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.

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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