similar investigations have been carried out with wheat, in which a 5 rnin exposure of etiolated leaf segments to red light also induces a large increase in endogenous GA-like activity which peaks after about 15 rnin and thereafter rapidly declines (Beevers et a/., I970 ; Loveys and Wareing, 1971a). The response has been associated with etioplasts and it has been proposed that the controlling influence of phytochrome is exerted through changes in the permeability of the etioplast envelope to GAS, the release of GAS from a “bound” form and an enhancement of de novo GA biosynthesis (Cooke and Saunders, 1975a,b; Cooke et al., 1975). Cooke and Kendrick (1976) further suggest that the etioplast envelope may be a site of GA metabolism and one effect of red light could be to induce the release and subsequent metabolism of “bound” GAS. However, when the experimental data are closely scrutinized, these proposals appear to be distinctly speculative. Estimates of GA-like activity in the wheat etioplast preparations are based on lettuce hypocotyl bioassay data. In most instances the bioassay responses are far too small to command confidence and the claimed variations in GA levels must therefore be of questionable significance (Graebe and Ropers, 1978). What the data from Triticum aestivum and Hordeum vulgare do demonstrate is that the red-far-red reversible changes in GA-like activity are controlled by phytochrome which seems to be located in the etioplast membrane. As Hedden et al. (1978) noted, it is unclear whether the increases in GA-like activity in etioplast preparations are due to an enhanced rate of synthesis, increased metabolism of specific precursor GAS, release of “bound” GAS from membranes, increased membrane permeability or a

132

ALAN CROZIER

combination of these and other factors. Progress in identifying the mechanisms involved is unlikely to be spectacular because the technical problems encountered require investigators to have not only expertise in GA analysis and metabolism but, also, a theoretical and practical knowledge of phytochrome and experience of procedures for the isolation of organelles from plant tissues. IV. STRUCTURE-ACTIVITY RELATIONSHIPS The relative activities of individual GAS in the barley aleurone, dwarf pea, lettuce hypocotyl, Tanginbozu dwarf rice and cucumber bioassays are presented in Table VII. The data are compiled from Crozier et a / . (1970), Yokota et a / . (1971), Fukui et a / . (1972), Yamane et a / . (1973), Reeve and Crozier (1975), Hoad et al. (1976) and Sponsel et a / . (1977). Most of these reports, as well as those of Brian et a / . (1964, 1967) contain data on other bioassays but the information is less comprehensive, at least as far as natural GASare concerned. There is, unfortunately, little or no data available on the biological activity of most of the more recently discovered GAS. It can be seen from Table VII that each GA exhibits a range of biological activities and that each bioassay responds to a characteristic spectrum of GAS. High activities are shown in most bioassays by GA1, GA3, GA, and GA32; GAS, GA6, GA36and GA3, all induce a good response but of a lower order. Other GAS, in particular GA9, GAlo, GAZ3,GA24 and GA3s show a tendency for species specificity, being highly active in some bioassays yet inducing poor response in others. Consistently low activity is exhibited by GAS, GA1 GAi2,GAi3rGA14,GAi7,GA21,GA25,GA27,GA2srGA29,GA33,GA34

and GA40; while GA26,GA40, GA43rGA46 and G A 5 1 are inactive in most test systems. Some GA glucosyl esters and ethers induce a response in certain bioassays. However, it is thought that they are probably inactive per se and that their activity is a consequence of the release of the aglycone following hydrolysis by plant enzymes and micro-organisms (Yokota er a / . , 1971; Sernbdner et a/., 1976). The individual bioassays vary in their specificity. The barley aleurone bioassay, for instance, responds only to a limited number of GAS while, in contrast, the dwarf rice bioassay responds in some degree to all the GAS that have so far been tested with the exception of GA21rGA25, GA26, GA43rGA46 and GA51. It has been hypothesized that there are two main factors playing an important role in GA structure-activity relationships (Brian et al., 1967; Crozier et a/., 1970). The “goodness of fit” theory contends that the activity of a GA arises from the degree to which it fits a hypothetical receptor molecule or site. Thus any alteration in the “ideal” GA structure will lead to a lowering of biological activity. Variations in the shape of the receptor molecule from one plant species to another will account for the different re-

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

133

sponse spectra of the assay systems. It is further proposed that the response of a bioassay can be affected by the ability of the plant tissues to metabolize the applied GA. In such circumstances the response induced by a GA can be influenced by the relative ease with which it is converted to the “active” structure. The main problem appears to be assessment of the degree to which GA interconversions participate in a particular test system. The two extremes are represented by the high specificity of the barley aleurone bioassay and the ubiquitous response of the Tanginbozu dwarf rice. Perhaps in the former instance, “goodness of fit” is the major selectivity-deciding factor, while in the latter, a capacity for extensive GA metabolism would seem more likely than a lack of specificity at the receptor site. The information obtained from a comparison of GA structures and biological activities is limited because of the fragmentary nature of the bioassay data. Slight variations in the bioassay techniques can radically affect the sensitivity of the response. A further complication is that GAS are rarely tested over a full concentration range from sub-threshold to saturation levels. It is therefore only strictly valid to compare the activities of GAS in a particular bioassay when all the compounds have been tested at the same time. An additional reason for exercising caution, is the purity of the GA standards. An inactive GA need only contain 0.1%of a highly active contaminant GA to erroneously exhibit low to moderate activity. Thus, it is not unexpected that certain GAS have shown widely varying relative activities when tested by different investigators. Table VII was drawn up by ranking GAS in five categories ranging from very high biological activity to inactive. Even this general comparison is very subjective and, where appropriate, details should be checked by reference to the original bioassay data, most of which are either presented by Hoad et al. (1976) and Sponsel et al. (1977) or have been compiled by Reeve and Crozier (1975). The greatest degree of structural diversity among the 62 characterized GAS, is mediated by the relative state of oxidation at C-20 (Fig. 2) and the presence or absence of 3p- and 13a-hydroxyl groups (Table I). Thus a comparison of the biological activity of the appropriate GA pairs, makes it possible to assess the effect of each substituent as well as interactions between groups of substituents. Table XVIII provides the basis for such a comparison. With the exception of 2a- and 2,O-hydroxy GASthere are usually insufficient analogs to allow firm conclusions to be drawn on the effects of other groups on biological activity. Table XIX shows the activities of 2a- and 2p-hydroxy GAS and their deoxy analogs. An estimate of the relative effects of the different GA configurations on biological activity, is summarized in Table XX. Comparison of the activities of the appropriate GA combinations listed in Table XVIII, shows that in the barley aleurone bioassay, a 3P-hydroxy-glactone structure is more or less mandatory if a GA is t c exhibit high activity.

134

ALAN CROZIER

TABLE XVlII Rdativr biological activities of C ,9- and Czo-GAswith and without hvdroxyl groups at the C-3 and C-13 positions" ~~~~

~~

Hydroxylation Bioassay Barley aleurone

Dwarf rice

Dwarf pea

Lettuce

Cucumber

___ GA configuration None

20-CH3 6-lactone 20-CHO y-lactone 20-COOH

0 0 0

30OH

0

13a-OH

38,13a-diOH 0

-

+

++ ++ +++ +

0

0

0

-

0

+ +++ +++ ++ +

0

0

0

0

0

0

+++ + +++ +++ + ++ +++ ++ +++

20-CH3 d-lactone 20-CHO y-lactone 20-COOH

0

0

-

20-CH3 b-lactone 20-CHO y-lactone 20-COOH

+ ++ +++ +++

20-CH 3 ii-lactone 20-CHO y-lactone 20-COOH 20-CH3 ii-lactone 20-CHO y-lactone 20-COOH

+

0

+ ++ +++ ++

+ + ++

++ 0 +++ 0

+

++ ++ +++

++ + ++ +

0

++f

+++ +++ 0

++ ++++

+

+++ +++ +

+

0

-

+ +++

+ + +++

0

0

0

0 0 0

0

Relative activities: + + + +, very high; + + +, high; + + , moderate; inactive. See Table I to identify individual GA configurations.

+ ++

0

0

+, low; 0, very low to

Neither group by itself contributes significantly to activity. Among other 3P-hydroxy GAS, only those possessing either a 8-lactone function or a C-20 aldehyde group, which may be in equilibrium with a d-lactol ring (Fig. 2) (Harrison et al., 1968),exhibit appreciable activity. It is possible that these GASare active because the six-membered h-lactone and h-lactol functions can partially substitute for a five-membered y-lactone ring at the receptor site. The activity of a GA is also considerably enhanced by the additional presence of a 13a-hydroxylgroup, That this less rigorous requirement

135

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

TABLE XIX Relnrive biological activities of 2a- and 2b-hydroxy G A Sm d their 2 - d e o x ~analogs

Bioassay Substitution Gibberellin

Barley aleurone

Dwarf pea

+

++

-

0 0

+++

+++

2-deoxy 2aQH 2fl-OH

-

2-deox y 2a-OH 2fl-OH

-

Lettuce hypocotyl

+++ +

Dwarf rice

++ +

Cucumber hypocotyl

+++ ++

0

0

0

+++ ++

0

++ +

++ +

0

++ + +

0

2-deoxy 28-OH

0 0

0 0

0 0

0 0

0

2-deox y 2j-OH

-

0 0

0

0

+++ + ++

+++ + +++ + +++ +

2-deoxy 2fl-OH 2-deoxy 28-OH 2-deoxy 2b-OH Relative activities: inactive.

+

++++ +++ + + ++ ++ + 0 + + + 0

+

0

+++ 0

+

+

0 0

++ 0

+++

0 0

0

+ + + +, very high; + + + , high; + + . moderate; +, low; 0, very low to

can be mimicked by a 16a-hydroxyl group, would appear to support the suggestion that “goodness of fit” is the dominant mechanism controlling biological activity in this bioassay. There is virtually no interaction between C-3 and C-I3 hydroxylation and the nature of the C-20 group in the Tanginbozu dwarf rice bioassay. Activity appears to be related to the degree of oxidation of the C-20 function irrespective of hydroxylation at C-3 and C-13. The activity of C20-GAs is much higher than in the barley aleurone bioassay. The y-lactonic CI9-GAs and C-20 aldehydic C2&iAs show broadly similar activities. Although &lactonic and C-20 methyl C 2 0 - G Aare ~ also active, the response is of a lower order in most instances. The C-20carboxyl CZo-GAsexhibit only low activity. Ifa GA metabolism pathway such as that illustrated in Fig. 36 were operating in Tanginbozu rice seedlings, it would be possible to visualize the activity of

136

ALAN CROZIER

TABLE XX Relative efSeect o f G A structural groups on hiologicd activity” Bioassay

Barley aleurone

Structural configurations Activating

Deactivating

y-lactone*3P-OH* 13a-OH (v. high) y-lactone*3/l-OH (high) y-lactone*d2, (moderate) 6-lactone*3/l-OH (low) 20-CHO*3p-OH (low)

la-OH (moderate) 2/1-OH (moderate)

18-COOH (high) 20-COOH (high) 2p-OH (high) 2a-OH (moderate) 13a-OH (low)

Dwarf pea

y-lactone*3P-OH (high) y-lactone*’d 2 , (high) y-lactone (low) 3P-OH*13a-OH (high)

Lettuce hypocotyl

y-lactone*d (high) y-lactone (high) h-lactone (low)

Dwarf rice

Almost all configurations except those listed in next column

la-OH (high 2P-OH (high) 3-keto (high) 18-COOH (high) 20-COOH (high) 2a-OH (moderate)

Cucumber hypocotyl

y-lactone (high) 6-lactone (high) 20-CHO (high)

la-OH (high) 12a-OH (high) 13a-OH (high) 2a-OH (moderate)

18-COOH (high) 2P-OH (high) la-OH (moderate) 2a-OH (moderate) 12a-OH (moderate)

“An asterisk * between groupings refers to the combined presence of the groups and in practice often bears no relation to the sum of the individual effects.

both C-20 methyl and aldehydic C2,,-GAs being determined by both the efficiency with which they are converted to CI9-GAs and their ability to substitute for the optimized y-lactone structure at the receptor site. In the case of &lactonic GAS, which may be artifacts that are not metabolized to C19-GAs, activity would be determined by “goodness of fit”. The isomeric 20,4y-lactone of GA4 (Fig. 44) is known to induct: a similar response to GA4

137

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

HO

.pJa CH3

COOH

20,4y-lactone isomer of GA4

CHZ

0

CH3

COOH

CH2

1 9 . 2 ~ - l a c t o n eA'.'o isomer of GA3

Fig. 44. Structures of the 20,4y-lactone isomer of G A L and the 19,2y-lactone d'.''isomei of GA,.

in the dwarf rice bioassay (Sponsel et al., 1977), while the 19,2y-lactone A's "isomer of GA, (Fig. 44) is only marginally less active than GA3 (Hoad eta!., 1976).This implies that a 19,lOy-lactone bridge per se is not an absolute requirement for high activity and that other structures of similar configuration can serve as effective substitutes. Oxidation of the C-20 aldehyde to form a C-20 carboxylic acid, would be a side branch in the synthesis of C19-GAs(Fig. 36). It may be that C-20 carboxyl C20-GA~are relatively inactive because they do not match the receptor site and further oxidation of the carboxylate ion does not occur. Other GAS showing low activity in the dwarf rice bioassay are also higher oxidation products. That they may likewise represent metabolic "dead ends" could be an explanation for their negligible activity. For instance, oxidation of GAZ0at the C-18 locus to give GAzl would be analogous to the CZo-GAs mentioned above. However, one cannot discount the possibility that the greater polarity of the tricarboxylic GAS adversely affects their ability to penetrate plant tissues. Leaf sheath elongation in rice is stimulated by a much wider range of GAS than a-amylase production in the aleurone cells of the same species (Ogawa, 1967). This may indicate that the GA receptor sites in the two tissues have different configurations. Alternatively, the receptor molecules may be similar but the leaf sheath responds to more GAS because it is capable of carrying out more extensive GA conversions than the aleurone layer. Evidence of the possible involvement of GA metabolism in leaf sheath elongation comes from studies on two dwarf varieties of rice, Tanginbozu and Waito C (Murakami, 1970a). GAS with a 38-hydroxyl group show similar relative activities when applied to the two varieties. However, the response of Waito C to 3-deoxy GAS is significantly lower than that of Tanginbozu. This suggests that the possession of a 38-hydroxyl group is obligatory if a GA is to fit the receptor site and that Waito C, unlike Tanginbozu, is unable to 38hydroxylate 3-deoxy GAS. Pseudo GAL (3a-hydroxy GA20) is much less active than GA1 in both Tanginbozu and Waito C (Murakami, 1970b). It therefore seems that a 3a-hydroxyl group cannot substitute for a 38-hydroxyl

138

ALAN CROZIER

function at the receptor site. The low activity of pseudo GA, compared to that of GA20.further suggests that the 3a-hydroxyl function is reasonably stable as far as dehydroxylation and conversion of pseudo GA, to GAIOis concerned. On purely theoretical grounds, it is improbable that further hydroxylation of pseudo GA, at the C-3 locus would occur. 2P-hydroxy GAS are relatively inactive in the dwarf rice and other bioassays. The deactivation is fairly stereospecific since 2a-hydroxylation is not as effective in reducing biological activity (Table XIX) (Sponsel et ul., 1977). GA, and GA4 both undergo 2,&hydroxylation in rice seedlings to yield GAB and GA34 respectively (Durley and Pharis, 1973; Railton et a/., 1973). 2f2-hydroxylation of GAS is frequently observed in plant tissues including those used for bioassays. GA, is metabolized to GA8 by barley aleurone layers (Nadeau and Rappaport, 1972; Nadeau et ul., 1972), lettuce hypocotyls (Silk rt ul., 1977), and cucumber hypocotyls (Rudich et a/., 1976) as well as pea seedlings (Durley et ul., 1974b) which also convert GAzo to GA29 (Railton et ul., 1974a,c). The fact that the 2P-hydroxy y-lactonic C19-GAs are much less active than their immediate precursors, indicates that they do not fit the receptor site and that the 2P-hydroxylation step may be a means whereby plant tissues deactivate GAS.In order to study further the biological effects of C-2 hydroxylation MacMillan and co-workers (unpublished data quoted by Hedden, 1979) investigated the biological activity of GA derivatives in which 2P-hydroxylation was blocked. In the d 5 maize mutant bioassay (Phinney, 1956),2P-methyl GA4 was found to be more active than GA4, although 2P-methoxy GA9 exhibited reduced activity equivalent to that of its 2P-hydroxy analog, GAS The response to 2,2 dimethyl GA4 was quite dramatic as it was 100 times more active than GA3with the growth rate being enhanced for a prolonged period of time. Further results are awaited with interest. C19-GAs,GA3 and G A 7 ,are the most active GAS in the dwarf The pea bioassay. The data in Table XVIII show that high activity is confined to 3fl-hydroxy and 3b,13a-dihydroxy GAS which, surprisingly, have not so far been identified as native constituents of Pisum sutivum tissues. Within these two groups, the best response is elicited by y-lactonic C ,9-GAs.C-20 methyl, 6-lactonic and C-20 aldehydic C20-GAsalso exhibit activity but of a lower order. Once again the C-20 carboxylic acids are inactive. These broad trends can be interpreted in terms of either a “goodness of fit” hypothesis or a GA interconversion mechanism. The conversion of [17-3H]GA,4 to CAI by dwarf pea seedlings (Durley et ul., 1974a,b) does not necessarily lend support for the latter proposal because GA14 is inactive in the dwarf pea bioassay at the 1 pg seedling- level, although Brian et ul. (1967) report that higher doses do elicit a response. A small portion of [1,2-3H]GA,fed to dwarf pea epicotyls becomes noncovalently bound to two protein fractions with estimated molecular weights

,.

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

139

of c. 60,000 and 500,000 daltons. The binding is specific in so far as GAS and GAsglucosyl ether, which are metabolites of G A , , are not associated with either protein fraction (Stoddart rt ul., 1974). In view of the high biological activity of G A 1 in the dwarf pea bioassay, it is tempting to speculate that the GA ,-protein complexes could contain the primary GA receptor. Alternatively, enzymes responsible for the metabolism of C A I may be present and the observed binding could represent enzyme-substrate complexes. At present, critical evidence is lacking and on the basis of the available data it is impossible to assess whether or not the GA-protein complexes have any physiological relevance. The lettuce hypocotyl bioassay appears to be fairly specific. It can be seen from Table XVIII that activity is almost completely restricted to the ylactonic C19-GAs,although in some instances the h-lactonic C20-GAsact as effective substitutes. Unlike the barley aleurone bioassay, there does not appear to be any significant interaction between the lactone requirement and C-3 and C-13 hydroxylation. However, data obtained by Nash rt u / . (1978) with excised lettuce hypocotyls, imply that the response to GA9 may be dependent upon its conversion by 13a-hydroxylation to GA20. Although some of the h-lactone GAS are moderately active, their C-20 aldehyde equivalents show only low activity. This perhaps suggests that applied C z O GASare not efficiently converted to active CI9-GAs,and that the activity of the S-lactone compounds results from the six-membered ring mimicking the y-lactone at the receptor site. Brian et a / . (1967) tested over one hundred GAS and GA derivatives and observed that the presence of a 13a-hydroxyl group resulted in a very marked reduction in activity in the cucumber hypocotyl bioassay. This finding contrasts with other assay systems where the effect of 13a-hydroxylation is less clear cut. At first sight, the obvious explanation for the unusual specificity of the cucumber seedlings is that the main requirement for activity is the presence of a lactonic bridge (the y-lactone displays similar activities to both the S-lactone and the C-20 aldehyde which, as mentioned earlier, may be in equilibrium with a 6-lactol structure) and for steric reasons a 13a-hydroxyl group results in a marked mismatching of the GA and the receptor molecule. With perhaps more surety it can be said that in the cucumber bioassay, 13a-hydroxy GAS per se possess low activity, and any mechanism to convert them to their more active 13-deoxy counterparts is apparently lacking. Further support for this view can be obtained from the data of Yaniane et ul. (1973).GA30 is the 12a-hydroxy analog of GA, and the presence of the 12ahydroxyl group is sufficient to strongly deactivate the molecule in the cucumber bioassay. Similarly, GA3 induces no response, whilst deoxy GAS (A2, 3GA9)is quite active. Brian et ul. (1967) showed that the 3-keto derivatives of GA4 and GA7 are active in the cucumber hypocotyl test but not in the dwarf pea or lettuce hypocotyl bioassays. This illustrates the cucumber

140

ALAN CROZIER

system’s lack of dependence on the nature of the C-3 substituent. The 3-keto derivatives of GA1 and GA3, on the other hand, are inactive, once again demonstrating the powerful deactivating influence of the 13a-hydroxyl group. In keeping with this reasoning, it is likely that the inactivity of GA33 in the cucumber bioassay, results from the presence of the 12a-hydroxyl group rather than the keto group at the C-3 position. However, as Yamane et a / . (1973) point out, in this instance the possible influence of the 1/3hydroxyl group cannot be discounted. Thus far it has been shown that the activities of GAS in cucumber hypocotyl elongation can be predicted on the grounds that either a 19,lOor 19,20 lactone bridge is essential for activity. This activity is in turn severely reduced by the presence of either a 12a- or 13ahydroxyl group. An exception to this rule, which defies a ready explanation, is the high activity of GA32since the presence of both 12a- and 13a-hydroxyl groups should in theory render this GA completely inactive. It seems too simple an explanation to suggest that the 15P-hydroxyl group reactivates the molecule. Despite the fact that large numbers of GASand GA derivatives have been tested in the various bioassay systems, only the broadest of trends in structure-activity relationships can be observed. This is perhaps to be expected in view of compounding factors such as penetration of the applied GA to the site of action, effects of the solvent in which the GA is dissolved and the relative stability of the exogenous GA in plant tissues. Although relatively little detailed information is available on the cellular and subcellular localization of GAS, it does seem unavoidable that application of exogenous GA will destroy this compartmentation to some extent. It is therefore possible that at least a part of the bioassay response results from an abnormal chemical modification of the applied GA that takes place before a normal state of compartmentation can be re-established. For instance, the biological activity of certain GA glucosides may be due to the release of free GASby the action of non-specific glucosidases which usually would not come into contact with endogenous GA glucosides. Despite these complications, if the general trends in bioassay specificity are borne in mind, it is possible to make reasonably accurate predictions about the potential activity of untested compounds of known structure.

V. CONCLUSIONS It is evident, if judged only by the proliferation in the number of GAS isolated from natural sources, that there has been significant progress in GA biochemistry in recent years. Techniques such as SICM and HPLC have the potential to routinely analyse sub-nanogram quantities of endogenous GAS and methods have been proposed that will enable the accuracy of such estimates to be assessed. The use of these procedures should permit substantially

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better measurements of GA pool sizes than have previously been possible. In vivo and in vitro metabolism studies have enabled details of G A biosynthesis pathways to be unravelled to varying degrees in Gibberella jujikuroi and several higher plants. This is however only the beginning, as a comprehensive study of G A biosynthesis must embrace the kinetics of the system under study and to this end methods will have to be devised to ascertain rates of turnover of individual G A pools. Studies on the sub-cellular compartmentation of GAs are still in their infancy although preliminary experimentation indicates that G A Sare located in both etioplasts and chloroplasts. The ultimate aims and aspirations of many gibberellinologists are focused on the means whereby G A S affect plant growth and development. Despite the progress that has been made in elucidating G A biosynthesis pathways and the generalized correlations that can be made between the structure, metabolism and biological activity of GAS, an understanding of the mechanisms through which G A Sinfluence growth is as far away as ever. At present nothing is known about either sites of G A action or the nature of the G A growth interface. The underlying problem appears to be that growth is an ill-defined multifarious phenomenon and the sheer vagueness of our concept of the process limits attempts to relate precise knowledge of G A levels, metabolism, catabolism and compartmentation to events such as stem elongation and leaf unrolling. This situation is unlikely to change until growth can be reduced to a number of simple, well defined sub-phenomena such as mechanical properties of the cell wall, microfibril orientation and the axial rate component of cell surface expansion. Without this information advances in G A biochemistry are likely to remain an enigma as far as the control of plant growth and development is concerned.

REFERENCES Alves, L. M. and Ruddat, M. (1979). Plant and Cell Physiol. 20, 123-130. Anderson, J. D. and Moore, T. C. (1967). Plant Physiol. 42, 1527-1534. Arpino, P. J. and Guiochon, G. (1979). Analyt. Chem. 51,682-701. Baeyer, A. and Villiger, V. (1899). Ber. Dtsch. Cheni. Ges. 32, 3625. Bailiss, K . W. and Hill, T. A. (1971). Bot. Rev. 37,437479. Baldev, B., Lang, A. and Agatep, A. 0.(1965). Science 147, 155-157. Bearder, J. R. and Phinney, B. 0.(1979). Abstracts of Tenth International Corlference on Plant Growth Substances. Madison, WI., p p . 42. Bearder, J . R. and Sponsel, V. M. (1977). Biochem. Soc. Trans. 5,569-582. Bearder, J . R., MacMillan, J. and Phinney, B. 0.(1973a). Phyrochern. 12,2655-2659. Bearder, J. R., MacMillan, J. and Phinney, B. 0.(1973b). Phyrocheni. 12,2173-2179. Bearder, J . R., MacMillan, J . , Wels, C . M., Chaffey, M. B. and Phinney, B. 0.(1974). Phytochern. 13,911-917. Bearder, J. R., MacMillan, J. and Phinney, B. 0. (1975a). J . Chem. Soc. Perkin 1, 72 1-726. Bearder, J . R., MacMillan, J., Wels, C. M. and Phinney, B. 0.(1975b). Phytochem. 14, 1741-1748.

142

ALAN CROZIER

Bearder, J . R.. Frydman, V. M.. Gaskin, P., Hatton, I. K., Harvey, W. E., MacMillan, J . and Phinney, B.O. (1976a). J . Cficm. Soc. Perkin 1, 178-183. Bearder, J. R . , MacMillan. J . and Phinney, B. 0.(l976b). J. Cheni. Soc. Cheni. Cornm., pp. 834835. Beeley, L. J., Gaskin, P. and MacMillan, J. (1975). Phytochem. 14, 779-783. Beevers, L., Loveys, B.. Pearson. J . A . and Wareing, P. F. (1970). Plurita 90,286-294. Binks, R., MacMillan, J . and Pryce, R . J. (1969). Phytochem. 8, 271-284. Bombaugh, K. J. (1971). In “Modern Practice of Liquid Chromatography” (J. J. Kirkland, Ed.), pp. 237-285. Wiley-Interscience, New York. Borrow, A., Brian, P. W., Chester, V. E. Curtis, P. J., Hemming, H. G., Henehan, C., Jefferies, E. G., Lloyd, P. B., Nixon, I . S., Norris, G. L. F. and Radley, M . (1955). J . Sci. Food Agric. 6, 34&348. Bowen. D. H., MacMillan, J. and Graebe, J. E. (1972). Phytocheni. 11,2253-2257. Bowen, D. H., Crozier, A,, MacMillan. J. and Reid, D. M. (1973). Phytochern. 12, 2935-2941. Bown, A. W . , Reeve, D. R. and Crozier, A. (1975). Planta 126,83-91. Brian, P. W . , Hemming, H. G. and Lowe, D. (1964). Ann. Bot. 28, 369-389. Brian, P . W., Grove, J. F. and Mulholland, T. P. C. (1967). Phytochem. 6, 1475-1499. Brown, S. A . and Wetter, L. R. (1972). In “Progress in Phytochemistry” (L. Reinhold and Y . Liwschitz, Eds), Vol. 3, pp. 1 4 5 . Interscience Publishers Inc., New York. Browning, G . and Saunders, P. F. (1977). Nature 265,375-377. Buggy, M . J., Britton, G . and Goodwin, T. W. (1974). Phytochern. 13, 125-129. Carr, D. J. and Reid, D. M. (1968). In “Biochemistry and Physiology of Plant Growth Regulators” (F. Wightman and G. Setterfield, Eds), pp. 1169-1 185. Runge Press, Ottawa. Carr, D. J., Reid, D. M. and Skene, K. G. M. (1964). Planra 69, 382-392. Cathey, H. M. and Stuart, N. W. (1961). Bot. Gaz. 123,51-57. Cavell, B. D., MacMillan, J., Pryce, R . J . and Sheppard, A. C. (1967). Phyrochem. 6, 867-874. Cockburn, B. J. and Wellburn, A. R . (1974). J. exp. Bot. 25,3&49. Cooke, N. H. C. and Olsen, K. (1979). American Laboratory 11 (8), 45-60. Cooke, R. J. and Kendrick, R . E. (1976). Planta 131,303-307. Cooke, R. J. and Saunders, P. F. (1975a). Planta 123, 299-302. Cooke, R. J. and Saunders, P. F. (1975b). PIanra 126, 151-160. Cooke, R. J., Saunders, P. F. and Kendrick, R. E. (1975). Planta 124,319-328. Coolbaugh, R. C. and Moore, T. C. (1971). Phytochem. 10,2395-2400. Coolbaugh, R. C., Moore, T. C., Barlow, S. A. and Eckland, P. R. (1973). Phytochem. 12, 1613-1618. Coolbaugh, R. C., Hirano, S. S. and West, C. A. (1978). Plant Physiol. 62,571-576. Cross, B. E. (1968). In “Progress in Phytochemistry” (L. Reinhold and Y . Liwschitz, Eds), Vol. 1, pp. 195-222. Interscience Publishers Inc., New York. Cross, B. E. and Norton, K. (1966). Tetrahedron Letters, pp. 6003-6007. Cross, B. E., Grove, J. F., MacMillan, J., Moffatt, J. S., Mulholland, T. P. C., Seaton, J. C. and Sheppard, N. (1959). Proc. Chem. Soc., pp. 302-303. Cross, B. E., Galt, R. H. B. and Hanson, J. R. (1964). J . Chem. SOC.,pp. 295-300. Cross, B. E., Galt, R. H. B. and Norton, K. (1968a). Tetrahedron 24,231-237. Cross, B. E., Norton, K. and Stewart, J. C. (1968b). J . Chem. SOC.Cornm., pp. 10541063. Cross, B. E., Stewart, J. C. and Stoddart, J. L. (1970). Phytochem. 9, 1065-1071. Crozier, A. and Reeve, D. R. (1977) In “Plant Growth Regulation” (P. E. Pilet, Ed.), pp. 67-76. Springer-Verlag, Heidelberg.

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

143

Crozier, A. and Reid, D. M. (1971). Canad. J . Bot. 48, 967-975. Crozier, A . and Reid, D. M. (1972). In “Plant Growth Substances 1970” (D. J. Carr, Ed.), pp. 414419. Springer-Verlag, Heidelberg. Crozier, A,, Aoki, H. and Pharis, R. P. (1969). J . exp. Bot. 20, 786795. Crozier, A., Kuo. C. C., Durley, R. C. and Pharis, R. P. (1970). Canad. J . Bot. 48, 867-877. Crozier, A., Bowen, D. H., MacMillan, J., Reid, D. M. and Most, B. H. (1971). Plaiira 97, 142-154. Crozier, A., Reid, D. M. and Reeve, D. R . (1973). J . exp. Bot. 24,923-934. Crozier, A., Zaerr, J . B. and Morris, R. 0. (1980). J . Chromatogr. 198,57-63. Crozier, A., Zaerr, J. B. and Morris, R. 0. (1982). J . Chromutogr. (in press). Dennis, D. T. and West, C. A. (1967). J . B i d . Chem. 242, 3293-3300. Dockerill, B. and Hanson, J . R. (1978). Phyrochem. 17,701-704. Dockerill, B., Evans, B. and Hanson, J. R . (1977). J . Chem. Soc. Chem. Comm., pp. 919-921. Douglas, T. J. and Paleg, L. G . (1974). Plant Physiol. 54, 238-245. Diinges, W. (1977). Analyt. Chem. 49,442445. Durley, R. C. and Pharis, R. P. (1972). Plunta 109, 357-361. Durley, R. C., MacMillan, J. and Pryce, R. J. (1971). Pliytochem. 10, 1891-1908. Durley, R. C., Crozier, A., Pharis, R. P. and McLaughlin, G . E. (1972). Phytochem. 11, 3029-3033. Durley, R. C., Railton, I . D. and Pharis, R. P . (1973). Phytochem. 12, 1609-1612. Durley, R. C., Railton. I. D. and Pharis, R . P. (1974a). In “Plant Growth Substances 1973”, pp. 285-293. Hirokawa Publishing Co., Tokyo. Durley, R. C., Railton, I. D. and Pharis, R . P. (1974b). Phytochem. 13,547-551. Durley, R . C., Sassa, T. and Pharis, R. P. (1979). Plant Physiol. 64,214-219. Durst, D., Milano, M., Kitka, E. J., Connelly, S. A. and Grushka, E. (1975). Analyr. Chem. 47, 1797-1801. Ecklund, P. R. and Moore, T. C. (1974). Plant Physiol. 53, 5-10. Evans, A . and Smith, H. (1976a). Proc. Nut. Acad. Sci. U S A 73, 138-142. Evans, A. and Smith, H. (1976b). Nature 259,323-325. Evans, R. and Hanson, J. R. (1972). J . Chem. Soc. Perkin I , 2382-2385. Evans, R. and Hanson, J. R. (1975). J . Chem. Soc. Perkin I , 633-666. Evans, R., Hanson, J. R. and White, A. F. (1970).J . Chem. Soc. Comm., pp. 2601-2603. Fall, R. R. and West, C. A. (1971). J . Biol.Chem. 246,6913-6928. Frankland, B. and Wareing, P. F. (1960). Nature 185,255-256. Frost, R. G. and West, C. A. (1977). Plant Physiol. 59, 22-29. Frydrnan, V. M. and MacMillan, J. (1975). Pluntu 125, 181-195. Frydman, V. M., Gaskin, P. and MacMillan, J. (1974). Plunta 118, 123-132. Fuchs, S. and Fuchs, Y . (1969). Biochim. Biophys. Acta 192, 528-530. Fukui, H., Ishii, H. Koshimizu, K., Katsumi, M., Ogawa, Y. and Mitsui, T. (1972). Agric. Biol. Chem. 36, 1003-1012. Ganguly, S. N. and Sircar, S . M. (1974). Phytochem. 13,1911-1913. Gaskin, P. and MacMillan, J. (1975). Phytochem. 14, 1575-1578. Gaskin, P. and MacMillan, J. (1978). In “Isolation of Plant Growth Substances” (J. R. Hillman, Ed.), pp. 79-95. Society for Experimental Biology Seminar Series No. 4. Cambridge University Press, Cambridge. Gaskin, P., MacMillan, J. and Zeevaart, J. A. D. (1973). PIunta 111, 347-352. Giddings, J. C. (1967). Anulyt. Chern. 39, 1027-1028. Glenn, J. L., Kuo, C. C., Durley, R. C. andPharis, R. P. (1972). Phytochem. 11,345-351. Grabner, R., Schneider, G. and Sembdner, G. (1976). J . Chromatogr. 121, 110-115.

144

ALAN CROZIER

Graebe, J . E. (1968). Phytochem. 7, 2003-2020. Graebe, J. E. (1969). Planta 85, 171-174. Graebe, J. E. (1972). In “Plant Growth Substances 1970” (D. J. Carr, Ed.), pp. 151-157. Springer-Verlag, Heidelberg. Graebe, J. E. and Hedden, P. (1974). In “Biochemistry and Chemistry of Plant Growth Regulators” (K. Schreiber, H . R. Schiitte and G. Sembdner, Eds), pp. 1-16. h a d , Sci. German Democratic Republic Inst. Plant Biochem., Halle (Saale). Graebe, J . E. and Ropers, H. J . (1978). In “Phytohormones and Related Compounds: A Comprehensive Treatise” (D. S. Letham, P. B. Goodwin and T. J. V. Higgins, Eds), Vol. I , pp. 107-203. Elsevier/North-Holland, Biomedical Press, Amsterdam. Graebe, J . E., Dennis, D. T., Upper, C. D. and West, C. A. (1965). J . Biol. Cheni. 240, 1847-1854. Graebe, J. E., Bowen, D. H. and MacMillan, J . (1972). PIanta 102,261-271. Graebe, J. E., Hedden, P., Gaskin, P. and MacMillan, J. (1974a). Phytochem. 13, 1433-1 440. Graebe, J. E., Hedden, P., Gaskin, P. and MacMillan, J. (1974b). Planta 120,307-309. Graebe, J. E., Hedden, P. and MacMillan, J. (1974~).In “Plant Growth Substances 1973”, pp. 26&266. Hirokawa Publishing Co., Tokyo. Graebe, J. E., Hedden, P. and MacMillan, J. (1975). J . Chem. SOC.Chem. Comm., pp. 161-162. Graebe, J. E., Hedden, P. and Rademacher, W. (1980). In “Gibberellins- Chemistry, Physiology and Use” (J. R. Lenton, Ed.). British Plant Growth Regulator Group, Monograph No. 5 , pp. 3147. Grove, J. F. (1961). J . Chem. SOC.,pp. 3345-3347. Halevy, A. H. and Shilo, R. (1970). Physiol. Plant 23 82&827. Hanson, J. R. (1966). Tetrahedron 22, 701-703. Hanson, J. R. (1971). Fortschr. Chem. Org. Naturst. 29, 395416. Hanson, J. R. and Hawker, J. (1972). Tetrahedron Letters, pp. 42994302. Hanson, J . R. and White, A. F. (1969). J . Chem. SOC.Comm., pp. 981-985. Hanson, J. R., Hawker, J . and White, A. F. (1972). J . Chem. SOC.Perkin. 1 , 1892-1895. Harada, A. and Nitsch, J. P. (1967). Phytochem. 6, 1695-1703. Harrison, D. M., MacMillan, J . and Galt, R. H. B. (1968). Tetrahedron Letters 27, 3 137-3 139. Hedden, P. (1978). In “Proceedings of the Plant Growth Regulator Working Group” (M. Abdel-Rahman, Ed.), pp. 3344. Plant Growth Regulator Working Group, Syracuse, New York. Hedden, P. (1979). In “Plant Growth Substances” (B. Mandava, Ed.), pp. 19-56. ACS Symposium Series 111, Amer. Chem. SOC.,Washington, D.C. Hedden, P. and Phinney, B. 0. (1979). Phytochem. 18, 1475-1479. Hedden, P., MacMillan, J. andPhinney, B. 0.(1974). J . Chem. SOC.Perkin 1,587-592. Hedden, P., Phinney, B. O., MacMillan, J. and Sponsel, V. M. (1977). Phytochem. 16, 1913-191 7. Hedden, P., MacMillan, J . and Phinney, B. 0. (1978). Ann. Rev. Plant Physiol. 29, 149- 192. Heftmann, E., Saunders, G. A. and Haddon, W. F. (1978). J. Chromatogr. 156,71-77. Hiraga, K., Yokota, T., Murofushi, N. and Takahashi, N. (1972). Agric. Biol. Chem. 36,345-347. Hiraga, K., Kawabe, S., Yokota, T., Murofushi, N., and Takahashi, N. (1974a). Agric. Biol. Chem. 38,2521-2527. Hiraga, K., Yokota, T., Murofushi, N. and Takahashi, N. (1974b). Agric. Biol. Chem. 38,251 1-2520.

METABOLISM AND PHYSIOLOGY OF GIBBERELLINS

145

Hoad, G . V. and Bowen, M. R. (1968). Planta 82,22-32. Hoad, G. V., Pharis, R. P., Railton, I. D. and Durley, R. C. (I 976). Planfa 130,113-120. Ikegawa, N., Kagawa, T. and Sumiki, Y. (1963). Proc. Japan. Acad. 39,507-512. Ingram, T. J. and Browning, G. (1979). Planta 146,423432. Jones, D. F. (1964). Nature 202, 1309-1310. Jones, M . G., Metzger, J. D. and Zeevaart, J. A. D. (1980). Plant Physiol. 65,218-221. Jones, 0 .P. and Lacey, H. J. (1968). J . Expr. Bot. 19, 52653 1. Jones, R. L. (1968). Planta 81,97-105. Jones, R. L. (1973). Ann. Rev. Plant Physiol. 24,571-598. Jones, R. L. and Lang, A. (1968). Plum Physiol. 43,629-634. Jones, R. L. and Phillips, I . D . J. (1966). Plant Physiol. 41, 1381-1386. Jones, R. L. and Phillips, I . D. J. (1967). Planta 72, 53-59. Jones, R . L. and Varner, J. E. (1967). Planta 72, 155-161. Kagawa, T., Fukinbara, T. and Sumiki, Y. (1963). Agric. Biol. Chem. 27,598-599. Kamienska, A,, Durley, R. C. and Pharis, R. P. (1976). Phytochem. 15,421424. Karger, B. L. (1971). In “Modern Practice of Liquid Chromatography” (J. J. Kirkland, Ed.), pp. 3-53. Wiley-Interscience, New York. Karger, B. L., Kirby, D. P., Vouros, P., Foltz, R. L. and Hidy, B. (1979). Analyt. Chem. 51, 2324-2328. Katsumi, M., Phinney, B. O., Jefferies, P. R. and Hendrick, C. A. (1964). Science 144, 849-850. Kawarada, A. and Sumiki, Y. (1959). Bull. Agric. Chem. SOC.Jap. 23,343-344. Kende, H. (1967). Plant Physiol. 42, 1612-1618. Kende, H. and Lang, A. (1964). Plant Physiol. 39,435440. Knapp, D. R. and Krueger, S. (1975). Analyt. Letters 8,603-610. Knofel, H. D., Miiller, P. and Sembdner, G. (1974). In “Biochemistry and Chemistry of Plant Growth Regulators” (K. Schreiber, H. R. Schiitte and G. Sembdner, Eds), pp. 121-124. Acad. Sci. German Democratic Republic Institute Plant Biochem., Halle (Saale). Kohler, D. (1965). Planta 67,4&54. Kohler, D . (1970). Z . P’anzenphysiol. 62,42&435. Kohler, D. and Lang, A. (1963). Plant Physiol. 38, 555-560. Krishen, A. (1977). J . Chromatogr. Sci. 15,434439. Kurogochi, S., Murofushi, N., Ota, Y. and Takahashi, N . (1978). Agric. Biol. Chem. 42, 207-208. Kurogochi, S., Murofushi, N., Ota, Y. and Takahashi, N. (1979). Planta 146,185-191. Kurosawa, E. (1926). Trans. Hist. Soc. Formosa. 16, 213-227. Lang, A. (1970). Ann. Rev. Plant Physiol. 21, 537-570. Leopold, A. C . (1971). Plant Physiol. 48,537-540. Letham. D. S., Higgins, T. S. V., Goodwin, P. B. and Jacobsen, J. V. (1978). In “Phytohormones and Related Compounds- A Comprehensive Treatise” (D. S. Letham, P. B. Goodwin and T. J. V. Higgins, Eds), Vol. 1, pp. 1-27. Elsevierporth Holland, Biomedical Press, Amsterdam. Lew, F. T. and West, C. A. (1971). Phytochem. 10,2065-2076. Locke, D. C . (1973). J . Chromatogr. Sci. 11, 12C128. Lockhart, J. A. (1956). Proc. Nat. Acad. Sci. USA 42,841-848. Lockhart, J. A. (1959). Plant Physiol. 34,457-460. Lockhart, J. A. (1962). Plant Physiol. 37, 759-764. Lorenzi, R., Horgan, R. and Heald, J. K. (1976). Phytochern. 15,789-790. Lorenzi, R., Saunders, P. F., Heald, J. K . and Horgan, R. (1977). Plant Sci. Letters 8, 179-1 82. Loveys, B. R. and Wareing, P. F. (1971a). Planta 98, 109-116.

