Inducible Plant Proteins: Their Biochemistry and Molecular Biology (Society for Experimental Biology Seminar Series)

Plants are able to respond and adapt to changing environmental and endogenous signals by the induction of the synthesis of specific proteins which act to modify cellular metabolism. Environmental signals are diverse and include, for instance, nutrient availability, temperature, light, anaerobiosis, and pathogen attack amongst others, whilst endogenous signals include changes in the level of plant growth regulators. In this text, leading researchers discuss the role that inducible proteins play in cellular metabolism, and the approaches being used to delineate the underlying molecular events which lead to their synthesis. The use of both classical methods, such as protein purification and characterisation, as well as molecular methods, such as the use of antisense RNA to down-regulate the synthesis of specific target enzymes, are considered as approaches to investigate the role particular proteins play in cellular metabolism. Other chapters discuss molecular approaches to the study of gene expression, the identification and characterisation of trans-acting transcription factors and attempts to dissect other parts of the signal transduction pathway by the search for pathway mutants. This timely review volume will be of great value and interest to final year undergraduates, graduate students and researchers in the fields of plant biochemistry and molecular biology.

SOCIETY FOR EXPERIMENTAL BIOLOGY SEMINAR SERIES: 49

INDUCIBLE PLANT PROTEINS: THEIR BIOCHEMISTRY AND MOLECULAR BIOLOGY

SOCIETY FOR EXPERIMENTAL BIOLOGY SEMINAR SERIES A series of multi-author volumes developed from seminars held by the Society for Experimental Biology. Each volume serves not only as an introductory review of a specific topic, but also introduces the reader to experimental evidence to support the theories and principles discussed, and points the way to new research. 2. Effects of pollutants on aquatic organisms. Edited by A.P.M. Lockwood 6. Neurones without impulses: their significance for vertebrate and invertebrate systems. Edited by A. Roberts and B.M.H. Bush 8. Stomatal physiology. Edited by P. G. Jarvis and T.A. Mansfield 10. The cell cycle. Edited by P.C.L. John 11. Effects of disease on the physiology of the growing plant. Edited by PC Ayres 12. Biology of the chemotactic response. Edited by J. M. Lackie and P. C. Williamson 14. Biological timekeeping. Edited by J. Brady 15. The nucleolus. Edited by E.G. Jordan and C.A. Cullis 16. Gills. Edited by D.F. Houlihan, J.C. Rankin and T.J. Shuttleworth 17. Cellular acclimatisation to environmental change. Edited by A.R. Cossins and P. Sheterline 19. Storage carbohydrates in vascular plants. Edited by D.H. Lewis 20. The physiology and biochemistry of plant respiration. Edited by J.M. Palmer 21. Chloroplast biogenesis. Edited by R.J. Ellis 23. The biosynthesis and metabolism of plant hormones. Edited by A. Crozier and J'.R. Hitlman 24. Coordination of motor behaviour. Edited by B.M.H. Bush and F. Clarac 25. Cell ageing and cell death. Edited by I. Davies and D.C. Sigee 26. The cell division cycle in plants. Edited by J.A. Bryant and D. Francis 27. Control of leaf growth. EditedbyN.R. Baker, W.J. Davies and C. Ong 28. Biochemistry of plant cell walls. Edited by C.T. Brett and J.R. Hillman 29. Immunology in plant science. Edited by T.L. Wang 30. Root development and function. Edited by P.J. Gregory, J. V. Lake and D.A. Rose 31. Plant canopies: their growth, form and function. Edited by G. Russell, B. Marshall and P.G. Jarvis

32. Developmental mutants in higher plants. Edited by H. Thomas and D. Grierson 33. Neurohormones in invertebrates. Edited by M. Thorndyke and G. Goldsworthy 34. Acid toxicity and aquatic animals. Edited by R. Morris, E.W. Taylor, D.J.A. Brown and J.A. Brown 35. Division and segregation of organelles. Edited by S.A. Boffey and D. Lloyd 36. Biomechanics in evolution. Edited by J.M. V. Rayner and R.J. Wootton 37. Techniques in comparative respiratory physiology: An experimental approach. Edited by C. R. Bridges and P.J. Butler 38. Herbicides and plant metabolism. Edited by A.D. Dodge 39. Plants under stress. Edited by H.G. Jones, T.J. Flowers and M.B. Jones 40. In situ hybridisation: application to developmental biology and medicine. Edited by N. Harris and D.G. Wilkinson 41. Physiological strategies for gas exchange and metabolism. Edited by A.J. Woakes, M.K. Grieshaber and C.R. Bridges 42. Compartmentation of plant metabolism in non-photosynthesis tissues. Edited by M.J. Ernes 43. Plant growth: interactions with nutrition and environment. Edited by J.R. Porter and D.W. Lawlor 44. Feeding and the texture of foods. Edited by J.F.V. Vincent and P.J. Lillford 45. Endocytosis, exocytosis and vesicle traffic in plants. Edited by G.R. Hawes, J.O.D. Coleman and D. E. Evans 46. Calcium, oxygen radicals and cellular damage. Edited by C.J. Duncan 47. Fruit and seed production: aspects of development, environmental physiology and ecology. Edited by C. Marshall and J. Grace 48. Perspectives in plant cell recognition. Edited by J.A. Callow and JR. Green

INDUCIBLE PLANT PROTEINS: THEIR BIOCHEMISTRY AND MOLECULAR BIOLOGY

Edited by

John L. Wray Plant Molecular Genetics Unit, Department of Biochemistry and Microbiology, University ofSt

CAMBRIDGE UNIVERSITY PRESS

Andrews

Published by the Press Syndicate of the University of Cambridge The Pitt Building, Trumpington Street, Cambridge CB2 1RP 40 West 20th Street, New York, NY 10011-4211, USA 10 Stamford Road, Oakleigh, Victoria 3166, Australia © Cambridge University Press 1992 First published 1992 Printed in Great Britain at the University Press, Cambridge A catalogue record for this book is available from the British Library Library of Congress cataloguing in publication data Inducible plant proteins: their biochemistry and molecular biology / edited by John L. Wray. p. cm. - (Seminar series / Society for Experimental Biology; 48) Includes index. 1. Plant enzymes. 2. Enzyme induction. 3. Plant proteins. I. Wray, John L. (John Langford), 1942. II. Series: Seminar series (Society for Experimental Biology (Great Britain)); 49. QK898.E58B56 1992 582'.01925-dc20 91^6320 CIP ISBN 0 521 40170 4 hardback

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Contents

List of Contributors

ix

Preface

xv

Metal-binding proteins and metal-regulated gene expression in higher plants A.B. TOMSETT, A.K. SEWELL, S.J. JONES, J.R. de MIRANDA, and D.A. THURMAN

1

Phosphate starvation inducible enzymes and proteins in higher plants A.H. GOLDSTEIN

25

Nitrate reduction in higher plants: molecular approaches to function and regulation P. R O U Z E and M. CABOCHE

45

Inducibility of the glutamine synthetase gene family in Phaseolus vulgaris L. J.V. CULLIMORE, J.M. COCK, T.J. DANIEL, L., R. SWARUP and M. J. BENNETT Expression and manipulation of genes involved in phenylpropanoid biosynthesis W. SCHUCH Biochemistry and molecular biology of CAM H.J. BOHNERT, D.M. VERNON, E.J. DeROCHER, C.B. MICHALOWSKI and J.C. CUSHMAN

79

97

113

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Contents

ABA- and GA-responsive gene expression F.L. OLSEN, K. SKRIVER, F. MULLER-URI, N.V. RAIKHEL, J.C. ROGERS and J. MUNDY Regulation of gene expression, ethylene synthesis and ripening in transgenic tomatoes D. GRIERSON, A.J. HAMILTON, M. BOUZAYEN, M. KOCK, G.W. LYCETT and S. BARTON Induction of nodulin genes and root nodule symbiosis D.P.S. VERMA and G.-H. MIAO Systemic acquired resistance: an inducible defence mechanism in plants J. RYALS, E. WARD, P. AHL-GOY and J.P. METRAUX Biochemistry and molecular biology of the anaerobic response E.S. DENNIS, M. OLIVE, R. DOLFERUS, A. MILLAR, W.J. PEACOCK and T.L. SETTER The heat shock response in transgenic plants: the use of chimaeric heat shock genes F. SCHOFFL, V. DIEDRING, M. KLIEM, M. RIEPING, G. SCHRODER and K. SEVERIN Biochemistry and molecular biology of cold-inducible enzymes and proteins in higher plants L. CATTIVELLI and D. BARTELS

139

155

175

205 231

247

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GBF-1, GBF-2 and GBF-3: three Arabidopsis b-Zip proteins that interact with the light-regulated rbcS-lA promoter 289 U. SCHINDLER, A.E. MENKENS and A.R. CASHMORE Index

305

Contributors

AHL-GOY, P. Department of Molecular Genetics, Agricultural Biotechnology Research Unit, CIBA-GEIGY Corporation, PO Box 12257, Research Triangle Park, North Carolina, USA. BARTELS, D. Max Planck Institut fiir Ziichtungsforschung, Carl von Linne"weg 10, D-5000 Koln 30, Germany. BARTON, S. AFRC Research Group in Plant Gene Regulation, Department of Physiology and Environmental Studies, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough LE12 5RD, UK. BENNETT, M.J. Department of Plant Sciences, The University of Arizona, Tucson, AZ 85721, USA. BOHNERT, H.J. Departments of Biochemistry, Molecular and Celullar Biology and Plant Sciences, The University of Arizona, Tucson, AZ 85721, USA. BOUZAYEN, M. AFRC Research Group in Plant Gene Regulation, Department of Physiology and Environmental Studies, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough LE12 5RD,UK. CABOCHE, M. Laboratoire de Biologie Cellulaire, INRA, Centre de Versailles, F-78026 Versailles Cedex, France. CASHMORE, A.R. Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. CATTIVELLI, L. Experimental Institute for Cereal Research, Section of Fiorenzuola d'Arda (PC), 29017, Via S. Protaso 302, Italy.

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List of contributors

COCK, J.M. Laboratoire de Biologie Moleculaire des Relations PlantesMicroorganismes, INRA-CNRS, Bp. 27, F-31326 Castenet-Tolosan Cedex, France. CULLIMORE, J.V. Laboratoire de Biologie Moleculaire des Relations PlantesMicroorganismes, INRA-CNRS, Bp. 27, F-31326 Castenet-Tolosan Cedex, France. CUSHMAN, J.C. Department of Biochemistry, The University of Arizona, Tucson, AZ 85721, USA. DANIELL, T.J. Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. DENNIS, E.S. CSIRO Division of Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. de MIRANDA, J.R. Department of Genetics and Microbiology, Donnan Laboratories, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK. DeROCHER, E.J. Department of Molecular and Cellular Biology, The University of Arizona, Tucson, AZ 85721, USA. DIEDRING, V. Department of Genetics, University of Tubingen, Auf der Morgenstelle 28, D-7400 Tubingen, Germany. DOLFERUS, R. CSIRO Division of Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia. GOLDSTEIN, A.H. Department of Biology, The California State University, Los Angeles, CA 90032-8201, USA. GRIERSON, D. AFRC Research Group in Plant Gene Regulation, Department of Physiology and Environmental Studies, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough LE12 5RD,UK. HAMILTON, A J . AFRC Research Group in Plant Gene Regulation, Department of Physiology and Environmental Studies, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough LE12 5RD, UK.

List of contributors

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J O N E S , S.J. Department of Genetics and Microbiology, Donnan Laboratories, University of Liverpool, P O Box 147, Liverpool L69 3BX, U K . K L I E M , M. Department of Genetics, University of Tubingen, Auf der Morgenstelle 28, D-7400 Tubingen, Germany. K O C K , M. A F R C Research Group in Plant Gene Regulation, Department of Physiology and Environmental Studies, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough LE12 5RD, UK. L Y C E T T , G.W. A F R C Research Group in Plant Gene Regulation, Department of Physiology and Environmental Studies, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough LE12 5 R D , U K . MENKENS, A.E. Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA METRAUX, J.P. Agricultural Division, CIBA-GEIGY Limited, CH-4002 Basel, Switzerland. MIAO, G.-H. Department of Molecular Genetics and Biotechnology Center, The Ohio State University, Columbus, OH 43210, USA. MICHALOWSKI, C.B. Department of Biochemistry, The University of Arizona, Tucson, AZ 85721, USA. MILLAR, A. CSIRO Division of Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia. MULLER-URI, F. Carlsberg Research Laboratory, Gl. Carlsberg Vej 6, Copenhagen, DK-2500, Denmark. MUNDY,J. Carlsberg Research Laboratory, Gl. Carlsberg Vej 6, Copenhagen, DK-2500, Denmark. OLIVE, M. CSIRO Division of Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia. OLSEN, F.L. Carlsberg Research Laboratory, Gl. Carlsberg Vej 6, Copenhagen, DK-2500, Denmark.

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List of contributors

PEACOCK, W.J. CSIRO Division of Plant Industry, GPO Box 1600, Canberra ACT 2601, Australia. RAIKHEL, N.V. MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA. RIEPING, M. Department of Genetics, University of Tubingen, Auf der Morgenstelle 28, D-7400 Tubingen, Germany. ROGERS, J.C. Division of Haematology and Oncology, Washington University Medical School, 660 S. Euclid, St Louis, MO 63110, USA. ROUZE, P. Laboratoire de Biologie Cellulaire, INRA, Centre de Versailles, F-78026 Versailles Cedex, France. RYALS, J. Department of Molecular Genetics, Agricultural Biotechnology Research Unit, CIBA-GEIGY Corporation, PO Box 12257, Research Triangle Park, North Carolina, USA. SCHINDLER, U. Plant Science Institute, Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA. SCHOFFL, F. Department of Genetics, University of Tubingen, Auf der Morgenstelle 28, D-7400 Tubingen, Germany. SCHRODER, G. Department of Genetics, University of Tubingen, Auf der Morgenstelle 28, D-7400 Tubingen, Germany. SCHUCH, W. ICI Seeds, Plant Biotechnology Section, Jealott's Hill Research Station, Bracknell RG12 6EY, UK. SETTER, T.L. University of Western Australia, Nedlands, Western Australia, Australia. SEVERIN, K. Department of Genetics, University of Tubingen, Auf der Morgenstelle 28, D-7400 Tubingen, Germany. SEWELL, A.K. Department of Genetics and Microbiology, Donnan Laboratories, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK. SKRIVER, K. Carlsberg Research Laboratory, Gl. Carlsberg Vej 6, Copenhagen, DK-2500, Denmark.

List of contributors

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SWARUP, R. Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. THURMAN, D.A. Department of Genetics and Microbiology, Donnan Laboratories, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK. TOMSETT, A.B. Department of Genetics and Microbiology, Donnan Laboratories, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK. VERMA, D.P.S. Department of Molecular Genetics and Biotechnology Center, The Ohio State University, Columbus, OH 43210, USA. VERNON, D.M. Department of Molecular and Cellular Biology, The University of Arizona, Tucson, AZ 85721, USA. WARD, E. Department of Molecular Genetics, Agricultural Biotechnology Research Unit, CIBA-GEIGY Corporation, PO Box 12257, Research Triangle Park, North Carolina, UK.

Preface

Plants are able to respond and adapt to changing environmental and endogenous signals by the induction of net synthesis of specific proteins which act to modify cellular metabolism. This text, based on papers presented at the Birmingham meeting of the Society for Experimental Biology in April 1991 in a two-day Symposium entitled 'Biochemistry and Molecular Biology of Inducible Enzymes and Proteins in Higher Plants', attempts to discuss the role these inducible proteins play in the biochemistry of the cell and the approaches being used to delineate the underlying molecular events which lead to their synthesis. The topics included in this text do not exhaustively cover all the known responses of plants to environmental and endogenous signals, but a wide range are discussed. Whilst classical approaches, such as protein purification and characterisation, are of great importance in understanding the biochemistry of inducible proteins the molecular cloning of the encoding genes means that transgenic approaches can also be used to unravel the role particular proteins play in metabolism. In particular, the use of antisense RN A to down-regulate the synthesis of specific target enzymes is a particularly powerful technique and its use is discussed here in chapters on nitrate reduction, heat shock, phenylpropanoid biosynthesis and fruit ripening. The way in which transgenic techniques might be used in a broader sense to modify cellular metabolism to man's advantage is also discussed (see for example the chapter on anaerobiosis). Attempts to understand the molecular basis for the induction of synthesis of specific proteins/enzymes are discussed in most of the chapters, but some topics are more advanced in this respect than others. In most cases induction is due to activation of gene expression, as measured by increased steady state levels of mRNA, but in some cases, for instance nitrate reductase, post-transcriptional events may also be important. The eventual aim of course is to understand the series of events - the so-called signal transduction pathway - which lead from perception of the signal to increased mRNA levels and hence increased enzyme synthesis. In a

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Preface

number of cases, as discussed in chapters on heat shock, CAM, anaerobiosis, gibberellic acid/abscisic acid and light, so-called responsive elements, that is stretches of DNA located at the 5' end of the gene which are probably involved in signal transduction via binding of trans-acting transcription factors, have been identified by promoter-deletion analysis. Identification and characterisation of such transcription factors (b-Zip proteins) in the context of light regulation of gene activity is discussed in thefinalchapter. The use of transgenic approaches to dissect other parts of the signal transduction pathway by a search for pathway mutants is discussed in the chapter on the heat shock response. I thank the contributors for their cooperation, in most cases given promptly, and in particular the Professor of Applied Chaos who added a little light relief whilst I gathered together and edited the manuscripts. Unfortunately two of the invited speakers at the Symposium were unable to attend owing to circumstances beyond their control and were unable subsequently to contribute chapters, one of which would have strengthened the light-related content of this text. Despite this I hope readers willfindthe volume a useful contribution to this rapidly developing field. John L. Wray

A.B. TOMSETT, A.K. SEWELL, S.J. JONES, J.R. de MIRANDA and D.A. THURMAN

Metal-binding proteins and metalregulated gene expression in higher plants Introduction The growth of plants in nature depends on their ability to respond to their environment. For the metabolism of metals, plants require a balance between the uptake of sufficient essential metal ions to maintain growth and development and the ability to protect sensitive cellular activity from excessive concentrations of essential and non-essential metals. Although phytotoxic amounts of metal occur more frequently from industrial and agricultural pollution than in soils under natural conditions, nevertheless, survival mechanisms are required to detect not only external/internal concentrations of metals, but also essential from non-essential metal ions. Plants thus have the ability to 'sense' metal ions since it is central to normal metal metabolism, protection from metal toxicity, and adaptation to metal tolerance. Such recognition can be envisaged to occur by a number of physiological processes, but at the molecular level it is likely to be the binding of metal ions to a protein, which directly or indirectly changes the pattern of cellular activity, usually by changing gene expression. Evidence is now emerging that this molecular recognition is 'programmed': evolution has fashioned proteins either to have rigid binding sites which accept some ions while rejecting others, or to have flexible binding sites in which the stereochemistry of the ion determines the final shape of the protein. In either case, evolution has given the organism the capability to distinguish metals and partition them in different ways. In this chapter we examine the relationship between metal homeostasis (i.e. essential metal metabolism and protection from metal toxicity) and metal tolerance, and the role that metal-binding proteins and metalregulated gene expression play in these processes.

Society for Experimental Biology Seminar Series 49: Inducible Plant Proteins, ed. J. L. Wray. ©Cambridge University Press, 1992, pp. 1-24.

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A.B. TOMSETT et al.

Heavy metals The term 'heavy metal' has been used classically in a number of contexts. Here, it will be used to define those metals with a specific gravity of greater than 5 which forms ligands containing nitrogen and sulphur centres rather than ligands containing oxygen: Nieboer & Richardson (1980) defined these as the Class B/Borderline heavy metals. This group includes mercury, cadmium, lead, copper, zinc, silver and nickel; they are the major toxic elements in biological systems. Copper (Cu) and zinc (Zn) are essential micronutrients, acting as prosthetic groups in a wide range of enzymes, but like all of the other metals, at elevated concentrations they reduce the vigour of plant growth or, in the extreme, totally inhibit growth. The specific effects of metals on biological systems vary because these depend upon the chemistry of the individual metal. However, the toxic effects are always complex because of the wide range of ligands with which they interact. For this reason, the most effective defence against metal toxicity requires a mechanism with a blanket effect rather than specific alteration of sensitive sites. The degree of such protection may determine whether we classify such plants as sensitive or tolerant to heavy metals, although metal tolerance in plants is generally polygenic, indicating that more than one mechanism is operating. Protection against metal toxicity There are a variety of ways in which plants could protect themselves against heavy metal toxicity; tolerant populations may have adapted a number of these to achieve colonisation of soils with phytotoxic levels of metal. In essence, there are two major strategies: avoidance maintains a low intracellular concentration, either by preventing metal ions from entering the plant (e.g. by extracellular precipitation), or by reduced uptake/active efflux; sequestration allows high intracellular concentrations to be tolerated, either by compartmentation (e.g. into vacuoles), or by binding the metal to ligands, thus separating the ions from sensitive cellular metabolism. It is the latter that has received most attention, and recently metal-binding proteins and peptides, collectively termed metallothioneins, have been the focus of most research. Metal-binding polypeptides Metallothioneins (MTs) are low molecular mass, cysteine-rich metalbinding proteins, which have been divided into three classes (see Kagi &

Metal-binding proteins

3

Kojima, 1987). The original molecules defined as MTs, now termed the class I MTs, possessed arrangements of cysteine residues, as Cys-Cys and Cys-x-Cys (where x is an amino acid other than cysteine), which aligned with those of equine renal MT (the first isolate). Class II MTs are remarkably similar molecules containing Cys-Cys and Cys-x-Cys clusters but which cannot be aligned easily with equine renal MT. Both class I and II MTs are gene-encoded polypeptides synthesised by transcription and translation. This is in marked contrast to a third group of polypeptides, now termed class III MTs, which are more commonly referred to as phytochelatins (PCs) or by their structural name poly[gamma-glutamylcysteinyljglycine ([gammaEC]nG). These molecules are not geneencoded; their synthesis is from glutathione (gammaECG) by a specific enzyme gamma-glutamyl cysteine dipeptidyl transpeptidase (common name phytochelatin synthase) (Loeffler et al., 1989): glutathione +

glutathione —> phytochelatin + glycine or phytochelatin

i.e. [gammaEC]G + [gammaEC]nG-> [gammaEC]n+1G + G PCs have been described with chain lengths varying between 5 amino acids ([gammaEC]2G) and 23 amino acids ([gammaEC]nG) (Grill et al., 1987). Their high cysteine content (40-47%) compares favourably with that of class I and II MTs (30%) and their metal-binding capacity has led to the suggestion that they fulfil a similar function in cellular metabolism (Grill etal., 1987). Animal metallothioneins Class I and/or II MTs have been described in all animals examined. Mammalian MTs have been some of the most extensively studied: of the 61 or 62 amino acids, 20 are cysteine residues. Metal ions are bound to the MT exclusively through thiolate bonds involving all 20 cysteines (see Hamer, 1986). They associate with a wide range of metals in vitro, 18 different metals in the case of rat liver MT (Nielson et al., 1985). Divalent and trivalent metals exhibit saturation binding at 7 mole equivalents forming M7-MT, whereas copper (Cu(I)) and silver (Ag(I)) bind as monovalent ions forming M12-MT. The structure of these molecules is such that two metal-binding domains are formed: an a-cluster from the carboxy-terminal portion of the protein, contains 11 cysteines which bind either 4 divalent or 6 monovalent ions; the P-cluster, the amino-terminal

4

A.B. TOMSETT et al.

half, binds 3 divalent or 6 monovalent ions through 9 cysteines. Nielson et al. (1985) predict different tertiary structures for the M7- and M12-proteins, which provide a differential specificity for Zn and Cu, the two major essential metals. Such differential structures are a key component of the ability of cells to 'sense' metals. Fungal metallothioneins Unlike animals, not all fungi contain class I or II MTs. Neurospora crassa, Agaricus bisporus and Saccharomyces cerevisiae each contain geneencoded MTs (Munger & Lerch, 1985; Winge et al., 1985; Munger et al., 1987); Schizosaccharomyces pombe, for example, does not (Murasugi et al., 1981; Butt & Ecker, 1987). Furthermore, these fungal MTs are Cuthioneins, being unable to bind the wide range of metals of their animal counterparts. S. cerevisiae has a 53 amino acid class II protein with 12 cysteine residues which bind 8 Cu(I) ions in thiolate clusters (Winge et al., 1985). The N. crassa and A. bisporus class I MTs are much smaller, only 25 amino acids, and bind 6 moles of Cu(I) to 7 cysteines (Munger & Lerch, 1985; Munger et al., 1987). Although 3 moles of divalent metal can be bound in vitro, there is no evidence that such ions bind in vivo. Once again metal/protein specificity provides the organism with the ability to differentiate between ions.

Fungal phytochelatins PCs have not been described in animals (although their non-existence is only assumed since there has been no determined search for them). PCs were first discovered as Cd-binding peptides in 5. pombe (Murasugi et al., 1981; Kondo et al., 1985) and called cadystins. The simple structure of these molecules, [gammaEC]3G of cadystin A and [gammaEC]2G of cadystin B, received little attention until Grill et al. (1985) isolated PCs from plant cell cultures. Later, these workers (Grill et al., 1986a) confirmed that cadystins and PCs were identical. It is curious that 5. cerevisiae and 5. pombe differ so markedly in the synthesis of metalbinding polypeptides. There is no evidence to suggest that 5. cerevisiae produces PCs in response to Cd; equally there is no evidence that 5. pombe encodes a Cu-thionein. This begs the question: how do these organisms cope with Cd and Cu, respectively? What is clear is that these yeasts recognise Cu and Cd differently, and respond differently. As will be discussed below, a third yeast, Candida glabrata, synthesises different classes of polypeptides when challenged with these two metals.

Metal-binding proteins

5

Plant phytochelatins The description of PCs has been relatively recent and resulted from a search for MTs in higher plants. Initial reports of MT-like proteins from higher plants exposed to Cd or Cu were made from a number of species: mung bean (Premakumar et al., 1975), rice (Dabin et al., 1978; Kaneta et al., 1983), Agrostis gigantea (Curvetto & Rauser, 1979; Rauser, 1981, 1984a,b; Rauser et al., 1983), tomato (Bartoff et al., 1980; Wagner & Trotter, 1982), soybean (Casterline & Barnet, 1982), cabbage (Wagner & Trotter, 1982; Wagner, 1984), Silene cucubalus (Lolkema et al., 1983), Datura innoxia (Jackson etal., 1984), maize (Rauser & Glover, 1984) and Mimulus guttatus (Robinson & Thurman, 1986). Many of these descriptions were based on partially pure samples. Comparisons of their amino acid compositions, where given, with mammalian MT suggests that either these were substantially different to MT, or that these MTs were impure. Within a relatively short period the purification of these metal-binding components had been completed by several groups (Grill et al., 1985; Steffens et al., 1986; Reese & Wagner, 1987; Jackson et al., 1987). Unfortunately, this led to a series of different names for the same molecules (see Steffens, 1990); for clarity in this chapter, we are using the term phytochelatin as a contrast to gene-encoded MTs. Once PCs had been described, a number of researchers optimised their search for these molecules, and the number of reports increased. A wide survey (200 species) of the plant kingdom, including the taxonomic divisions Bryophyta, Pteridophyta and Spermatophyta, found PCs in all species tested (Gekeler et al., 1989). Grill et al. (19866) have described a group of related peptides in members of the Fabales which also bind Cd but have P-alanine at the C-terminus instead of glycine. Such plants do not contain glutathione (gammaECG), the substrate for PC synthesis, but instead contain its |3-alanine homologue, homoglutathione (Klapheck, 1988), and thus these peptides have been called homophytochelatins. These two findings strongly indicate that (i) PCs (or their P-alanine homologues) are ubiquitous in plants and (ii) early reports of MT-like proteins were in fact impure PCs; this led to the suggestion that only PCs, and not MTs, occur in plants and that 'a fundamental evolutionary divergence in heavy metal sequestration has occurred between animals and plants' (Grill et al., 1987). This suggestion may well be correct if animals do not synthesise PCs; that is, the presence of PCs may allow plants to use different polypeptides from animals to achieve metal homeostasis and/or metal tolerance. However, this was no argument for plants to lack gene-encoded MTs; until plants have been carefully examined for MTs, their existence cannot be ruled out.

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A.B. TOMSETT et al.

Plant metallothioneins The presence of gene-encoded MTs, in both the structural and functional sense, in higher plants is still not proven, but the evidence is getting stronger. There were some early indications that PCs could not account for all the 'MT-like' protein reports. For example, Reese & Wagner (1987) reported that a Cu complex, isolated from suspension cultures of tobacco, did not appear to have the same properties as PC even though its apparent molecular weight was similar to a Cd PC isolated from the same tissue. This led us to examine metal-binding components of M. guttatus for evidence of MTs as well as PCs. Metal-binding polypeptides in roots of copper-tolerant

Mimulus It is difficult to interpret many of the data on metal-binding components of plants because these have been determined from experiments on Cdresistant, Cd-grown cell cultures. This presents two problems. First, plant cell cultures do not necessarily respond physiologically in the same way as intact plants, and secondly, Cd is not an essential metal and hence may not elicit the same responses as an essential one. Our studies thus aimed to examine whole plants, in particular roots, since this is the normal route of metal entry into the plant, and to compare essential with non-essential metal responses. After the initial description of Cu-binding proteins in M. guttatus by Robinson & Thurman (1986), we have extended the purification by standard chromatography and HPLC techniques. Salt et al. (1989) reported that after Sephadex G-50 gel permeation, and HPLC-DEAE anion-exchange chromatography, two Cu-containing fractions could be isolated from Cu-tolerant roots of M. guttatus grown in the presence of 10 UM Cu for 4 days. These peaks were not detected in similar roots grown in micronutrient levels of Cu. The amino acid analyses of these two fractions are shown in Table 1. It was clear that these represented PC3 and PQ, as described in 5. pombe and other higher plants. More detailed examination, however, revealed that these were not the only Cu-containing components identified by anion exchange. Initially, we identified four Cu-containing fractions after Sephadex G50 gel filtration and HPLC-DEAE anion-exchange chromatography (Tomsett et al., 1989), but the yield of some components was too low for subsequent purification. By making the initial isolation step DE-52 anionexchange chromatography, greater yields were obtained (S. Jones, unpublished data; A.K. Sewell, unpublished data). Figure \A shows the

Metal-binding proteins

1

Table 1. Amino acid compositions of cadmium (CdBS2) and copper (CuBS3) containing peptides from roots of M. guttatus grown in the presence of cadmium or copper Total residues (%)

Cys Asx Thr Ser Glx Pro Gly Ala Val Met He Leu Tyr Phe His Trp Lys Arg

CdBS2a

CdBS2b

CuBS3a

CuBS3b

CdPC

28.6 3.9 3.5 6.1 22.2 0.9 13.9 4.8 3.0 1.7 0.9 0.9 2.2 0.0 3.9 0.0 2.6 0.9

49.1 1.5 0.7 2.7 32.1 0.0 12.3 1.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.0

29.7 4.5 2.7 5.4 29.7 1.8 16.2 3.3 0.0 0.0 1.9 2.2 0.0 0.6 0.0 0.0 1.5 0.4

39.1 1.7 0.7 1.5 39.1 0.0 13.4 1.8 0.0 0.0 0.4 0.8 0.0 0.5 0.0 0.0 0.8 0.0

28.0 3.4 1.2 5.1 36.5 0.0 16.5 2.6 1.1 0.0 1.2 1.3 0.5

0.6 0.8 0.6 0.9 0.4

Note: CdBS2 and CuBS3 (see Fig. 2) yielded two derivatives (a & b) which are PC2 and PC3, respectively (A.K. Sewell, unpublished data). The CdPC column is the Cd-containing peptide isolated from tobacco (Reese & Wagner, 1987).

DE-52 elution profile of an extract of 250 g of Cu-tolerant M. guttatus roots initiated and grown in the presence of 4 UM CU for 4 weeks, the growth medium being changed every 4 days to maintain the metal concentration. After pooling the fractions which eluted off this column at conductivities between 6 and 16 mmho (as shown), the sample was applied to an HPLC-DEAE anion-exchange column; the results are shown in Fig. 2A. Four Cu-containing fractions were observed (CuBSl to CuBS4), eluting with a similar profile to that observed previously (Tomsett et ai, 1989), but with substantially more Cu associated with each. For comparison, Fig. 16 shows the DE-52 elution profile of an extract of 250 g of roots of Cu-tolerant M. guttatus initiated and grown in the

A.B. TOM SETT et al. .

10

o

I N

'

T3 O

U

20

40

60

80

100

Fraction Number

B

-50

-40

E o

I

60

U

O

-30

E

-20

73

3

1 -10

0

20

40

60

80

100

Fraction Number Fig. 1. DE-52 elution profile of extracts of copper-tolerant Mimulus guttatus roots grown in j$ Long Ashton Solution plus added metal ions, for 4 weeks (solutions changed every 4 days to maintain the metal concentration). The column was eluted with a linear gradient of 0 to 1 M KC1 in 10 mM HEPES, pH6.5. Fractions were collected and measured for metal (-•-) and conductivity (-<>-)• Metal ions were added as sulphates. A, From roots grown in 4 UM copper. B, From roots grown in 2 (XM cadmium.

Metal-binding proteins

A

50

1000

o

c

§o o

U

0

10

20

30

40

50

60

70

80

90

100

110

Time (minutes)

B 1.5 r

1000

800

- 600

5

- 400

§ 5 o

200

20

30

40

50

60

70

80

90

100

HO

Time (minutes) Fig. 2. HPLC-DEAE anion-exchange elution profiles of material which eluted off the DE-52 column at a conductivity between 6 and 16 mmho. The column was eluted with a gradient between 0 and 1 M KC1 in 10 ITIM HEPES, pH6.5. A, From roots grown in 4 UM copper (Fig. 1A). B, From roots grown in 2 U.M cadmium (Fig. IS).

10

A.B. TOMSETT et al.

presence of 2 UM Cd for 4 weeks, with regular changes of growth medium as before. Fractions eluting at conductivities between 6 and 16 mmho (as shown) were pooled and applied to HPLC-DEAE anion-exchange chromatography. Figure 2B shows that this sample contains only two major Cd-containing fractions (CdBSl and CdBS2), although previously we had detected only one (Tomsett et al., 1989). CuBS3 and CdBS2 both elute at 300 mM KC1 and contain a mixture of PC, and PC3 as described previously (Salt et al., 1989; A.K. Sewell, unpublished data). In addition, both CuBS3 and CdBS2 appear to contain approximately the same amounts of P Q and PC3 and in the same proportion, i.e. 4 PC,:1 PC3 (data not shown; A.K. Sewell, unpublished data). Other metal-binding components of Mimulus CdBSl has not been extensively analysed but p-chloromercuribenzoic acid derivatisation yielded a peak on reverse phase HPLC corresponding to glutathione (A. Sewell, unpublished data). Although CuBS2 elutes at approximately the same position in the salt gradient as CdBSl, this peak is not exclusively PC or glutathione. The amino acid composition of essentially pure CuBS2 (Table 2) reveals a complex mixture of amino acids; although high in Glx, Cys and Gly (50%), the amino acid composition is not characteristic of other impure or purified PCs which have 75-90% Glx, Cys and Gly (Table 1; Salt et al., 1989; Tomsett etal., 1989). Furthermore, it is estimated to have a molecular mass of 2700 Da and is remarkably similar to the class I MT from A. bisporus (Table 2). CuBSl has no equivalent peak from Cd-grown roots. It is eluted at approximately 125 ^M KC1 from the gradient and has been shown to be 6000-7000 Da (A.K. Sewell, unpublished data). After further gel filtration and HPLC-DEAE anion-exchange steps, the amino acid composition of this substance was determined (Table 2). As with CuBS2, this peak is relatively high in Glx, Cys and Gly (55%) but its composition is not characteristic of PC fractions. Furthermore, when compared with proteins of similar size (Table 2), the amino acid composition suggests that this is also a MT. CuBS4 has an apparent molecular mass of approximately 13 000 Da. Attempts to determine the amino acid analysis of this material have revealed that it contains very little amino nitrogen; after performic acid oxidation, however, the sample was shown to contain abundant amino nitrogen. Further evidence to suggest that CuBSl and CuBS2 are not related to

Metal-binding proteins

11

Table 2. Comparison of the amino acid compositions by residues of (CuBSl) and (CuBS2) with the Mimulus putative MT gene product and Agaricus and rat liver MT Number of residues

Cys Asx Thr Ser Glx Pro Gly Ala Val Met He Leu Tyr Phe His Trp Lys Arg

CuBSl"

CuBS2*

Agaricus MT

Rat Mt

Mimulus Mt

9 13 2 5 12 1 15 4 2 1 1 2 1 1 1 1 1 0

5 3 1 4 3 1 6 2 1 0 0 1 0 0 0 0 1 0

7 1 2 5 1 0 6 2 0 0 0 0 0 0 0 0 1 0

20 4 4 11 1 2 6 3 2 1 0 0 0 0 0 0 7 0

12 8 4 10 5 3 11 2 2 5 1 1 2 1 0 0 5 0

Notes: "Molecular mass 7000 Da. * Molecular mass 2700 Da.

PCs comes from a study of the behaviour of PC and Cu when mixed in vitro: metal-free PC and Cu only produce CuBS3. This indicates that the other fractions (CuBSl, 2 and 4) detected in vivo, at the very least, must contain other components. What is clear from the analysis of metal-binding components in M. guttatus roots is: 1. Cu and Cd stimulate the synthesis of, and bind to, different combinations of components, as can be seen by comparing Fig. L4 with IB, and Fig. 24 with IB. 2. Cu, but not Cd, induces polypeptides (CuBSl and CuBS2)

12

A . B . TOMSETT et al.

whose amino acid compositions are similar to known geneencoded MTs of animals and fungi. Such data suggest that plant cells may distinguish essential and nonessential metal ions, both through their chelation to different polypeptides, and by the different conformations of the complexes when bound to the same polypeptide.

Metal regulated gene expression Cellular responses to metal ions include changes of patterns of gene expression. Many of these changes are non-specific 'shock' effects: for example, subsets of the heat shock proteins are synthesised in response to Cd (Czarnecka et al., 1984; Lin et al., 1984; Delhaize et al., 1989). However, some mRNAs are specifically induced by Cd (Delhaize et al., 1989). Thus, by examining metal-regulated gene expression, whether specific or non-specific, it may be possible to determine the relative roles of proteins and polypeptides specific to metal homeostasis and metal tolerance, as opposed to functions involved in general stress responses. Metallothionein gene regulation in animals Mammalian MTs are known to accumulate after administration of various metal salts. This control is not exclusive, however, as a variety of other stimuli also trigger MT synthesis, including several hormones, tissue injury, bacterial endotoxin and interferon (see Karin, 1985; Hamer, 1986). Each of these factors relates directly or indirectly to various acute stresses. This could indicate that MT is a general stress protein. Such a definition is incomplete because MT levels also change during embryogenesis and tissue differentiation. This has prompted the suggestion that the primary role of MT is as a modulator of cellular activity (Karin, 1985). Mammalian MT synthesis is regulated at the transcriptional level. A series of as-acting control sequences has been identified and can be attributed to the range of effectors described above (see Hamer, 1986). Among these are sequences defined as metal-responsive elements (MREs). In the mouse MTI gene is a pair of MREs which are imperfectly duplicated 12 bp sequences, at —46 (relative to the start of transcription) and —114; further partial repeats are centred at positions —160, —134 and -62. The requirement for such sequences in metal regulation is proven: for the mouse MTI promoter, Zn and Cd induction requires two or more MREs(Searleeffl/., 1985). Since mammalian MT is a metal-inducible, metal-binding protein, it is

Metal-binding proteins

13

clearly a candidate to fulfil a role in protection against metal toxicity and in some instances to confer metal tolerance. The fact that its expression is modulated during growth and development indicates a futher role in metal homeostasis. Metallothionein gene regulation in fungi Unlike mammalian MTs, fungal MTs are (essentially) only Cu-inducible (see Tomsett & Thurman, 1988). Analyses of the upstream regions of the N. crassa MT gene reveal no homology to mammalian MREs or to sequences adjacent to the yeast gene (Munger et al., 1987). In this case, transcription is induced specifically by Cu. In 5. cerevisiae, six Cu regulatory elements can be recognised upstream of the MT (CUP1) gene, which are unrelated to the mammalian MREs (see Butt & Ecker, 1987). A regulatory protein, ACEl, binds to these upstream sequences in the presence of Cu ions but not in their absence. Cu activates the ACEl protein by inducing a change in its conformation that stimulates its binding to DNA, activating transcription. It is interesting to note that Ag can also bind to ACEl in vitro and activate MT gene expression in vivo: other divalent cations tested (Zn, Cd, Ni, Fe, Pb, Sn) could not induce DNA-binding activity (Furst et al., 1988). Once again, the interaction between the chemical properties of the metal with the recognition protein determines the metal specificity of the response; here only monovalent ions elicit the functional conformation. The fact that the ACEl protein gene appears to have evolved from the MT gene (Furst et al., 1988) accounts for the unusual properties of this unique DNA binding protein. Although the 5. cerevisiae MT gene is also glucose-repressible (see Butt & Ecker, 1987), the primary role of this protein appears to be in metal homeostasis. This conclusion must also be drawn for the N. crassa MT, the expression of which does not appear to be regulated by anything other than Cu (Munger et al., 1987). Metal regulation of phytochelatin synthesis In contrast to MT induction, the available evidence suggests that PC synthesis is not stimulated by direct transcriptional activation. PCs can be synthesised very rapidly (within 5 min) in response to metals entering the cells, even when protein synthesis is totally inhibited (Scheller et al., 1987; Robinson et al., 1988). Phytochelatin synthase thus appears to be a constitutive enzyme. Loeffler et al. (1989) have shown that, for the enzyme purified from Silene cucubalus, metal ion 'induction' is a post-

14

A.B. TOMSETT et al.

translational control mechanism, whereby metal ions activate pre-existing protein. There is an absolute requirement for both the substrate, glutathione, and the effector, metal ions, for the enzyme to be active: no PC synthesis occurred when purified enzyme was incubated under standard conditions in the absence of metal. Furthermore, in vitro studies reveal that even active enzyme can be instantaneously prevented from further PC synthesis by the addition of EDTA (or even excess metal-free PC) to chelate any remaining metal ions. The regulation of PC synthesis is thus a very simple negative feedback loop. The enzyme is inactive until metal ions activate the synthesis of PCs, which chelate the metal preventing further activation of the enzyme, thus preventing further PC production. Grill et al. (1989) have shown that the best activator of PC synthase is Cd, which correlates with the best inducer of PC synthesis in plant cell cultures (Grill et al., 1987), and the activation series is reported to be Cd > Ag>Bi > Pb > Zn > Cu > Hg. This constitutive, post-translationally activated enzyme enables plant cells to respond very rapidly to metal ions entering the cell. Metal-regulated genes from Mimulus We are investigating metal-regulated gene expression in Cu-tolerant M. guttatus in an attempt to examine the relationship between metal homeostasis and metal tolerance. A lambda gtlO cDNA library was made from poly (A) + RNA from the roots of plants exposed for 24 h to 10 (XM CuSO4. This library was differentially screened with first-strand cDNA prepared from the roots of Cu-treated and untreated plants. From 30 000 primary recombinant plaques screened, we isolated 250 putative Curegulated clones, of which 40 (20 inducible and 20 repressible) were selected for further analysis by northern blotting and partial DNA sequencing. The partial DNA sequences have been run through the Geneman/NBRF computer database to search for DNA or protein homology of predicted open-reading frames (ORFs). Although many of the cDNAs did not reveal significant homology to any sequence in the database, several interesting observations were made. Of the Cu-inducible clones, two showed protein homology to known sequences: one appeared to be related to horseradish and turnip peroxidase, which are stress-inducible proteins; the other had features characteristic of cytochrome oxidase, a Cu-containing enzyme. The repressed clones have so far revealed the most significant data. Of 20 repressed clones isolated from the cDNA library, 18 were from a single class of message (c. 500 bases), as shown by northern blot analysis and cross-hybridisation. Five of these were completely sequenced to reveal

Metal-binding proteins

15

that they had identical ORFs and predicted protein products (de Miranda et al., 1990), even though their DNA sequences indicated that they represented transcripts from two distinct genes in the genome of the plant. Analysis of the predicted protein sequencing using the database showed that 19 of the top 23 matches were for a series of animal and fungal MTs. This homology was limited to the two major MT domains at the N- and C-termini of the protein: the central region showed no homology to MTs or any other sequence (Figs 3 and 4). This, together with the description of a similar pea gene (see below), was the first report of a higher plant MT gene (de Miranda et al., 1990). The first report of a plant MT protein was based on a partial amino acid sequence of the wheat Ec protein (Lane et al., 1987). Although this failed to reveal significant homology to any sequence in the database, it has been classified as a class II MT because of its high cysteine content, some of which are present as Cys-x-Cys clusters, and its ability to bind Zn (Kagi & Schaffer, 1988). Ec is a cysteine-rich protein present early in the germination of wheat and hence it could fulfil a cysteine-storage function: the ability to bind Zn might be anticipated of any protein containing so many SH groups. The hypothetical 72 amino acid M. guttatus MT protein, predicted from the cDNA, has several features in common with MTs: low molecular mass (7348 Da); an absence of aromatic amino acids in the MT domains; and usually serine or lysine as the bridging amino acid in the Cys-x-Cys clusters. The overall structure of the molecule is, however, different from previously reported MTs. As described above, the strong similarity of this protein to class I and II MTs is due to the two domains of 14/15 amino acids, each of which contains six cysteine residues as Cys-x-Cys clusters. These N- and C-terminal domains are separated by a large region (39 amino acids) which is devoid of cysteine and which has no homology to other MTs. This unusual structure is also characteristic of three other proteins predicted as products of cDNAs from Pisum sativum (Evans et al., 1990), Zea mays (de Framond, 1991), and Glycine max (Kawashima et al., 1991), as shown in Figs 3 and 4. Structural predictions of the Mimulus protein indicate random coil for the MT-like domains, which is consistent with the mammalian MT folding, with a largely extended configuration for the intervening region. It is the regulation of these genes that has been surprising. Each of the above sequences is abundant in the mRNA populations of roots; in Cutolerant M. guttatus, addition of metal (Cu, Cd or Zn) to the growth medium reduces the steady-state level of transcript in the roots (de Miranda et al., 1990). This 'repression' appears to be a shock effect since roots initiated and grown continuously in Cu have levels of transcript

MT domain 1

MT domain 2

Mimulus Pisum

MSS—G—CSCGSGCKCGDNCS-CSM-YPD METNTTVTMIEGVAPLKMYS-EGSEKSFGA-EGGNGCKCGSNCKCDPCNC :: :—:G:::S:N:::S:K-:NKRSSGLSYS:ME::E:V:L::G:A:IQF-::A:M:AAS-:D:-:::::D::T::::::K

Glycine max Zea mays

::-CC:GN:G:::S::::NG:GG:K:-:::LSYT:ST::E:LVM::::V:AQF-::A:MGVP:-:ND-:::::P::S:N: :T:K :: :::::S:G::SS:K-:GKK:::LEETS:AAQP:WL::::E:KAAP:FV:AAAESG:AAH::S:::G::::::::

consensus

MS

CxCGS CXCG I

CXC i

T

G

V

P

K

E

E

E

GCxCG I

CXC PCxC i

Fig. 3. Amino acid alignment of the putative MTs from M. guttatus (de Miranda et al., 1991), Pisum sativum (Evans et al., 1990), Zea mays (de Framond, 1991) and Glycine max (Kawashima et al., 1991). The inclusion of (—) in the sequence indicates a gap introduced for optimal alignment, (:) denotes amino acid homology, and x is used in the consensus sequence to highlight the Cys-x-Cys clusters.

Metal-binding proteins

17

Ml domains Equss MTla a domain

(33)

v

K

(51)

c

p t G G S C T C A g S C K

K

(22)

C S C c p G G c a r C A q G

Equus MTla fi domain

(4)

C S

Neurospora MT

(3)

C G

c

s g a S S C N C G S G C S

C S

(26)

C s

(20)

Mimuhts MT domain 1

(4)

C S

c

G S G C K C G D n C S

Zea MT domain 1

(2)

C S

c

G S S C G C G S S C K

c

g

(18)

(3)

C G

c

G S S C N C G D S C K

c

N

(19)

(7)

C G

c

G S S C K C G n g C gg

c

k

(24)

C N

c

Pisum MT domain 1 Glycine domain 1

(57)

C K

c

G S N C K

c

- D P

Zea MT domain 2

(63)

C S

c

G S G C K

c

- D P C N

c

Pisum MT domain 2

(62)

C K

c

G d N C T

c

- D P C N

c

K

(76)

(64)

C K

c

G P N C s

c

- n P C t

c

K

(79)

Mimttlus MT domain 2

Glycine MT domain 2

(72) (77)

Fig. 4. Amino acid sequence alignment of the MT-like domains of the putative plant MTs (Fig. 3) with the a and P domains of Equine MT and the single domain of N. crassa MT. The numbers refer to the residue positions within the complete protein.

similar to those grown without metal: continuous growth in Zn or Cd, however, still results in lower levels of message. In M. guttatus, pea and maize, these genes appear to be expressed in a root-specific pattern. Levels of transcript are barely detectable in leaves, developing cotyledon, pith, seed and anther (de Miranda et al., 1990; Evans et al., 1990; de Framond, 1991). However, the soybean gene is expressed constitutively in both roots and leaves (Kawashima et al., 1991). For the pea and maize genes, the upstream sequences of genomic clones have been examined (Evans et al., 1990; de Framond, 1991). In maize, no sequence matching the consensus MRE of animal MT genes was found, whereas in pea a related sequence was found between —241 and —248 upstream of the transcription start site. As yet, there is no evidence that this element is active. It is interesting to note that the four described plant MT genes have been isolated by three different methods. The pea and maize genes were selected as root-specific cDNAs by differential screening of a root cDNA library (Evans et al., 1990; de Framond, 1991). The Mimulus gene was identified as a metal-repressible sequence in a root cDNA library by differential screening (de Miranda et al., 1990). The soybean cDNA was isolated by probing a library with a synthetic oligonucleotide correspond-

18

A.B. TOMSETT et al.

ing to a consensus nucleotide sequence at the N-terminus of the mammalian MT genes (Kawashima et al., 1991). It is clear, then, that there is significant circumstantial evidence to indicate that these are indeed plant MT genes. Metal homeostasis and metal tolerance: role of MTs and PCs If the available evidence suggests that both MTs and PCs are present in plant cells, what function(s) do these fulfil? Do they act as alternatives in metal homeostasis, or do they have separate roles in the storage/detoxification of essential/non-essential metal ions? Until recently, no organism had been investigated which had both PCs and MTs. Examination of the yeast C. glabrata, however, broke the apparent dichotomy. This organism produces both MTs and PCs in a metal-specific manner: exposure to Cu stimulates synthesis of two MT-like proteins; in the presence of Cd, the cells form PCs (Mehra et al., 1988). The two MTs have 62 and 51 amino acids, of which 18 and 14 are cysteines, respectively (Mehra et al., 1989). Both genes are inducible by Cu and to some extent by Ag, but no MT message is present when cells are treated with Cd, indicating not only that there is no induction by Cd, but also that Cd repression of the low level constitutive expression is seen in the absence of metal. This situation is not entirely equivalent to the higher plant data in which both Cu and Cd stimulate PC synthesis and the MT gene is not inducible but constitutive in roots. Nevertheless, the same principle may apply: namely, that the essential ion, Cu, is processed differently from the non-essential metal, Cd. The role of PCs It has been suggested that PCs may act as the sulphur carrier during sulphate reduction because of the similarities in their size and synthesis (Steffens et al., 1986). However, even if this is so, the absolute requirement for metal ions of PC synthase must indicate a role in metal homeostasis (Loeffler et al., 1989). Vogeli-Lange & Wagner (1990) have suggested that this role might be a Cd-transport function. By examination of protoplasts and vacuoles, these authors showed that all the PCs and all the Cd were located in the vacuolar sap, rather than the tonoplast membrane. Since it is probable that PCs are synthesised in the cytoplasm, these data suggest that PCs transport Cd to the vacuole. Such a role for PCs would be consistent with a function in protection against metal toxicity, since the result is an effective sequestration of the

Metal-binding proteins

19

metal. It will be interesting to await results on whether Cu-PCs are also transported to the vacuole. Since the chemistry of Cu(I) binding and Cd(II) binding will be different, this may determine different conformations of PC-metal complexes which could affect their subcellular localisation. It is not difficult to imagine that metal tolerance could result from an increased transport capacity for PCs to the vacuole. Since PCs are present in all plant species examined, tolerance could be determined solely by the individual's capacity to produce PCs. We have examined metal-tolerant individuals to determine whether PCs are involved in cross-tolerance to a range of metals. In both a Zn-tolerant Deschampsia cespitosa and Cutolerant M. guttatus, tolerance to a range of metals could be identified relative to their respective non-tolerant clones: this tolerance could be destroyed by the treatment of plants with buthionine sulphoximine (BSO), an inhibitor of PC synthesis (A.K. Sewell et ai, unpublished data). These data indicate that while they may not be the sole component, PCs must play a key role in metal detoxification, and therefore metal tolerance in plants. The role of MTs At present, it is not possible to define the role of plant MTs; that will await final purification, and both in vivo and in vitro studies of metalbinding. The evidence to date, however, indicates a role in Cu metabolism but not Cd metabolism: non-PC-like components bind Cu but not Cd; MT genes from a range of plants are constitutive and abundant in roots, indicating a key role in normal cellular activity. The inference of this is that these proteins function in essential metal metabolism. The evidence available from mammalian and fungal systems indicates that overproduction of MT can confer tolerance. When mammalian cell cultures are subjected to stepwise selection to increasing concentrations of Cd, the resultant Cd-resistant cells have both a higher capacity for MT synthesis and amplification of the MT genes (see Hamer, 1986). In 5. cerevisiae, MT gene amplification confers Cu-tolerance (see Hamer, 1986); Cu-resistant strains of C. glabrata also showed chromosomal amplification of the MT-II gene but not the MT-I gene (Mehra et al., 1990). Whether the synthesis/regulation of plant MTs can be adapted to confer metal tolerance awaits further analysis of tolerant and non-tolerant individuals of the same species.

20

A.B. TOMSETT et al.

Conclusion Higher plants produce two distinct MT-like polypeptides, gene-encoded proteins with similar properties to certain class I and II MTs, and PCs (class HI MTs). The synthesis of both types of molecule is regulated by metal ions. PC synthase is directly activated by metal ions by a posttranslational mechanism. The gene-encoded MTs have constitutive abundant mRNA levels in roots which may be repressible by 'metalshock' in some species. Since the synthesis of both classes of these MTs has not yet been shown to be specifically regulated by one metal rather than another, any specificity of function may result from molecular recognition in the binding of ions to these polypeptides. It is clear that in M. guttatus, Cu, but not Cd, is bound to certain proteins perhaps because of the different chemical properties of the ions (although as yet transcriptional/translational control of synthesis of these proteins cannot be totally rejected). Furthermore, although both Cu and Cd form PC complexes, it is possible that PC complexes with essential metal ions could be processed differently from those with non-essential metal ions. References Bartoff, M., Brennan, E. & Price, C.A. (1980). Partial characterization of a cadmium-binding protein from the roots of cadmium-treated tomato. Plant Physiology 66, 438-41. Butt, T.R. & Ecker, D.J. (1987). Yeast metallothionein and applications to biotechnology. Microbiological Reviews 51, 351-64. Casterline, J.L., Jr & Barnett, N.M. (1982). Cadmium-binding components in soybean plants. Plant Physiology 69, 1004-7. Curvetto, N.R. & Rauser, W.E. (1979). Isolation and characterization of copper-binding proteins from roots of Agrostis gigantea tolerant to excess copper. Plant Physiology 63, 55-9. Czarnecka, E., Edelman, L., Schoffl, F. & Key, J.L. (1984). Comparative analysis of physical stress responses in soybean seedlings using cloned heat shock cDNAs. Plant Molecular Biology 3, 45-58. Dabin, P., Marafante, E., Mousny, J.M. & Myttenaere, C. (1978). Absorption, distribution and binding of cadmium and zinc in irrigated rice plants. Plant and Soil Science 50, 329-41. de Framond, A.J. (1991). A metallothionein-like gene from maize (Zea mays): cloning and characterization. FEBS Letters 290, 103-6. Delhaize, E., Robinson, N.J. & Jackson, P.J. (1989). Effects of cadmium on gene expression in cadmium-tolerant and cadmium-sensitive Datura innoxia cells. Plant Molecular Biology 12, 487-97.

Metal-binding proteins

21

de Miranda, J.R., Thomas, M.G., Thurman, D.A. & Tomsett, A.B. (1990). Metallothionein genes from the flowering plant Mimulus guttatus. FEBS Letters 260, 277-80. Evans, I.M., Gatehouse, L.N., Gatehouse, J.A., Robinson, N.J. & Croy, R.R.D. (1990). A gene from pea (Pisum sativum) with homology to metallothionein genes. FEBS Letters 262, 29-32. Furst, F.S., Hu, S., Hackett, R. & Hamer, R. (1988). Copper activates metallothionein gene transcription by altering the conformation of a specific DNA-binding protein. Cell 55, 705-17. Gekeler, W., Grill, E., Winnacker, E.-L. & Zenk, M.H. (1989). Survey of the plant kingdom for the ability to bind heavy metals through PCs. Zeitschrift fiir Naturforschung 44c, 361-9. Grill, E., Gekeler, W., Winnacker, E.-L. & Zenk, M.H. (19866). Homo-phytochelatins are heavy metal binding peptides of homoglutathione containing Fabales. FEBS Letters 205, 47-50. Grill, E., Loeffler, S., Winnacker, E.-L. & Zenk, M.H. (1989). Phytochelatins, the heavy metal-binding peptides of plants are synthesised from glutathione by a specific gamma-glutamyl cysteine dipeptidyl transpeptidase (phytochelatin synthase). Proceedings of the National Academy of Sciences (USA) 86, 6838-42. Grill, E., Winnacker, E.-L. & Zenk, M.H. (1985). Phytochelatins: The principle heavy metal complexing peptides in higher plants. Science 230, 674-6. Grill, E., Winnacker, E.-L. & Zenk, M.H. (1986a). Synthesis of seven different homologous phytochelatins in metal-exposed Schizosaccharomyces pombe cells. FEBS Letters 197, 115-20. Grill, E., Winnacker, E.-L. & Zenk, M.H. (1987). Phytochelatins, a class of heavy metal binding peptides from plants are functionally analogous to metallothioneins. Proceedings of the National Academy of Sciences (USA) 84, 439-43. Hamer, D.H. (1986). Metallothionein. Annual Review of Biochemistry 55, 913-51. Jackson, P.J., Roth, E.J., McClure, P.R. & Naranjio, CM. (1984). Selection, isolation and characterisation of cadmium-resistant Datura innoxia suspension cultures. Plant Physiology 75, 914-18. Jackson, P.J., Unkefer, C.J., Doolen, J.A., Watts, K. & Robinson, N.J. (1987). Poly(gamma-glutamylcysteinyl) glycine: its role in cadmium resistance in plant cells. Proceedings of the National Academy of Sciences (USA) 84, 6619-23. Kagi, J.H.R. & Kojima, Y. (1987). Chemistry and biochemistry of metallothionein. In Metallothionein II, ed. J.H.R. Kagi & Y. Kojima, pp. 25-61. Basel: Birkhauser Verlag. Kagi, J.H.R. & Schaffer, A. (1988). Biochemistry of metallothionein. Biochemistry 27, 8509-15. Kaneta, M., Hikichi, H., Endo, S. & Sugiyama, N. (1983). Isolation of

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A.B. TOMSETT et 0.1. cadmium-binding protein from cadmium-treated rice plants (Oryza sativa L.). Agricultural and Biological Chemistry 47, 417-18. Karin, M. (1985). Metallothioneins: proteins in search of function. Cell 41, 9-10. Kawashima, I., Inokuchi, Y., Chino, M., Kimura, M. & Shimizu, N. (1991). Isolation of a gene for a metallothionein-like protein from soybean. Plant Cell Physiology 32, 913-16. Klapheck, S. (1988). Homoglutathione: isolation, quantification and occurrence in legumes. Physiologia Plantarum 74, 727-32. Kondo, N., Isobe, M., Imai, K. & Goto, T. (1985). Synthesis of metallothionein-like peptides cadystin A and B occurring in a fission yeast, and their isomers. Agricultural and Biological Chemistry 49, 71-83. Lane, B., Kajioka, R. & Kennedy, T. (1987). The wheat-germ Ec protein is a zinc-containing metallothionein. Biochemistry and Cell Biology 65, 1001-5. Lin, C.-Y., Roberts, J.K. & Key, J.L. (1984). Acquisition of thermotolerance in soybean seedling. Plant Physiology 74, 152-60. Loeffler, S., Hochberger, A., Grill, E., Winnacker, E.-L. & Zenk, M.H. (1989). Termination of the phytochelatin synthase reaction through sequestration of heavy metals by the reaction product. FEBS Letters 258, 42-6. Lolkema, P.C., Donker, M.H., Schouten, A.J. & Ernst, W.H.O. (1983). The possible role of metallothioneins in copper tolerance of Silene cucubalis. Planta 162, 172-9. Mehra, R.K., Garey, J.R., Butt, T.R., Gray, W.R. & Winge, D.R. (1989). Candida glabrata metallothioneins, cloning and sequence of the genes and characterization of proteins. Journal of Biological Chemistry 264, 19747-53. Mehra, R.K., Garey, J.R. & Winge, D.R. (1990). Selective and tandem amplification of a member of the metallothionein gene family in Candida glabrata. Journal of Biological Chemistry 265, 6369-75. Mehra, R.K., Tarbet, E.B., Gray, W.R. & Winge, D.R. (1988). Metalspecific synthesis of two metallothioneins and gamma-glutamyl peptides in Candida glabrata. Proceedings of the National Academy of Sciences (USA) 85, 8815-19. Munger, K., Germann, U.A. & Lerch, K. (1987). The Neurospora crassa metallothionein gene. Journal of Biological Chemistry 262, 7363-7. Munger, K. & Lerch, K. (1985). Copper metallothionein from the fungus Agaricus bisporus: chemical and spectroscopic properties. Biochemistry 24, 6751-6. Murasugi, A., Wada, C. & Hayashi, Y. (1981). Cadmium-binding peptide induced in fission yeast, Schizosaccharomyces pombe. Journal of Biochemistry (Tokyo) 90, 1561^. Nieboer, E. & Richardson, D.H.S. (1980). The replacement of the nondescript term 'heavy metals' by a biologically and chemically sig-

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nificant classification of metal ions. Environmental Pollution, Series Bl 3-26. Nielson, K.B., Atkin, C.L. & Winge, D.R. (1985). Distinct metalbinding configurations in metallothionein. Journal of Biological Chemistry 260, 5342-50. Premakumar, R., Winge, D.R., Wiley, R.D. & Rajagopalan, K.V. (1975). Copper chelatin: isolation from various eucaryotic sources. Archives of Biochemistry and Biophysics 170, 278-88. Rauser, W.E. (1981). Occurrence of metal-binding proteins in plants. In Heavy Metals in the Environment, ed. W.H.O. Ernst, pp. 281-4. Edinburgh: CEP Consultants Ltd. Rauser, W.E. (1984a). Isolation and partial purification of cadmiumbinding protein from roots of the grass Agrostis gigantea. Plant Physiology 74, 1025-9. Rauser, W.E. (1984b). Copper-binding protein and copper-tolerance in Agrostis gigantea. Plant Science Letters 33, 239-47. Rauser, W.E. & Glover, J. (1984). Cadmium-binding proteins in roots of maize. Canadian Journal of Botany 62, 1645-50. Rauser, W.E., Hartmann, H.-J. & Weser, U. (1983). Cadmium-thiolate protein from the grass Agrostis gigantea. FEBS Letters 164, 102-4. Reese, N.R. & Wagner, G.J. (1987). Properties of tobacco (Nicotiana tabacum) cadmium-binding peptides. Biochemical Journal 241, 641-7. Robinson, N.J., Ratcliff, R.L., Anderson, P.J., Delhaize, E., Berger, J.M. & Jackson, P.J. (1988). Biosynthesis of poly (gamma-glutamyl cysteinyl) glycines in cadmium-resistant Datura innoxia cells. Plant Science 56, 197-204. Robinson, N. J. & Thurman, D. A. (1986). Isolation of a copper complex and its rate of appearance in roots of Mimulus guttatus. Planta 169, 192-7. Salt, D.E., Thurman, D.A., Tomsett, A.B. & Sewell, A.K. (1989). Copper phytochelatins of Mimulus guttatus. Proceedings of the Royal Society of London Series B 236, 79-89. Scheller, H.V., Haung, B., Hatch, E. & Goldsbrough, P.B. (1987). Phytochelatin synthesis and glutathione levels in response to heavy metals in tomato cells. Plant Physiology 85, 1031-5. Searle, P.F., Stuart, G.W. & Palmiter, R.D. (1985). Building a metalresponsive promoter with synthetic regulatory elements. Molecular and Cellular Biology 5, 1480-9. Steffens, J.C. (1990). The heavy metal-binding peptides of plants. Annual Review of Plant Physiology 41, 553-75. Steffens, J.C, Hunt, D.F. & Williams, B.G. (1986). Accumulation of non-protein metal-binding polypeptides (gamma-glutamyl-cysteinyl)nglycine in selected cadmium-resistant tomato cells. Journal of Biochemistry 261, 13879-82. Tomsett, A.B., Salt, D.E., de Miranda, J. & Thurman, D.A. (1989). Metallothioneins and metal tolerance. Aspects of Applied Biology,

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A.B. TOMSETT et al. Roots and the Soil Environment 22, 365-72. Association of Applied Biologists. Tomsett, A.B. & Thurman, D.A. (1988). Molecular biology of metal tolerances in plants. Plant, Cell and Environment 11, 383-94. Vogeli-Lange, R. & Wagner, G.J. (1990). Subcellular localization of cadmium and cadmium-binding peptides in tobacco leaves. Plant Physiology 92, 1086-93. Wagner, C.J. (1984). Characterisation of a cadmium-complex from the roots of cabbage leaves. Plant Physiology 76, 797-805. Wagner, G.J. & Trotter, M. (1982). Inducible cadmium-binding complexes of cabbage and tobacco. Plant Physiology 69, 804-9. Winge, D.R., Nielson, K.B., Gray, W.R. & Hamer, D.H. (1985). Yeast metallothionein: sequence and metal-binding properties. Journal of Biological Chemistry 260, 14464-70.

A.H. GOLDSTEIN

Phosphate starvation inducible enzymes and proteins in higher plants

Introduction In this chapter, I discuss the current state of knowledge about the biochemistry and molecular biology of phosphate starvation inducible (psi) enzymes and proteins in higher plants. Special attention will be paid to the excreted phosphate starvation inducible (epsi, pronounced ee *P) acid phosphatase (APase) of higher plants. A significant amount of data now exists to support the theory that the epsi-APase, an epsi-RNase, several intracellular RNases and other proteins of unknown function form a family of co-induced proteins that act, at least in part, as a phosphate starvation rescue mechanism for higher plants. In addition, we have conducted experiments to show that some of these proteins can apparently affect phosphate use-efficiency metabolism under non-starvation conditions. While crucial experiments remain to be done, our data further suggest that the epsi-APase genes as well as genes for other psi proteins can be regulated at the mRNA level (possibly by transcriptional activation). We have proposed, as a working model, that these psi genes are part of the higher plant pho stimulon and may be co-regulated by the same transacting element(s) to form a pho regulon (Goldstein et al., 1989a).

Phosphorus is an essential nutrient for all cells. For organisms that absorb their mineral nutrients directly from the external medium, ionic inorganic phosphate (Pi, usually H2PO4" or HPO42") is the preferentially absorbed form of phosphorus. A macronutrient based on its contribution to biomass, Pi is one of the least available mineral nutrients in many environments. For example, the level of Pi in the solution phase of soils is often below those of many micronutrients (Fried & Brosehart, 1967; Epstein, 1972). In soils, all major nutrient ions except Pi are normally present at concentrations from 1.0 to 0.1 ITIM whereas the Pi concentration is commonly 1.0 ^M or less. In many natural ecosystems, phosphorus Society for Experimental Biology Seminar Series 49: Inducible Plant Proteins, ed. J. L. Wray. © Cambridge University Press, 1992, pp. 25-44.

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is the growth-limiting element owing to its low concentration (Ozanne, 1980). In contrast to low levels of soluble Pi, ecosystems often contain large amounts of both organic and insoluble mineral phosphate. This ecological paradox has resulted in the evolution of a number of gene systems that function to enhance phosphate availability. These coordinately regulated phosphate starvation inducible (psi) genes, collectively called a psi regulon, have been studied extensively in both bacteria and yeast. Psi metabolism has been an extremely useful model system in yeast and bacteria. Consequently, the regulation of genes controlling phosphate mobilisation, uptake and metabolism are well described for these microorganisms (see below). In contrast, little is known about phosphateregulated gene expression in higher plants despite the fact that plants depend almost exclusively on Pi absorbed from the external solution (Fried & Brosehart, 1967; Epstein, 1972). It is probable that plants also have evolved starvation rescue systems composed of psi genes that, in part, act to enhance the concentration of exogenous Pi. Elucidation of the molecular bases for the psi response in higher plants may have important applications for agriculture and biotechnology. Major problems associated with current phosphate fertiliser technology have an impact on both the quality of our environment and the conservation and use of non-renewable natural resources. The success of modern agriculture is based on the application of large amounts of highly soluble phosphate salts. While this approach is efficacious, it is also energyintensive and environmentally destructive. Unlike atmospheric nitrogen, there is no inexhaustible, easily available store of mineral phosphate. Highly soluble phosphate salts (e.g. potassium phosphate) are chemically unstable and tend to revert to poorly soluble mineral phosphates (e.g. hydroxyapatite) when applied to the soil (Barrow, 1980; Englestad & Terman, 1980). Up to 75% of applied fertiliser material may be reprecipitated; therefore, farmers often apply four times the fertiliser necessary for crop production. In the time between application and reprecipitation, a large percentage of this fertiliser material either washes into surface waters with soil erosion or percolates into the groundwater. Therefore, fertiliser P is the largest non-point source of agricultural phosphate pollution. As with nitrogen fixation, a biorational approach to phosphate fertilisation could lay the foundation for a renewable biorational fertiliser technology (Goldstein, 1986; Goldstein & Liu, 1987). Towards this end, this laboratory has pursued molecular genetic and tissue culture approaches with the goal of developing highly phosphateefficient crop plants. The rationale for taking a genetic approach to Pi use-efficiency in higher plants is based, to a large extent, on the seminal

Phosphate starvation inducible enzymes

27

work conducted with microbial systems. Phosphate-regulated gene expression has been one of the most useful and productive model systems for molecular genetic studies in both bacteria and fungi. The pho regulon of Escherichia coli includes a minimum of 20-4 genes that share a common, positive regulatory element, phoB. The phoB protein (of molecular mass 29kDa), which is itself psi, binds specifically to DNA fragments containing the promoters of several genes in the pho regulon. It activates in vitro transcription of these genes (Torriani & Ludtke, 1985; Shinigawa et al., 1987). The phoB gene in turn is regulated by two genes, phoR and phoM. A number of genes in this regulon have been cloned. Another 40-60 genes, located in other regulons, are also controlled by Pi so that the E. coli stimulon involves about 3% of the entire genome. Many of these genes have additional regulatory elements that allow them to act as parts of other stress-induced regulons such as the SOS and oxidative stress systems (Torriani & Ludtke, 1985). A similar pho regulon has been described in Saccharomyces cerevisiae (Bergman et al., 1986). Many of the E. coli psi genes function to enhance Pi availability in, and uptake from, the external medium. For example, phosphate starvation induces phoA whose product is alkaline phosphatase, a hydrolytic enzyme that is excreted into the periplasmic space where it acts to cleave extracellular organic P to Pi. A second psi gene system, the phosphatespecific transport (Pst) operon uses energy to transport Pi across the E. coli membrane. The affinity of this four-gene transport system is much greater than that of the constitutive Pi shuttle. Many of these same molecular starvation rescue mechanisms have been characterised in yeast. Phosphate starvation inducible acid phosphatases in higher plants There have been numerous studies demonstrating induction of APases in higher plants as a result of Pi depletion in the external medium. In 1960, Hewitt and Tatham showed that APase activity in tomato leaf tissue increased up to 18 times normal under Pi deficiency conditions. Phosphate starvation inducible enhancement of APase activity has also been shown for Euglena gracilis (Sommer & Blum, 1965), Nicotiana tabacum (Ueki & Sato, 1971; Ueki, 1978, 1979), Spirodela oligorrhiza (Bieleski & Johnson, 1972), Ochromonas dancia (Patni & Aaronson, 1974), Ipomoea sp. (Zink & Veliky, 1979), Triticum aestivum (Hirata etal., 1982), Oryza sativa (Hirata et al., 1982), Zea mays (Dick et al., 1983; Kummerova, 1986), Agrostis capillaris (McCain & Davies, 1984) and Arabidopsis thalania (A.H. Goldstein, unpublished data). De Jong (1965) localised

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A . H . GOLDSTEIN

APase in onion roots. Enzyme activity was mainly extracellular with the heaviest concentration in corner spaces between the epidermal and hypodermal layers. He suggested the possibility of a subcutaneous pore through which the enzyme could be released to the root surface. Bieleski and co-workers (Reid & Bieleski, 1970; Bieleski & Johnson, 1972) studied the psi induction and location of APase in duckweed {Spirodela oligorrhiza). APase in control plants was located primarily in and around the vascular strands. In P-deficient plants psi-APase activity was 10-20 times the control value. Enzyme activity was primarily located in the epidermis of the root and undersurface of the frond, the tissue locations most likely to provide access to phosphate esters in the medium. These workers further demonstrated that hydrolysis of organic phosphates occurred in the external medium and/or the apoplast followed by Pi uptake into the cell. In 1971, Ueki and Sato demonstrated that omitting inorganic phosphate from the medium resulted in an increase in the acid phosphatase activity in tobacco cells growing in suspension culture. In later studies, Ueki (1978, 1979) showed that excretion of acid phosphatase was the result of an energy-dependent transport system, inhibited by exogenous Pi and regulated by divalent cations. Ninomiya et al. (1977) resolved the extracellular APase activity of tobacco cells in suspension culture into three fractions by sequential chromatography. Two of these fractions were neutral pyrophosphatases with diesterase activity, with a pH optimum at 6.8. The third fraction showed the psi enhancement in activity. This APase had a broad substrate specificity with a pH optimum at 5.8. A number of other workers have identified APase isozymes in higher plants but have not explored the relationship between phosphate deficiency and enzyme activity (compare Paul & Williamson, 1987). We have studied the regulation of the epsi-APase in tomato and shown that, while APase activity in the external medium increases with phosphate starvation, the total intracellular (symplastic) + cell wall-associated (apoplastic) activity remained the same in both Pi sufficient and deficient cells (Goldstein et al., 1988a). We have raised antibodies to the epsiAPase. Our most recent experiments showed that enhancement in epsiAPase activity was accompanied by an increase in excreted protein (Goldstein et al., 19896). These data support the hypothesis that plants have specific epsi-APase genes with unique regulatory and targeting sequences. These data also point out the possibility that plants may be able to target secretion products differentially to both the cell wall space and the external environment. The increased secretion of the epsi-APase is one of the earliest and most dramatic responses to phosphate starvation. The time frame and

Phosphate starvation inducible enzymes

29

degree of induction may not appear rapid to workers used to looking at phenomena such as light-induced photosynthetic genes. However, it is important to keep in mind that the psi response is occurring against the complex background of integrated cellular phosphate metabolism. We have previously shown that our suspension cultured cells can grow for up to 8 days without exogenous Pi. However, within 48 h of transfer to -Pi medium, the level of epsi-APase was significantly higher than in control cultures (Fig. L4; see also Goldstein etal., 1988a), indicating that there is a signal-sensing apparatus to provide an early warning and response. We have clearly shown that secretion of the epsi-APase is proportional to the level of Pi in the media. The level of exogenous Pi, in turn, affects the internal P pool as measured by the number of days until growth ceases and the total biomass that can be accumulated. The overall behaviour of the system indicates continuous feedback to monitor and adjust the psi responses to maintain some optimal level of cell growth. Studies of this type of system will provide important insights into the regulation of cellular metabolism. Peak epsi-APase induction in these cultures corresponded to the point where a lack of phosphate inhibited further growth (Goldstein et al., 1988a). We routinely observed a 10-fold enhancement in epsi-APase levels in our cell cultures at that point. Recently we have shown that the increase in activity was the result of an increase in protein secretion (Fig. 1C; see also Goldstein et al, 19896). Fully 50% of the total acid phosphatase activity in the culture was extracellular under these conditions with increases of 50-fold or more having been observed (Bieleski & Johnson, 1972; A.H. Goldstein et al., unpublished data on Chlamydomonas reinhardtii). Root secretion data were less dramatic, being averages across all tissues. Using the visible indicator dye XP, we observed that the earliest response was concentrated in the root hair tips. Under severe starvation, the entire root system was affected (Goldstein et al., 1989a). Once we have the tools to look at epsi-APase expression at the subcellular level in differentiated, Pi absorbing cells, even more dramatic responses may be observed. By controlling Pi concentration, it should be possible for us to study the psi response at the cellular, tissue and organ level under varying amounts of stress. Data from this laboratory, as well as from others (Clarkson & Scattergood, 1982; Nurnberger et al., 1990), shows clearly that Pi starvation metabolism induces integrated cellular, tissue and organ responses that may result from pho regulon-like coordinate gene expression. Tomato cells regulate the amount of a specific epsi-APase isozyme excreted into the external medium in response to the concentration of Pi in the medium (Goldstein etal., 19886). Cells in early log phase show enhanced extracel-

30

4-

A.H. GOLDSTEIN

4+

4 - 4+ 4 -

4+

3-

3+

Fig. 1. Comparison of several methods used to study the induction of the epsi-APase during the early stages of phosphate starvation. A, SDSPAGE/activity stained gel of epsi-APase in the medium of 4-day-old tomato cells, L. esculentum cv. VF36, grown —Pi or +Pi. Simultaneous quantitative analysis using /;-nitrophenyl phosphate as substrate (not shown but see Goldstein et al., 1988a) gave a psi enhancement of about two-fold for the -Pi treatment. All - / + numbers and/or effects shown in the figures or discussed in the text are normalised to an equivalent biomass basis (see Table 1). (This gel is not supposed to be physically aligned with B, C or D.) B, Immunoblot of AP3 to a SDS-PAGE separation of proteins excreted by 4-day-old cultures growing -Pi or +Pi. The protein sample is identical to that shown in C and the figures are superimposable. Only the non-epsi 54.5 kDa protein could be visualised at this time point using antiserum AP3. C, Total excreted proteins from 4-day-old cells grown -Pi or +Pi were separated via SDS-PAGE and silver stained. The position of the epsi-APase is indicated by the arrow. Laser scanning densitometry allowed resolution of this protein (apparent molecular mass 53.6 kDa) from the major non-psi protein band which ran just above the epsi-APase (apparent molecular mass 54.5 kDa). At this time point, the -Pi treatment had excreted about twice the total epsi-APase protein of the +Pi treatment (Table 1; see also Goldstein et al., 19896)- Psi enhancement of epsi-APase levels becomes more dramatic later in starvation (see Fig. 6). D, Steady-state labelling of culture medium proteins from 3-day-old cells grown -Pi or +Pi was carried out with (35S)-methionine. This figure shows an autoradiogram of these labelled culture-medium proteins after separation via SDS-PAGE. Although growth was not affected until day 8, cells showed

Phosphate starvation inducible enzymes

31

lular APase activity within 24 h of transfer to medium lacking Pi. This enhancement in secreted APase activity reaches a maximum under severe Pi starvation, i.e. after the cells have stopped growing. Whole plant data from the same study (Goldstein et al., 1988a) show that psi excretion of APase may act as one component of a phosphate starvation rescue system. In the early stages of Pi starvation, enhancement of APase activity occurred mainly in the roots. Under longer term Pi stress both stem and leaf growth were inhibited while root growth in the Pi deficient plants remained the same as in the Pi sufficient plants. Severely starved plants reached only one-third of the biomass of the unstressed control but, because of a combination of psi-APase excretion by roots and a shift in biomass to this organ, they excreted 5.5 times the APase activity of the unstressed control. Under severe stress, up to 9% of the APase produced by the roots accumulated in the external hydroponic medium. Other workers have shown that Pi starvation also induces dramatic increases in both the rate of Pi uptake and bidirectional transport (Clarkson & Scattergood, 1982). To characterise the psi response further we examined the proteins excreted into the external medium by 3-day-old suspension cultured tomato cells grown -Pi or + Pi. It is important to emphasise that, while lacking in dramatic visual effect (compare Fig. 1C), significant differences observed long before growth was inhibited allowed us to be sure that we were looking at specific psi phenomena and not generalised stress responses resulting from starvation and/or growth inhibition. Proteins identified in these early stages were then tracked through the course of starvation (compare also Fig. 6; Goldstein et al., 19896). In one series of experiments, cells were labelled with (35S)-methionine and extracellular proteins were precipitated (Goldstein et al., 1988i»). This fraction contained 90% of the extracellular APase activity. Several proteins were labelled to a greater degree under —Pi than under +Pi conditions. The protein with the apparent molecular mass of the epsi-APase (identified via western Caption to Fig. 1 (cont.) both secretion of new proteins and enhanced levels of existing proteins including the psi-APase which was just below the major constitutive protein of 54.5 kDa (Goldstein et al., 19886). Scanning laser densitometry showed an approximate two-fold enhancement in a band that appeared as a shoulder on the low molecular weight side of this major constitutive protein (data not shown). This shoulder is seen more clearly in the immunoblot shown in Fig. 4. The induced 23 kDa protein recently identified by Nurnberger et al. (1990) as a psi excreted RNase is also shown. (This autoradiogram is not intended to be physically aligned with CorD.)