146

ALAN CROZIER

Loveys, B . R. and Wareing, P. F. (1971b). Planta 98, 117-127. Luckwill, L. W. and Whyte, P. (1968). In “Plant Growth Regulators”, pp. 87-101. SOC. Chem. Ind. Monograph No. 31. Staples, London. MacMillan, J. (1972). In “Plant Growth Substances 1970” (D. J. Cam, Ed.), pp. 790-797. Springer-Verlag, Heidelberg. MacMillan, J. (1974). In “Biochemistry and Chemistry of Plant Growth Regulators” (K. Schreiber, H. R. Schutte and G. Sembdner, Eds), pp. 3347. Acad. Sci. German Democratic Republic Institute Plant Biochem., Halle (Saale). MacMillan, J. and Pryce, R. J. (1973). In “Phytochemistry” (L. P. Miller, Ed.), Vol. 3, pp. 283-326. Van Nostrand-Reinhold, New York. MacMillan, J. and Suter, P. J. (1958). Naturwissenschaften 46,46. MacMillan, J. and Suter, P. J . (1963). Nature 197, 190. MacMillan, J. and Takahashi, N. (1968). Nature 217, 17&171. MacMillan, J. and Wels, C. M. (1973). J. Chromatogr. 87,271-276. MacMillan, J. and Wels, C. M. (1974). Phytochern. 13, 1413-1417. McFadden, W. H. (1979). J. Chromogr. Sci.17,2-17. McFadden, W. H., Bradford, D. C., Gaines, D. E. and Gower, J . L. (1977). International Laboratory Oct., 5544. McInnes, A. G., Smith, D. G., Durley, R. C., Pharis, R. P., Arsenault, G. P., MacMillan, J., Gaskin, P. and Vining, L. C. (1977). Canad. J . Biochem. 55,728-735. Majors, R. E. (1975). International Laboratory. Nov./Dec., 11-35. Majors, R. E. and MacDonald, F. R. (1973). J . Chromatogr. 83, 169-179. Metzger, J. D. and Zeevaart, J. A. D. (1980). Plant Physiol. 66,844-846. Mishra, D. and Pradham, G. C. (1968). Curr. Sci.,pp. 263-264. Moore, T. C. and Coolbaugh, R. C. (1976). Phytochem. 15,1241-1247. Morris, R. 0.and Zaerr, J. B. (1978). Anafyt. Letters. A l l (l), 73-83. Murakami, Y. (1968). Bot. Mag. (Tokyo) 79,3343. Murakami, Y. (1970a). Jap. Agric. Res. Quart. 5, 5-9. Murakami, Y. (1970b). Bot. Mag. (Tokyo) 83,211-213. Murofushi, N., Irinchijima, S., Takahashi, N., Tamura, S., Kato, J., Wada, Y., Watanabe, E., Aoyama, T. (1966). Agric. Bid. Chem. 30,917-924. Murofushi, N., Nagura, N. and Takahashi, N. (1979). Agric. Bid. Chem. 43, 11591161. Murphy, G. J. P. and Briggs, D. E. (1973). Phytochem. 12,1299-1308. Murphy, G. J. P., and Briggs, D. E. (1975). Phytochem. 14, 429433. Murphy, P. J . and West, C. A. (1969). Arch. Biochem. Biophys. 133,395407. Musgrave, A. and Kende, H. (1970). Plant Physiol. 45,53-61. Nadeau, R. and Rappaport, L. (1972). Phytochem. 11, 1611-1616. Nadeau, R., Rappaport, L. and Stolp, C. F. (1972). Planta 107,315-324. Nakano, H., Sato, H. and Tamoahi, B. H. (1968). Biochim. Biophys. Acta, 164,585-595. Nash, L. J. (1976). Ph.D. thesis, University of Glasgow. Nash, L. J. and Crozier, A. (1975). Planta 127,221-231. Nash, L. J., Jones, R. L. and Stoddart, J. L. (1978). Planta 140, 143-150. Nash, L. J., Reeve, D. R. and Crozier, A. (1982). In preparation. Nicholls, P. B. and Paleg, L. G. (1963). Nature 199,823-824. Ogawa, Y. (1967). Bot. Mag. (Tokyo) 80,27-32. Oster, M. 0. and West, C. A. (1968). Arch. Biochem. Biophys. 127, 112-123. Paleg, L. G. (1960). Plant Physiol. 35,902-906. Patterson, R. J. and Rappaport, L. (1974). Planta 119, 183-191. Patterson, R. J., Rappaport, L. and Breidenbach, R. W. (1975). Phytochem. 14, 363-368. Pharis, R. P. and Kuo, C. C. (1977). Canad. J . For. Res. 7,299-325.

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

147

Phillips, I. D. J. (1971). Planta 107, 277-282. Phillips, I. D. J. and Jones, R. L. (1964). Planta 63, 269-278. Phinney, B. 0. (1956). Proc. Natl. Acad. Sci. U.S.A.42, 185-189. Phinney, B. 0.(1961). In “Plant Growth Regulation” (R. M. Klein, Ed.), pp. 489-501. Iowa State College Press, Ames. Phinney, B. 0. (1979). In “Plant Growth Substances” (B. Mandova, Ed.), pp. 57-78. ACS Symposium Series 111, Amer. Chem. SOC.,Washington, D.C. Phinney, B. 0. and West, C. A. (1961). In “Encyclopedia of Plant Physiology” (W. Ruhland, Ed.), Vol. 14, pp. 1185-1227. Springer-Verlag, Heidelberg. Phinney, B. O., West, C. A,, Ritzel, M. B. and Neeley, P. M. (1957). Proc. Nut. Acad. Sci. U.S.A. 43,398-404. Pitel, D. W., Vining, L. C. and Arsenault, G.P. (1971a). Canad. J . Biochem.49,185-193. Pitel, D. W.,Vining, L. C. and Arsenault, G. P. (1971b). Canad. J . Biochem.49,194200. Powell, L. E. (1963). Nature 200,79. Powell, L. E. and Tautvydas, K. J. (1967). Nature 213,292-293. Pryce, R. J. (1973). Phytochem. 12,507-514. Pryde, A. and Gilbert, M. T. (1979). “Applications of High Performance Liquid Chromatography”. Chapman and Hall, London. Rademacher, W. and Graebe, J. E. (1979). Biochem. Biophys. Res. Comm. 91,3540. Radley, M. (1956). Nature 178, 1070-1071. Radley, M. (1967). Planta 75, 164-171. Railton, I. D. (1974a). Plant Sci. Letters 3, 207-212. Railton, I. D. (1974b). Plantn 120, 197-200. Railton, I. D. (1976). S. Afr. J . Sci. 72, 371-377. Railton, I . D. (1977). Z. PJanzenphysiol. 81, 323-329. Railton, I . D. (1979). Z . PJanzenphysiol. 91,283-290. Railton, I. D. and Reid, D. M. (1974a). PLant Sci. Letters 2, 157-163. Railton, I. D. and Reid, D. M. (1974b). Plant Sci. Letters 3,303-308. Railton, I. D. and Rechav, M. (1979). Plant Sci.Letters 14,75-78. Railton, I . D., Durley, R. C. and Pharis, R. P. (1973). Phytochem. 12,2351-2352. Railton, I . D., Durley, R. C. and Pharis, R. P. (1974a). In “Plant Growth Substances 1973”, pp. 294-304. Hirokawa Publishing Co., Tokyo. Railton, I . D., Durley, R. C. and Pharis, R. P. (1974b). Plant Physiol. 5 4 , 6 1 2 . Railton, I. D., Murofushi, N., Durley, R. C. and Pharis, R. P. (1974~).Phytochem. 13, 793-796. Rappaport, L. and Adams, D. (1978). Phil. Trans R. Soc. London B.284,521-539. Reeve, D. R. and Crozier, A. (1974). J . Expt. Bot. 25,431-445. Reeve, D. R. and Crozier, A. (1975). In “Gibberellins and Plant Growth” (H. N. Krishnamoorthy, Ed.), pp. 35-64. Wiley Eastern Ltd., New Delhi. Reeve, D. R. and Crozier, A. (1976). Phytochem. 15,791-793. Reeve, D. R. and Crozier, A. (1977). J . Chromatogr. 137,271-282. Reeve, D. R. and Crozier, A. (1978). In “Isolation of Plant Growth Substances” (J. R. Hillman, Ed.), pp. 41-77. Society for Experimental Biology Seminar Series No. 4. Cambridge University Press, Cambridge. Reeve, D. R. and Crozier, A. (1980). In “Hormonal Regulation of Development I. Molecular Aspects of Plant Hormones” (J. MacMillan, Ed.). Encyclopedia of Plant Physiology, New Series Vol. 9, pp. 203-280. Springer-Verlag, Heidelberg. Reeve, D. R., Yokota, T., Nash, L. J. and Crozier, A. (1976). J. exp. Bot. 27,1243-1258. Reid, D. M. and Burrows, W. J . (1968). Experimentia 24,189-190. Rkid, D. M. and Carr, D. J. (1967). Planta 73, 1-1 1. Reid, D. M . and Crozier, A. (1970). PLanta 94,95-106. Reid, D. M. and Crozier, A. (1971). J . Expt. Bot. 22, 3948.

148

ALAN CROZIER

Reid, D. M. and Crozier, A. (1972). In “Plant Growth Substances 1970” (D. J. Carr, Ed.), pp. 420-427. Springer-Verlag, Heidelberg. Reid, D. M., Clements, J. B. and Carr, D. J. (1968). Nature 217, 580-582. Reid, D. M., Crozier, A. and Harvey, B. R. M. (1969). Plunta 89,37&379. Robinson, D . R. and West, C. A. (1970a). Biochem. 9,7&79. Robinson, D. R. and West, C.A. (1970b). Biochem. 9,8&89. Rogers, L. J . , Shah, S. P. J. and Goodwin, T. W. (1966). Biochem. J . 99,381-388. Ropers, H. J., Graebe, J. E., Gaskin, P. and MacMillan, J . (1978). Biuckem. Biophys. Res. Comun. 80,69&697. Ruddat, M., Lang, A. and Mosettig, E. (1963). Narurwissetischrlften 50,23. Ruddat, M., Heftman, E. and Lang, A . (1965). Arch. Biuchem. Biophys. 111, 187- 190. Rudich, J., Sell, H. M. and Baker, L. R. (1976). Plant Physiol. 57, 734-737. Sawada, K. (1912). Formosan Agr. Rev. 36, 10-16. Schreiber, K . , Weiland, J., and Sembdner, G. (1970). Phytochem. 9, 189-198. Sembdner, G. and Schreiber, K. (1965). Phytochem. 4, 49-56. Sembdner, G., Weiland, J., Aurich, 0. and Schreiber, K. (1968). In “Plant Growth Regulators”, pp. 70-86. SOC.Chem. Ind. Monograph No. 31. Staples, London. Sembdner, G., Borgmann, E., Schneider, G., Liebisch, H. W., Miersch, O., Adam, G., Lishchewski, M. and Schreiber, K. (1976). Planta 132,249-257. Shechter, I. and West, C. A. (1969). J. Biol. Chem. 244, 3200-3209. Silk, W. K., Jones, R. L. and Stoddart, J. L. (1977). Plant Physiol. 59,211-216. Simcox, P. D., Dennis, D. T. and West, C. A. (1975). Biochem. Biophys. Res. Comm. 66, 166172. Simpson, T. H. (1968). J . Chromatog. 38,23-34. Sitton, D., Richmond, A. and Vaadia, Y. (1967). Phytochem. 6,1101-1105. Skene, K. G. M. (1967). Planta 74,250-262. Smith, L. L. (1974). Terpenoid Steroids 4 , 394-530. Spector, C. (1964). Ph.D. thesis. University of California, Los Angles. Spector, C. and Phinney, B. 0. (1966). Science 153, 1397-1398. Spector, C. and Phinney, B. 0. (1968). Physiol. Plant 21, 127-136. Sponsel, V. M. and MacMillan, J. (1977). Planta 135, 129-136. Sponsel, V. M. and MacMillan, J. (1978). Planta 144, 69-78. Sponsel, V . M., Hoad, G. V. and Beeley, L. J. (1977). Plunta 135, 143-147. Sponsel, V. M., Gaskin, P. and MacMillan, J. (1979). Planta 146, 101-105. Stoddart, J. L. (1969). Phytochern. 8, 831-837. Stoddart, J. L. (1976). Nature 261,454455. Stoddart, J. L., Breidenbach, R. W., Nadeau, R. and Rappaport, L. (1974). Proc. Nut. Acad. Sci.USA 71,3255-3259. Stodola, F. H., Raper, K. B., Fennell, D. I., Conway, H. F., Sohns, V. E., Langford, C. T. and Jackson, R. W. (1955). Arch. Biochem. 54,240-245. Takahashi, N., Kitamura, H., Kawarada, A., Seta, Y., Takai, M., Tamura, S. and Sumiki, Y. (1955). Bull. Agr. Chem. Soc. Japan 19,267-277. Upper, C. D. and West, C. A. (1967). J . Biol. Chem. 242, 3285-3292. Van Bragt, J. (1969). Neth. 1.Agric. Sci. 17, 183-188. Vining, L. C. (1971). J . Chromatogr. 60, 141-143. Watanabe, N., Yokota, T. and Takahashi, N. (1978). Plant and Cell Physiol. 19, 1263-1 270. Weaver, R. J. (1972). “Plant Growth Substances in Agriculture”. W. H. Freeman and Co., San Francisco. Wellburn, A. R. and Hampp, R. (1976). Biochem. J . 158,231-233.

METABOLISM A N D PHYSIOLOGY OF GIBBERELLINS

149

West, C . A . (1973). In “The Control of Biosynthesis in Higher Plants”(B. V. Milborrow, Ed.), pp. 143-169. Academic Press, New York and London. 81, 24242427. West, C. A. and Phinney, B. 0. (1958). J . A m . Chen7. SOC. Whyte, P. and Luckwill, L. C. (1966). Nuture 210. 1360. Wiinsche, V . (1969) Planta 85, 108-1 10. Yabuta, T. (1935). Agr.. and Hort. 10, 17-22. Yabuta, T. and Hayashi, T. (1939). J . Agric. Chem. Soc. (Japan) 15,257-266. Yabuta, T. and Sumiki, Y . (1938). J . Agric. Chem. Soc. Japan 14, 1526. Yamaguchi, I., Yokota, T., Murofushi, N., Ogawa, Y. and Takahashi, N. (1970). Agric. Biol. Chrm. 34, 1439-1441. Yamane, H.. Yamaguchi, I., Yokota, N., Murofushi, N., Takahashi, N. and Katsumi, K. (1973). Phytochem. 12, 255-261. Yamane, H., Murofushi, N. and Takahashi, N. (197.5). Phytochrni. 14, 1195-1200. Yamane, H., Murofushi, N . Osada, H. and Takahashi, N . (1977). Ph)Toc/wni. 16, 83 1-835. Yamane, H., Takahashi, N., Takeno, K. and Furuya, M. (1979). Plutiru 147,251-256. Yokota. T., Murofushi, N., Takahashi, N . and Katsumi, M. (1971). Phyrochem. 10. 2943-2949. Yokota, T., Yamazaki, S., Takahashi, N. and Iitaka, Y. (1974). Tetrahedron Letters pp. 2957-2960. Yokota, T., Yamane, H. and Takahashi, N . (1976). Agric. Biol. Cheni. 40, 2507-2508. Yomo. H. (1960). Hukko Kyoktri Shi 18, 603-606. Yomo, H. and Iinuma, H. (1966). Pllrr7t~i71. 113-1 18. Zeevaart, J. A . D. (1966). Plunr Physiol. 41, 856862. NOTE A D D E D I N P R O O F Recent studies by Hedden and Graebe (1981) reveal that in cell-free preparations from Cucurbita maxima liquid endosperm 7b-hydroxykaurenolide is not formed from enf7a-hydroxykaurenoic acid as illustrated in Fig. 24. Instead the pathway branches at ent-kaurenoic acid and proceeds to 7P-hydroxykaurenolide via ent-kauradienoic acid ( m f - A 6 . ’ kaurenoic acid). In turn, 7b-hydroxykaurenolide undergoes 12a-hydroxylation to yield 7p, 12a-dihydroxykaurenolide. Cell-free preparations derived from the suspensor of the embryo of Phasrolus coccinetu seed at a very early stage of development have been shown to convert (1) mevalonic acid to ent-kaurene (Ceccarelli et a / . , 1979), (ii) ent-kaurene to ent-kaurenol, ent-kaurenol, ent-kaurenoic acid and ent-7a-hydroxykaurenoic acid (Ceccarelli ef a/., 1981a) and (iii) ent-7a-hydroxykaurenoic acid to G A L ,G A Sand GAS (Ceccarelli et al., 1981b). G A I is an established endogenous component of the Phaseolus cotcineus suspensor (Alpi et al., 1979). Alpi, A,, Lorenzi, R., Cionini, P. G., Bennici, A. and D’Amato, F. (1979). Planta 147, 225-228. Ceccarelli, N., Lorenzi, R. and Alpi, A. (1979). Phytochem. 18, 1657-1658. Ceccarelli, N., Lorenzi, R. and Alpi, A. (1981a). Planr Sci. Letters 21. 325-332. Ceccarelli, N., Lorenzi, R. and Alpi, A. (1981b). 2. Pflanxnphysiol. 102, 37-44. Hedden, P. and Graebe, J . E. (1981). Phyrochem. 20, 1011-1015.

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The Control of the Patterned Differentiation of Vascular Tissues

TSVI SACHS Department of Botany. The Hebrew University. Jerusalem. Israel

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I1 . A Flux from the Leaves to the Roots Which Controls Differentiation . A . Introductory Summary . . . . . . . . . . . . . B. The Induction of Differentiation by Leaves and by Auxin . . . C . The Orienting Effect of Roots on the Flux of the Signals for Differen. . . . . . . . . . . . . . . . . tiation D . A Relation of Vascular Differentiation to a Flux of Inductive Signals E . Evidence for Additional Controls . . . . . . . . . .

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I11. Cell Polarization by a Flux of Signals . . . . . . . . . A . Introductory Summary . . . . . . . . . . . . B. Facilitation of Signal Transport as a Basis for Strand Formation C . The Stability of Polarity and its Possible Basis . . . . . D . The Formation of Vascular Networks . . . . . . .

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IV . Cellular Responses Involved in Oriented Differentiation . . . . . A . Introductory Summary . . . . . . . . . . . . . B. Early Events Indicating Determination and Differentiation . . . C . Is Cellular Differentiation Dependent on the Gradient or the Flux of Signals? . . . . . . . . . . . . . . . . .

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V . Special Developmental Processes in the Cambium . . . . . . . A . Introductory Summary . . . . . . . . . . . . . B . Quantitative Controls of Cambial Activity . . . . . . . C . The Constant Changes in the Cambium . . . . . . . . D . Ray Formation and the Radial Polarity of the Cambium . . .

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VI. The Cellular Complexity of the Vascular System . . A. Introductory Summary . . . . . . . . B. The Relation Between the Xylem and the Phloem C. The Controls of Fibre Differentiation . . . . D. The Controls of Parenchyma Formation . . .

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VII. The Relation of the Controls of Vascular Differentiation to other Aspects of Plant Morphogenesis . . . . . . . . . . . . . . 246 VIII. The Major Characteristics ofthe Hypothesis IX. Summary . . . Acknowledgements References . . .

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I . THE PROBLEMS The shoots and roots of land plants occupy different habitats and are connected by vascular systems (Fig. 1 ; for descriptions see Fahn, 1974; Esau, 1977; Cutter, 1978). These systems are complex, since different mechanisms are used for the transport of organic materials and water; in addition the same systems are also major sources of mechanical support. Vascular differentiation, which continues as long as the plant grows, may be broadly classified into three types : (a) Primary differentiation, which occurs by direct changes in the cells formed in the apices of the shoots and roots, the intervening meristematic stages being known as the procambium (Figs 2 and 4a). (b) Secondary differentiation, which occurs in cells formed by a special meristem, the vascular cambium (Fig. 6). This meristem, which is part of the vascular system, increases the width of the organs in which it is situated, whereas the primary meristems increase their length. (c) Regenerative differentiation, which occurs by a re-differentiation of parenchyma cells or changes in the cambium (Fig. 3). Though this latter process occurs most often when a plant is wounded, it can also result from such internal developmental changes as the growth of lateral roots. This complex developing system raises many questions which can be roughly classified under the headings of the exact description of the system, the mechanisms of transport, the controls of development, the relation to ecological adaptations and the phylogeny of the system. All these questions require further work; even the descriptive knowledge is far from complete, especially at the level of individual vessels and sieve tubes (Aloni and Sachs, 1973; Zimmermann, 1976). The questions to be dealt with here will be restricted to one aspect of development, the controls of patterned, orderly differentiation. Though the

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major stress is on the xylem of dicotyledons, and especially on pea and bean seedlings, this is due only to the limitations of the available information, and the discussion is meant to refer to the patterns of the vascular tissues in plants in general. Thus the topics considered will include the controls of the relation between organ development and vascular differentiation (Fig. l), the controls which cause the processes of sieve and vessel element differentiation to result in the formation of elongated tubes rather than isolated cells (Figs 10d, 17c), and the determination of the relative location and size of the various component tissues and cells of the vascular system. This formulation of the subject means that the ability of one genome to direct differentiation into a variety of cell types will be taken for granted, and the questions asked will concern the location of the various differentiation processes relative to other cells, tissues and organs. Information about the processes of vascular differentiation rather than the controls of their patterns may be found, for example, in Torrey et ul. (1971), Brant (1976) and Roberts (1976). It might be useful to clarify the type of answer being sought to these questions of patterned differentiation. The structure of the vascular system suggests that interactions between the various parts are essential in determining the relative location of each event of cellular differentiation. The phenomena of regeneration (Fig. 3) must be due to a disruption of the normal interactions and they thereforeconfirm that such interactions are taking place. It would thus be desirable to know the nature of the intercellular signals involved. Such knowledge would not, however, suffice for an understanding of the basis for the pattern of differentiation; the signals alone could not establish the directional relations between cells and the stability of the pattern must require the highly specific reactions of the cells. These could include directional-transport, use, and production of the same and additional signals, which could be the basis for a patterned distribution both of the signals and the associated differentiation (Sachs, 1978a). In addition, internal developmental programmes may be switched on, and these may be both complex and stable. These statements are vague because they are so general; the central purpose of the discussion which follows is to be more specific and to suggest a conceptual framework and hypotheses for the study of the patterns of vascular differentiation. The questions considered below are but a specific case of the general biological problem of orderly differentiation in contrast to tumorous growth (Fig. 4). These problems were relatively neglected in the past, possibly because of the lack of the necessary concepts for their study, but they now receive growing attention in a wide variety of organisms; for example, see Wolpert (1971). Plants in general, and their vascular systems in particular, have important experimental advantages for the study of patterned differentiation ; these include the continued formation of new organized tissues not only in fragile embryos but also in large, rapidly growing plants, the lack of cell movement and the relative simplicity of the structures which yet form

Fig. 1. Contacts of vascular strands in a pea seedling. These seedlings, used for many of the experiments considered here, are unusual in having vascular tissues in the stem centre (Fig. Sb). (a) Simplified drawing of the seedling. Only the side facing the viewer was marked, the stipules were removed along the dotted lines and the phloem fibres were omitted. (b) Pea leaf petiole (for the network of the lamina see Fig. 16e). Note that the vascular system of the seedling is complex and orderly, though a variability of details can be seen by comparing the two sides of the petiole. Vascular development can be followed from leaf primordia of different ages (numbered); it is closely correlated with apical growth. (Based o n seedlings cleared with Lactic Acid when leaf number 1 was a third of its final size and on a cleared mature leaf.)

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Fig. 2. Cellular structure of vascular strands. (a) Early stages of vascular differentiation (a procambium) at the base of a young leaf. (b) Mature strand that can, however, expand by the activity of the cambium (absent in monocotyledons). Note that the first stages of vascular differentiation include oriented cell growth and division. The mature strand is a complex array of different cell types. The transporting channels (vessels and sieve tubes) are long files of specialized cells (Figs 10d; 17c). The drawings are diagrammatic, since in living tissues there are many overlapping cells, the cell files are not oriented in one plane and no section includes all the details (for a photograph, see Fig. 4c). ( ( a ) is from a section through a Coleus blumei apex and (b) is based on many radial sections of a bean hypocotyl. Drawn with a projection microscope.) (a) x 400; (b) x 125.

complex patterns. This chapter will attempt to show that as a result of these advantages the vascular development of plants is in some ways the best understood biological pattern of cellular differentiation. 11. A FLUX FROM THE LEAVES TO THE ROOTS

WHICH CONTROLS DIFFERENTIATION A. INTRODUCTORY SUMMARY

The first set of problems concern the mechanisms that correlate the location of vascular differentiations with the various plant organs. Two possibilities should be considered first : (a) organ development induces vascular differentiation, and (b) vascular tissues determine the initiation of the various organs. These possibilities are not mutually exclusive and can be studied by following the developmental correlations, interrupting tissue continuity by wounds, removing parts of the plant and producing new relations between

Fig. 3. Xylem regeneration in wounded stems. (a, b) From a section through a wounded pea stem, where regeneration occurs by a redifferentiation of parenchyma cells. (b) Is a detail of(a) in which the dark cells are the new xylem and the phloem, not clearly seen, is further away from the wound. ( c x e ) Regeneration from cambial derivatives in bean seedlings. (d) and (e) are two magnifications of xylem above a hole in the stem. Note the general form of the new xylem, which indicates a flux of differentiation signals around wounds. The influence of polar auxin transport can be seen by comparing the upper and lower side of the wounds in (c), (Thick sections cleared with Lactic Acid. (c)-(e) were then dissected to uncover the xylem.) (a) x 31; (b) x 90; (c) x 9; (d) x 30; (e) x 2 16.

Fig. 4. Normal and tumorous tissues of Heliunthus unnuus. (a, b) Cross-sections through healthy and Crown Gall tissues of a stem. (c, d) Similar longitudinal sections. Note that vascular and other differentiation occurs in the tumors, but it lacks consistent orientations and relations between cells. This stresses the question of the control of normal patterned differentiation. (Sections of embedded hypocotyls a month after the plants were wounded and infected with Crown Gall bacteria.) (aHd) x 80.

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parts by grafting. Attempts to identify the inductive signals can be made by replacing plant parts by substances they are known to produce. There is a very general (though not invariable) correlation between leaf development and vascular differentiation. Wounds, leaf removal and grafts prove that leaves, especially developing leaves, induce vascular differentiation oriented in the direction of the roots. Localized auxin sources replace many of the effects of leaves on vascular differentiation; auxin is unique among known substances in controlling the location and orientation of new vascular tissues. Since auxin is known to be produced by leaves and it replaces some of their effects, it follows that auxin is part of the inducing signals, or evocators, by which leaves influence vascular differentiation. Roots, unlike leaves, do not induce vascular differentiation. Roots are not even essential for this process, but when they are present new vascular tissues are so oriented as to connect the roots with the rest of the plant. These facts can be understood if the roots act as sinks into which the signals flow from the shoot. These conclusions suggest the hypothesis that is the basis of much of the following discussion : all aspects of vascular differentiation occur along the channels of the flux of inductive signals from the shoots to the roots. A major source of these signals are the young leaves and other developing organs of the shoot, but they may be produced in smaller amounts by all parts of the plant, including the vascular tissues themselves. This hypothesis is supported by the general form of vascular differentiation and by experimental evidence that auxin is effective only under conditions in which its flux can be expected. This may not be the only control of vascular differentiation, but there is not much evidence at present for the existence of other controls, nor for a reciprocal effect on organ initiation by the vascular tissues. B . THE INDUCTION OF DIFFERENTIATION BY LEAVES AND BY AUXIN

1 . The Correlation Between Leaf Development and Vascular Diferentiation There is a clear correlation between leaf development and occurrence and orientation of vascular tissues. This correlation is both spatial and temporal and is expressed in all aspects of vascular differentiation.

The pattern of the primary strands in stems is always correlated with the presence of leaves (Fig. 1; Esau, 1965). Changes in phyllotaxis during development are associated with a corresponding change in the pattern of vascular tissues (Philipson and Balfour, 1963 ;Pulawska, 1965). The course of leaf development is reflected in the vascular pattern of the leaf (Deinege, 1898). The initiation of vascular differentiation occurs at the same time as leaf development. This correlation, which was found long ago for primary tissue (Bonnier, 1900; for review see Esau, 1965) is often very precise (Jacobs and Marrow, 1957; De Sloover, 1958; Larson, 1979). This

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correlation is real even if it is not invariable (see Section 1I.E for discussion). New cambial divisions and secondary vascular differentiation usually begin at the time when new leaves are formed in spring (for reviews see Priestley, 1930a; Studhalter et a/., 1963; Reinders-Gouwentak, 1965; Jacquiot, 1968). This has also been observed in trees growing outside the temperate region (Coster, 1927; Fahn 1959) and in plants with an anomolous cambium (Dobbins, 1970). The correlation has been demonstrated even in trees in which vascular differentiation occurs very early (Wareing, 195l), and relatively recent work has extended the observations to include the formation of secondary phloem (Evert, 1963; Davis and Evert, 1970). Regenerative vascular differentiation is also associated with leaf development. This may be expressed by the reorientation of cambial initials and new vascular tissues (discussed in Section 1II.C) at the base of buds released from apical inhibition (Neeff, 1914, 1922; Sachs, 1970) and adventitious buds (Brown, 1935). Regenerative differentiation is also expressed by the differentiation of parenchyma cells to form vascular contacts to new adventitious leaves or buds (Boodle, 1920; Crooks, 1933; Galavazi, 1964). It may be concluded that leaf development and all aspects of vascular differentiation are often correlated with respect to their location and time of formation. The exceptions to this generalization, which do not contradict either its general validity or any of the conclusions reached below, will be considered in Section 1I.E. Since this correlation is found in cases of regeneration as well as normal development, it could not depend solely on an early pre-determination of both events. It follows therefore that some developmental signals must pass between the developing leaves and the differentiating vascular tissues. These correlative signals could be of two types (Fig. 5a,b): they might originate in the leaves and induce vascular differentiation or they might be produced or channelled by the vascular tissues and control leaf initiation and development. Note that these two possibilities are not mutually exclusive; they could even reinforce one another. The first question considered here is the evidence for correlative signals originating in the leaves and whether they can be identified. Controls which might operate in the opposite direction will be considered below (Section 1I.E). 2. The Induction of Differentiation by Leaves The evidence that leaves cause an oriented differentiation of vascular tissues may be summarized as follows. ( a ) Disrupting the contact with the leaves reduces all aspects of vascular diflerentiation. This effect of the leaves is polar, in the sense that it extends morphologically downwards, towards the roots. The removal of the leaves reduces cambial activity and secondary vascular

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Fig. 5. Possible interactions between pea seedling organs and vascular differentiation. (a) Signals originating in the shoot induce vascular differentiation. The root directs these signals by acting as their sink. (b) The same relation operating in the opposite direction. (c) A control by signals of both the shoot and the root. The possible production of signals by the vascular tissues themselves was omitted for brevity. The experiments discussed in the text prove that the major, if not the only, control is the one represented in (a).

differentiation in both bean seedlings and trees (Jost, 1891, 1893). This effect is also seen when the leaves remain on the plant but the downward contact between them and the observed region of the stem is cut (Figs 6 and 7a-c; Jost, 1893; Amer and Neville, 1979). Girdling, the removal of the cambium and the tissues external to it, is sufficient (Janse, 1914). Though the effect of developing leaves is the most general and the most pronounced (Amer and Neville, 1979), secondary vascular differentiation is also correlated with the presence of mature leaves (Fig. 31; Munch, 1938; Wareing and Roberts, 1956; Hess and Sachs, 1972; Benayoun and Sachs, 1976). Procambial and primary vascular differentiation are reduced when very young leaf primordia are removed (Fig. 8 ; Helm, 1932; Wardlaw, 1946, 1950; Young, 1954; Pellegrini, 1963; Wangermann, 1967; Sachs, 1972a). The influence of a primordium is limited to the leaf traces which connect it to the stem and to the morphologically downward direction. When the primordium

Fig. 6 . Cross-sections showing the polar effects of leaves and auxin on vascular differentiation. Treatments explained in Fig. 7a-q where the broken lines show the location of the sections. (a) With direct contact with both the leaves and the roots. (b) No direct contact with the leaves. (c) No direct contact with the roots. (d) As (c), but with an auxin source (1% Indole Acetic Acid in lanolin). Note that xylem differentiation and cambial activity depended on contact with the leaves while contact with the roots tended to decrease differentiation. The phloem does not show the same responses clearly, but only because much of its differentiation was completed before the experiments were started. (Hand cut sections cleared with Lactic Acid and stained with Lacmoid. Week-old plants treated; sectioned three weeks later after being kept in room conditions.)

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is removed at an early stage of its development, the differentiation of its vascular contacts may be completely prevented Vascular regeneration around wounds (Fig. 3) is promoted by the presence of leaves (Jost, 1942; Jacobs, 1952, 1956; La Motte and Jacobs, 1963; Robbertse and McCully, 1979). The promotive effect is found only if the leaves are present above, and not below, the wound (Aloni and Jacobs, 1977a). ( b ) Grafting or theformation of additional leaves is always associated with the differentiation of new vascular contacts. Grafting tissues which include buds (and thus also developing leaves) causes the differentiation of vascular tissues from parenchyma cells and activates the cambium below the graft (Timmel, 1927; Simon, 1930; Jost, 1931; Nickell, 1948; Homes, 1965; Sachs, 1968a). Grafts of plants belonging to different phyletic groups, when they are successful, are associated with vascular differentiation, indicating that the signals produced by the leaves are not species-specific (Simon, 1930; Nickel, 1948). The growth of higher plant parasites may be considered a

Fig. 7. Vascular differentiation from bean cambium. Only new xylem and phloem, formed after the experiment was started, are indicated by wavy lines. (a-d) Influence of polar contact with leaves, roots and a source of auxin (dots) on vascular differentiation. The tissues shown were seen after two to three days; cross-sections along the broken lines of a longer experiment can be seen in Fig. 6 . Note that a polar contact with the leaves or a source of auxin causes vascular differentiation, but even a complete isolation of the cambium by girdling does not prevent the formation of some new vessels, presumably in response to signals of the cambium itself. (e-g) Tissues cut and grafted, in (g) after an inversion of one of the two members of the graft (arrow shows the original orientation relative to the roots). The new differentiation follows the original polarity (towards the roots) whenever possible, and it occurs preferentially in the upper member ofthe graft, so that (e) and (f)are not symmetrical. Vessels which reverse their direction in response to polarity inversion (9) are shown in Fig. 15a. (h) Cambial differentiation tends to spread to relatively isolated regions. This can involve a departure from the shortest shoot-to-root contacts and is another indication (see (d) )for activity of the cambium inducing vascular differentiation. (i-k) Cuts prevented differentiation except in new orientations relative to the original polarity. Regeneration of new shoot-to-root contacts in these conditions occurs readily, even when it involves a complete reversal of the original polarity of vessel differentiation for a distance of a few mm (k). (i) Was cleared two days after wounding, and the new vessels are not the shortest shootto-root contact, being influenced by the original polarity. Later vessels (shown in ('j)) do follow the shortest route. (1-p) The influence of auxin sources (dots) in relation to flux. Auxin applied to an intact plant (I) causes vascular differentiation, though this is polar and limited. (This contrasts with parenchyma redifferentiation which requires a neighbouring wound and is inhibited when the existing strands are in contact with the leaves; see Fig. 9.) Localized auxin has a clear effect on cambial regions when they are completely girdled (m). A cambium surrounded by the auxin source (n), however, so no gradient or flux are expected, shows no differentiation. In large regions of cambium an internal flux may occur, and limited differentiation is found (0). A narrow contact with the cambium of the rest of the plant causes the differentiation of many vessels in the form of the expected flux (p). (The diagrams are based on many experiments with week-old bean seedlings cut from their roots and kept in closed dishes. They were cleared with Lactic Acid three days after treatment except for (e), (0, (9) and 6).which required a week or longer for clear results.)

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Fig. 8. Influence of leaf primordia on stem differentiation. (a) Explanation of the experiment. A cut along the broken line removes the youngest leaf and the promeristem or apical dome of a pea bud. leaving two older leaves (numbered) intact. The removed leaf is about 0.1 mm in diameter; it was removed together with the promeristem to prevent the complications due to new leaves formed after the experiment started, but this removal had no direct effect on the result. (b) Crosssection o f a n untreated control stem. Stripes show the vascular tissues and dots the fibres. (c) The stem below the second leaf primordia after a cut as shown in (a). Note that the vascular tissues which would have led t o the missing leaf were not formed. When the leafwas removed at thisearly stage. furthermore, the adjoining parenchyma and epidermis tissues were also missing (drawn from Sachs, 1972a).

special type of graft, and it causes the expected differentiation (Thoday, 1961). Gall development acts in the same way (Jost, 1931 ; Meyer, 1969). Buds grafted on tissues grown in culture cause the differentiation of vascular tissues below the graft (Camus, 1949; Wetmore and Sorokin, l955; Wetmore and Rier, 1963). Buds grafted on young meristematic tissue have the same effect (Gulline and Walker, 1957; Sachs, 1972a), but since these grafts develop only after a week or more, the induced vascular tissues are best considered as a result of a re-differentiation of parenchyma. Two mature leaves may develop in the place of one when a leaf primordium is split at a sufficiently early stage (Sachs, 1969b). This regeneration is correlated with the formation of a double set of primary vascular strands (Sachs, 1972a). The removal of the leaves of a tree results in bud growth and the formation of new leaves, and these processes are correlated with renewed cambial activity and secondary vascular differentiation (Jost, 1893 ; Bernstein and Fahn, 1960). The relation between renewed leaf growth and cambial activity is, however, not always simple (for review see Studhalter et a/., 1963). ( c ) Mutations qf leaf development also influence vascular diferentiation. At least in one case (Caruso and Cutter, 1970) a mutation that influences vascular differentiation does so indirectly. When normal leaves are grafted on to a mutated stem differentiation is normal. ( d ) The direction in which vascular digerentiation proceeds indicates an efect ofyoung leaves. Cambial activity and vascular differentiation associated with bud break in spring proceed from the bud downwards (for reviews see

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Wilcox, 1962; Studhalter et al., 1963; Reinders-Gouwentak, 1965) as one would expect a leaf-signal to spread. A similar direction of the spread of differentiation is seen when adventitious buds are formed (for review see Esau, 1965). This directionality is not found in normal primary differentiation, where it is influenced by the local age of the tissue more than by the origin of the signals for differentiation (Section 1I.E). From this evidence it can be concluded that leaves are a source of signals which induce vascular d@erentiution. The effect of these signals is morphologically downwards; or, in other words, it is polar. The signals control all aspects of vascular differentiation, both in terms of the different tissues and the type of differentiation (primary, secondary and regenerative). 3. Replacing the Leaves by Auxin The conclusion reached above raises the question of the nature of the correlative signals produced by the leaves. The various general possibilities (water movement, nutrients, hormones or electrical signals) were the subject of much discussion in the early literature (for example see Jost, 1891, 1893; Simon, 1908a). Most of the facts listed above do not exclude even the possibility that the leaves signal the differentiation of the tissues leading to them by acting as a sink for some critical substances. The experiments first performed by Jost (1893; see Fig. 7a-c), however, show that the effect of the leaves is polar, towards the morphologically lowest part of the plant, even when this direction is not that of the roots or any important part of the plant (Fig. 9e,@ This result can be readily repeated for phloem as well as xylem differentiation (Sachs, in preparation). The demonstration by Snow (1933) that the cambium-activating effect will cross a water gap, showed for the first time that the leaves act by being a source of hormones. Since the discovery of auxin many experiments have shown that it will partially replace the leaves in controlling vascular differentiation. The various relevant facts may be grouped as follows. ( u ) A source of auxin replaces the leaves in inducing most types of vascular diferentiation. This effect of auxin was first found for the promotion of cambial activity (Snow, 1935) and, together with the promotion of xylem vessel differentiation has since been found to be general (Figs 6 and lOa,e; for review see Wilcox, 1962; Reinders-Gouwentak, 1965; Goodwin, 1978). Auxin promotes cambial activity in roots as well as shoots (Torrey, 1963; Torrey and Loomis, 1967). A source of auxin replaces a young leaf in promoting the late stages of primary differentiation (Wangermann, 1967). Auxin does not replace the leaves in causing procambial differentiation (Young, 1954). Regeneration around wounds is promoted by auxin as well as by leaves (Fig. 9 b d ; Jost, 1942; Jacobs, 1952, 1956). This is true for the phloem as well as the xylem (La Motte and Jacobs, 1963; Jacobs, 1970). Auxin causes

0

i

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xylem differentiation when it is applied directly to parenchyma close to a wound (Figs 9g,h, 10e,f; Kraus et ul., 1936; Sachs, 1968b). Phloem differentiation is also induced in these conditions (Fig. log; Sachs, in preparation). The differentiation induced in tissue cultures by buds can also be elicited by a source of auxin (Camus, 1949; Wetmore and Rier, 1963; Clutter, 1960; Earle, 1968; for review see Roberts, 1969, 1976). The replacement of the effect of leaves by auxin is thus very common. Auxin does not replace leaves in causing the early stages of primary differentiation nor does it cause the formation of xylem or phloem with many fibres (see Section V1.C). Auxin does induce sieve tube differentiation but the generality and completeness of this effect are not yet known ; this topic will be considered in Section V1.B. ( b ) The influence of an auxin source, like that of leaves, follows a polar course and orients diJerentiation. Leaves do not merely induce vascular Fig. 9. Vascular differentiation from parenchyma of wounded pea seedlings. All experiments were on stems in contact with cotyledons and roots. Half of each stem was removed by a longitudinal cut, exposing a wounded surface on which the experiments were performed. The location of this cut determined the presence of existing vascular strands (straight lines); new vascular tissues (vessels and, in those cases where they were checked, sieve tubes as well) are indicated by wavy lines. Dots show the location of sources of auxin (1% Indole Acetic Acid in lanolin). (a-f) The effect of wounds. When these do not damage the vascular tissue there is no regeneration (a). Such regeneration (b) (see also Fig. 3a,b) is prevented if the shoot above is removed (c) and is restored by a source of auxin (d). Regeneration follows the original polarity of the tissue (e) (the direction towards the roots) even when this polarity does not lead to functional contacts with the cotyledons and the roots. This is not because the contacts cannot be formed (f). (g-I) Induction of vascular differentiation by an auxin source. Photographs of these tissues appear in Fig. 1Oe-g; such photographs are limited to one focal plane and the diagrams here present a more general picture. Auxin applied laterally induces, within four days, a redifferentiation of parenchyma cells to vascular tissues which connect the auxin source to the vascular system of the plant (g, h). The size of this new connecting system is a function of auxin concentration (there was a ten-fold difference between the concentrations applied in (g) and in (h)). The new differentiation is oriented by tissue polarity and the existing vascular strands. Polarity in terms of vessel differentiation relative to the auxin source may even be reversed if no other course is available (i). Differentiation towards the existing vascular tissues are inhibited if these are in contact with young leaves (i),or supplied with auxin (k). This indicates that the vascular tissues orient the new differentiation by acting as a sink for auxin. A cut preventing auxin flux from taking any other route than towards the existing strand (I) causes differentiation even when the latter are in contact with the leaves or supplied by exogenous auxin. (m-p) The influence of one auxin source on the differentiation induced by another. A comparison of (m) and (n) shows a diversion of differentiation when two neighbouring sources are present. This diversion is found even if the lower source in (n) is applied two days after the upper one, showing that induction requires a long-term presence of auxin and is not a process that becomes determined and continues in the absence of signals external to the cells. When diversion is impossible (p), or when induction occurs through the polarized cells of a vascular system (not shown), an auxin source induces a strand passing right through another source. This can be understood on the basis of a relation of differentiation to auxin flux, but not to its gradient. (Based on numerous experiments performed on five-day-old pea seedlings and cleared with Lactic Acid four days later. Some ofthese experiments or their variations were reported by Sachs, (1968a, 1968b, 1969a, 1974).)