32

A . H . GOLDSTEIN

Table 1. Quantitative comparison of the level of enhancement of epsi-APase in the medium 3" or 4b days after transfer to —Pi medium

Parameter Enhancement

APase specific activity*

(35S)met labelled protein"

Silver stained total protein6

1.9

2.2

2.0

blots as discussed below) showed an approximate two-fold enhancement in incorporated label at 3 days of growth -Pi (Fig. ID; Table 1). As shown in Fig. 1 and Table 1 specific enzyme activity and total protein also showed a two-fold enhancement at this time. At least two radiolabelled proteins were found only in the medium from starved cells. We have isolated the epsi-APase from 3-day-old phosphate starved tomato cell cultures and resolved two peaks of activity via size exclusion chromatography (Goldstein et al., 1988&). The lower molecular mass peak showed most of the psi enhancement of activity. This low molecular mass peak, in turn, resolved into two isozymes on native PAGE. Interestingly, the two chromatography peaks showed different pH vs activity profiles. The high molecular mass peak exhibited phosphatase activity at pH8.1 (the highest pH assayed), functionally defining this enzyme as both an acid and an alkaline phosphatase. The lower molecular mass peak showed the expected APase activity with a sharp decline in activity above pH6. Both fractions produced identical activity-stained bands when run on SDS-PAGE (enzyme activity was retained in the absence of betamercaptoethanol and if the samples are not boiled; Fig. L4). Later experiments showed that this band was really a doublet, as seen on native gels. Using desalting/resalting experiments, we have shown that the epsiAPase can reversibly form high molecular mass (>300 kDa) aggregates that show the acid and alkaline phosphatase activities. Interestingly, the specific activity of the high molecular mass aggregate for both alkaline and acid phosphatase activity was in the same range as that of the (presumably) disaggregated form that shows only acid phosphatase activity. The (apparently) disaggregated lower molecular weight peak of APase activity described above was partially purified by anion-exchange column chromatography (Goldstein et al., 19896). Further purification of the APase via chromatofocusing resolved two closely eluting peaks of activity (probably corresponding to the isozymes seen on the native gels) with

Phosphate starvation inducible enzymes

33

apparent pi values of 5.2 and 5.3. The fractions containing these two peaks were pooled and reconcentrated via ultrafiltration. The chromatofocused fraction was further purified by Con-A Sepharose chromatography and SDS-PAGE and a purified protein of apparent molecular mass of 54 kDa was used for production of antisera in rabbits. A polyclonal antiserum (designated AP3) was produced that recognised the epsiAPase on protein immunoblots. This antiserum also recognised several other secreted proteins and was ultimately shown to be almost entirely directed against the terminal xylose of the N-linked oligosaccharide moiety of the enzyme (Goldstein et al., 1989a). Figure IB shows a SDS-PAGE of total extracellular proteins from cells growing - P or +Pi for 4 days used for an immunoblot with AP3. Figure \C shows an identical gel silver stained to visualise the proteins. The epsiAPase (seen in Fig. \C at 53.6 kDa) was identified by comigration with, and identical immunoblotting characteristics to, the purified protein used to produce AP3. Visual quantitative analysis of the silver stained gel as well as the autoradiogram shown in Fig. ID was confounded by the position of the epsi-APase directly adjacent to a major non-psi protein (apparent molecular mass 54.5 kDa). The 54.5 kDa protein had a xylose moiety (Goldstein et al., 19896) and therefore bound AP3. The amount of the non-psi 54.5 kDa protein remained high through the induction period. As a result this protein gave the only visible signal on the immunoblot performed with AP3 on day 4 (Fig. 15). (Figure IB is included as a visual reference to and is superimposable onto Fig. 1C.) By day 8, the amount of epsi-APase had dramatically increased relative to the 54.5 kDa protein so that a comparison of the immunoblot in Fig. 6/4 with that in Fig. IB showed the emergence of the large epsi-APase band at 53.6 kDa. Autoradiograms of the immunoblots were not sufficiently resolved for quantitative analysis. However, laser scanning densitometry of gels such as those shown in Fig. \C showed that the epsi-APase had approximately twice the total protein in the -Pi treatment at day 4 (Table 1). More dramatic enhancement levels were seen by day 8 (see below). Using the same laser scanning technology, we were able to measure a two-fold psi enhancement in a (35S)-methionine labelled protein of the appropriate molecular mass with the same position relative to the major non-psi protein (Fig. ID; Table 1). Therefore, the same two-fold enhancement was seen in specific activity, total protein and steady-state labelled protein. This is significant because at this early stage there is no reduction in growth rate attributable to depletion of Pi in the external medium. Based on these data, we propose that the epsi-APase has an apparent molecular mass of approximately 53.6 kDa and that psi enhancement of enzyme activity results

34

A . H . GOLDSTEIN

+ DMM DMM

Fig. 2. Treatment of cell cultures with deoxymannojirimycin (DMM) inhibited processing of the complex carbohydrate moiety of the epsiAPase but did not affect excretion into the medium. Three-day-old cells grown —Pi as previously described (Goldstein etal., 1988a) were treated with 0.1 miu DMM for 24 h which resulted in inhibition of processing of the complex carbohydrate moiety of the epsi-APase. Culture-medium proteins from +DMM or -DMM treatments were precipitated with 50% acetone, separated via SDS-PAGE and activity stained (also as previously described, Goldstein etal., 19886). As shown here, inhibition of carbohydrate processing caused a visible increase in apparent molecular mass but did not inhibit the excretion of epsi-APase into the medium.

from enhanced protein excretion into the medium. It is of interest to note that excretion of this enzyme is apparently unaffected by final processing of the carbohydrate moiety. Inclusion of deoxymannojirimycin (DMM), a compound that acts to inhibit final Golgi processing thereby causing retention of the high mannose configuration, in the medium resulted in the expected increase in apparent molecular mass but did not decrease the enzyme activity excreted into the medium (Fig. 2). While we have not yet cloned an epsi-APase cDNA or gene our data do show that psi regulation of protein synthesis involves changes in mRNA levels. Both - / + screening of cDNA libraries and cell free translation studies have been conducted with mRNA isolated from 3-day-old tomato cells grown —Pi or +Pi. At this growth stage, both treatments would be accumulating biomass at the same rate. Three non-homologous cDNA clones have been isolated that show induction in northern blot analyses. Figure 3 shows the results from one of these northern blots. Likewise, cell

Phosphate starvation inducible enzymes

3 - 3+ 6 - 6 + 9 -

35

9+

Fig. 3. A northern blot of poly(A)+ RNA probed with a psi cDNA clone showed enhanced levels of psi messenger RNA as phosphate starvation became more severe. Poly(A)+ RNA was isolated from cultures at 3, 6 and 9 days after transfer to —Pi or +Pi medium (A. Danon et al., unpublished data in preparation). Equal amounts of RNA (1 ug per treatment) were separated on a denaturing formaldehyde/agarose gel and used for a northern blot. The filter was probed using a psi cDNA clone (identified by —/+ screening using standard methods) as the probe. Enhanced levels of mRNA for this clone are seen as early as 3 days after transfer to —Pi medium although cell growth equivalent to the unstressed control continued until day 8. free translation studies have shown an enhancement in the synthesis of a

protein with an apparent molecular mass in the range of the epsi-APase (Fig. 4), as well as others. One of the proteins induced at day 3 is known to be an epsi-RNase (Glund & Goldstein, 1992). It is interesting to speculate that the RNase and APase act in tandem to cleave and subsequently dephosphorylate RNA in the rhizosphere as part of the phosphate starvation rescue strategy. In the process of developing an in vitro system to select phosphate starvation resistant cell lines we simultaneously selected for a line that was constitutively induced for APase excretion. Tissue cultured tomato cells were plated onto solid medium containing starvation levels of phosphate. While most cells died, we identified isolated clumps of callus capable of near-normal rates of growth. Starvation-resistant cells were used to start suspension cultures that were kept under phosphate starva-

36

A.H. GOLDSTEIN 3-

3+

B

MW BMV

Fig. 4. A, Cell-free translation of poly(A)+ RNA from 3-day-old cells grown in —Pi medium showed enhancement (over cells grown +Pi) in message translatable into proteins with apparent molecular masses approximating the epsi-APase (upper arrow) and epsi-RNase (lower arrow). Other lanes are B (no added RNA), molecular weight standards and brome mosaic virus (BMV) positive control (left to right). B and C, enlargements of the epsi-APase and epsi-RNase molecular weight regions of the gel shown in A. The reduced intensity of most bands in B indicated an apparent degradation of the higher molecular weight mRNA in the —Pi treatment. We now consider that the degradation of message in the —Pi treatment resulted from the induction of intra- and extracellular RNases. Based on this obvious degradation, it is probable that there was an even greater enhancement in the psi-translatable messages that were seen in this autoradiogram. tion conditions. The selected cell line showed constitutively enhanced secretion of the epsi-APase even under +Pi conditions (Fig. 5), and greatly increased rates of phosphate uptake. These pleiotropic effects suggest modification of a regulatory apparatus that controls coordinated changes in the expression of a multigene system. The somaclonal variant cell line grew normally under phosphate sufficient conditions but did significantly better than unselected cells under phosphate limited conditions (Goldstein, 1991).

Phosphate starvation inducible enzymes

PSR

37

VF36

Fig. 5. A cell line selected for phosphate starvation resistance was constitutively induced for the excretion of APase into the medium. Threeday-old tomato cells selected for phosphate starvation resistance (PSR) and unselected cells (L. esculentum cv. VF36) were grown under Pisufficient conditions. Proteins excreted by the cells were separated by SDS-PAGE and immunoblotted with AP3 antiserum from which the Xylose-binding component had been removed via stem bromalin treatment (Goldstein, 1991). The selected cells showed constitutive excretion of high levels of APase protein based on the large signal obtained from the immunoblot. Measurement of enzyme activity gave a similar result (not shown).

Other PSI proteins This laboratory has shown that at least three and possibly as many as seven excreted proteins identified by SDS-PAGE are psi (Goldstein et al., 198%). One of these proteins is, of course, the epsi-APase while another has been identified by K. Glund and co-workers as an epsiRNase (Nurnberger et al., 1990). In addition, these workers have identified four intracellular RNases (K. Glund et al., unpublished data) that are induced by Pi starvation of tomato cells, and at least one high molecular weight non-secreted phosphoprotein apparently induced by Pi starvation conditions (see below). Therefore, it is clear that the Pi stimulon in higher plants involves a larger number of inducible proteins. In one study, excreted proteins were separated by SDS-PAGE and silver stained

38

A.H. GOLDSTEIN

8- 8+

8-

8+ -67.2

.60.2 -59.5 -57.7 -54.5 •53.6 -48.2

-45.3 -42.9

B Fig. 6. Under severe starvation conditions the epsi-APase and several other excreted proteins were present in the medium at very high levels relative to unstressed controls. Proteins excreted by cells growing 8 days —Pi or +Pi were separated via SDS-PAGE and immunoblotted using AP3 (A) or silver stained to show total protein (B). The psi enhancement of the epsi-APase (53.6 kDa) was clearly shown. The apparent molecular masses of several proteins selected for further study are indicated. Significant psi enhancement was shown for several of these proteins (Goldstein etal., 198%).

(Fig. 6; see also Goldstein et al., 198%). Analysis via scanning laser densitometry showed that severe Pi starvation induced secretion of at least six proteins. These same six proteins (identified by superposition of the immunoblot onto the silver stained gel) also showed enhancement in antibody binding (Fig. 6). We have concluded that phosphate starvation induced enhanced protein secretion (almost five times the amount of protein is secreted from 8 day —Pi vs +Pi cells per unit biomass) and that this enhancement was the result of Golgi-mediated secretion. Psi enhancement of APase activity (-Pi/+Pi) in one experiment was 1.4,1.9, 2.4 and 7.1 times at 2, 4, 6 and 8 days respectively. Psi enhancement in the amount of the 53.6 kDa protein (identified by AP3 binding) was 2.9, 2.0, 5.9 and 7.8 in the same experiment. The 53.6 kDa protein was one of only three proteins that showed psi enhancement at day 2. Other proteins (e.g. the 59.5 and 42.9 bands shown in Fig. 6) were induced later so that Pi starvation effects could not be unequivocally distinguished from more global pleiotropic effects that resulted from changes in rates of growth

Phosphate starvation inducible enzymes

39

and/or cell division. We also demonstrated psi enhancement of Pi uptake which may have been caused by increased levels of a constitutive Pi transporter or the induction of a high affinity transporter protein(s). The 22.3 kDa epsi-RNase was not identified in this experiment although poorly resolved psi bands were seen in the lower molecular mass range (Goldstein et al., 19896). A protein with the correct apparent molecular mass to be the epsi-RNase was seen clearly in the pulse-labelling data shown in Fig. 1. Glund and co-workers showed that induction of synthesis and secretion of the epsi-RNase was rapid and detectable by antibody staining and fluorography within 3 h after transferring cells from +Pi to —Pi conditions. Studies using 31P-NMR showed that the induction process occurred at intracellular Pi concentrations comparable with those of noninduced cells. These data are in agreement with previous observations showing that epsi-APase in tomato cells was induced well in advance of growth reduction as a result of depletion of the intracellular pool of available phosphate (Goldstein et al., 1988a). Depletion of the tomato cells for nitrate, addition of sodium chloride (salt stress), addition of 0.3 M mannitol or 10% polyethyleneglycol (osmotic stress), incubation for 15 min at 42 °C (heat shock) or addition of 0.1 ITIM salicylic acid neither decreased nor enhanced excreted RNase activity under high and low Pi, respectively. Recently, K. Glund and co-workers have identified, purified and studied this epsi-RNase from cultured tomato cells. The enzyme consists of 205 amino acid residues with a molecular mass of 22 666 Da and an isoelectric point of 4.24. Glund & Goldstein (1992) have recently reviewed data on induction and synthesis of the epsi-RNase genes in tomato. We have used the techniques of molecular biology to study phosphate-regulated gene expression in tomato. While a great deal of work remains to be done, we have presented data that suggest that phosphate starvation induces the expression of epsi-RNase and/or other messages with sequence homology to the epsi-RNase (Glund & Goldstein, 1992). Cell free translation data shown in Fig. 4 provide evidence for an increase in the level of translatable message coding for a protein of apparent molecular mass in the range of 20 kDa. A lambda gtlO cDNA library was screened with oligonucleotide probes to the amino-terminal sequence of the epsi-RNase. We identified a 0.7 kb cDNA fragment and subcloned it into a plasmid (designated pGLAD2). This fragment was used both in northern blot analyses and to probe a lambda EMBL3 genomic library. A genomic clone identified in this study was used in nuclear runoff experiments. Northern blot analyses showed that, 3 days after transfer to —Pi medium, there was a dramatic enhancement in the amount of poly(A)+ RNA that hybridised to the 0.7 kb insert of pGLAD2. Likewise,

40

A . H . GOLDSTEIN

the nuclear runoff experiments clearly showed an increase in the amount of material that hybridised to the genomic clone. Unfortunately, severe compression problems have so far made it impossible to verify that the nucleotide sequence of pGLAD2 codes for the epsi-RNase. These results are discussed in detail in the review of Glund & Goldstein (1992). When viewed as a whole, these data provide evidence for the proposal that phosphate starvation can regulate expression of epsi-RNase genes in tomato. However, based on the complete amino acid sequence of this enzyme and the isolation of genomic clones, additional experiments are currently under way that will allow us quickly to confirm or disprove this thesis. Currently we do not have functional roles for the other epsi proteins identified in cultured tomato cells. In microbial systems, a number of proteins play a role in enhancing the movement of Pi to the plasma membrane. In E. coli these proteins include porins through which Pi diffuses with enhanced efficiency and an outer membrane Pi binding protein that is coded for by a multigene operon that also codes for a high efficiency Pi membrane transporter (Torriani & Ludtke, 1985). It is interesting to speculate that some of these excreted proteins may have analogous functions in higher plants. In addition to regulation involving quantitative changes in protein levels, we have identified at least one apparent psi protein phosphorylation event (Fig. 7). This high molecular weight phosphoprotein appears early in Pi starvation and is not excreted into the medium. The function of this molecule in psi metabolism is unknown. Conclusion The data presented above and those from previous studies are consistent with the existence of a higher plant pho stimulon that acts at the level of enzyme and protein induction. Further biochemical and molecular biology studies are required to address questions of gene regulation (i.e. a higher plant pho regulon). Given that plants have evolved in environments where exogenous solution phase Pi is often limited, it is reasonable to propose that they have developed starvation rescue systems comparable to those of bacteria and fungi. Psi metabolism provides an outstanding system with which to study the feedback interaction between the external environment, metabolism and gene expression. The plant's response to depletion of Pi in the external environment is both rapid and dramatic. Psi metabolism in higher plants involves integrated responses of cells, tissues and organs. In addition to complex changes in the distribution of intracellular P pools (Bieleski, 1973), higher plants

Phosphate starvation inducible enzymes 3+

3-

41

3+3-3+3"

14.3 ~

Fig. 7. Evidence for protein phosphorylation as an early event in the psi response. 2 ml of three-day-old cells growing —Pi or +Pi were washed for 2 h in +Pi consisting of 20 uCi of carrier-free (32P)-orthophosphate. Cells were pelleted and total proteins extracted and separated via SDSPAGE. A, Autoradiogram with equal cpm loaded per lane. The arrow indicates the position of a putative phosphorylated protein that showed psi enhancement. B, The same as A except that the lanes were loaded to represent equal amounts of cell biomass (dry weight). Dramatic enhancement in phosphorylation of all bands in B may be the result of increased rates of Pi transport in the —Pi treatment or these data may imply that several proteins show enhanced phosphorylation under psi conditions. C, As A and B except total proteins were visualised via silver staining. (All gels are aligned by apparent molecular mass.) apparently have an emergency rescue system for scavenging exogenous phosphates. We now know that plants respond quickly to depletion of exogenous Pi via enhancements in root growth, Pi uptake, bidirectional Pi transport, APase excretion, RNase excretion, intracellular RNase activity and protein phosphorylation. All of these physiological and biochemical data indicate that psi metabolism in higher plants is complex and highly regulated. Current data argue against changes in the cytoplasmic Pi pool as a major part of the signal transduction pathway, as previously suggested (Bieleski & Ferguson, 1983). In fact, the concept of an earlywarning, signal-sensing and transduction system (i.e. preceding significant changes in intracellular Pi pools) appears to be firmly established as a component of the higher plant psi stimulon. Elucidation of the molecular mechanisms that underlie this system may provide important insights into

42

A.H. GOLDSTEIN

strategies employed by plants for sensing and responding to environmental stress. It is important to note that we have not even begun to address at the molecular level the complex sequence of events that regulate and mediate the whole plant changes associated with psi metabolism. For example, the ability of severely starved tomato seedlings to produce an essentially unstressed root system while simultaneously exhibiting severe stunting of both stem and leaves (Goldstein et al., 1988a) clearly requires an interface between psi metabolism and the more global regulatory metabolic networks that control plant growth and development. Given that the E. coli stimulon involves 3% of the entire genome, it is not unreasonable to propose that the higher plant Pi stimulon will involve the induction (as well as the repression) of a large number of proteins. Studies of the higher plant Pi stimulon will continue to give an exciting experimental system for basic research and may provide the basis for a new P fertiliser technology as we look towards a future that will require more emphasis on energy efficiency and renewable agricultural practices. References Barrow, N.J. (1980). Evaluation and utilization of residual phosphorus in soils. In The Role of Phosphorus in Soils, ed. F.E. Khasawneh et al., pp. 333-55. Madison, WI: American Society of Agronomy. Bergman, L.W., McClinton, D.C., Madden, S.L. & Preis, L.H. (1986). Molecular analysis of the DNA sequences involved in the transcriptional regulation of the phosphate-repressible acid phosphatase gene (PHO5) of Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences (USA) 83, 6070-4. Bieleski, R.L. (1973). Phosphate pools, phosphate transport, and phosphate availability. Annual Review of Plant Physiology 24, 225-51. Bieleski, R.L. & Ferguson, J.B. (1983). Physiology and metabolism of phosphate and its compounds. In Inorganic Plant Nutrition, Encyclopedia of Plant Physiology, New Series, Vol. 15A, ed. A. Lauchli & R.L. Bielski, pp. 422-49. Berlin: Springer-Verlag. Bieleski, R.L. & Johnson, P.N. (1972). The external location of phosphatase activity in phosphorus-deficient Spirodela oligorrhiza. Australian Journal of Biological Sciences 25, 707-20. Clarkson, D.T. & Scattergood, C.B. (1982). Growth and phosphate transport in barley and tomato plants during development of and recovery from phosphate stress. Journal of Experimental Botany 33, 865-75. De Jong, D.W. (1965). Histochemical demonstration of extracellular distribution of acid phosphatase in onion roots. Phyton 22, 141-6.

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Dick, W.A., Juma, N.G. & Tabatabi, M.A. (1983). Effects of soils on acid phosphate and inorganic pyrophosphatase of corn roots. Soil Science 136, 19-25. Englestad, O.P. & Terman, G.L. (1980). Agronomic effectiveness of phosphate fertilizers. In The Role of Phosphorus in Agriculture, ed. F.E. Khasawneh et al., pp. 311-29. Madison, WI: American Society of Agronomy. Epstein, E. (1972). Mineral Nutrition of Plants. New York: John Wiley. Fried, M. & Brosehart, H. (1967). The Soil-Plant System in Relation to Inorganic Mineral Nutrition. New York: Academic Press. Glund, K. & Goldstein, A.H. (1992). Regulation, synthesis and secretion of a phosphate starvation inducible RNase by cultured tomato cells. In Control of Plant Gene Expression, ed. D.P.S. Verma. Boca Raton: CRC Press. Goldstein, A.H. (1986). Bacterial solubilization of mineral phosphates: historical perspective and future prospects. American Journal of Alternative Agriculture 1, 57-63. Goldstein, A.H. (1991). Plant cells selected for resistance to Pi starvation show enhanced P use-efficiency. Theoretical and Applied Genetics (in press). Goldstein, A.H., Baertlein, D.A. & McDaniel, R.G. (1988a). Phosphate starvation inducible metabolism in L. esculentum I. Plant Physiology 87, 711-15. Goldstein, A.H., Baertlein, D.A. & Danon, A. (1989a). Phosphate starvation stress as an experimental system for molecular analysis. Plant Molecular Biology Reporter 7, 7-16. Goldstein, A.H., Danon, A., Baertlein, D.A. & McDaniel, R.G. (19886). Phosphate starvation inducible metabolism in L. esculentum. II. Plant Physiology 87, 716-20. Goldstein, A.H. & Liu, S.-T. (1987). Molecular cloning and regulation of a mineral phosphate solubilizing (mps) gene from Erwinia herbicola. Bio/technology 5, 72-4. Goldstein, A.H., Mayfield, S.P., Danon, A. & Tibbot, B.K. (1988ft). Phosphate starvation inducible metabolism in L. esculentum. III. Plant Physiology 91, 175-82. Hewitt, E.J. & Tatham, P. (1960). Interaction of mineral deficiency and nitrogen source on acid phosphatase activity in leaf extracts. Journal of Experimental Botany 11, 367-76. Hirata, H., Hisaka, H. & Hirata, A. (1982). Effects of phosphorus and potassium deficiency treatment on root secretion of wheat and rice seedlings. Soil Science and Plant Nutrition 28, 543-52. Kummerova, M. (1986). Localization of acid phosphatase in maize root under phosphorus deficiency. Biologia Plantarum (Praha) 28, 270-4. McCain, S. & Davies, M.S. (1984). Effects of pretreatment with phosphate in natural populations of Agrostis capillaris. New Phytologist 96, 589-99.

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Ninomiya, Y., Ueki, K. & Sato, S. (1977). Chromatographic separation of extracellular acid phosphatase of tobacco cells cultured under Pisupplied and omitted conditions. Plant and Cell Physiology 18, 413-20. Nurnberger, T., Abel, S., Jost, W. & Glund, K. (1990). Induction of an extracellular ribonuclease in cultured tomato cells upon phosphate starvation. Plant Physiology 92, 970-6. Ozanne, P.G. (1980). Phosphate nutrition of plants - a general treatise. In The Role of Phosphorus in Agriculture, ed. E. Khasawneh et al., pp. 559-85. Madison, WI: American Society of Agronomy. Patni, N.J. & Aaronson, S. (1974). Partial characterization of the intraand extracellular acid phosphatase of an alga, Ochromonas dancia. Journal of General Microbiology 83, 9-20. Paul, E.M. & Williamson, V.M. (1987). Purification and properties of acid phosphatase-1 from a nematode-resistant tomato cultivar. Plant Physiology 84, 399-403. Reid, M.S. & Bieleski, R.L. (1970). Changes in phosphatase activity in phosphorus-deficient spirodela. Planta 94, 273-81. Shinigawa, K., Amemura, M. & Nakata, A. (1987). Structure and function of regulatory genes for the phosphate regulon in E. coli. In Phosphate Metabolism and Cellular Regulation in Microorganisms, ed. A. Torriani-Gorini et al., pp. 20-5. Washington, DC: American Society of Microbiology. Sommer, J.R. & Blum, J.J. (1965). Cytochemical localization of acid phosphatases in Euglena gracilis. Journal of Cell Biology 24, 235-48. Torriani, A. & Ludtke, D.N. (1985). The Pho regulon of Escherichia coli. In The Molecular Biology of Bacterial Growth, ed. M. Schaechter et al., pp. 224-43. Boston: Jones and Bartlett, Inc. Ueki, K. (1978). Control of phosphatase release from cultured tobacco cells. Plant and Cell Physiology 19, 385-92. Ueki, K. (1979). Stimulation of phosphatase release from cultured tobacco cells by divalent cations. Plant and Cell Physiology 20, 789-96. Ueki, K. & Sato, S. (1971). Effect of inorganic phosphate on the extracellular acid phosphatase activity of tobacco cells cultured in vitro. Physiologia Plantarum 24, 506-11. Zink, M.W. & Veliky, I.A. (1979). Acid phosphatases of Ipomoea sp. cultured in vitro. 1. Influence of pH and inorganic phosphate on the formation of phosphatases. Canadian Journal of Botany 57, 739-53.

P. ROUZEand M. CABOCHE

Nitrate reduction in higher plants: molecular approaches to function and regulation

Introduction Nitrate from the soil is the main nitrogen source for most higher plants (Beevers & Hageman, 1969; Guerrero, Vega & Losada, 1981; Blevins, 1989). To increase both the growth and yield of those many crops that are unable to fix atmospheric nitrogen, farming has made use of nitrateproviding biological by-products or chemical fertilisers. There is a need to improve the control of the level of nitrate in the soil, to avoid water and atmospheric environmental pollution, as well as to lower production costs (Crawford & Campbell, 1990). For health concerns in humans and animals, there is also a need to maintain a low level of nitrate in food and forage plants. For these reasons, besides soil science, understanding which factors are involved in nitrate uptake and assimilation by plants and how these systems operate is of importance. Nitrate absorbed by roots is assimilated inside the plant cell in the cytoplasm (see Fig. 1) either after transport from the outside through the plasma membrane, or through the tonoplast from the vacuole, where large amounts of nitrate can be accumulated. Both processes are active, and unfortunately remain poorly understood at a molecular level (Wray, 1988; Crawford & Campbell, 1990). Nitrate assimilation inside the cell involves two enzymes, nitrate reductase (NR), which reduces nitrate to nitrite, and nitrite reductase (NiR), which reduces nitrite to ammonium. This overall process is an 8-electron reduction step. Ammonium is then utilised for the biosynthesis of amino acids primarily through the action of glutamine synthetase (GS) and glutamate synthase (GOGAT). As ammonium can be taken up directly (especially in plants growing on acid soils), or come from nitrogen fixation in some species, or from photorespiration and other secondary sources, the NR and NiR steps are the only ones to be nitrate assimilation specific. Nitrate reduction has long been shown to be regulated by many Society for Experimental Biology Seminar Series 49: lnducible Plant Proteins, ed. J. L. Wray. © Cambridge University Press, 1992, pp. 45-77.

46

P. ROUZE AND M. CABOCHE

1 •+* N H j - ^ G S - ^ G I r v ^ - GOGAT - * - G | u 1 Glu (ATP)

/

(F<0

2-oxoglutarate """""TK

Fig. 1. The nitrate assimilation pathway in higher plants. The pathway of nitrate assimilation in the tobacco leaf is illustrated. In some other species an additional cytosolic GS is found in the leaf. The pathway in plant roots is more poorly documented and more variable: GS in roots is mostly cytosolic, and some enzymes such as GOGAT are found as isoforms utilising alternate reducing substrates. T, expected nitrate carrier; NR, nitrate reductase; NiR, nitrite reductase; GS, glutamine synthetase; GOGAT, glutamate synthase; Fd, ferredoxin; Gin, glutamine; Glu, glutamate.

environmental factors, among which are the nitrogen source, light, O 2 / CO2, pH and temperature, and by endogenous factors, such as metabolites and phytohormones (Guerrero et al., 1981). Nitrate reductase is usually considered to perform the rate-limiting step of the nitrate assimilation pathway, owing to the low turnover of NR compared with the other enzymes in the pathway, and to play a central role in regulation, a common function of the first enzyme in a pathway (Beevers & Hage-

Molecular approaches to nitrate reduction

47

man, 1969). Special attention has thus been paid to NR by plant physiologists, biochemists and breeders. The recent isolation of cDNA sequences and genes encoding the NR and NiR apoenzymes from a variety of species now allows us to look at the molecular features of these enzymes and the mechanisms of their regulation. In addition, NR is a very convenient genetic marker, that has the potential to be selected or counter-selected in many organisms, whether they be plants (Caboche & Rouze, 1990; Nussaume et al., 1991) or microorganisms (Unkles et al., 1989). NR-deficient (NR") mutants will not grow on nitrate, but unlike wild-type organisms will not be killed by chlorate, a nitrate analogue that is toxic when reduced by NR. Recent reviews and proceedings from meetings have covered different aspects of nitrate reduction in plants (Rajasekhar & Oelmiiller, 1987; Campbell, 1988; Wray, 1988; Wray & Kinghorn, 1989; Caboche & Rouze, 1990; Campbell & Kinghorn, 1990; Kleinhofs & Warner, 1990; Solomonson & Barber, 1990; Ullrich et al., 1990). In the light of the most recent data from biochemistry, molecular biology and genetics, this chapter will deal first with the structure and function of plant nitrate reductase, and secondly with the molecular analysis of the regulation of nitrate reduction in higher plants. Structure and function of plant nitrate reductases and their encoding genes

Reducing substrates and isoforms Assimilatory nitrate reductase from plants is a soluble, multicentre redox enzyme that catalyses the two-electron reduction of nitrate to nitrite using NADH or NADPH or both as reducing substrate. Despite the initial isolation of NR from soybean nearly 40 years ago (Evans & Nason, 1953), our knowledge of its biochemistry remained limited, due both to the low level of the enzyme in planta and its low stability in vitro (Wray, 1988). NADH:NR (EC 1.6.6.1) appears to be the most common isoform in higher plants and algae, and also the best characterised, mostly resulting from studies on the Chlorella enzyme, which is more abundant and stable than most higher plant NRs (Solomonson & Barber, 1990). Two other NR isoforms have been described in plants. A bispecific NAD(P)H:NR (EC 1.6.6.2) has been found in some mosses (e.g. Sphagnum) and eukaryotic algae (e.g. Chlamydomonas) and in many higher plant species, either as a second isoform in the roots of plants having NADH:NR in their leaves or, in a few cases, as the sole isoform, as in Betula pendula (Friemann et al., 1991), Psophocarpus tetragonolobus and Erythrina senegalensis (Kleinhofs & Warner, 1990). NADPH:NR (EC 1.6.6.3) occurs in

48

P. ROUZE AND M. CABOCHE

fungi and in mosses (Padidam et al., 1991) but has never been found in higher plants. In addition, peculiar isozymes with lower pH optima for activity, and referred to as 'constitutive' NRs as they are seen even in the absence of nitrate, have been found in nitrogen-fixing plants. Thus in soybean, three NR isoforms have been identified: 'constitutive' NADH: and NAD(P)H:NRs and the usual inducible NADH:NR (Streit et al., 1987). Surprisingly, the intracellular localisation of NR in higher plants is still debated (Kleinhofs & Warner, 1990; Solomonson & Barber, 1990). Most evidence argues for a cytosolic location, but it has recently been proposed that NR may also be plasma membrane-bound and be involved in nitrate uptake (Ward etal., 1988). However, barley and N. plumbaginifolia NR~ mutants are still able to accumulate nitrate (Warner & Huffaker, 1989; Caboche & Rouze, 1990), and this argues against a role for NR as a nitrate carrier. Moreover, recent experiments have shown that the plasma membrane-associated NR activity may be soluble NR trapped inside vesicles (Askerlund et al., 1991). Nevertheless, a loose attachment of NR to membranes, which might be of physiological importance, cannot be ruled out. Most higher plants have been shown to possess several genes coding for the NR apoenzyme (Caboche & Rouze, 1990), as partly predicted from the biochemical data. Even Arabidopsis thaliana, chosen as a genetic model for its small genome size, has at least two NR genes (Wilkinson & Crawford, 1991). In contrast, solanaceous species such as Nicotiana spp. and tomato have been shown to possess only one NR structural gene (nia) per haploid genome, and are thus well suited for genetic studies of nitrate reductase function and regulation.

Structure and function of plant NR: a common model Comparison of NR protein sequences obtained from the purified enzyme (Neurospora: Le & Lederer, 1983; corn: Gowri & Campbell, 1989; Chlorella: Solomonson & Barber, 1990) or deduced from the cloning of mRNA and genes encoding plant assimilatory NR apoenzymes from fungi (Aspergillus: Johnstone et al., 1990; Neurospora: Okamoto et al., 1991), algae (Chlamydomonas: Fernandez et al., 1989; Chlorella: Cannons et al., 1991), dicots (tobacco: Calza et al., 1987; Vaucheret et al., 1989; Arabidopsis: Crawford etal., 1988; Cheng etal., 1988; Wilkinson & Crawford, 1991; tomato: Daniel-Vedele et al., 1989; squash: Hyde et al., 1990; spinach: Prosser & Lazarus, 1990; bean: Hoff et al., 1991; birch: Friemann et al., 1991) and monocots (barley: Cheng et al., 1988; Schnorr etal., 1991; corn: Gowri & Campbell, 1989; rice: Choi et al., 1989), shows

Molecular approaches to nitrate reduction

49

them to be very similar (see Fig. 3), whatever the isoform. This argues strongly for a very similar 3-dimensional architecture and catalytic mechanism and thus an essentially common structural and functional model (Caboche & Rouze", 1990; Solomonson & Barber, 1990) can probably be applied to all plant assimilatory NRs. Subunits, domains and activities From biochemical studies after in vitro isolation, NADH:NR appears to be a soluble homodimeric enzyme (Pan & Nason, 1978; Solomonson & Barber, 1990), aggregating into tetramers at high concentration, as has been described in Chlorella and occasionally observed in higher plants. Both subunits (molecular mass 100-110 kDa) have recently been shown to be linked by a disulphide bond which can be reduced without loss of activity (Hyde et al., 1989). There is no direct evidence that a monomer subunit alone is active. Nevertheless, radiation inactivation analysis (Solomonson et al., 1987) provided a target size for Chlorella and spinach NADH:NR of 100 kDa, suggesting that indeed each subunit would behave independently as a functional NADH:NR unit. Three redox cofactors, FAD, haem and a molybdenum cofactor (MoCo), are bound to each monomer in a 1:1:1 ratio, and are used in that order for the catalytic transfer of electrons from NADH to nitrate (Solomonson & Barber, 1990). In addition to the 'full' physiological NADH:NR activity, NR can perform various in vitro activities using different electron donors in place of NADH (nitrate-reducing activities, often referred as 'terminal' activities) or electron acceptors in place of nitrate (dehydrogenase activities, sometimes termed diaphorase activities). They are referred to as 'partial' activities since they utilise only one or two of the three prosthetic groups. Using mostly artificial substrates, these partial reactions have no physiological significance except, perhaps, the reduction by NR of ferric salts (Campbell & Redinbaugh, 1984) and molecular oxygen (Ruoff & Lillo, 1990). Limited proteolysis (Solomonson et al., 1986; Kubo et al., 1988; Notton et al., 1989) and radiation inactivation experiments (Solomonson et al., 1987) have shown that NR is organised in functional domains, as initially suggested by Brown etal. (1981). Each of the prosthetic groups is held in one of three catalytic domains which are linked one to another by protease sensitive hinges. Intersubunit interactions occur via the MoCo domain. After cleavage of either of the two hinges, NADH:NR activity is lost but each domain, or pair of domains, retains its specific functional redox properties. This approach has revealed that each of the partial enzymatic activities of NR can be ascribed to one specific domain, or to

50

P. ROUZE AND M. CABOCHE

two of them linked together. The biochemical analysis of the Nicotiana plumbaginifolia NR~ mutants from the nia complementation group (mutated in the nia structural gene encoding the NR apoenzyme) allows the classification of these mutants into four groups, according to the remaining NR partial activities (see Fig. 2). This classification reinforces the results of the limited proteolysis experiments and reveals which domain is probably affected by each mutation (Cherel et ai, 1990). Thus for dehydrogenase partial activities of NR, the 28 kDa FAD domain alone is a NADH:ferricyanide reductase (NADH:FR) and the FAD domain linked to the haem domain (40 kDa) is a NADHxytochrome c reductase (NADH:CR) as well as a NADH:dichlorophenolindophenol reductase (NADH:DR). As for the nitrate-reducing activities, the methylviologen:nitrate reductase (MV:NR) and the reduced flavin:nitrate reductase (FMN:NR) activities involve the MoCo domain (75 kDa) linked to the haem domain, whereas reduced brom-

ELISA test with 96.9.25

test

PRESENCE OF DEHYDROGENASE AND TERMINAL ACTIVITIES

NADH:CR MV:NR FMN:NR BP:NR

test NO NO NO NO

test

NADH:CR MV:NR FMN:NR BP:NR

NO VES VES YES

NADH:CR MV:NR FMN:NR BP:NR

test YES NO NO NO

NADH:CytcR NO MVHlNR NO FMN:NR NO BPB:NR YES

PREDICTED LOCALISATION OF THE MUTATED DOMAINS (hatched) ON A STRUCTURAL MODEL OF PLANT NITRATE REDUCTASE MONOMER (grey: protein or mRNA absent)

Classified as :

Class 1

Class 2

Complementing with :

None of tne other classes

Classes 3 S 4

Class 3

Class 4

Fig. 2. Biochemical classification of the nia mutants, mutated domains and intragenic complementation. A collection of Nicotiana plumbaginifolia nia mutants (defective for NR apoenzyme) was tested for NR protein and activities (Cherel et al., 1990) and checked for in vivo and in vitro intragenic complementation (Pelsy & Gonneau, 1991). NR protein amounts were measured by a sandwich ELISA test using as first antibody the anti-corn NR monoclonal antibody 96.9.25 (Che'rel etal., 1985) shown to be directed against the haem domain (M. Kavanagh, personal communication; Meyer etal., 1991). The result is shown here as + or —, when a positive or negative ELISA test, respectively, was observed. Many class 4 mutants, most probably frameshift and deletion mutants, complement class 3 but not class 2 mutants.