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differentiation, they also cause it to be oriented so that it forms downward connections with the rest of the plant. A source of auxin has the same effect (Fig. 10; Sachs, 1968b). As will be discussed below (Section 1I.E) many substances other than auxin influence vascular differentiation, but no other substance is known to induce organized orientated strands. Leaves can also re-orient cambial initials so that they conform to a new shoot-to-root axis imposed by wounds and girdles (Fig. 7i-k; the process is discussed in Section 1II.C). Local sources of auxin have been found to have the same effect (Kirschner et al., 1971). The effect of leaves on vascular differentiation is in the morphologically downward direction, and thus follows the well known polarity of auxin transport (for review see Goldsmith, 1969, 1977). Vascular tissues induced by auxin also follow the same pattern (Sachs, 1969a). This can be seen for auxin-induced phloem as well as xylem (Sachs, in preparation). ( c ) Auxiii is known to be formed in the leuves und it is present and tratibported in dijerentiating vascular tissues. There is much evidence that leaves, and especially young leaves, are a source of auxin that affects the rest of the plant. This statement is based both on studies of auxin synthesis (Thimann and Skoog, 1934; Avery et a / . , 1937; for review see Sheldrake, 1973a; Goodwin et al., 1978) and on the possibility of replacing the various developmental effects of young leaves by a source of auxin (Sachs, 197%; see Section VII). The differentiation of xylem associated with young developing leaves shows a simple quantitative relation to the development of these leaves, and, possibly, to auxin production as well (Jacobs and Marrow, 1957). Auxin is present in differentiating vascular tissues, and there is a general quantitative relation between the amount of auxin and the rate of differentiation (Soding, 1937; Digby and Wareing, 1966b; Sheldrake, l971,1973a,b; Bourbouloux and Bonnemain, 1979). A correlation has also been found between the rate of auxin transport and cambial activity (Hollis and Tepper, 1971). It is thus known that leaves cause vascular differentiation, that leaves Fig. 10. Induction of vascular differentiation by auxin. (a-d) Local auxin causes vessels and sieve tube differentiation from storage root cambium of Swede (Brassicu nupus). A general view of such treatments is illustrated in Fig. 1 1. The new vessels can be formed in all possible angles (a) and careful observation shows that new sieve tubes (c) are also present. The auxin-induced vessel members have the shape ofthe original cambial cells, and when these would have formed ray cells in the intact plant (b) they are readily distinguishable from normal vessels (d). (e-g) Vessels and sieve tubes formed in response to auxin from pea stem parenchyma near a wound. Many neighbouring cells can be induced by a 1% auxin preparation (e). The first auxininduced vessel members have the shape of the neighbouring parenchyma cells (f). After a week or longer, sieve tubes can also be found (g). (Material cleared with Lactic Acid, stained with Lacmoid and viewed in Sodium Lactate); (a) x 8; (b), (f), (8) x 192; (c-e) x 80.

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produce auxin and that auxin causes vascular differentiation. These facts must mean that part of the signal by which leaves induce vascular differentiation is auxin. It should be noticed that this conclusion is presented as a logical necessity, not a hypothesis. It does not, however, imply that other controls of differentiation are not important. Nor does it imply that other substances do not control differentiation ; as will be discussed below, some do (see Sections II.E, VI.B, VLC), though the argument made above can at present be made only for auxin. The conclusion concerns the role of auxin as a signal and does not state anything about the mechanism of auxin action at the cell level (see Sections III.B, 1V.C). The importance of the conclusion concerning the role of auxin as a signal is that it means that part of the chemical nature of an inducing principle (Spemann, 1938) or evocator (Waddington, 1962; Needham, 1942) is known. This fact is of great importance for the discussions which follow. It should be noted that auxin is not specific to any one process of vascular differentiation : it is known to cause cambial divisions, xylem differentiation and phloem differentiation. The specificity of the reaction resides in the cells and in additional factors discussed in Section VI. C. THE ORIENTING EFFECT OF ROOTS ON THE FLUX OF THE SIGNALS FOR

DIFFERENTIATION

I . The Possible Mechanisms for Root Control of Differentiation The discussion of the effect of leaves on vascular differentiation raises similar questions concerning the effects of roots. In view of the conclusions reached above, two possible mechanisms by which differentiation may proceed into the roots can be suggested (Fig. 5a,c). These mechanisms are not mutually exclusive. (a) The roots resemble the leaves in being the source of signals for vascular differentiation. This would require some special interaction between the leaf and root signals to account for the organized pattern of contacts. (b) The roots may influence vascular differentiation by serving as a sink for the signals originating in the shoot. In this case vascular differentiation would occur along the channels of an inductive flux (Janse, 1914; Neeff, 1922). This would not mean that the roots are not an active source of hormones and other signals (see Section VII), but only that these influence vascular differentiation indirectly, by their effect on the shoot and its development. 2. The Evidence f a ) Roots do not induce vascutar diferentiation though they genera fly, though not invariably, orient its course. The presence of roots on cuttings or on callus in culture does not necessarily result in vascular differentiation. In

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short-term experiments, pieces of radish storage tissue or bean hypocotyls differentiate less new vascular tissue if roots are present (Sachs, unpublished). When vascular differentiation is induced by leaves or by auxin, it is oriented so that it connects to any existing roots, or to tissues which connect morphologically downwards, towards the roots (Figs 7,9). This connection with the roots is not always found. Plants can be cut so that differentiation may proceed either along the polarity of the tissue (in the morphologically downward direction, that which originally led to the roots) or in a transverse orientation which connects to the roots (Fig. 9e). In such experiments the polar orientation is preferred, even though control plants in which the polar tissue is removed show that the transverse differentiation can occur readily (Fig. 9f; Sachs, 1969a). This is true for phloem as well as xylem (Sachs, in preparation). ( b ) Roots are not essential for the induction of vascular diferentiation. Differentiation has been found in small seedlings, from which the roots were completely removed (Digby and Wangermann, 1965; Sachs, 1968a; Peterson, 1973; Robbertse and McCully, 1979). Differentiation above girdles, in regions connected to the roots only through the xylem, has been observed many times (Janse, 1914; Munch, 1938). It is also found in flaps of tissue which point downwards and have no direct contact with the roots (Fig. 7b; Jost, 1893). Vascular differentiation both of xylem and phloem occurs in cultured tissues which are devoid of roots (Camus, 1949; Clutter, 1960; Wetmore and Rier, 1963). Differentiation is also found in isolated stem segments of Coleus (Thompson and Jacobs, 1966)and of many other plants (Sachs, unpublished). Pieces of storage tissues which include cambium respond readily to auxin by vascular differentiation (Figs 10a-c, l l a ) ; the same response is found in cambial regions of bean which are completely girdled on all sides (Fig. 7m). ( c ) Hormones known to be produced by the roots do not orient vascular diferentiation. Roots are known to form cytokinins and, at least in some cases, they are also a source of gibberellins (see Section VII). Both these groups of substances have been shown to promote vascular differcntiation (for reviews see Roberts, 1976; Goodwin, 1978; Sections II.E, VI.B, V1.C) and in the case of cytokinins, Houck and La Motte (1977) have reported a replacement of the effect of the roots on phloem regeneration. The localized application of these substances does not, however, replace the root in orienting vascular differentiation (Sachs, unpublished) and it is therefore likely that their effects are indirect, though this does not mean that they are unimportant (see Sections VI, VII).

3. Conclusions and Discussion The results above show that roots orient vascular diferentiation without being essential for its occurrence. It thus appears that roots act as sinks, injuencing

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differentiation by orienting the j ? u x o f the inducing signals originating in the shoot. This is a very clear choice between the two possibilities suggested above (Fig. 5a,c). The following reservations concerning this conclusion may be raised : (a) The evidence considered above depends heavily on the differentiation of vessels, which are easy to observe. Sieve tube differentiation has also been observed in a variety of situations in which roots were either absent or not directly connected with the region of differentiation (Figs IOc, 11; Wetmore and Rier, 1963; Sachs, in preparation). The study of sieve tube differentiation has certainly not been thorough and quantitative effects of the roots may still be found. (b) It might be necessary to qualify the statement that no roots are present in various cuttings. When the roots are removed from bean hypocotyls new, adventitious roots may be seen within a few days, and it is therefore possible that from a physiological point of view, such as the production of hormones, new roots are present within a shorter time. The physiological functions of roots may also be supplied by callus development, which can be viewed as partially-organized root tissues (Kirschner et al., 1971; see Section VII). It is still true, however, that vascular differentiation proceeds preferably in a polar direction even when functional roots can be contacted in a transverse direction (Figs 7b, 9e; Sachs, 1969a). This preference for the polar direction is true, at least in peas, for phloem as well as xylem differentiation (Sachs, in preparation). (c) Even if the signals originating from the roots are not essential they may still have unknown effects on vascular differentiation and its orientation. These considerations do not change the conclusion that roots influence vascular differentiation by serving as a sink for auxin and other possible signals for differentiation ; they do suggest, however, that further work is required to establish whether this is the only mechanism by which the correlation between roots and vascular differentiation is established and maintained. D . A RELATION OF VASCULAR DIFFERENTIATION TO A FLUX OF INDUCTIVE SIGNALS

I . Introduction The previous two sections presented evidence that vascular differentiation occurs in response to signals which originate in the leaves and find their sink in the roots. It follows from these conclusions that vascular differentiation

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is correlated in some way with the flux of the signals through the plant, and that this flux determines the pattern of differentiation. The nature of this possible correlation at a cellular level will be considered below (Sections III.B, 1V.C); the purpose of the present section will be to review the evidence which supports this concept. The relation of differentiation to signal flux is a partial answer to the questions raised in the first chapter and the basis for much of the following discussion. 2. The Form and the DifSerentiation of the Vascular System ( a ) Both normal and regenerative vascular systems bear a general resemblance to drainage patterns, connecting the various parts of the shoot with the roots. This resemblance is most clearly seen in regeneration around wounds (Fig. 3; Section 1I.B). Vessels forming close to partial girdles (Janse, 1914; Kirschner et al., 1971) and at the base of adventitious buds (Brown, 1935) always exhibit a pattern resembling the flow of a liquid. This is also true of both primary and secondary differentiation (Section III.C), though in leaves a complex network which includes closed loops is formed. These leaf networks could not form by a simple directional flux of signals, but as will be seen below (Section 1II.D)they can be accommodated to the general concept. There are also rare cases of isolated xylem elements in leaves which do not form part of any recognizable strand (Herbst, 1972). These might be a local response to a temporary inductive flux, the cells maturing early while the rest of the strand reverts to parenchyma. They might also depend on controls normally involved in the formation of the sclereids of leaves and fruits rather than on the normal controls of vascular tissue differentiation. Therefore, while these isolated vascular elements require further study, they are the rare exception stressing the rule that vascular elements are found in strands which could form in response to some form of flux. ( b ) The temporal sequence of secondary vascular diferentiation shows that aflux of signals is involved. When secondary vascular differentiation is followed carefully (see Section 1I.B) it is found to proceed from the buds downwards, as it would if it depended on a flux of a finite rate (Tepper and Hollis, 1967). Primary differentiation, on the other hand, occurs by the sequential addition of cells at the apices and could be expected to depend on the age of these cells rather than on any flux which might control the differentiation. Thus the appearance of the procambium and the mature phloem in the shoot apices proceeds acropetally and presumably reflects the relative times at which these cells are formed. Xylem maturation follows a more complex course, as isolated elements are temporarily found at the base of leaves. Further maturation later incorporates these elements into continuous strands. This pattern might well depend, however, on the local cessation of division at the nodes while the petioles and internodes continue to elongate. Thus easily observed final stages of maturation need not reflect anything con-

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cerning the controls of differentiation in cases where a non-uniform tissue, such as a developing stem apex, is observed. 3 . The Relation of Experimentally Induced Vessels to a Flux of Auxin ( a ) A local application of auxin to wounded plants induces vessels and sieve tubes which appear to flow away from the source in the direction of the roots (Figs 7 , 9). The pattern of the induced vessels (Wareing et al., 1964; Fayle and Farrar, 1965) certainly suggests a relation to flux. This is also supported by the influence of tissue polarity (Section III.C), which is known to influence the flux of auxin. Triiodobenzoic acid, a known inhibitor of auxin transport, inhibits the induction of vessel differentiation by auxin (Fosket and Roberts, 1964). ( b ) Auxin applied to isolated pieces of a plant causes vessel diflerentiation only when an auxin flux can be expected (Figs 7m-p, 1 Id,e). A relation to auxin flux, as suggested by Torrey (1966), can be readily demonstrated by applying auxin in various ways. A girdled region of bean cambium (or an isolated piece of turnip) usually do not differentiate any vessels when surrounded by auxin in lanolin (Fig. 7n). When the isolated region is large, however, some vessels do differentiate, and these are found when internal polar transport can be expected (Fig. 70). When the same isolated cambium is allowed an “outlet”, through which the auxin may flow to other tissues, a large strand of vessels is induced (Fig. 7p). The interactions of the fluxes of two sources of auxin applied in close proximity can be seen in the vessel pattern (Figs 9n, llc,e; Sachs, 1974). When the only possible flux from one source is not polar but transverse it can be blocked by another source and vessel differentiation is inhibited (Fig. lle). Polar flux can occur even against a concentration gradient, and direct vessel contacts differentiate between two sources which are one above the other (Figs 9p, 1l i ; Section V.C). ( c ) Callus, in culture and on cut plants, forms xylem whose relation toflux is not clear. A common form of differentiation in tissue cultures is nodules in which the exact relations between the cells are not clear (Gautheret, 1959). Similar nodules often form on callus at the base of a plant (Fig. 20a; Mosse and Labern, 1960),and they may indicate the absence of an oriented flux and, possibly the differentiation of localized sinks for auxin (Section VII). The importance of localized application of auxin, which might indicate a need for flux, was noticed by Wetmore and Sorokin (1955), who found that auxin was not effective when it was added to the culture medium. Patterned differentiation in culture was found by Dalessandro (1973), whose observations might be understood as a result of a transport of auxin from the periphery to the centre of relatively large pieces of callus. Observations which cannot be explained by a relation to flux are the differentiation of isolated parenchyma cells in culture (Kohlenbach and

(e)

3 ......

Fig. 11. Differentiation in pieces of storage root. Wavy lines indicate the location of vessels and sieve tubes formed after the experiment started (for photographs see Fig. I0a-d). Existing vascular tissues (see Figs IOd; 17c) were omitted for clarity. Dots show the location of auxin sources. (a-e) Relation of differentiation to applied auxin. A localized single source (a) induces considerable differentiation within three days. The form of the new vessels shows the influence of polar transport (towards the original direction of the root; not downwards, as the sections were horizontal during the experiment). Cuts which prevent this transport (b) increase differentiation in other directions. Two neighbouring sources (c) interact in terms of the tissues they induce. At some distance from such sources their vessels generally form joint strands. A comparison of (d) and (e) shows that one source can also inhibit the differentiation induced by another. Such inhibition is found even when the source present in (e) and not in (d) is applied almost two days after the other, showing that induction requires a continuous auxin effect (a similar experiment and result appear in Fig. 9m, n). (f, g) Differentiation in the absence of external auxin. Limited differentiation, always restricted to the root-side, is found after a few days (f). In experiments kept for a week or longer (9) this differentiation is extensive and many vessels and sieve tubes have the form ofrings and whirlpools (see Fig. 20d, for photograph). (h-j) Induction by auxin applied to the root-side of the tissue. Such induction involves a reversal of polarity as it is expressed by the relation between the vessels and the source ofauxin. It is both limited and slower than the induction expressed in (a). When auxin sources are present on the two opposite sides of the tissue (i) some vessel contacts are formed between them. These contacts, discussed in the text, are not readily understood in terms of an auxin flux though this readily accounts for all other observations. (Unpublished experiments on Swede, Brussica nupus, storage roots. Pieces whose area was about 2 x 2 cm and thickness of0.5 cm were kept horizontal in closed moist dishes at room temperature. Auxin sources were 0.05-1% Indole Acetic Acid in lanolin. After three days, or a week for (g) and sieve tube observations, the material was trimmed, cleared in Lactic Acid and dissected through the cambium. Sieve tubes were stained with Lacmoid and observed in Sodium Lactate.)

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Schmidt, 1975; Fukuda and Komamine, 1980). These may be an expression of other controls of lignification mentioned earlier, such as those of leaf sclereids and the lignified tissues of fruits. The occurrence of isolated elements does not contradict the proposed relation of most vascular tissues to a flux of signals, though it does suggest that this is not the only way in which lignification can be induced. 4 . Conclusion and Discussion The facts considered above show that the pattern of vascular differentiation, especially in cases of regeneration, can be readily accounted for by some relation between a flux of signals and differentiation. In addition, it was shown that the induction of vascular tissues by auxin, a natural signal or evocator, must involve some relation to a flux of this signal. It may be concluded, therefore, that the natural pattern of vascular strands is largely or completely dependent on theflux of signals through the diferentiating cells. One may ask, however, whether there are any other mechanisms which could account for the observed pattern. Since the conclusion stated above does not specify the nature of the signals nor their direction of movement, the only other possibility could be local interactions between neighbouring cells. These interactions would have to be localized along one axis of each cell, but they need not have any determined direction so as to account for the formation of strand. A mathematical model which incorporates localized interactions rather than over-all flux of signals has been developed by Meinhardt (1978). The evidence favouring an over-all flux of signals consists of the results summarized in the last two sections rather than any characteristics of the vascular strands themselves. It will be shown below (especially Section II1.B) that the flux of differentiation signals can be the basis of the local interactions necessary to account for strand formation. It is, of course, possible that additional local interactions control differentiation, but there is no evidence at present for their existence. E. EVIDENCE FOR ADDITIONAL CONTROLS

1. Introduction The previous three sections dealt primarily with positive evidence showing that vascular differentiation can depend on signals originating in the leaves and moving along the polarity of the plant into the roots. The existence of this control system does not, however, exclude the possibility of differentiation independent of it. Almost all the evidence considered above was concerned with relative degrees of vascular differentiation and not with its presence or absence, and the almost impossible task of determining that no other controls exist was not even attempted. The present section, therefore, will consider those cases of differentiation which are not clearly dependent

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on the flux of differentiation signals from leaves to roots. The attitude of the following discussion will be that much of this differentiation may indicate additional sources and additional traits of the signals whose existence was proven above. An attempt will be made to identify the observations which indicate additional controls, both internal and external to the differentiating cells. 2. The Apparent Absence of a Correlation Between Leaf Development and Cambial Activity Primary differentiation in the shoot is always correlated with the development of new leaves or other organs, such as reproductive structures, which might be expected to have effects similar to those of leaves. The correlation in the case of secondary differentiation is by no means clear. Cambial activity can be associated with mature rather than young leaves (Fig. 3 1 ; Munch, 1938; Wareing and Roberts, 1956; Hess and Sachs, 1972; Benayoun and Sachs, 1976) and can be found even in petioles of mature leaves which have been rooted (Jost, 1893; Simon, 1929). Thus there is no reason to doubt that mature leaves can produce signals for vascular differentiation, and the possible role of these signals in controlling the type of wood formed by trees will be considered in Section V1.C. It is not clear, however, how much of this signal production goes on in intact plants (see Section V1.C). The mature leaves also cause formation of abnormal xylem in some plants (Simon, 1929; Shininger, 1970; Benayoun and Sachs, 1976). The role of mature leaves in controlling cambial activity in intact plants therefore deserves further study, though there is no reason to assume that their effect differs from that of young leaves by anything more than quantitative characteristics. The correlation between leaf development and cambial activation is very general (see Section 1I.B) but it is not invariable (Chowdhury and Tandan, 1950; Studhalter et al., 1963; Casperson, 1965; Amobi, 1972). Some of these observations may be misleading, because leaf development must be judged using dissection and microscopy so as to include all the changes which can affect vascular differentiation. For this reason, statements that cambial activity precedes leaf development (Zasada and Zahner, 1969) require further work and a definition of what leaf development is. The cessation of cambial activity has also been rarely studied together with the events in the apices, where next season’s leaf primordia are being initiated. Another important reason for a complex relation between cambial activity and leaf development might be the effect of the functional vascular system, which can transport the signals for differentiation and thus prevent their expression (see Section V.B). Any conclusions based on the correlation of leaf development and cambial activity will therefore require additional work which must include experimental treatments, such as leaf removal.

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3. Vascular Diferentiation in the Absence of Leaves A variety of experiments demonstrate clearly that vascular differentiation can continue when all leaves and other shoot organs are removed, or when the direct contact with the leaves is completely disrupted. The removal of the leaves reduces cambial activity and regeneration around wounds (II.B), but it does not completely prevent their occurrence. Cambial activity has also been observed in trunks from which the entire shoot was removed (Neeff, 1922), below complete girdles (Soding, 1937; for review see Wilcox, 1962) and in regions of the cambium which have been completely girdled from all sides (Fig. 7d; Evert and Kozlowski, 1967; Kirschner rt al., 1971 ; Evert er al., 1972). Vascular differentiation has also been found in pieces of tobacco stem grown in culture (Sheldrake and Northcote, 1968), in cultured root tips, in which primary development continues in the absence of exogenous auxin (Torrey, 1963), and the cambium of small pieces of storage tissues kept in moist conditions (Fig. 1 I f ) . Differentiation in the absence of leaves may depend on the formation of the normal leaf signals in all parts of the plant, especially in the differentiating vascular tissues (Soding, 1937; Jost, 1940; Sheldrake, 1973a) rather than on other cpntrols such as a determinate state of the cambium which could continue to divide in the absence of external signals. There are direct measurements showing that auxin can be produced in the cambial region, possibly in the differentiating vascular elements (Sheldrake, 1973a), and other developmental events also indicate the presence of auxin when vascular differentiation is taking place (Sheldrake and Northcote, 1968). Cambial activity in isolated storage tissues resembles that induced by low concentrations of auxin and it can be inhibited by Triiodobenzoic acid (Sachs, in preparation). It thus appears necessary to expand the hypothesis developed in the preceding sections and to suggest that the differentiation signals are produced in all parts of the plant. Leaves, and especially developing leaves, may have a special role not because of unique signals they produce but because they produce them in relatively large quantities. It is still unclear, however, how much of the signal production found, when leaves are removed or in isolated sections, also occurs in intact plants. Sheldrake (1973a) suggests that auxin is produced whenever cells die, including during vascular differentiation. This hypothesis might be true and might have considerable evolutionary significance, but the evidence from the controls of vascular differentiation and other developmental phenomena (see Section VII) is that most of this auxin does not influence the rest of the plant; certainly roots and senescing leaves do not have auxin-like effects. Kirschner et al. (1971) suggest, on the other hand, that auxin production in cut plants is a first stage of a regenerative process, a sort of general “regeneration” of the physiological tip (Went and

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Thimann, 1937). This possibility, which was mentioned above concerning the effect of mature leaves, requires further work. 4 . The Injluence of Substunces other than Auxin Many substances have been found to influence cambial activity in cut plants or the differentiation of tracheary elements in tissue cultures (Kiinning, 1950; Robards et ul., 1969; Torrey rt ul., 1971 ; Roberts, 1969,1976). As mentioned above, the effect of auxin is unique in being directional and in replacing the effect of organs known to produce auxin. Some of the other substances may be effective through their influence on auxin transport (Hejnowicz and Tomaszewski, 1969). Gibberellins may have an important role as indicated by their specific influence on the type of cells which differentiate (see Sections VI.B, V1.C) and by their action in early stages of shoot development, before auxin is effective (Siebers and Ladage, 1973; see Section V1.D). But the question as to whether the effects of other substances indicate additional controls of vascular differentiation remains unanswered at present. It should be remembered that if a substance elicits vascular differentiation it does not mean that it is present, or present as a limiting factor, in the developing plant. Differentiation in tissue culture might be limited by any of the numerous factors which influence the normal activity of the cells and not only by controls of the specific processes of differentiation. Most tissue culture work, furthermore, has concentrated on elucidating the processes of differentiation and not on the controls of pattern, which are the topic of this review. 5. Do Vuscular Tissues Control Leaf' Initiation und Development :) The control of vascular differentiation by leaves does not exclude a reciprocal effect. This would form a feedback loop and one would expect it to be a likely means for the control of development. The influence of vascular tissues on leaves is, however, rather hard to investigate, since these tissues cannot be readily removed or grafted, and the ability to form new leaf primordia is restricted to minute regions inconvenient for experimental manipulation. It should be noted that the problem considered here does not include the influence of vascular tissues on vascular differentiation, a topic to be considered in another context (Section 1II.B). Leaf development can be expected only if all the nutrients, possibly including hormones, are moved in the right direction from the rest of the plant. Vascular tissues are known to be the channels through which such transport can occur rapidly. Thus, leaf development both induces vascular differentiation and depends on its presence, and the two processes must be connected by a positive feedback control. This conclusion does not, however, apply to leaf initiation and the early stages of development, which require only limited transport. It has in fact been shown that early stages of leaf development

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continue even when the vascular tissues leading to the primordia are cut (Snow and Snow, 1947; Wardlaw, 1956). The first signs of procambial differentiation may be seen in some plants before the appearance of the primordia they would supply in the intact plant (De Sloover, 1958; Esau, 1965; Larson, 1975); but this evidence depends on the available methods of recognizing the procambium and the leaf primordia. Leaves which are not seen microscopically may be present, in a physiological sense, as centres of specialized metabolism and sources of hormones and other signals. The decision as to which process occurs first depends on the method of observation, and thus cannot be used as evidence concerning the influence of one process or another. The direction of the differentiation of the procambium and the primary phloem is from the stem towards the young leaf (Esau, 1965). This has been understood to mean that the vascular strands determine the location of leaf primordia (Esau, 1965; Larson, 1975). The observations cannot, however, be made on the unknown earliest stages of differentiation. As was discussed above, the time at which the cells reach relatively late stages of differentiation depends primarily on the maturity of these cells, and will thus occur earlier in the stem than right next to the primordium (Section 1I.D). The observed “direction of differentiation” is thus no indication that vascular tissues determine leaf primordia or vice versa. An experimental study of the relation of leaf primordia to the procambial strands was carried out by Snow and Snow (1947). They found that cutting the procambium does not prevent the initiation of leaves in the expected location. This alone would not prove that the leaves were not determined by the procambium before the cuts were made (Esau, 1965). But the Snows showed that the presumptive location of the very same primordia could be shifted by microsurgery. It follows, therefore, that primordia whose location had not yet been irrevocably determined did not depend on the procambium to specify their location. It may be concluded that while a feedback relation between leaf development and vascular differentiation is very likely to exist, there is no evidence at present that the location of leaves is determined by vascular tissues. The discussions in this review will depend only on the induction of vascular differentiation by leaves and not on the presence or absence of a reciprocal process. It is possible that favourable material may yet provide evidence for vascular determination of leaf initiation. 111. CELL POLARIZATION BY A FLUX OF SIGNALS A. INTRODUCTORY SUMMARY

The problems dealt with in this chapter concern the control of organization of discrete strands of primary tissues and the formation of vessels and sieve

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tubes from specialized cells, arranged in defined rows. This pattern of cells shows that relations between cells along the axis of the strand are different from relations in other directions. Since vascular tissues are capable of regeneration these relations must involve oriented interactions. The basic characteristics of these interactions are the subject of the present chapter. If vascular differentiation depends on a shoot-to-root flux it must be asked how this flux becomes canalized in discrete channels. This canalization could occur if the process of differentiation itself increased the ability of cells to transport the signals for differentiation. This would mean a control by a positive feedback : differentiation depending on a flux of signals and the flux depending on differentiation. Since differentiating cells would increase the flux along their own file and drain it away from neighbouring files, this control would provide the necessary oriented interactions. Experiments on the control of contacts between new and existing strands show that a strand orients vascular differentiation towards itself but only as long as it is not connected to leaves or loaded with auxin. Other evidence supporting the hypothesis concerns the transport of radioactive auxin, which occurs most readily and rapidly in intact vascular tissues and can be maintained and even increased by auxin treatments. The suggestion that differentiation involves polarization raises the question of the relation of this process to the determined polarity of plant tissues. The original polarity, as expressed by differentiation, can be remarkably stable, for example in re-oriented cambial grafts. Vascular differentiation follows this polarity in most cases, but differentiation in new orientations can occur readily, even in cases which can be considered as complete polarity reversals. The differences in the conditions which lead to these results indicate that polarity is stable only as long as the flux of the differentiation signals continues; otherwise a flux in a new direction can re-orient the cells. It is thus suggested that the flux of auxin, and presumably other signals, both induces and maintains polarity. Induction by a canalized flux should be always directional, and it is therefore asked whether a directionality or polarity can be found for all vascular tissues. The general form of the vascular tissues is that of a drainage system, in which all parts can be assigned a clear shoot-to-root orientation. The networks found in leaves, however, include neighbouring vessels with opposite shoot-to-root polarities, and it is therefore suggested that though induction has a given direction at any given time, this direction can be reversed, even within a given cell. This suggestion is supported by observations of leaf growth and experiments in which the location of auxin application was repeatedly changed. Thus, it is the axis rather than the polarity of the cells which might be the first determined characteristic during vascular differentiation.

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B. FACILITATION OF SIGNAL TRANSPORT AS A BASIS FOR STRAND FORMATION

1 . The Problem und the Working Hypothesis When a bud, grafted or adventitious, induces the differentiation of parenchyma cells or a cambium, the new vascular elements differentiate as organized strands of cells and form vessels and sieve tubes. The same orderly arrangement of the differentiating cells is found when a localized source of auxin is applied (Figs 10, 11). The orderly formation of strands must therefore be due to some properties of the cells themselves and not to any external cue other than the presence of a localized source of inductive signals. The problem to be dealt with here is the nature of the properties responsible for the major longitudinal pattern of vascular differentiation. If differentiation were to depend on an exact concentration of a diffusing signal, the new vascular elements would have the form of a cup surrounding the source of auxin. The relation of differentiation to a flux of signals, one of which is auxin (see Section II.D), explains why this does not occur, but it still does not account for the formation of discrete rows or strands of cells such as vessels or sieve tubes. For discrete strands to be formed the flux must be canalized to narrow channels. This canalization cannot be due only to some predetermined traits of the cells for it occurs around wounds (Fig. 3) and in completely arbitrary locations and orientations relative to the original polarity of the tissues (see next section). The canalization of auxin transport must therefore depend on processes which occur during vascular differentiation. Canalized signal flux during differentiation would mean that differentiation increases the ability of the cells t o transport the very same signals which induce differentiation (Fig. 12; Sachs, 1968a,b, 1969a, 1975b, 1978a). This hypothesis would account for the general form of vascular strands; an analogy would be the way that erosion causes the formation of discrete channels for the flow of water. Increased transport ability, or polarization, would influence the flux of cells situated along the same channel while draining the signal away from neighbouring cells and thus inhibiting their differentiation. The hypothesis has been subjected to mathematical analysis (Mitchison, 1980)which has shown, among other things, that it could operate by changes of either the diffusion or the polar transport through the cells. Here, canalization will be treated in the broadest sense and the main task will be to review the evidence which supports the working hypothesis.

2 . Evidence from the Relations Between Strands When a strand is wounded, the surrounding cells form new, regenerative vascular elements (Figs 3,9b; Simon, 1908a). A wound that does not cut the vascular elements does not have the same effect, even though it does make the surrounding cells competent to respond to auxin by vascular differentiation

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(a

1

(b)

( c )

( d )

Fig. 12. Hypothesis that canalization of flux determines vascular patterns. Cut pea stems to which auxin sources were applied, as in Fig. 9g and k. Auxin flux, dependent on diffusion, polar flux and preferred transport through vascular tissues, is indicated by arrows. Plus signs indicate auxin accumulation that inhibits flux. ( a d ) Are stages in the canalization of the flux to future vascular strands. The preferred channels inhibit further induction and differentiate to vascular strands. In (d) the existingvascular cylinder was saturated by an auxin source and does not act as a sink. This saturation inhibits the flux from the lateral source and, therefore, inhibits new differentiation.

(Fig. 9 a g ; Sachs, unpublished). It follows that a strand inhibits the differentiation of additional vascular elements nearby. This same effect is also seen when the treatments shown in Figs 9e,f and lla,b are compared: preventing the differentiation of vessels in the normal morphologically downward direction by cutting promotes the formation of vessels in other directions. These facts are most readily accounted for if the differentiating vessels are the preferred channels for the transport of the signals which induce their own differentiation. Exposed ends of vascular strands, on the other hand, induce the differentiation of additional cells of their own types. This “homeogenetic induction” (Lang, 1973) has been observed many times (Janse, 1921; Jost, 1931; Rudiger, 1953; Gautheret, 1957; Kuroda, 1960) and is implicit in the very form of strand differentiation (Fig. 2). This phenomenon is again to be expected if differentiating strands are the best transporters of inductive signals, and thus their free ends are either the best sources or the preferred sinks of these signals. The “sink” effect of vascular strands on the flux of signals for vascular differentiation is seen in the attractive influence of cut strands on the course of new vascular tissues. Regenerative strands formed around wounds always connect to the cut strands not only above but also below the wounds (Fig 9b). When auxin is applied to a cut stem, the new vessels join the existing strands along the shortest route (Fig. 9g). The working hypothesis outlined above suggests that the movement of auxin is first determined by diffusion (Fig. 12). The presence of a “sink” which transports auxin away actively will

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create a flux in its own direction. This flux will be reinforced by the autocatalytic nature of the differentiation process, the increase in transport capacity as differentiation proceeds, draining the signals away from other cells. The attractive influence of a cut strand on the course of new differentiation can be reduced or prevented by supplying the existing, cut strand with a source of auxin (Figs. 9k, 12d; Sachs, 1968b, 1969a). This result supports the working hypothesis considered here, for a loaded strand would not be expected to be a sink for auxin. As one would expect from the hypothesis, the auxin applied to the existing strand does not have a clear, threshold effect; the formation of contacts depends on the relative concentrations of the two auxin sources, one inducing vascular differentiation and the other inhibiting its contacts with the existing strands (Sachs, 1969a). Contacts are also promoted by cuts below the inductive auxin, which increase its effective concentration by preventing its polar flow down the stem (Fig. 91; Sachs, unpublished). Evidence favouring the hypothesis is thus found in the control of contacts between new and old vascular strands. This raises the question whether similar controls operate for naturally induced vascular tissues in intact plants. A major feature of the vascular systems of most plants which might depend on these controls is the leaf gap, a region of parenchyma rather than vascular tissues located above the vascular strands which diverge to leaves (Figs la, 13a; Fahn, 1974; Esau, 1977). There is, to state it differently, a general inhibition of direct contacts with vascular strands connecting to developing leaves. That this is in fact an effect of the young leaves is shown by the formation of contacts across the leaf gap when the leaf primordia are removed at an early stage (Fig. 13b; Wardlaw, 1946, 1968; Sachs, 1968b, 1972a). Differentiation in the leaf gap region also occurs when the leaf abscisses, by an extension of the neighbouring cambium ; this again, would be expected if the leaf gap is formed and maintained by an active influence of the leaf. The vascular contacts of lateral buds are another example of the operation of the controls found in auxin application studies. Laterals growing on intact plants do not make direct contacts with the strands leading to the leaves above and below them (Sachs, 1970). Laterals growing as a result of the removal of the shoot above do, on the other hand, make direct contacts with the neighbouring strand, a process which can involve a re-orientation of the cambium (Fig. 15b; Neeff, 1914, 1922; Sachs, 1970; see also the next section). These contacts of the released buds may be made specifically to the strands leading to leaves which have been removed (Sachs, 1968b). The possible role of this control of vascular differentiation in apical dominance is considered in Section VII.

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( b )

Fig. 13. Leaf gaps and the control of their formation. Vascular contacts at a pea stem node. (a) From an untreated plant, showing that the strands connecting to the leaf on the left are not contacted by strands leading to the leaves above. In other words, there is a gap in vascular continuity. (b) The leaf was damaged when it was very young but after its strands were induced (later than the removal shown in Fig. 8). The gap is bridged by contacts made to the strands leading to the damaged leaf. As in Fig. 9h and j, the intact leaf inhibits contacts to its vascular supplies (drawn from Sachs, 1968b).

3 . Evidence from the Transport of Radioactive Auxin The hypothesis suggested above makes specific predictions concerning the transport of a signal, part of which is the known molecule auxin. Thus, results concerning the transport of radioactive auxin can be used to test the hypothesis. It is evident, therefore, that while auxin can be transported through parenchyma, the preferred channels are cells of the vascular system (Sheldrake, 1973b; Wangermann, 1974, 1977). The preparation of sections for studies of transport damages the phloem, at least temporarily (Gersani et al., 1980b), and the transport of auxin has been found to be much more rapid in intact plants (Morris et af., 1973; Goldsmith et al., 1974). These facts agree with the assumption that vascular differentiation is associated with an increase in auxin transport, though they do not give the essential information about when during the process of differentiation this increase occurs. Both long-term and short-term studies have shown that treating plant sections with auxin increases their ability to transport radioactive auxin as compared with untreated controls (Leopold and Lam, 1962; Hertel and Flory, 1968; Osborne and Mullins, 1969; Rayle et al., 1969). These results can be interpreted, however, to mean that auxin treatments maintain the activity of transport channels present at the initiation of the experiments but

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do not cause the differentiation of new channels. Sheldrake (1973b), however, found an increase in transport associated with cambial activity and thus with the formation of new vascular tissues. An attempt to demonstrate completely new channels was based on observations of a new orientation found above a transverse wound (Fig. 14; Sachs, 197513). An auxin treatment above such a wound evokes obvious differentiated vessels observable after two days. Auxin transport through such differentiating channels was judged by the amount of radioactivity found in the cut hypocotyl below the wound. Auxin treatment had an effect on radioactive auxin transport within 16 h ; this effect was a significant, though small, increase compared with all the obvious controls, which included intact plants cut just before the measurements were started.

([I)

( b )

( C )

Fig. 14. Induction of auxin transport by auxin. Sections of bean stems with one cotyledon to which 'H Indole Acetic Acid was applied above lateral cuts (indicated by arrows). The numbers are the c.p.m. above and below the cotyledon and the percentage of the radioactivity transported to the lower part. (a) The stem was cut from a seedling just before the radioactivity was applied. (b) Stem cut 16h earlier. (c) As (b), but cold auxin present during the 16 h before being replaced by radioactive auxin. Vascular tissues appeared in (c) above the wound two days after the treatment was started. The early increase in auxin transport seen in the numbers, though of limited capacity, was repeatable (from Sachs, 1975b).

These results are the ones to be expected if auxin transport were increased during differentiation. The available experiments do not, however, supply any information about the parameters of the changes. These parameters cannot be measured readily because they describe the behaviour of relatively narrow channels, while auxin transport can be measured only for the tissue as a whole. Autoradiography might supply useful information but it could not measure the auxin that is actually moving. Thus while the transport results fully accord with the hypothesis, they do not test it in a rigorous way.

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4 . Conclusion and Discussion It may be concluded that the available evidence supports the hypothesis that the processes of vascular difierentiation are associated with an increased transport of the signals which induce this diferentiation. This hypothesis accounts for the major traits of the pattern of vascular strands and the contacts between them. It is thus relevant to ask whether other hypotheses would fit the facts. Though more complicated explanations are possible, they would not take account of all the various facts known about vascular differentiation, especially those considered in the previous section. The one published suggestion (Meinhardt, 1978) has been shown by computer modelling to be a possible control system for strand differentiation. This suggestion would require separate signals for the induction of differentiation and the inhibition of similar processes in neighbouring cells, and would require differentiation to be an all-or-none determinate event, rather than the gradual change proposed here which is stabilized by positive feedback. As will be seen in Section IV.B, there is clear evidence that the determination of vascular tissues occurs gradually. At present, therefore, the hypothesis outlined above is the only one which fits the available evidence, and it is attractive because of its simplicity and its various implications, considered below. C. THE STABILITY OF POLARITY AND ITS POSSIBLE BASIS

I . Introduction The hypothesis that differentiation is associated with a flux of signals, one of which is auxin, was supported by observations of a relation between vessel induction and the known shoot-to-root polarity of auxin transport (Figs 9e, 1la; Section 1I.D). The discussion of the previous section further suggests that differentiation involves a polarization of the cells for auxin transport. It is therefore of interest to find out to what extent the pdarization involved in differentiation can change the determined polarity of the tissues. This information could be relevant to an understanding both of the canalized transport considered in the previous section and of the basis for the determinate nature of the polarity of plant tissues. Tissue polarity can be expressed in various ways (Bloch, 1965): the axis of cell elongation, the planes of cell division, the location of regenerated buds and roots, the success of graft unions, the course of vascular differentiation and the transport of auxin. The orientation of vascular strands and the shape and wall structures of their component cells, especially the vessel members, define an axis, though not a direction, and can therefore be used to study polarity. The advantage of these anatomical criteria is that they can show polarity changes even when the tissues or organs as a whole maintain their original state. This section, therefore, will consider polarity as seen by vessel differentiation.

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2 . The Evidence for the Stability of Polarity The concept of a completely determined cytoplasmic polarity was originally suggested by Vochting (1878, 1892, 1906, 1918) and has been the subject of much discussion (Bunning, 1957; Sinnott, 1960; Bloch, 1965).One basis for Vochting’s suggestion was the polar regeneration of buds and roots. This regeneration can be readily changed by external conditions, but it has been argued that this is an alteration of the expression of polarity without a change in its cytoplasmic basis (Vochting, 1906,1918; see also Sinnott, 1960). Vochting also based his ideas on the limited development and unusual vascular differentiation of inverted cuttings -cuttings in which buds were allowed to grow only morphologically below the roots, so that at least a section of the original stem must function in a reversed shoot-to-root orientation. Inverted cuttings have been grown successfully (Castan, 1940; Went, 1941;Sheldrake, 1974)but theseare exceptions rather than the rule(Kirschner and Sachs, unpublished). Thus the facts concerning regeneration and plant development point to a determined polarity, though not necessarily to the absolute stability which Vochting assumed. Auxin transport has been found to be polar in numerous experiments using all types of plant material (for review see Goldsmith, 1969, 1977). This polarity is not changed by external conditions, and there is good evidence that it is the basis of other expressions of polarity (Warmke and Warmke, 1950; Gautheret, 1959; see Section VII). There is only one report of a clear reversal of the polarity of auxin transport, in inverted cuttings of Tagetes (Went, 1941) and these results could not be repeated (Sheldrake, 1974). The effect of polarity on vascular differentiation was seen in inverted cuttings by Vochting (1892,1918),and its stability was an important basis for his suggestion that individual plant cells have a determined polarity which cannot be changed. The general conformity of vascular differentiation to polarity has been mentioned earlier and can be seen even in work which stresses the deviations from this polarity, which will be considered below (Brown, 1936; Rehm, 1936). The differentiation response to experimental applications of auxin has been found to depend on tissue polarity, including cases where the auxin was applied to the basal rather than apical end of stem sections (Thompson and Jacobs, 1966; Thompson, 1970; Aloni and Jacobs, 1977a). A clear demonstration of the stability of polarity expressed by vascular differentiation is found when tissues that include a cambium are grafted so that the new shoot-to-root orientation does not conform to the original polarity. The new vascular tissues follow a complex course, showing clear effects of the determined polarity of the tissue, and whirlpool patterns are often found (Vochting, 1892, 1918; Rothe, 1924; Sax and Dickson, 1956; Thair and Steeves, 1976; Kirschner and Sachs, unpublished). The effect of polarity is also seen when an inverted stem section is grafted at the base of a

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cut plant (Figs 7g, 1Sa; Sachs, 1968a). A small piece of inverted tissue grafted between the root and the shoot of pea seedlings eventually differentiates continuous xylem and does not prevent normal development; at least some of its cells, therefore, can be assumed to have been inverted (Sachs, 1968a). Polarity therefore has a clear determined nature, but this determination neednot be absolute. 3 . Changes in Polarity Seen by Vascular Diferentiation It is now necessary to summarize evidence that changes of polarity can occur rather readily. The relation of these changes to the determination considered above will be deferred to the following discussion. A change of polarity may be seen in bridges left in cut stems (Fig. 7i). These bridges connect the shoot and the root along a new orientation, and the new vascular tissues are formed to conform to this new orientation (Janse, 1914; Priestley, 1930b; Kirschner et al., 1971). The changes in the cambium can be followed readily because they are recorded in the successive layers of the xylem cells formed by the cambium (see Section V.C).The major steps in this cambial reorientation (Priestley, 1930b; Kirschner et al., 1971) are the rapid differentiation of new vessels from cells whose longitudinal axis still conforms to the original polarity, the division of all cambial initials into short cells and the intrusive growth of these cells along the new shoot-root orientation, establishing a normal cambium with a new orientation. As this reorientation progresses the new vessels become straighter and the relations between the individual vessel members are more orderly. These changes have been carefully described for a 90" reorientation, but girdles and cuts involving a complete reversal of polarity are also successful (Figs 7k, 1%; Janse, 1914; Sachs, in preparation). The reorientation seen most clearly in partial girdles is also found in other cases. The growth of laterals following their release from apical dominance is associated with a change in the shoot-to-root orientation of the cells at the base of the growing buds (Fig. 15b). When a cambium is present, its cells reorientate in the same way as they do in a partial girdle (Neeff, 1914, 1922). A similar change is found at the base of adventitious buds growing from a cambium. The development of adventitious roots (Fig. 15c),and even normal roots, is associated with the same processes. Even regeneration around wounds requires new differentiation at an angle with the original polarity of the unwounded plant (Figs 3, 15d). A change of polarity of this general type can be seen near any inductive source of auxin (Fig. 10a). Though it is true that the general course of differentiation follows the polarity of the tissue (see Section I1 .D), there are many local exceptions close to the source of auxin where the vessels fan out at various angles. This raises the question whether complete reversal can be caused by auxin. The vessels themselves have a clear axis but no direction ; polarity reversal becomes meaningful, however, if one defines vessel polarity

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as the direction away from the inductive source of auxin. According to this definition complete reversal of polarity is common when auxin is applied to isolated stem segments or pieces of storage tissue (Figs 1 lh, 150. It is not found as readily when auxin is applied to intact plants. A limited reversal, involving cambial activity but not vascular maturation, was observed next to buds on cut plants or ringed roots (Brown, 1936; Rehm, 1936). Auxin applied so that only reverse polarity induction can occur causes the differentiation of vessels of considerable length a few millimetres in pea epicotyls (Fig. 9i; Sachs, 1969a) or in isolated Coleus cambium (Fig. 150 and over a centimetre in storage tissues (Fig. 1 l h ; Sachs, in preparation). It may be asked, therefore, whether this differentiation involves a reversal of polarity in any sense other than that defined above. It is thus significant that vessels involving a change of polarity are not formed if a polar possibility is available (Fig. 9e,f; Sachs, 1969a). The rate of differentiation of these vessels is far slower than that of vessels formed along the original axis of polarity of the plant (Sachs, in preparation). All types of vessel induction are inhibited by triiodobenzoic acid, a compound known to inhibit polar auxin transport (Niedergang-Kamien and Skoog, 1956), which indicates that polarity can in fact be changed. Though it would be desirable to have direct evidence for changes in the polarity of auxin transport, vessel differentiation and the changes cell division and cell elongation (Neeff, 1914, 1922; and Section VII) show clearly that polarity changes are possible and that they are induced by the same signal flux which controls vascular differentiation (Janse, 1914; Neeff, 1914). ~

Fig. 15. Vessel differentiation in relation to polarity. (a) Graft where one member was inverted as in Fig. 7g. The sharp angle in the vessels shows a dependence of their orientation on the determined polarity of the two tissues. (b-d) Regenerative vessels involving a reorientation of polarity. (b) shows the vessels connecting to a lateral bud that grew after the removal of the shoot above it. The new vessels contact all the strands of the removed leaves. These contacts require differentiation to occur at various angles to the original polarity. Adventitious roots on a cutting, such as the one on the lower right of (c), involve the same phenomena. Vessels requiring tissue reorientation connect to this root at least half way across the stem. Vessels formed around wounds (as in Fig. 3d) involve the same phenomenon, and (d) shows that these vessels can be composed ofcells whose shape and cell wall thickenings reflect the original axis of the tissue. (e, f) Reversal of polarity by wounds and auxin. (e) is a stem cut as in Fig. 7k, so that the contact between the shoot and the root requires a reversal of the original orientation of the tissues. Vascular differentiation occurs readily across such regions, provided they are not longer than a few mm. An auxin source also induces tissues whose formation is a reversal of the original polarity, provided no other possibility is available. In (0auxin was applied below the base of the photographed region to a region cut as in Fig. 91, so the only contact with the rest of the plant was on the upper left side. The photograph shows the vessel differentiated from the cambium along this tissue flap. ( (f) is a Coleus stem and all the rest are from bean hypocotyls. Experiments cleared with Lactic Acid 5-10 days after the first treatment, Stained with Lacmoid and viewed in Sodium Lactate, except for (d), which is a permanent mount). (a and r) x 80; (b) x 27; (c and e) x 8: (d) x 192.

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4 . Discussion: A Possible Basis for Polar Determination The facts summarized above are apparently contradictory : the polarity of the cambium, as it is expressed by vessel differentiation, can be maintained in reoriented grafts and other experiments for long periods, and yet the very same polarity can be readily changed by simple wounds, by bud growth and by localized auxin. There is a consistent difference, however, between the conditions which lead to the two different results : changes of polarity were found only when no polar auxin flux could be expected. Whenever such a flux continued, even along a tortuous path, as in reoriented grafts, polarity was found to be very stable. It follows, therefore, that polar auxin transport maintains the polarity which is expressed by the very same transport. This suggestion was made by Sheldrake (1974) on the basis of his study of inverted cuttings and is supported, though by no means proven, by the experiments which show that auxin maintains the ability of a tissue to transport auxin, considered in the previous section. This suggestion may now be related to the canalization hypothesis developed in the previous section. It appears that auxin flux can both polarize the cells and maintain their polarity. In the absence of this flux, polarity is labile and can be influenced by auxin coming from other directions. This feedback relationship could account for both the stability and the changes of polarity as they are expressed in vessel differentiation. The results considered above, therefore, both confirm and widen the scope of the hypothesis concerning the role of canalized auxin flux in cell polarization and vessel differentiation. D. THE FORMATION OF VASCULAR NETWORKS

1. Are All Vascular Tissues Polar?

A control of vascular differentiation by a shoot-to-root flux would suggest that a polarity can be assigned to each vascular element. The question therefore arises whether this polarity is always present. As mentioned above (Section II.D), the vascular system of the plant axis, as well as the major veins of the leaves, have the form of a drainage system leading from the shoot tissues to the root apices (Figs 1, 16). The individual vessels in these vascular strands do not contradict the principle of polarity, though their course may meander, especially within the secondary system (Braun, 1959 ;Zimmermann, 1976). The sieve tubes have hardly been studied, for technical reasons, but the available evidence shows that while they may bend and change their direction (Aloni and Sachs, 1973) a shoot-to-root polarity can be readily assigned to each cell. Primary vascular systems often form a network in which individual strands make repeated contacts with their neighbours. This network aspect is readily seen in the stems of herbaceous plants (Fig. 1 ; Esau, 1965), in which many of the phloem contacts are not readily observed (Aloni and Sachs, 1973). Networks of this type are also commonly found in fern leaves (Fig.

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16b) and they are also a feature of the secondary system, where the contacts form a complex mesh of vessels or sieve tubes, depending on the tissue (Fig. 17c). From the point of view of control of differentiation by directional flux, these networks are clearly polar. They do indicate, however, that constant changes may occur in the inductive streams, a phenomenon to be considered below. Complex networks in which the individual cells do not have an obvious shoot-to-root polarity are found in the leaves of many ferns and almost all angiosperms (Fig. 16c-f). Any rules chosen for assigning a polarity to the cells of such networks soon lead to opposite polarities within many strands and even in individual cells (Sachs, 1975a). Complex networks, therefore, are not polar and would not be expected to be formed by the directional control discussed in the previous sections. Complex networks may thus indicate the operation of a control system not considered previously. They might, however, indicate a form of polar control that can operate in a manner not suggested by studies of differentiation in stems and roots. Both these possibilities are of obvious interest and the evidence relating to them is the subject of the present section. 2. The Formation of Vascular Loops by Induction in Opposite Directions The differentiation of complex networks has been carefully followed (Esau, 1965; Lersten, 1965; Blackman, 1971), but these descriptions of the order of appearance of the mature, observable vascular elements do not offer any suggestions concerning the control of their formation. It is therefore necessary to define a clear case of vascular differentiation with no polarity. The simplest case is the presence of loops of vascular tissues with both ends connecting towards the roots (Fig. 17b; Sachs, 1975a). Regardless of how the distance from the roots is measured, there must be a point along each loop that divides it into two equal arms as measured from the roots. No flux of shoot-to-root signals would be expected through 'such a point. Yet vascular loops with no observable gap, or region of special differentiation, are common (especially in petals). Large vascular loops do, however, have a special structure which suggests an answer to the problem of strand differentiation through regions which are equidistant from the roots. Such loops occur at the base of some leaves, as well as in some stems at the junction of two opposite leaves (Fig. 17d,e; Sachs, 1975a). The structure of these large loops is complicated by the presence of strands joining them from above, but the argument concerning their polarity still applies. The advantage of large loops is that individual vessels within them can be followed and their contacts used to determine polarity (see previous section). The observation of these vessels shows that they are all polar in the sense of connecting shoot tissues with the direction of the roots; this polarity may, however, have opposite directions

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within the very same strand (Fig. 17a; Sachs, 1975a). Thus though the loops have a region that is equidistant from the roots on both its sides, the alignment of the vessels suggests that there was an inductive flux through this region but not in any one direction. The conclusion that induction can occur in opposite directions within strands is also required by the “vessel triangles” common in leaves (Fig. 17a). Flux in two opposite directions could occur at the same or at different times. Observations of various stages in the development of large loops show that the vessels first have one clear polarity and indicate that vessels of opposing polarities are added alternately (Sachs, 1975a). This observation suggests that the inductive flux has a given direction at any given moment, but the direction is reversed repeatedly and the complexity of the strand increases as it matures (Fig. 17e). It can be suggested that in small strands of leaf networks changes in flux direction occur even within the same cells, and the effect of the changes can therefore not be seen in individual vessels. 3 . Further Evidence for Reversals in the Direction of the Inductive Flux An instability in the inductive flux is evident from the vascular structure even when all the differentiation is clearly polar. As mentioned above there are repeated contacts between neighbouring strands and neighbouring vessels even when there is no apparent reason for any deviation from a straight longitudinal course (Fig. 17c). Thus there may well be some oscillatory aspect to the inductive process itself. Actual reversal of the inductive direction, however, probably requires changes in the sources of the signals. It is thus significant that complex vascular networks are not found in leaves of many ferns, where the relatively simple vascular pattern is correlated with orderly development along the leaf margins (Goebell, 1905). Leaves that have complex vascular networks develop over their entire surface. The following evidence indicates that this development is not uniform, various parts of the leaf expanding, and possibly producing signals as well, at different times: Fig. 16. Polarity of leaf veins. (a-d) Four examples of fern leaves, with vein systems of increasing complexity. In (a) the veins branch only in one direction and a clear polarity, or direction relative to the roots, can be assigned to all ofthem. Contacts in both directions, as in (b), do not rule out polarity but require changes of the flux with time to account for their formation (see text). In (c) the strands have free ends in the middle of the lamina. These complicate the network and show that inductive effects are not restricted to the growing leaf edges. Further complication, probably due to shifting sources of differentiation signals (see text), results in systems such as the one in (d), where there are veins with no defined polarity. (e, fl Complex networks of angiosperm leaves. Those of dicotyledons (e) are generally less orderly than those of monocotyledons (0, but in both cases many of the small veins have no clear polarity relative to the roots. Note that the differences in vein density between ferns (a-d) and angiosperms (e, fl are not due to magnification. An inhibition of direct contacts with the main veins in (e) is presumably similar to that demonstrated experimentally in Figs 9g, j, k and 13. ( (a) Adiantum. (b) Aneimia, (c) Cirtomium, (d) Agioomorpha, (e) Pisum, (f) Zea. Leaves cleared with boiling Ethanol followed by Lactic Acid.) (a-f) x 7 .

Fig. 17. Polarity changes during normal vascular differentiation. (a, b) Defined cases of strands that could not depend on a flux with a fixed polarity. Triangles of vessels (a) are found in many leaves. Regardless of the polarity assigned to each vessel, there must be a region of a vein with two opposite polarities. Veins which connect to the roots on both sides (at the base of photograph (b) ) must have some point which is equidistant from the roots and has no defined polarity. The vein in (b), however, appears continuous, even at high magnifications. (c-e) Vascular systems that indicate changes of the inductive flux during differentiation. Sieve tubes of a storage root (c) form a polar network similar to that of Fig. 16b. Such networks could result from repeated changes in the preferred channel of the inductive flux (explanation in text). (d) and (e) are two stages in the development of vessel contacts across a bean stem node; leaves were on the two sides of the photograph and the roots connected to its base. These contacts resemble the strand in (b), and should have a point with no one polarity in relation to the roots. The individual vessels, however, all have a defined polarity, connecting a leaf with the roots, but vessels with opposite polarities run side by side within the strand. As the system develops gradually, there might be repeated changes in the direction of the inductive flux that would have a defined polarity at any given time and place (see text). ( (a, b) Pea stipule; (c) Secondary phloem of Brassica napus; (d, e) First node above the cotyledons of four- and ten-day-old bean seedlings. Material cleared by boiling in Ethanol followed by Lactic Acid. Phloem was then strained with Lacmoid and viewed in Sodium Lactate. Except for (c) the photographs repeat observations made by Sachs, 1975a.) (a, b, d) x 70; (c) x 7; (e) x 31.5.

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(a) These leaves often have a wrinkled, repeatedly changing form during development, unlike the flat surfaces of leaves which develop at their margins. (b) Measurements of the growth in area of squares marked on leaves (Avery, 1933) show that the growth of neighbouring regions does not occur at the same time (though this was not pointed out by the author). (c) Observations of the development of stomata show a complex, unsynchronized development of the leaf epidermis (Sachs, 1978b, 1979).This complex development could thus be a source of repeated change in the formation and local flux of the signals inducing vascular differentiation. 4 . Experimental Observations Concerning the Possible Reversal of Flux Polar ity If flux is to be reversed within a strand, the early differentiation of the cells must facilitate movement along a given axis but not necessarily in a given direction. This prediction can be tested experimentally for the known part of the inducing signals, namely for auxin. As was shown in the previous section, the direction of auxin induction is influenced by polarity, but induction in other orientations is possible. It could therefore be expected that a complete reversal of polarity (180") would be preferred to other deviations from polar induction. Careful observations of the effect of auxin applied to isolated pieces of turnip indicate that this is in fact so (Sachs, unpublished). This result, however, is ambiguous, since any differentiation not located along the axis of polarity must involve changes in the ray initials rather than the elongated, fusiform initials. Further experimentation on this question is clearly required. Auxin can also be used to test the prediction that reversals of flux can induce strands with no defined polarity. Flux reversals can be expected if an auxin source is applied temporarily and then applied again at a different point along the expected course of a differentiating vascular loop. As will be seen below, when two auxin sources are placed so that one is morphologically above the other there are often direct vessel contacts between them (Figs 9p; 1li). The probable reason for these vessels is that polar auxin transport from the upper source continues (Sections 1V.C and V.C). The experimental induction of a horizontal loop was therefore attempted above a horizontal wound (Fig. 18), where the expected changes in the direction of the flux are at right angles to the determined polarity of the tissue. The results of such experiments show that the formation of vascular loops by auxin is possible (Sachs, 1975a). When the location of the auxin was changed repeatedly, about a third of the plants formed the expected vascular contacts; these are not polar. Such contacts occasionally formed, however, even when the location of the auxin was not changed. These loops, and the formation of contacts between auxin sources on opposite sides of an isolated cambium (Fig. 1li), indicate that changes in flux may be due to the processes of differentiation

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( b )

( C )

Fig. 18. Induction of non-polar strands by auxin. Pea stems in contact with the cotyledons and roots but with the shoots above removed. These stems were cut longitudinally in a plane that exposed n o vascular strands and wounded as shown by the darkened rectangle. Auxin sources (dots) were applied above the wound. In (c) these sources were applied alternately on two sides, as shown, and continuous vessel strands were often formed. Some point along these new vessels has no polarity relative to the roots (as in Fig. 17b). The results shown are only the most common ones, and continuous strands were sometimes found in treatment (b) (drawn from Sachs, 1975a).

and not only to changes in the sources of signals for differentiation. This was also indicated by the prevalence of polar networks considered above, in which strands repeatedly form contacts with one another (Figs 16b, 17c). The polarity of induction thus requires further study, part of which should be a mathematical analysis of the possible controls (Mitchison, 1980). 5 . Discussion and Conclusion

It is thus possible to account for complex networks on the basis of a facilitated flux of inductive signals and two additional assumptions. The first is that the demonstrated lack of synchrony in leaf development is associated with changes in the localized production of differentiation signals. The second is that the facilitation of the movement of signals is initially for a given axis and not for a determined direction along such an axis. There is some evidence for this axial preference from experiments using auxin. It is likely, however, that it functions primarily at the time networks are formed, which is at the procambial stage of differentiation when there is no evidence that auxin acts as an inductive signal (Section I1.B). This means that there is no need to assume a polar determination, which is known only for auxin, so as to account for the formation of strands. Diffusion and a facilitation of movement along an axis (Mitchison, 1980) would suffice, and this raises the question how much axial preference, rather than polar determination, can be found for the transport of hormones such as the gibberellins. An axial preference is, of course, a characteristic of phloem, where transport in opposite directions can occur within the same strand (Eschrich, 1975). Is there any other way of accounting for the formation of complex networks? They could, of course, involve other, independent controls; the ad-

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vantage of the suggestion made here is that it brings such networks into line with other manifestations of vascular differentiation in plants, and it would appear to be the only way this could occur. The one published model of network formation (Meinhardt, 1978) uses directional, cell-to-cell interactions. The joining-up of strands to form complex networks is assumed to result from a forced progression of the induction, which is inhibited from occurring in any other way, rather than to a positive attraction mechanism. As was pointed out by Mitchison (1980), this model would not predict the formation of networks in three-dimensional organs, though such networks certainly do occur, for example in the fruits of the Cucurbitaceae (Sinnott and Bloch, 1943). It may therefore be suggested, at least as a working hypothesis, that an early response to the differentiation signals is a ,facilitation of their movement along the axis, not the direction, of the originalflux. This modification of the hypothesis discussed in the previous two sections accounts for the pattern of the vascular tissues in the leaves and suggests that the unknown signals for differentiation will be characterized by this trait. IV. CELLULAR RESPONSES INVOLVED IN ORIENTED DIFFERENTIATION A. INTRODUCTORY SUMMARY

The hypothesis that oriented facilitated transport is an early and critical stage of vascular differentiation raises many questions about events at the cellular level. The distinction between cellular and strand levels is, of course, arbitrary and only a matter of focus. The central cellular questions deal with the molecular events and their interdependence and these fall outside the scope of this chapter since they are not concerned directly with the relations between cells. The basic parameters of the oriented changes occurring in the cells, however, are an essential factor in the determination of pattern. It would be desirable to know, for example, whether the facilitation of signal transport is a threshold or a gradual process and at what stage it occurs, whether these processes can be recognized in any way other than the observation of mature vascular elements, and whether cells respond as the signal for further differentiation to a static gradient across them or to the facilitated flux itself. Removing a source of auxin at various times after application provides evidence about the minimal time during which the inductive signal must be present to cause differentiation. Such experiments show that auxin is necessary for at least a day, while differentiation leading to cell death does not take much more than two days. The influence of an auxin source on the course of the differentiation induced by a neighbouring source provides another means for timing the early events of vascular differentiation. This influence

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on neighbouring differentiation is found even if auxin application is delayed for about two days, almost as long as it takes vessel differentiation to be completed. It is thus clear that differentiation is a gradual process which requires continuous intercellular signalling, rather than a determinate process which follows an early threshold event. Changes in cell orientation can be observed microscopically. They are expressed by the presence and orientation of cytoplasmic strands through the vacuole which are visible in certain cells two hours after differentiation is first induced by a wound. These changes can also be seen more clearly and in a wider range of cells by the pattern of the plasmolysed cytoplasm, but this response is only found after 16 h of differentiation. The development of such methods provides evidence about the controls of differentiation that does not lead to mature vascular elements. The specification of the cell axis by the signals for differentiation could be a response of the cells either to a static gradient of signal concentration across the cells or to the actual flux of the signals. Using the polar transport of auxin, which depends on a determinate character of the cells, it is possible to set up conditions in which no long-term gradient of auxin can be expected and yet a flux will continue; such treatments do not prevent vessel differentiation. Vessels and sieve tubes also differentiate in the form of rings and whirlpools, which could not depend on any long-term gradient. The common bends found in vessels and sieve tubes also could not result from a long-term gradient and are readily understood as an expression of a flux whose axis can become determined. The same is true for the very long vessels in the trunks of trees. It is concluded, therefore, that cells must be sensitive to the actual flux of signals. B . EARLY EVENTS INDICATING DETERMINATION A N D DIFFERENTIATION

1. The Gradual Determination of Vascular Diflerentiation

( a ) The concept of determination. Vascular differentiation has been shown to depend on cues from the surrounding tissues (the flux of signals). There could therefore be some stage at which the signals from the surrounding tissues are no longer required for the continuation of the processes of differentiation; this stage will be referred to here as the time of determination. It should be noted that it will be convenient to use the term determination to refer not only to the stability of the changes which have already occurred but also to the internally-controlled continued maturation of the vascular elements. Since the programme of differentiation must be present in the cells rather than in the signals, which only induce its expression and determine its orientation, it is possible that short, all-or-none signals would suffice for the entire programme to unfold, leading to mature vascular elements. The hypothesis outlined in the previous chapter, however, would indicate that mature elements are formed only in response to a long-term flux of signals,

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whose effects are not dependent on an all-or-none switch mechanism but rather on a positive feedback with the cellular response of facilitated flux. It is thus important to test whether determination occurs as predicted by the working hypothesis. It is also important to gain some knowledge of the meaning of the "long-term" relative to the time required for vascular differentiation, which is two to three days in the systems illustrated in Figs 7,9 and 1 1 . When the term determination is defined as it is here, various tests of its timing are possible, and these have different implications. The tests considered will be an interruption of the source of differentiation signals and a change in the orientation of their flux. ( h ) How long must the signals be present to cause vessel muturatiorr :) The question refers to vessels for convenience only ; sieve tubes are much harder to observe, and their mature, or functional, stage is not well defined. One way to answer this question is to remove a source of auxin at various times after its application and follow differentiation that could have started only at the same time as auxin application, such as regeneration around a wound. Roberts (1960) found that the shoot apex must be present for more than a day to cause any noticeable effect on vessel differentiation. This is a relatively long period, but it should be noticed that it is a lower time limit only, as the removal of the apex could not be expected to have an immediate effect on the transport of signals through the differentiating elements. Another way to measure the same thing is to observe vessel differentiation induced directly by the applied auxin (Fig. 10). Such experiments (Sachs, unpublished) show that the differentiation signals must be present for between one and two days. Since the first vessels, composed of dead cells, can be seen in these systems after two days, the result means that signals for differentiation must act during most or all of the time that the cells are differentiating. Much shorter treatments are necessary when auxin is placed just above a transverse wound (Fig. 1 lb), but this is presumably because the auxin diffuses into the tissue and the wound prevents its polar transport away from the affected area. This auxin can therefore be distributed in the tissue only by slow diffusion, or by the flux associated with the processes of differentiation, the results of which can be observed. ( c ) The disruption ofdi'erentiation b y added auxin. The time of determination can also be measured by the effect of later treatments which could change the course of differentiation. Examples are shown in Figs 9n and 1 Ic, in which the lower source of auxin diverts the vessels induced by the upper source. It is possible to add the lower source at various times after the upper source and observe how late the second application can still have a diverting effect. The result is that a delay of two days in the application of the lower source still influences at least most of the vessels induced by the upper one (Sachs, 1974). This experiment can also be done with the treatment shown in Fig. 1Id and e, where one auxin source prevents the effects of the other. The

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results are the same: a delay of almost the entire time required for differentiation does not prevent a change in the effects of the first source (Sachs, unpublished). It is thus clear that vascular differentiation, or at least vessel differentiation, is labile rather than determined throughout most or all of the time it is taking place. ( d ) The additive efect of short-term treatments. This conclusion does not mean that short-term treatments cause no changes that are determined, or stable, for as long as a signal flux of another orientation does not affect the cells (see Section 1II.C). A way of observing these possible changes is to check whether short auxin treatments are additive in their effect upon vessel differentiation, even when separated by periods of a day or longer. Using Swede storage roots (Figs IOa-d, 1 l), it was found that auxin treatments of a few hours, which have no observable effect, still change the cells so that they respond to another short-term treatment by the clear maturation of vessels (Sachs, in preparation). It would be desirable to know whether these changes also involve a preference for some oriented path of signal flux and vessel differentiation. This could be tested by experiments in which conditions are altered after various periods, so that a flux in a new direction would be the shortest, though not the only, path towards the roots. Experiments of this type have not yet yielded clear results. Auxin transport experiments (Section 1II.B) and the microscopy considered below would indicate that such a preference for a certain path can be induced. ( e ) Conclusion. The available evidence thus suggests that the process of differentiation requires a flux of signals throughout the time the cells are changing. Changes that have already occurred are readily disrupted by a flux of auxin with a new orientation, but otherwise they may be relatively stable. These conclusions are in agreement with the working hypothesis developed in the previous chapter, according to which the determinate aspects of vascular differentiation depend on a positive feedback between the flux of signals and the response of the cells, which in turn facilitates this flux. 2 . The Observation of Cell Orientation During Early Stages of DifSerentiaiion ( a ) The problem of recognizing changes connected with cell orientation. The results described above, and the working hypothesis developed in the previous chapter, suggest that there could be structural changes involved in the oriented facilitated flux that is an early stage of vascular differentiation. It must therefore be asked whether such structural changes can be seen. The structure of differentiating vascular elements has been described at both the light and electron microscope levels (Torrey et al., 1971; O’Brien, 1974; Roberts, 1976), but these descriptions, though valuable for considering the cellular controls of differentiation, do not answer the question considered here. This is because the recognition of changes associated with cell orientation requires not only special emphasis upon them but also the use of cells

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in the process of re-orientation (Section 1II.C); in cells that are differentiating normally there is no way of recognizing changes specific to cell orientation or the facilitation of signal flux. As was pointed out in Section III.C, cell divisions and cell elongation are stages in the re-orientation of the cambium in cut or partially girdled plants with a transverse section (Fig. 79. This suggests that the orientation of normal cell division and elongation are also aspects of the response to a flux of signals (see Section VII). The results to be considered here concern the observation of orientation and re-orientation within the cells, in terms of the distribution and contacts of the cytoplasm. ( b ) Plusmolysis patterns as indicators of cytoplasmic orientation. Ruge (1937) reported that directional treatments with auxin influenced the axis of plasmolysed cytoplasm, or the choice of cell walls from which the cytoplasm was first detached. Ruge did not report any untreated controls, but it is logically possible that the degree of maintenance of cytoplasmic contacts between cells during plasmolysis might be an indication of their relative strength, and thus an indication of cell orientation. Sachs (1972b) studied the repeatability of patterns of plasmolysis and the conditions which maximize this repeatability. It was found that slow but severe plasmolysis resulted in a clear oriented response that depended on the tissue observed. In vascular tissues and early stages of the primary meristems the plasmolysed cytoplasm was oriented along the axis of the plant; maturing and mature parenchyma, on the other hand, showed a transverse orientation (Fig. 19a-c). The expected change in plasmolysis patterns during re-oriented differentiation around wounds was also observed. This included a diagonal orientation of the plasmolysed cytoplasm (Fig. 19c), a result not found in any other conditions. It thus appears that a microscopic expression of the cellular re-orientation preceding certain types of vascular differentiation can be observed. Some indications of the control of the axis of plasmolysis by auxin reported by Ruge (1937) were found by Sachs (1972b), but the conditions in which this effect is clear and repeatable were not defined. Plasmolysis patterns could thus be useful as indicators of oriented interactions between cells, particularly for the study of interactions that do not lead to mature, readily observable vessels (see Section V1.D). The limitations of the method are indicated, however, by the finding of plasmolysis changes only 16 h after the plants were wounded in a way which could be expected to cause vascular regeneration. On the basis of the hypothesis developed in the previous chapter, and the results reported below, one would expect observable changes after a much shorter period. This limitation could perhaps be overcome by improving the techniques of gentle plasmolysis and an understanding of why the cytoplasm stays close to some walls. Though the observed patterns may depend on the relative strength of the plasmodesmata, this is not necessarily so, since contacts of plasmolysed cytoplasm to casparian strips are strong in the absence of plasmodesmata (Bonnett, 1968).

Fig. 19. Cytoplasmic reorientation preceding vascular differentiation. Pea seedlings were wounded as in Fig. 9b, and the parenchyma cells near the wound were observed at various times. ( a x ) The preferred contacts ofthe cytoplasm with the cell walls after severe plasmolysis. (a) and (b) are from control plants, and the difference between them is that (b) was next to the vascular strand. Diagonal contacts, as in (c). were found only in the region of future vascular regeneration, 16 h after the plants were wounded. Absence of contacts between neighbouring cells results at least partially from the limited focal depth of the microscope. (d, e) Diagonal cytoplasmic strands in the region which would have formed vascular tissues. These strands are seen earlier than the plasmolysis patterns of (c), but are restricted to cells of an appropriate age. ( (a+) drawn from Sachs, (19724. (d, e) are unpublished pictures of Kirschner and Sachs, (1978). (d) 11 h after the plants were wounded and auxin (0.004% Indole Acetic Acid in lanolin) replaced the shoot, as in Fig. 9d. (e) 25 h after wounding; the shoot was intact.) (d)x240; (e) x 380.

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( c ) The orientation of cytoplasmic strands. The plasmolysis studies raise the question of whether there are any oriented cytoplasmic events that can be observed without such drastic treatments. Sinnott and Bloch (1945) reported that the appearance of oriented cytoplasmic strands preceded vascular regeneration around wounds. No illustrations of these strands were presented, however, and their work centred on the cytoplasmic pattern occurring prior to the formation of wall thickenings. Evidence for a cytoplasmic scaffolding for wall thickening comes also from experiments using colchicine (Roberts and Baba, 1968) and electron microscope studies (Torrey et al., 1971), but this is a different topic from that of the orientation of entire cells considered here. An oriented appearance of cytoplasmic strands was found by Kirschner and Sachs (1978) and was considered an early stage of vascular regeneration around wounds (Fig. 19d,e). In the most sensitive region these strands appeared within two hours after the wounding of vascular tissues of pea seedlings in both fresh and fixed sections. They were considered an early stage of vascular differentiation because they appeared in the same locations and orientations that would be predicted for vascular tissues and depended on the same controls. The diagonal strands were absent when the plants were not wounded or when the wounds did not cut the vascular tissues. They were reduced when all the leaves above the wound were removed. The effect of the leaves could be replaced by auxin in lanolin. ( d ) Conclusion and discussion. It may be concluded that early stages of vascular diflerentiation can be observed in the orientation of the cytoplasm. It may be objected that the evidence is based entirely on the regeneration of vascular tissues around wounds, but it is this regeneration that provides both a clear starting time for differentiation and defined control treatments with no differentiation. This conclusion could be important for understanding the processes of vascular differentiation and their control, and could also be the basis for recognizing early stages of differentiation that do not lead to mature readily observable vessels. It is possible, for example, that the inductive flux may initially influence more cells than the ones which later become the facilitated flux channel, and some evidence for this was found by Kirschner and Sachs (1978). Inductive effects involving flux may also play additional roles in plant development (see Sections V1.D and VII) and their recognition will require methods that could perhaps depend on observations of cytoplasmic localization and orientation. C. IS CELLULAR DIFFERENTIATION DEPENDENT ON THE GRADIENT

OR THE FLUX OF SIGNALS?

1. The Problem It was concluded in Sections I1 and I11 that vascular differentiation occurs along the flux of shoot-to-root signals. This flux determines the organization

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of the cells in longitudinal files, but response at strand level need not mean that the individual cells respond to the same control. They could not be reacting only to signal concentration since it alone would not suffice to specify cell orientation as expressed in shape and wall structures. Since the signals are at least partially hormonal, the logical possibilities for cell control would be either the signal gradient, the signal flux, or both. The problem to be considered here, therefore, is that of distinguishing between these possibilities; the question of the mechanism of the response is outside the scope of this review (see Section I) and will be considered only briefly in the discussion below. 2. The Experimental Separation of the Gradient and the Flux of Auxin For most substances flux is a function of the gradient of concentrations, and thus the effect of the two cannot be readily separated. This is true even when the flux does not depend only on diffusion; the rapid transport of sucrose through the phloem, for example, appears to be controlled by source-sink relationships (Reinhold, 1974). Auxin is, however, a special case since it is transported according to a determined polarity of the cells rather than along its observable gradient (for review see Goldsmith, 1969,1977). There are thus conditions where a flux could exist between two sources of auxin even in the absence of a persistent gradient of concentrations. The results of experiments in which two sources of auxin were placed one above the other are illustrated in Fig. 9m,n. Differentiation and the expected flux are clearly influenced by the gradient, and one source of auxin may divert the vessels induced by another source (Sachs, 1974). When no diversion around the lower source of auxin is possible, however, differentiation still occurs (Fig. 9p; Sachs, 1974). Thus differentiation is possible where there could be no long-term gradient but there could be a flux of auxin. Two possible reservations concerning the meaning of this result should be considered. The first is that the observed differentiation in Fig. 9p might be induced by the lower rather than the upper source of auxin, and represents a “reversal of polarity” (see Section I11 .C). Since the differentiation reaches the upper source, however, it would still represent induction where no gradient is expected. Furthermore, when upper source of auxin is omitted there is less rather than more vessel differentiation (Fig. 90). The second possible reservation is that due to active polar transport and depletion of auxin sources gradients do develop and it is these that are expressed by vessel differentiation. Such gradients, however, could not have the long-term effect which is necessary to induce vascular differentiation (see the first part of the previous section). The evidence of the experiments described in Fig. 9m-p, therefore, points to a relation between the flux of auxin and differentiation.

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3 . Evidence from the Form of Vessels and Sieve Tubes The course of vessels and sieve tubes sometimes follows a tortuous path which is by no means the shortest connection between two points (Fig. 20f). Both these structures can, furthermore, sometimes form closed rings (Fig. 20a,b,d,e). Gradients can only exist between two different points and not in a circle. Gradients would be steepest, furthermore, and thus presumably most effective, along the shortest connection between a source and a sink. Flux, on the other hand, can depend on polar transport, which is a determined state of the cells and can therefore exist in a closed ring. Furthermore, if sharp bends are formed, through a chance process or for some other reason, they could become determined by a facilitated flux while a gradient would always tend to revert to the shortest possible route regardless of the previous determination of the cells. It might be objected that these structures are abnormal and could depend on processes other than those responsible for normal differentiation of vascular tissues. Vessels and sieve tubes in the form of rings are found, however, in a variety of situations (Vochting, 1892; Timmel, 1927; Sachs, in preparation). They commonly occur when normal polarity is disturbed, as in the formation of basal callus tissue, and they can be induced experimentally by auxin and inhibited by triiodobenzoic acid (Sachs, in preparation). Sharp bends and tortuous paths are common phenomena of plant nodes and are not associated with abnormal development. 4 . Evidence from the Size of Plants The direct involvement of flux in the control of differentiation is supported by the scale in which strand differentiation may occur. Gradients, in contrast to flux, may be expected to be dependent on size; as the distance between source and sink increases, the slope across individual cells will decrease. A flux, on the other hand, could remain unchanged. It is significant therefore that cambial activity and the differentiation of both vessels and sieve tubes occur in trees over distances of many metres. Individual vessels as long as 18 m have been measured (Braun, 1970). Even the process most clearly induced by the young leaves, the activation of the cambium in spring, moves down the stem at considerable speed. A rate of 6 cm per day, for example, was measured by Tepper and Hollis (1967). If the inducing influence must persist for at least two days, as was concluded in the previous section, it must be active over a distance of 12 cm at any given time. While this would imply only a very weak gradient between the two sides of any cell, it presents no problems if considered as a response to an active polar flux. 5 . Discussion and Conclusion

Are there any possibilities other than those of flux and gradient? None suggest themselves concerning the orienting effect of a hormone like auxin.

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Flux and gradient, however, are not necessarily always separable. A signal travelling in the form of waves might subject the cells to repeated gradients. This possibility would remove the objections to a gradient response, and at the same time would not be distinguishable from a response to flux. Such waves could convey additional information (Goodwin and Cohen, 1969), important in specifying the different types of cells that compose the vascular system (Section VI). There is some evidence that auxin travels in waves, though these were not observed in careful work in which pulses of radio activity were followed down simple coleoptiles (Goldsmith, 1969, 1977). It is possible that the problem is a lack of synchrony among individual files of cells, but this suggestion requires further work. A response to flux which depends upon temporary gradients has been considered for slime mold aggregation (Robertson et a/., 1972; Parnas and Segel, 1978). A direct response to the flux of a signal raises the question of a possible underlying molecular mechanism. A definitive answer to this question is not available, but a few suggestions might demonstrate that a response to flux is plausible. (a) An external polar flux may result from and depend on the development of gradients within each cell (Mitchison, in press). This would mean that an overall flux could create in the cells sharp intercellular differences of concentrations which in turn could have a direct, “orthodox” effect on metabolic processes. (b) The intercellular flux of the signal could be coupled to another molecule to which the cell membranes are impermeable. A local intracellular accumulation of such molecules would provide a mechanism for translating flux into concentration differences. (c) Passage of the signal through cell membranes could cause a change, Fig. 20. Circular vessels. ( a x ) New vessels formed in a bean cutting. In the callus (a) and the intact cambium (b) of the basal or root-side the new vessels have various complex forms. Among these there are closed rings, larger in the callus than in the more organized products of the cambium. The callus closer to the removed shoot (c) includes meandering vessels but none with the form of rings or whirlpools. Vessels with special forms are not found in the upper part of the cambium. (d, e) Circular vessels in isolated pieces of storage roots. Such vessels appear at the basal side of the cambium about a week after the plant is cut ( (d); see also Fig. 1 Ig). These vessels can also be induced away from the basal side by auxin (e). They appear only at some distance from the auxin source, to which they are connected by relatively straight vessels. (f) Vessels with a sharp bend from a node of an untreated bean seedling. Such vessels are not rare. Their formation, and the formation of circular vessels, could depend on auxin flux, but not on a long-term gradient. (The bean seedlings used for (a-c) were cut when they were a week old and kept in closed dishes for two weeks. (d) is from a piece of radish (Raphanus sativus) kept in a closed dish for 10 days, and (e) is from a Swede (Brassicu napus) treated laterally with a 1% auxin preparation and kept for about a week. (f) is from a bean seedling, and a lower magnification of the same node appears in Fig. 17d.)(a-c)x80; (d)x27; (c)x 128; ( 0 x 3 0 4 .