Molecular approaches to nitrate reduction

51

phenol blue:nitrate reductase (BP:NR) activity would involve the MoCo domain alone. This last conclusion does not come from proteolysis experiments but relies on immunological tests and NR mutant analyses in Nicotiana spp. A monoclonal antibody (ZM 96.9.25), subsequently shown to recognise an epitope on the haem domain (M. Kavanagh et al., unpublished data), was shown to have an opposing effect on NR activities, inhibiting NADH:NR, NADH:CR, FMN:NR and MV:NR while activating BP:NR (Meyer et al., 1987). On the other hand, mutants affected in the haem domain (Meyer et al., 1991) no longer show MV:NR and FMN:NR activities but retain BP:NR activity (Cherel et al., 1990). NR catalytic domains and sequences The three catalytic domains are clearly defined in the NR sequences (see Fig. 3), since each of them shows significant homology with redox proteins of known sequence and function (Galza et al., 1987; Crawford et al., 1988). The 81-83 amino acid (aa) long haem domain, shown by Le & Lederer (1983) to be a member of the cytochrome 65 superfamily, occupies a central position in the approximately 900 aa-long NR polypeptide (the shorter sequence, from Aspergillus, comprised 873 aa and the longer one, from Neurospora, 982 aa). The 270-275 aa-long FAD domain, defined by overall homology with the hydrophilic domain of human NADH:cytochrome 65 reductase (Cb5R) is totally C-terminal in the NR polypeptide. The 430-460 aa MoCo domain is located, as expected, in the /V-terminal half of the NR polypeptide by homology with the MoCo domain of sulphite oxidase (SO). The three domains are linked one to another by two hydrophilic hinge regions, which were shown to be less conserved between NR proteins (Daniel-Vedele et al., 1989; Campbell & Kinghorn,1990) and to be the sites for inter-domain cleavage by proteolytic enzymes (Solomonson et al., 1986; Kubo et al., 1989). Finally, the most N-terminal parts of the NR sequences vary in length from 43 to 119 aa from one NR to another (the extremes again being for Aspergillus and Neurospora, respectively) and have few residues in common, but they are always hydrophilic and share a striking acidic stretch. They show no overall homology with any other known sequence and are thus suggested not to play a role in catalysis (Daniel-Vedele et al., 1989). This domain structure, which is remarkably conserved for every assimilatory NR coding sequence, is a good example of evolution by gene fusion. It is often claimed that this results from exon shuffling, each exon coding for a functional unit (Campbell & Kinghorn, 1990; Dorit et al., 1990). The observed location of introns does not favour such a hypo-

Fig. 3. A : N-TEBM1NAL DOMAIN

fl;;;;;;:.

, , [HRSD 17?] IHSD VP - - -MSER

W-(p] \? 3SlNGlF T S L I IP|NG]VGCS

iGSiiG I j - - • • • • • RlRlRlAD - I S P V H l - GClGlF [T LLY_EJH N D(N]D I DNIPJL ASTJRTLAJ Iv RT

Rlc* Bar I Nauro L E TJSivfNiYfslH 3 SlN|T|N|T N TfSJC [R)V - - AtfMf

B : MoCo DOMAIN .110. .120. .90. .100. •OOP oo o LMHHGF 1 D E G T A D N W I E RTNTFIS M I R L T G K H«P F • N Sm E P Pt LlNlR I TAONW I E R NIF|S[L1I R L T O K H P F N S E P P L S R L U H H O F I T P V L O R H D E G T A D N W I E R N|A]s UfV]R L T G K H P F N S E P P L[N]R L M H H G F I T P V 5]0[|1R D E G T A 0[~ I E R N P S M I R L T G K H P F N S E P P L T R L M H H O F [ L 1 T P V I E R N P S kivlR L T G K H P F N S E|A]P L[N]R L M H H G F I T P V L 0 P R 0 ElylT A D| IvlEH N(A|f]l4yjR L T G K H P F N S E|AJP L T R L M H H G F I T P F A L D P fl D EIAJT A D FlD P R DlDlG TtSiL ^ ^ _JSM I R L T G K H P F 5D(A]R|NJE G T A Dffl [TJFR N P S ( L 1 I R L T G K H P £ "BDEGTA £JJPS|L I R L T G K H _ PIRL i RLTGISIHPF rjLipjTiDirKiTrpioET] S|L|O|KT[ PIR LI I R L T GMH P F

TobI Tom* Squ. Spin Aril 8irch Baan Rio

.160. O H PMKFTMDO L R PMKFTMDO L R pfAlK FTMDQ L RPQlFTMDOL R PMKFTMDO L R P(AR'|FTMD[R1L R P|TC|F TM]EQ L R P W R LlTMD E|L R PTAR LITMDIEIL

.200. AGNR R AGNRR AGNR R AGNRR AGNRR AGNR R AGNRR AGNR R AGNRR AGNRR

.140. t o f VRNHGPVPK[G fVRNHOPVPKA t VRNHG[V)VPKA 1VRN HG PVP§A f v RNHG[H]VP K A rVRNHGPVPKA ifSlM G P V PfHlA _JN H G[A|V PlRG * VRNHGIA VPBG i V R N H G PQJPIS'S V * VRNHGP VPlHlv

210

230.

.240.

K EONMV KOT G F N WG A AlAlV S TjTt^WRG V P L RIA LIL K RCGIVIF L N V C FEGAJD V I I P G G K E O N M V KOX G FNWG A A | A | V S T | T [ V W R G V P L R A L L K R C G I V E I |A L N V C F E G[3 D VlL P G G KEONMVK. G FNWG A AG V S T S VWR[R]v P L|CJD[LJL K R C G I T L I S R K K LNVCFEGAEDLPGG KEON»|K G F N W G ( S ] A ( A 1 V S T S V W R G V P L R D V L K R C G L V g s l s T l K , ^ A L N V. C. F. E G A E O L P G G K E O N M V K f i f S J JG FNWG[S|AG V S T S VWRG VP L © D V L[R]RCG I F S R ] - | K G G A L N V C F EG[^E 0 L P G G K E O N M V K[l G FNWG A AG V S T S V W R G V P LR D V L K R C G I F S R p RlGHAfFiN V C F E G A E O L P G G K E O N M VJ^I G F N W G A g j G V S T S V W R G V g | L R g ] t ] L K R C G I I Y I S R|A[K C • A UBlV C F E G A E O L P G G KEONU' '" F N W G A A G V S T S V W R G ( A R | L R 0 V L | 1 ! R C G i J l M P S K G • A L N V C F E G A E D L P G G K EONM' FNWOAAGVSTSVWRGIARIL HDVLILIRCGIVMISIKI • IK GIOIA L N V C F E G A E D L P G G

g

Fig. 3 (cont.) Tob2 Tomi Squa Spin Ara2 Birch Baan Corn Rlc«1 Barli Chlor Nwiro A«p«r C.SO a.so

Tob2 Toma Squa Spin Ara2 Birch Bun Corn Rlc*1 BarM Chi Of Naur a A«p*r C.SO R.SO

250. 260 •Ol KYGTSIKKE AMDPtAjRD KYGTSIKKE AMD P S R D A M D PJAJR 0 KYGTSIKKE iEFA AMDP[AJRO KYGTS0C KYGTS I K.K.E A M D P S R O AMD P[A]RD J^YGTSP GTSjr AfDD P S R D

.270.

.280.

••

• pmoo • •

IVlA Y M O N G E K L[AjP D H G F P V R lyjA Y M O N G ESS] L S P O H G F P V R L A Y M O N G E|OJ L[A]P D H G F P V R L A Y M Q N G E K L S P D H G 0 P VR

MIL A Y M O N G E — "AIQNGEP

.340. Q AWWY KP EY I AWWY KP EY I AWWYKPE0I AWWYK@EYI G^WWYKPEY I AWWY KP E[H]l 5)WWY K P E Y I AWWY KP E Y I AWWY KP EY I AWWY|H]P E Y I Y KPfDpI

.370. N AWTTOR P NAWTTORP NAWTTORP NAWTTORP

.320. GGRM V GGRMV GGRM V GGRMV GGRMV GGRMV GGRMV GGRMV GGRMV GGRMV

»o oo

E S D|S Y Y H F K D N KWUJKR E SfE SJY Y H 0 K D N KWL KR KWL KR ESEWYVHFKDN KWL KR KWL K R _ _ , NjON KWL KR _ KWL K R [V1V|SN|Q|Q|S|QS1H|Y H[YJK D N KWLKR I V TJPJAE S O N Y Y H F K D N VTKR I V T T A E S O N Y Y H F K D N VTTAESONYYHFKDN PJV TfTvlE sf^N Y Y H FJH]D N VTKGP1S[E]NMY H|V F D N Hi I

.380. .390. JL_Q L R G Y[SjY S G G G K YT L RG Y A Y S O G G K K V T R V E V T L YT L R G Y [ i l Y S G G G K K V T R V E V T L K V T R VEVTF Y TfriiRG Y A Y S G G G{R] YTL1K]GYA YSGGGK |KVTRVEVT YT L R G Y A Y S G G GJRlK V T R V E V T JK V T R V E [ Q T L . G Y A Y S G G G[R| IKVTRVEVT L YA YSGGGK K V T R V E V T L Y TKJ'KIG Y A Y S G G G K |[RT]T R V E V T L Y JITIKJO Y A Y S G G G K I IT R v E v T L |R G Y f ~ i f ] r irTlR[c|EV[s]L

.460. Tob2 Toma Squa Spin Ara2 Birch Baan Corn FUni Bar II Chtor Nwro [ | C.SO

WCWCFWSLEVEVLDLLSAKE WCWCFWSLEVEVLD LLSAKE WCWCFWSLE._. W C W C F W S L E V E V L D L L(G]A K E WCWCFWSLEVEVLDLL WC WC F W S L E V E V L O L L [ | WCWCFWSLE V E V L D Q ] L I G J A K E I WCWCFWSLE V E V L D L L S A K E I

AVRAWDETLNTOPEKL AVRALXIDETLNTQPEKL si T O P E K I. V R A W D ElSJL N T Q P E K L A V R A W D E l iNTOPE TOP E K L A V R A W D EP A V R A WD E AR51N T O P E K L A V R A W 0 EJS[TN T O P E K L

WN VMGMMNNCW IWN VMGMMNNCW i WN[L]MGMMNNCW IWN VMGMMNNCW I WNJTjMGMMNNCW IWN VMGMMNNCW i WN[T>«GM{T]NNCW IWN VMGMMNNCW IWNVMGMMNNCW _WN[DMGMMNNCW

J V MMN

MMN ISJVlLlGMMN * W A HR I HO L K /

Fig. 3 (cont.) C : HINGE 1

GE I 0 I VFEHPTOPONOSGGWUgjRERHl O I VFEHPTfUPON|ESGGWMA[K]E|R

D : HEME DOMAIN

• | K M Y S M S E V R K K | S | S A D S A W I I V H O |H|||YD|A|TRF L K D H P G O T D S •[KMY 3 M 9 E V R K M N S J s l O S A W I I V H O OIAISIRF L K D H P G O V O S - NTIYIT U S E V|T<|K H N S P O l s A W I I V H O I ^ V Y D C T R F L K O H P G G S D S • [KM Y S M S E V|KJK H N | T U D S A W I @ V H 0 Y|N]AJTRF L K D H P G G S D S - KMY s Q s E V R K H N | T | A D S A W I I V H O TIYDCTRFLKOHPGGTDS " ( H N S A D s[5)w I I V H O IYDCTRF L[M]D H P G G[|}D 3 -IKMIFISMSEVIKIKHNSAIEISAWI I VHO I|YDCT(H]FLKOHPQGAO3

wifvjv SE V H K H [ 9 | S | 0 ( D 3 A W | | V I V H Q SEVRKHE1S!KIE]SAWIMVHG V[ET1HITIT1MIE|3 A W[F VIVIDIO

o

-DM

n

L INAOTDCTEEFDAI L INAOTOCTEEFDAI L I NAGTDCTEEFDAI L INAQTDCTEEFDA I NAGTDCTEEFlIlA I N AGTDCT E EF|EJA I

_ NAGTDCTEEFDA I CTRF LKDHPGG[§DS L[T]N AGTDCT E E F | I ) A I NAGTDCTEEFDA I NAGTDCTEEFDAI N A G T D C T E E F DA I

H VYD@T(A|F L K D H P G G A D S H V Y D C T(«|F L K D H P 0 G A 0 S KIVYO[A1TIP1F L K O H P O O A D S

ZE L SE L

E : HINGE 2

PIGIN 3 V H GI3IS PNHSVH P21N3|T]HO(A]S{N

SVHOGS PNIVI3 VHOfAlSN F

Fig. 3 (cont.) F : FAD DOMAIN MO.

.700.

- I E D O V L G L P V G K H I F L C A[V | E 0 Q V . I O L P VG KM I F_L_CAT|v _ SLPVGKHIl_ • I f O O V L G L P V G K H I FLCAlNV ]

|

felv L G L P V G K H MDQVLOLPVQK • EDO V@G L P VG|N]l.^. - pTTilVLGLPfiiGKH I Fj •SDOVLG -SDOVIQLPVGKHIF

LGHIEY LGHIEY LGH I E Y LGHIEYL LGHIEYK L G H [ V ] E Y[L

LCMRAYTPTS1 LCMRAYTPTS1 LCMRAYTPTS; LCURAYTPglS' L CftlR A Y T P T S g L C[L|R A Y T Pg]S T LCMRAYTPTST LCMRAYTPT 3 2 LCURPRITIRITJ

KIFAKKlLAM I AGGTG

TPIVIYOV TP|V|YOV

L G H|V|E Y T G R G N

LGHIEYTGRGN LGH[V]E YTGRGlg L G H M E YTGRGg] LGHIVIEYTGRGN

I|H]»IS|RLAM I U M L A H I AGGl IIAIRIR L A M I IclG Glsl

E V G Y FIEIL V V K I I I Y F KGL1JH P K F

3ITEMY VVYANRTEDD _a)TEMYVVYANRTEDO - PEDETEMYVVYANRTEDO ANRTED! PED[K]TEU[H]VVY ANR T E[i][ PEDETEMYVVYANRTEDD P E O E T E M Y vQ]Y A N R T E [ U D P E 0 E T E M[f]v V Y A N R T E D D P E D[R]T E MY V V Y A N R T E D D OA|VlyjR|D O P E D M T E M [ H T ] V Y A N R T E D D dfsy U R D O P E DLTJT E MJH LJV Y » N R T E D D o AIVILIRID O P E D E T E M I H L I V Y A N R[S|E D D LUFIANIT

.190. Tob2 Torn* Squa Spin Aral Ar.I •Irch Baan Coin Rlcal Barli CMant Nauro Aapar

I|PI£IR|V|KVWYVVO(PJS I VWYVVOES I KVWYVVOE3 I VY VV VYVV

UKVWYKV

EKIL K VWY vv JD R L K VWY V ]RLKVWYV R LKVWYVI W A A E Y P D R L K VWYV

|SHT|T L A L A C G P P P M lT L A L A C G P P P M LALACGPP|ApJ

OFAVINIPN L E K M OFAQJNJPN LEKM OFAVOPN LEK OFAVOPN L© ~ Lll O F A[QO P N L E LALAOQPPPM |£JLASACGPPPM O F A V O P N L E [EEIA VIRILIN I E OF A V O P N L E .., F A[TS|P N L E K |0T L» L 0 C G P P P M Q D D T L A L A C Q P P P M l IKIF £ A3S PN L E G D D T L A L A C O P P P M IKIFAM SIPN LE ODTLALACGPPPM

l

oHLALACOSpg^l

56

P. ROUZE AND M. CABOCHE

thesis. Indeed, there are no introns between domains and there is no indication that they would cut between functional blocks. On the contrary, there is an example of two introns cutting in the proximity of a codon for an essential residue (His587). Furthermore, intron number and position in NR and homologous proteins (Cb5R) show considerable variation between distantly related organisms. Most higher plant NR genes have three introns of variable size but conserved position (Caboche & Rouze, 1990), although the Arabidopsis nia2 gene has no third intron (Wilkinson & Crawford, 1991), and the barley gene has only the second one (Schnorr et ai, 1991). In fungi the Aspergillus niaD gene has six introns, whilst the Neurospora nit-3 gene retains only the last one. The Chlamydomonas gene has at least two introns and the mammalian Cb5R gene has eight introns. Moreover, there is no common position between these different sets of introns. Haem domain: 3-dimensional structure and site-directed mutants From active site labelling experiments and from sequence comparison of NRs with homologous proteins, functionally important residues can be predicted and localised on the NR sequences.

Caption to Fig. 3. Domains, sequences and conserved residues of plant nitrate reductases and homologous redox proteins. The sequences were aligned and gaps (dashes) were introduced when necessary to maximise homology and keep homologous segments in blocks. Identical residues for a given position are boxed. The positions that are conserved for all NRs are shown above the alignment by a circle that is filled if the position is also conserved for the homologous protein. Numbering is given according to the tobacco NR sequence. The cleavage sites of trypsin and Staphylococcus aureus V8 protease, as observed for spinach NR, are indicated by arrows above the alignment. Intron positions are given by flags below the alignment: 'a' and 'n' indicate introns from Aspergillus and Neurospora NRs, 'p' indicates introns from higher plants NRs, 'c' indicates introns from Chlamydomonas NR and 'h' indicates introns from mammalian Cb5Rs. Tob2, tobacco nia2 NR; Toma, tomato NR; Squa, squash NR; Spin, spinach NR; Aral and Ara2, Arabidopsis NR1 and NR2; Barll, barley NADH:NR; Ricel, Rice NR1; Chlor, Chlorella NR; Neuro, Neurospora NR; Asper, Aspergillus NR; C.SO and R.SO, chicken and rat liver sulphite oxidase; b5, bovine cytochrome bs; CR, human cytochrome b5 reductase. References for the sequences are given in the main text.

Molecular approaches to nitrate reduction

57

In the haem domain, 11 positions are conserved in every member of the cytochrome 65 superfamily, among which is the characteristic stretch HPGG. They are two histidines, two aromatic amino acids (Trp, Phe) and seven small amino acids occurring in turns (Pro, Ala, Gly). From the X-ray crystallography coordinates of bovine cytochrome 65 (Mathews et al., 1971), we have established the 3-dimensional structure of the tobacco NR haem domain by homologous replacement of amino acids and free energy minimisation, and showed it to be very similar to the model cytochrome 65 (Meyer et al., 1991). The two conserved histidines (His39, His63) are shown to bind to the haem Fe. Replacement of His63 in cytochrome 65 by Ala results in a protein which cannot bind haem (Beck von Bodman et al., 1986). Sequencing of the nia gene from some Nicotiana plumbaginifolia NR-apoenzyme mutants presumably affected in the haem domain (Cherel et al., 1990) has shown that in one of them (maE56) the first Fe-binding histidine, His564 (//PGG), was replaced by an asparagine (/VPGG), a direct demonstration of the essential role of that histidine (Meyer et al., 1991). Some charged residues on the surface of cytochrome b5 were being shown to be involved in ionic interaction with the reducing partner, cytochrome 65 reductase (Hackett & Strittmatter, 1984). They are often replaced in the NR haem domain by hydrophobic amino acids, suggesting that the interaction with the FAD domain would be hydrophobic rather than ionic. One can speculate that ionic interaction would be necessary to allow long-range interaction between separate molecules (cytochrome 65 and cytochrome 65 reductase), whereas short-range hydrophobic interactions would be more appropriate to allow close association of two domains which are covalently linked, [see note 1 added in proof]. FAD domain: functional sites and conserved residues Several residues have been demonstrated to be involved in the binding site for NADH in Cb5R or in NR: a lysine (Hackett et al., 1988), a serine (Yubisui et al., 1991), a cysteine (Hackett et al., 1986; Barber & Solomonson, 1986) and an arginine (Baijal & Sane, 1988). The essential lysine was identified in Cb5R as LysllO (Hackett et al., 1988). There is only one conserved lysine in the FAD domain of NR (Lys717 in tobacco NR) which precisely occupies a position homologous to LysllO. Near this lysine, Serl27 was shown from analysis of natural and site-directed mutants to play an essential structural role in the NADH-binding site of Cb5R, probably through its hydroxyl side chain (Yubisui et al., 1991). It is interesting to observe that the homologous position in NR is always occupied by either a serine (Ser734 in tobacco)

58

P. ROUZE AND M. CABOCHE

or a threonine, both of which provide an aliphatic hydroxyl side chain. This strongly argues that Lys717 and Ser/Thr734 are involved in the NADH binding site, and consequently play identical roles to their Cb5R counterparts. Only one cysteine (Cys273 in human Cb5R, Cys876 in tobacco NR) is conserved among every member of the family, and was thus predicted to be the essential one (Crawford et al., 1988). Nevertheless, labelling experiments suggested a different location for this cysteine: Cys283 in Cb5R (Hackett etal., 1986) and a cysteine outside the FAD domain in NR (Barber & Solomonson, 1986). A more recent site-directed approach with cytochrome b5 reductase (Shirabe et al., 1991) comes to the conclusion that indeed Cys273, the sequence-conserved cysteine, is in close contact with NADH and would facilitate catalysis. However, this mutation does not completely inactivate the enyme, arguing against a direct role for this thiol in catalysis. Cys876 is located close to the C-terminus of the FAD domain, whilst Lys717 and Ser734 are in the N-terminal half of the domain. This suggests that folding is necessary to form the nucleotide binding sites. From X-ray analysis of ferredoxin NADP:reductase (FNR), NR is suggested to be a member of a new family of flavoenzymes (Karplus et al., 1991). A lysine homologous to Lys717 in NR is essential for activity of all members of the family and in the case of FNR has been shown to bind NADH, albeit within the FAD subdomain. Baijal & Sane (1988) have shown that arginine-specific reagents inactivate NADH:NR and the partial activities of the Amaranthus NR. Inactivation of NADH dehydrogenase activity occurs more slowly than that of FMN:NR activity and can be prevented by NADH. Similarly, they have shown that inhibition of the dehydrogenase activity of NR by a histidine reagent is also protected by NADH (Baijal & Sane, 1987). This suggests that at least one arginine and one histidine would be involved in the NADH-binding site. One histidine, His683, and two arginines, Arg697 and Arg861 in tobacco NR, are conserved in the FAD domain and are thus putative candidates. Group-specific modifications of Cb5R have shown similarly that a tyrosine participates in FAD binding (Strittmatter, 1961). Two tyrosines are conserved (Tyr719 and Tyr757 in tobacco) both of which lie in the Nterminal half of the FAD domain. In addition, it would be of special interest to pinpoint those residues which determine the choice of reducing substrate. From the structure of FNR, one may predict that residues of the NAD(P)H subdomain numbered 842 and 843 in the NR tobacco sequence (Fig. 3) are probably important for NADH or NADPH specificity. X-ray analysis of the Cb5R 3-dimensional structure is in progress

Molecular approaches to nitrate reduction

59

(Yubisui et al., 1991) and this knowledge will be of great help in obtaining further insights into the structure-function relationships of NR. A similar 3-dimensional structure is expected, because of the mean 50% sequence homology between Cb5R and the NR FAD domain. Active cytochrome b5 (Beck von Bodman et al., 1986) and Cb5R (Shirabe et al., 1989) as well as the NR haem domain (Cannons et al., 1991) and the NR FAD domain (Hyde & Campbell, 1990) have been expressed in Escherichia coli, alone or linked to the other NR domains (M. Kavanagh et al., unpublished data) and also in Saccharomyces cerevisiae (Truong et al., 1991). A site-directed mutagenesis approach is now possible which will show the effect of replacing the tagged and conserved residues in these domains, as has already been done for the haem axial His (Sligar et al., 1987) and for the Ser and Cys in the NADH • binding site (Yubisui et al., 1991; Shirabe et al., 1991). MoCo, MoCo synthesis and MoCo domain active sites The molybdenum cofactor from two animal molybdoenzymes, sulphite oxidase (SO) and xanthine dehydrogenase (XDH), has been shown to be a complex between molybdenum and a unique pterin component called molybdopterin (Kramer et al., 1987). The structure of the molybdopterin was thought to be the same for all molybdoenzymes, except nitrogenase, as it was shown that upon acidification molybdoenzymes from diverse sources can reconstitute NR activity in extracts from MoCo-deficient mutants of Neurospora crassa (Nason et al., 1971). However, we now know that the MoCo of bacterial molybdoenzymes contains molybdopterins which are substituted by nucleotides (Johnson et al., 1990; Crawford & Campbell, 1990). Furthermore, the Mo ligands as well as the redox state of the pterin ring may differ between molybdoenzymes, as observed between SO and XDH (Gardlik & Rajagopalan, 1990). MoCo from plant NR appears to be mostly similar to MoCo from animal SO, as indicated by EXAFS spectroscopy of the Mo centre of both enzymes (see Fig. 4). In agreement with cofactor identity, a high sequence homology is observed between NR and SO MoCo domains (Neame & Barber, 1989), whereas no clear homology with any other molybdoenzyme, including XDH and NR from E. coli, has been found [see note 2]. The molybdenum cofactor is often assumed to be involved in dimerisation of NR, either as a bridge as in an early structural model (Pan & Nason, 1978), or more indirectly. This hypothesis is no longer valid, since it has been shown that in bakers yeast, an organism which lacks the MoCo biosynthetic pathway, a transgenic, MoCo-less tobacco NR is nevertheless dimeric (Truong et al., 1991). This is in agreement with the observa-

60

P. ROUZE AND M. CABOCHE

A

O

HN

H

^ j . ^ ^ j — C=C-CHOH-CH2OPO5

/ \

HoN

HN

/

N

N H

O

H

II

J*

^.f N

X

/ \ , / N

H

s s

— C=C-CHOH-CH2OPO^

s s

x JHo'

Fig. 4. Structure of molybdopterin and MoCo and its suggested attachment to plant NR apoenzyme. This model is adapted from Kramer et al. (1987) for the molybdopterin, Gardlik & Rajagopalan (1990) for the pterin reduction state (shown here as a 5,6-dihydropterin, one of the three possible structures) and Neame & Barber (1989) for the hypothetical thiol ligand from the side chain of Cysl80 of tobacco NR. A, Molybdopterin. B, MoCo. tion that NR is also dimeric in most Nicotiana plumbaginifolia NR~ cnx mutants (Marion-Poll et al., 1991) that are affected in the biosynthetic pathway of MoCo. This pathway is still poorly understood. Genetic analysis of MoCo-deficient NR mutants in N. plumbaginifolia has shown that at least six independent loci (cnx A to F) are involved. Mutants in the likely final Mo-insertion step of the pathway (cnxA) can be 'repaired' by high molybdate levels and show high amounts of NR protein. NR proteins from other cnx mutants appear to be unstable, show a modified structure and are often degraded. The phenotype of these mutants is similar to some nia mutants affected in the MoCo domain. This suggests that the integrity of the MoCo domain, and not only MoCo itself, may be required for stability of the NR dimer. A single cysteine (Cysl86 in chicken liver SO, Cysl80 in tobacco NR) is conserved in the MoCo domain of every NR and SO sequenced to date (Barber & Neame, 1990), and this residue is found within the very con-

Molecular approaches to nitrate reduction

61

served sequence TLXCAGNRR(K/S)E(Q/M) which appears to be a signature of this molybdoenzyme family. From NR reconstitution experiments with the nit-] MoCo-deficient Neurospora mutant, it was expected that a thiol would be involved in MoCo binding (Wahl et al., 1984). Barber & Neame (1990) suggested that Cysl86 would play this role by directly liganding molybdenum in the cofactor. The experiment of Baijal & Sane (1988) shows that one arginine (or more) is probably involved in the activity of the MoCo domain. If it is, it could be one (or more) of the four conserved arginines of the domain, two of which are in the conserved sequence encompassing Cysl86 (as above). One might expect to find at least two binding sites in the MoCo domain, one for MoCo itself and one for nitrate. Unfortunately, the structural data for molybdoenzymes are too limited at present to allow further prediction to be made. Furthermore, attempts both to express NADH:NR in an active form, and the NR MoCo domain, in E. coli have so far been unsuccessful, the expressed polypeptide being shorter than expected (M. Kavanagh et al., unpublished data). When transfected in yeast by means of an appropriate vector, the NR cDNA is translated into a full-length polypeptide and is assembled into a dimeric apoenzyme that shows NADH:NR activity after complementation by exogeneous MoCo (Truong et al., 1991). This is nevertheless a rather inefficient process; as a consequence, testing the role of particular sequences or amino acids in plant NR through a reverse genetic approach requires to be done. Intragenic complementation between mutated NRs Intragenic complementation has been observed between N. plumbaginifolia nia mutants (Pelsy & Gonneau, 1991). NADH:NR activity was restored in vivo in extracts from Fl crosses between some nia mutants and in vitro by mixing extracts from each of these mutants, although the levels of activity obtained were lower than wild-type. Complementation only occurred between partners from different mutant classes (determined according to Cherel et al., (1990)), mutated in different redox domains (see Fig. 2). This suggests that electron transfer from NADH to nitrate can occur through an interaction between the redox domains of different NR molecules. One may conclude that electron transfer need not necessarily take place within a single NR subunit. Regulation of the nitrate reduction pathway Availability of nucleic acid probes for the NR and NiR mRNA and of specific antibodies for the proteins, in addition to measurements of activi-

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ties, have provided the necessary tools to allow the study of when and at which level (transcription/translation) nitrate reduction is regulated.

Regulation by nitrate availability and light It has been established that the activity of both NR and NiR is induced by nitrate in plants (Rajasekhar & Oelmiiller, 1987), even if in a few species constitutive NR and NiR isoforms are found in addition to the inducible ones. Nitrate supply leads to an increase in the steady-state level of mRNA encoding NR in a variety of plant species and tissues (Cheng et al., 1986, 1991; Crawford etai, 1986; Smarrelli etai, 1987; Galangau et al., 1988; Gowri & Campbell, 1989; Hamat et al., 1989; Hoff et al., 1991; Friemann et al., 1991). This in turn results in NR protein synthesis and increased activity, shortly after nitrate supply to the roots, and after a lag in leaves (Melzer et al., 1989). Where performed, nuclear runoff transcription assays have indicated that the high mRNA level results from increased transcriptional activity (Callaci & Smarrelli, 1991; Lillo, 1991). Similarly, it has been demonstrated that mRNA for nitrite reductase increases rapidly following nitrate supply in several species including spinach, corn, rice, mustard and N. plumbaginifolia (Gupta & Beevers, 1985; Ogawa & Ida, 1987; Back et al., 1988; Kramer et al., 1989; Schuster & Mohr, 1990; Faure et al., 1991). The 5' promoter sequence of the spinach NiR gene has been shown to confer nitrate-inducibility to a GUS reporter gene (Back etai., 1991) demonstrating with certainty that nitrate controls the transcription of the NiR gene. Thus nitrate co-regulates the levels of NR and NiR transcripts in plants (Faure et al., 1991) as in fungi, where nitrate regulation is controlled by a pathway-specific positive regulator, the product of the nirA gene in Aspergillus (Burger et al., 1991) and of the nit-4 gene in Neurospora (Fu et al., 1989). These regulatory genes were first identified by mutant analysis and have recently been cloned. Unfortunately, up to now no regulatory mutant has ever been obtained in higher plants, and no regulatory gene has been cloned. In fungi, some NR" mutants show constitutive expression of NR protein and NiR activity in the absence of nitrate. This observation has led to the autogenous regulation hypothesis (Cove & Pateman, 1969), which suggests that NR interacts with the positive regulator protein nirA/nit-4 when nitrate is absent, thus suppressing the activation of the promoter (Marzluf, 1981). In contrast, it has been demonstrated in N. plumbaginifolia, that nitrate induction of transcription of the NR and NiR genes relies neither on NR activity (Deng et al., 1989a) nor on NR protein, since mRNA for NiR as well as mutant NR is still induced by nitrate in

Molecular approaches to nitrate reduction

63

NR~ plants, even in those where no NR protein is found (Pouteau et al., 1989; Faure et al., 1991). Similarly, no barley NR~ mutant was shown to be constitutive for NR expression (Melzer et al., 1989). Thus, although NR autoregulation is a valid model in fungi, a similar model is unlikely to apply to higher plants. When tomato plants are nitrogen-starved, both NR protein and activity show a dramatic decline in less than 24 h while NR mRNA remains at a high level for at least 12 days (Galangau etal., 1988). This indicates either a block in translation or a rapid degradation of NR protein as the result of starvation, which in turn may be explained by a positive effect, on either process, by the nitrogen source. Oaks et al. (1988) observed that in corn plantlets, NR protein was present at very low nitrate level, but that NR activity appeared only when the nitrate supply was increased. Both observations, and others (Small & Wray, 1980; Schuster & Mohr, 1990), suggest that in addition to the transcription control, nitrate may also be involved in the regulation of NR and NiR activities by acting either on translation of their mRNA, or on stability and activation of the proteins, or even on cofactor availability, as suggested in Chlorella for MoCo (Zeiler & Solomonson, 1989). Light is also well known to be involved in NR and NiR expression (Duke & Duke, 1984; Rajasekhar & Oelmuller, 1987) but its role is intricate and remains far from clear, since several distinct mechanisms are probably acting in concert. As extreme examples, blue light has been shown to reactivate the NR protein (Aparicio et al., 1976) and strong red light photooxidises plastids, destroying their ability to send a 'plastidic signal' that appears to be involved in NR and NiR regulation (Oelmuller et al., 1988). Neither aspect will be reviewed here. Light induces NR and NiR protein synthesis by increasing mRNA steady-state levels. This is at least in part a transcriptional process separate from nitrate induction, both factors being required for full induction although the relative importance of one or the other appears to vary with the species, the tissue and the experimental setting. In cotyledons and in etiolated plantlets, the light effect on NR and NiR activities and transcript levels was suggested to be mediated by phytochrome (Sharma & Sopory, 1984; Rajasekhar et al., 1988; Gowri & Campbell, 1989; Melzer et al., 1989; Schuster & Mohr, 1990), significant variations being observed between isoforms and species (Seith etal., 1991). Cytokinin was shown to enhance light induction of NR gene transcription in etiolated barley leaves (Lu et al., 1990). In light-grown plants treated with nitrate, the levels of NR and NiR mRNAs, proteins and activities slowly decrease after 2 days in the dark (Deng et al, 1990; Bowsher et al., 1991). When plants receive light again, NR and NiR are induced following a sharp

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increase in specific mRNAs (Deng et al., 1990; Cheng et al., 1991; Faure et al., 1991), the response in green tissues appearing to be phytochromeindependent (Gowri & Campbell, 1989; Melzer etal., 1989). Induction of transcription of NR and NiR genes by light is independent of NR protein structure or activity, since it was also observed in NR" mutants (Faure et al., 1991). The differential involvement of phytochrome in the light effect was confirmed in tomato by comparing the induction of NR and NiR in etiolated or light-grown wild-type plants and aurea phytochrome-deficient mutants (T. Becker, personal communication). It has been reported that recessive copl mutants from Arabidopsis that do not etiolate in darkness show a high level of nia2 mRNA in the dark (Deng et al., 1991a). This opens a way to look at the light transduction process, and tells us that in this process a repressor is acting during darkness, maybe in addition to activator(s) acting in the light. NR~ plants deficient in the structural gene may be complemented by transformation with a wild-type NR gene. This has been done in tobacco and N. plumbaginifolia using either of the tobacco nia genes (Vaucheret et al., 1990), in N. plumbaginifolia using the tomato nia gene (Dorbe et al., 1992), and in Arabidopsis, using the homologous nia2 gene (Wilkinson & Crawford, 1991), resulting in restoration of NR activity that was always partial compared to wild-type plants. NR~ N. plumbaginifolia plants have also been transformed with a tobacco cDNA under the control of a 35S-CaMV constitutive promoter (Vincentz & Caboche, 1991). These plants were viable and fertile and grew as well as the wild-type plants in a greenhouse. They expressed from one-fifth to three times the wild-type NR activity in their leaves. The chimaeric NR gene in these plants is no longer regulated by nitrate and light and expresses NR mRNA constitutively (Vincentz & Caboche, 1991). On the other hand, the tomato nia gene transferred to NR~ plants was shown to be correctly regulated by nitrate and light (Dorbe et al., 1991), offering additional proof that nitrate and light control transcription. However, NR protein and activity decreased in the dark by a factor of 3 to 4 in plants expressing NR mRNA constitutively (Vincentz & Caboche, 1991). This observation supports previous reports in higher plants (Remmler & Campbell, 1986) which suggest that light, like nitrate, also acts post-transcriptionally.

Circadian rhythm and regulation by nitrogen metabolites Diurnal variation of nitrate reductase activity was first observed many years ago (Hageman et al., 1961), and was shown to be maintained for several days in continuous light, but to disappear during continuous dark-

Molecular approaches to nitrate reduction

65

ness (Lillo, 1984). When measurements were made in tobacco and tomato leaves, a dramatic oscillation of NR mRNA was observed, from a nearly undetectable level during the day to a transient sharp peak at the end of the night, resulting in a concomitant rhythm in NR protein levels (Galangau et al, 1988). A similar rhythm is observed for both NR mRNAs in Arabidopsis, the NR2 transcript accumulating slightly earlier than that for NR1 (Cheng et al., 1991). The observed circadian rhythm in NR transcript level was retained in continuous light and faded slowly in the dark (Deng et al., 1990). Other studies have shown that in N. plumbaginifolia leaves NiR mRNA fluctuates, albeit to a lower extent, in phase with NR mRNA (Faure et al., 1991). A circadian rhythm in mRNAs for NR and NiR has also been observed in corn shoots, but not in corn roots (Bowsher et al., 1991) and runoff experiments suggest that the increase in NR mRNA comes from a change in transcription rate (Lillo, 1991). The oscillation of NR activity was not totally coincident with the oscillation of NR protein (Galangau et al., 1988, Lillo, 1991), indicating an (in)activation process of the NR protein in addition to the regulation of the mRNA level. The day-night fluctuation of NR mRNA is no longer seen in plants fed on tungstate instead of molybdate, and which contain an inactive NR (Deng et al., 1989a), nor in NR" plants, irrespective of the type of mutation (Pouteau etal., 1989). These plants show not only high constitutive levels of NR mRNA but also of NiR mRNA (Faure et al., 1991), showing again that both genes are co-regulated. Expression of NR and NiR genes in NR" transgenic plants complemented for NR activity either by a NR gene controlled by its own promoter (Dorbe et al., 1992) or by a NR-coding sequence controlled by a constitutive promoter (Vincentz & Caboche, 1991) indicates that the circadian rhythm of NR mRNA is controlled at the transcriptional level. Moreover, these experiments taken together suggest that the 3 kb upstream sequence from the tomato NR gene contains all the necessary as-acting elements to allow light, nitrate, and circadian rhythm regulation. Overexpression of NiR and NR mRNAs was always observed in plants deficient for NR activity. Inhibition of NiR activity in tobacco by expression of an antisense mRNA leads similarly to overexpression of NR mRNA (H. Vaucheret, personal communication). These results demonstrate that nitrate reduction is involved in control of NR and NiR gene expression, and suggest that a reduced nitrogen metabolite may down-regulate the transcription of both genes. In agreement with this hypothesis, it was observed that the concentration of amino acids varies in tobacco leaves according to a circadian rhythm, the glutamine pool showing the most important variations (see Fig. 5), being highest during

66

P. ROUZE AND M. CABOCHE 120

16

24

32

48

56

Time (hours) Fig. 5. The steady-state levels of NR mRNA and glutamine in tobacco leaves follow a circadian rhythm in opposite phase. Tobacco plants were grown with 10 mM nitrate as sole nitrogen source in a light/dark regime (16/8 h; 25/18 °C) until the 6-leaf stage. After a final dark period (0-8 h), plants were kept in continuous light and constant temperature (25 °C, 856 h). Leaves were sampled at intervals for northern blot analyses of NR mRNA and measurements of the glutamine content. The effective last night of the light/dark regime is shown by a black rectangle at the top of the graph. The dark rectangles indicate the positions of the night periods if the light/dark regime had been maintained. Adapted from Deng et al., 1991ft.

the day when the NR mRNA was very low, and lowest during the night when NR mRNA increased (Deng et al., 19916). Furthermore, spraying tobacco leaves with phosphinothricine to inhibit GS activity led to a large increase in NR mRNA in those leaves in which the glutamine level was falling and ammonium was accumulating (Deng et al., 19916). Finally, it has been shown in tobacco roots (Deng et al, 19916) and in squash cotyledons (Martino & Smarrelli, 1989) that exogenous glutamine supply reduces NR mRNA levels. In higher plants ammonium does not repress nitrate induction as has been observed in many lower organisms, and regulation by reduced Nmetabolites is presently open to debate. In fungi, nitrogen catabolite repression is well established, and is performed by one or several Nmetabolites, the most likely candidate being glutamine (Marzluf, 1981). Genetic analysis in both A. nidulans and N. crassa has shown that nitro-

Molecular approaches to nitrate reduction

67

gen catabolite repression involves a positive regulator encoded by the areA and nit-2 genes, respectively, members of the zinc-finger transcription factor family (Fu & Marzluf, 1990; Kudla et al., 1990). If Nmetabolites also regulate nitrate reduction in higher plants, one would expect that similar regulatory genes may be found. Conclusion and prospects The relationship between NR structure and function is now better understood, thanks to biochemical, genetic and sequence data. Advances in the future will depend on the application of techniques such as reverse genetics and site-directed mutagenesis, coupled with the ability to produce large amounts of the protein in E. coli, yeast or some other organism. Determination of the structural elements responsible for NR stability, for activity modulation, reducing substrate selectivity, enzyme turnover and affinity for each substrate is of fundamental and practical interest. Besides its intrinsic importance in plant physiology, agriculture, biotechnology and the environment, nitrate reductase, as a soluble protein with several covalently linked redox domains, constitutes an interesting model of a short electron transfer chain, well suited to study protein interactions involved in electron transfer between redox centres. Recent molecular studies on the regulation of nitrate reduction have already provided important lessons, some of them quite unexpected. It is clear that nitrate and light are involved in the control of transcription of the NR and NiR structural genes, although light induction covers several distinct phenomena and deserves further analysis. In addition to these two factors, metabolic regulation by a reduced N-metabolite, perhaps glutamine, is also involved in the control of transcription of both genes. This regulation is probably part of the cross-talk mechanism between nitrogen and carbon metabolism (Deng et al., 19896), and as such deserves specific and close attention. Since transcription of the NR and NiR genes appears to be co-regulated by all three factors, it is likely that similar as-acting, and identical trans-acting, factors remain to be found. Some of the mechanisms of light regulation of NR genes appears to be shared with several other light-regulated genes. With the exception of autoregulation by NR, transcriptional regulation of nitrate reduction by nitrate and N-metabolites in higher plants bears some resemblances to regulation in fungi, for which a regulatory model has been propounded and regulatory genes isolated. Having obtained clear evidence of transcriptional regulation, it is very puzzling to observe that transgenic tobacco plants, relying on a constitutively expressed NR gene, are able to grow and develop in a green-

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house as well as the wild-type plant. Several explanations can be offered to account for this result: (i) NR and NiR activities are not limiting, their excess has no deleterious effects and the rate of nitrate assimilation is controlled by availability of their substrate. Hence enzyme regulation would not be essential. In partial agreement with this explanation it has been observed that NR~ mutant or transformed plants with very low NR activity are able to grow, albeit more slowly, (ii) Fine tuning of the posttranscriptional regulation of NR and NiR dominates or compensates for coarse transcriptional regulation. In both hypotheses one has to explain why transcriptional control is dispensible and why it has been kept during evolution. One suggestion would be that coarse transcriptional control plays a major role in stress or near-stress conditions, such as obtain under low nutritional supply, high or low light, drought or cold, and plays only a minor role in a controlled environment such as obtains in a greenhouse, where nutrient supply, light and temperature have been optimised for growth. References Aparicio, P.J., Roldan, J.M. & Calero, F. (1976). Blue light photoreactivation of nitate reductase from green algae and higher plants. Biochemical and Biophysical Research Communications 70, 1071-7. Askerlund, P., Laurent, P., Nakagawa, H. & Kader, J.-C. (1991). NADH-ferricyanide reductase of leaf plasma membranes. Partial purification and immunological relation to potato tuber microsomal NADH-ferricyanide reductase and spinach leaf NADH-nitrate reductase. Plant Physiology 95, 6-13. Back, E., Burkhart, W., Moyer, M., Privalle, L. & Rothstein, S. (1988). Isolation of cDNA clones coding for spinach nitrite reductase: complete sequence and nitrate induction. Molecular and General Genetics 212, 20-6. Back, E., Dunne, W., Schneiderbauer, A., de Framond, A., Rastogi, R. & Rothstein, S.J. (1991). Isolation of the spinach nitrite reductase gene promoter which confers nitrate inducibility on GUS gene expression in transgenic tobacco. Plant Molecular Biology 17, 9-18. Baijal, M. & Sane, P.V. (1987). Chemical modification of nitrate reductase from Amaranthus. Indian Journal of Biochemistry and Biophysics 24, 75-9. Baijal, M. & Sane, P.V. (1988). Arginine residue(s) at the active site(s) of the nitrate reductase complex from Amaranthus. Phytochemistry 27, 1969-72. Barber, M.J. & Neame, P.J. (1990). A conserved cysteine in molybdenum oxotransferases. Journal of Biological Chemistry 265, 20912-15.