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such as activation of an H' pump in the case of auxin (Rayle and Cleland, 1977). The membranes might well be polarized, so that the change occurs only when auxin moves in one direction and not in the other. A directional flux would then result in a difference of concentration between the two sides of each cell. It may be concluded that both experimental results with auxin and patterns of vessel and sieve tube diflerentiation indicate that thef7ux of signals. and not

their static gradient, is both the stimulus for diferentiation and the signal which determines its axis. This flux could act by establishing gradients within the cells, but the conclusion would still hold for intercellular relations. An attractive possibility, for which hardly any evidence exists, is that the flux is in the form of pulses whose periodicity could be an additional factor in specifying differentiation.

V . SPECIAL DEVELOPMENTAL PROCESSES IN THE CAMBIUM A. INTRODUCTORY SUMMARY

The terms cambium and cambial region have been used in different ways, and may mean either a wide zone of dividing and differentiating cells or an unidentifiable central layer of initial cells, one product of which may continue to divide indefinitely (Wilson et al., 1966; Philipson et al., 1971). The first and wider use of the term will be more appropriate for this chapter. In the previous discussion no separation of the controls of primary and secondary differentiation was warranted, and much of the evidence came from studies on the cambium. There are, however, processes which are unique to the cambium and require a separate chapter. The most obvious one is the differentiation that results not only in mature vascular tissues but also in meristematic cells that can continue to divide indefinitely. Another special trait is the presence of two types of meristematic cells (fusiform and ray initials, see Fahn, 1974; Esau, 1977) whose relations raise further problems. A general problem, most readily considered in relation to the cambium, is the control of the volume of vascular tissues formed in any part of the plant. The facilitation of signal transport during differentiation suggests that the formation of new vascular tissues would depend only on that part of the signal flux that cannot be transported by the available vascular tissues. This possibility, with its clear functional advantages, is supported by the effect of wounds, which cause the formation of a completely new vascular system and, at least in some conditions, increase the total activity of the cambium. Other possible controls may involve local effects on the competence of the cells (as in many storage tissues) and the selective use and release of signals in various parts of the plant, including the differentiating tissues themselves. The cellular patterns of the cambial initials are in a constant state of

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21 1

change which is recorded in the structure of the xylem. These changes are also expressed in the vessels, with their repeated irregular contacts with one another. The responses of cambia in isolated parts of plants indicate that they are able both to synthesize and to consume signals for differentiation, processes that might be first stages in regeneration. Vessels induced by auxin also indicate an instability in the processes of differentiation which results in complex networks and even in “a-polar vessels”. These are not readily explained by the flux hypothesis. The rays also form a constantly changing pattern which has, nevertheless, some underlying orderly aspects. The vertical fusiform initials are dependent on contacts with the rays for their continued activity. The rays, on the other hand, are initiated and grow in relation to the differentiation of new xylem and phloem, even when they interfere and compete for space with these vertical tissues. The most plausible control of ray formation and maintenance is a hypothetical flux of signals between the differentiating phloem and xylem tissues. The pattern of the rays would then depend on its own past history and on the constantly changing pattern of the differentiating vertical tissues. Graft union and regenerative joining of cambia occur only when the phloem-to-xylem orientations of the cut ends correspond. This expression of transverse polarity, which has hitherto been explained on the basis of gradients, might in fact depend upon the same signal flux which controls the rays. B. QUANTITATIVE CONTROLS OF CAMBIAL ACTIVITY

1. Introduction The activation of the cambium was considered in the previous chapters as one aspect of the control of vascular differentiation. A full understanding of the controls of the cambium must, however, include the quantitative aspects of the absolute and relative rates of cambial divisions and the various differentiation processes. The quantitative questions can also be posed concerning the primary tissues (see Section VI.D), but the only concrete information available at present concerns the controls of secondary differentiation. Even for this latter process the discussion will be limited to effects on the crosssectional area of the mature xylem, though there are descriptive observations of the rates of the component processes (Philipson e l af., 1971). If cambial activity were a simple additive function of the differentiation signals produced above, the cross-sectional area of the xylem at any given level would be equal to the sum of the cross-sectional areas of the organs it supports. This relation would be expected to hold for both the shoot and the root. In many cases there is a quantitative and simple relation between the size of an organ and the size of the vascular system which connects it to the plant (Rehm, 1936; Waring et al., 1976; for reviews see Wilcox, 1962; Studhalter et al., 1963) but one may sense intuitively that no simple additive

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relation holds for the entire shoot or root systems. A logarithmic relation between the xylem areas at different heights of trees was found by Murray (1927), but observations on a smaller scale reveal other relations including a decrease in differentiation in the downward direction (Alexandrov et al., 1927). An example of measurements showing the complexity of the relations among amounts of xylem at different heights on the plant is shown in Fig. 21.

6 . 0 ( 0 . 7 ; 125 1

/” 3.6 (0.4; 6 5 )

4 2 . 0 (1.1 ; 1 4 0 )

5 0 . 0 (2.0;300 1

Fig. 21. Quantitative aspects of the xylem of a Ricinus seedling. The numbers are the crosssectional area of the xylem in lo6 pm2 at the heights marked by the double horizontal lines and, in parentheses, the area (in the same units) and number of the vessels. Note the lack ofclear relations between the sizes of the xylem system in different parts of the seedling. (The petioles without a lamina had carried the cotyledons. Areas were obtained by drawing the cross-sections with a projection microscope and weighing the cut paper. Xylem was defined by the presence of lignin. From Sachs and Fahn, unpublished.)

These observations could indicate that controls independent of the shootto-root differentiation signals are involved, but as was seen above (Section 1I.E) there is little or no evidence that such controls have an important role. It may be suggested, therefore, that the magnitude of the signal flux and its effects on differentiation are dependent not only on source-sink relations but also on additional factors. This section will deal primarily with the effect on differentiation of functional, mature vascular tissues as implied by the hypothesis developed in the previous chapters. For if differentiation is

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canalized by an increased transport capacity for inductive signals, it follows that maturing and even mature tissues (in the case of the phloem and parenchyma) should reduce further differentiation by diverting the signals through their own channels. It is in fact known that polar auxin transport occurs most readily through the vascular region (Wangermann, 1974, 1977) and that auxin moves most rapidly in the functional phloem (Bonnemain, 1971 ; Goldsmith rt 01.. 1974). Thus the hypothesis of facilitated flux suggests a dual control of vascular differentiation, both by the developing shoot which the tissues form to supply and by the functional vascular tissues which are already present. This suggestion has the advantage of a testable prediction : damage to the vascular system should promote cambial activity. The present section will deal primarily with evidence concerning this point. Other controls of the quantitative aspects of vascular differentiation, not excluded by the proposed role of functional vascular tissues, will be considered only briefly in the discussion of this section. These latter controls are important but evidence about them is incomplete and does not fall directly within the topics covered by this chapter. 2. How Loc~ilize~l is the Damage Caused by Woutids? As mentioned in the first chapter, it has been known since the beginning of the century that wounds of the vascular system result in regeneration (Fig. 3). In relation to the suggested control of differentiation by functional vascular tissues, however, it is important to distinguish between two possible types of regeneration (Fig. 22). The new tissues might only replace the damaged regions, restoring the continuity of the original vessels and sieve tubes, or entirely new strands of cells may be formed primarily by the cambium. A general promotion of cambial activity by damage to the functional vascular tissues is to be expected only if the second possibility is correct. The weak regeneration of the vascular tissues of monocotyledons (Simon, 1908a) could be due to the absence of a cambium, but it could also be explained as due to differences in cell competence. Eschrich (1953) found that phloem regeneration replaced entire sieve tubes, not only the region of the immediate wound. His work was limited, however, to small strands consisting only of a few sieve tubes and no xylem. Homes (1961) also found that vascular regeneration connected only to new elements formed by the cambium. Benayoun et a / . (1975) observed that the vessels and sieve tubes formed around wounds were not continuous with similar strands that had been cut by the wound. This has also been observed in studies of vessels connecting to new lateral buds and to adventitious roots, in a variety of plants (Benayoun et al., 1975; Sachs, unpublished). Regeneration around wounds is thus associated with cambial activity in the undamaged region, possibly along the entire plant axis.

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Fig. 22. Possible connections of transport channels around a wound. (a) The regenerative vessels and sieve tubes form direct contacts with the corresponding channels that were cut by the wound. (b) The regenerative vessels and sieve tubes are continuous only with new, uncut channels formed from the cambium after the plant was wounded. The regenerative system thus joins the old strands but not the individual old channels. Observations show that plants regenerate according to the possibility diagrammed in (b). (Based on Benayoun ef al., 1975.)

The transport of radioactive substances is another and an independent method for judging the extent of damage caused by wounds. Patrick and Wareing (1973) followed the transport of sucrose in intact and cut bean plants, and concluded that wounds do not have a large effect on the activity of the phloem in the rest of the plant. Other observations are in accordance with this conclusion. Callose formation indicated a rapid response to wounds -limited, however, to their immediate vicinity (Engelman, 1965). Removal of roots of small bean plants had a large but temporary (8 h) effect on the transport of sugar towards the intact shoots (Gersani et al., 1980b).Transport of Fluorescein in the same plants showed that the entire phloem was reactivated and moved this substance in the direction of the shoots. There is an apparent contradiction, therefore, between the microscopic observation of regeneration and the effects of wounds on functional transport. The difference might be due to use of different plants or to different time scales, and possibly even the phloem reactivated following wounds is later replaced during regeneration. The problem clearly merits further work. From the point of view of the controls of differentiation it is significant that regeneration around wounds involves cambial activity, but this does not prove that an increased rate of this activity is involved. 3. Quantitative Eflects of Wounds on Xylem Formation A simple experiment demonstrating that a wound may promote the formation of more xylem than would be formed in its absence can be performed on

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21 5

isolated pieces of turnip or radish storage tissues to which auxin is applied (Sachs, unpublished). If auxin in lanolin is placed on such tissues and removed after a few hours, no new xylem is formed. The same treatment above a horizontal cut, however, causes considerable xylem differentiation (Fig. 1lb). It appears, therefore, that a wound which prevents the transport of auxin in the normal polar channels increases its effect on xylem differentiation. A local increase of cambial activity in the vicinity of wounds has been found in many experiments (for example see results in Sections V.D and V1.D). The effect is often a many-fold increase over the local cambial activity found in unwounded controls. In all these cases, however, the wound confines cambial activity to a limited region, and the local effect does not necessarily imply that there has been an overall increase in differentiation. The success of grafts, and thus the establishment of vascular channels, has been observed to reduce xylem differentiation (Shimomura and Fujihara, 1978). This is the expected effect, but again there is no evidence that total differentiation was reduced, only that it was diverted away from the wound callus. The only report of measurements of the effect of wounds on xylem differentiation, based on a comparison with intact plants, appears to be that of Benayoun et al. (1975; Fig. 23). Wounding pea plants was found to cause a significant increase in the cross-sectional area of the xylem, even at some distance from the wound. This result, however, was not found in all experiments. The reason for the variability could be a double effect of wounds: they may increase differentiation by diverting the signals to the cambium but they will also damage the plants, and especially the developing shoot apices, thus reducing the production of signals. In order to separate these two effects, plants with no shoot apices were also wounded. In these plants the major source of differentiation signals were the mature leaves (Fig. 23b,c), which can be expected to be less sensitive to wounds than developing leaves in shoot apices. The effect of wounds on xylem differentiation below these mature leaves was clear-cut and repeatable. Doubling of the cross-sectional area of the xylem formed after the first treatment was found repeatedly. 4 . Conclusion It may be concluded that the degree of cambial activity is dependent not only on signals from the shoot above, but also on the extent to which existing functional vascular tissues transport these signals and thus prevent them from inducing further differentiation. This dual control is attractive because it has a clear adaptive value, regulating differentiation in accord with both the demand for vascular tissues and their supply. This conclusion raises the questions of what these “functional tissues” are, how they can be recognized and what influences change their effectiveness as transporters of signals. It

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186f9

124f15

. .

(a)

(b)

(C)

Fig. 23. Wounds increase xylem differentiation. The numbers are the cross-sectional area of the xylem (in lo3 pmZ)at the levels of the double horizontal lines on the pea seedlings. The upper number ofeach pair is for unwounded plants and the lower one for plants from which a triangle of tissue was removed, as shown. Two weeks before the experiment started the plants in (b) had their shoot apex and all lateral buds removed, while in (c) the cut was below the top leaf. Note that the wound increased xylem differentiation except when there were no leaves above it. This influence on new differentiation is larger than appears at first, since most of the tissues measures above the leaf in (c) were present in all plants before the experiment started. The results of treatment (a), however, were not always repeatable, presumably because the wounds had variable effects on apical growth and thus on the formation of differentiation signals. (Measurements as in Fig. 22. Based on Benayoun et al., 1975.)

is by no means clear that only the sieve tubes are involved, and knowledge of functional changes is limited even in the case of sieve tubes. Variations in the relation between extension growth and cambial activity (Studhalter et al., 1963) might depend therefore not only on the relation of this growth to hormone production (see below) but also on differences in the amount and functional state of mature vascular tissues. 5 . Other Possible Factors Influencing Vascular Dierentiation

The above conclusion does not preclude the existence of other factors important in determining the quantitative aspects of vascular differentiation. It should be noted that it is not proposed that these factors control the location of vascular strands, which was the major topic of the previous chapters. For lack of evidence and space these factors will not be discussed in any depth, but it might none the less be useful to mention the major possibilities.

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( a ) The number ofdiferentiated cells may not depend only on the quantity of the signalspassing through the tissue. A simple quantitative relation between the magnitude of signal flux and the cross-sectional area of the differentiating tissues was implied in the discussion above and has been found in experiments using various concentrations of auxin (Fig. 9g,h; Jacobs and Marrow, 1957; Sachs, 1974). The localized formation of reaction wood and storage tissues by the vascular cambium indicates that this simple relation is not an invariable rule. It may be expected, therefore, that the reaction of cells may depend on their competence to respond to differentiation signals, and this competence will be influenced both by the developmental history of the tissue and by environmental factors. This topic will be considered briefly in Section V.D, but little is known about it at present. ( b ) The size and developmental rate of organs may have a complex relation to their activity in terms of signaljhx. There are new precise measurements of the production of hormones, and the relation of auxin synthesis to developmental rate is not clear (Goodwin et al., 1978). It is also possible that while production of factors such as gibberellins may depend on environmental conditions this may not be reflected in the final size of the organs. These factors could contribute to the complex relation between extension growth and cambial activity (Studhalter et a / . , 1963; see Section 1I.E). In general, however, a simple relation is found between the size of an organ and the crosssectional area of the stem supplying it, indicating that hormonal and other correlative effects are related to the rate and duration of development, though the poor vascularization of etiolated tissues shows that this is not an invariable rule. ( c ) The signals may be produced, consumed or changed during the processes of diferentiation. The first question related to these possibilities is whether vascular differentiation is necessarily correlated with signal change. It is thus relevant that a strand of xylem induced by a localized source of auxin may have a uniform width along a distance of a centimetre or more (Sachs, 1974). Very long vessels also differentiate without any branching or attenuation in the trunks of trees (Braun, 1970). These observations indicate that the signals are neither consumed nor produced, but this may not always be true. Vascular strands often become attenuated in the direction from the leaves downwards (Fourcroy, 1943; Fukuda, 1967) and a similar attenuation is found in the effect of auxin when it is applied to a cambial region (Fig. 11). These observations could be accounted for by postulating transport of signals through mature vascular tissues rather than consumption or change of signals during differentiation. Experiments with isolated plant parts including a cambium, however, indicate that the cambial region can serve as a sink for exogenous auxin (see Fig. 11 and next section), so that both transport through mature tissues and local sink effects might be important. Evidence for the production of hormones by differentiating vascular cells

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has been presented by Sheldrake (1973a). This may be of considerable biochemical and evolutionary importance, but the constant width of induced strands, mentioned above, suggests that these hormones may not be released in a form effective for controlling vascular differentiation. Isolated cambial tissues certainly do show xylem differentiation (Figs 7d, 1lf,g; Sheldrake and Northcote, 1968), but, as discussed in the next section, this might be related to early stages of regeneration rather than to normal controls of differentiation. C . THE CONSTANT CHANGES IN THE CAMBIUM

I . Introduction Unlike preceding sections, this section is not based on a defined problem. Instead, results are summarized that appear important in relation to the structure of the cambium as a meristem. These do permit a clearer understanding of the processes which occur in the cambium and they point to problems demanding further study. The observations under consideration were made not on the dividing cambium itself but rather on the mature xylem. This would appear to limit their interpretation. Any direct observations, however, can relate only to a situation at a given time and not to a process, and the cambium is so variable that observations made in different locations cannot be used to reconstruct processes. The structure of the xylem, on the other hand, reflects the structure of the cambium at the time it was formed, and, unlike the cambium, is very easy to study. Changes in the cambium, furthermore, are recorded in the inert xylem and can be reconstructed from serial tangential sections (Fig. 24; see Philipson et al., 1971). 2. The Relations Between Cambial Initials The activity of the cambium results in an increase in the diameter of the plant axis and thus in the surface area of the cambium itself. New cambial initials must therefore form and this occurs by divisions of existing initials. The number of such initials formed, however, exceeds the demand created by surface growth (Bannan, 1951,1968; Evert, 1961; Hejnowicz, 1961;Cheadle and Esau, 1964; Cumbie, 1969; for review see Philipson et al., 1971). The balance in the cambium is maintained by a loss of initials, which occurs by differentiation of all their derivative cells to mature vascular elements. These processes as recorded in the xylem portray a constant competition between initials, where much of the outcome depends on chance. The one rule emerging from these studies, that initials in contact with rays are more likely to continue dividing, will be considered in the next section. Contacts between cells are also in a constant state of change, due not only to the formation and loss of initials but also to the elongation of initials and to changes connected with the rays (see next section and Philipson et al. (1971)

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for review). These contact changes must reflect a certain independence of the individual cells of the cambium. There is no evidence, however, that the cells can slide one upon the other, or, in other words, that adjacent points on neighbouring cells can change their relative positions. The changes in cell contacts appear to be due entirely to intrusive growth, in which only the tip of a cell elongates independently of the surrounding tissues (Neeff, 1914; Sinnott, 1960). The resistance of the bark to the growth of the cambium along the plant radius and the excessive production of initials can be expected to generate considerable physical pressures within the cambial region. Experiments carried out on tissue cultures show that such pressure might have a morphogenetic role (Brown and Sax, 1962). Evidence for the existence of such pressures in living trees was published recently (Hejnowicz, 1980). The constant changes in the cambium presumably have an orderly basis, but except for those associated with the pattern of the rays, to be considered in the next section, only two regular aspects have been documented. The formation of new cambial initials occurs by diagonal, or “pseudotransverse”, divisions of existing initials. These divisions may thus have one of two orientations, and, like other changes in the cambium, they can be followed in tangential serial sections of the xylem. An extensive study of these divisions has revealed that they have the same orientation over relatively large regions of the cambium (Hejnowicz, 1973). These regions, or “domains”, do not occupy permanent locations in the cambium ; rather, they slowly migrate over the surface and their behaviour may be described as that of waves with a period of 10 to 20 years. The meaning of these changes is unknown. On a different time scale, the vessels formed by the cambium do not follow a completely vertical course. Their meanders result in repeated contacts between all neighbouring vessels (Braun, 1959, 1970). The same is true for the sieve tubes (Fig. 17c). Presumably these contacts have a functional value; their control must involve repeated changes during vessel induction (see Secion 1II.D). 3 . Special Responses of the Cambium t o Inductive Signals The cambium responds to signals from the leaves and to auxin by divisions and by differentiation of xylem and phloem (see Chapter 11). These are the same responses as those of the procambium and of parenchyma close to a wound. The cambium, however, displays a number of special traits, at least in terms of the differentiation of the vessels which are readily observed.

(a) The cambium may serve as a source of differentiation signals. Vessel formation continues in the cambium even in tissues isolated from any contact with the shoot‘or the root (Figs 7d, 1lf,g; Sheldrake and Northcote, 1968 and see Section 1I.E). This differentiation is probably due to the same

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signals as those produced by the leaves, since it is inhibited by triiodobenzoic acid (Sachs, unpublished) and measurements show that auxin can be formed in the cambial region (Sheldrake, 1973a). It was suggested in the previous section, on the basis of the quantitative relations of the new xylem, that this auxin release may be, at least partially, a response to isolation of the tissue rather than a process occurring in the intact plant. (b) In isolated pieces of radish and other storage tissues new vessels often end blindly (Fig. 11). This differs from the normal pattern found in wounded parenchyma or in the highly polar cambium of bean seedlings (Fig. 7c). Blind endings not next to other vessels are also uncommon in the intact plant. In terms of a control by a signal flux, dead-end vessels indicate that the cambium or adjoining tissues may serve as sinks for the differentiation signals. As in the case of the cambium as a signal source, it is possible that part or all of this sink activity is found only in isolated tissues. (c) Vessel differentiation induced in the cambium by a localized auxin source tends to spread to all available cambial tissues (Figs 7h, 1 la). The inhibition of the formation of contacts to regions loaded with auxin (Fig. 9j,k; Section 1II.B) is generally masked by the tendency of the induced vessels to spread in the cambium. But a quantitative inhibition can still be noticed, for example in a comparison of the differentiation at the base of the wound in Fig. 7a and c. This weak inhibition of contacts does not contradict any of the earlier conclusions since it can be expected that the cambium will divide and thus expand to accommodate the available signals, so that it can be overloaded only in a region of direct application of auxin. Inhibition of contacts in such regions is readily demonstrated (Fig. 1 lc,e). The tendency of induction to spread is, on the other hand, less expected, and presumably is due to the ability of all isolated parts of the cambium to serve as sinks for auxin. This effect is possibly augmented by the local release of signals, causing partial differentiation and leading to the formation of facilitated channels which would attract any additional flux of signals nearby. (d) Vessels formed from the cambium often contact each other, and these contacts can even be expressed in a polar differentiation. As may be expected from the hypothesis of facilitated transport, strands induced in wounded parenchyma do not form contacts, even when they are forced next to each other by wounds (Sachs, 1968b). Vessels induced in a cambial region, on the other hand, form many contacts. When the effects of two neighbouring sources are observed (Fig. 1lc,i) these contacts are always found, except in the immediate vicinity of the auxin sources (where the cambium is presumably overloaded). As was discussed in Section III.D,

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these repeated contacts probably indicate an instability, or an oscillatory aspect, in the inductive flux of the signals. Contacts between vessels need not raise any problems concerning the direction, or polarity, of the auxin flux. But a problematic result does arise when auxin is applied on both the shoot and the root side of an isolated cambial region, either in a girdled part of a bean or Coleus stem or in a cut piece of radish (Fig. 1 li). The differentiating vessels show a clear effect of both auxin sources, the effect of the one on the shoot side being more rapid and more pronounced (see Section 1II.C). There are also vessels that form a direct contact between the upper and lower source (Fig. 1 li), and these vessels have no polarity, or no defined direction, as would be expected if they are caused by an inductive flux (Secion 1II.D). These “a-polar” vessels were found repeatedly, and careful microscopy showed that they have no interruptions or other special features. They are not, however, formed in preference to other vessels in the neighbouring cambium, so there is no reason to think that one auxin source exercises any attractive or other effect on the vessels induced by another. The formation of these vessels was not affected in any clear way by the concentration of auxin sources or the size of the isolated cambial region. From the point of view of the hypothesis of facilitated transport developed in the previous chapters these “a-polar’’ vessels are unexpected and are the most difficult result that has been found. Yet there is nothing in the observed patterns ofvessels (Figs 9n, 1 lc,e) to indicate that differentiation might occur preferentially along a “ridge” of auxin concentration between two sources. A possible explanation must depend on the changes in the inductive flux and on the possibility of a reversal of its orientation, at least in early stages of differentiation (Section 1II.D).Unlike the loops found in leaf networks, however, these flux changes would not be due to variable sources of inductive signals but rather to the processes of facilitation and differentiation. This suggestion is not based on any direct evidence, but the contacts between vessels mentioned above make it a likely hypothesis. 4 . Conclusion and Discussion The meaning of the various observations have already been considered individually, and what remains now is to attempt a general synthesis. The principle that can unite all the observations considered above is that the cambium is not a stable meristem which is either active or inactive. Instead, every aspect of the cambium is in a constant state of change: the identity of the initial cells, the relationships between cells, the channels of thepux of signals and possibly also the release and change ofthese diflerentiation signals. As will be seen in the next section, this principle of change includes also the pattern of the rays. The basis for these variations could not be found only in varying stimuli from neighbouring tissues or from the environment, and must there-

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fore be sought in instability in the developmental processes themselves. This instability could readily arise from the facilitation of the flux of differentiation signals (Mitchison, 1980). The changes may thus both control and be controlled by differentiation, but these possibilities require further work, It may be asked, finally, whether these changes have any functional, adaptive roles. The value of vessel and sieve tube systems composed of complex networks is clear for conditions where damage is likely. So is the ability of the cambium to regenerate in a way which may lead to the formation of new shoot and root apices (Section VII). The apparent competition between initials may be also important, for the cambium is a meristem which continues to divide for hundreds and even thousands of years, and an internal selection of cells may be essential in reducing the effect of random mutations. D. RAY FORMATION AND THE RADIAL POLARITY OF THE CAMBIUM

I . Introduction All the topics considered above were concerned with the controls of differentiation along the plant axis. In the cambium, more than in any other tissue, there are, however, also expressions of another axis or polarity, that of the radius of the stem and the root. The purpose of this section is to consider two such expressions, the formation of vascular rays and limitations on the grafting of the cambium. An attempt will be made here to define the controls for these phenomena and their relation to one another as well as to the shoot-to-root flux considered above. A third related phenomenon, the location of phloem and xylem, will be considered in Section VI.

2. The Pattern of the Rays It is a general rule that differentiation of secondary vascular tissues from the cambium is accompanied by the formation of rays of parenchyma. There are only exceptional plants in which rays do not appear (Aymard, 1968 ;Gibson, 1978) and it is probably significant that these plants have only limited cambial activity. There are also many trees in which more than one type of ray is found (Philipson et al., 1971). In tangential sections the rays often appear to be fairly uniformly distributed, and it was suggested by Bunning (1965) that this is due to an inhibition by existing rays of the formation of additional rays in their vicinity. The tangential distribution of the rays would then be a spacing pattern in the sense used by Wolpert (1971). It is certainly true that new rays are added as the surface of the cambium increases, for otherwise their density would decline and they would be virtually undetectable in mature trees. The formation of new rays, however, is but one of the processes occurring during cambial activity. As with other changes in the cambium, it is possible to follow the ontogeny of any group of rays in serial tangential sections of the xylem. Such serial sections (Fig. 24) show that in some trees the rays are always changing; there are constant processes of

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1

51!plO

14 2 r n m

22 7 m m

25.2mrn

Fig. 24. Changes of a specific ray pattern during development. Selected examples from a complete series of tangential sections through Ailunthirs uliissima wood in which individual rays could be followed. The distances in millimetres show the location of each section relative to the centre of the branch. Note that many of the changes do not increase the orderly spacing of the rays. (From Lipman, Fahn and Sachs, unpublished.)

ray initiation, growth, fusion, splitting, diminution and disappearance (for review see Braun, 1955; Philipson et al., 1971). These changes are not only in the rays themselves but also in the elongated cells which separate them (Bannan, 1951) and are, therefore, part of the constant flux in the state of the cambium which was considered in the previous section. 3. Controls of Ray Distance and Ray Initiation Though the ontogeny of the cambium as it appears in tangential sections of the xylem yields a confusing picture, one significant generalization has been reported a number of times (Bannan, 1951 ; Evert, 1961 ; Cheadle and Esau, 1964): elongated or fusiform cambial initials not in contact with rays tend to disappear by the differentiation of all their derivatives much more readily than other initials. This observation can be a mechanism for maintaining a

Fig. 25. Location of newly initiated rays. Examples of new rays (darkened) identified by their first appearance in a complete series of tangential sections through the xylem of Ailanthus altissima (see Fig. 24). Note that initiation did not necessarily occur in the centre of the largest area free of rays. (From Lipman, Fahn and Sachs, unpublished.)

I

0 0

14o Horizontal direction A Vertical direction

12 12h

0

L

10-

f 0 c

m 00 ._c V

c

r

60 0 c

0-

;4 A

A A

810

O 1:

1& Distance ( p m

O 2;

260

O 2;

O:3

1

Fig. 26. Ray occurrence as a function of distance from newly initiated rays. The curves are an analysis of the location of ray initiation seen in Fig. 25. Note that there is no definite relation between a new ray and its neighbourhood other than the absence of other rays in its immediate vicinity. (Newly initiated rays were identified by their first appearance in a series of tangential sections. Distances are measured from 76 such rays according to the method of Sachs (1978b). From Lipman, Fahn and Sachs, unpublished.)

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relatively orderly distance between rays, and it indicates an important interaction between the ray and the fusiform initials of the cambium. A control of the distance between rays would still not account for the initia:ion ofnew rays, a common process during cambial activity. The identification of new rays cannot be based on their size but only on the examination of serial sections through the xylem. Examples of the pattern of rays determined unequivocally from such serial sections appear in Fig. 25. On the basis of Bunning’s hypothesis, or of any other mechanism that would predict a simple interaction between rays, it might be expected that a new ray would appear at the greatest possible distance from neighbouring rays. This is clearly not the case for the initiation events shown in Fig. 25. Using a method developed for the study of stomata spacing patterns (Sachs, 1978b) it is possible to average the relations of many new rays. As may be seen from Fig. 26, there is a limited region near existing rays in which new ones are unlikely to form. This is an “inhibitory region” of the type suggested by Bunning (1965). It is, however, limited relative to the average distance between rays, and beyond its borders the location of a new ray seems to be a chance event. A final result relevant to the control of ray initiation is its relation with cambial activity. Normal activity causes an increase in the surface area of the cambium and ray initiation keeps up with this increase in surface area so that the spacing pattern is maintained. This fact, which was the basis of Biinning’s (1965) suggestion, led Carmi et a / . (1972) to girdle trees and leave a confined cambial bridge in which divisions could be expected to be rapid but increase in surface was physically prevented. The unexpected result was that the initiation and growth of rays not only continued in the absence of an increase in the available surface but were even greatly enhanced (Fig. 27). This result is surprising because it could be expected that a competition for space between the vertical and radial system would prevent any increase in the size of the rays, which must be at the expense of vertical transport tissues. This led to the observation of a general positive correlation, not yet quantitatively documented, between the rate of cambial activity and the fraction of the tangential area occupied by rays, which was found regardless of the normal increase in the surface area of the cambium. This positive correlation can also be noticed when trees growing in different conditions are compared (Linnemann, 1953; Gregory and Romberger, 1975). 4 . Conclusion: A Suggested Control of’ Ray Formation What mechanism could account for all these diverse facts? It is clear that an active control is involved that is important enough to compete for cambial surface with the vertical system, even when this surface is limiting. This control also influences the survival of fusiform initials, and it must always be active, causing repeated fine adjustments whenever new vascular tissues are formed. The control is not very precise at the level of the relation between

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A

Fig. 27. Relation of ray development to rapid cambial growth. Tangential views of the wood of Ailunfhusulrissima. The average number of rays (in 1.8 mm’) and the percentage of the tangential area they occupy appear below the sections. (a) Wood formed in a narrow bridge across a girdle. This bridge was confined so its tangential area could not increase but its radial development was unusually rapid. (b) Control wood from the same tree. Note that rapid radial growth was associated with a great increase in the ray system. This increase occurred at the expense of the space available for longitudinal channels, limited by the treatment (drawn from Carmi et a/., 1972).

individual rays but it does maintain a repeatable overall pattern. All this suggests that the rays are both induced and serve as channels for the transport of signals passing between the new phloem and the new xylem (Carmi et al., 1972). These signals or their drainage are also essential for the fusiform initials of the cambium. Wherever new vertical tissues form in the absence of sufficient ray tissues, the signals cause the induction of new rays from the cells through which they pass. Rays, therefore, inhibit the formation of new rays by draining away the signals for their initiation. The tangential area of the rays would then depend on the volume of the differentiating phloem and xylem, and thus on cambial activity. The developing pattern of the rays at any given time would depend on two factors : the rays which were already present, whose pattern would gain stability from their ability to drain away signals, and the pattern of the various elements differentiating in the vertical system which produce the ray-inducing signals. It is this second factor which is completely unknown and deserves further work. Clearly this suggested control is but a transverse extension of the hypothesis concerning the differentiation of vascular strands. It does, however, fit the known facts and its relation to the vertical control is not a weakness. It is not suggested here, of course, that the same signals are involved; the signals for ray maintenance and formation are not known, nor is there direc-

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tion of movement, but there is evidence that a variety of substances can be transported through the rays (HO11, 1975). An important advantage of the hypothesis is the clear relation between the presence of the rays and other phenomena, such as the spatial arrangement of the phloem and the xylem (Section V1.B) and the polarity of the cambium considered below. 5 . Radial Limitations of Cambial Grafts

The vertical polarity associated with vascular differentiation has only a limited effect on the initial success of grafts (Section 1II.C). Radial limitations, however, can be readily demonstrated (Janse, 1921; Rzimann, 1932; Snow, 1942; Warren Wilson and Warren Wilson, 1961b) and there are no reported exceptions to their applicability. These limitations are illustrated in Fig. 28 : cambial tissues always join readily, either directly or by a regeneration of the cambium in intervening tissues, but only when they have the same phloem-to-xylem orientation. There are also general tendencies for the cambium to spread (see previous section) and to form a closed ring.

Fig. 28. Possible grafts between cut cambia. Diagrammaticcross-sections in which double lines represent the cambium and, where broken, a regenerative cambium. Arrows mark the phloem-toxylem orientation. Cambial union occurs as shown in (a), even when this requires considerable regeneration through callus tissue. The more direct grafts shown in (b) do not occur, presumably because they would involve adjoining regions with opposite radial orientations.

What controls could be responsible for these observations? There must be interactions between cells, and these might have two possible orientations. (a) Stimuli could circle around the axis of the plant and favour the formation of a closed cambial circle (Snow, 1942). (b) The interactions may have a transverse orientation, operating between the phloem and the xylem. The fusion of cambia with opposite phloem-to-xylem orientations or polarities could be prevented (Warren Wilson and Warren Wilson, 1961a; Warren Wilson, 1978).

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There is no evidence in favour of the first possibility, other than the tendency of the cambium to form closed circles. Development of cambial strips, however, is both common and normal in many petioles, in stems of desert plants (Fahn, 1974) and in many damaged trees. Strips of cambium unite readily even if they do not form a closed circle. provided they have the same phloem-to-xylem orientation. Evidence against a circular interaction is also the successful grafting of inverted cambial regions (Section I1I.C). If the circular interactions are to prevent cambial joining they must have a consistent, determined direction, and they would therefore prevent inverted grafts from uniting. The presence of the phloem and the xylem on opposite sides of the cambium suggests that it has a transverse polarity (Janse, 1921). This transverse aspect could readily account for the results of grafts, and the question therefore arises as to the nature of the interactions involved. Two basic possibilities may be distinguished : (a) relatively static gradients along the transverse axis (Warren Wilson and Warren Wilson, 1961a, 1961b; Warren Wilson, 1978), and (b) a polar movement of signals from the differentiating phloem to the xylem or vice versa (Carmi et ul., 1972; and the discussion of ray formation above). Warren Wilson (1978) considers not only graft unions but also the location of any new cambium (a subject briefly discussed in Section V1.D) to depend on gradients. He used a computer simulation to show that gradients could determine the location of a regenerative cambium (and thus of cambial grafts as well). O n the basis of a wide literature review he suggests that there are two opposite gradients, of auxin and of sucrose acting as morphogens. The shortcoming of the gradient hypothesis is that it does not take into account the transverse differentiation of the vascular rays which was considered above. Since these rays do appear to be formed in response to some pressure of “demand” and to be inhibiting the formation of additional rays, their formation could not depend on a static gradient. The transverse polarity they reveal might well control the union of cambial grafts in which case the exact location of the cambium would depend on other factors, and should be viewed as part of a three-dimensional rather than a cross-sectional phenomenon (see Section V1.D). It is, of course, possible that neither or both hypotheses are correct and additional studies are clearly needed. VI. THE CELLULAR COMPLEXITY OF THE VASCULAR SYSTEM A. INTRODUCTORY SUMMARY

In much of the discussion above the vascular tissues were treated as a unit, and often observations of the xylem were used to represent the vascular system as a whole. There are, however, three different vascular tissues

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(phloem, cambium and xylem), each of which consists of more than one type of cell (Fig. 2b). The relations between these tissues, and the quantitative and spatial relations between their component cells, are orderly in the sense of being repeatable for plants of any given species. Furthermore, the primary vascular system is always formed together with the neighbouring parenchyma, the cortex and the pith. It must therefore be asked what controls the formation of the different tissues and cell types. Three possible answers to this problem that are not mutually exclusive and differ in the relation between the longitudinal inductive flux and the local interactions between the cells, are outlined in Fig. 29. As will be seen below, the general problem is unanswered though the evidence points to the operation of different controls, with a dominant role played by the one shown in Fig. 29a.

Fig. 29. Possible controls of tissue differentiation within a strand. The three hypotheses represented are not mutually exclusive and they all involve the known polar induction of vascular tissues. (a) The inductive signal includes a number of components. After an initial stage in which the cells are equivalent different parts of the signal are preferentially transported through different cells and induce specific processes. The location of the tissues depends on an additional radial gradient or flux. (b) Tnteractions between the cells of the differentiating strand are responsible for the specific processes in the tissues. (c) An early radial influence predetermines the cells so they respond differently to the polar inductive signals. Evidence considered in the text supports the existence of controls of the type shown in (a) and (b).

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The first case to be considered is the relation between the xylem and the phloem, or the vessels and sieve tubes. Both the normal and regenerative development of these tissues are closely correlated. The xylem does not normally differentiate without the presence of phloem, and only small strands of phloem differentiate without the xylem. Auxin, alone, can determine the formation and the orientation of the phloem, the xylem and the cambium between them, at least in some plants. In other cases there is evidence that gibberellins promote the formation of the cambium and sometimes the phloem as well, and sucrose, directly or indirectly, promotes the formation of the phloem. These results can be understood on the basis of the hypothesis that the different tissues start their differentiation together and specialize to transport different parts of a complex signal. The previous section, however, also provided evidence for a direct, transverse interaction which could also serve to control the relations between the phloem and the xylem. The fibres are a distinctive cell type and can readily be studied. The ratio between the fibres and the vessels of ring-porous trees varies in an annual cycle, and this appears to be related to the control of the cambium either by expanding leaves or by a combination of mature and primordial leaves. Phloem fibres have also been found to depend on a polar flux of signals from the leaves; the most effective age of the leaves varies depending on the species. There are indications that gibberellins promote all types of fibre differentiation, usually (possibly always) acting in combination with auxin. No clear role for gibberellins within the plant can be assigned at present, but their effect is likely to be an important clue for future work. The best working hypothesis concerning fibre differentiation is the same one considered here as a major basis for the relations of the phloem, cambium and xylem, illustrated, in Fig. 29a. The formation and maturation of parenchyma occurs at the same time as that of the vascular tissues, of which it forms both an integral part and a surrounding matrix. The possibility of inducing vascular differentiation in parenchyma cells by auxin suggests that they, too, share the common early stages with the various vascular cells. The canalization of the signal flux to the centrally located conducting and supporting elements would then drain the signals away from the parenchyma and determine its fate. The limited period during which parenchyma tissues are able to regenerate and the gradual change in their competence to respond to auxin support this suggestion. There is evidence, however, that parenchyma development is controlled by signals from the leaves even when the vascular tissues are mature, so that they might be more than “partially induced” vascular tissues. The importance of the suggestion, however, is that in a modified form it may account for the formation of the entire plant axis as a function of the signal flux considered here.