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Barber, M.J. & Solomonson, L.P. (1986). The role of the essential sulfhydryl group in assimilatory NADH:nitrate reductase of Chlorella. Journal of Biological Chemistry 261, 4562-7. Beck von Bodman, S., Schuler, M.A., Jollie, D.R. & Sligar, S.G. (1986). Synthesis, bacterial expression, and mutagenesis of the gene coding for mammalian cytochrome b5. Proceedings of the National Academy of Sciences (USA) 83, 9443-7. Beevers, L. & Hageman, R.H. (1969). Nitrate reduction in higher plants. Annual Review of Plant Physiology 20, 495-522. Blevins, D.G. (1989). An overview of nitrogen metabolism in higher plants. Recent Advances in Phytochemistry 23, 1-41. Bowsher, C.G., Long, D.M., Oaks, A. & Rothstein, S.J. (1991). Effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots. Plant Physiology 95, 281-5. Brown, J., Small, I.S. & Wray, J.L. (1981). Age-dependent conversion of nitrate reductase to cytochrome c reductase species in barley leaf extracts. Phytochemistry 20, 389-98. Burger, G., Tilbur, J. & Scazzocchio, C. (1991). Molecular cloning and functional characterization of the pathway-specific regulatory gene nir A, which controls nitrate assimilation in Aspergillus nidulans. Molecular and Cellular Biology 11, 795-802. Caboche, M. & Rouz6, P. (1990). Nitrate reductase: a target for molecular and cellular studies in higher plants. Trends in Genetics 6, 187-92. Callaci, J.J. & Smarrelli, J., Jr. (1991). Regulation of the inducible nitrate reductase isoform from soybeans. Biochimica et Biophysica Ada 1008, 127-30. Calza, R., Huttner, E., Vincentz, M., Rouz6, P., Galangau, F., Vaucheret, H., Cherel, I., Meyer, C , Kronenberger, J. & Caboche, M. (1987). Cloning of DNA fragments complementary to tobacco nitrate reductase mRNA and encoding epitopes common to the nitrate reductases from higher plants. Molecular and General Genetics 209, 552-62. Campbell, W.H. (1988). Nitrate reductase and its role in nitrate assimilation in plants. Physiologia Plantarum 74, 214-19. Campbell, W.H. & Kinghorn, J.R. (1990). Functional domains of assimilatory nitrate reductases and nitrite reductases. Trends in Biochemistry 15, 315-19. Campbell, W.H. & Redinbaugh, M.G. (1984). Ferric-citrate reductase activity of nitrate reductase and its role in iron assimilation by plants. Journal of Plant Nutrition 7, 799-806. Cannons, A.C., Iida, N. & Solomonson, L.P. (1991). Expression of a cDNA clone encoding the haem-binding domain of Chlorella nitrate reductase. Biochemical Journal 278, 203-9. Cheng, C.-L., Acedo, G.N., Dewdney, J., Goodman, H.M. & Conkling, M.A. (1991). Differential expression of the two Arabidopsis nitrate reductase genes. Plant Physiology 96, 275-9.

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Cheng, C.-L., Dewdney, J., Kleinhofs, A. & Goodman, H.M. (1986). Cloning and nitrate induction of nitrate reductase mRNA. Proceedings of the National Academy of Sciences (USA) 83, 6825-8. Cheng, C.-L., Dewdney, J., Nam, H.-G., den Boer, B.G.W. & Goodman, H.M. (1988). A new locus (NIA1) in Arabidopsis thaliana encoding nitrate reductase. The EMBO Journal 7, 3309-14. Cherel, I., Gonneau, M., Meyer, C , Pelsy, F. & Caboche, M. (1990). Biochemical and immunological characterization of nitrate reductasedeficient nia mutants of Nicotiana plumbaginifolia. Plant Physiology 92, 659-65. Cherel, I., Grosclaude, J. & Rouze, P. (1985). Monoclonal antibodies identify multiple epitopes on maize leaf nitrate reductase. Biochemical and Biophysical Research Communications 129, 686-93. Choi, H.K., Kleinhofs, A. & An, G. (1989). Nucleotide sequence of rice nitrate reductase genes. Plant Molecular Biology 13, 731-3. Cove, D.J. & Pateman, J.A. (1969). Autoregulation of the synthesis of nitrate reductase in Aspergillus nidulans. Journal of Bacteriology 97, 1374-8. Crawford, N.M. & Campbell, W.H. (1990). Fertile fields. The Plant Cell 2, 829-35. Crawford, N.M., Campbell, W.H. & Davis, R.W. (1986). Nitrate reductase from squash: cDNA cloning and nitrate regulation. Proceedings of the National Academy of Sciences (USA) 83, 8073-6. Crawford, N.N., Smith, M., Bellissimo, D. & Davies, R.W. (1988). Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains. Proceedings of the National Academy of Sciences (USA) 85, 5006-10. Daniel-Vedele, F., Dorbe, M.F., Caboche, M. & Rouze", P. (1989). Cloning and analysis of the tomato nitrate reductase-encoding gene: protein domain structure and amino acid homologies in higher plants. Gene 85, 371-80. Deng, X.-W., Caspar, T. & Quail, P.H. (1991a). copl: a regulatory locus involved in light-controlled development and gene expression in Arabidopsis. Genes and Development 5, 1171-82. Deng, M.-D., Moureaux, T. & Caboche, M. (1989a). Tungstate, a molybdate analog inactivating nitrate reductase, deregulates the expression of the nitrate reductase structural gene. Plant Physiology 91, 304-9. Deng, M.-D., Moureaux, T., Cherel, I., Boutin, J.-P. & Caboche, M. (1991b). Effects of nitrogen metabolites on the regulation and circadian expression of tobacco nitrate reductase. Plant Physiology and Biochemistry 29, 239^*7. Deng, M.-D., Moureaux, T. & Lamaze, T. (19896). Diurnal and circadian fluctuation of malate levels and its close relationship to

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nitrate reductase activity in tobacco leaves. Plant Science 65, 191-7. Deng, M.-D., Moureaux, T., Leydecker, M.-T. & Caboche, M. (1990). Nitrate-reductase expression is under the control of a circadian rhythm and is light inducible in Nicotiana tabacum leaves. Planta 180, 257-61. Dorbe, M.-F., Caboche, M. & Daniel-Vedele, F. (1992). The tomato NIA gene complements a Nicotiana plumbaginifolia nitrate reductasedeficient mutant and is properly regulated. Plant Molecular Biology 18,363-75. Dorit, R.L., Schoenbach, L. & Gilbert, W. (1990). How big is the universe of exons? Science 250, 1377-82. Duke, S.H. & Duke, S.O. (1984). Light control of extractable nitrate reductase activity in higher plants. Physiologia Plantarum 62, 485-93. Evans, H.J. & Nason, A. (1953). Pyridine nucleotide nitrate reductase from extracts of higher plants. Plant Physiology 28, 233-54. Faure, J.D., Vincentz, M., Kronenberger, J. & Caboche, M. (1991). Co-regulated expression of nitrate and nitrite reductases. The Plant Journal 1, 107-13. Fernandez, E., Schnell, R., Ranum, L.P.W., Hussey, S.C., Silflow, C D . & Lefebvre, P. (1989). Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. Proceedings of the National Academy of Sciences (USA) 86, 6449-53. Friemann, A., Brinkmann, K. & Hachtel, W. (1991). Sequence of a cDNA encoding bi-specific NAD(P)H-nitrate reductase from the tree Betula pendula and identification of conserved protein regions. Molecular and General Genetics 227, 97-105. Fu, Y.-H., Kneesi, Y.Y. & Marzluf, G.A. (1989). Isolation of Nit-4, the minor nitrogen regulatory gene which mediates nitrate induction in Neurospora crassa. Journal of Bacteriology 171, 4067-70. Fu, Y.-H. & Marzluf, G.A. (1990). nit-2, the major positively-acting nitrogen regulatory gene of Neurospora crassa, encodes a sequencespecific DNA-binding protein. Proceedings of the National Academy of Sciences (USA) 87, 5331^. Galangau, F., Daniel-Vedele, F., Moureaux, T., Dorbe, M.-F., Leydecker, M.-T. & Caboche, M. (1988). Expression of leaf nitrate reductase genes from tomato and tobacco in relation to light-dark regimes and nitrate supply. Plant Physiology 88, 383-8. Gardlik, S. & Rajagopalan, K.V. (1990). The state of reduction of molybdopterin in xanthine oxidase and sulfite oxidase. Journal of Biological Chemistry 265, 13047-54. Gowri, G. & Campbell, W.H. (1989). cDNA clones for corn leaf NADH: nitrate reductase and chloroplast NAD(P) + : glyceraldehyde-3phosphate dehydrogenase. Characterization of the clones and analysis of the expression of the genes in leaves as influenced by nitrate in the light and dark. Plant Physiology 90, 792-8.

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Guerrero, M.G., Vega, J.M. & Losada, M. (1981). The assimilatory nitrate-reducing system and its regulation. Annual Review of Plant Physiology 32, 169-201. Gupta, S.C. & Beevers, L. (1985). Regulation of synthesis of nitrate reductase in pea leaves: in-vivo and in-vitro studies. Planta 166, 89-95. Hackett, C.S., Novoa, W.B., Kensil, C.R. & Strittmatter, P. (1988). NADH binding to cytochrome 65 reductase blocks the acetylation of lysine 110. Journal of Biological Chemistry 263, 7539^3. Hackett, C.S., Novoa, W.B., Ozols, J. & Strittmatter, P. (1986). Identification of the essential cysteine residue of NADH-cytochrome 65 reductase. Journal of Biological Chemistry 261, 9854-7. Hackett, C.S. & Strittmatter, P. (1984). Covalent cross-linking of the active sites of vesicle-bound cytochrome 65 and NADH-cytochrome 65 reductase. Journal of Biological Chemistry 259, 3275-82. Hageman, R.H., Flesher, D. & Gitter, A. (1961). Diurnal variation and other light effects influencing the activity of nitrate reductase and nitrogen metabolism in corn. Crop Science 1, 201—4. Hamat, H.B., Kleinhofs, A. & Warner, R.L. (1989). Nitrate reductase induction and molecular characterization in rice (Oryza saliva L.). Molecular and General Genetics 218, 93-8. Hoff, T., Stummann, B.M. & Henningsen, K.W. (1991). Cloning and expression of a gene encoding a root-specific nitrate reductase in bean (Phaseolus vulgaris). Physiologia Plantarum 82, 197-204. Hyde, G.E. & Campbell, W.H. (1990). High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH-nitrate, reductase and its comparison to human NADH-cytochrome 65 reductase. Biochemical and Biophysical Research Communications 168, 1285-91. Hyde, G.E., Crawford, N.M. & Campbell, W.H. (1990). CMNITRA, Accession Number M33154. EMBL Data Base. Hyde, G.E., Wilberding, J.A., Meyer, A.L., Campbell, E.R. & Campbell, W.H. (1989). Monoclonal antibody-based immunoaffinity chromatography for purifying corn and squash NADH:nitrate reductases. Evidence for an interchain disulfide bond in nitrate reductase. Plant Molecular Biology 13, 233-46. Johnson, J.L., Bastian, N.R. & Rajagopalan, K.V. (1990). Molybdopterin guanine dinucleotide: a modified form of molybdopterin identified in the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides forma specialis denitrificans. Proceedings of the National Academy of Sciences (USA) 87, 3190-4. Johnstone, I.L., McCabe, P.C., Greaves, P., Gurr, S.J., Cole, G.E., Brow, M.A.D., Unkles, S.E., Clutterbuck, A.J., Kinghorn, J.R. & Innis, M.A. (1990). Isolation and characterization of the crnA-niiAniaD gene cluster for nitrate assimilation in Aspergillus nidulans. Gene 90, 181-92. Karplus, P.A., Daniels, M.J. & Herriott, J.R. (1991). Atomic structure

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of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family. Science 251, 60-6. Kleinhofs, A. & Warner, R.L. (1990). Advances in nitrate assimilation. In The Biochemistry of Plants. A Comprehensive Treatise, Vol. 16. Intermediary nitrogen metabolism, ed. B.J. Miflin & P.J. Lea, pp. 89120. San Diego, CA: Academic Press. Kramer, S.P., Johnson, J.L., Ribeiro, A.A., Millington, D.S. & Rajagopalan, K.V. (1987). The structure of the molybdenum cofactor. Journal of Biological Chemistry 262, 16357-63. Kramer, V., Lahners, K., Back, E., Privalle, L.S. & Rothstein, S. (1989). Transient accumulation of nitrite reductase mRNA in maize following the addition of nitrate. Plant Physiology 90, 1214-20. Kubo, Y., Ogura, N. & Nakagawa, H. (1989). Limited proteolysis of the nitrate reductase from spinach leaves. Journal of Biological Chemistry 263, 19684-9. Kudla, B., Caddick, M.X., Langdon, T., Martinez-Rossi, N.M., Bennett, C.F., Sibley, S., Wayne Davies, R. & Arst, H.N. (1990). The regulatory gene areA mediating nitrogen metabolite repression in Aspergillus nidulans. Mutations affecting specificity of gene activation alter a loop residue of a putative zinc finger. The EM BO Journal 9, 1355-64. L£, K.H.D. & Lederer, F. (1983). On the presence of a heme-binding domain homologous to cytochrome b5 in Neurospora crassa assimilatory nitrate reductase. The EMBO Journal 2, 1909-14. Lillo, C. (1984). Orcadian rhythmicity of nitrate reductase activity in barley leaves. Physiologia Plantarum 61, 219-23. Lillo, C. (1991). Diurnal variations of corn leaf nitrate reductase: an experimental distinction between transcriptional and post-transcriptional control. Plant Science 73, 149-54. Lu, J.-L., Ertl, J.R. & Chen, C.-M. (1990). Cytokinin enhancement of the light induction of nitrate reductase transcript levels in etiolated barley leaves. Plant Molecular Biology 14, 585-94. Marion-Poll, A., Ch6rel, I., Gonneau, M. & Leydecker, M.-T. (1991). Biochemical characterization of cnx nitrate reductase-deficient mutants of Nicotiana plumbaginifolia. Plant Science 76, 201-9. Martino, S.J. & Smarrelli, J., Jr. (1989). Nitrate reductase synthesis in squash cotyledons. Plant Science 61, 61-7. Marzluf, G.A. (1981). Regulation of nitrogen metabolism and gene expression in fungi. Microbiological Reviews 45, 437-61. Matthews, F.S., Levine, M. & Argos, P. (1971). Structure of the calf liver cytochrome b5 at 2.8A resolution. Nature New Biology 233, 15-16. Melzer, J.M., Kleinhofs, A. & Warner, R.L. (1989). Nitrate reductase regulation: effects of nitrate and light on nitrate reductase mRNA accumulation. Molecular and General Genetics 217, 341-6. Meyer, C , Cherel, I., Moureaux, T., Hoarau, J., Gabard, J. & Rouze,

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P. (1987). Bromphenol blue:nitrate reductase activity in Nicotiana plumbaginifolia. An immunological and genetic approach. Biochimie 69, 735^12. Meyer, C , Levin, J.M., Roussel, J.M. & Rouze, P. (1991). Mutational and structural analysis of the nitrate reductase haem domain of Nicotiana plumbaginifolia. Journal of Biological Chemistry 266, 20561-6. Nason, A., Lee, K.Y., Pan, S.-S., Ketchum, P.A., Lamberti, A. & Davies, J. (1971). In vitro formation of assimilatory reduced nicotinamide adenine dinucleotide phosphate:nitrate reductase from a Neurospora mutant and a component of molybdenum-enzymes. Proceedings of the National Academy of Sciences (USA) 68, 3242-6. Neame, P.J. & Barber, M.J. (1989). Conserved domains in molybdenum hydroxylases. Journal of Biological Chemistry 264, 20894-901. Notton, B.N., Fido, R.J., Whitford, P.N. & Barber, M. (1989). Effect of proteolysis on partial activities and immunological reactivity of spinach nitrate reductase. Phytochemistry 28, 2261-6. Nussaume, L., Vincentz, M. & Caboche, M. (1991). Constitutive nitrate reductase: a dominant conditional marker for plant genetics. The Plant Journal!, 761-1 A. Oaks, A., Poulle, M., Goodfellow, V.J., Cass, L.A. & Deising, H. (1988). The role of nitrate and ammonium ions and light on the induction of nitrate reductase in maize leaves. Plant Physiology 88, 1067-72. Oelmiiller, R., Schuster, C. & Mohr, H. (1988). Physiological characterization of a plastidic signal required for nitrate-induced appearance of nitrate and nitrite reductases. Planta 174, 75-83. Ogawa, M. & Ida, S. (1987) Biosynthesis of ferredoxin nitrite reductase in rice seedlings. Plant and Cell Physiology 28, 1501-8. Okamoto, P.M., Fu, Y.-H. & Marzluf, G.A. (1991). Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover. Molecular and General Genetics 227, 213-23. Padidam, M., Venkatesvarlu, K. & Johri, M.M. (1991). Ammonium represses NADPH-nitrate reductase in the moss Funaria hygrometrica. Plant Science 75, 184-94. Pan, S.-S. & Nason, A. (1978). Purification and characterization of homogeneous assimilatory reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase from Neurospora crassa. Biochimica et Biophysica Ada 523, 297-313. Pelsy, F. & Gonneau, M. (1991). Genetic and biochemical analysis of intragenic complementation events among nitrate reductase apoenzyme-deficient mutants of Nicotiana plumbaginifolia. Genetics 127, 199-204. Pouteau, S., Cherel, I, Vaucheret, H. & Caboche, M. (1989). Nitrate reductase mRNA regulation in Nicotiana plumbaginifolia nitrate

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reductase-deficient mutants. The Plant Cell 1, 111-20. Prosser, I.M. & Lazarus, CM. (1990). Nucleotide sequence of a spinach nitrate reductase cDNA. Plant Molecular Biology 15, 187-90. Rajasekhar, V.K., Gowri, G. & Campbell, W.H. (1988). Phytochromemediated light regulation of nitrate reductase expression in squash cotyledons. Plant Physiology 88, 242-A. Rajasekhar, V.K. & Oelmiiller, R. (1987). Regulation of nitrate reductase and nitrite reductase in higher plants. Physiologia Plantarum 71, 517-21. Remmler, J.L. & Campbell, W.H. (1986). Regulation of corn leaf nitrate reductase. II. Synthesis and turnover of the enzyme's activity and protein. Plant Physiology 80, 442-7. Ruoff, P. & Lillo, C. (1990). Molecular oxygen as electron acceptor in the NADH-nitrate reductase system. Biochemical and Biophysical Research Communications 172, 1000-5. Schnorr, K.M., Juricek, M., Huang, C , Culley, D. & Kleinhofs, A. (1991). Analysis of barley nitrate reductase cDNA and genomic clones. Molecular and General Genetics 227', 411-16. Schuster, C. & Mohr, H. (1990). Appearance of nitrate reductase mRNA in mustard seedling cotyledons is regulated by phytochrome. Planta 181, 327-34. Seith, B., Schuster, C. & Mohr, H. (1991). Coaction of light, nitrate and a plastidic factor in controlling nitrate-reductase gene expression in spinach. Planta 184, 74-80. Sharma, A.K. & Sopory, S.K. (1984) Independent effects of phytochrome and nitrate on nitrate reductase and nitrite reductase activities in maize. Photochemistry and Photobiology 39, 491-3. Shirabe, K., Yubisui, T., Nishino, T. & Takeshita, M. (1991). Role of cysteine residues in human NADH-cytochrome b5 reductase studied by site-directed mutagenesis. Journal of Biological Chemistry 266, 7531-6. Shirabe, K., Yubisui, T. & Takeshita, M. (1989). Expression of human erythrocyte NADH-cytochrome b5 reductase as a thrombin-cleavable fused protein in Escherichia coli. Biochimica et Biophysica Acta 1008, 189-92. Sligar, S.G., Egeberg, K., Sage, J.T., Morikis, D. & Champion, P. (1987). Alteration of heme axial ligands by site-directed mutagenesis: a cytochrome becomes a catalytic demethylase. Journal of the American Chemical Society 109, 7896-7. Small, I.S. & Wray, J.L. (1980). NADH nitrate reductase and related NADH cytochrome c reductase species in barley. Phytochemistry 19, 387-94. Smarrelli, J., Jr, Malone, M., Watters, M.T. & Curtis, L.T. (1987). Transcriptional control of the inducible nitrate reductase isoform from soybeans. Biochemical and Biophysical Research Communications 146, 1160-5.

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P. ROUZE AND M. CABOCHE Solomonson, L.P. & Barber, M.J. (1990). Assimilatory nitrate reductase: functional properties and regulation. Annual Review of Plant Physiology and Plant Molecular Biology 41, 225-53. Solomonson, L.P., Barber, M.J., Robbins, A.P. & Oaks, A. (1986). Functional domains of assimilatory NADH:nitrate reductase from Chlorella. Journal of Biological Chemistry 261, 11290-4. Solomonson, L.P., McCreery, M.J., Kay, C.J. & Barber, M.J. (1987). Radiation inactivation analysis of assimilatory NADHrnitrate reductase: apparent functional sizes of partial activities associated with intact and proteolytically modified enzyme. Journal of Biological Chemistry 262, 8934-9. Streit, L., Martin, B.A. & Harper, J.E. (1987). A method for the separation and purification of the three forms of nitrate reductase present in wild-type soybean leaves. Plant Physiology 84, 654-7. Strittmatter, P. (1961). The nature of the flavin binding in microsomal cytochrome b5 reductase. Journal of Biological Chemistry 236, 2329-35. Strittmatter, P., Kittler, J.M., Coghill, J.E. & Ozols, J. (1992). Characterization of lysyl residues of NADH-cytochrome b5 reductase implicated in charge-pairing with active-site carboxyl residues of cytochrome b5 by site-directed mutagenesis of an expression vector for the flavoprotein. Journal of Biological Chemistry 267, 2519-23. Truong, H.-N., Meyer, C. & Vedele, F. (1991). Characteristics of Nicotiana tabacum nitrate reductase protein produced in Saccharomyces cerevisiae. Biochemical Journal 278, 393-7. Ullrich, W.R., Rigano, C , Fuggi, A. & Aparicio, P.J. (ed.) (1990). Inorganic Nitrogen in Plants and Microorganisms. Uptake and Metabolism. Berlin: Springer-Verlag. Unkles, S.E., Campbell, E.I., de Ruiter-Jacobs, M.J.T., Broehhuisen, M., Macro, J.A., Carrez, D., Contreras, R., Van den Hondel, C.A.M.J.J. & Kinghorn, J.R. (1989). The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway. Molecular and General Genetics 218, 99-104. Vaucheret, H., Chabaud, M., Kronenberger, J. & Caboche, M. (1990). Functional complementation of tobacco and Nicotiana plumbaginifolia nitrate reductase deficient mutants by transformation with the wild-type alleles of the tobacco structural genes. Molecular and General Genetics 220, 468-74. Vaucheret, H., Kronenberger, J., Rouze, P. & Caboche, M. (1989). Complete nucleotide sequence of the two homeologous tobacco nitrate reductase genes. Plant Molecular Biology 12, 597-600. Vincentz, M. & Caboche, M. (1991). Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants. The EMBO Journal 10, 1027-35. Wahl, R.C., Hageman, R.V. & Rajagopalan, K.V. (1984). The relationship of Mo, molybdopterin, and the cyanolyzable sulfur in the Mo

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cofactor. Archives of Biochemistry and Biophysics 230, 264—73. Ward, M.R., Tischner, R. & Huffaker, R.C. (1988). Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings. Plant Physiology 88, 1141-5. Warner, R.L. & Huffaker, R.C. (1989). Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings. Plant Physiology 91, 947-53. Wilkinson, J.Q. & Crawford, N.M. (1991). Identification of the Arabidopsis CHL3 gene as the nitrate reductase structural gene NIA2. The Plant Cell 3, 461-71. Wootton, J.C., Nicolson, R.E., Cock, J.M., Walters, D.E., Burke, J.F., Doyle, W.A. & Bray, R.C. (1991). Enzymes depending on the pterin molybdenum cofactor: sequence families, spectroscopic properties of molybdenum and possible cofactor-binding domains. Biochimica et Biophysica Ada 1057, 157-85. Wray, J.L. (1988). Molecular approaches to the analysis of nitrate assimilation. Plant, Cell and Environment 11, 369-82. Wray, J.L. & Kinghorn, J.R. (ed.) (1989). Molecular and Genetic Aspects of Nitrate Assimilation. Oxford: Oxford Science Publications. Yubisui, T., Shirabe, K., Takeshita, M., Kobayashi, Y., Fukumaki, Y., Sakaki, Y. & Takano, T. (1991). Structural role of serine 127 in the NADH-binding site of human NADH-cytochrome i»5 reductase. Journal of Biological Chemistry 266, 66-70. Zeiler, K.G. & Solomonson, L.P. (1989). Regulation of Chlorella nitrate reductase: control of enzyme activity and immunoreactive protein levels by ammonia. Archives of Biochemistry and Biophysics 269, 46-54.

Notes added in proof 1. Three lysine-containing sequences in Cb5R have been shown to interact with carboxyl-containing sequences from cytochrome b5 (Strittmatter et al., 1992). One of these charge-pairs, which is Glu581-Lys647 in the tobacco sequence (Fig. 3), is conserved in NRs. 2. Wootton et al., (1991) have classified the molybdoenzymes according to their sequences into a prokaryotic family, which includes the respiratory NRs, and a eukaryotic family comprising assimilatory NRs, SOs and XDHs. Despite their very low homology, the MoCo domains of these proteins have been aligned, and a consensus sequence (PROSITE motif Molybdopterin Euk) has been deduced by Amos Bairoch (University of Geneva). Only five residues are completely conserved in the family and these are probably involved in MoCo binding; in the tobacco NR sequence (Fig. 3), they are Glull4, Cysl80, Argl84, Arg295 and Gly397.

J.V. CULLIMORE, J.M. COCK, T.J. DANIELL, R. SWARUP and M.J. BENNETT

Inducibility of the glutamine synthetase gene family of Phaseolus vulgaris L. Introduction Glutamine synthetase (GS) plays a key role in the nitrogen metabolism of higher plants. This enzyme works in conjunction with the second enzyme of the pathway, glutamate synthase (GOGAT), to form the major, if not sole, route of ammonium assimilation in higher plants (Miflin & Lea, 1980). This pathway is often referred to as the glutamate synthase cycle or GS/GOGAT pathway. The physiological importance of this pathway is that ammonium derived from the plant's primary nitrogen sources is assimilated into glutamine and glutamate from which all other nitrogenous compounds are derived (see Fig. 1). In addition, a number of other pathways in the plant produce ammonium, such as photorespiration, phenylpropanoid metabolism and amino acid catabolism and, at least for photorespiration, this ammonium also appears to be reassimilated by GS and GOGAT (see Givan et al., 1988). These enzymes are therefore in a key position to control the flux of nitrogen into the plant and its recycling during metabolism. amino acids proteins nucleotides

N nucleic acids chlorophyll photorespiration phenylpropanoid metabolism amino acid catabolism

^v 2

metaDOlltes

Fig. 1. The central importance of the GS/GOGAT pathway in the nitrogen metabolism of higher plants. Society for Experimental Biology Seminar Scries49: Inducible Plant Proteins, ed. J. L. Wray. © Cambridge University Press, 1992, pp. 79-95.

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GS has been well studied in higher plants and the enzyme is known to be located in two subcellular compartments, the plastid and cytosol, as separate isoenzymes which can be separated by ion-exchange chromatography (McNally & Hirel, 1983). The active enzyme is octameric in both compartments but the subunit sizes differ slightly: about 39 kDa for cytosolic GS and 42 kDa for plastid-located GS (Forde & Cullimore, 1989). GOGAT also occurs as separate isoenzymes but with different cofactor specificities (Suzuki & Gadal, 1984), dependent on either NADH (NADH-GOGAT) or ferredoxin (Fd-GOGAT) as reductant. Both GOGAT isoenzymes appear to be located in plastids (Suzuki & Gadal, 1984; Chen & Cullimore, 1989). They are both monomeric but can be separated owing to their distinctive molecular weights: c. 140 kDa for Fd-GOGAT and c. 230 kDa for NADH-GOGAT. Recently, work has begun to uncover the individual roles of the different GS and GOGAT isoenzymes. In legumes the primary nitrogen source for growth may either be nitrate (as for most plants), or dinitrogen, because of the symbiosis with the diazotroph Rhizobium. Dinitrogen is fixed by the Rhizobium bacteroids enclosed within the peribacteroid membranes in the cells of the central tissue of the root nodule. Ammonium produced by this reaction is excreted into the plant fraction of the nodule and assimilated mainly by a cytosolic GS and plastidlocated NADH-GOGAT (Robertson & Farnden, 1980; Chen & Cullimore, 1989). Nitrate may be assimilated in the roots or leaves, most probably by plastid-located enzymes as the ammonium from nitrate reduction is produced in this subcellular location (Miflin & Lea, 1980). In leaves, however, the major flux of ammonium is attributable to photorespiration; this pathway is estimated to produce 10-fold higher amounts of ammonium than primary nitrogen assimilation and there is now strong genetic evidence that the released ammonium is then reassimilated by a chloroplast-located GS and ferredoxin-dependent GOGAT (Givan et al., 1988). In this chapter we describe the genetic control and regulation of GS in higher plants with particular reference to the legume Phaseolus vulgaris. In order to study this enzyme we set out to clone the encoding genes and to establish the molecular basis for the different GS isoenzyme activities. This work then allowed us to address the question of whether the GS genes are inducible and how important GS gene expression is in effecting changes in the activities of the isoenzymes. Note that in this chapter we have used the term gene expression to refer solely to the production of mRNA related to the gene, rather than to the production of the encoded polypeptide or isoenzyme.

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The GS genes and isoenzymes of P. vulgaris Through a collaboration between Dr B. G. Forde's group at Rothamsted Experimental Station and the group at Warwick University it has been established that there are five GS (gin) genes in P. vulgaris, all of which are in the nuclear genome. Four of these genes are known to be functionally expressed since they were initially identified by the isolation of fulllength cDNA clones (Cullimore et al., 1984; Gebhardt et al., 1986; Lightfoot et al., 1988; Bennett et al., 1989). The fifth GS gene, gln-e was identified on a genomic clone containing gln-y (Forde et al., 1989) but as mRNA for this gene has not been detected in a search through a number of organs at different developmental stages, it appears that this gene may not be expressed (Swarup et al., 1990). In vitro transcription/translation of the cDNA clones followed by characterisation of the protein products (Bennett et al., 1989) has determined that the gln-a, gln-(5 and gln-y genes encode the cytosolic a, (3 and y polypeptides which had previously been identified in extracts of plant tissue following separation on denaturing two-dimensional gels (Lara et al., 1984). The DNA sequences show that these three polypeptides contain 356 amino acids corresponding to molecular masses of about 39 kDa and they are 85-89% identical. The nucleic acid sequences of these three genes are about 79-86% identical in the coding regions but show very little homology in the 5' and 3' flanking regions. The fourth expressed GS gene, gln-d, encodes a polypeptide of about 47 kDa which, in comparison to the cytosolic GS polypeptides, has extensions to the amino- and carboxy-termini of 57 and 16 amino acids, respectively. Within the co-linear region the coding sequence of this gene is about 78% homologous at the amino acid level to each of the three cytosolic GS polypeptides and about 70% identical at the DNA level. The N-terminal extension has been shown to encode a chloroplast targeting presequence which is cleaved during transport of the precursor polypeptide into its subcellular compartment (Lightfoot et al., 1988). The resulting chloroplast-located GS polypeptide, termed 6, is about 42 kDa in size and assembles into the chloroplast-located isoenzyme. As in pea (Tingey et al., 1987) there is only one gene encoding the chloroplast GS and therefore the resulting isoenzyme is made up of a single subunit type. By contrast the three cytosolic GS polypeptides of P. vulgaris appear to be able to assemble, perhaps randomly, into a range of isoenzyme activities containing differing proportions of the a, |3 and y polypeptides (Bennett & Cullimore, 1989; Cai & Wong, 1989). These isoenzymes can be partially separated by ion-exchange high-performance liquid chromatography (IEX-HPLC), with activities composed primarily of the Y, P, a and 6 polypeptides eluting in this order from the column and with

82

j . v . CULLIMORE etal. g/n-ot

g/n-fi

9ln-T

\r


gln-b

gln-Z

chloroplast

^ r (leaf/root)

GS n 2 (nodule)

GS n1 (nodule)

Fig. 2. The genetic control of GS isoenzymes in nodules, roots and leaves of Phaseolus vulgaris (from Forde & Cullimore, 1989).

mixed isoenzymes eluting at the intermediary positions (Bennett & Cullimore, 1989). In this way IEX-HPLC of plant extracts can be used to detect the isoenzyme activities produced from each GS gene. A model for the genetic control of GS isoenzymes in P. vulgaris is shown in Fig. 2. The isoenzyme profiles and the subunit composition of GS activity from nodules, roots and leaves are shown in Fig. 3.

Expression of the GS genes gln-y An RNase protection technique has been used to analyse the abundance of mRNA of each GS gene in a specific and quantitative manner. The glny mRNA was found to be highly abundant in nodules but not in roots and leaves. Surveying a number of other organs revealed that the expression of this gene is not nodule-specific as originally suggested (Cullimore et al., 1984) as its mRNA was detected at lower levels in stems, petioles and cotyledons of germinating seeds (Bennett et al., 1989). Further studies on cotyledons revealed that the gln-y gene is expressed transiently about 2 days after sowing and produces a major GS polypeptide at this time (Swarupefa/., 1990).

83

Inducibility of the glutamine synthetase gene family Y

5

ii

Nodules

.GS

4 3

6

0-4

Y-

0-3

2

0-2

1

01

0

0

2-5 . Roots

GS,

20

110 ~ 0 x

I12 .

02 U

7.

> 0

Leaves

0-4 0-3 02 01 ^ i

0

4

^ i

8 12 16 20 2428 32 36 40 44 Fraction number

Fig. 3. GS isoenzymes and their subunit composition in nodules, roots and leaves of P. vulgaris. The figure shows the GS isoenzyme activity profiles following separation of crude organ extracts by IEX-HPLC (adapted from Cullimore et al., 1990). The subunit composition of the isoenzyme activity peaks was determined following separation on 2-D denaturing gels. GS,=GS transferase activity. The position of elution of octameric GS isoenzymes composed of a single subunit type is shown above the figure.

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J.V. CULLIMORE

etal.

During nodulation the abundance of gln-y mRNA increases strongly from a barely detectable level in roots to become the major GS mRNA 10 days after Rhizobium inoculation (Gebhardt et al., 1986; Bennett et al., 1989) (see Fig. 4/1). Experiments with promoter fusions linked to the reporter gene P-glucuronidase (GUS) have revealed that this promoter is expressed only in the infected cells of the central tissue of transgenic Lotus corniculatus nodules (Forde et al., 1989). Using a number of Rhizobium mutants that arrest nodulation at different stages of development it was shown that the induction oigln-y did not occur in nodule-like protuberances lacking intracellular bacteroids but did occur in Fix" nodules which contained intracellular bacteria (Cock et al., 1990). It thus appears that the release of Rhizobium may lead to the production of factors specific to the infected cells which are required for the expression of this gene in nodules. Studies on the gln-y gene have shown that the upstream regions contain sites that bind nodule nuclear proteins but it is not yet known whether these proteins are involved in regulating gene transcription (Forde et al., 1990). In soybean it has been proposed that the increased expression of plant GS genes in nodules is caused by a substrate induction by ammonium derived symbiotically from dinitrogen fixation (Hirel era/., 1987; Miao et al., 1991). In P. vulgaris the gln-y gene is still induced in nodules formed with certain Fix" nodules (see above) and also in nodules grown under an 80% Ar/20%O2 atmosphere. In the latter situation nitrogenase is expressed normally but there is no ammonium produced owing to substrate limitation. However, in both types of non-fixing nodules the abundance of the gln-y mRNA reached levels only 2 to 4-fold lower than in nitrogenfixing nodules, suggesting that nitrogen fixation is a positive factor increasing the abundance of this mRNA during the induction. Adding ammonium exogenously to the non-fixing nodules failed to increase the abundance of the gln-y mRNA, and in fact caused a substantial decline, implying that the positive effect of dinitrogen fixation is not attributable directly to the level of ammonium (Cock et al., 1990). However, ammonium added exogenously may not be mimicking the localised supply of ammonium from the bacteroids and, moreover, appeared to initiate the senescense of the nodule. More work therefore needs to be done

Fig. 4. Changes in A, GS mRNAs and B, GS isoenzymes, during nodule development. The abundances of the mRNAs were measured by an RNase protection technique and the isoenzymes following separation by IEX-HPLC (adapted from Cullimore et al., 1990). GSS=GS synthetase activity. The isoenzymes are as defined in Fig. 3.

Inducibility of the glutamine synthetase gene family

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to investigate the role of ammonium, if any, in increasing gln-y expression in nitrogen-fixing nodules.