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23 1

B. THE RELATION BETWEEN THE XYLEM A N D THE PHLOEM

I . The Problem and the Possible Answers The previous sections depended primarily on observations of the xylem, though the conclusions referred to the vascular tissues, in which the phloem is a major if not a dominant component. The observations made on the xylem that support the hypothesis of a control by a facilitating flux of signals, one of which is auxin have, however, been made on the phloem as well. The limiting factor for work on phloem differentiation has been the problem of its observation. Though methods for staining the phloem so that its threedimensional structure can be studied have been developed (La Motte and Jacobs, 1962; Peterson and Fletcher, 1973a; Aloni and Sachs, 1973) it is still much easier to study the xylem. These methods, furthermore, depend on staining the callose, and early, though functional, stages of differentiating phloem may stain very poorly (Sachs, in preparation). There is therefore only limited information about the control of phloem formation and its spatial relation to xylem. The same is true for the related questions concerning the appearance of the cambium between the xylem and phloem of many plants. For this reason the present section is more an attempt to list all the facts likely to be relevant than a statement of a clear hypothesis. It should still be useful, however, to consider the possible answers consistent with the induction of both xylem and phloem differentiation by a shoot-toroot flux of signals. The three basic types of controls that could be involved are outlined in Fig. 29; it should be noted that these possibilities are not mutually exclusive and the problem is, therefore, what the exact mechanisms and their relative roles are rather than which of the three is correct. 2. The Normal Developmental Relutions Between the Phloem and the Xylem ( a ) Primury tissues. The phloem and the xylem normally develop at the same time, though the first phloem often completes its maturation earlier, or closer to the promeristems (Esau, 1965). There are also differences in the direction in which the final stages of differentiation take place, but, as discussed in Section II.E, these are not likely to be very meaningful for control of differentiation. A more important difference is that the phloem is found in small strands lacking xylem, and these are much more common than is generally realized (Aloni and Sachs, 1973). The converse, xylem in the absence of phloem, has rarely been reported for untreated plants and even then only for very short distances (Tomlinson and Zimmermann, 1968). In stems of some families the phloem is found both on the inner and the outer side of the xylem, and in these cases there are contacts between the sieve tubes on the two sides (Bonnemain, 1969). Plants in which the phloem occurs only on the inner side of the xylem are not known. Neither the phloem nor the xylem differentiate as large masses of tissue

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distant from one another. This could be a reason why the vascular tissues of stems of pteridophytes, which may be concentrated as solid masses, become convoluted and broken-up in large organs (Bower, 1930; Wardlaw, 1968). The same is true for the roots of virtually all plants, where the xylem and the phloem form alternating groups. The number of xylem and phloem poles is not constant in plants in which the size of the roots can vary greatly, especially the monocotyledons. The number of poles is related to the size of the root (Fig. 30; Jost, 1931; Torrey, 1965; Wardlaw, 1968; Feldman, 1977) and no phloem group becomes very large. ( b ) Secondary tissue. The orientation of the phloem and xylem relative to one another is determined in the primary tissues and is maintained by the cambium. This is true not only for the most common relation, where the phloem is external, but also for that between the xylem and internal phloem (Warren Wilson and Warren Wilson, 1961b). Such cambia which form phloem internally are also found in some petioles and in continuity with a convoluted cambium in cases of unusual development (Janse, 1921; Rudiger, 1953). A cambium also appears in interfasicular regions with no xylem or phloem (Fahn, 1974; Esau, 1977). This cambium appears to "spread" from the vascular strands and it maintains their phloem-to-xylem orientation. The formation of phloem and xylem on opposite sides of the common type of cambium occurs at about the same time, though in deciduous trees phloem formation may start earlier in spring (Catesson, 1964; Davis and Evert, 1970). "Anomolous cambia", whose activity results in strands of phloem embedded in the xylem, are found in various unrelated families (Philipson et al., 1971). Many, if not all, anomolous cambia form the phloem and the vessels of the xylem on opposite sides and at the same time, as do the common cambia; the special structure results from a differentiation of all the cells of the anomolous cambium to mature vascular elements and the continuation of cambial activity outside the phloem. The formation of xylem by this new cambium is internal, but external to the previously formed phloem. This process is repeated many times. The controls of the formation of new cambia or islands of phloem are not known ; cross-sections through various plants show that they are not related to the development of new leaves (Zamski, in press). This is also shown by the occurrence of many regions of embedded phloem at the base of single beet leaves that had been rooted and allowed to grow (Spence et al., 1972). 3 . The Experimental Induction of Phloem Diferentiation ( a ) Regeneration experiments. As mentioned in Section 11, regeneration of phloem around wounds is both similar to and closely correlated with xylem regeneration (Von Kaan Albest, 1934; Eschrich, 1953; La Motte and Jacobs, 1963; Thompson, 1967; Aloni and Jacobs, 1977b). The differentiation of phloem is promoted by the presence of leaves above the wound, and the

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effect of these leaves can be replaced by auxin with no additional substances (La Motte and Jacobs, 1963; Thompson, 1967; Jacobs, 1970; Houck and La Motte, 1977). Reports that the leaves are not effective (Von Kaan Albest, 1934) are probably due to the unrealized presence of phloem contacts between the vascular strands even in the intact controls (Aloni and Sachs, 1973). The relation between the controls of xylem and phloem differentiation has been studied by observing regeneration when only one of these two tissues is wounded. These experiments are technically difficult, but the general result is that cutting the phloem of large strands causes both phloem and xylem regeneration while cutting the xylem alone causes no regeneration (Roberts, 1960; Roberts and Fosket, 1962; Thompson, 1967; Aloni, unpublished). Only Von Kaan Albest (1934) reported that cutting one of the two tissues caused its own regeneration. Wounding very young tissues, however, would necessarily include the cambium, and the regeneration of this cambium would appear as a regeneration of its products (see Section V.A). The usual result might thus not be very significant, but it does agree with the hypothesis that the differentiation signals for both xylem and phloem can be transported through the mature phloem (Section V.B). ( b ) The effects c!f' hornione applications to iiituct utid wounded plarrts. As was discussed in Section II.B, auxin by itself causes the differentiation of xylem from the cambium or from parenchyma tissues near a wound. Most studies of this induced differentiation neglected the phloem, which is technically difficult to follow. Kraus et ul. (1936) noted that auxin causes xylem differentiation even from phloem parenchyma ; this type of differentiation can also be found as part of regenerative processes around wounds (Timmel, 1927; Sachs, unpublished). Absence of any induced phloem in the derivatives of a cambium activated by auxin was found by Digby and Wareing (1966a). In other plants, however, careful observation of cleared material shows that auxin by itself induces the differentiation not only of vessels but also of typical sieve tubes; this was found in wounded pea parenchyma and the derivatives of turnip cambium (Fig. lOc,g; Sachs, in preparation). As mentioned above, auxin alone could be limiting for the differentiation of phloem around wounds in Coleus stems (for review see Jacobs, 1970). Gibberellins added in the presence of auxin greatly enhance cambial activity (Bradley and Crane, 1957; Wareing, 1958; Wareing rt ul., 1964; Shininger, 1971; Hess and Sachs, 1972). In some plants the number of cell layers formed on the phloem side of the cambium increased as the concentration of the applied gibberellins was increased (Wareing et ul., 1964; Digby and Wareing, 1966a; Harrison and Klein, 1979). Sieve tubes have been identified in the cambial products induced in the presence of gibberellins (Digby and Wareing, 1966a). Gibberellin alone induced the differentiation of sieve cells from a cambium of pine trees, as judged by polarized light observations (De Maggio, 1966). The effect of gibberellins in promoting phloem

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differentiation, however, is not always found (Bradley and Crane, 1957; Waisel er al., 1966; Hess, Fahn and Sachs, unpublished) and they commonly cause fibre formation (Section V1.C). It is not likely, therefore, that gibberellins are specific to one type of differentiation (Wareing et al., 1964). ( c ) Organ and tissue culture experiments. A unique case in which experimental manipulations control the relations of the phloem and the xylem has been reported by Torrey (1957, 1963). The number of xylem and phloem poles in pea roots was varied by auxin treatments; this effect might well be related to a control of the size of the apical meristems (Torrey, 1965). Cytokinins, known to inhibit the formation of root meristems, were found to disrupt the normal pattern (Torrey, 1963). The hormonal treatments were made more effective by studying regenerating roots, in which the pattern is presumably more labile. Though in untreated pea roots the number of xylem poles does not vary, the results are probably related to some developmental requirement for a close contact (considered above) between the differentiating xylem and phloem which prevents the phloem tissue from differentiating in large masses. This is also supported by the results of Feldman and Torrey (1975) who varied the size of maize roots by using different sugar concentrations and by observations of untreated roots of different sizes (Fig. 30). The normal orientation of the phloem outside the xylem is generally preserved in culture. This orientation may be extremely stable, even when tissues are grafted into the plant axis in a reverse orientation (Siebers, 1971a). The transverse polarity (see previous section) of the cambium may be determined very early, even before the cambium is microscopically apparent (Siebers, 1971b). A reversal of the phloem-to-xylem orientation has, however, been found for new vascular tissues induced in cultured material, and the effect depends on the origin of the tissue (Gautheret, 1959). This reversal, often found in cultured crown gall tissues, also occurs in callus developing on a wounded plant (Mosse and Labern, 1960). The conditions limiting phloem formation in tissue cultures have not generally been studied, in contrast with the voluminous information about xylem differentiation (Roberts, 1976). Sieve tubes, however, have been identified in culture (Galavazi, 1964; Hanson and Edelman, 1970). The influence of gibberellins applied with auxin, which together increase cambial activity and phloem formation in wounded plants, has also been reported for cultured callus tissues (Gautheret, 1961). The best studied control of phloem formation, however, has been the concentration of external sugars. A promotion of phloem differentiation by added sucrose was found long ago for intact plants (Molliard, 1907), and it has been more recently confirmed for cultured callus (Wetmore and Rier, 1963; Wetmore et al., 1964). This effect of sugars has been confirmed in a number of laboratories (for review see Warren Wilson, 1978) though it has not been invariably found (Forest and McCully, 1971). The chemical nature of the sugars effective in influencing

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Fig. 30. Pattern and size in root xylem. Vessels and endodermis of cross-section of three roots of one Urginea maritima plant. As in many other monocotyledons the large variations in the size of the vascular system are not paralleled by the size of the vessel groups and the phloem between them. This results in a linear correlation between the number of xylem groups (or poles) and the diameter of the vascular cylinder (and the root as a whole).

phloem differentiation was studied by Jeffs and Northcote (1967), who found that a-glycosyi disaccharides are necessary and of these sucrose is the most effective. The sucrose has not been demonstrated to determine the location of the new vascular elements (Wetmore et al., 1964); rather, it influences the ratio of the phloem and the xylem induced by auxin, and at high sucrose concentrations no xylem may be formed (Wetmore et al., 1964).

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4. Disciission uiid Conclusion

The review above included many facts that are apparently unconnected. It is therefore necessary to list the relevant generalizations which can be made on the basis of these facts : (a) The formation of the phloem, cambium and xylem is closely correlated, both in normal and in regenerative differentiation. The phloem, however, may appear in small amounts without the xylem, but not vice versa. (b) The rays are evidence for a transverse interaction between the phloem and the xylem (see previous section). (c) The location of the phloem, cambium and xylem depends on a polar inductive effect of the leaves. This effect is at least partially replaceable by auxin, which by itself can induce the differentiation of all three tissues.

(d) Additional treatments modify the influence of the leaves and the auxin. Gibberellins and sucrose are the best studied and most effective, and they can greatly increase the proportion of the phloem and the activity of the cambium. The possible working hypotheses outlined in Fig. 29 may now be considered. N o specific inducers of phloem, cambium and xylem have emerged, and it is quite possible that they do not exist. At least two substances, however, modify the inductive effect of auxin. This might suggest that the signal originating in the leaves is complex ; all its components are initially transported through all the cells but a later stage of specialization, both in transport and in structural differentiation, soon sets in. The various specialized cells need not transport a specific signal; they may be specialized in terms of different proportions of the same signals. This mechanism, an obvious extension of the facilitated flux hypothesis developed in Section 111, would account for the close correlation, both in time and in space, of the differentiation of the various vascular tissues. Can it be suggested that any known substances are components of this complex signal? This is certainly a valid working hypothesis for the gibberellins. The case for sucrose is ambiguous, since it would influence all processes in the tissue, including the synthesis of both known and unknown hormones. Sucrose is, of course, transported in the sieve tubes of the phloem, and may thus act as a morphogen. The leaves induce polar differentiation, however, even at a stage of development in which they must be importers of sucrose, and this polar differentiation can proceed, in cut plants, away from the sources of stored nutrients (Fig. 9e; Sachs, in preparation). The precise role of sucrose, therefore, remains unclear. The evidence presented in Section V.D, especially concerning vascular rays, indicates that there are transverse interactions between the differentiating phloem and differentiating xylem. This does not contradict any of the

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considerations raised above, and may well be an additional major factor in maintaining the relations and integrity of the vascular system. Such interactions and a transverse polarity of the cambium would also account for the stability of location of phloem and xylem found by Siebers (1971a). The transverse polarity of new tissues may, however, be influenced by local gradients within a callus (Gautheret, 1959). A predetermined state of the cells, which could control the formation of phloem, cambium and xylem is most unlikely in view of the readiness with which regenerative processes occur, including the induction of xylem vessels in phloem tissues. The working hypothesis which emerges, therefore, involves the two axes of polarity proposed by Janse (1921). In addition, this polarity depends on facilitated transport, and the transported signals may be complex. Specific transport of parts of this complex signal would be the key to the relations between the tissues, For this, of course, there is no direct evidence. C. THE CONTROLS OF FIBRE DIFFERENTIATION

I . The Experimental Systems Fibres, unlike sieve elements, have thick (often lignified) walls and a distinctive elongated form; they can therefore be readily identified. They differ from vessel elements, the other common cells with thick lignified walls, in the narrowness of the lumen and the absence of bordered pits. The fibres are thus a convenient object for the study of questions concerning the control of the differentiation of various cell types which occur together. Fibres are found in both the xylem and the phloem, and their distribution varies according to the species and its growth habit, so that many patterns of distribution are readily available for study. The systems that offer special experimental possibilities are the seasonal occurrence of fibres in the secondary xylem of many trees and the occurrence of strands of phloem fibres which are isolated, or relatively isolated, from the vascular tissues. Since there is no a priori reason to suppose that these different types of fibres depend on the same controls, they will be considered separately, at least from the factual point of view. As in the previous section, it will be necessary to list the facts which appear intuitively relevant rather than to present clear hypotheses or answers. 2. The Diflerence Between Early and Late Wood ( a ) The possible controls of vessel andjibre distribution in ring porous trees. The most convenient objects for the study of the problem considered here are the extreme ring porous trees, where wood formed early in the growing season includes many wide vessels while that formed in late summer consists primarily of fibres, with a few narrow vessels (Fahn, 1974; Esau, 1977).This is a predictable and readily observable shift in the types of cells formed during xylem differentiation. Three different factors, not necessarily independent of one another, may contribute to this difference in the products ofthe cambium :

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(1) Early wood may form in response to signals from rapidly expanding leaves. This is supported by a correlation between the presence of extreme ring porous wood and the rapidity and synchrony of leaf expansion (Priestly et al., 1935; Wareing, 1951 ; Wareing and Roberts, 1956). The differentiation of the vessels may occur even before leaf expansion becomes apparent, but there is no reason to assume that the leaves are not already active in terms of their hormonal effects (Wareing, 1951). Leaf expansion is known to be associated with high auxin synthesis, and exogenous auxin induces xylem with many wide vessels (Wareing, 1958; Hess and Sachs, 1972). The best evidence is, however, that a second flush of leaf expansion is often (though not always) associated with the formation of a new ring of wide vessels (Studhalter et al., 1963; Bernstein and Fahn, 1960). (2) The change in environmental conditions may influence the production of differentiation signals and the activity of the cambium. An indirect effect of the environment through its influence upon leaf initiation and development would account for many results which show that photoperiodic conditions are especially important (Wareing and Roberts, 1956; Larson, 1964; Waisel and Fahn, 1965). Such indirect effects are outside the scope of this chapter, though they do stress the importance of the expansion of young leaves. There is, however, some evidence from experiments with decapitated conifers that photoperiodic conditions may influence signal production in the leaves independently of their influence on leaf development (Wodzicki, 1961). Experiments with rooted leaves, in the complete absence of apices, could be used to study this possibility (promising indications were obtained by Benayoun, Fahn and Sachs, unpublished). (3) The new, functional phloem could influence the type of wood produced later in the season (see Sections V.B and V1.B). There appear to be no publications dealing with this possibility. ( b ) The influence of mature leaves on late wood production. The discussion above shows that xylem of the late wood type is formed in the absence of young, rapidly expanding leaves. Since in the absence of leaves cambial activity is generally not very pronounced (see Section 11), it follows that some influence of the mature leaves alone, or in combination with that of slowly developing apices, controls the differentiation of the fibres typical of late wood. Experiments in which young leaves were removed (Munch, 1938) and the orientation of vessels which connect directly with mature leaves (Wareing and Roberts, 1956)show clearly that mature leaves can have a xylem-inducing effect. The problem, therefore, is the identification of these signals and the comparison of their properties with those of auxin. In some plants the xylem formed below mature leaves in the absence of any

( a 1 1 2 2 0 5 4 9 ; 23%

(C)1634?365; 8 %

( b

1 6 3 0 2 4 9 ; 29 Yo

( d 1189k75; 4170

Fig. 31. Control of vessel and fibre differentiation by bean leaves. The numbers are the crosssectional area of the xylem (in lo3 pmZ)and the percentage of this area occupied by vessels at the level of the double horizontal lines. The treatments indicated by the drawings are intact plants (a), only young leaves above the studied region (b), only a mature leaf (c) and no leaves (d). The results for (d) were not very different from those found at the time the experiment was started. Note that young leaves induce many vessels while mature leaves induce a large amount of xylem (even compared with intact plants) but relatively few vessels. (Plants kept growing for a month before they were sectioned. (Based on Hess and Sachs, 1972.)

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buds is abnormal, and consists of parenchyma with isolated groups of vessels (Simon, 1929; Shininger, 1970; Benayoun and Sachs, 1976).The presence of young leaves, or of auxin, causes normal differentiation (Shininger, 1970, 1971, 1979). In other plants, however, single mature leaves cause the formation of a fully lignified xylem, consisting primarily of fibres (Fig. 31; Hess and Sachs, 1972; Benayoun and Sachs, 1976). This effect of mature leaves, which was found in a convenient experimental system using seedlings and also in some trees, was not limited in its duration and was clearly polar, extending only in the direction of the roots. Concentrating the effect of the mature leaves by means of wounds which confine it to a narrow section of stem did not change the cellular composition of the xylem. The cross-section of the xylem formed in beans with mature leaves only was greater than that found in intact plants (Fig. 31), indicating that even when the xylem appears normal its differentiation is not identical with that occurring in the presence of intact apices. The interaction between young and mature leaves, however, may be at two possible levels : a direct interaction of the signals influencing the cambium and an indirect effect of the young leaves on the production of signals by the mature leaves. The second possibility is supported by observable changes in mature bean leaves in the absence of growing apices and by observations on plants where the young and the mature leaves were on different stems (Hess and Sachs, 1972). The nature of the influence of mature leaves in intact plants is thus far from clear. It is possible, however, to attempt to replace their effect by known hormones. A detailed theory of how abscissic acid could influence the diameter of conifer tracheids has been proposed by Wodzicki and Wodzicki (1 980). There is no clear evidence, however, that abscissic acid acts directly on the type of cells produced by the cambium, and it did not promote fibre production in the experiments of Hess and Sachs (1972). Gibberellic acid, on the other hand, has been found to promote fibre formation in various plants (Bradley and Crane, 1957; Wareing, 1958; and other reports quoted by Hess and Sachs, 1972). In leaf replacement experiments, gibberellic acid caused the formation of a fibre-rich xylem when applied together with auxin. It is not known how relevant this gibberellin effect is to the events in the intact plant, but it could prove an important key for the study of the controls of fibre differentiation.

3 . The Formntioii of’ Phloeni Fibres The fibres of the phloem system have the experimental advantage of being completely or relatively isolated from the rest of the vascular system, and thus amenable to specific treatments. Primary phloem strands surrounded by cortical parenchyma occur in peas, and their control was studied experimentally by Sachs (1972a). It was found that their location was dependent on the presence of leaf primordia, and this process was already complete when

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the primordia were still extremely small, before the formation of most of the primary phloem and xylem. This effect of the leaves. which could not be replaced by auxin, was also demonstrated by surgical treatments resulting in the formation of two primordia, each one inducing its own fibre strand. Fibre strands could regenerate across grafts, provided these were made in very young tissue. The removal of a young primordium caused its fibre strand to be joined by the new strand induced by the next primordium above (see Fig. 13 and Section 1II.B for a similar elimination of a leaf gap in the case of xylem differentiation). Thus there was a general similarity between the control of xylem and the control of fibre differentiation, and it can be suggested that the same facilitated flux hypothesis applies in this case as well. The difference is in the nature of the signal, unknown for the case of fibres; it is produced very early in the development of leaf primordia and might be related to the unknown signal controlling procambium differentiation (see Chapter 11). The differentiation of phloem fibres in Coleus, which are an integral part of the vascular system, was studied by Aloni (1976, 1978). He found that this differentiation depended on the presence of leaves, whose effect was strictly polar. Unlike peas, however, both young and mature leaves induced fibre differentiation. Coleus fibres were also found to regenerate from relatively mature parenchyma cells around wounds. This novel type of differentiation, also observed in tissue culture, required a period of a few weeks. Gibberellins are effective in replacing the effect of leaves on fibre differentiation. There is also evidence that gibberellins influence fibre differentiation when they are sprayed on intact plants of various species (Stant, 1963). 4. Discussion and Conclusion All available information points to a control of fibre differentiation by the same type of inductive flux that controls the differentiation of vessels and sieve tubes. The difference lies in the nature of the signals, since auxin alone does not appear to cause much fibre differentiation. There are strong indications that gibberellins are part of the fibre-inducing signal, but proof would consist not only of the effects of external applications but also evidence that gibberellins replace organs known to be the cause of their flux (either as a source or a sink). Such evidence, available for auxin (Jacobs, 1970; Jacobs and Marrow, 1957), is completely lacking concerning gibberellins. Of the possible scenarios considered above (Fig. 29) the most probable, therefore, would consist of an early common differentiation stage of fibres and other vascular elements followed by specialization of fibres for the transport of a specific component of the inductive signals (Sachs, 1972a). This hypothesis points to the need for information concerning transport capacities of the fibre cells during early stages of differentiation, before they form thick walls. The available evidence does not require any assumptions

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about direct interactions between the future fibres and neighbouring cells other than those which would result from specific transport and the draining of signals. Suggestions that the differentiation of fibres depends on transverse gradients (Prat, 195 1) ignore the longitudinal pattern. Finally, the different patterns of fibre distribution found in plants should be mentioned. It is not known at present whether all fibres depend on the same longitudinal flux, but it may be significant that gibberellins increase the differentiation of both xylem fibres (Hess and Sachs, 1972) and phloem fibres (Aloni, 1978). It is thus possible that the precise distribution of fibres relative to other cell types depends on small differences in signal transport capacity and its specific facilitation in various differentiating cells. There is little to support this working hypothesis at present, but it does emphasize the phenomena requiring further study. D. THE CONTROLS OF PARENCHYMA FORMATION

1. The Problem and a Working Hypothesis Vascular strands consist not only of sieve tubes, vessels and fibres (Fig. 2b), they also include many cells referred to by the general term “parenchyma”. These cells do not have any obvious special traits distinguishable by microscopy, but this, of course, cannot be taken as strong evidence for a uniformity either of structure or function. In addition, cortical and pith parenchyma form and differentiate at the same time as the vascular tissues. This means that the consideration of strands might be extended to include virtually all of the plant axis, though the presence of the epidermis, collenchyma and cortical fibres will not be discussed here. The problem, therefore, is how the location and quantity of parenchyma is determined, and what relation its controls bear to the formation of vascular tissues. The control of vascular differentiation by facilitation of an inductive flux of signals suggests a working hypothesis for the formation of parenchyma. If facilitation is a gradual process, as the evidence seems to suggest (Section IV.B), the flux must pass through more cells (in cross-section) at early stages of differentiation than in later ones. There would thus be cells which are “partially induced” : cells formed in response to the flux, and polarized by it, before it becomes confined to the more specialized, central cells that later differentiate as conducting and supporting elements. This hypothesis can make clear predictions concerning the control of parenchyma formation and the response of its cells to inductive signals.

2. The Evidence ( a ) The induction of parenchyma formation by leaves. The removal of young leaves reduces all aspects of vascular differentiation in the stem below (see Section 11). The removal of very young leaf primordia, however, prevents the formation not only of the vascular tissues but also of the entire

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stem region surrounding them, or, in other words, the parenchyma (Fig. 8; Sachs, 1972a). This observation is supported by experiments in which the number of leaves varies; although this cannot be done by grafting young tissues (see Section 11), splitting a very young leaf primordium does result in the formation of two leaves (Sachs, 1969b), and the two vascular strands formed below them are surrounded by normal parenchyma (Sachs, 1972a). This induction of parenchyma by leaves is restricted to tissues very close to the shoot apex. Adventitious leaves formed on mature tissues or on callus do not have the same effect. Cells close to the shoot apex might not respond to auxin, but a non-auxin signal must be assumed to account for the formation of the procambium (Young, 1954; Section 11) and this signal would, at first, pass through all the stem tissues. At a later stage auxin is the signal for the radial growth of the young parenchyma (Sachs, in preparation). ( h ) The competence of the purenchynin to foriw vascular elements. When auxin is applied to parenchyma cells near a wound they form vessels and sieve tubes (Fig. 1Oe-g). This response indicates that parenchyma differentiation does not exclude a “continued differentiation” to mature transporting elements, and in this sense it fits, though does not prove, the working hypothesis outlined above. There has been much discussion of the question whether parenchyma cells must divide before vessel members are formed (for review see Roberts, 1976). Interest in this problem has been primarily in relation to questions of the mechanisms of gene regulation (Shininger, 1979) and the subject therefore lies outside the scope of this chapter. There is no doubt that cell division during formation of vessel members is common, though it may not be an invariable rule. The requirement of a wound near redifferentiating parenchyma cells (Shimomura and Fujihara, 1978 ; Sachs, unpublished) indicates that they are not simply “partially differentiated” transporting elements. The wounds have a fairly persistent effect on the competence of parenchyma cells, and auxin is effective even when applied more than two days after young pea stems are wounded (Sachs, unpublished). Auxin applied to a cambium induces cells even in the absence of an immediate wound (Fig. 11) except when the cambium is dormant (Gouwentak, 1941). It is more significant to the topic considered here that parenchyma will also respond in the absence of a wound if it is very young, before the cells have completed most of their growth (Sachs, in preparation). Parenchyma in completely mature organs, furthermore, does not respond by vessel formation to any known treatment. It may thus be concluded that parenchyma cells undergo a gradual change during which they lose their competence to respond to auxin by vessel formation and that there is a period during which this competence can be restored by the effects of a wound. (c) The relative polarity of different parenchyma cells. The working hypothesis outlined above predicts that the signal flux would gradually be canalized

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to increasingly limited strands of cells. Thus the parenchyma close to the vascular tissues should be subjected to the inductive flux for longer periods and, barring threshold effects, might be more “polarized”. Apparently this effect can be observed, because the parenchyma next to the vascular tissues is considerably more elongated than the central parts of the cortex or the pith, and the most elongated are the parenchyma cells within the vascular tissues. Polar auxin transport, furthermore, proceeds most readily in the region of the vascular strands (see Section 1II.B). Parenchyma that is not part of the vascular strands is often inactive in mature stems, and, especially in the pith, often collapses and disappears. ( d ) The timing ojparenc.hymaformttion andgrowth. If parenchyma formation were to depend on signals that become canalized in vascular strands, it would be expected to occur before these tissues are fully differentiated from the surrounding cells. The ability of a tissue to regenerate is an indication that the interactions determining its presence are still taking place, and by this criterion the formative regions of parenchyma lie very close to both the shoot and the root tips. The decline in regenerative capacity of parenchyma as the distance from the apices increases is gradual (Sachs, unpublished) and could be dependent on the formation of the procambium and vascular tissues. There is no direct evidence, however, that the negative correlation between ability of the parenchyma to regenerate and differentiation of the vascular system has any causal basis. In contrast to the process of their formation, the growth of the parenchyma tissues continues long after the vascular tissues are complete. Unequal growth of this parenchyma is the basis of various morphogenetic events in relatively mature organs, such as the opening of the seedling “hook” in many dicotyledons. These cases of growth do not occur if the apex and the young leaves are removed, and they are often, if not always, stimulated by auxin. Late parenchyma growth is rare in roots (though it does occur in contractile roots). All this leads to the conclusion that the parenchyma is not only a tissue “left over” after the formation of vascular strands. There must be specific signals actively controlling the parenchyma, though the hypothesis that early stages of parenchyma formation depend on an uncanalized flux of general signals for vascular differentiation might still be valid. ( e ) The relation between the procambium and the cambium. The transition from primary to secondary vascular differentiation (Fahn, 1974; Esau, 1977) is a topic related only indirectly to parenchyma development, but it does involve changes in competence of cells to respond to signals. The procambium responds to signals from the leaves by growing both in length and in width, while the cambium is limited to growth at right angles to the signal flux. Yet the anatomical evidence indicates not only that the two meristematic tissues are continuous but also that there is no clear dividing line between them (Cumbie, 1967; Fahn et al., 1972; Larson, 1976). Furthermore, the evidence

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summarized in Section I1 suggests that the two issues are controlled by the same signal flux, though the early stages of the procambium are not sensitive to the auxin component. Wounds which can be expected to confine the signal flux to a narrow sector of a Ricinus stem cause local early formation of a cambium continuous with the procambium on both its upper and its lower sides (Fahn er al., 1972). The conclusion relevant to the discussion of parenchyma formation is that the same flux of signals may cause growth along its axis or at right angles to it, and the response of cells depends on local conditions and their past history. 3. Conclusion and Discussion The facts summarized above tend to support the hypothesis that parenciiymu, both within and surrounding the vascular system, is dependent on the sume controls as vuscular diflerentiation. The signal flux, therefore, is involved in the formation of the entire plant axis and not just the vascular strands. The canalization of the signals to the procambium, leaving the future parenchyma “partially induced”, may well play a role in determining the nature and location of the future parenchyma cells, but this cannot be the whole story. There is clear evidence that signals from the shoot influence the development of relatively mature parenchyma, though the role of these signals and of internal programmes in the gradual changes of parenchyma cells are not known. The relation of parenchyma to the vascular system, therefore, resembles that of the phloem to the xylem and the fibres to the vessels; in all these cases the most likely hypothesis is that differentiation involves a specialization for component signals of the inductive flux (Fig. 29a). In parenchyma this specialization is for transport not canalized to limited strands. This hypothesis would account for the general structure of the plant axis, each vascular strand being induced together with the parenchyma which surrounds it. The uniform distance between primary strands would not depend on inhibitory interactions between strands (Biinning, 1965) but rather on their being formed as one unit together with parenchyma separating them from other strands. A similar control, on a different scale, appears to determine the spacing pattern of the stomata (Sachs, 1978b). The difference between the organization of the vascular tissues in the stem and the root would then be a result primarily of the influence of the leaves (Wardlaw, 1968). The difference in the direction of auxin flux relative to the apex should not necessarily influence the vascular pattern, which could be determined according to the same rules. The root, then, would be one large strand with radial rather than bilateral symmetry; the pattern of the phloem and the xylem relative to each other would produce their local interactions (see Section V1.B).

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VII. THE RELATION O F THE CONTROL O F VASCULAR DIFFERENTIATION TO OTHER ASPECTS OF PLANT MORPHOGENESIS 1 . The Problem The preceding sections suggested that a leaves-to-roots signal flux controls vascular differentiation at the organ, tissue and cellular levels. The preceding section attempted to enlarge the scope of this control to the entire plant axis. This raises the general topic of this section: what other roles might this signal flux play and what is its relation to other controls of plant development? This question has obvious intrinsic interest; it could also be important in pointing to additional characteristics and experiments concerning the signal flux considered above. 2 . Other Correlative EfSects of Leaves and of’Auxin The most prominent control of vascular differentiation is the effect of the leaves; this can be partially replaced by auxin (Section 11). It may be asked, therefore, what other processes are influenced by leaves and by auxin in similar fashion? In addition to the various aspects of stem development considered above, the presence of leaves inhibits the development of new leaves and of lateral buds, inhibits the formation of an abscission zone on the axis connecting them with the roots while promoting the formation of abscission zones lateral to this axis, and promotes the formation of both lateral and adventitious root apices (Sachs, 1972c, 1975c; Leopold and Kriedmann, 1975; Goodwin et al., 1978; Thimann, 1977). All these effects are at least partially replaceable by auxin. These facts suggest that auxin is not a hormone that is “typically” involved in cell elongation and has a confusing array of other effects, but rather that it can be characterized as a major hormonal signal of the leaves, especially developing leaves, which elicits different responses according to the nature and past history of the tissue it reaches (Sachs, 1975~). How are the effects on bud growth, abscission and root initiation related to the signal flux that controls the formation, growth and differentiation of the stem tissues? The case of root initiation is the clearest : roots are formed wherever the tissues are competent to respond and the signals accumulate; they do so close to a root tip or where a wound, tissue polarity or environmental conditions prevent their further transport. The initiation of roots is, therefore, a response to the concentration of signals whose flux causes growth and vascular differentiation. Abscission depends on a balance of auxin concentrations applied on the two sides of the responding zone (Addicott, 1970), and may thus be a specialized response to the cessation of the flux considered in the previous sections. The promotion of abscission by other leaves would then be due to a relatively direct effect on this flux and to competition for root factors, considered below.

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Concerning lateral bud growth, it has been suggested that the ability of these buds to form vascular contacts is a critical control (Munch, 1938; Sorokin and Thimann, 1964). The dependence of vascular contact formation on the direction of auxin flux (Figs 9g,k, 12, 13; Section 1II.B) can logically be expected to play some role in bud growth (Sachs, 1970; Kirschner and Sachs, 1972). This is supported by the finding that buds growing on intact plants have vascular contacts different from those of buds released by decapitation, and only the latter make direct contacts to the strands leading to the organs above them (Sachs, 1968b, 1970). The objections to the role of vascular differentiation in bud inhibition have been based on the rapidity with which bud growth begins following decapitation and on the occurrence of inhibition of buds with differentiated vascular connections (Cutter, 1972; Peterson and Fletcher, 1973b). There are a number of reasons, however, for thinking that vascular contacts might none the less be important. It would be the increase, rather than the mere presence, of vascular contacts which could be expected to control bud growth. The effect of the signal flux on the oriented transport of critical factors (such as cytokinins, see below) might be much more rapid than the maturation of vascular tissues (Sachs, 1972b, 1975b; Kirschner and Sachs, 1978). Finally, the argument in favour of the importance of vascular contacts is a logical one (Sachs, 1970) but it does not stipulate that this is the only control of a process so complex as bud growth. It may be concluded, therefore, that a polar diversion of essential factors (Phillips, 1969) towards a strong source of a signal flux is an important, though not unique, control of bud growth. It may be noted that this conclusion implies some form of “hormone directed transport” (for reviews see Reinhold, 1974; Patrick, 1976). Hormones, including auxin, have been shown to direct the transport of radioactive molecules towards the region to which they are applied. There is evidence, however, that much of this effect is indirect and is due to the influence of the hormones on the formation ofsinks for the various metabolites. The orienting influence of auxin on vascular differentiation (Fig. lo), on the other hand, is a long-term form of directed transport (Gersani et al., 1980a). It is suggested above that this demonstrable effect may not only play a role in bud growth but could also be more rapid than its microscopic manifestations. 3. A Hormonal Feedback Between the Shoot and the Root It was concluded in Section I1 that roots orient and control vascular differentiation by acting as sinks for auxin and other signals from the leaves. This raises the problem as to whether the roots act as actual sources of hormones which influence other morphogenetic events. Since sink activity can change the hormone balance as much as an actual source, this problem could only be answered on the basis of positive evidence that the roots produce hormones which replace their correlative effects on the plant. Some such evidence,

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however, is available (for reviews see Sachs, 1 9 7 5 ~Goodwin ; rt “ I . , 1978). Roots are an important source of cytokinins (Letham, 1978) though it is not clear that they are the only source (Wang and Wareing, 1979). Cytokinins replace the correlative effect of the roots in maintaining the activity of leaves, promoting bud initiation and bud growth and inhibiting the formation of lateral and adventitious roots. All these processes also influence vascular differentiation, but there is no evidence for a direct effect of cytokinins on the location and orientation of vascular strands (Section 1I.C). There is also evidence that the roots produce gibberellins whose role is not clear though it might well be very important (Frydman and Wareing, 1974). Since shoots produce auxin which controls the initiation of new root apices and the root apices produce cytokinins which are necessary for shoot development, a positive feedback system must be set up (Sachs, 1972c, 197%; Goodwin and Morris, 1979; Gersani rt d., 1980a). This feedback is also expressed in the general maintenance of plant form, though this may also depend on other factors, not necessarily hormonal. There is evidence that bud growth may not always depend on cytokinins from the root (Wang and Wareing, 1979) though at least some experimental results may be complicated by regenerative processes, where callus tissues could be forming both shoot and root hormones (see Section 1I.E and Van Staden, 1979). The quantitative relations between roots and shoots are further complicated by the operation of the hormonal control on root initiation (Keeble et al., 1930) rather than on the growth of the roots, which is necessary for the maintenance of even non-developing shoots. The most important, and as yet unknown, aspects of the shoot/root hormonal feedback, however, are the quantitative relations between the hormones received by an organ and the hormones it produces (Goodwin rt al., 1978) and the role of gibberellins as correlative factors in the plant. 4 . Tlic. Firndanirriral Rolr of tlir Horrnoriul Fredbmck

Vascular differentiation thus appears t o be but one expression of a control system with very diverse functions. This control suggests that plants should be viewed as a branched, bipolar axis and the growth of the opposite sides is correlated by their different hormonal requirements. Apices on the same side of the axis are not uniform since they differentiate to form the various types of root and shoot structures, but this differentiation is influenced by the general hormonal feedback of the plant (for example, see Wooley and Wareing, 1972). Callus, or unorganized growth on cut surfaces may also be of two types (Simon, 1908b) and depend on this hormonal system (Kirschner et l i l . , 1971). One hormonal control system is thus important not only for the maintenance of plant form but also for apical differentiation and the intercellular relations involved in vascular organization. Some aspects of this hormonal

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feedback might even be responsible for apical organization (Sachs, 1972~). All this would appear to put undue stress on the role of the hormonal relations between the shoots and the roots and their expression in vascular differentiation. There certainly are other control systems in the plant. It is significant, however, that an auxin and a cytokinin are the only molecules necessary for the growth of tissues in culture which do not have a clear role as building blocks for the cytoplasm. Relatively unorganized growth, such as that of crown gall (Fig. 4). is associated with and can be accounted for by the unregulated formation of the same two hormones (see Sachs, 1975~). Other unusual substances are formed in crown gall, but unorganized growth on wounded plants can be obtained by the application of only auxin and cytokinins (Sachs, unpublished). Some domination of the plant in terms of vascular tissue differentiation is essential for the development of galls (Meyer, 1969) as well as higher plant parasites (Tsivion, 1978). I t is also significant that the sensitivity of the plant to a variety of environmental conditions is mediated by the same hormones of the shoot/root feedback control (Leopold and Kriedmann, 1975; Thimann, 1977).Thus the emphasis on these hormonal relations, though perhaps exaggerated, is a useful working hypothesis and brings some order into the endless lists of “effects” of known plant hormones.

VIII. THE MAJOR CHARACTERISTICS OF THE HYPOTHESIS The preceding sections outlined a hypothesis that accounts for vascular patterns, at cellular, tissue and whole plant levels, on the basis of one control system. It is based on signals that are partially known and cellular responses that have been observed. While many aspects of this hypothesisare far from proven, vascular tissues of plants are still one of the best understood biological patterns, especially at the level of the relations between neighbouring cells of the same type. It is therefore of interest to ask what are the general principles of the suggested control system and whether they could be relevant to other cases of biological development, in both plants and animals. In common with other controls of development, the hypothesis suggests that the pattern depends on factors of two types : interactions between cells (or other developing parts) and internal programmes which can be stable, or determined. The cellular interactions involved in vascular differentiation appear to be due, at least partially, to a channelling of signals and thus a restriction of their effects to cells arranged in long files. As a result, the same signals, which are a t least partially hormonal, are involved in both the interactions between organs and the local interactions between cells. The determination of internal programmes is expressed not only by the stability of changes which have already occurred but also in two other ways: the possibility of continued development independently of environmental in-

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structions and the dependence of the responses of cells on their developmental history. Determination is thus essential for pattern formation not only because it stabilizes changes and magnifies small differences through autocatalytic responses, but also because it means that a cell develops according to some integral of the environmental instructions received during its entire development. This means that much more environmental information can be used than would be possible if the cellular environment had to be specified for one given moment. Feedback relations, furthermore, are possible and can correct chance mistakes. The unusual aspect of the hypothesis developed here is, therefore, not the role played by determined programmes of cell polarization but the suggestion that determination depends on the very same factors which are concerned with intercellular relations. These aspects of the hypothesis make it very economical in terms of the molecules and principles which need be involved; whether they have any significance for other developmental systems remains to be seen. The response of the cells to the signals for vascular differentiation is gradual, and since the processes continue only in those cells which differentiate to become the best transporters, it is under constant feedback control. This aspect of the hypothesis is in clear contrast to the rapid, unidirectional control of cell determination which is assumed or implied in many published hypotheses of pattern formation (for example, Wolpert, 1971; Meinhardt, 1978). The control suggested for vascular differentiation does not produce very precise patterns, which could be readily obtained with a strict all-or-none programme. The pattern of veins in plants, however, is not precise and no two sides of a leaf are exactly the same. The advantage of the gradual feedback control is that it prevents the occurrence of mistakes, and there are no regions of a leaf which are under-supplied or over-supplied with vein connections. Observations of biological structure suggest that plant vascular tissues are by no means unique and that approximate controls, corrected by long term feedback relations may be common. Wolpert (1971) suggests that biological patterns may depend on “positional information” : this is a grid of signal gradients which can be used by the cells for a precise interpretation of their location without any interactions with their neighbours. This suggestion stresses the generality of the signals and places the specificity at the level of their interpretation by the cells. The opposite extreme would be a pattern formed by many local interactions between cells, involving specific signals for each type of differentiation. The hypothesis suggested here is in some senses a compromise between the two extremes and avoids both the problems of very precise interpretation of local differences and the need for a large number of specific signals. The control of vascular differentiation depends primarily on a general signal system which involves the whole plant; the local pattern of these signals, however, has very sharp differences which permit no mistakes in their interpretation. These

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local differences arise from the transport changes that are a major component of the processes of differentiation, and they could in other cases involve the use and production of signals. The control of the pattern of vascular differentiation is thus “differentiation dependent” (Sachs, 1978a) and its principles could be of general significance. Current hypotheses of pattern formation assume that the cells respond to the concentration or the gradient of “morphogenes”, the substances that serve as signals for differentiation. The evidence summarized above points to a dependence of differentiation on the flux rather than the gradient of signals. An advantage of a control by flux is that it enables one mechanism to account for two basic phenomena : the differentiation of cells along a strand and the inhibition of similar differentiation by neighbouring cells. The competence of neighbouring cells for this differentiation is shown by the response to wounds, and no dependence on gradients alone would account for their not differentiating to vascular elements in intact organisms. A dependence on flux is, therefore, an economical control, and it may play a role in other strand systems. Such systems in plants include laticifers and collenchyma about which there is no information at present. Though cell determination and responses to the local environment are probably very important in these cases, the flux of some morphogen is still likely to be a primary control. Animal development, unlike that of plants, involves cell motility and a great degree of cell determination. There is, however, evidence for directional induction of development in both blood vessels and nerves, and a response to flux could be a control that not only specifies the orientation of differentiation but also limits its extent. A determined polarity of the kind known for auxin has not been found in animals, but as was pointed out above (Section 1II.D) it is the preferred axis of transport, rather than its determined direction, which is the underlying principle of the pattern of vascular differentiation in plants.

IX. SUMMARY This review is concerned with the interactions and internal programmes that result in the patterned diferentiation of vascular tissues. The term pattern is used to refer to the high degree of order, or predictability, of the system at all levels : the orientation and contacts of the strands forming a coherent system supplying all the organs of the plant, the organization of the transporting cells in files which are functional vessels and sieve tubes, and the relations between the different vascular tissues (phloem, cambium and xylem), and their component cells. The stress is thus on relations between cells of the same and of different types and not on the processes of differentiation and controlled gene expression. Much of the evidence comes from observations of xylem differentiation in dicotyledons, but this only reflects our present

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knowledge and an attempt is made to consider the different vascular tissues in relation to all land plants. It is shown that all aspects of the development and maintenance of the vascular tissues might depend on one control system which is at least partially hormonal. The way this control could operate is in the working hypothesis that, together with the problems and possibilities it poses, is the central conclusion of this work. All aspects of vascular difeerentiation ure found to depend on u continuous f l u x ofsignals, one ofwhich is the hormone uuxin, from the various tissues of the shoots to the root apices. The evidence for this flux comes from a wide range of experiments on organ removal, wounds, girdles and grafts, involving many different plants as well as tissue cultures. The absence of a direct, polar contact with the various organs of the shoot, and especially the developing leaves, causes a reduction of all aspects of vascular differentiation. Many of the effects of leaves, including the induction of differentiation of both vessels and sieve tubes, can be replaced by a source of auxin. Since auxin is also known to be produced by young leaves, it must be part of the inductive signal in the intact plant. Roots are not essential for vascular differentiation, though when present they orient its course towards themselves. These effects of leaves and roots suggest a dependence of vascular differentiation on a flux of signals, and this suggestion is confirmed by the pattern of vessels and sieve tubes and by various experiments which show that vessels are induced only when an auxin flux can be expected. Vascular differentiation also occurs independently of the presence of leaves, or other growing parts of the shoot; this differentiation might be an indication of additional controls but it might also be the response to a signal flux originating in the plant axis, either as a normal process or an early stage of regeneration. The inductive flux follows and maintains the predetermined polarity of the tissue, but it can also reorient the cells, inducing a new polarity. Vessels and sieve tubes induced either by growing leaves or exogenous auxin follow the original polarity and are directed towards the roots. The polar direction is chosen even in tissues where cuts separate this route from the only possible connection with the cotyledons and the roots. When no polar induction is possible, however, differentiation at any angle with the original polarity occurs readily. In the cambium such induction along a new axis is followed by cell division and cell growth, leading to a complete reorientation of the tissue. The orienting effect of the signal flux could account for the relation of new organs to the differentiation of vascular contacts connecting them with the rest of the plant. A central problem concerning vascular patterns is why differentiation is limited to defined strands of cells. The hypothesis suggests that the cells respond to the signal flux by gradually differentiating to become the preferred or facilitated channels for thisflux, along its original axis. As a result of this gradual, autocatalytic process the signals become canalized to discrete

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channels which differentiate to vascular strands and, as the process of facilitation continues within these strands, vessels and sieve tubes are formed. The initial facilitation is for a given axis rather than a direction or polarity, and in angiosperm leaves, where the location of growth centres changes repeatedly, complex vascular networks are formed, rather than polar “drainage” systems. The evidence for this facilitation of transport is that existing vascular strands orient the differentiation of new strands towards themselves, thus acting as sinks for signal flux. This orienting effect is replaced by an inhibition of contacts when the existing strands are supplied by young leaves or an exogenous source of auxin. Further evidence comes from experiments on the transport of radioactive auxin, which occurs most rapidly in vascular tissues and is both maintained and, at least in one case, induced by exogenous auxin. The suggestion that axis rather than the direction of polarity is facilitated first is based on the presence of large vascular loops in which the vessels with opposite shoot-to-root orientations occur side by side. Differentiation is a gradual, long-term response of the cells to the actualflux of the signal and not to any short-term gradients. The presence of a source of auxin is essential during most, if not all, of the time during which differentiation takes place. The lack of an early determination of genetic programmes is also shown by the diverting effects of additional sources of auxin, even when they are added at a time when differentiation can be expected to be almost complete. Early stages of differentiation along a new axis can be recognized, however, by the orientation of the cytoplasm after severe plasmolysis or, in some living cells, by the appearance and orientation of cytoplasmic strands passing through the vacuole. Vessels form when no gradient of auxin can be expected though the determined polarity of the cells can maintain auxin flux. The appearance of vessels and sieve tubes with sharp bends, and even in the form of rings and whirlpools, can also be the response to a determined flux but not to any long-term gradient. New differentiation depends not only on the magnitude of signaljux but also on the diverting influence of mature tissues, which can transport the signals without responding to them by further diflerentiation. This suggestion follows from the hypothesis suggested above, and it is also supported by the effect of wounds, which can cause an increase in the rate of cambial activity when they do not have too large an effect on shoot development (and thus on the production of signals). There must be other, unknown factors that control the volume of vascular differentiation, though the uniform diameter of strands induced by a local source of auxin shows that differentiation need not result in the use or release of inductive signals. The rays formed by the cambium are a response to radial signals passing between the diflerentiating vertical tissues. This is suggested by a correlation between ray formation and growth and the rate of vertical differentiation, a

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correlation found even when the rays compete with the conducting elements for limiting space. The exact pattern of the rays at any given time depends on the ontogenic history of the rays and on other unknown factors, probably associated with the differentiation of the vertical tissues. The structure of the xylem records constant changes in the rays which are but one aspect of changes in the cambium. These changes include an apparent competition between the dividing cells and variations in the orientation of the initials and their differentiating products, and must reflect some instability in the response of the meristematic cells to the inductive flux. The tissue and cellular complexity of the vascular system raises major problems requiring much future work. In addition to interactions between the tissues, such as those indicated by the presence of rays, a control of the relation between the diflerent tissues and cell types might be a joint early facilitation of the transport of a complex signalfollowed by a specialization of the different cell types to transport the components of the signal. There is no evidence for this hypothetical control other than the general similarity in the controls for the components of the vascular system. Small strands of phloem form independently of the xylem, but otherwise the formation of the two tissues is closely correlated, both in time and in space. Auxin alone can induce the differentiation, and determine the location and orientation, of xylem, phloem and a cambium between them, though its effects in terms of the type of the tissues formed can be modified by gibberellins and by sucrose. The fibres of both phloem and xylem form in response to a signal flux which is similar, or partially identical, with the flux controlling vessel formation. An experimental observation, whose meaning is unclear, is that a combination of auxin and gibberellin often induces fibre differentiation. Not only the vascular tissues but the entire plant axis may be formed in response to the same signal flux. The removal of very young leaf primordia influences not only the vascular elements but also the formation of the parenchyma, both within and outside the vascular strands. This parenchyma, when young, can be induced by wounds and by auxin to redifferentiate to all the different types of vascular elements. It is possible, therefore, that the parenchyma consists of those cells from which the inductive signals, or most of them, were diverted by the formation of the Frocambium and the early vascular tissues where the transport of these signals was facilitated. The signals for vascular diflerentiation are part of ageneral positive feedback between shoot and root tissues that controls many essential features of plant development. The feedback involves not only the source-sink relationship on which vascular differentiation depends but also complementary signals from the roots; one of these is cytokinins. Hormonal controls can have many roles in development because the responses of the tissues depend on their developmental history and not on the nature of the hormone. Auxin is therefore not a cell-elongation factor but a major signal of the developing parts of the

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shoot, and cytokinins are not a cell division factor but a signal of the root apices. The results considered in this work suggest that plant hormones, and especially auxin, play an essential role both in the relation between plant organs and in the relations between neighbouring cells. A major characteristic of the hypothesis considered is that a gradual, feedback relation between diflerentiation and signal distribution creates a reliable pattern. Controls of this type may not result in very precise patterns, but they would be simple and dependable in avoiding mistakes of functional significance. They could thus account for the vascular patterns found in plants, where no two halves of the same leaf are exactly the same. These controls also have the virtue of depending on known signals and cellular responses. It is thus possible that the principles on which these controls are based have a wide relevance, both in plants and in other cases of biological development. ACKNOWLEDGEMENTS I would like to thank D. Cohen, A. Fahn, A. M. Mayer, G . J . Mitchison, L. Reinhold, L. W. Sachs and A. R. Sheldrake for stimulatingdiscussions. Most of this review was written during a sabbatical year spent at the MRC Laboratory of Molecular Biology, Cambridge, and supported by grants of the British Council and the Sherman Fund.

REFERENCES Addicott, F. T. (1970). B i d . Rev. 45, 485-524. Alexandrov, W. G., Alexandrova, 0. G. and Timofeev, A. S. (1927). Planta 3,6&76. Aloni, R. (1976). Ann. Bot. 40,395-397. Aloni, R. (1978). Ann. Bot. 42, 1261-1269. Aloni, R . and Jacobs, W. P. (1977a). A m . J . Bot. 64,395403. Aloni, R . and Jacobs, W. P. (1977b). A m . J . Bot. 64,615421. Aloni, R . and Sachs, T. (1973). Planta 113,345-353. Amer, M . and Neville, P. (1979). Revue gkn. Bot. 86,203-220. Amobi, C . C. (1972). Ann. Bot. 36, 199-205. Avery, G. S. (1933). Am. J . Bot. 20, 565-592. Avery, G. S., Burkholder, P. R . and Crighton, H. B. (1937). Am. J. Bot. 24,51-58. Aymard, M . (1968). Bull. SOC.bot. Fr. 115, 187-196. Balatinez, J . J. and Farrar, J. L. (1966). Can. J . Bot. 44, 1108-1110. Bannan, M. W. (1951). Can. J . Bot. 29,421437. Bannan, M . W. (1968). Can. J . Bot. 46, 1005-1008. Benayoun, J. and Sachs, T. (1976). Israel J . Bot. 25, 184194. Benayoun, J., Aloni, R . and Sachs, T. (1975). Ann. Bot. 39,447454. Bernstein, Z . and Fahn, A. (1960). Ann. Bot. 24, 159-171. Blackman, E. (1971). Ann. Bot. 35,653-665. Bloch, R. (1965). Handb. P’Physiol. 15 (l), 234-274. Bonnemain, J. L. (1969). Revue&. Bot. 76,5-36. Bonnemain, J. L. (1971). C. r . hebd. S a n c . Acad. Sci., Paris. D273, 1699-1702. Bonnett, H. T. (1968). J . Cell Biol. 37, 199-205.

256

TSVI SACHS

Bonnier, G. (1900). C. r . hebd. Seanc. Acad. Sci., Paris. 131, 1276-1286. Boodle, L. A. (1920). A m . J . Bot. 34,431437, Bourbouloux, A. and Bonnemain, J. L. (1979). Physiologia PI. 47,26&268. Bower, F . 0.(1930). “Size and Form in Plants”. MacMillan and Co., London. Bradley, M. V. and Crane, J. C. (1957). Science, N . Y . 126,972-973. Brant, J. A,, Ed. (1976). “Molecular Aspects of Gene Expression in Plants”. Academic Press, New York and London. Braun, H. J. (1955). Bot. Stud. Jena,4, 73-131. Braun, H. J. (1959). Z . Bot. 47,421434. Braun, H. J. (1970). “Funktionelle Histologie der skundaren Sprossachse. I Das Holze” 2nd ed. Hundb. PfiAnat. 9 (1). Brown, A. B. (1935). New Phytol. 34, 163-179. Brown, A. B. (1936). Can. J . Res. C14, 74-88. Brown, C. L. and Sax, K . (1962). A m . J . Bot. 49,683-691. Bunning, E. (1957). Protoplasmatologiu 8, 1-86. Biinning, E. (1965). Hundb. PfiPhysiol. 15 (l), 383408. Camus, G. (1949). Revue Cytol. Biol. V i g . 11, 1-199. Carmi, A,, Sachs, T. and Fahn, A. (1972). New Phytol. 71, 349-353. Caruso, J. L. and Cutter, E. G. (1970). Am. J . Bot. 57, 420-429. Casperson, V. G. (1965). Flora. Jena 155, 515-543. Castan, R. (1940). Revue gkn. Bot. 52, 285-304. Catesson, A. M. (1964). Annls. Sci. nut. Bot. 12 ser., 5, 229498. Cheadle, V. I . and Esau, K. (1964). Univ. CaliJ Publs. Bot. 36, 143-252. Chowdhury, K . A. and Tandan, K . N. (1950). Nature, Lond. 165,732-733. Clutter, M. E. (1960). Science, N . Y . 132,548-549. Coster, C. (1927). “Zur Anatomie und Physiologie der Zuwachszonen und Jahrsbildung in den Tropen”. E. J. Brill, Leiden. Crooks, D. M . (1933). Bot. Gaz. 95,209-239. Cumbie, B. G. (1967). Bull. Torrey bot. Club 94, 162-175. Cumbie, B. G. (1969). A m . J . Bot. 56, 139-146. Cutter, E. G. (1972). Adv. exp. M e d . Biol. 18, 51-73. In “The Dynamics of Meristem Cell Populations” (M. M. Miller and C. C. Kuehnert, Eds), pp. 51-73. Plenum, New York. Cutter, E. G. (1978), “Plant Anatomy”, 2nd ed. Edward Arnold, London. Dalessandro, G. (1973). PI. Cell Physiol. Tokyo 14, 1167-1 176. Davis, J. D. and Evert, R. F (1970). Bot. Gaz. 131, 128-138. Deinege, U. (1898). Flora, Jena 85,452498. De Maggio, A. E. (1966). Science, N . Y . 152, 370-372. De Sloover, J . (1958). Cellule 59, 55-202. Digby, J. and Wangermann, E. (1965). New Phytol. 64, 168-170. Digby, J. and Wareing, P. F. (1966a). Ann. Bot. 30, 539-548. Digby, J. and Wareing, P. F. (1966b). Ann. Bot. 30,607-622. Dobbins, D. R. (1970). A m . J . Bot. 57, 735 (abstr.) Earle, E. D. (1968). A m . J . Bot. 55, 302-305. Engelman, M. E. (1965). Ann. Bor. 29,83-101. Esau, K. (1965). “Vascular Differentiation in Plants”. Holt, Rinehart and Winston, New York. Esau, K . (1977). “Anatomy of Seed Plants” 2nd ed. John Wiley and Sons, New York. Eschrich, K. (1953). PIanta 43,37-74. Eschrich, K . (1975), Handb. PfiPhysiol. N.S. 1 ( l ) , 245-255. Evert, R . F. (1961). A m . J . Bot. 48,479488. ~

PATTERNED DIFFERENTIATION OF VASCULAR TISSUES

257

Evert, R . F. (1963). A m . J . Bot. 50, 149-159. Evert, R. F. and Kozlowski, T. T. (1967). A m . J . Bot. 54, 1045-1054. Evert, R. F., Kozlowski, T. T. and Davis, J. D. (1972). A m . J . Bot. 59,632-641. Fahn, A. (1959). Bull. R K S .Council Israel, D7, 122-129. Fahn, A. (1974). “Plant Anatomy” 2nd ed. Pergamon, Oxford. Fahn, A., Ben Sasson, R. and Sachs, T. (1972). In “Research Trends in Plant Anatomy” (A. K . M. Ghouse and M. Yunus, Eds), pp. 161-170. Tata McGraw-Hill, New Delhi. Fayle, D . C. F. and Farrar, J. L. (1965). Can. J . Bot. 43, 1004-1007. Feldman, L. J. (1977). Bot. Gaz. 138, 393401. Feldman, L. J. and Torrey, J. G. (1 975). Can. J . Bot. 53,2796-2803. Forest, J . C. and McCully, M . E. (1971). Can. J . Bor. 49,449452. Fosket, D. E. and Roberts, L. W. (1964). Am. J . Bot. 51, 19-25. Fourcroy, M . (1943). C. r. hrbd. S u n c . Acad. Sci., Paris. 216, 125-127. Frydrnan, V. M. and Wareing, P. F. (1974). J . exp. But. 25,42&429. Fukuda, H. and Komamine, A. (1980). PI. Physiol. 65,57-60. Fukuda, Y. (1967). J . Fuc. Sci. Univ. Tokyo Univ. 3, 313-375. Galavazi, G. (1964). Acta bor. need. 13,42@421. Gautheret, R . J. (1957). J . natn. Cancer Inst. 19,555-573. Gautheret, R. J. (1959). “La Culture de Tissus Vegetaux”. Masson et Cie, Paris. Gautheret, R . J . (1961). C. r . hebd. S a n e . Acad. Sci., Paris, 253, 1381-1385. Gersani, M., Lips, S. H. and Sachs, T. (1980a). J . K X ~ Bot. . 31, 177-184. Gersani, M., Lips, S. H . and Sachs, T. (1980b). J . K X ~ Bot. . 31,783-789. Gibson, A. C . (I978). J . Torrey bot. Club, 105,39-44. Goebell, K. (1905). “Organography of Plants” Part 2. Oxford. Goldsmith, M. H. M . (1969). In “Physiology of Plant Growth and Development” (M. B. Wilkins, ed.), pp. 127-162. McGraw-Hill, New York. Goldsmith, M . H . M . (1977). A . Rev. PI. Physiol. 28,439478, Goldsmith, M. H . M., Cataldo, D . A,, Karn, J., Brenneman, T . and Trip, P. (1974). Planta 116, 301-317. Goodwin, B. and Cohen, M . H. (1969). J . theor. Biol. 25.49-107. Goodwin, P. B. (1 978). In “Phytohormones and Related Compounds. A Comprehensive Treatise” (D. S. Letharn, P. B. Goodwin and H. J . V. Higgins, Eds), Vol. 2, pp. 3 1-1 74. Elsevier/North-Holland, Amsterdam. Goodwin, P. B. and Morris, S . C . (1979). Aust. J . PI. Physiol. 6, 195-200. Goodwin, P. B., Gollnow, B. I. and Letham, D. S. (1978). In “Phytohormones and Related Compounds. A Comprehensive Treatise (D. S. Lelham, P. B. Goodwin and H . J. V. Higgins, Eds), Vol. 2, pp. 215-250. Elsevier/North-Holland, Amsterdam. Gouwentak, C . A. (1941). Proc. See. Sci. K . ned. Acud. Wet. 44,654-663. Gregory, R . A. and Romberger, J. A. (1975). Bot. Gaz. 136,246-253. Gulline, H. F. and Walker, R. (1957). Aust. J . Bot. 5 , 129-136. Hanson, A. D . and Edelman, J. (1970). PIanta 93, 171-174. Harrison, M. A. and Klein, R. M. (1979). Bot. Caz. 140,2&24. Hejnowicz, Z. (1961). Acta Soc. Bot. Pol. 30,729-749. Hejnowicz, Z . (1973). PI. Sci. Letters 1, 359-366. Hejnowicz, Z. (1980). Am. J . Bot. 67, 1-5. Hejnowicz, Z. and Branski, S . (1966). Acta Soc. Bor. Pol. 35, 3 9 5 m . Hejnowicz, Z. and Tomaszewski, M. (1969). Physiologia PI. 22,984-992. Helm, J. (1932). Planta 16,607-621. Herbst, D. (1972). Am. J . Bot. 59,843-850.

258

TSVI SACHS

Hertel, R. and Flory, R. (1968). Planta 82, 123-144. Hess, R . and Sachs, T. (1972). New Phytol. 71,903-914. HOI1, W. (1975). In Handb. PJiPhysiol. N . S . , Vol. 1 (M. H. Zimmermann and J. A. Milburn, Eds), pp. 432450. Hollis, C. A. and Tepper, H. B. (1971). PI. Physiol. 48, 146-149. Homes, J. L. A. (1961). Bull. Soc. r . Bot. Belg. 93, 75-92. Homes, J. L. A. (1965) In Proc. Int. Conf. PI. Tissue Culture (P. R. White and A. R. Grove, Eds), pp. 243-249. McCutchan Publ. Corp., Berkeley, California. Houck, D. F. and La Motte, C. E. (1977). Am. J . Bot. 64,799-809. Jacobs, W. P. (1952). Am. J . Bot. 39, 301-309. Jacobs, W. P. (1956). Am. Nut. 90,163-169. Jacobs, W. P. (1970). fnt. Rev.Cytot. 28,239-273. Jacobs, W. P. and Marrow, I. B. (1957). Am. J . Bot. 44, 823-842. Jacquiot, C. (1968). Bull. SOC.bot. Fr. 1966, 129-136. Janse, J. M. (1914). Annls. Jard. bot. Buitenz. 28, 1-90. Janse, J. M. (1921). Annls. Jard. bot. Buitenz. 31, 167-180. Jeffs, R. A. and Northcote, D. H. (1967). J . Cell Sci. 2, 77-88. Jost, L. (1891). Bot. Ztg. 49,485-630 (interrupted). Jost, L. (1893). Bot. Ztg. 51,89-138. Jost, L. (1931). Z . Bot. 25,481-522. Jost, L. (1940). Z . Bot. 35, 114-148. Jost, L. (1942). 2.Bot. 38, 161-215. Keeble, F., Nelson, M. G . and Snow, R. (1930). Proc. R.SOC.B106, 182-188. Kirschner, H. and Sachs, T. (1972). Israel J . Bot. 21, 129-134. Kirschner, H. and Sachs, T. (1978). Israel J . Bot. 27, 131-137. Kirschoer, H., Sachs, T. and Fahn, A. (1971). Israel J . Bot. 20, 184-198. Kohlenbach, H. W. and Schmidt, B. (1975). Z . PJiPhysiol. 75,369-374. Kraus, E. J., Brown, N. A. and Hamner, K. C. (1936). Bot. Gaz. 98, 37W20. Kiinning, H. (1950). Planta 38, 36-64. Kuroda, K. (1960). C.r . hebd. Seane. Acad. Sci., Paris 250,582-584. La Motte, C. E. and Jacobs, W. P. (1962). Stain Technol. 37, 63-73. La Motte, C. E. and Jacobs, W. P. (1963). Devl. Biol. 8, 8Cb98. Lang, A. (1973). Brookhaven Symp. B i d . 25, 129-144. Larson, P. R. (1964). In “Formation of Wood in Forest Trees” (M. H. Zimmermann, Ed.), pp. 345-365. Academic Press, New York and London. Larson, P. R. (1975). Am. 1.Bot. 62, 1084-1099. Larson, P. R. (1976). Am. J. Bot. 63,369-381. Larson, P. R. (1979). Am. J . Bot. 66,452462. Leopold, A. C. and Kriedmann, P. E. (1975). “Plant Growth and Development” 2nd ed. McGraw-Hill, New York. Leopold, A. C. and Lam, S. L. (1962). Physiologia Pi. 15,631-638. Lersten, N. (1965). Am. J . Bot. 52,767-774. Letham, D. S. (1978). In “Phytohormones and Related Compounds. A Comprehensive Treatise” (D. S. Letham, P. B. Goodwin and H. J. V. Higgins, Eds, Vol. 1, pp. 205-264. Elsevierrnorth-Holland, Amsterdam. Linnemann, G. (1953). Ber. dt. bot. Ges. 6 6 , 3 7 4 3 . Meinhardt, H. (1978). Rev. Physiol. Biochem. Pharmacol. 80,47-104. Meyer, J. (1969) MPm. Soc. bot. Fr. 1969,75-97. Mitchison, G . J. (1980). R o c . R . SOC.B207,79-109. Molliard, M. (1907). Revue @n. Bot. 19,241-391 (interrupted). Morris, D. A., Kadir, G. 0. and Barry, A. J. (1973). Planta 110, 173-182.

PATTERNED DIFFERENTIATION OF VASCULAR TISSUES

259

Mosse, B. and Labern, M. V. (1960). Ann. Bot. 24,50&507. Munch, E . (1938). Jb. Wiss.Bot. 86,581-673. Murray, C. D. (1927). J . gen. Physiol. 10, 725-739. Needham, J. (1942). “Biochemistry and Morphogenesis”. Cambridge University Press, Cambridge. Neeff, F. (1914). Z . Bot. 6,465-547. Neeff, F. (1922). Jb. Wiss. Bot. 61,205-283. Nickell, L. G . (1948). Science N . Y . 108, 389. Niedergang-Kamien, E. and Skoog, F. (1956). Physiologia P1.9,6&73. O’Brien, T. P. (1974). In “Dynamic Aspects of Plant Ultrastructure” (A. W. Robards, Ed.), pp. 414-440. McGraw-Hill, New York. Osborne, D. and Mullins, M. G . (1969). New Phytol. 68,977-991. Parnas, H. and Segel, L. A. (1978). J . theor. Biol. 71, 185-207. Patrick, J. W. (1976). In “Transport and Transfer Processes in Plants” (I. F. Wardlaw and J. B. Passioura, Eds), pp. 433446. Academic Press, New York and London. Patrick, J . W. and Wareing, P. F. (1973). J . exp. Bot. 24, 1158-1171. Pellegrini, 0.(1963). Delpinoa 5, 17-24. Peterson, C. A. and Fletcher, R. A. (1973a). Stain Techno[.48,23-27. Peterson, C. A. and Fletcher, R. A. (197313). J . exp. Bot. 24,97-103. Peterson, R. L. (1973). Can. J . Bot. 51,475480. Philipson, W. R. and Balfour, E. E. (1963). Bot. Rev. 29, 382-404. Philipson, W. R., Ward, J . M. and Butterfield, B. G. (1971). “The Vascular Cambium. Its Development and Activity”. Chapman and Hall, London. Phillips, I. D. J . (1969). In “Physiology of Plant Growth and Development” (M. B. Wilkins, Ed.), pp. 163-202. McGraw-Hill, New York. Prat, H. (1951). Bot. Rev. 17,693-746. Priestley, J . H. (1930a). New Phytol. 29, 316354. Priestley, J . H . (1930b). New Phytol. 29,96140. Priestley, J . H., Scott, L. I. and Malins, M. E. (1935). Proc. Leeds. phil. lit. SOC.2, 365-374. Pulawska, J. (1965). Acta SOC.Bot. Pol. 22,697-712. Rayle, D. L. and Cleland, R. (1977). Curr. Topics Devl. Biol. 11, 187-214. Rayle, D. L., Ouitrakull, R. and Hertel, R. (1969). Planta 87,49-53. Rehm, S . (1936). Planta 26,255-274. Reinders-Gouwentak, C. A. (1965). Handb. PflPhysiol. 15 (l), 1077-1 105. Reinhold, L. (1974). In “Phloem Transport” (S. Aronoff, J. Dainty, P. R. Gorham, L. M . Srivastava and C. A. Swanson, Eds), pp. 367-388. Plenum Press, New York. Robards, A. W., Davidson, E. and Kidawai, P. (1969). J . exp. Bot. 20,912-920. Robbertse, P. J. and McCully, M. E. (1979). Planta 145,167-173. Roberts, L. W. (1960). Bot. Gaz. 121,201-208. Roberts, L. W. (1969). Bot. Rev. 35,201-250. Roberts, L. W. (1976). “Cytodifferentiation in Plants”. Cambridge University Press, Cambridge. Roberts, L. W. and Baba, S. (1968). Pl. Cell Physiol. 9, 315-323. Roberts, L. W. and Fosket, D. (1962). Bot. Gaz. 123,247-254. Robertson, A., Drage, D. and Cohen, M. H. (1972). Science N . Y . 175,333-335. Rothe, G . (1924). Bibltca Bot. (Stuttgart) 93, 1-23. Riidiger, W. (1953). Z . Bot. 41,373-382. Ruge, U . (1937). Z . Bor. 31, 1-56. Rzimann, G. (1932). Gartenbauwissenschafr 6,612-636. Sachs, T. (1968a). Ann. Bot. 32,391-399.

260

TSVI SACHS

Sachs, T . (1968b). Ann. Bot. 32,781-790. Sachs, T. (1969a). Ann. Bot. 33,263-275. Sachs, T. (1969b). Israel J . Bot. 18, 21-30. Sachs, T. (1970). Israel J . Bot. 19,484498. Sachs, T. (1972a). Ann. Bot. 36,189-197. Sachs, T. (1972b). Israel J . Bot. 21,9&98. Sachs, T. (1972~).J . theor. B i d . 37,353-361. Sachs, T. (1974). In Proc. 8th Int. Conf. Plant Growth Substances, pp. 9W906. Hirokawa, Tokyo. Sachs, T. (1975a). Ann. Bot. 39, 197-204. Sachs, T. (1975b). Pluntu 127,201-206. Sachs, T. (1975~).J . theor. Biol. 55,445453. Sachs, T. (1978a). Diflerentiation 11,65-73. Sachs, T. (1978b). Symp. Soc. Devl. Biol. 36, 161-183. Sachs, T. (1979). Ann. Bot. 43,693-700. Sax, K. and Dickson, A. Q. (1956). J . Arnold Arbor. 37, 173-179. Sheldrake, A. R . (1971). J . exp. Bot. 22, 735-740. Sheldrake, A. R. (1973a). Biol. Rev. 48,509-559. Sheldrake, A. R . (1973b). J . exp. Bot. 24, 87-96. Sheldrake, A. R . (1974). New Phytol. 73, 637-642. Sheldrake, A. R. and Northcote, D. G . (1968). New Phytol. 67, 1-13. Shimomura, T. and Fujihara, K . (1978). PI. Cell Physiol. 19, 877-886. Shininger, T. L. (1970). Am. J . Bot. 57, 769-781. Shininger, T. L. (1971). PI. Physiol. 47,417422. Shininger, T. L. (1979). A . Rev. Pl. Physiol. 30,313-337. Siebers, A. M. (1971a). Acta bot. neerl. 20,211-220. Siebers, A. M. (1971b). Acta bot. neerl. 20, 343-355. Siebers, A. M. and Ladage, C. A. (1973). Acta bot. neerl. 22,41&432. Simon, S . (1908a). Ber. dt. bot. Ges. 26 (festsch.) 364-396. Simon, S. (1908b). Jb. Wiss.Bot. 45, 351478. Simon, S. V. (1929). Jb. Wiss. Bot. 70,368-388. Simon, S. V. (1930). Jb. Wiss.Bot. 72, 137-160. Sinnott, E. W. (1960). “Plant Morphogenesis”. McGraw-Hill, New York. Sinnott, E. W. and Bloch, R. (1943). Bot. Gaz. 105, 9&92. Sinnott, E. W. and Bloch, R. (1945). A m . J . Bot. 32, 151-156. Snow, M. and Snow, R . (1947). New Phytol. 46,5-19. Snow, R. (1933). New Phytol. 32,288-296. Snow, R. (1935). New Phytol. 34,347-360. Snow, R. (1942). New Phytol. 41, 101-107. Soding, H. (1937). Jb. Wiss. Bot. 84,639470. Sorokin, H. P. and Thimann, K. V. (1964). Protoplasma 59,326350. Spemann, H. (1938). “Embryonic Development and Induction”. Yale University Press, New Haven. Spence, J . A., Soff, R . W. and Humphries, E. C. (1972). Planta 104,352-356. Stant, M. Y. (1963). Ann. Bot. 27, 185-196. Studhalter, R. A., Clock, W. S. and Agerter, S. R. (1963). Bot. Rev. 29,243-365. Tepper, H. B. and Hollis, C. A. (1967). Science N . Y . 156, 1635-1636. Thair, B. W. and Steeves, T. A. (1976). Can. J . Bot. 54, 361-373. Thimann, K . V. (1977). “Hormone Action in the Whole Life ofPlants”. University of Massachusetts Press, Amherst. Thimann, K . V. and Skoog, F. (1934). Proc. R . SOC.B114,317-339.

PATTERNED DIFFERENTIATION OF VASCULAR TISSUES

26 1

Thoday, D. (1961). Proc. R. Soc. B155, 1-25. Thompson, N. P. (1967). Am. J . Bot. 54, 588-595. Thompson, N. P. (1970). Am. J . Bot. 57,39&393. Thompson, N . P. and Jacobs, W. P. (1966). PI. Physiol. 41,673-682. Timmel, H. (1927). Flora, Jena 122,203-241. Tomlinson, P. B. and Zimmermann, M. H. (1968). J . Arnold Arbor. 49,307-316. Torrey, J. G. (1957). Am. J . Bor. 44, 859-870. Torrey, J. G. (1963). Symp. Soc. e x p . Biol. 17,285-314. Torrey, J . G. (1965). Handb. PJPhysiol. 15 (l), 1257-1327. Torrey, J. G. (1966). Adv. Morphogenesis 5,39-91. Torrey, J. G. and Loomis, R. S. (1967). Am. J. Bot. 54, 1098-1106. Torrey, J. G., Fosket, D. E. and Hepler, P. K. (1971). Am. Scient. 59, 338-352. Tsivion, Y. (1978). Israel J . Bot. 27, 103-121. Van Staden, J . (1979). Bot. Gaz. 140, 138-141. Vochting, H. (1878). “Uber Organbildung im Pflanzenreich”. Erster Teil. Max Cohen und Sohn, Bonn. Vochting, H. (1892). “Uber Transplantation am Pflanzenkorper”. Verlag H . Laupp’schen Buchhandlung, Tubingen. Vochting, H. (1906). Bot. Ztg. 64 (Erste abt.), 101-148. Vochting, H. (1918). Untersuchungen zur experimentallen Anatomie und Pathologie des Pflanzenkorpers. I1 Die Polaritat der Gewasche. Verlag H. Laupp’schen Buchhandlung, Tubingen. Von Kaan Albest, A. (1934). Z . Bot. 27. 1-92. Waddington, C. H . (1962). “New Patterns in Genetics and Development”. Columbia University Press, New York. Waisel, Y. and Fahn, A. (1965). New Phytol. 64,43&442. Waisel, Y., Noah, I. and Fahn, A. (1966). New Phytol. 65,319-324. Wang, T. L. and Wareing, P. F. (1979). New Phytol. 82, 19-28. Wangermann, E. (1967). New Phytol. 66,747-754. Wangermann, E. (1974). New Phytol. 73,623-636. Wangermann, E. (1977). New Phytol. 79, 501-504. Wardlaw, C. W. (1946). Ann. Bot. 10,97-107. Wardlaw, C. W. (1950). Ann. Bot. 14,435455. Wardlaw, C. W. (1956). Ann. Bot. 20, 121-132. Wardlaw, C. W. (1968). “Morphogenesis in Plants”. Methuen, London. Wareing, P. F. (1951). Physiologia PI. 4, 546562. Wareing, P. F. (1958). Nature, Lond. 181, 1744-1745. Wareing, P. F. and Roberts, D. L. (1956). New Phytol. 55, 356366. Wareing, P. F., Hanney, C. E. A. and Digby, J. (1964). In “The Formation of Wood in Forest Trees” (M. H. Zimmermann, Ed.), pp. 323-344. Academic Press, New York and London. Waring, R. H., Gholz, H. L., Grier, C. C. and Plummer, M. L. (1976). Can. J . Bot. 55, 14741477. Warmke, H. E. and Warmke, G . L. (1950). Am. J . Bot. 37,272-280. Warren Wilson, J. (1978). Proc. R . Soc. B203, 153-176. Warren Wilson, J. and Warren Wilson, P. M . (1961a). New Phytol. 60,63-73. Warren Wilson, P. M. and Warren Wilson, J. (1961b) Ann. Bot. 25, 104115. Went, F. W . (1941). Bot. Gaz. 103, 386390. Went, F. W. and Thimann, K. V. (1937). “Phytohormones”. MacMillan, New York. Wetmore, R. H. and Rier, J. P. (1963). Am. J . Bot. 50,418430. Wetmore, R. H. and Sorokin, S. (1955). J . Arnold Arbor. 36,305-324.

262

TSVI SACHS

Wetmore, R. H.. De Maggio, A. E. and Rier, J. P. (1964). Phytomorphology 14, 203-2 17. Wilcox. H. (1962). I n “Tree Growth” (T. T. Kozlowski, Ed.), pp. 57-88. Ronald Press, New York. Wilson, B. F., Wodzicki, T. J . and Zahner, R . (1966). Forest Sci. 12,438440. Wodzicki. T. J . (1961). Acta Soc. Bot. Pot. 30,293-306. Wodzicki, T. J . and Wodzicki, A. 8 . (1980). Physio!ogia PI. 48, 443447. Wolpert. L. (1971). Curr. Topics Devl. B i d . 6,183-224. Wooley, D. J . and Wareing, P. F. (1972). Plunta 105,3342. Young, B. s. (1954). New Phytol. 53,445460. Zasada, J . and Zahner, R. (1969). Can. J . Bot. 47, 1965-1971. Zirnrnerrnann. M. H. (1976) In “Transfer and Transport Processes in Plants” (I. F. Wardlaw and J . B. Passioura, Eds), pp. 221-235. Academic Press, London and New York.

AUTHOR INDEX The numbers in italics indicate the pages on which names are mentioned in the reference lists

A

Adam, G., 132,148 Adams, D., 129,147 Addicott, F. T., 246,255 Agatep, A. O., 42,127,141 Agerter, S. R.,159,164,165,177,211,

216,217,238,260

Basha, S. M. M., 6,7,28 Baumgartner, B., 17,21,28 Baussiimer, R.,23,30 Beachy, R.N., 24,28 Bearder, J. R.,69,88,94,95,96,99,122,

123, 124,125,126,141,142

Beeley, L. J., 62,132,133, 137,138,148 Beevers, L.,6,7,9,28,30,131, 142 Benayoun, J., 160, 177,213,214, 215,

Alexandrov, W. G., 212,255 Alexandrova, 0. G., 212,255 216,240,255 Aloni, R.,152,163,188,192,213,214, Bennici, A., 24,28 215,216,231,232,233,241,242,255 Ben Sasson, R.,244,245 Alves, L.M., 43,81,82,141 Bergfeld, R.,11, 28 Amer, M., 160, 255 Bernstein, Z., 164,238,255 Amobi, C. C., 177,255 Bewley, J. D., 3,21,24,28 Anderson, J. D., 85, 141 Binks, R.,41,69,142 Aoki, H., 51, 143 Black, M., 3,21,24,28 Aoyama, T., 43,146 Blackman, E., 193,255 Arpino, P. J., 78,141 Bliss, F. A,, 24,29 Arsenault, G. P.,58, 94,96,146,147 Blobel, G., 8,28 Aurich, O., 23,30,1 1 1, 115, 148 Bloch, R.,187,188,199,205,255,260 Avery, G. S., 169,197,255 Bollini, R.,7,21,28 Aymard, M., 222,255 Bombaugh, K . J., 59,142 Bonnemain, J. L., 169,213,231,255,256 Bonnett, H. T.,203,255 B Bonnier, G., 158,256 Boodle, L.A., 159,256 Baba, S., 205,259 Borgmann, E., 132,148 Baeyer, A,, 124,141 Borrow, A., 35,142 Bailey, C. J., 4,7,16,26,28 Boulter,D.,1,2,4,5,6,7,8,10,11,16,17, Bailiss, K. W., 62,141 18,20,21,22,23,24,25,26,28,29,30, Bain, J. M., 10,11, 16,28 31 Baker, L.R.,138,148 Bourbouloux, A,, 169,256 Baldev, B., 42,127,141 Bowen, D.H., 43,67,69,90,96. 1 15, 120, Balfour, E. E.,158,259 127,142,143,144 Bannan, M.W., 218,223,255 Bowen, M.R.,128,144 Barlow, S. A., 85,142 Bower, F. O., 232,256 Barr, A. J., 185,259 Bowles, D.J., 9,30 Barton, K.A,, 24,28 Bown, A. W., 120,121,142

264

AUTHOR INDEX

Bradford, D. C., 78, 146 Bradley, M. V., 233, 234. 240, 256 Brant, J. A., 153, 256 Braun, H. J., 192, 207, 217,219, 223,256 Breidenbach, R. W., 115, 139, 146, 148 Brenneman, T., 185, 213, 257 Brewster, V., 24, 29 Brian, P. W., 35. 64, 132, 138, 139, 142 Briarty, L. G., 10, 16, 17, 28 Briggs, D. E., 88, 110, 128, 146 Britton, G., 110. 142 Brown, A. B., 159, 173, 188, 191, 256 Brown, C. L., 219, 256 Brown, N. A.. 167, 233, 258 Brown, P., 23. 29 Brown, S. A.. 110, 142 Browning, G., 46, 101, 142, 14.5 Brownrigg, A.. 18, 30 Buchbinder, B. U., 24, 29 Buggy, M. J., 110, 142 Bunning, E.. 188, 222. 225, 245, 256 Burkholder. P. R., 169, 255 Burrows, W. J., 128, 147 Burton, K., 19, 28 Butterfield. B. G., 210,21 I , 218,222,223, 232, 259 Buttrose, M. S., 10, 30

Cobb, A,, 7. 16, 28 Cockburn, B. J., 129, 142 Cohen, M . H., 209, 257, 259 Connelly, S. A., 73, 143 Conway, H. F., 35, 148 Cooke, N. H. C., 57, 142 Cooke, R. J., 131, 142 Coolbaugh, R. C., 85, 90, 110, 127, 142, 146

Coster, C., 159, 256 Coult, D. A., 10. 16, 17, 28 Craig, S., 8, 10, 1 I, 28 Crane, J. C., 233, 234, 240, 256 Crighton, H. B., 169, 255 Crocomo, 0. J., 2, 28 Crooks, D. M., 159, 256 Cross, B. E., 35, 90, 92, 94, 95, 122, 123, 142

Croy, R. R. D., 5,6,7,8, 10, 17,22,23,25, 29.30

Crozier, A., 33, 42, 43, 44, 45, 46, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 59, 62, 66, 67, 72, 73, 80, 81, 83, 84, 86, 106, 108, 115, 116, 117, 118, 119, 120, 121, 128, 132, 133, 142. 143, 146, 147, 148 Cullis, C. A., 3, 24, 29 Cumbie, B. G., 218, 244, 256 Curtis, P. J., 35, 142 Cutter, E. G., 152, 164, 247, 256

C

Camus, G., 164, 167, 171, 256 Carlier, A. R., 18, 30 Carmi, A.. 225, 226, 228, 2.56 Carr. D. J., 128, 130, 142, 147, 148 Caruso, J. L., 164, 256 Casey, R., 6, 7, 20, 24, 28, 29 Casperson, V. G., 177, 256 Castan, R., 188, 256 Cataldo, D. A., 185, 213, 257 Catesson, A. M.. 232, 2.56 Cathey, H. M . , 86, 142 Cavell, B. D., 68, 142 Chaffey. M. B.. 69, 94. 141 Cheadle, V. I . , 218, 223, 256 Chester, V. E.. 35, 142 Chowdhury, K. A,, 177, 256 Chrispeels, M . J., 6, 7,9, 17, 21,28,29, 31 Cionnini, P. G., 24, 28 Cleland, R., 210, 259 Clements, J. B., 130, 148 Clutter, M. E., 167, 171, 256

D Dalessandro, G., 174, 256 D’Amato, F., 24, 28 Davey, R. A., 6, 7, 29 Davidson, E., 179, 25Y Davies, D. R., 20, 24, 25, 26, 29, 30 Davies, H. M., 9, 29 Davis, J. D., 159, 178, 232, 256, 257 Davis, 0 . J., 18, 28 De Duve, C., 16, 29 Deinege, U., 158, 256 Delmer, D. P., 9, 29 De Maggio, A. E., 233,234,235,256,262 Dennis, D. T., 85, 88, 1 10, 129, 143, 144, 148

Derbyshire, E., 4, 5, 6, 7, 25, 28, 29 De Sloover, J., 158, 180, 256 Dickson, A. Q., 188, 260 Dieckert, J . W., 11, 29 Dieckert, M . C., 11, 29