The mRNA for the gln-/3 gene has been detected in all organs so far examined including leaves, roots, nodules, stems, petioles, cotyledons and embryos from germinating seeds (Forde & Cullimore, 1989; R. Swarup et al., unpublished data). However, its abundance differs considerably in these organs suggesting that this gene can not be considered as a 'housekeeping' gene. This conclusion is borne out by studying the expression of these gene during organ development. For example, in roots the gln-/3 gene provides the major GS mRNA but this is strongly induced in abundance during the early stages of radicle emergence (Ortega et al., 1986). The gin-ft gene appears to be orthologous to the gene from soybean that is strongly induced by addition of ammonium to roots (Hirel et al., 1987; Miao etal., 1991). In similar experiments with P. vulgaris no change in the mRNA abundance of this gene was observed (Cock et al., 1990), suggesting that gln-fi is not ammonium-inducible. During nodulation the abundance of the gln-P mRNA remains at a fairly high and constant level in the organ as a whole (Gebhardt et al., 1986; Bennett et al., 1989) (Fig. 4A). Experiments with GUS fusion in L. corniculatus suggest that in fact complex changes are occurring at the cellular level. Initially the promoter was expressed in both the cortical and infected regions of the nodule, but as the nodules matured expression became restricted to the vascular tissue (Forde et al., 1989). Again in this organ there was no evidence that this gene is induced by ammonium derived either from dinitrogen fixation or by exogenous addition, but this gene did not show the dramatic decline in mRNA abundance during ammonium-induced nodule senescence observed for the gln-y gene (Cock etal., 1990). The gln-/3 gene is also expressed in leaves but studies on primary leaves suggest that this occurs at a specific time during leaf development, with the mRNA being at maximum abundance about 5 days after germination (Cock et al., 1991) (Fig. 5A).

gln-a To date very little is known of the regulation of the gln-a gene. This gene must be expressed strongly during seed development as its mRNA is present at quite high levels in both the cotyledons and embryonic axis of dry seeds and is the predominant or perhaps only GS mRNA present in

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these desiccated organs (Swarup et al., 1990). During germination the gln-a mRNA remains the most abundant GS mRNA in the cotyledons (Swarup etal., 1990), radicles (Ortega etal., 1986) and plumules (Cock et al., 1991) for up to 2 days and this is most likely caused by renewed transcription as the mRNA often increases slightly in abundance. As the organs mature the abundance of the mRNA declines to a barely detectable level. In nodules the gln-a mRNA is present at the very early stages of development but then declines (Bennett etal., 1989) (Fig. 44). However, in nodules that were induced to senesce early, either by the addition of exogenous ammonium or by growth in an Ar/O2 atmosphere, a renewed induction was observed (Cock et al., 1990). The reasons behind this induction and the regulation of this gene are presently being investigated further (T. J. Daniell & J. V. Cullimore, unpublished data). gln-d The gln-d gene, encoding the plastid-located GS, is highly expressed in chlorophyllous organs but weakly in roots. During nodulation the mRNA from this gene increases significantly at about the same time as the gln-y induction (Fig. 4A), suggesting that there may be similar regulatory elements in these two genes controlling nodule-specific expression. However, the gln-d mRNA always remains a minor species in nodules suggesting a quantitative difference in expression in comparison to the gln-y gene. To date it is not clear what physiological role the plastidlocated GS plays in this organ. During primary leaf development the gln-d gene is expressed starting about 2 days after germination and after about day 7 it rapidly produces the major GS mRNA (Fig. 5^4). As the organ matures the abundance of the mRNA declines to a much lower level. The initial induction has been shown to be attributable to the differential expression of two transcripts from this gene. One transcript appears later than the other and increases to a much higher abundance. Induction from this promoter does not require chloroplast development as this transcript also accounts for the increase in gln-d mRNA during nodule development (Cock etal., 1991). It therefore appears that the controls governing the organ-specific expression of the gln-d gene are complex, perhaps involving multiple controls on two promoter sites. In leaves experiments have been performed to determine the roles of light and photorespiration in the regulation of expression of this gene (Cock et al., 1991). Light is clearly a major regulatory factor as there is very low expression of this gene in leaves of dark-grown plants and

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transfer of etiolated plants to the light causes a massive induction. However, it is likely that there is an underlying developmental regulation of this gene as the low-level expression in the dark occurs at about the same time as the gene is induced during leaf development in the light. As described earlier, the major role of the chloroplast GS in leaves is to assimilate the ammonium from photorespiration; it was reasonable, therefore, to address the question of whether the expression of this gene is related to the rate of this process. The results have proved complicated to interpret but suggest that there is no direct induction of this gene by photorespiratory ammonium as there was no increase in the abundance of the gln-d mRNA following transfer of plants grown in a high CO2 atmosphere (conditions that suppress photorespiration) to photorespiratory conditions (air). Moreover, the light induction of gln-d occurred whether etiolated plants were transferred to photorespiratory conditions or not. Surprisingly, we found, in common with work on pea (Edwards & Coruzzi, 1989), that the abundance of the chloroplast-GS mRNA was higher in plants grown continuously in photorespiratory conditions than when the atmosphere was supplemented with CO2. We feel that this difference may reflect a more indirect effect of photorespiration, perhaps on the growth rate of the plant or on the intracellular pH. Does GS gene induction lead to changes in isoenzyme activities? The preceding section has shown that substantial changes occur in the expression of each GS gene in different organs and during development. In this section we discuss what happens to the GS polypeptides and isoenzymes and whether gene induction is a major factor regulating isoenzyme activity. During nodule development there are major changes in the abundance, of all four GS mRNAs but the most pronounced is the induction of the gln-y gene (Fig. 4/4). The production of the gln-y mRNA is closely followed by the appearance of the y polypeptide which becomes the major GS polypeptide by 12 days after inoculation (Bennett et al., 1989). In uninoculated roots and at early stages during infection the GS isoenzymes are composed mainly of the P polypeptide. Following induction of the gln-y gene the y-rich isoenzymes became predominant and isoenzymes composed of mixtures of the y/P polypeptides were also observed (Fig. AB). The latter observation suggests that the gln-y and glnft genes are, at least to some extent, expressed in the same cell types. However, the abundance of the (3 isoenzyme always remains high and may be explained by the observation that expression of the gln-fi gene

90

j . v . CULLIMORE etal.

also occurs in the cortex of the nodule where gln-y is not expressed (Forde et al., 1989; Chen & Cullimore, 1989). These changes in the y and P isoenzymes closely follow the changes occurring in the gln-y and gln-fi mRNAs. The same is also true for the less abundant a and 5 isoenzymes (Fig. 46), suggesting that changes in GS isoenzymes during nodule development are effected largely by regulating expression of the four GS genes. In experiments where the rate of dinitrogen fixation was manipulated by altering the atmospheric concentration of dinitrogen around the root systems, again a correlation was observed between changes in the abundance of the GS mRNAs and the corresponding polypeptides and isoenzymes (Cock et al., 1990; Chen et al., 1990). Thus switching the plants from non-fixing (Ar/O2) to fixing conditions (N2/O2) promoted an increase in the abundance of the gln-y mRNA, polypeptide and isoenzyme which was visible by 24 h (Fig. 6). The opposite switch (fixing to non-fixing conditions) led to a decrease in the mRNA after one day but it was several days before this was reflected in a decrease in the y isoenzymes (Fig. 6). This lag is most probably due to a greater stability of the protein products of the gene compared to the mRNA. In leaves the sequential induction of the gln-a, gln-fi and gln-5 mRNAs during germination was mirrored by changes in the isoenzymes. Note that the overlapping expression of the two cytosolic-GS genes led to the production of mixed isoenzymes containing a and |3 polypeptides (Cock et al., 1991) (Fig. 5). Eventually the high level induction of the gln-d gene resulted in over 90% of the GS activity in mature leaves being chloroplastic. Following maximum induction of the chloroplast GS the abundance of the gln-d mRNA declined to very low levels but this was not reflected in a decrease in the isoenzyme activity. There are two explanations for this observation: either the enzyme is much more stable than the mRNA, or a lower level of the mRNA is able to sustain a greater translational activity. At present we have not distinguished between the two possibilities. In plants grown in the dark the low-level expression of the gln-d gene surprisingly led to a substantial production of the 5 polypeptide and about 50% of the GS activity was attributable to the chloroplastic form. The large induction of the gln-d gene following illumination further increased the level of this isoenzyme to over 70% of the total GS activity within 24 h (Cock et al., 1991). Thus in these experiments on nodules and leaves it is clear that changes in GS activity occur mainly as a result of changes in the abundances of the mRNAs, particularly during organ development. However, there are situations, particularly in mature organs, where there is not a close correlation between the abundance of the mRNA and the corresponding

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isoenzyme, suggesting that other factors are also involved in regulating GS isoenzyme activity. Conclusion The genetic control and regulation of GS activity has attracted much attention because of the important physiological role of this enzyme. In pioneering work on enteric bacteria it has been shown that there is a single GS gene whose expression is controlled by two regulatory proteins in a manner which is sensitive to the nitrogen status of the cell. In addition the enzyme itself is regulated by a reversible activation/deactivation mechanism sensitive to the same cellular stimulus (Magasanik, 1988). The work on P. vulgaris has shown that the regulation of GS in higher plants is quite different, involving multiple GS genes which are individually controlled. This increase in complexity, compared to the prokaryote, may reflect the greater compartmentation and differentiation of the higher plant cells and organs. This is clearly seen by the presence of GS activity in two subcellular compartments and the requirement for separate genes to encode the different isoenzymes. The main control on the GS genes appears to be developmental, leading to the expression of specific genes only in certain organs. However, in addition there appear to be controls that alter the level of expression in relation to the metabolism of the cell. Unlike the situation in bacteria we at present know very little of the components that regulate GS gene expression, but work on the identification of cis- and trans- acting factors represents a start on this analysis. By devising methods to follow the changes in the mRNA, polypeptide and isoenzyme of each GS gene we have been able to show that changes in GS mRNA abundance are a major factor regulating changes in the production of GS activity, particularly during organ development. These alterations during development operate over a time scale of hours or days and, in contrast, it is not clear whether there are also mechanisms acting on the enzyme itself, in fully differentiated cells, which afford a more rapid regulation of GS activity in response to changing environmental conditions. Clearly there is much to be done to uncover the mechanisms regulating gene expression and enzyme activity before we are able to understand fully the importance of GS in regulating nitrogen metabolism in higher plants. Acknowledgements We are grateful to the Agricultural and Food Research Council for grants to support J.M.C., M.J.B. and the link with Rothamsted Experimental

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Station, and to the Science and Engineering Research Council for a grant to support R. S. and a studentship to support T. J. D. References Bennett, M.J. & Cullimore, J.V. (1989). Glutamine synthetase isoenzymes of Phaseolus vulgaris L.: subunit composition in developing root nodules and plumules. Planta 179, 433-40. Bennett, M.J., Lightfoot, D.A. & Cullimore, J.V. (1989). cDNA sequence and differential expression of the gene encoding the glutamine synthetase y polypeptide of Phaseolus vulgaris L. Plant Molecular Biology 12, 553-65. Cai, X. & Wong, P.P. (1989). Subunit composition of glutamine synthetase isoenzymes from root nodules of bean (Phaseolus vulgaris L.). Plant Physiology 91, 1056-62. Chen, F.-L., Bennett, M.J. & Cullimore, J.V. (1990). Effect of the nitrogen supply on the activities of isoenzymes of NADH-dependent glutamate synthase and glutamine synthetase in root nodules of Phaseolus vulgaris L. Journal of Experimental Botany 41, 1215-21. Chen, F.-L. & Cullimore, J.V. (1989). Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L. Planta 179, 441-7. Cock, J.M., Brock, I.W., Watson, A.T., Swarup, R., Morby, A.P. & Cullimore, J.V. (1991). Regulation of glutamine synthetase genes in leaves of Phaseolus vulgaris. Plant Molecular Biology 17, 761-71. Cock, J.M., Mould, R.M., Bennett, M.J. & Cullimore, J^V. (1990). Expression of glutamine synthetase genes in roots and nodules of Phaseolus vulgaris following changes in the ammonium supply and infection with various Rhizobium mutants. Plant Molecular Biology 14, 549-60. Cullimore, J.V., Cock, J.M., Robbins, M.P. & Bennett, M.J. (1990). Glutamine synthetase of French bean: from genes to isoenzymes. In Inorganic Nitrogen in Plants and Microorganisms, ed. W.R. Ullrich, C. Rigano, A. Fuggi & P.J. Aparicio, pp. 273-80. Berlin: Springer-Verlag. Cullimore, J.V., Gebhardt, C , Saarelainen, R., Miflin, B.J., Idler, B.K. & Barker, R.F. (1984). Glutamine synthetase of Phaseolus vulgaris L.: organ-specific expression of a multigene family. Journal of Molecular and Applied Genetics 2, 589-99. Edwards, J.W. & Coruzzi, G.M. (1989). Light and photorespiration act in concert to regulate the expression of the nuclear gene for chloroplast glutamine synthetase. The Plant Cell 1, 241-8. Forde, B.G. & Cullimore, J.V. (1989). The molecular biology of glutamine synthetase in higher plants. In Oxford Surveys of Plant Molecular and Cell Biology, vol. 6, ed. B.J. Miflin, pp. 247-96. Oxford: Oxford University Press.

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Forde, B.G., Day, H.M., Turton, J.F., Shen, W.-J., Cullimore, J.V. & Oliver, J.E. (1989). Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants. The Plant Cell 1, 391-401. Forde, B.G., Freeman, J., Oliver, J.E. & Pineda, M. (1990). Nuclear factors interact with conserved A/T-rich elements upstream of a nodule-enhanced glutamine synthetase gene from French bean. The Plant Cell 2, 925-39. Gebhardt, C , Oliver, J.E., Forde, B.G., Saarelainen, R. & Miflin, B.J. (1986). Primary structure and differential expression of glutamine synthetase genes in nodules, roots and leaves of Phaseolus vulgaris. The EMBO Journal 5, 1429-35. Givan, C.V., Joy, K.W. & Kleczkowski, L.A. (1988). A decade of photorespiratory nitrogen cycling. Trends in Biochemical Sciences 13, 433-7. Hirel, B., Bouet, C , King, B., Layzell, D., Jacobs, F. & Verma, D.P.S. (1987). Glutamine synthetase genes are regulated by ammonia provided externally or by symbiotic nitrogen fixation. The EMBO Journal 6, 1167-71. Lara, M., Porta, H., Padilla, J., Folch, J. & Sanchez, F. (1984). Heterogeneity of glutamine synthetase polypeptides in Phaseolus vulgaris L. Plant Physiology 76, 1019-23. Lightfoot, D.A., Green, N.K. & Cullimore, J.V. (1988). The chloroplast-located glutamine synthetase of Phaseolus vulgaris L.: Nucleotide sequence, expression in different organs and uptake into isolated chloroplasts. Plant Molecular Biology 11, 191-202. Magasanik, B. (1988). Reversible phosphorylation of an enhancer binding protein regulates the transcription of bacterial nitrogen utilisation genes. Trends in Biochemical Sciences 13, 475-9. McNally, S.F. & Hirel, B. (1983). Glutamine synthetases of higher plants. Physiologia Vegetale 21, 761-74. Miao, G.-H., Hirel, B., Marsolier, M.C., Ridge, R.W. & Verma, D.P.S. (1991). Ammonia-regulated expression of a soybean gene encoding glutamine synthetase in transgenic Lotus corniculatus and tobacco. The Plant Cell 3, 11-22. Miflin, B.J. & Lea, P.J. (1980). Ammonia assimilation. In The Biochemistry of Plants, vol. 5, ed. B.J. Miflin, pp. 169-202. New York: Academic Press. Ortega, J.L., Campos, F., Sanchez, F. & Lara, M. (1986). Expression of two different glutamine synthetase polypeptides during root development in Phaseolus vulgaris L. Plant Physiology 80, 1051^4. Robertson, J.G. & Farnden, K.J.F. (1980). Ultrastructure and metabolism of the developing legume root nodule. In The Biochemistry of Plants, vol. 5, ed. B.J. Miflin, pp. 66-113. New York: Academic Press.

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Suzuki, A. & Gadal, P. (1984). Glutamate synthase: physicochemical and functional properties of different forms in higher plants and in other organisms. Physiologia Vegetate 22, 471-86. Swamp, R., Bennett, M.J. & Cullimore, J.V. (1990). Expression of glutamine-synthetase genes in cotyledons of germinating Phaseolus vulgaris L. Planta 183, 51-6. Tingey, S.V., Walker, E.L. & Coruzzi, G.M. (1987). Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules. The EMBO Journal 6, 1-9.

W. SCHUCH

Expression and manipulation of genes involved in phenylpropanoid biosynthesis

Introduction The phenylpropanoid pathway leads to the biosynthesis of a large number of phenolic compounds in plants. The importance of this diverse class of chemicals originating from phenylalanine has been recognised for some time, as it plays a key role in plant development and protection against environmental stress. The variety of chemical structures synthesised includes compounds like quinones involved in electron transport (French et al., 1976),flavonoidpigments responsible for flower coloration (Ebel & Hahlbrock, 1982) and protection against UV irradiation (Hahlbrock et al., 1982), and cinnamic acid esters and phytoalexins involved in disease resistance (Dixon et al., 1983). Phenolic metabolites like acetosyringone activate bacterial genes such as the Agrobacterium virulence genes or Rhizobium nodulation genes in the rhizosphere (Downie & Johnston, 1986; Stachel & Zambryski, 1986). Salicylic acid, which is involved in systemic induced resistance, is induced after wounding. Lastly, lignin, a major cell wall polymer found in close association with cellulose fibres and hemicellulose in the xylem, is a product of the phenylpropanoid pathway (Lewis & Yamamoto, 1990). The biosynthesis of some of these compounds, for example those involved in flower pigment biosynthesis, is well understood. In addition, the structure and pattern of expression of genes encoding these enzymes has been studied extensively. The application of novel techniques which permit the modulation of gene expression has recently led to the manipulation of genes involved in flavonoid biosynthesis in Petunia. In these experiments the chalcone synthase gene encoding a key enzyme in flavonoid biosynthesis was the target for down-regulation via antisense RNA (van der Krol et al., 1988), and for modulation via a co-suppression mechanism (Napoli et al., 1990; van der Krol et al., 1990). Inhibition of the expression of chalcone synthase and dihydroflavonol reductase has Society for Experimental Biology Seminar Series49: Inducible Plant Proteins, ed. J. L. Wray. ©Cambridge University Press, 1992, pp. 97-111.

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added to the natural variation in petal pigmentation in Petunia and is now the focus of commercial interest. Owing to the importance of phenolic compounds in plant defence and plant pigmentation, the pathways leading to phytoalexin and pigment biosynthesis have attracted most attention and have led to the purification of several pathway enzymes and the cloning of their genes from several plant species (Mol et al., 1988; Hahlbrock & Scheel, 1989). However, other pathways have been characterised only at the level of the chemical structures of pathway intermediates. One example of this is the lignin-specific pathway in plants. The structure of the phenolic precursors which are condensed into lignin is well documented (Freudenberg & Neish, 1968). However, the enzymes and their genes have not been analysed in any detail. Thus, the first step in any attempt to manipulate and improve lignin composition will be the identification and purification of the enzymes involved in lignin biosynthesis pathways and the molecular cloning of the cognate genes. Methods for the manipulation of gene expression are now available which should open avenues for the production of increased phenolics through the overexpression of specific genes (Lagrimini et al., 1990), the reduced production of end products through the use of antisense RNA or 'sense' RNA strategies (Smith et al., 1988, 1990), or the production of novel chemical structures through the insertion of enzymes which can use the phenolic substances as precursors, but are not normally found in the plant of choice. One of the major features of phenylpropanoid metabolism is the diversity of end products. The set of enzymic reactions leading from phenylalanine to 4-coumaroyl coenzyme A is common to pathways which lead to these diverse end products and is known as the general phenylpropanoid pathway (Fig. 1). Those biochemical reactions which lead to the synthesis of specialised products are known as branch pathways. L-phenylalanine Phenvlalanine ammonia lyase rrans-cinnamic acid Cinnamate-4*hydroxylase 4-coumaric acid 4-coumarate: CoA ligase 4-coumaroyl-Co A

Fig. 1. Enzymes of the general phenylpropanoid pathway.

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Table 1. Genes encoding enzymes of the general phenylpropanoid pathway cDNA source

Enzyme Phenylalanine ammonia lyase (PAL)

4-coumarate:CoA ligase (4CL)

Gene source

Reference

French bean Parsley Rice Arabidopsis Parsley

Edwards et al., 1985 Lois etal., 1989 Tanakaefa/., 1989 Ozekie/o/., 1990 Cramer etal., 1989 Lois eta/., 1989 Minamiefa/,,1989 Ohl etal., 1990 Douglases al., 1987

French bean Parsley Sweet potato Carrot

In this chapter I will focus on biochemical and molecular aspects leading to lignin production. We have studied in detail phenylalanine ammonia lyase (PAL; EC 4.3.1.5), the first enzyme of the general phenylpropanoid pathway, and cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195), an enzyme specific to the branch pathway leading to lignin formation.

The general phenylpropanoid pathway The synthesis of phenylpropanoids is achieved via a complex set of biochemical reactions which differ depending on the specific end product. The general phenylpropanoid pathway, on the other hand, is common to all higher plants (Fig. 1). The first enzyme of this pathway, phenylalanine ammonia lyase (PAL), catalyses the deamination of phenylalanine to fra/w-cinnamic acid and represents the most widely studied enzyme of the phenylpropanoid pathway (Jones, 1984). Work carried out on the bean enzyme and its genes will be reviewed below. The only other enzyme of the general phenylpropanoid pathway which has been purified and for which gene probes are available is 4-coumarate:CoA ligase (4CL) (Kuhn et al., 1984; Douglas et al., 1987). Table 1 summarises the sources of gene probes of the general phenylpropanoid pathway.

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Biochemistry of phenylalanine ammonia-lyase PAL deaminates phenylalanine to yield trans-cinnamic acid. The enzyme is encoded by a small gene family (see below), each member of which produces multiple subunits which are generated through secondary modifications at the peptide level. Eleven isoforms have been detected after in vivo labelling of bean suspension culture cells (Bolwell et al., 1985). These multiple forms are encoded by three genes (see below). It is not clear whether the enzyme is found as a homomer or a heteromer in vivo. It is possible that in different cell types PAL has a different subunit composition. In tissue culture cells multiple forms of the native enzyme, composed of multiple subunits, can be extracted (Bolwell et al., 1985). It is not clear whether subcellular localisation is also an important facet of the regulation of the enzyme. On the other hand, it has also been demonstrated that expression of a single full-length cDNA in Escherichia coli cells leads to the production of active enzyme in bacteria, thus indicating that homotetramers of the enzyme may also be found in vivo (Schulz et al., 1989). This structural diversity is likely to play an important role in the in vivo regulation of the enzyme which is the first and regulatory step of the phenylpropanoid metabolism, both during plant development and in response to environmental signals. The enzyme is under strict genetic and biochemical control. This is seen in the low levels of expression in tissues which are not heavily committed to the production of phenolics. However, in response to environmental signals such as fungal elicitor treatment of suspension culture cells, PAL enzyme biosynthesis is rapidly initiated (Lawton et al., 1983; Bolwell et al., 1985). The multiplicity of different genes coding for different subunits leads to the formation of enzyme forms with different Kms for phenylalanine and enables a rapid modulation of the flux through the pathway (Bolwell et al., 1985; Liang et al., 19896). It has been demonstrated that the pattern of isoforms changes after elicitor treatment. Forms with a lower Km for substrate increase, illustrating the demand for increased flux through the pathway required for phytoalexin production (Bolwell et al., 1985; Liang et al., 19896). However, detailed analysis of the subunit composition of individual PAL isoforms in specific cell types during changes in plant development has not been undertaken. In addition, the subcellular localisation of different enzyme forms may play an important role in the regulation of the phenylpropanoid pathway but this has not yet been studied. So far the developmental and tissue specific expression of PAL genes has been analysed through PAL promotermarker gene fusion (Bevan et al., 1989; Liang et al., 1989a; see below) and by whole plant tissue RNA analysis (Liang et al., 19896).

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The rapid response in reaction to environmental signals is caused by the transcriptional activation of PAL genes (Lawton & Lamb, 1987) and the synthesis of new PAL mRNA (Edwards et al., 1985), leading to de novo synthesis of the enzyme (Cramer et al., 1985). This increase in gene activation is extremely rapid: using sensitive molecular methods an activation of PAL gene transcription has been measured within 5 min of elicitor treatment of bean suspension cultures (Lawton & Lamb, 1987). Once peak levels of PAL mRNA have been reached (after 2 h in the bean suspension culture system), transcript accumulation from the PAL genes is reduced, causing a transient accumulation of PAL mRNA (Edwards et al., 1985). This in turn leads to a reduced rate of enzyme synthesis (Bolwell et al., 1985). However, even several hours after elicitor treatment PAL enzyme accumulation has been observed (Cramer et al., 1985). This implies that a pool of stable PAL mRNA is maintained in these cells. Thus, a sensitive regulation of PAL gene expression is achieved, reflecting the requirement for the control of the flux through the phenylpropanoid pathway. In addition to this up-regulation, PAL is also under strict negative control. It has been postulated that the reaction product of PAL, cinnamic acid, is itself an inhibitor of PAL enzyme activity (Lawton et al., 1980). It has been demonstrated that after addition of exogenous cinnamic acid to suspension cultures, PAL enzyme activity is rapidly lost owing to an inactivation of the active site dehydroalanine of the enzyme (Bolwell et al., 1986). In addition, cinnamic acid has also been shown to inhibit PAL mRNA appearance (Bolwell et al., 1988) with a differential effect being exerted on the different members of the bean PAL family (Mavandadef a/., 1990). One aspect shared with several other genes of the phenylpropanoid pathway is the transient induction after environmental challenge. This has also been demonstrated for chalcone synthase (Ryder et al., 1984) and chalcone isomerase (Cramer et al., 1985; Mehdy & Lamb, 1987), enzymes involved in phytoalexin production, and for cinnamyl alcohol dehydrogenase (CAD) an enzyme of lignin biosynthesis, in response to elicitor treatment of bean tissue culture cells (Grand et al., 1987). The developmental regulation and tissue-specific expression of PAL has been analysed in a whole plant study in bean and has shown that differential expression of PAL genes can be detected in different tissues (Liang et al., 19896). However, this study has not been complemented by a detailed analysis of PAL biochemistry at the cellular or tissue level.

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Molecular biology of phenylalanine ammonia-lyase

Isolation of PAL cDNAs and genes PAL cDNA clones have been isolated from a number of species (Table 1). The first PAL cDNA clone was isolated from French bean suspension culture cells treated with fungal elicitor (Edwards et al., 1985). The clones were preselected by differential hybridisation to RNA from non-treated and elicitor-treated suspension culture cells. Further identification involved in vitro translation of hybrid-selected RNA and immunoprecipitation (Edwards etal., 1985), using an antibody raised against the purified bean PAL enzyme (Bolwell et al., 1985). This work identified pPAL5 as a PAL cDNA clone. Using this clone the genes encoding PAL have been isolated from genomic libraries of French bean (Cramer et al., 1989). In this plant PAL is encoded by a small multigene family. There are three classes of gene, each of which displays limited polymorphism when analysed in different bean varieties. The classes are represented by the gene encoding the pPAL5 cDNA, gPALl (only truncated versions of this gene have been isolated), and by gPAL2 and gPAL3 (Cramer et al., 1989). The latter two genomic clones encode proteins of 710 and 712 amino acids, respectively. At the protein level sequence similarity of 72% is seen. The differences are likely to contribute to the isoform diversity observed. Use of marker gene fusion to study temporal and spatial expression of PAL gPAL2 Transcriptional and translational fusions between 5' upstream sequences of gPAL2 and the bacterial gus reporter gene have been generated (Bevan et al., 1989; Liang et al., 1989a). These constructs were transferred to tobacco and potato (Bevan et al., 1989) via Agrobacteriummediated transformation and a detailed whole plant cytological analysis was undertaken. This has demonstrated that the regulatory sequences of this gene activate marker gene expression in developing xylem cells, phloem fibre cells, epidermal and gland hair cells, the root tips and root hair cells, in the pigmented regions of the flower petals and in response to wounding. It has also been demonstrated that high levels of expression of the marker gene can be observed in the anthers and stigma whereas low levels of expression of the gus reporter gene can be observed in the sepals, ovaries and leaves (Liang et al., 1989a). These observations are in good agreement with the pattern of expression expected from the known

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Table 2. Expression o/GUS marker gene driven by gPAL2 and gPAL3 promoters

Xylem Flowers Root tip Trichomes Cortex Epidermis Pericycle Pith Wounding

gPAL2-gus

gPAL3-gi«

+ + + + + +

ND ND +

ND ND

+

+ ND

+ + + +

Notes: Plants were transformed with gPAL2-gus and gPAL3-gus vectors and the tissue-specific expression determined using a histochemical assay. ND, not detectable.

biochemical specialisation of these cell types: lignin biosynthesis in the xylem, pigment biosynthesis in flower petals, low molecular mass phenolic compounds and various flavonoids in root hairs and the root apical region, flavonoid UV protectants in the epidermis, and phytoalexin and lignin-like synthesis after wounding. gPAL3 Another member of the PAL gene family, gPAL3, has also been described by Cramer et al. (1989). This clone has 261 bases 5' upstream from the granscription start site. Additional clones homologous to gPAL3 have been isolated (W. Schuch and K.J. Edwards, unpublished data) which contain approximately 3-9 kb of 5' upstream sequences. This promoter fragment was fused to the bacterial gus reporter gene and the vector transferred to transgenic tobacco plants (D. Shufflebottom et al., unpublished data). Histochemical analysis of these plants revealed a different pattern of expression. The promoter of gPAL3 does not lead to the expression of the gus reporter gene in the developing or mature xylem (Table 2). Instead, expression is seen in the central pith area of the stem and, particularly, in a single cell layer surrounding the vascular cylinder which is believed to represent the endodermis. The high level of expression seen in the endodermis is likely to be correlated with suberin deposi-

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tion in this tissue. Suberin is a polyphenolic compound made up of phenylpropanoid monomers. Low level expression is also seen in the epidermis, the pericycle and the root hair cells. This, a clear functional division of these two members of the PAL gene family is observed. In addition gus reporter gene expression is also detected in some other tissues in which gPAL2 is expressed (Table 1). It has been demonstrated by in vitro antisense experiments that gPAL3 encodes the most basic isoform subunits of PAL (Liang et al., 19896). These basic isoforms of the PAL enzyme have been shown to have a lower Km than the acidic isoforms (Bolwell et al., 1985). If the regulation of the endogenous tobacco PAL genes and enzyme forms is equivalent to those in bean, it is likely that for the production of suberin in the endodermis and other tissues the gPAL3-encoded enzyme is used in preference to the gPAL2 encoded subunit. A similar conclusion has been reached by Liang et al. (19896). Thus, combining promoter-marker gene fusion analysis of whole transgenic plants with a single cell biochemical approach promises to unravel the biochemical specialisation and complexities of the phenylpropanoid pathway. It is clear that a complex set of signals must operate at the DNA level which lead to cell-specific activation and control of expression of the PAL gene family. Deletion analysis of gPAL2 promoter In order to gain an insight into the molecular recognition architecture of the gPAL2 promoter and to identify cw-elements responsible for expression during xylogenesis, an extensive deletion analysis was carried out (M. Bevan etal., unpublished data). 5' deletions and internal deletions of the gPAL2 promoter have been constructed. These have been fused to the gus reporter gene and have been transferred to tobacco plants. Populations of individual transgenic plants containing these vectors have been analysed for expression in xylem, cortex, root tips and flowers using the histochemical method. This has demonstrated that 5' deletions to — 123 bp relative to the transcription start site abolished promoter activity in all tissues. Deletions which left 253 or more bases of the 5' region gave tissue-specificity of expression identical to the 11 kb promoter fragment used previously (Bevan etal., 1989). In addition the quantitative levels of expression were identical to those of the longer promoter. A fragment of 148 bases between -150 and —252 was identified which alone gave xylem- and petal-specific expression. An overlapping fragment covering the region between —212 and —252 gave petal-specific expression only. Thus it appears that the as-acting region responsible for xylem-specific expression is located in this region between —212 to -252. Further work is now in progress to clarify this.

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Lignin biosynthesis Biochemistry of cinnamyl alcohol dehydrogenase (CAD) Lignin biosynthesis is of crucial importance in plant growth and development. Lignin(s) are an essential component of cell walls in tissues like the sclerenchyma and xylem of vascular plants. They play an important role in the conducting function of the xylem by reducing the permeability of the cell wall to water. They are also responsible for the rigidity of the cell wall, being deposited during secondary cell wall thickening. In woody tissue, lignins act as a bonding agent between cells giving the plant resistance to impact, compression and bending. Finally, they are involved in mechanisms of resistance to pathogens by impeding the penetration or propagation of the pathogen. The branch pathway of lignin biosynthesis is shown in Fig. 2. The first steps are shared with the general phenylpropanoid pathway. Cinnamic acid is transformed by hydroxylation and methylation to produce acids with different substitutions on the aromatic ring. The 4-coumaric, ferulic and sinapic acids are then esterified by hydroxycinnamate:CoA ligase to produce cinnamyl-CoAs, which are reduced by cinnamyl-CoA reductase (CCR) to produce the three aldehydes. These in turn are reduced by CAD to the three cinnamyl alcohols which are then polymerised into lignins. Recently, the cloning of CAD has been reported from elicitor-treated bean tissue culture cells (Walter et al., 1988). In these experiments CAD 4-coumaric acid

I

*- ferulic acid

•

I

-»-

sinapic acid

\

hydroxycinnamate CoA ligase

-f-

-r

coumaraldehyde

coniferaldehyde

sinapaldehyde

I

I

I

4-

cinnamyl alcohol dehydrogenase coumaryl alcohol

coniferyl alcohol peroxidase

I lignins

Fig. 2. Enzymes of lignin biosynthesis.

sinapyl alcohol

106

w. SCHUCH

cDNA clones were identified using a polyclonal antibody raised against the purified poplar enzyme. However, in a later publication the authors pointed out that the proposed CAD clone contains considerable nucleotide and amino acid sequence homology to maize malic enzyme (Walter et al., 1990). In order to be able to clone authentic CAD we have decided to purify CAD to homogeneity, partially sequence the enzyme and use oligonucleotide probes for the cloning of CAD from tobacco stems. Purification of tobacco CAD enzyme We have developed an efficient protocol for the purification of CAD from tobacco stems (Halpin et al., 1992). This organ was chosen as considerable secondary thickening, caused by extensive lignin deposition, takes place during the growth of the tobacco plant. Our purification protocol employs three chromatography matrices - blue Sepharose, MonoQ and 2'5' ADP-Sepharose - and has lead to the purification of CAD to homogeneity with a purification of approximately 1200-fold. Thus, in tobacco stems CAD protein represents approximately 0-05% of total protein. The purified tobacco CAD enzyme consists of two different subunits with apparent molecular masses of 44 and 42-5 kDA. The composition of CAD in vivo is not clear: it is possible that the protein is present as homoor heterodimers. It is also possible that, through the combination of different subunits, CAD enzyme forms are produced which have specificity for the different aldehydes. Further characterisation of the purified enzyme will be required to resolve this question. The two subunits have been purified by reverse phase HPLC and have been used to generate a series of tryptic peptides. This has yielded peptide sequence information for the two CAD peptides. Cloning of CAD The amino acid sequence information described above has been used to design mixed oligonucleotide probes which have been used to screen a cDNA library established from 6-week-old tobacco stems. 600 000 clones have been screened and six full-length clones identified (M.E. Knight et al., unpublished data). These fall into two classes encoding the two polypeptides identified during the protein purification. The clones are now being used for isolation of CAD genes and an analysis of their expression during plant development.

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Outlook Cell wall biosynthesis is of critical importance during plant growth and development. The incorporation of lignin building blocks into the cell wall of higher plants leads to complex polymeric structures which are of vital importance for plant development and productivity. Two enzymes, PAL and CAD, representing the general phenylpropanoid pathway (PAL) and the lignin-specific branch pathway (CAD), have been isolated and characterised. The bean PAL gene has been analysed in considerable detail with particular emphasis on expression during xylem development and differentiation. This has lead to the identification of ds-elements which promote expression of marker genes in the xylem. The availability of CAD cDNA probes will now enable us to clone the CAD gene and study the developmental expression of both genes which are coordinately regulated during xylogenesis. One of the interesting challenges will be the isolation and characterisation of trans-acting factors which are involved in this coordinate regulation of expression of these genes. In addition, the availability of the CAD cDNA will now enable us to generate novel plant mutants using antisense RNA in which the expression of this enzyme has been specifically modulated. This, combined with detailed structural analysis of the cell wall, will shed light on the role of lignin deposition during cell wall biogenesis, its effect on plant stature, shape, development and its interaction with pathogens. Acknowledgement Financial support from the EEC (AGRE 0021) is gratefully acknowledged for part of this work. References Bevan, M., Shufflebottom, D., Edwards, K., Jefferson, R. & Schuch, W. (1989). Tissue and cell-specific activity of a phenylalanine ammonia-lyase promoter in transgenic plants. The EMBO Journal 8, 1899-1906. Bolwell, G.P., Bell, J.N., Cramer, C.L., Schuch, W., Lamb, C.J. & Dixon, R.A. (1985). L-Phenylalanine ammonia-lyase from Phaseolus vulgaris: characterisation and differential induction of multiple forms from elicitor-treated cell suspension cultures. European Journal of Biochemistry 149, 411-19. Bolwell, G.P., Cramer, C.L., Lamb, C.J., Schuch, W. & Dixon, R.A. (1986). L-Phenylalamine ammonia-lyase from Phaseolus vulgaris:

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Modulation of the levels of active enzyme by trans-cinnamic acid. Planta 169, 97-107. Bolwell, G.P., Mavandad, M., Millar, D.J., Edwards, K.J., Schuch, W. & Dixon, R.A. (1988). Inhibition of mRNA levels and activities by /rans-cinnamic acid in elicitor-induced bean cells. Phytochemistry 27, 2109-17. Cramer, C.L., Bell, J.N., Ryder, T.B., Bailey, J.A., Schuch, W., Bolwell, G.P., Robbins, M.P., Dixon, R. & Lamb, C.J. (1985). Coordinated synthesis of phytoalexin biosynthetic enzymes in biologically-stressed cells of bean. The EMBO Journal 4, 285-9. Cramer, C.L., Edwards, K., Dron, M., Liang, X., Dildine, S.L., Bolwell, G.P., Dixon, R.A., Lamb, C.J. & Schuch, W. (1989). Phenylalanine ammonia-lyase gene organisation and structure. Plant Molecular Biology 12, 367-84. Dixon, R.A., Dey, P.M. & Lamb, C.J. (1983). Phytoalexins: enzymology and molecular biology. Advances in Enzymology and Related Areas of Molecular Biology 55, 1-135. Douglas, C , Hoffmann, H., Schulz, W. & Hahlbrock, K. (1987). Structure and elicitor or UV-light-stimulated expression of two 4coumarate:CoA ligase genes in parsley. The EMBO Journal 6, 1189-95. Downie, J.A. & Johnston, A.W.B. (1986). Nodulation of legumes by Rhizobium: the recognised root? Cell 47, 153-4. Ebel, J. & Hahlbrock, K. (1982). Biosynthesis of flavonoids. In The Flavonoids: Advances in Research, ed. J.B. Harborne & T.J. Mabry, pp. 641-79. London: Chapman and Hall. Edwards, K., Cramer, C.L., Bolwell, G.P., Dixon, R., Schuch, W. & Lamb, C.J. (1985). Rapid transient induction of phenylalanine ammonia-lyase mRNA in elicitor-treated bean cells. Proceedings of the National Academy of Sciences (USA) 82, 6731-5. French, C.J., Vance, C.P. & Towers, G.H.N. (1976). Conversion of pcoumaric acid to p-hydroxybenzoic acid by cell free extracts of potato tubers and Polydorus hispidus. Phytochemistry 15, 564-6. Freudenberg, K. & Niesh, A.C. (1968). Constitution and biosynthesis of lignin. Molecular Biology, Biochemistry and Biophysics 2,132-9. New York: Springer-Verlag. Grand, C , Sarni, F. & Lamb, C.J. (1987). Rapid induction by fungal elicitor of the synthesis of cinnamyl-alcohol dehydrogenase, a specific enzyme of lignin synthesis. European Journal of Biochemistry 169, 73-7. Hahlbrock, K., Kreuzaler, F., Ragg, H., Fautz, E. & Kuhn, D.N. (1982). Regulation of flavonoid and phytoalexin accumulation through mRNA and enzyme induction in cultured plant cells. In Biochemistry of Differentiation and Morphogenesis, ed. L. Jaenicke, pp. 34-43. Berlin: Springer-Verlag.

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Hahlbrock, K. & Scheel, D. (1989). Physiology and molecular biology of phenylpropanoid metabolism. Annual Review of Plant Physiology and Plant Molecular Biology 40, 347-9. Halpin, C , Knight, M.E., Boudet, A., Grima-Pettenati, J. & Schuch, W. (1992). Purification and characterisation of cinnamyl alcohol dehydrogenase from tobacco stems. Plant Physiology 98, 12-16. Jones, D.H. (1984). Phenylalanine ammonia-lyase: regulation of its induction and its role in plant development. Phytochemistry 23, 1349-60. Kuhn, D., Chappell, J., Boudet, A. & Hahlbrock, K. (1984). Induction of phenylalanine ammonia-lyase and 4-coumarate:CoA ligase mRNAs in cultured plant cells by UV-light or fungal elicitor. Proceedings of the National Academy of Sciences (USA) 81, 1102-6. Lagrimini, L.M., Bradford, S. & Rothstein, S. (1990). Peroxidaseinduced wilting in transgenic tobacco plants. The Plant Cell 2, 7-18. Lawton, M.A., Dixon, R.A., Hahlbrock, K. & Lamb, C.J. (1983). Rapid induction of the synthesis of phenylalanine ammonia-lyase and chalcone synthase in elicitor-treated plant cells. European Journal of Biochemistry 130, 131-9. Lawton, M.A., Dixon, R.A. & Lamb, C.J. (1980). Elicitor modulation of the turnover of L-phenylalanine ammonia-lyase in French bean suspension cultures. Biochimica et Biophysica Ada 633, 162-75. Lawton, M.A. & Lamb, C.J. (1987). Transcriptional activation of plant defense genes by fungal elicitor, wounding and infection. Molecular and Cellular Biology 7, 335-41. Lewis, N.G. & Yamamoto, E. (1990). Lignin: occurrence, biogenesis and biodegradation. Annual Review of Plant Physiology and Plant Molecular Biology 41, 455-96. Liang, X., Dron, M., Cramer, C.L., Dixon, R.A. & Lamb, C.J. (1989b). Differential regulation of phenylalanine ammonia-lyase genes during plant development and by environmental cues. Journal of Biological Chemistry 264, 14486-92. Liang, X., Dron, M., Schmid, J., Dixon, R.A. & Lamb, C.J. (1989a). Developmental and environmental regulation of a phenylalanine ammonia-lyase-|3-glucuronidase gene fusion in transgenic tobacco plants. Proceedings of the National Academy of Sciences (USA) 86, 9284-8. Lois, R., Dietrich, A., Hahlbrock, K. & Schulz, W. (1989). A phenylalanine ammonia-lyase gene from parsley: structure, regulation and identification of elicitor and light-responsive as-acting elements. The EMBO Journal 8, 1641-8. Mavandad, M., Edwards, R., Liang, X., Lamb, C.J. & Dixon, R.A. (1990). Effects of trans-cinnamic acid on expression of the bean phenylalanine ammoni-lyase gene family. Plant Physiology 94, 671-80.

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W. SCHUCH Mehdy, M.C. & Lamb, C.J. (1987). Chalcone isomerase complementary DNA cloning and messenger RNA induction by fungal elicitor, wounding and infection. The EMBO Journal 6, 1527-34. Minami, E.I., Ozeki, Y., Matsuoka, M., Koizuka, N. & Tanaka, Y. (1989) Structure and some characterisation of the gene for phenylalanine ammonia-lyase from rice plants. European Journal of Biochemistry 185, 19-25. Mol, J.N.M., Stuitje, T.R., Gerats, A.G.M. & Koes, R.E. (1988). Cloned genes of phenylpropanoid metabolism in plants. Plant Molecular Biology Reporter 6, 274-9. Napoli, C , Lemieux, C. & Jorgensen, R. (1990). Introduction of a chimeric chalcone synthase gene into Petunia results in reversible cosuppression of homologous genes in trans. The Plant Cell 2, 279-89. Ohl, S., Hedrick, S.A., Chory, J. & Lamb C.J. (1990). Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis. The Plant Cell 2, 837^18. Ozeki, Y., Komamine, A. & Tanaka, Y. (1990). Induction and repression of phenylalanine ammonia-lyase and chalcone synthase enzyme proteins and mRNAs in carrot cell suspension cultures regulated by 2,4-D. Physiologia Plantarum 78, 400-8. Ryder, T.B., Cramer, C.L., Bell, J.N., Robbins, M.P., Dixon, R.A. & Lamb, C.J. (1984). Elicitor rapidly induces chalcone synthase mRNA in Phaseolus vulgaris cells at the onset of phytoalexin defense response. Proceedings of the National Academy of Sciences (USA) 81, 5724-8. Schulz, W., Eiben, H.G. & Hahlbrock, K. (1989). Expression in E. coli of catalytically active phenylalanine ammonia-lyase from parsley. FEBS Letters 258, 335-8. Smith, C.J.S., Watson, C.F., Bird, C.R., Ray, J., Morris, P.C., Schuch, W. & Grierson, D. (1990). Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants. Molecular and General Genetics 224, 477-81. Smith, C.J.S., Watson, C.F., Ray, J., Bird, C.R., Morris, P.C., Schuch, W. & Grierson, D. (1988). Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334, 724-6. Stachel, S.E. & Zambryski, P.C. (1986). Agrobacterium tumefaciens and the susceptible plant cell: a novel adaptation of extracellular recognition and DNA conjugation. Cell 47, 155-7. Tanaka, Y., Matsuoka, M., Yamanoto, N., Ohashi, Y., KanoMurakami, Y. & Ozeki, Y. (1989). Structure and characterisation of a cDNA clone for phenylalanine ammonia-lyase from cut-injured roots of sweet potato. Plant Physiology 90, 1403-7. van der Krol, A.R., Lenting, P.E., Veenstra, J., van der Meer, I.M., Koes, R.E., Gerats, A.G.M., Mol, J.N.M. & Stuitje, A.R. (1988). An anti-sense chalcone synthase gene in transgenic plants inhibits flower pigmentation. Nature 333, 866-9.

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van der Krol, A.R., Mur, L.A., Beld, M., Mol, J.N.M. & Stuitje, A.R. (1990). Flavonoid genes in Petunia: addition of a limited number of gene copies may lead to suppression of gene expression. The Plant Cell 2, 291-9. Walter, M.H., Grima-Pettenati, J., Grand, C , Boudet, A.M. & Lamb, C.J. (1988). Cinnamyl alcohol dehydrogenase, a molecular marker specific for lignin synthesis: cDNA cloning and mRNA induction by fungal elicitor. Proceedings of the National Academy of Sciences (USA) 86, 5546-50. Walter, M.H., Grima-Pettenati, J., Grand, C , Boudet, A.M. & Lamb, C.J. (1990). Extensive sequence similarity of the bean CAD4 (cinnamyl-alcohol dehydrogenase) to a maize malic enzyme. Plant Molecular Biology 15, 525-6.

H.J. BOHNERT, D.M. VERNON, E.J. DeROCHER, C.B. MICHALOWSKI and J.C. CUSHMAN

Biochemistry and molecular biology of CAM Introduction Crassulacean Acid Metabolism (CAM) has been called a curiosity (Osmond, 1978), the importance of which has been long overlooked in the context of environmental adaptations of plants to cope with arid, hot environments. CAM has since been studied more intensely and our knowledge has been extended by focusing on physiological studies (Ting & Gibbs, 1982; Ting, 1985; Cockburn, 1985), on the biochemical characterisation of important enzymes (O'Leary, 1982; Nimmo et al., 1986), and on ecological aspects (Kluge & Ting, 1978; Liittge, 1987). As a result of the diurnal separation of night CO2 fixation by PEPCase, storage of the acidic product, malate, in the vacuole, and final carbon assimilation by Rubisco, CAM plants conserve water and hence can occupy ecological niches that have limited water or CO2 availability. To mention only a few examples (see Kluge & Ting, 1978), CAM is expressed in flowering plants that are continually exposed to sea water, in cacti that inhabit true deserts, in climbing vines in the rainforest (Ting et al., 1985), and in freshwater plants where CO2 is limiting (Keeley & Busch, 1984). CAM may be constitutive in a species, or the pathway may appear during ageing either throughout the plant, or specifically in the older leaves (Guralnick et al., 1984; Ting, 1985). In some plants the pathway may be induced by environmental factors, such as drought, high salinity or low temperature, which affect the availability of water (Winter, 1982). The fact that CAM has arisen in many different plant families would indicate that the genetic modifications required for CAM photosynthesis are relatively small. Assuming that CAM plants utilise genes and enzymes common to all (C3) plants (Cockburn, 1981) in a specialised manner, the study of CAM development or induction can be expected to reveal general molecular responses to environmental factors that affect water status and CO2 availability. Society for Experimental Biology Seminar Series 49: Inducible Plant Proteins, ed. J. L. Wray. ©Cambridge University Press, 1992, pp. 113-137.

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Because of its ability to switch to CAM under environmental stress, Mesembryanthemum crystallinum (common ice plant; Aizoaceae) has been used for some time as a physiological model to investigate CAM (Winter & von Willert, 1972; Heun et al, 1981; Winter & Gademann, 1991). The information gathered on the physiology of CAM induction has made the ice plant a good candidate for biochemical and molecular studies of the CAM pathway (Bohnert etal., 1989; Cushman etal., 1991). There are several advantages to using this plant. The ability to induce CAM purposely for experimental studies is probably the most important advantage. Plants may be switched to CAM, which also enhances the reproductive growth phase, most rapidly by salt stress, although drought and exposure of the plants to low temperature operate in addition. The characteristic growth pattern of the ice plant is also helpful since it is developmentally programmed and influenced by stress treatment. A variety of environmental stresses can be used either to retard development in young plants, or to enhance changes in CAM induction, growth habit and organ morphology at later growth periods. A further advantage of the ice plant is the relatively small size of its genome - 390 million kbp (Meyer et al., 1990; DeRocher et al., 1990) - which is approximately three times larger than the genome of Arabidopsis thaliana. This limited genome size has been helpful in establishing gene libraries and isolating specific genes. Finally, techniques are available to culture the plant in vitro, to regenerate plants through organogenesis (Meiners et al., 1991) and to grow cell suspension cultures (De Armond et al., 1991). We are interested in studying the induction of the CAM pathway in the Fig. 1. The pathway of carbon in CAM. Diagrammatic presentation of the CAM specific pathway of malate synthesis and storage from glycolysis to gluconeogenesis. The enzymes involved in the pathway whose activities are induced by salt stress (and which increase during the transition from C3-photosynthesis to CAM) in Mesembryanthemum crystallinum include: G-PI, phosphohexose isomerase; PGM, phosphoglyceromutase; GADPH, glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; enolase; PEPCase, phosphoeno/pyruvate carboxylase; MOD, NADP+ malic enzyme; MDH, NAD(P)+ malate dehydrogenase; and PPDK, pyruvate orthophosphate dikinase. Those enzymes that are not induced by salt stress include: Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase; aldolase; TPI, triose phosphate isomerase; PG1M, phosphoglucomutase; PFK, phosphofructokinase; Fl,6BPase, fructose 1,6-bisphosphatase. For simplicity, the intracellular compartmentation of these enzymes and their regulation has not been included. The genes encoding PEPCase, GAPDH, PPDK, MOD and MDH have been isolated and found to be induced. The vacuole as the malate storage compartment is indicated.

Biochemistry and molecular biology of CAM

115

ice plant to learn about the regulation of genes that lead to functional CAM. We have focused primarily on the expression and structure of two genes encoding the enzyme PEPCase, one specifying a C3-form of the enzyme and one gene specifying the CAM-form. In addition, genes for several other CAM-related enzymes, and genes for photosynthesisrelated functions, have been obtained and their regulation has been partially characterised. The CAM pathway The flow of carbon during CAM is schematically given in Fig. 1. Without attempting to review the solid, physiological information about CAM, LIGHT

DARK

(slomata open)

(slomata closed) • MALATE '.'•

O A A — ^ ••Mai

Mai

ATP*

" PEP

A M P

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PEP

\

t

2-PGA - RuBP •*—k

2-PGA

j

3-PGA '

3-PGA

ATP

^ <

ADP

1,3-DiPGA i^NADH.

\ NADP*

L-NAD. < 3-PGAId = ±

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L I

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NADP. "^

T

CALVIN BENSON CYCLE

3-PGAId ^ Z T DHAP

DHAP

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1 1 Amylopeclin Amylose

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UOPG \ Sucrose-P •

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Sucrose

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H . J . BOHNERT et al.

the figure shows the many interactions of enzymes and products of the pathway with basic plant energy metabolism. The reactions catalysed during functional CAM are, in principle, similar to the reactions catalysed by guard cells in all plants (Cockburn, 1981; Willmer, 1983). A view which we favour is that the evolution of CAM has included appropriate changes in the control of gene expression to allow expression of guard cell-specific genes of C3 plants in other cell types in CAM plants. Included in this figure are the enzymes whose activities change (abbreviations of induced enzymes are outlined in bold) during CAM induction (see below) in M. crystallinum, based on a number of physiological studies (Edwards et al, 1982; Holtum & Winter, 1982; Winter, 1982). Owing to its abundance and key role, the enzyme on which most attention has been focused in CAM and in C4 plants is PEPCase (O'Leary, 1982; Nimmo et al., 1986; Andreo et al., 1987). PEPCase isoforms All plants contain a PEPCase enzyme which, among other likely roles (Vidal et al., 1986), serves to replenish tricarboxylic acid cycle intermediates that are consumed during ammonium assimilation (Latzko & Kelly, 1983). The role of this enzyme, other than its presumed housekeeping function, has not been studied in any detail and its activity is probably not controlled by environmental factors. Isoforms of PEPCase have been observed in many plants (C3, C4 and CAM); for example, in rice five isoforms have been detected immunologically and in most plants two to four bands that react with anti-PEPCase antibodies are found (Matsuoka & Hata, 1987). It is not clear how these isoforms arise, e.g. by posttranslational modification of one form, or whether all of these forms are products of different genes. The housekeeping-type PEPCase enzyme, concerned with anaplerotic functions, is distinct from other PEPCase enzymes that function in plants with C4 and CAM metabolism (see below). In some plants, high PEPCase activities have been detected in specialised tissues or cells, other than the mesophyll cells of C4 and CAM plants. For example, PEPCase is found in guard cells where it is involved in stomatal opening and closing (Tarczynski & Outlaw, 1990). The authors concluded, however, that - based on the alterations in chemical environment during stomatal activity - the reactions catalysed could be accomplished by a C3-type PEPCase enzyme and that there was no reason to invoke the existence of isoforms. Less well known is the PEPCase activity and protein detected in reproductive tissues. For example, high PEPCase activity has been observed in seed pods of chickpeas (Singh,

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1989). It has been suggested that this activity may be responsible and essential for the reutilisation of respiratory CO2. Like chickpeas, the ice plant contains high PEPCase activity in the seed pods. This protein appears to be identical or very similar to the CAM-form of the enzyme as detected by immunoblotting analysis (C.B. Michalowski, unpublished data). However, no information is available on whether the housekeeping PEPCase is responsible for the activity, or whether different ppc genes are expressed in such organs or differentiated cells. Immunocytological staining, after reaction with anti-PEPCase antibodies of leaf sections of stressed ice plants, indicates that PEPCase enzyme is present in all leaf cells (EJ. DeRocher, unpublished data). Quantitation of relative amounts in specific cells was, however, not attempted. In Mesembryanthemum at least two isoforms exist, although the antibodies used for immunoblotting distinguish up to four bands (Hofner et al., 1987; Schmitt et al., 1988; Cushman et al., 1990a). There are two major bands with molecular masses of 109 000 and 110 000 kDa, and two minor bands of approximately 120 000 kDa. The latter two bands cannot be explained at present. They may be the result of ubiquitinylation or glycosylation, or they may be the products of genes that have not yet been detected. The PEPCase of 109 000 kDa is the product of the housekeeping gene ppc2 and represents the C3-form of the enzyme. This isoform is found in roots, shoots and leaves of young plants. The amount of Ppc2 protein diminishes during ageing of the plants and the amount of enzyme is not enhanced by environmental stresses that induce CAM. A second isoform of PEPCase with an apparent molecular mass of 110 000 kDa is the product of the ppcl gene which is induced during the transition to CAM (Cushman et al., 19906). The amount of Ppcl protein is low or not detectable in young unstressed plants; it accumulates slowly when the plants age and drastically when the plants are stressed. After stress the total PEPCase activity increases up to 40-fold (Holtum & Winter, 1982) and the amount of the enzyme increases at least 10-fold within approximately 6 days (Ostrem et al., 1987) to amount to more than 1% of the total soluble protein in stressed green tissue. No increase in the level of immuno-detectable enzyme or activity occurs in root tissue. Figure 2 shows a comparison of the C3- and CAM-forms of the ice plant PEPCase proteins. The C3-form, isolated from young, unstressed leaves, was compared with the CAM-form, isolated from old, stressed leaves. Enzymatically active fractions were subjected to partial proteolytic digestion using V8-protease (Cleveland et al., 1977). The digestion products were separated by SDS-PAGE, blotted to nitrocellulose filters and reacted with anti-PEPCase antibodies. Most of the digestion products are identical between the two enzymes reflecting the high degree of sequence

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2

3

97- '

66 -

29 -

kd Fig. 2. Biochemical differences between PEPCase isolated from the ice plant operating in C3-mode and in CAM-mode. PEPCase isolation was performed from either young (4-week-old plants or younger), unstressed leaves (C3-mode), and from leaves and shoots from 8-week-old plants after 10 days of salt stress (CAM-mode) as described (Schmitt et al., 1988) with minor modifications. On Sephacryl columns the PEPCase activity from stressed and unstressed plants eluted in different fractions (D.M. Vernon, unpublished data). Column fractions containing PEPcase activity were pooled, protein was concentrated, desalted and stored in aliquots at -70 °C in 50mM Tris/HCl, pH 8.0, 5itiM DTT, 5 mM MgCl2, 7.5% glycerol. The partially purified PEPCase preparations from either C3 or CAM plants were separated by SDS-PAGE (8% gels), briefly stained with Coomassie Blue, and the PEPCase bands, 109 kDa and 110 kDa, respectively, were excised. After digestion with V8-protease, essentially as described by Cleveland et al. (1977), the products of cleavage were separated by (12%) SDS-PAGE and visualised immunologically. Lane 1, mixture of the digestion products of the C3and CAM-forms; lane 2, C3-form; lane 3, CAM-form. The arrow points to the most obvious differences in the largely identical protease digestion patterns.

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identity which is obvious from the deduced amino acid comparisons (see below). The CAM-form PEPCase, for which full-length cDNA clones are available, has been cloned for expression in Escherichia coli (Fig. 3A). Expression of such constructions in a ppc~ E. coli strain led to the successful complementation of the mutant strain on selective media, indicating that the enzyme can function in E. coli (data not shown). Similar complementation had previously been used to identify PEPCase-encoding clones from Anacystis nidulans genomic DNA after introduction into a PEPCase-deficient E. coli strain (Kodaki et al., 1985). Using a fulllength cDNA clone, strong overexpression of the CAM protein could be achieved with the P,ac promoter system in E. coli. However, the enzymatically active Ppcl protein (CAM-form enzyme) accumulated in a number of different strains of E. coli only to a relatively low level, and most of the overexpressed protein was partially degraded (Fig. 3C) and found in inclusion bodies. To circumvent this problem, CAM-PEPCase was cloned, under the control of an insect promoter (Fig. 3B), into a baculovirus expression vector system (Smith et al., 1985) and transfected into cultured insect cells. Cell lines were obtained that contained nondegraded Ppcl protein (Fig. 3C) at moderate levels, as detected immunologically. Owing to the low steady-state levels of the mRNA for the C3-form of ice plant PEPCase (Ppc2 protein) in leaves, we have only recently obtained full-length ppc2 cDNAs from root libraries (J.C. Cushman and C.B. Michalowski, unpublished data). Sequencing of a number of these C3-form cDNAs indicated that all were derived from one gene. This C3specific cDNA will allow us to verify the genomic sequence obtained earlier (Cushman & Bohnert, 19896) and will provide clones for overexpression of the enzyme in insect cells. Obtaining PEPCase enzyme from the overexpression of these cDNAs will be an important achievement for structural studies and for the in-depth biochemical characterisation of the different PEPCase enzymes, which are difficult to separate from each other when isolated from plant material.

PEPCase regulation A comparison of the deduced amino acid sequences of seven available PEPCase genes indicates general homology over the entire coding region with a relatively lower degree of homology in the amino-terminal portions. In total, 14 regions can be identified that have significantly higher identity and similarity scores than average. These regions were initially outlined (Rickers et al., 1989) for four enzymes and the addition of the

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A

•MCS P lac = LacZ promoter MCS = Multiple cloning lite Ap = Ampicillin ruUtance

-Ppcl Pph = polyhcdrin promoter The initiation codpn of Ppc 1 is underlined.

B 1 2

3

4

1 2

3 4

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sequences published since has not changed this conserved (putative) domain structure. Only some of these regions have been associated with a function. One region has been identified as a regulatory site. A conserved threonine (position 223 in ice plant ppcl) indicates a site that is important for the regulatory effect of fructose, 1,6-bisphosphate (Sutton et al., 1986). Positions 629-655 (ice plant ppcl) have been implicated as part of the binding site for phosphoeno/pyruvate (reviewed by Andreo et al., 1987). Studies undertaken to identify the active site suggest that it should be located in the carboxy-terminal portion of the enzyme (where several conserved regions can be identified) and that two histidines, a cysteine, one arginine and one lysine should be active site residues (Andreo et al., 1987). Indeed, two histidine residues and several positively charged amino acids are conserved in all sequences in the more highly conserved 14 regions (above), although cysteine residues are conserved only among the higher plant sequences. Clearly, the availability of sequences has provided some impetus to focus more work on this enzyme, which is regulated by a number of different metabolites and by phosphorylation

Fig. 3. Gene constructions and over-expression of Ppcl protein (CAMPEPCase). A, Vector for the expression of Ppcl protein in E. coli. The full-length cDNA clone for ppcl (Rickers et al., 1989) was cloned into pKEN602 (Nakamura et al., 1980) for induction of protein expression in E. co//WM61on::tnlO(Mandeckie/a/., 1986). B, Vector for the expression of Ppcl protein in insect cells. The full-length cDNA clone for ppcl (Rickers et al., 1989) was cloned into pAC373 (Smith et al., 1985) for expression in insect cell cultures. C, Immunological detection of Ppcl protein by immunoblotting after separation of total cell extracts by SDSPAGE. CAM-PEPCase activity was observed only in extracts which contained Ppcl protein. Panel A, Expression of CAM-PEPCase in insect cells. Lane 1, non-transformed insect cells; lane 2, PEPCase expression in insect cells; lane 3, PEPCase in young, unstressed ice plants; lane 4, PEPCase in 7-week-old ice plants, after a 7-day stress period. Panel B, Expression of CAM-PEPCase in E. coli. Lane 1, PEPCase from salt-stressed ice plants; lane 2, E. coli protein extract containing vector DNA only; lane 3, overexpression in E. coli, indicating extreme degradation of PEPCase; lane 4, same as lane 1. The top band (lane 3) migrates at the same position as PEPCase in stressed ice plants (lanes 1 and 4). Procedures and techniques used have been described previously (Hofner et al., 1987; Schmitt et al., 1988; Ostrem et al., 1987, 1990; Cushman et al., 1989, 1990a,fo, 1992; Meyer et al., 1990; Michalowski et al., 1989a,b, 1991; Vernon etal., 1988, 1991; DeRocher et al., 1990, 1991; Rickers et al., 1989; McElwain et al., 1991; De Armond etal., 1991).

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status, and whose activity appears to be dependent on the aggregation state of the enzymes (Jawali, 1990; Jiao & Chollet, 1990; RodriguezSotres & Munoz-Clares, 1990; Wu et al., 1990; Jiao et al., 1991). The PEPCase enzyme found in the leaves of C4 plants, such as maize or sorghum, is among the best studied. The developmentally regulated biosynthesis (for a recent review see Nelson & Langdale, 1989), the genes, and kinetic parameters of these enzymes are known (e.g. Andreo et al., 1987; Hudspeth & Grula, 1989). Most significantly, the enzyme is photoregulated by a phosphorylation/dephosphorylation cycle (Jiao & Chollet, 1990). This cycle assures optimal integration of the competing activities of carbon assimilating enzymes when the plants are in a functional C4 mode. Recently, light activation was shown to be effected by phosphorylation of a single serine residue. The N-terminal serine-15 in the maize enzyme is targeted by a specific serine kinase (Jiao etal., 1991). The kinase may be replaced by a mammalian cAMP-dependent protein kinase (Terada et al., 1990). The substrate of the protein kinase appears to be determined by a sequence in which the phosphorylated serine is preceded by a pair of acidic and basic amino acids (Jiao & Chollet, 1990). This sequence, NH2-D/E-K/R-X-X-S15-COOH, is, indeed, found in the photoregulated C4 (Yanagisawa et al., 1988; Hudspeth & Grula, 1989; Matsuoka & Minami, 1989) and the ice plant CAM (Cushman & Bohnert, 1989a) enzymes, and is absent from the non-regulated ice plant C3-form (Cushman & Bohnert, 19896) of the enzyme. CAM-PEPCase from B. fedtschenkoi (Nimmo et al., 1986) is phosphorylated at a serine residue at night, which appears to confer low sensitivity to inhibition by malate, while the dephosphorylated day-form is more sensitive to malate inhibition. Thus, while this feature of the enzyme is similar to that found with the C4 enzyme, the mode of regulation by phosphorylation is different. It appears that CAM and C4 plants have evolved separate circuits to control the key enzymes and thus avoid futile cycles of carbon assimilation. The CAM-PEPCase enzyme, like that from C4 plants, appears to be controlled by its aggregation state. Dimer and tetramer forms have been observed, although some controversy exists as to their importance. Considering that CAM has appeared in different plant families and that the pathway may have evolved a number of times, it appears possible that different, pre-existing ppc genes were recruited which then gave rise to different CAM-PEPCase enzymes whose activity is controlled by different mechanisms. Much less is known about the biochemistry and molecular biology of other CAM/Q enzymes and their genes than for PEPCase enzymes. The activity of a number of these enzymes is up-regulated during CAMinduction in the ice plant (Foster et ah, 1982; Edwards et al., 1982;

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Holtum & Winter, 1982) and during C4 development (e.g. Nelson & Langdale, 1989). It is to be hoped that in addition to more work on gene structure and expression, additional work will be focused on the biochemistry and structure of the enzymes. Interest in the developmental aspects of the establishment of the C4 pathway, in plants such as Flaveria (Adams et al., 1986) that contain interfertile C3, C3/C4 intermediate, and C4 species, and in CAM as a water-conserving pathway, should be sufficient reason to do this.

PEPCase genes PEPCase is encoded by a multigene family of at least three (and probably up to five) genes in maize, with the C4-specific form being encoded by a single gene (Hudspeth & Grula, 1989; Matsuoka & Minami, 1989). Hermans & Westhoff (1990) distinguished four classes of hybridisation signals in two Flaveria species, one species being a C4-form and the other a C3/C4 intermediate, but an assignment of the genomic hybridisations to specific genes must await further studies. Two genes encoding different PEPCase enzymes have been identified and characterised in the ice plant (Cushman et al., 1989; Cushman &. Bohnert, 1989a,6). ppcl is the CAM-specific form, which is expressed predominantly in green tissue, leaves and shoots. A second isogene, ppc2, appears to be responsible for the housekeeping functions of PEPCase and is expressed constitutively at low levels in all tissues of the plant. A comparison of the deduced amino acid sequences of PEPCase enzymes from E. coli (Fujita et al., 1984), Corynebacterium glutamicus (Eickmanns et al., 1989), the cyanobacterium Anacystis nidulans (Katagiri et al., 1985), the C4 enzymes from Zea mays (Izui et al., 1986; Yanagisawa etal., 1988; Hudspeth & Grula, 1989; Matsuoka & Minami, 1989) and Sorghum vulgare (Cretin etal., 1990), the ice plant CAM-form (Rickers et al., 1989; Cushman & Bohnert, 1989a), the ice plant C3-form (Cushman & Bohnert, 19896) and the C3-form of Nicotiana tabacurn (Koizumi et al., 1991) has been performed and the resulting dendrogram (CLUSTAL multiple alignment: Higgins & Sharp, 1988, 1989) of PEPCase amino acid alignments is shown in Fig. 4. Separate from the higher plant and the E. coli sequence are the sequences of Corynebacterium and Anacystis. The higher plant C4-forms are very similar to one another and presumably are the result of an early gene duplication, whereas the C3form and the single CAM sequence diverged relatively late. This dendrogram must obviously be considered very preliminary owing to the limited number of sequences. For example, the separation of CAM and

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r

Mc2

C4

CAM

C3

Nt

Mc1

Zm

Sv

Ec

Cg

An

Fig. 4. Dendrogram of Q , C4, and CAM-PEPCase sequences. Amino acid sequences were deduced from either genomic or cDNA sequences. An, Anacystis nidulans; Cg, Corynebacterium glutamicus; Ec, Escherichia coli; Nt, Nicotiana tabacum, C3-form; Mel, Mesembryanthemum crystallinum, CAM-form; Mc2, Mesembryanthemum crystallinum, C3-form; Sv, Sorghum vulgaris, C4-form; Zm, Zea mays, C4form. For references see the text.

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C4-forms might simply reflect the dicot/monocot branches of the plant kingdom. It will be essential to obtain sequences for more CAM enzymes from different plant families, more sequences from C3-forms and, very importantly, also sequences from dicot C4-forms. CAM gene expression in the ice plant We have isolated several CAM-related genes from cDNA and genomic libraries of the ice plant in order to use them as molecular markers in the study of stress-induced expression changes (Table 1). So far, our results show that the steady-state levels of mRNAs for these genes increase drastically, by a factor of 20 to 40-fold, when plants are challenged with 500 mM NaCl (Michalowski et al., 1989a; Cushman et al., 1989, 19906; J.C. Cushman, unpublished data). However, this behaviour must be seen in the context of ice plant development. Optimal up-regulation of the amounts of mRNA is seen in 6-week-old plants (under our growth conditions). At earlier time points the plants respond more slowly; at later times, mRNA amounts have already increased to some degree in the absence of external stress (Cushman et al., 19906). Up-regulation of mRNAs is a complex process that involves not only increased transcription of PEPCase-genes (Cushman et al., 1989, 19906) and many other genes, but also presumably changes in the half-life of the mature mRNAs of PEPCase genes and their translational efficiency (Cushman et al., 19906, and unpublished data). We are at present investigating whether this behaviour can be generalised to include other CAM genes. Contrary to the up-regulation of CAM-genes, salt stress affects the mRNAs of photosynthesis-related genes, such as rbcS (small subunit of Rubisco: DeRocher et al., 1991), petH (ferredoxin NADP+-oxidoreductase: Michalowski et al., 19896), and prk (phosphoribulokinase: Michalowski et al., 1992) differently. Transcript amounts of these genes either decline (rbcS) or decline transiently (petH, prk) following salt stress. The (transient) decline of rbcS, prk and petH mRNAs is not caused by reduced rates of transcription (Cushman et al., 1989; Michalowski et al., 1992), but rather to a lower stability of the transcripts (DeRocher & Bohnert, 1991). Aside from PEPCase, a number of other CAM-related genes have been partially characterised (Table 1). These include cDNA clones for pyruvate, orthophosphate dikinase (PPDK), a specific NADP malate dehydrogenase (MDH), glyceraldehyde phosphate dehydrogenase (GaPDH) and NADP-dependent malic enzyme (MOD). Previous studies indicated that the enzymatic activities of these gene products increased upon salt stress in the ice plant (Holtum & Winter, 1982). As in the case

Table 1. Ice plant genes and their characteristics Gene

Clone

Size

Gene copy number

Enzyme/function/location

Expression characteristics

Organ specificity

ppcl

Genomic & cDNA

>7kb 3.4 kb

Single

Stress-induced

Leaves

ppc2

Genomic & cDNA

>7kb 3.2 kb

Single

Constitutive, low

Leaves & roots

gpdl"

>5kb 2.1kb >8kb 3.1kb 2.2 kb

Single

Constitutive, low & stressenhanced Stress-induced

Leaves & roots

modi"

Genomic & cDNA Genomic & cDNA cDNA

Stress-induced

Leaves

mdhl

cDNA

1.8 kb

ND

Stress-induced

Leaves

rbcSc (I-*)

Genomic & cDNAs

each > 1.5 kb 0.7-0.8 kb

Six in total

PEPCase CAM-form cytosolic PEPCase C3-form cytosolic NAD-GaPDH cytosolic PPDK chloroplast Malic enzyme NADP cytosolic MDH, NAD(P) chloroplast SSUof Rubisco chloroplast

Repressed by stress

Leaves

ppdkl

Single ND

Leaves

Notes: ND, not determined. Modi and mdhl were isolated using heterologous probes from Flaveria bidentis and Zea mays, respectively (J.C. Cushman, unpublished). PPDK (Schmitt et al., 1988; B. FifSlthaler and J.M. Schmitt, personal communication); GaPDH (Ostrem et al., 1990); PEPCase (Cushman et al., 1989; Cushman and Bohnert, 1989a,6). RbcSl^l (DeRocher et al., 1991a,fc; E.J. DeRocher and F. Quigley, unpublished data). "A single gene encoding cytosolic GaPDH was found to be expressed at a low level in the absence of salt stress and at high level after salt stress. *We suggest the name modi for the gene encoding this enzyme which is usually termed 'malic enzyme'. By its reaction it is 'malate dehydrogenase (decarboxylating)' or 'malate: NAD(P)+ oxidoreductase (decarboxylating)' (EC 1.1.1.40). c Six RbcS genes have been isolated. Transcripts have, however, been detected from only four of these genes.

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of PEPCase, these increases in enzyme activity are the direct result of increases in de novo enzyme synthesis (Hofner et al., 1987), resulting from mRNA accumulation (Ostrem et al., 1987) and increases in transcription rate (Cushman et al., 1989). We expect that the other CAMrelated enzymes will show a similar behaviour. This has since been confirmed for the transcriptional induction of the gene encoding GaPDH (D.M. Vernon, unpublished data). Having a collection of coordinately expressed genes available will enable us to identify cw-elements important to salt stress regulation and other common regulatory motifs which affect the expression of CAM genes, such as motifs for light induction, ABA, and cytokinins. Included in Table 1 are the four transcribed genes encoding the SSU of Rubisco in order to demonstrate the different behaviour of non-CAM genes in response to environmental stress.

CAM induction during development and environmental stresses A developmental component in the expression of the CAM phenotype has been recognised in the ice plant (von Willert et al., 1976; Winter et al., 1978; Bloom & Troughton, 1979; Ostrem et al., 1987; Chu et al., 1990; Winter & Gademann, 1991) and in other species (Brulfert et al., 1982; Guralnick et al, 1984; Sipes & Ting, 1985; Holthe et al., 1987). More recently, Chu et al. (1990) demonstrated increased activities for PEPCase and 'malic enzyme' as well-watered ice plants aged. Winter & Gademann (1991) showed that diurnal decreases in leaf turgor became increasingly more pronounced with leaf age. The cumulative effects of such diurnal water deficits are thought to precede the expression of the CAM phenotype. What our data (Cushman etal., 19906) contribute is a distinction between the capacity for induction of the pathway and the actual induction of the enzymes of the pathway. Gene activity follows an endogenous programme that is modulated only to a certain degree by the environment. We assume that this programme evolved to assure reproductive success of the plant in its particular ecological niche. Mesembryanthemum crystallinum is native to the Namibian Desert. A short period of winter rain enables the plant to establish itself. After several weeks, drought conditions and high salinity characterise the natural habitat (Winter et al., 1978; Bloom & Troughton, 1979). Unseasonably early drought conditions retard growth, although the plants eventually develop CAM inducibility. Drought conditions occurring later in the growth cycle are experienced by larger plants which can switch to CAM faster than young plants because the gene expression programme by which CAM-related transcripts accumulate has already been induced.

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The magnitude of the induction of the pathway enzymes in ice plants is clearly controlled by environmental factors (Cushman et al., 1990a,b). The developmental aspect that has been observed does not negate the importance of environmental stimuli for the expression of CAM, but rather shows that inducibility, not induction per se, nor the level of induction, is developmentally programmed. CAM is induced (at least the activity of some CAM enzymes is enhanced) by a number of factors, such as salt stress, drought, polyethylene glycol (PEG), low temperature, and application of the hormone ABA (Chu et al., 1990; Cushman etal., 1991; McElwain et al., 1991). It is too early to decide what the effects of the individual stress treatments are, in terms of changes in promoter activity (see below) leading to increased transcription, and in terms of the control of mRNA stability. What is clear, however, is that all response mechanisms must have a structural basis in the promoter sequences of CAM genes and in the complexity of trans-acting factors interacting with one another and with these promoters. CAM gene promoters in the ice plant The control elements of several CAM genes are being studied in our laboratory. Like other (plant) promoters that have been functionally characterised in some detail, a number of cw-acting elements to which trans-acting factors bind have been revealed (J.C. Cushman and H.J. Bohnert, unpublished data). In a discussion on DNA-protein interactions Benfrey & Chua (1990) have recently outlined the 'combinatorial complexity of a plant promoter'. Promoter characterisations have progressed to such an extent that specific enhancer and silencer elements in DNA, factor binding to DNA, multimerisation of factors, and development and environmental control over the complement of factors have been shown or suggested. In many instances, however, the in-depth analysis of a promoter and of promoter deletion mutants, in the homologous background, still needs to be performed. We have characterised promoters from the ice plant that are pertinent to the problem of gene induction during CAM build-up. Two ppc promoters, for the C3- and CAM-forms (Cushman et al., 1992 and unpublished data), and the gpdl promoter which determines the expression of GaPDH, have been sequenced. The sequences upstream of the transcription start sites, which presumably mediate transcriptional regulation, of the ppcl and ppc2 promoters are completely different. In contrast, the ppcl and gpdl promoters share several sequence motifs. Most analysis has been focused on the ppcl promoter. The regulatory regions appear to be confined to an approximately 1600 bp fragment upstream of

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the transcription start site. Easily distinguishable are TATA A and CAAT elements at approximately -35 and -108 bp upstream of the start of transcription. Further upstream, many inverted and tandem repeat elements (some of which possess internal palindromes) are found that may serve as potential binding sites for transcription factors. Preliminary analyses indicate that the promoter is negatively regulated in young, unstressed plants and that a number of different elements are involved in this control. Also indicated by in vitro analysis using gel retardation assays and DNA footprinting, is a change in the occupation of distinct elements of the promoter upon stress which appears to result in transcriptional enhancement. Such in vitro data must be confirmed by corresponding in vivo analyses before we can assign a specific role to any particular DNA binding activity. A common strategy used to test the specificity of promoter elements is to link these elements to a reporter gene and to introduce the constructs into transgenic hosts. Usually no problems are encountered when the species from which the promoter was derived, and the transgenic plant into which this promoter had been transferred, show similar use and regulation of the gene and gene product. In many instances tobacco is used, because of the ease with which this species is transformed and regenerated. The initial question to be answered was whether a CAM promoter would be expressed in tobacco with the same tissue- or cellspecificity as it is expressed in the ice plant. Fusion of the ppcl promoter to the coding region of Gus (Jefferson et al., 1987) drives the expression of Gus in transgenic tobacco at high levels in the absence of stress in most cell types of tobacco (J.C. Cushman, unpublished data). Similar patterns of expression have been observed for a Q-PEPCase promoter from maize expressed in transgenic tobacco (Matsuoka & Sanada, 1991). Since the expression of the ppcl promoter was thus different in tobacco from its expression in the ice plant, a homologous test system had to be developed that could express promoter constructions in a CAM-specific manner. The character of the ice plant ppcl promoter is faithfully mimicked in transient assays when fused to a reporter gene (Gus) and introduced into intact ice plant leaves by microprojectile bombardment. Unstressed leaves show little GUS activity, whereas GUS activity is significantly higher when constructs are shot into leaves from salt-stressed plants (J.C. Cushman and H.J. Bohnert, unpublished data). Since care was taken to include the 5'- untranslated leader region of the ppcl gene in all gene constructs, the entire promoter and deletions of the promoter, one can assume that salt stress-inducibility is sensed by the promoter of the ppcl gene. Using such promotor deletions, we were able to establish that only the region from the transcription start site to position —133 bp of the

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promoter is essential for the salt stress response. Presumably, regions further upstream are involved in positive and negative regulation of this promoter. Further c«-elements, and their corresponding mms-acting factors, are likely to be responsible for the fine tuning of PEPCase expression in response to the multitude of internal signals derived from second messengers or direct sensing mechanisms that communicate the external, environmental status of the plant. Most likely the greatest challenge, and the largest reward, will be to study and understand the signals and the signal transduction pathways that govern how ice plants acquire the developmental competence to become induced, and how the pathway is then expressed. Any hypothesis about competence acquisition must consider the whole plant developmental aspect: plants are gradually becoming competent after a specific time point in development (approximately 6 weeks of age). How do younger plants compare in this respect? Do they lack the frans-acting factors that allow older plants to respond to the environment, or is the signal transduction pathway between the promoters and the environment not yet expressed or disfunctional? Is DNA in young plants different from DNA in old plants, for example by degree of methylation? These are tissues in a 6-week-old plant that are six weeks old, but other tissues are much younger. How is communication accomplished between these tissues of different age? Does communication proceed by a diffusible endogenously produced agent, for example by a plant hormone, by environmental triggers, e.g. the amount or flux of NaCl in cells, or by the operation of a system that stabilises CAM-specific mRNAs? Induction of PEPCase gene transcription occurs to a limited extent in young plants (3 weeks) with induction being maximal at 6 weeks of age. When plants are induced at a later age (9 weeks) further PEPCase transcriptional activation no longer occurs. Transcriptional activation might be brought about by modification of the DNA, for example by methylation changes, or by changes in the long-range structure of chromatin. Transcription may also be enhanced by the modification of a set of pre-existing factors, or by changing the composition of ?ra/is-acting factors. Our preliminary data suggest that the complexity of frans-acting factors able to bind to the ppcl promoter changes during the transition from C3 to CAM (J. C. Cushman et al., unpublished data). Changes in complexity may be caused by several mechanisms. New factors may be synthesised, or existing factors may be altered by covalent modification. The distribution of factors between different genes may be altered, or existing factors could be turned over with an altered rate. It appears likely at present that most of the DNA-binding factors are ubiquitous and always present, but that their interactions are regulated by several, different mechanisms. Alter-

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ations in the interaction of factors would have to be brought about by externally imposed changes that lead either to a change in osmolarity (salt, soluble sugars) inside the nucleus, or to the physical alteration of some factors, by phosphorylation, reduction, or some other (covalent) modifications that establish a new equilibrium in the interaction of transacting factors. As much as we have already learned about the molecular events accompanying the switch from C3 to CAM in ice plant, it would appear that the more exciting part of the analysis is still ahead.