265

AUTHOR INDEX

Digby, J., 169, 171, 174, 233, 234, 256, 26 I Di Rienzo, J. M., 8, 29 Dobberstein, B., 8, 28 Dobbins, D. R., 159, 256 Dockerill, B., 122, 126, 127, 143 Domoney, C., 20, 29 Douglas, T. J., 86, 143 Drage, D., 209, 259 Dudman, W. F., 6. 7, 20, 29, 30 Diinges, W., 73, 143 Dure, L. S . , 4, 29 Durley, R. C., 43, 48, 50, 51, 52, 59, 62, 68, 94,96, 100, 103, 106, 107, 1 1 1 , 113, 132, 133, 137, 138, 143, 145, 146, 147 Durst, D., 73, 143 Dyer, T. A,, 17, 29 E Earle, E. D., 167, 256 Eaton-Mordas, C. A., 6, 7, 29 Ecklund, P. R., 85, 109, 142, I43 Edelman, J., 234, 257 Efron, D., 18, 30 Elbein, A. D., 9, 10, 29 Ellis, R. J., 17, 20, 28 Engelman, M. E., 214, 256 Ericson, M. C., 6, 29 Esau, K., 152, 158, 165, 180, 184, 192, 193, 210, 218, 223, 231, 232, 237, 244, 256 Eschrich, K., 198, 213, 232, 256 Evans, A., 130, 131, 143 Evans, B., 122, 143 Evans, 1. M., 5, 6. 8, 22, 23, 29 Evans, R., 85, 90, 91, 92, 94, 95, 143 Evert, R. F., 159, 178,218,223,232,256 F Fahn, A,, 152, 159, 164, 169, 172, 173, 178, 184, 189, 210, 225, 226, 228, 232, 234, 237, 238, 244, 245, 248, 255, 256, 257, 258, 261 Fall, R. R., 86, 143 Farrar, J. L., 174, 257 Fayle, D. C. F., 174, 257 Feldman, L. J., 232, 234, 257 Fennell, D. I., 35, 148

Fletcher, R. A., 231, 247, 259 Flory, R., 185, 258 Foltz, R. L., 78, 145 Forest, J. C., 234, 257 Fosket, D. E., 153, 174, 179, 202, 205, 233, 257, 259, 261 Fourcroy, M., 217, 257 Frankland, B., 64, 143 Frost, R. G., 86, 143 Frydman, V. M., 95, 99, 100, 108, 142, 143, 248, 257 Fuchs, S . , 68, 143 Fuchs, Y . , 68, 143 Fujihara, K., 215, 243, 260 Fukinbara, T., 51, 145 Fukuda, H., 176, 257 Fukuda, Y . , 217, 257 Fukui, H . , 62, 132, 143 Fukushima, D., 7, 2Y Furuya, M., 42, 149 G Gagnon, J., 8, 30 Gaines, D. E., 78, 146 Galavazi, G., 159, 234, 257 Galt, R. H. B., 40,90,94,95, 134,142,144 Ganguly, S . N., 43, 143 Gardiner, M., 9, 2Y Gaskin, P., 43, 71, 88, 90, 94, 95, 96, 98, 99, 100, 1 1 1 , 112, 115, 121, 127, 142, 143, 144, 146, 148 Gatehouse, J. A., 5, 6, 7, 8, 25, 29, 30 Gautheret, R. J., 174, 183, 188, 234, 237, 25 7 Gersani, M., 185, 214, 247, 248, 257 Gholz, H. L., 211, 261 Gibson, A. C., 222, 257 Giddings, J. C., 51, 143 Gilbert, M. T., 71, 147 Gilroy, J., 6, 25, 29 Glenn, J. L., 48, 50, 143 Glock, W. S., 159, 164, 165, 177,211,216, 217, 238, 260 Goebell, K., 195, 257 Goldsmith, M. H. M., 169, 185, 188,206, 209, 213, 257 Gollnow, B. I., 169, 217, 246, 248, 257 Goodchild, D. J., 8, 10, 11, 28 Goodwin, P. B., 44, 145, 165, 169, 171, 209, 217, 246, 248, 257

266

AUTHOR INDEX

Goodwin, T. W., 110, 129, 142, 148 Gordon, M . E., 21, 22, 29 Gouwentak, C. A., 243, 257 Gower, J. L., 78, I46 Grabner, R., 49, 143 Graebe, J. E., 34,41,42,44,69,85,88,90, 92, 96, 98, 99, 100, 121, 126, 127, 131, 142, 143, 144, 147, 148 Graham, T. A,, 8, 29 Grant, G., 10, 17, 30 Gregory, R. A., 225, 257 Grier, C. C., 21 1, 261 Groner, Y . , 18, 30 Grove, J. F., 35, 106, 132, 138, 139, 142, 144 Grushka, E., 73, 143 Guiochon, G., 78, 141 Gulline, H. F., 164, 257 Gunning, B. E . S., 8 , 29

Hepler, P. K., 153, 179, 202, 205, 261 Herbst, D., 173, 257 Herman, F. M . , 17, 31 Hertel, R., 185, 258, 259 Hess, R., 160, 177, 233, 238, 239, 240, 242, 258 Hidy, B., 78, 145 Higgins, T. S. V., 8 , 29, 44, 145 Hill, T. A., 62, 141 Hiraga, K., 11 1, 144 Hirano, S. S., 90, 142 Hoad, G. V., 62, 128, 132, 133, 137, 138, 144, 145, 148 HO11, W., 227, 258 Hollis, C. A., 169, 173, 207, 258, 260 Homes, J. L. A., 163, 213, 258 Horgan, R., 43, 145 Houck, D. F., 171, 233, 258 Humphries, E . C., 232, 260 Hurkmann, N. J., 16, 29 Hutchinson, P., 22, 29

H Halevy, A. H., 86, 144 Hall, T. C., 24, 29 Hamner, K . C., 167, 233, 258 Hampp, R., 129, 148 Hanney, C. E. A,, 174, 233, 234, 261 Hanson, A. D., 234, 257 Hanson, J. R., 85, 90,91, 92, 94, 95, 106, 122, 123, 126, 127, 142, 143, 144 Harada, A., 43, 144 Hardham, A. R., 11, 28 Harris, N., 10, 11, 16, 29 Harrison, D. M., 40, 134, 144 Harrison, M. A,, 233, 257 Harvey, B. R. M., 128, 148 Harvey, W. E., 95, 142 Hatton, I. K., 95, 142 Hawker, J., 90, 122, 144 Hayashi, T., 50, 149 Heald, J. K., 43, 145 Hedden, P., 34, 42, 71, 85, 86, 87, 88, 90, 91,92,94,95,96, 98,99, 108, 121, 126, 131, 138, 144 Heftman, E., 95, 144, 148 Hejnowicz, Z., 179, 218, 219, 257 Helm, J., 160, 257 Hemming, H . G., 35, 64, 132, 142 Hendrick, C. A., 86, 95, 145 Hendricks, S. B., 3, 31 Henehan, C., 35, 142

I Iinuma, H., 128, 149 Iitika, Y . , 41, 149 Ikegawa, N., 68, 145 Ingram, T. J., 101, 145 Inouye, M., 8 , 28 Irinchijima, S., 43, 146 Ishii, H., 62, 132, 143

J Jackson, P., 5, 6, 29 Jackson, R. J., 18, 30 Jackson, R. W . , 35, 148 Jacobs, W. P., 158, 163, 165, 169, 171, 188, 217, 231, 232, 233, 241, 255, 258, 261 Jacobsen, J. V., 44, 145 Jacquiot, C., 159, 258 Jamieson, J. D., 8, 9, 29 Janse, J. M . , 160, 170, 171, 173, 183, 189, 191, 227, 228, 232, 237, 258 Jarvis, N., 24, 28 Jefferies, E . G., 35, 142 Jefferies, P. R., 86, 95, 145 Jeffs, R. A,, 235, 258

267

AUTHOR INDEX

Jones, D. F., 111, 145 Jones, M. G., 57, 58, 14.5 Jones, 0. P., 128, 145 Jones, R. L., 34, 46, 64, 73, 94, 106, 108, 109, 119, 128, 130, 131, 138, 139, 145,

Kurogochi, S., 43, 145 Kurosawa, E., 34, 35, 145

L

146, 148

Jost, L., 160, 163, 164, 165, 171, 177, 178, 183, 232, 258 Juliano, B. O., 16, 29

K Kadir, G. O., 185, 259 Kagawa, T., 51, 68, 145 Kamienska, A., 43, I45 Karger, B. L., 53, 78, I45 Karn, J., 185, 213, 257 Kato, J., 43, 146 Katsumi, M., 62, 86, 95, 132, 139, 140, 143, 14.5, 149

Kawabe, S., 111, 144 Kawarada, A., 35, 43, 145, 148 Keeble, F., 248, 258 Kemp, J. D., 24, 29 Kende, H., 108, 109, 145, 146 Kendrick, R. E., 131, 142 Khan, Md. R. I., 6, 29 Kidawai, P., 179, 259 Kirby, D. P., 78, 145 Kirschner, H., 169, 172, 173, 178, 189, 204, 205, 247, 248, 258 Kitamura, H., 35, 148 Kitka, E. J., 73, 143 Klein, R. M., 233, 257 Knapp, D. R., 73, 145 Knofel, H. D., 48, 145 Kohlenbach, H. W., 174, 258 Kohler, D., 64, 108, 109, 145 Komamine, A., 176, 257 Koshimizu, K., 62, 132, 143 Koshiyama, I., 7, 29 Kozlowski, T. T., 178, 257 Kraus, E. J., 167, 233, 258 Kriedmann, P. E., 246, 249, 258 Krishen, A., 49, 145 Krueger, S., 73, I45 Kiihne, T., 11, 28 Kunning, H., 179, 258 Kuo, C. C., 42, 43, 48, 50, 62, 132, 143, 146

Kuroda, K., 183, 258

Labern, M. V., 174, 234, 258 Lacey, H. J., 128, 145 Ladage, C. A., 179, 260 Lam, S. L., 185, 258 La Motte, C. E., 163, 165, 171, 231, 232, 233, 258 Lang, A., 42, 64, 86, 95, 106, 108, 109, 127, 141, 145, 148, 183, 258 Langford, C. T., 35, I48 Larkins, B. A , , 16, 29 Larson, P. R., 158, 180, 238, 244, 258 Lehle, L., 9, 30 Leigh, B. A., 17, 30 Lennarz, W. J., 9, 31 Leopold, A. C . , 90, 145, 185, 246, 249, 258

Lenten, N., 193, 258 Letham, D. S., 44,145, 169,217,246,248, 257, 258

Lew, F. T., 88, 145 Liebisch, H. W., 132, 148 Linnemann, G., 225, 258 Lips, S. H., 185, 214, 247, 248, 257 Lishchewski, M., 132, 148 Lloyd, P. B., 35, 142 Locke, D. C., 57, I45 Lockhart, J. A,, 86, 106, 145 Lonsdale, D., 18, 30 Loomis, R. A,, 165, 261 Lorenzi, R., 43, 145 Lott, J. N. A., 10, 30 Loveys, B. R., 130, 131, 142, 145, 146 Lowe, D., 64, 132, 142 Luckwill, L. C . , 64, 128, 146, 149

M MacDonald, F. R., 52, 146 MacMillan, J., 34, 35, 40, 41, 43, 51, 59, 67, 68, 69, 71, 85, 88,90,91,92,94,95, 96,98,99, 100, 101, 103, 104, 108, 11 1, 112, 113, 115, 120, 121, 122, 123, 124, 125, 127, 131, 134, 141, 142, 143, 144, 146, 148

Madison, J. T., 24, 28

268

AUTHOR INDEX

Majors, R. E., 52, 57, I46 Malins, M. E., 238, 259 Manteuffel, R., 23, 30 Marcus, A., 18, 30 Marks, G. E., 24, 30 Marrow, 1. B., 158, 169, 217, 241, 258 Matile, P., 11. 16, 17, 30 Matta, N., 5, 30 McCully, M. E., 163, 171, 234, 257, 259 McFadden, W. H., 78, 146 McInnes, A. G., 94, 96, 146 McIntosh, R., 25, 29 McLaughlin, G. E., 51, 52, 59, 106, 143 Meinhardt, H., 176, 187, 199, 250, 258 Mense, R. M., 9, 30 Mercer, F. V., 10, 11, 16, 28 Metzger, J. D., 43, 57, 58, 145, 146 Meyer, J., 164, 249, 258 Miege, M., 10, 30 Miersch, O., 132, 148 Milano, M., 73, 143 Millerd, A., 4, 6, 8, 10, 20, 23, 24, 28, 29, 30, 31 Milton, J. M., 5, 6, 29 Mishra, D., 86, 146 Mitchison, G. J., 182, 198, 199, 222, 258 Mitsui, T., 62, 132, I43 Moffatt, J. S., 35, 142 Molliard, M., 234, 258 Molnar, J., 8, 30 Moore, R. G., 6, 7, 29 Moore, T. C., 85, 109, 110, 127, I41,142,

Murphy, P. J., 90, I46 Murray, C. D., 212, 259 Musgrave, A., 108, 146

N Nadeau, R., 115, 138, 139, 146, 148 Nagahashi, J., 9, 30 Nagura, N., 95. 146 Nakamura, K., 8, 29 Nakano, H., 124, 146 Nash, L. J., 52, 53, 56, 57, 73, 115, 116, 118, 119, 120, 139, 146, 147 Needham, J., 170, 259 Neeff, F., 159, 170, 178, 184, 189, 191, 219, 259 Neeley, P. M., 35, 147 Nelson, M. G . , 248, 258 Neumann, D., 10, 11, 30 Neville, P., 160, 255 Nicholls, P. B., 64, I46 Nickell, L. G., 163, 259 Niedergang-Kamien, E., 191, 259 Nierlich, D. P., 20, 30 Nitsch, J. P., 43, 144 Nixon, I. S., 35, 142 Noah, I., 234 Norris, G. L. F., 35, 142 Northcote,D. G., 178,218,219,235,258, 260

Norton, K., 90, 92, 94, 95, 122, 123, 142

143, 146

Morre, D. J., 16, 30 Morris, D. A., 185, 259 Morris, G. F. I., 8, 30 Morris, R. O., 49, 73, 74, 143, 146 Morris, S. C., 248, 257 Mosettig, E., 95, 148 Mosse, B., 174, 234, 258 Most, B. H., 115, 120, I43 Mulholland, T. P. C., 35, 132, 138, 139, 142

Miiller, P., 48, 145 Mullins, M. G., 185, 259 Munch, E., 160, 171, 177, 238, 247, 259 Miintz, K., 7, 23, 30 Murakami, Y . , 64, 119, 137, 146 Murofushi, N., 43, 48, 62, 95, 111, 113, 114, 132, 138, 139, 140, 144, 145, 146, 147, I49 Murphy, G. J. P., 88, 110, 128, I46

0 O’Brien, J. P., 202, 259 Ogawa, Y . , 48, 62, 132, 137, 143, 146 Olsen, K., 57, 142 O’Malley, B. W., 2, 30 Opik, H., 7, 16, 30 Osada, H., 111, 113, 114, 149 Osborne, D., 185, 259 Oster, M. O., 85, 146 Ota, Y . , 43, 145 Ouitrakull, R., 185, 259 P Paleg, L. G., 64, 86, 128, 143, 146 Palmiter, R. D., 8, 30

269

AUTHOR INDEX

Parnas, H., 209, 259 Parthier, B., 23, 30 Patrick, J. W., 214, 247, 259 Patterson, R. J., 115, 146 Paul, C., 25, 29 Payne, E. S . , 18, 30 Payne, P. I., 7, 17, 20, 21, 22, 29, 30 Pearson, J. A., 131, 142 Pelham, H. R. B., 18, 30 Pellegrini, O., 160, 259 Pernollet, J. C . , 16, 30 Peterson, C. A., 231, 247, 259 Peterson, R. L., 171, 259 Peumans, W. J., 18, 30 Pharis, R. P., 42,43,48,50,51,52,59,62, 68, 94, 96, 100, 103, 106, 107, 132, 133, 137, 138, 143, 145, 146, 147 Philipson, W. R., 158,210, 21 1,218,222, 223, 232, 259 Phillips, I. D. J., 128, 129, 145, 146, 247, 259 Phinney, B. O., 34, 35, 51, 64, 69, 85, 86, 87, 88, 91,92, 94, 95, 96, 99, 108, 111, 122, 123, 124, 125, 131, 138, 141, 142, 144, 145, 147, 148, 149 Pitel, D. W., 58, 96, 147 Plummer, M. L., 211, 261 Polacco, J. C . , 26, 30 Powell, L. E., 51, 52, 53, 54, 58, 62, 119, 147 Pradham, G. C., 86, 146 Prat, H., 242, 259 Priestly, J. H., 159, 189, 238, 259 Pryce, R. J . , 41, 50, 68, 69, 92, 111, 113, 142, 143, 146, 147 Pryde, A., 71, 147 Piichel, M., 23, 30 Pulawska, J., 158, 259 Pusztai, A., 6, 10, 17, 30 Pyne, J. W., 24, 29

Railton, I. D., 34, 46, 62, 68, 106, 107, 108, 109, 110, 111, 120, 132, 133, 137, 138, 143, 145, 147 Raper, K . B., 35, 148 Rappaport, L., 115, 129, 138, 139, 146, 147, 148 Rayle, D. L., 185, 210, 259 Rechov, M., 46, 147 Reeve, D. R., 42,44,45,46,48,50,52,53, 54, 55, 56, 57, 62, 66, 72, 73, 80, 81, 83, 84,86,108,115,116, 118,119,120,121, 132, 133, 142, 146, 147 Rehm, S., 188, 191, 211, 259 Reid, D. M., 42, 43, 46, 67, 86, 109, 11 1, 115, 117, 120, 128, 130, 142, 143, 147, 148 Reinders-Gouwentak, C. A., 159, 165, 259 Reinhold, L., 206, 247, 259 Revel, M., 18, 30 Richmond, A,, 128, 148 Rier, J. P., 164, 167, 171, 172, 234, 235, 261, 262 Ritzel, M. B., 35, 147 Robards, A. W., 179, 259 Robbertse, P. J., 163, 171, 259 Roberts, D. L., 160, 177, 238, 261 Roberts, L. W., 153, 167, 171, 174, 179, 201, 202, 205, 233, 234, 243, 257, 259 Robertson, A., 209, 259 Robinson, D. R., 42, 85, 148 Rogers, L. J., 129, 148 Romberger, J. A., 225, 257 Ropers, H. J., 34,41,42,44,88,90,92,99, 100, 127, 131, 144, 148 Rothe, G., 188, 259 Ruddat, M., 43, 81, 82, 95, 141, 148 Riidiger, W., 183, 232, 259 Rudich, J., 138, 148 Ruge, U., 203, 259 Rzimann, G., 227, 259

Q S Quail, H., 10, 16, 17, 30

R Rademacher, W., 42, 98, 121, 126, 144, I47 Radley, M., 35, 128, 142, 147

Sabatini, D., 8, 28 Sachs, T., 151, 152, 153, 159, 160, 163, 164, 167, 169, 171, 172, 173, 174, 177, 178, 182, 184, 185, 186, 189, 191, 192, 193, 195, 196, 197, 198, 201, 203, 204, 205, 206, 213, 214, 215, 216, 217, 220, 224, 225, 226, 228, 231, 233, 234, 238,

270

AUTHOR INDEX

Sachs- continued 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 251, 255, 256, 257, 258, 259, 260 Sassa, T., 100, 103, 143 Sato, H., 124, 146 Saunders, P. F., 46, 131, 142, 145 Sawada, K., 35, 148 Sax, K., 188, 219, 256, 260 Scharpe, A., 24, 30 Schmidt, B., 176, 258 Schmidt, P., 23, 30 Schneider, G . , 49, 132, 143, 148 Schopfer, P., 11, 28 Schreiber, K., 43, 111, 115, 132, 148 Schreurs, J., 18, 30 Schroeder, H. E., 20, 25, 31 Schwartz, R. J., 2, 30 Scott, L. I., 238, 259 Seaton, J. C., 35, 142 Segel, L. A., 209, 259 Sell, H. M., 138, 148 Sembdner, G . , 43, 48, 49, 1 11, 115, 132, 143, 145, 148 Seta, Y., 35, 148 Shah, S. P. J., 129, 148 Shechter, I., 42, 85, 148 Sheldrake, A. R., 169, 178, 185, 186, 188, 192, 218, 219, 220, 260 Sheppard, A. C., 68, 142 Sheppard, N., 35, 142 Shibasaki, K., 6, 31 Shilo, R., 86, 144 Shimomura, T., 215, 243, 260 Shininger, T. L., 177, 233, 240, 243, 260 Siebers, A. M., 179, 234, 237, 260 Silk, W. K., 138, 148 Simcox, P. D., 110, 129, 148 Simon, S. V., 163, 165, 177, 182,213,240, 248, 260 Simpson, T. H., 68, 148 Sinnott, E. W., 188, 199, 205, 219, 260 Sircar, S. M., 43, 143 Sitton D., 128, 148 Skene, K. G. M., 128, 142, 148 Skoog, F., 169, 191, 259, 260 Smith, D. G . , 94, 96, 146 Smith, D. L., 24, 30 Smith, H., 130, 131, 143 Smith, L. L., 124, 148 Smith, M. H., 4, 30 Snow, M., 180, 260

Snow, R., 165, 180, 227, 248, 258, 260 Soding, H., 169, 178, 260 Soff, R. W., 232, 260 Sohns, V. E., 35, 148 Sorokin, H. P., 247, 260 Sorokin, S., 164, 174, 261 Spector, C., 92, 148 Spemann, H., 170, 260 Spence, J. A., 232, 260 Spencer, D., 8, 20, 23, 24, 29, 30 Sponsel, V. M., 62, 71, 85, 99, 100, 101, 103, 104, 112, 115, 124, 125, 126, 127, 132, 133, 137, 138, 141, 144, 148 Stafford, A., 20, 30 Stant, M. Y., 241, 260 Steeves, T. A,, 188, 260 Stewart, J. C., 90, 92, 95, 122, 142 Stiller, M., 20, 30 Stoddart, J. L., 73,90, 111, 130, 138, 139, 142, 146, 148 Stodola, F. H., 35, 148 Stolp, C. F., 138, 146 Stuart, N. W., 86, 142 Studhalter, R. A., 159, 164, 165, 177,211, 216, 217, 238, 260 Sumiki, Y., 35, 42, 51, 68, 145, 148, 149 Sun, S. M., 24, 29 Suter, P. J., 35, 51, 146 Szybalski, W., 3, 30

T Takahashi, N., 35, 41, 42, 43, 48, 62, 95, 111, 113, 114, 132, 139, 140, 144, 145, 146, 148, 149 Takai, M., 35, 148 Takeno, K., 42, 149 Tamoahi, B. H., 124, 146 Tamura, S., 35, 43, 146, 148 Tandan, K. N., 177, 256 Tanner, W., 9, 30 Tautvydas, K. J., 51, 52, 53, 54, 58, 62, 119, 147 Taylorson, R. B., 3, 31 Tepper, H. B., 169, 173, 207, 258, 260 Thair, B. W., 188, 260 Thanh, V. H., 6, 31 Thimann, K. V., 169,179,246,247,249, 260, 261 Thoday, D., 164, 261

27 I

AUTHOR INDEX

Thompson, J. A., 20, 24, 25, 28, 31 Thompson,N. P., 171, 188,232,233,261 Thurman, D. A,, 5, 6, 8, 28, 29, 30 Timmel, H., 163, 207, 233, 261 Timofeev, A. S., 212, 255 Tomaszewski, M., 179, 257 Tomlinson, P. B., 231, 261 Torrey, J. G., 153, 165, 174, 178,179,202, 205, 232, 234, 2.57, 261 Towle, H. C., 2, 30 Trip, P., 185, 213, 257 Tsivion, Y., 249, 261 Tyler, M., 29

U Upper, C. D., 85, 88, 144, 148 V Vaadia, Y., 128, 148 Van Bragt, J., 86, 148 Van der Wilden, W., 17, 31 Van Paris, R., 24, 30 Van Staden, J., 248, 261 Varner, J. E., 64, 94, 119, 130, 131, 145 Villiger, V., 124, 141 Vining, L. C., 58,59,94,96,146,147,148 Vochting, H., 188, 207, 261 Von Kaan Albest, A., 232, 233,261 Vouros, P., 78, 145

Wareing, P. F., 64, 130, 131, 142, 143, 145, 146, 159, 160, 169, 174, 177, 214, 233, 234, 238, 240, 248, 256, 257, 259, 261, 262 Waring, R. H., 211, 261 Warmke, G. L., 188, 261 Warmke, H. E., 188, 261 Warren Wilson, J., 227,228,232,234,261 Warren Wilson, P. M., 227,228,232,261 Watanabe, E., 43, 146, 148 Watt, W. B., 6, 30 Weaver, R. J., 34, 148 Weber, E., 10, 11, 30 Weeks, D. P., 18, 30 Weiland, J., 11 1, 115, 148 Wellburn, A. R., 129, 142, 148 Wels, C. M., 59, 69, 94, 95, 99, 141, 146 Went, F. W., 178, 188,261 West, C. A., 35,42,51,85,86,88,90, 110, 111, 127, 129, 142, 143, 144, 145, 146, 147, 148, 149 Wetmore, R. H., 164, 167, 171, 172, 174, 234, 235, 261, 262 Wetter, L. R., 110, 142 Wheeler, C. T., 8, 24, 31 White, A. F., 90, 91, 92, 124, 143, 144 Whyte, P., 64, 128, 146, 149 Wilcox, H., 165, 178, 211, 262 Wilson, B. F., 210, 262 Wodzicki, A. B., 240, 262 Wodzicki, T. J., 210, 238, 262 Wolpert, L., 153, 222, 250, 262 Wooley, D. J., 248, 262 Wright, D. J., 4, 6, 7, 25, 29 Wiinsche, V., 86, 149

W

I

Wada, Y., 43, 146 Waddington, C. H., 170, 261 Waechter, C. J., 9, 31 Waisel, Y., 234, 238, 261 Walker, P. R., 27, 31 Walker, R., 164, 257 Walsh, K. A., 8, 30 Wang, T. L., 248, 261 Wangermann,E., 160,165,171,185,213, 256, 261 Ward, J. M., 210,211,218,222,223,232, 259 Wardlaw, C. W., 160, 180, 184,232,245, 261

Y Yabuta, T., 35, 50, 149 Yamaguchi, I., 48, 62, 132, 139, 140, 149 Yamane, H., 41, 42, 62, 111, 113, 114, 132, 139, 140, 149 Yamazaki, S., 41, 149 Yarwood, A., 17, 18, 20, 28, 30, 31 Yarwood, J. N., 18, 30, 31 Yokota, N., 62, 116, 132, 139, 140, 149 Yokota, T., 41, 43, 48, 52, 53, 56, 57, 62, 111, 132, 144, 147, 148, 149 Yomo, H., 128, 149 Young, B. S., 160, 165, 243, 262

272

AlJTHOR INDEX

2 Zaerr, J. B., 49, 73, 74, 143, 146 Zahner, R., 177, 210, 262 Zasada, J., 177, 262

Zeevaart, J. A. D., 42,43,57,58,143,145, 146, 149 Zimmermann, M. H., 152, 192,231,261, 262

SUBJECT INDEX A

B

Abscissic acid chromatography, 49 influence on conifer tracheids, 240 N-acetylglucosamine and glycoprotein formation, 9 Adiantum, polarity of leaf veins, 194 Aglaomorpha, polarity of leaf veins, 194 Ailanathus altissima, vascular ray development in relation to cambial growth, 226 pattern of development, 223, 224 Althea rosea, identification of gibberelIins, 43 a-Amylase, induction by gibberellins, 34, 62 Aneimia, polarity of leaf veins, 194 Arachis hypogea, storage protein, 2, 7 Auxin and pattern of vascular differentiation, 174, 175, 179, 199-200 and tissue polarity, 188, 189, 190, 191, 192, 197, 198, 220-221, 252 and vascular differentiation, 158, 161, 162, 184, 233 auxin production in cambium, 220 in differentiating cells, 178 auxin transport canalization, 181-182,183,185-186, 192 induced by auxin, 186 polarity, 156, 167-170, 174-1 76, 181, 188, 192, 206-207, 209-210, 244, 247, 251, 252 control of parenchyma differentiation, 243-244 replacing the effect of leaves in differentiation, 165, 170, 246

Baeyer-Villiger reaction, in gibberellin biosynthesis, 124-125, 126 Barley aleurone bioassay for gibberellins, 62, 63, 64, 65, 67, 94, 119, 131, 132, 133, 134, 135, 136, 138, 139 Bioassays for gibberellins, 60-68, 128-129, 132-140 Bondapak separation of gibberellins, 57-58 Brassica napus occurrence of circular vessels, 208 vascular differentiation induced by auxin, 168, 175 polarity changes, 196 Bryophyllum daigremontianum, identification of gibberellins, 43 C Cajanus cajan, crop protein yield, 2 Callose, staining of phloem, 23 1 Calonyction gladiata, presence of gibberellins, 41 Cambium, developmental processes dynamic aspects of cambium conclusions, 221-222 introduction, 2 18 relations between cambial initials, 218-2 19 responses of cambium to inductive signals, 219-221 quantitative controls of cambial activity conclusions, 215-2 16 introduction, 21 1-213 magnitude of wound effect, 213-215 ray formation control, 223-227

274

SUBJECT INDEX

Cambium continued introduction, 222 pattern of the rays, 222-223 radial limitations of cambial grafts, 227-228 summary, 2 10-2 1 1 Canavalia ensiformis, urease storage, 4 Capsicum ,frutescens, modification of sex expression by gibberellins, 34 Carbohydrate in storage protein of legume seeds, 6-7,

D

~

8-10

transport in wounded plants, 214 Chloroplasts and gibberellin biosynthesis, 110-111, 129 Cicer arietinum and crop protein yield, 2 Citrus retirulata, identification of gibberellins, 43 Cladesporium resinae, Baeyer-Villiger reaction, 124 Colchicine and cytoplasmic reorientation, 205 Coleus

auxins and reversal of polarity, 190, 191 vascular differentiation, 171, 221, 241 C. hlumei, procambium structure, 155 Convicilin in legume seeds, 6, 20, 25 Crown gall, effect on vascular differentiation, 157, 234, 249 Cucumber hypocotyl bioassay for gibberellins, 60, 62, 63, 64, 65, 132, 134, 136, 138, 139, 140 Cucumis sativus, modification of sex expression by gibberellins, 34 Cupressus arizonica, gibberellins identification, 43 modification of sex expression, 34 Curcurbita maxima, gibberellin biosynthesis, 85, 88, 90, 92, 96-99, 121-122, 126, 127 C. pepo, see C. masima Curcurbitaceae modification of sex expression by gibberellins. 34 vascular network formation, 199 Cyrtomium, polarity of leaf veins, 194 Cytokinin production by roots, 248, 254 promotion of vascular differentiation, 171

Dextran gel, separation of gibberellins, 58-59 DNA, in developing legume seeds, 18, 19, 24 Dolichol phosphate, involvement in glycoprotein formation, 9 Dwarf maize leaf sheath bioassay for gibberellins, 64 Dwarf pea bioassay for gibberellins, 62, 63, 64, 65, 132, 134, 135, 136, 138 Dwarf rice bioassay for gibberellins, 67, 132, 134, 135, 136, 137, 138, 139 E Echinocvstis macrocarpa, see Marah macrocarpus

Endoplasmic reticulum, in legume seeds and origin of protein bodies, 11-17 and storage protein synthesis, 8, 9 Etioplast permeability and gibberellins, 129-130, 131 F Feedback control theory of vascular differentiation, 247-249, 250, 254 Fern leaves, polarity of veins, 193, 194, 195 Fibre differentiation effect of gibberellin, 240 in phloem, 240-241 in xylem, 237-240 G Gibberella fujikuroi

effect on growth of rice, 62 gi bberellin biosynthesis, 42, 85, 88-90, 92-96, 99, 122-127 production, 35, 36, 46 Gibberellins analytical methods extraction and partitioning, 46-48 general observations, 44-46 group purification procedures

275

SUBJECT INDEX

ion-exchange, 49-50 reverse-phase, 50 steric-exclusion, 48-49, 50 identification procedures bioassays, 62-68 physicochemical methods, 68-79 radioimmunological assays, 68 separatory techniques high performance liquid chromatography, 52-58 silica gel chromatography, 5 1-52 verification of accuracy, 79-85 and vascular differentiation differentiation of fibres, 240, 241, 242, 254 differentiation of sieve tubes, 233-234, 236 enhancement of cambial activity, 233 production by roots, 248 promotion of differentiation, 17 1, 179, 230, 234 biosynthesis ent-kaurene to GA , 2aldehyde, 88-92 mevalonic acid to mi-kaurene, 85-88 pathways beyond G A , ,aldehyde, 92-121 sites of biosynthesis and compartmentation, 127-132 discovery, 34-35 distribution in tissue, 4 1 4 3 effect on plant growth, 33-34 structure, 35-41 structure-activity relationships, 132140 Glycine mux crop yield, 2 glycinin storage protein, 6, 7 urease-rich mutants, 26 Glycosylation and protein synthesis in legumes, 8-9, 18, 26 Golgi apparatus and formation of protein bodies, 1 I , 16

High performance liquid chromatography of gibberellins detection of derivatives benzyl esters, 72-73 p-bromophenacyl esters, 73, 74 methoxycoumaryl esters, 73, 74. 75, 76. 77, 78 methyl esters, 76 normal phase, 52-57 reverse phase, 57-58 Hordwm distichon, gibberellin biosynthesis, 88, 110 H . vulgure, gibberellin biosynthesis, 110-1 1 I , 128, 129, 130, 131 Hurniilus lupulus, identification of gibberellins. 43 1

lmmunofluorescent light microscopy of protein bodies, 8 Indole acetic acid and vascular differentiation, 161 column chromatography, 51 Isotopically labelled hormones auxin transport, 185-186,247-248,253 gibberellin detection, 54,55, 56,61,69, 71 J Juniperus scopulorum, identification of gibberellins, 43

K eni-kaurene, involvement in gibberellin biosynthesis, 85, 86, 87, 109, 110, 127, 128 1

H Heliunlhus unnuus gibberellin synthesis, 128 vascular differentiation, 157

Leaf development and cambial activity, 177 effect of vascular tissues on leaf initiation, 179-180 effect on vascular differentiation, 158-161,163-170,230,242,246,252

276

SUBJECT INDEX

Leaf-continued influence in ring porous trees, 238,240 polarity of veins, 193, 194 primordia, effect on location of phloem fibres, 240-241 parenchyma formation, 242-243, 254 stem differentiation, 160, 163, 164, 242-243 Legume seeds biology, 3-4 storage proteins convicilin, 6, 20, 25 legumin, 4, 5 , 6, 7, 8, 20, 23, 25 separation techniques, 5 synthesis and deposition biochemistry, 17-27 intracellular sites, 7-8 post-translational modifications, 8-10 protein bodies, 7-8, 10-17 vicilin, 4, 5 , 6, 7, 8, 20, 23, 25, 26 Legumin storage protein deposition, 8 structure, 4, 5, 6, 7, 25 synthesis, 8, 20, 23 Lens culinaris and storage protein yield, 2 Lettuce hypocotyl bioassay for gibberellins, 61, 62, 63, 67, 132, 134, 135, 136, 138, 139 Light effects on gibberellins, 120-121, 130-131 Lupinus spp., storage protein, 6, 7 Lycopersicum esculentum, modification of sex expression by gibberellins, 34 Lygodium japonicum, presence of gibberellin, GA9, 42

M Mallinckrodt CC-4, chromatography of gibberellins, 51-52, 53 Marah macrocarpus, gibberellin biosynthesis, 85, 88, 90, 127, 129 Mass spectrometry of gibberellins, 68-71, 7&78 Melanconiales, gibberellins, 42 Merckogel, chromatographic efficiency, 53 Mevalonic acid and gibberellin biosynthesis, 85, 86, 87,94,96, 109, 110, 127, 129

N

Nicotiana tabacum, gibberellins biosynthesis, 86 identification, 43 Nucleic acids in legume seeds changes during seed development, 18-2 1 control of protein synthesis, 21-24

0 Octadecylsilane, separation of gibberellins, 5 1 Oryza sativa effect of gibberellins on growth, 35 identification of gibberellins, 43

P Parenchyma, control of formation effect of leaf primordia, 242-243, 254 relation between procambium and cambium, 244-245 relative polarity of different parenchyma cells, 243-244 timing of parenchyma formation and growth, 244-245 Partisil, chromatographic efficiency, 53-54 Pharbitis nil, gibberellins chromatography, 57-58 function in seeds, 42 Phaseolus auxin transport, 186 carbohydrate transport in wounded plants, 214 gibberellin extraction, 46 vascular system circular vessels, 208 differentiation, 160, 162, 163, 190, 239, 240 regeneration, 156 structure, 155 P. aureus, see Vigna radiata P. coccineus, gibberellin biosynthesis, 11 1-121 effect of light, 120-121 identification, 35, 41, 43, 62, 66, 67

277

SUBJECT INDEX

P. vulgaris gibberellin biosynthesis, 111-121 storage protein in seeds, 6, 9, 17 Phenol, chromatography, 53 Phloem auxin transport, 213 development and leaf formation, 159, 230, 232-233 differentiation, 165, 171, 172, 173, 230, 233 regeneration and cambial activity, 2 13-2 14 relation between phloem and xylem development, 213-237 Phosphatase in legume protein bodies, 17 Phyllostachys edulis, identification of gibberellins, 43 Phyllotaxis and pattern of vascular tissue, 158 Phytochrome and gibberellins, 130-131 Picea sitchensis, identification of gibberellins, 43 Pinus attenuata, gibberellin extraction, 49-50 identification, 43 Pisum seed morphology, 3 vascular differentiation anatomy of vascular system, 154 differentiation from parenchyma, 166 effect of auxin, 166, 183, 198, 243 influence of leaf primordia, 164, 240-241 interaction with seedling organs, 160 polarity of leaf veins, 194 wound production of cytoplasmic strands, 204, 205 phloem fibres, 240-241 sieve tubes, 233 xylem, 215, 216 P. legurnin, genetics of storage protein formation in seeds, 25 P. sativum gibberellins and growth, 34 biosynthesis, 85, 88, 90, 99-1 11 effect of light, 106, 108-109 extraction, 46, 109 function in seeds, 42 identification, 43, 102, 103, 138 sites of biosynthesis, 127, 129

storage protein in seeds convicilin, 6, 20 legumin, 5, 7, 19, 20 nucleic acid changes during development, 18, 19, 20 protein body formation, 10, 11, 12-13 structure, 5-6 synthesis, 18-20, 22 Polarity of tissue in relation to vascular differentiation, 187-199 Protein bodies in legume seeds formation, 10-16 occurrence, 7-8 proteolysis, 16, 17 Protein synthesis in developing legume seed biochemical mechanism, 17-2 1 control, 21-24 Pseudotsuga menziesii, identification of gibberellins, 43

R Raphanus sativus circular vessels in storage root, 208 effect of wounding on xylem formation, 215 Rhizophera mucranata, identification of gibberellins, 43 Ribes nigrum, identification of gibberellins, 43 Ricinus, vascular tissue in seedling quantitative aspects of xylem, 212 wound formation of cambium, 245 R. communis, gibberellin biosynthesis, 85, 129 Ring porous trees, control of vessel and fibre distribution, 237-240 RNA in legume seeds changes during germination, 18-21 control of storage protein synthesis, 21-24 Roots cytokinin production, 248 effect on vascular differentiation, 170-172, 246 Rumex leaf senescence bioassay for gibberellins, 64

278

SUBJECT INDEX

S

V

Selected ion current monitoring for analysis of gibberellins, 69 Sieve tube differentiation and auxins, 167,168,175,210,233,252 and gibberellins, 233-234, 236 formation of closed rings, 207 polarity, 192, 196 regeneration, 21 3 Silica gel chromatography of hormones, 51 Solanum melongena, modification of sex expression by gibberellins, 34 Sonneratia aperala, gibberellins, 43 Sphaceloma manihoticola, gibberellins, 42 Spinacea oleracea, gibberellins, 43 Steric exclusion chromatography of gibberellins, 48-49 Sfevia rebaudiana, gibberellins, 43,81,82, 83, 95-96 Stomata1 development and vascular differentiation, 197 Sucrose, effect on phloem differentiation, 234-235, 236

Vascular loops, occurrence, 193, 195, 197, 253 Vascular rays control of formation, 21 1, 225-227 control of ray distance and initiation, 223-225 pattern, 225, 254 radial limitations of cambial grafts, 227-228 radial polarity of cambium, 222 Vascular strands control of tissue differentiation, 229-230 “sink effect”, 182-185 Vascular tissue differentiation cambial developmental processes dynamic changes, 218-222 quantitative controls, 21 1-218 ray formation, 222-228 summary, 210-21 1 cell polarization by a flux of signals stability of polarity, 187-192 strand formation, 182-1 87 summary, 180-181 vascular networks, 192-1 99 cellular complexity of the vascular system control of fibre differentiation, 237-242 control of parenchyma formation, 242-245 relation between xylem and phloem, 231-237 summary, 228-230 cellular responses involved in orientation of differentiation determination and differentiation, 200-205 signal flux, 205-210 summary, 199-200 control by flux from leaves to roots additional controls, 176-1 80 induction by leaves and auxin, 158-170 orientation of signal flux by roots, 170-1 72 signal flux, 172-176 summary, 155, 158 problems, 152, 153, 155

T Tanginbozu dwarf rice bioassay for gibberellins, 60, 62, 63, 64, 66, 133, 135 Trifolieae, storage proteins, 5 Triiodobenzoic acid, inhibition of auxin transport, 174 cambial activity, 178, 220 closed vascular ring formation, 207 vessel induction, 191 Triticum aestivum, gibberellins, 46, 131 Turnip, vascular differentiation, 215,233

U

Urease in Canavalia ensiformis, 4 in Glycine max, 26 Urginea maritima, pattern and size of root xylem, 235

279

SUBJECT INDEX

relation to other aspects of plant morphogenesis effects of leaves and auxins, 246-247 hormonal feedback, 247-249 the problem, 246 summary, 251-255 Vascular tissue regeneration cambium, 2 13, 227-228 effect of auxin, 165, 167, 201-203, 205 effect of leaves, 163, 165, 167, 178,233 parenchyma differentiation, 159, 243 polarity, 188 relation between xylem and phloem, 232-237 signal flux, 153, 173-176, 182-183 xylem regeneration, 156, 214-216 Vicia fuba seeds protein bodies formation, 10, 11, 14-15 proteolysis, 16-17 protein synthesis during development DNA, 24 protein changes, 18 RNA, 23 storage protein legumin, 5, 6, 7 vicilin, 5 Vicieae, storage protein, 5, 6 Vicilin, storage protein deposition, 8 structure, 4, 5, 6, 7, 10, 25 synthesis, 8, 20, 23, 26

Vignu radiuta, storage protein, 6, 9, 17 V . unguiculata, storage protein, 2, 6, 10, 11 W

Woelm silica gel chromatography of gibberellins, 51-52

x Xylem development differentiation, 153, 156, 165-167, 172 172 maturation pattern, 173-174 quantitative aspects, 21 1-212,214-216 relation between xylem and phloem, 231-237

Z Zeu polarity of leaf veins, 194 root size and sugar concentration, 234 Z . mays, gibberellin biosvnthesis. 86-88 effec; on growth, 34, 109 ~

I

I

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