Acknowledgements We wish to thank Drs W. Taylor (CSIRO) and T. Nelson (Yale) for providing MOD and MDH gene probes. Supported by USDA-CRGP (Environmental Stress Program), USDA-CRSR (SW Consortium on Plant Genetics) and, in part, the National Science Foundation. We are indebted to Dr John L. Wray for suggestions that improved the manuscript. References Adams, C.A., Leung, F. & Sun, S.S.M. (1986). Molecular properties of phosphoeno/pyruvate carboxylase from C3, C3-C4 intermediate, and C4 Flaveria species. Planta 167, 218-25. Andreo, C.S., Gonzales, D.H. & Iglesias, A.A. (1987). Higher plant phosphoeno/pyruvate carboxylase. FEBS Letters 213, 1-8. Benfey, P.N. & Chua, N.-H. (1990). The cauliflower mosaic virus 35S promoter: combinatorial regulation of transcription in plants. Science 250, 959-66. Bloom, A.J. & Troughton, J.H. (1979). High productivity and photosynthetic flexibility in a CAM plant. Oecologia 38, 35-43. Bohnert, H.J., Cushman, J.C., Meyer, G. & Ostrem, J.A. (1989). Changes in gene expression in response to salt stress. In Plant Water Relations and Growth under Stress, ed. M. Tazawa, pp. 143-50. New York: MYU K.K. Press. Brulfert, J., Guerrier, D. & Queiroz, O. (1982). Photoperiodism and crassulacean acid metabolism. Planta 154, 332-8. Chu, C , Dai, Z., Ku, M.S.B. & Edwards, G.E. (1990). Induction of crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum by abscisic acid. Plant Physiology 93, 1253-60. Cleveland, D.W., Fisher, S.G., Kirschner, M.W. & Laemmli, U.K. (1977). Peptide mapping by limited proteolysis in sodium dodecylsulfate and analysis by gel electrophoresis. Journal of Biological Chemistry 252, 1102-6.

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H.J. BOHNERT et al. Cockburn, W. (1981). The evolutionary relationship between stomatal mechanism, crassulacean acid metabolism and C4 photosynthesis. Plant, Cell and Environment 4, 417-18. Cockburn, W. (1985). Variation in photosynthetic acid metabolism in vascular plants: CAM and related phenomena. New Phytologist 101, 3-24. Cretin, C , Keryer, E., Tagu, D., Lepiniec, L., Vidal, J. & Gadal, P. (1990). Complete cDNA sequence of sorghum phosphoeno/pyruvate carboxylase involved in C4 photosynthesis. Nucleic Acids Research 18, 658. Cushman, J.C. & Bohnert, H.J. (1989a). Nucleotide sequence of the Ppcl gene from M. crystallinum, encoding the CAM-form of phosphoeno/pyruvate carboxylase. Nucleic Acids Research 16, 6745-6. Cushman, J.C. & Bohnert, H.J. (1989b). Nucleotide sequence of the Ppc2 gene from M. crystallinum, encoding a housekeeping isoform of phosphoeno/pyruvate carboxylase. Nucleic Acids Research 16, 6743-4. Cushman, J . C , DeRocher, E.J. & Bohnert, H.J. (1990a). Gene expression during adaptation to salt stress. In Environmental Injury to Plants, ed. F.R. Katterman, pp. 173-203. New York: Academic Press. Cushman, J.C, Meyer, G., Michalowski, C.B., Schmidt, J.M. & Bohnert, H.J. (1989). Salt stress leads to differential expression of two isogenes of PEPCase during CAM induction in the common ice plant. Plant Cell 1, 715-25. Cushman, J . C , Michalowski, C B . & Bohnert, H.J. (19906). Developmental control of crassulacean acid metabolism inducibility by salt stress in the common ice plant. Plant Physiology 94, 1137-42. Cushman, J . C , Vernon, D.M. & Bohnert, H.J. (1992). ABA and the transcriptional control of CAM induction during salt stress in the common ice plant. In Control of Plant Gene Expression, ed. D.P. Verma. Boca Raton: CRC Press (in press). De Armond, R., Bohnert, H.J. & Thomas, J.C. (1991). Growth, amino acid composition, and PEPCase in ice plant suspension cultures. Plant Physiology 96S, 96. DeRocher, E.J. & Bohnert, H.J. (1991). Post-transcriptional regulation of rubisco small subunit gene expression during environmental stress and development. Plant Physiology 96S, 83. DeRocher, E.J., Harkins, K.R., Galbraith, D.W. & Bohnert, H.J. (1990). Developmental^ regulated systemic endopolyploidy in succulents with small genomes. Science 250, 99-101. DeRocher, E.J., Michalowski, C B . & Bohnert, H.J. (1991). cDNA sequences for transcripts of the ribulose-l,5-bisphosphate carboxylase/oxygenase small subunit gene family of Mesembryanthemum crystallinum. Plant Physiology 95, 976-8. Edwards, G.E., Foster, J.G. & Winter, K. (1982). Activity and intracellular compartmentation of enzymes of carbon metabolism in CAM

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plants. In Crassulacean Acid Metabolism, ed. I.P. Ting & M. Gibbs, pp. 92-111. Rockville, MD: American Society of Plant Physiologists. Eickmanns, B.J., Follettie, M.T., Griot, M.U. & Sinskey, A.J. (1989). The phosphoeno/pyruvate carboxylase gene of Corynebacterium glutamicum: Molecular cloning, nucleotide sequence, and expression. Molecular and General Genetics 218, 330-9. Foster, J.G., Edwards, G.E. & Winter, K. (1982). Changes in the levels of phosphoeno/pyruvate carboxylase with induction of CAM in M. crystallinum L. Plant and Cell Physiology 23, 585-94. Fujita, N., Miwa, T., Ishijima, S., Izui, K. & Katsuki, H. (1984). The primary structure of phosphoeno/pyruvate carboxylase of Escherichia coli. Nucleotide sequence of the ppc gene and deduced amino acid sequence. Journal of Biochemistry 95, 909-16. Guralnick, L.J., Rorabaugh, P.A. & Hanscom, Z. Ill (1984). Seasonal shifts of photosynthesis in Portulacaria afra (L.). Plant Physiology 76, 643-6. Hermans, J. & Westhoff, P. (1990). Analysis of expression and evolutionary relationships of PEPCase genes in Flaveria trinervia (C4) and F. pringlei (C3). Molecular and General Genetics 224, 459-68. Heun, A.-M., Gorham, J., Liittge, U. & Wyn Jones, R.G. (1981). Changes in water-relation characteristics and levels of organic cytoplasmic solutes during salinity induced transition of M. crystallinum from Q-photosynthesis to crassulacean acid metabolism. Oecologia 50, 66-72. Higgins, D.G. & Sharp, P.M. (1988). CLUSTAL: a package for performing multiple sequence alignments on a microcomputer. Gene 73, 237-44. Higgins, D.G. & Sharp, P.M. (1989). Fast and sensitive multiple sequence alignments on a microcomputer. CABIOS Communications 5, 151-3. Hofner, R., Vazquez-Moreno, L., Winter, K., Bohnert, H.J. & Schmitt, J.M. (1987). Induction of crassulacean acid metabolism in M. crystallinum: Mass increase and de-novo synthesis of PEP-carboxylase. Plant Physiology 83, 915-19. Holthe, P.A., Sternberg, L.S.L. & Ting, I.P. (1987). Developmental control of CAM in Peperomia scandens. Plant Physiology 84, 743-7. Holtum, J.A.M. & Winter, K. (1982). Activity of enzymes of carbon metabolism during the induction of crassulacean acid metabolism in M. crystallinum. Planta 155, 8-16. Hudspeth, R.L. & Grula, J.W. (1989). Structure and expression of the maize gene encoding the phosphoeno/pyruvate carboxylase isozyme involved in C4 photosynthesis. Plant Molecular Biology 12, 579-89. Izui, K., Ishijima, S., Yamaguchi, Y., Katagiri, F., Murata, T., Shigesada, K., Sugiyama, T. & Katsuki, H. (1986). Cloning and sequence analysis of cDNA encoding active phosphoe/io/pyruvate carboxylase of the C4-pathway from maize. Nucleic Acids Research 14, 1615-28.

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H.J. BOHNERT et al. Jawali, N. (1990). The dimeric form of PEPCase isolated from maize: physical and kinetic properties. Archives of Biochemistry and Biophysics 277, 61-8. Jefferson, R.A., Kavanagh, T. & Bevan, M.W. (1987). GUS fusions: pglucuronidase as a sensitive and versatile fusion marker in higher plants. The EMBO Journal 6, 3901-7. Jiao, J. & Chollet, R. (1990). Regulatory phosphorylation of serine-15 in maize phosphoeno/pyruvate carboxylase by a C4-leaf protein-serine kinase. Archives of Biochemistry and Biophysics 283, 300-5. Jiao, J., Vidal, J., Echevarria, C. & Chollet, R. (1991). In vivo regulatory phosphorylation site in C4-leaf phosphoeno/pyruvate carboxylase from maize and sorghum. Plant Physiology 96, 297-301. Katagiri, F., Kodaki, T., Fujita, N., Izui, K., Katsuki, H. (1985). Nucleotide sequence of the phosphoeno/pyruvate carboxylase gene of the cyanobacterium Anacystis nidulans. Gene 38, 265-9. Keeley, J.E. & Busch, G. (1984). Carbon assimilation characteristics of the aquatic CAM plant Isoetes howellii. Plant Physiology 76, 525-30. Kluge, M. & Ting, I.P. (1978). Crassulacean acid metabolism - analysis of an ecological adaptation. Ecological Studies, Vol. 30. Heidelberg: Springer-Verlag. Kodaki, T., Katagiri, F., Asano, M., Izui, K. & Katsuki, H. (1985). Cloning of phosphoeno/pyruvate carboxylase gene from a cyanobacterium, Anacystis nidulans, in Escherichia coli. Journal of Biochemistry 97, 533-9. Koizumi, N., Sato, F., Terano, Y. & Yamada, Y. (1991). Molecular analysis of phosphoeno/pyruvate carboxylase of a C3 plant, Nicotiana tabacum. Plant Molecular Biology 17, 535-9. Latzko, E. & Kelly, G.J. (1983). The many-faceted function of phosphoeno/pyruvate carboxylase in C3 plants. Physiologie Vegetale 21, 805-15. Liittge, U. (1987). Carbon dioxide and water demand: CAM, a versatile ecological adaptation exemplifying the need for integration in ecophysiological work. New Phytologist 106, 593-629. McElwain, E.F., Bohnert, H.J. & Thomas, J.C. (1991). Endogenous ABA levels and the CAM response in M. crystallinum during salt stress. Plant Physiology 96S, 17. Mandecki, W., Powell, B.S., Mollison, K.W., Carter, G.W. & Fox, J.L. (1986). High-level expression of a gene encoding the human complement factor C5a in Escherichia coli. Gene 43, 131-8. Matsuoka, M. & Hata, S. (1987). Comparative studies of phosphoeno/pyruvate carboxylase from C3 and C4 plants. Plant Physiology 85, 947-51. Matsuoka, M. & Minami, E. (1989). Complete structure of the gene for phosphoeno/pyruvate carboxylase from maize. European Journal of Biochemistry 181, 593-8.

Biochemistry and molecular biology of CAM

135

Matsuoka, M. & Sanada, Y. (1991). Expression of photosynthetic genes from the C4 plant, maize, in tobacco. Molecular and General Genetics 225, 411-19. Meiners, M.S., Thomas, J.C., Bohnert, H.J. & Cushman, J.C. (1991). Regeneration of multiple shoots and plants from M. crystallinum. Plant Cell Reports 9, 563-6. Meyer, G., Schmitt, J.M. & Bohnert, H.J. (1990). Direct screening of a small genome: Estimation of the magnitude of gene expression changes during adaptation to high salt. Molecular and General Genetics 224, 347-56. Michalowski, C.B., DeRocher, E.J., Bohnert, H.J. & Salvucci, M. (1992). Phosphoribulokinase from ice plant: Transcription, transcripts and protein expression during environmental stress. Photosynthesis Research 31 (in press). Michalowski, C.B., Olson, S.W., Piepenbrock, M., Schmitt, J.M. & Bohnert, H.J. (1989a). Time course of mRNA induction elicited by salt stress in the common ice plant (M. crystallinum). Plant Physiology 89, 811-16. Michalowski, C.B., Schmitt, J.M. & Bohnert, H.J. (1989b). Expression during salt stress and nucleotide sequence of cDNA for ferredoxinNADP+ reductase from M. crystallinum. Plant Physiology 89, 817-23. Nakamura, K., Iwasaki, Y. & Hattori, T. (1980). An improved Escherichia coll expression vector for the construction and identification of full-length cDNA clones. Gene 44, 347-51. Nelson, T. & Langdale, J. A. (1989). Patterns of leaf development in C4 plants. The Plant Cell 1, 3-13. Nimmo, G.A., Nimmo, H.G., Hamilton, I.D., Fewson, C.A. & Wilkins, M.B. (1986). Purification of the phosphorylated night form and dephosphorylated day form of phosphoeno/pyruvate carboxylase from Bryophyllum fedtschenkoi. Biochemical Journal 239, 213-20. O'Leary, M.H. (1982). Phosphoeno/pyruvate carboxylase: An enzymologists' view. Annual Review of Plant Physiology 33, 297-315. Osmond, C.B. (1978). CAM - a curiosity in context. Annual Review of Plant Physiology 29, 379^14. Ostrem, J.A., Olson, S.W., Schmitt, J.M. & Bohnert, H.J. (1987). Saltstress increases the level of translatable mRNA of phosphoeno/pyruvate carboxylase in M. crystallinum. Plant Physiology 84, 1270-5. Ostrem, J.A., Vernon, D.M. & Bohnert, H.J. (1990). Stress increases the expression of a gene coding for NAD-glyceraldehyde 3-phosphate dehydrogenase during the transition from C3 photosynthesis to crassulacean acid metabolism in M. crystallinum. Journal of Biological Chemistry 265, 3497-502. Rickers, J., Cushman, J.C, Michalowski, C.B., Schmitt, J.M. & Bohnert, H.J. (1989). Expression of the CAM-form of phos-

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H.J. BOHNERT et dl. phoeno/pyruvate carboxylase and nucleotide sequence of a full-length cDNA from M. crystallinum. Molecular and General Genetics 215, 446-54. Rodriguez-Sotres, R. & Munoz-Clares, R.A. (1990). Kinetic evidence of the existence of a regulatory PEP binding site in maize leaf PEPCase. Archives of Biochemistry and Biophysics 276, 180-90. Schmitt, J.M., Hofner, R., Abou-Mandour, A.A., Vazquez-Moreno, L. & Bohnert, H.J. (1988). CAM-induction in M. crystallinum: protein expression. Photosynthesis Research 17, 159-71. Singh, R. (1989). Carbon dioxide fixation by PEP carboxylase in podwalls of chickpea. In Photosynthesis, Molecular Biology and Bioenergetics, ed. G.S. Singhal et al., pp. 315-29. New Delhi: Narosa Publishing House. Sipes, D.L. & Ting, I.P. (1985). Crassulacean acid metabolism and crassulacean acid metabolism modifications in Peperomia camptotricha. Plant Physiology 77, 59-63. Smith, G.E., Ju, G., Ericson, B.L., Moschera, J., Lahm, H.W., Chizzonite, R. & Summers, M.D. (1985). Modification and secretion of human interleukin 2 produced in insect cells by a baculovirus expression vector. Proceedings of the National Academy of Sciences (USA) 82, 8404-8. Sutton, F., Butler, E.T. Ill & Smith, E.T. (1986). Isolation of the structural gene encoding a mutant form of E. coli phosphoerco/pyruvate carboxylase deficient in regulation by fructose 1,6bisphosphate. Journal of Biological Chemistry 261, 16078-81. Tarczynski, M.C. & Outlaw, W.H. (1990). Partial characterization of guard-cell phosphoeno/pyruvate carboxylase: kinetic datum collection in real time from single-cell activities. Archives of Biochemistry and Biophysics 280, 153-8. Terada, K., Kai, T., Okuno, S., Fujisawa, H. & Izui, K. (1990). Maize leaf PEPCase: phosphorylation of Ser15 with a mammalian cAMPdependent protein kinase diminishes sensitivity to inhibition by malate. FEBS Letters 259, 241^. Ting, I.P. (1985). Crassulacean acid metabolism. Annual Review of Plant Physiology 36, 595-622. Ting, I.P. & Gibbs, M. (1982). Crassulacean Acid Metabolism. Rockville, MD: American Society of Plant Physiologists. Ting, I.P., Lord, E.M., Sternberg, L.S.L. & DeNiro, M.J. (1985). Crassulacean acid metabolism in the strangler Clusia rosea Jacq. Science 229, 969-71. Vernon, D.M., Ostrem, J.A. & Bohnert, H.J. (1991). The regulation of genes involved in salt tolerance and CAM induction in ice plant: a complex web of molecular responses to environmental stimuli. Plant Physiology 96S, 18. Vernon, D.M., Ostrem, J.A., Schmitt, J.M. & Bohnert, H.J. (1988).

Biochemistry and molecular biology of CAM

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PEPCase transcript levels in M. crystallinum decline rapidly upon relief from salt stress. Plant Physiology 86, 1002^t. Vidal, J., Nguyen, J., Perrot-Rechenmann, C. & Gadal, P. (1986). Phosphoerco/pyruvate carboxylase in soybean root nodules: an immunochemical study. Planta 169, 198-201. von Willert, D.J., Kirst, C O . , Treichel, S. & von Willert, K. (1976). The effect of leaf age and stress on malate accumulation and phosphoeno/pyruvate carboxylase activity in Mesembryanthemum crystallinum. Plant Science Letters 7, 341-6. Willmer, CM. (1983). Phosphoeno/pyruvate carboxylase activity and stomatal operation. Physiologie Vegetale 21, 943-53. Winter, K. (1982). Regulation of PEP Carboxylase in CAM plants. In Crassulacean Acid Metabolism, ed. I.P. Ting & M. Gibbs, pp. 153-69. Rockville, MD: American Society of Plant Physiologists. Winter, K. & Gademann, R. (1991). Daily changes in CO2 and water vapor exchange, chlorophyll fluorescence, and leaf water relations in the halophyte M. crystallinum during the induction of CAM in response to high NaCl salinity. Plant Physiology 95, 768-76. Winter, K., Luttge, U. & Winter, E. (1978). Seasonal shift from C3 photosynthesis to CAM in M. crystallinum growing in its natural environment. Oecologia 34, 225-37. Winter, K. & von Willert, D.J. (1972). NaCI-induzierter Crassulaceen Saurestoffwechsel bei Mesembryanthemum crystallinum. Zeitschrift fur Pflanzenphysiologie 67, 166-70. Wu, M.-X., Meyer, C.R., Willeford, K.O. & Wedding, R.T. (1990). Regulation of the aggregation state of maize PEPCase: evidence from dynamic light-scattering measurements. Archives of Biochemistry and Biophysics 281, 324-9. Yanagisawa, S., Izui, K., Yamaguchi, Y., Shigesada, K. & Katsuki, H. (1988). Further analysis of cDNA clones for maize phosphoeno/pyruvate carboxylase involved in C4 photosynthesis. FEBS Letters 229, 107-10.

F.L. OLSEN, K. SKRIVER, F. MULLER-URI, N.V. RAIKHEL, J.C. ROGERS and J. MUNDY

ABA- and GA-responsive gene expression Introduction Abscisic acid (ABA) mediates embryo maturation during late seed development. Maturation involves various morphogenic and biochemical changes including the programming of embryo dormancy and desiccation tolerance. Genetic analysis implicate ABA in the control of dormancy (Koorneef et al., 1984; McCarty et al., 1989). Molecular studies suggest that certain ABA-responsive genes expressed during late embryogenesis are part of a developmental programme leading to desiccation tolerance (Bartels et al, 1988; Dure et al., 1989). Certain of these genes are also expressed in vegetative tissues during osmotic stress (Mundy & Chua, 1988; Gomez et al., 1988), at which time ABA levels rise and growth is inhibited. At present, the connection between ABA effects during embryogenesis and in vegetative tissues is unclear, in part because we do not know the function(s) of most of the major ABA-responsive genes (for reviews, see Skriver & Mundy, 1990; Galau et al., 1991; McCarty & Carson, 1991). However, the hormone mediates responses to osmotic stress, and causes developmental or growth inhibition in both embryonic and vegetative tissues (Smart & Trewavas, 1984; Bensen et al., 1988; Creelman, 1989). In many fruits, embryo dormancy is broken by environmental cues and physiological factors which initiate germination. In cereal seeds, gibberellic acid (GA) appears to be one such factor by promoting this expression of genes encoding a-amylase and other hydrolases. This effect of GA can be inhibited by ABA (Jacobsen & Beach, 1985; Nolan & Ho, 1988; Huttly & Baulcombe, 1989), an antagonism which may mediate physiological changes controlling the switch from seed quiescence or dormancy to germination. GA also affects leaf and stem morphology and growth in many plants (Lanahan & Ho, 1988; Boother et al., 1991). Interestingly, certain vegetative responses to GA can be repressed by ABA (Metzger, Society for Experimental Biology Seminar Series 49: Inducible Plant Proteins, ed. J. L. Wray. ©Cambridge University Press, 1992, pp. 139-153.

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etal.

1988), suggesting that the antagonism between these phytohormones modulates diverse plant processes.

Osmotic-stress and ABA-responsive genes Studies on osmoregulation in bacteria suggest that bacterial cells sense osmotic changes via activation of primary turgor-responsive genes (Higgins et al., 1988; Forst & Inouye, 1988). In plant cells, the expression of ABA-responsive and other genes may occur after such a primary turgor response, enabling the plant to adapt and/or recover from the stress. Continued stress may later induce expression of genes involved in adaptive homeostasis or stress avoidance (Cushman et al., 1990; Austin et al., 1982). Figure 1 outlines potential early steps in this process. Recent work on turgor-responsive genes in plants has identified clones encoding proteins with homology to proteins known to be involved in osmoregulation. Most notable are (i) aldehyde reductases, involved in the synthesis of sugar alcohols (non-metabolisable osmolytes), and (ii) putative membrane-spanning, ion-channel proteins, perhaps involved in osmotic adjustment via ion flow (Guerrero et al., 1990; Bartels et al., 1991). Table 1 presents an updated list of cDNAs/genes whose expression is promoted by osmotic stress and ABA. The precise function of most of these genes remains unknown and preliminary studies of their temporal and spatial patterns of expression yield few clues. Several of the /ate embryogenesis abundant {lea) and j4BA-responsive {rab) genes are developmentally expressed during embryogenesis, and are also expressed in osmotically stressed vegetative STRESS

drought, salt cold, wounding

STIMULUS

reduced turgor

EFFECT

increased cytoplastic and apoplastic ABA

RESPONSE

increased osmolytes protectant/recovery proteins

Fig. 1. Potential early steps in the response to osmotic stress.

A BA - and GA -responsive gene expression

141

tissues. The pattern of expression and biochemical properties of the proteins encoded by the rabl6H7 genes may be typical of this gene set (Mundy & Chua, 1988; Vilardell et al., 1990). Crude cellular fractionations and other biochemical tests showed that these proteins are soluble in aqueous buffers, indicating that they are soluble cytosolic or vacuolar proteins. Dure et al. (1989) suggested that such proteins may be involved in protein and/or membrane protection during osmotic stress via stabilising interactions analogous to those posited for proline (Csonka, 1989) and heat shock proteins (Pelham, 1986). Recent work, however, on the maize RAB17 protein indicates that it is associated in scutellar cells with phytate bodies, implying that the protein is involved in phytate/phosphate mobilisation or metabolism (M. Pages, personal communication). This suggests that LEA and RAB proteins have very specific cellular roles. Interestingly, in situ hybridisations performed at late stages in the development of rice and wheat seeds indicate that high levels of rabl6 mRNA accumulate in the depleted cells (Fig. 2). These cells, whose ontogeny is poorly understood, appear to form a barrier in mature seeds between terminally differentiated, highly desiccated cells of the starchy endosperm, and quiescent epithelial cells of the scutellum. Further work is required to determine whether the deposition of RAB16 proteins mirrors this mRNA distribution. If so, this novel spatial pattern of expression suggests that this ABA-responsive gene may be involved in desiccation tolerance in seeds. The antagonism between ABA and GA During germination in cereal seeds, GA promotes the transcription of genes encoding a-amylase and other germinative enzymes. ABA represses this induction, primarily at the level of transcription (Salmenkallio et al., 1990; Huttly & Baulcombe, 1989). The inhibition may also occur at: the level of a-amylase mRNA stability (Rogers, 1988; Khursheed & Rogers, 1989), or at the level of enzyme activity via a specific a-amylase inhibitor, the a-amylase/subtilisin inhibitor (ASI: see Table 1). The antagonism of GA action by ABA can be seen in studies of steadystate mRNA accumulation in aleurone cells, a major site of a-amylase synthesis in germinating cereal seeds (Fig. 3). GA treatment promotes the accumulation of high levels of a-amylase, shown by immunoprecipitation, while ABA, alone or with GA, completely represses this accumulation. In contrast, ABA clearly promotes the accumulation of aamylase inhibitor mRNA, as determined by specific immunoprecipitation, while GA alone slightly reduces the level of a-amylase inhibitor mRNA. However, GA, together with ABA, does not decrease the level

Table 1. ABA-responsive genes Clone name

Stress induced

pHVAl pLea76 LEAD7 RAB16

7 D 7

RAB17 LEA Dll Dehydrin TAS14 p8B6

O, D,C

Em

LEAD 19 cor, several lti 140 pG22-69 pLEA, several pN24 Osmotin LEA D34 LEA D113 SalT pcC, several Glbl p511 Napin Conglycinin DC8

DC59 WGA pMAH9 ASI

PI-2 HS70

O,D,C, 7 D

O, D D D 7 C C D D O O ? ? O,D D 7 ? D 7 ?

7 O

D, W D W

O,D,H, W

Species

Function

Organ specific

Reference

Barley Rape Cotton Rice

7 7 ? 7

7 7 ? -

Maize Cotton Barley Tomato Radish Wheat Cotton Arabidopsis Arabidopsis Barley Tomato Tomato Tobacco Cotton Cotton Rice Craterostigma Maize Wheat

7 ? ? 7 7

7 7 7 Sd Sd 7 ? St Sd Sd

Hong et al., 1988 Harada et al., 1989 Baker et al., 1988 Mundy & Chua, 1988 Hahn & WaLbot, 1989 Vilardelle/a/.,1990 Baker et al., 1988 Close etal., 1989 Godoy etal., 1990 Raynal etal., 1989 MarcotteetaL, 1988 Baker etal., 1988 Hajelaefa/., 1990 Nordinefa/., 1991 Bartels etal., 1991 Cohen & Bray, 1990 King etal., 1988 Singh etal., 1989 Baker et al., 1988 Baker etal., 1988 ClaesefG/., 1990 Bartels etal., 1990 Kriz #<*/., 1990 Williamson & Quatrano, 1988

7 7 ? 7

Aldose reductase ? ?

Pase inhibitor ? 7 ? ? ?

7S globulin 7S globulin

Rape Soybean Carrot Carrot Wheat Maize

7S globulin 7S globulin Lipid body protein Lectin RNP?

Sd Sd Sd Sd Sd -

Barley Potato Maize

Amylase/Pase inhibitor Pase inhibitor Heat shock protein

Sd —

7

Finkelstein et al, 1985 Bray & Beachy, 1985 Hatzopoulos et al., 1990 Hatzopoulos et al., 1990 Cammue et al., 1989 Gomez et al., 1988, Bandziulis etal., 1989 Leah & Mundy, 1989 Pena-Cortes et al., 1990 Heikkila etaL, 1984

Notes: Data are adapted from compilations of Dure et al. (1989). O, high osmoticum (PEG or salt); D, desiccation; C, cold; W, wounding; H, heat; ?, untested or unknown. Sd, seed; St, stem; Pase, protease; ASI, a-amylase/subtilisin inhibitor; WGA, wheat germ agglutinin; RNP, ribonuclear protein; - , not organ specific. Table updated from Skriver & Mundy (1990).

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A

etal.

B

Fig. 2. In situ localisation of rabl6 mRNA in developing wheat seeds. A, Scanning electron micrograph of scutellar/starchy endosperm boundary. DC, depleted cells; SE, scutellar epithelial cells. B, Dark field micrograph of in situ hybridised rabl6 sense RNA probe. No specific hybridisation is visible. C, Dark field micrograph of in situ hybridised rabl6 antisense RNA probe. Note specific hybridisation to depleted cells. Scanning electron microscopy was performed according to Mundy et al. (1986); in situ hybridisation after Raikhel et al. (1989).

of mRNA encoding the a-amylase inhibitor {ASP). Similarly, levels of rabl6 mRNA, encoding the major ABA-responsive protein of molecular mass 16 kDa seen among the total products, appear to be unaffected by GA. This kind of antagonism is apparently not restricted to embryonic tissues (Metzger, 1988), suggesting that varying levels of the two hormones may modulate different physiological processes throughout the life of a plant.

ABA and GA DNA response elements To begin to understand how ABA and GA regulate specific gene expression, we used a transient gene expression assay in barley aleurone protoplasts to delineate the hormone response elements (HREs) of the ABA-responsive rice gene, rabl6 (Mundy et al., 1990) and of the GAresponsive barley gene a-Amy 1/6-4 (Khursheed & Rogers, 1988). Sequences of their 5' upstream regions were fused to the Cat reporter

ABA-

and GA-responsive gene expression

TOTAL o

145

IMMUNO

Si

• • amylase

rab 16

— inhibitor

12 3 4 5 6 7 8 Fig. 3. Translation products of barley aleurone layer mRNAs. Lanes 14, total products from layers treated in water (control), 1 JXM ABA, 1 piM GA3, or both hormones, respectively. The rabl6 protein is marked with an arrow. Lanes 5-8, immunoprecipitation of a-amylase (upper bands) and the a-amylase/subtilisin inhibitor (lower bands) from the translation products shown in lanes \-A. mRNA extraction, translation and immunoprecipitation were performed as previously described (Mundy era/., 1986).

gene to identify positively the response elements in 'gain of function' experiments. Initially, long promoters containing several hundred base pairs of upstream sequence with TATA boxes of the native genes were assayed for hormone responsiveness. Later, chimaeric promoters con-

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etal.

taining tandemly repeated copies of shorter sequences, placed upstream of the 35S CaMV gene TATA box, were used to delineate the HREs. Delineation of the ABA-response DNA element (ABRE) was facilitated by previous work which indicated that conserved 5' sequences of various ABA-responsive genes may be ABREs (Marcotte et al., 1989; Guiltinan et al., 1990; Yamaguchi-Shinozaki, 1990). In rabl6A these regions contain the sequence -179 ACGTGGC and two copies of a degenerate sequence (-168 CCGCCGCGCCT and -130 CCGCCGCGCT) that are within sites for nuclear protein binding (Mundy et al., 1990). In barley aleurone protoplasts treated with 1 UM ABA, a long fragment of the rabl6A promoter (-442/+8), including the homologous TATA box, conferred 40-fold induction of CAT reporter enzyme activity compared with control treatments. This induction was unaffected by treatment with ABA in the presence of an equimolar GA. A chimaeric promoter containing six copies of the sequence —181 GTACGTGGCGC —171 conferred six-fold ABA-responsive expression over control. Like the long promoter fragment, transcription from this ABRE was unaffected by equimolar GA. GA-responsive sequences in the a-Amy 1/6-4 gene were delineated in similar experiments, again facilitated by earlier studies describing conserved sequences in the promoters of numerous a-amylase and other GAresponsive genes (Huang et al., 1990; Whittier et al., 1987). In GAtreated protoplasts, a long fragment of this promoter (-639/+43), including its own TATA box, conferred eight-fold induction on CAT expression over controls. Co-treatment with equimolar ABA abolished this induction. In the same way, a chimaeric promoter containing six copies of the sequence -148 GGCCGATAACAAACTCCGGCC -128 conferred four-fold GA-responsive expression on the reporter gene. Transcription from this GA response element (GARE) was repressed by co-treatment with equimolar ABA. The core of this element, TAACAAA, and related sequences are found in the promoters of other GA-responsive genes. Models of gene regulation by ABA and GA The ABRE and GARE are probably recognised by different regulatory proteins because their sequences are not related. Furthermore, in the case of the ABRE, this protein may be constitutively expressed because rab mRNAs accumulate independently of protein synthesis (Mundy & Chua, 1988). In contrast, GA-induction of a-amylase mRNA requires protein synthesis (Muthukrishnan et al., 1979), indicating that the factors) controlling this response are newly synthesised. These results suggest that the antagonistic effect of ABA on GA-responsive gene

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expression is probably attributable to decreased activity or expression of the GA-regulatory protein(s). Other regulatory factors may, however, be involved in modulating the effects or activities of putative ABA and/or GA regulatory proteins. For example, McCarty et al. (1989) have cloned a maize gene by genetic techniques, which is involved in the control of embryo dormancy. This gene is thought to encode a transcription factor. It will be interesting to see whether it controls dormancy by positively affecting developmental events controlled by ABA, or by negatively affecting germinative events enhanced by GA (Wilson et al., 1973). The sequences of the ABA- and GA-response elements may also shed light on an older question in plant biology: do ABA and GA, both hydrophobic terpenoids, affect specific gene expression in the same way as the animal steroid hormones? Recent work on the DNA response elements of steroid hormones in insects and vertebrates shows that they all contain a spaced palindrome motif (Martinez et al., 1991). This structure is a key molecular landmark along the pathway between steroid hormones and their target genes. The ABRE does not appear to contain or to be associated with a spaced palindrome, although its sequence is part of an imperfect palindrome. This suggests that the mechanism by which ABA affects target gene expression is fundamentally different from that used by the animal steroid hormones. Furthermore, Guiltinan et al. (1990) have recently characterised a DNA-binding protein containing a leucine zipper domain which interacts with the ABRE of the wheat Em gene. This protein may be a member of a family which recognises 'Gbox' DNA motifs, thought to be enhancers in various genes (DeLisle & Ferl, 1990). If this protein is the 'ABA-factor', then the expression of ABA-responsive genes is presumably induced by interactions between dimeric leucine zipper proteins. As is the case for gene regulation by members of the Jun and Fos protein family, heterodimer formation may also modulate transactivation by these proteins (Vinson et al., 1989). In contrast, the putative GARE of the a-Amy 1/6-4 is part of a spaced palindrome, although this motif bears no sequence resemblance to the animal steroid response elements. We are currently analysing mutations within the GARE sequences to see whether the palindrome is functionally important. In conclusion, it now appears unlikely that ABA and GA affect specific gene expression by mechanisms similar to that involved in animal gene regulation by animal steroid hormones. Continued work on the characterisation of plasmalemma receptors for ABA (Hornberg & Weiler, 1984) and GA (Hooley et al., 1991) seems likely to confirm these, findings.

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Acknowledgements This study was supported by Eureka grant 270 (ABIN) to F.L.O., K.S. and J.M., an F. Lynen stipend from the A. von Humboldt Foundation to F.M.-U., and in part by a contract (DE-AC02-76ERO-1338) from the US Department of Energy to N.V.R., and a grant (#90-37262-5350) from the US Department of Agriculture to J.R. References Austin, R.B., Henson, I.E. & Quarrie, S.A. (1982). Abscisic acid and drought resistance in wheat, millet and rice. In Drought Resistance in Crops with Emphasis on Rice, ed. M.R. Vega, pp. 171-80. Los Banos, The Philippines: International Rice Research Institute. Baker, J., Steele, C. & Dure, L. Ill (1988). Sequence and characterization of 6 Lea proteins and their genes from cotton. Plant Molecular Biology 11, 277-91. Bandziulis, R.J., Swanson, M.S. & Dreyfuss, G. (1989). RNA-binding proteins as developmental regulators. Genes and Development 3, 431-7. Bartels, D., Engelhardt, K., Roncarati, R., Schneider, K., Rotter, M. & Salamini, F. (1991). An ABA and GA modulated gene expressed in the barley embryo encodes an aldose reductase related protein. The EMBO Journal 10, 1037-43. Bartels, D., Schneider, K., Terstappen, G., Piatkowski, D. & Salamini, F. (1990). Molecular cloning of abscisic acid-modulated genes which are induced during desiccation of the resurrection plant Craterostigma plantagineum. Planta 181, 27-34. Bartels, D., Singh, M. & Salamini, F. (1988). Onset of desiccation tolerance during development of the barley embryo. Planta 175, 485-92. Bensen, R.J., Boyer, J.S. & Mullet, J.E. (1988). Water deficit-induced changes in abscisic acid, growth, polysomes and translatable RNA in Soybean hypocotyls. Plant Physiology 88, 289-94. Boother, G.M., Gale, M.D., Gaskin, P., MacMillan, J. & Sponsel, V.M. (1991). Gibberellins in shoots of Hordeum vulgare. A comparison between cv. Triumph and two dwarf mutants which differ in their response to gibberellin. Physiologia Plantarum 81, 385-92. Bray, E.A. & Beachy, R.N. (1985). Regulation by ABA of P-conglycinin expression in cultured developing soybean cotyledons. Plant Physiology 79, 746-50. Cammue, B.P.A., Broekaert, W.F., Kellens, J.T.C., Raikhel, N.V. & Peumans, W.J. (1989). Stress-induced accumulation of wheat germ agglutinin and abscisic acid in roots of wheat seedlings. Plant Physiology 91, 1432-5.

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Claes, B., Dekeyser, R., Villarroel, R., Van den Bulcke, M., Bauw, G., Van Montagu, M. & Caplan, A. (1990). Characterization of a rice gene showing organ-specific expression in response to salt stress and drought. The Plant Cell 2, 19-27. Close, T.J., Kortt, A.A. & Chandler, P.M. (1989). A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn. Plant Molecular Biology 13, 95-108. Cohen, A. & Bray, E.A. (1990). Characterization of three mRNAs that accumulate in wilted tomato leaves in response to elevated levels of endogenous acid. Planta 182, 27-33. Creelman, R.A. (1989). Abscisic acid physiology and biosynthesis in higher plants. Physiologia Plantarum 75, 131-6. Csonka, L.N. (1989). Physiological and genetic responses of bacteria to osmotic stress. Microbiological Reviews 53, 121-47. Cushman, J.C., Michalowski, C.B. & Bohnert, H.J. (1990). Developmental control of crassulacean acid metabolism inducibility by salt stress in the common ice plant. Plant Physiology 94, 1137-42. DeLisle, A.J. & Ferl, R.J. (1990). Characterization of the Arabidopsis Adh G-box binding factor. The Plant Cell 2, 547-57. Dure, L. Ill, Crouch, M., Harada, J., Ho, T.-H.D., Mundy, J., Quatrano, R., Thomas, T. & Sung, Z.R. (1989). Common amino acid sequence domains among the LEA proteins of higher plants. Plant Molecular Biology 12, 475-86. Finkelstein, R.R., Tenbarge, J.M., Shumway, J.E. & Crouch, M.L. (1985). Role of ABA in maturation of rapeseed embryos. Plant Physiology 78, 630-6. Forst, S. & Inouye, M. (1988). Environmentally regulated gene expression for membrane proteins in Escherichia coli. Annual Review of Cell Biology 4, 21^t2. Galau, G.A., Jakobsen, K.S. & Hughes, D.W. (1991). The controls of late dicot enbryogenesis and early germination. Physiologia Plantarum 81, 280-8. Godoy, J.A., Pardo, J.M. & Pintor-Toro, J.A. (1990). A tomato cDNA inducible by salt stress and abscisic acid: nucleotide sequence and expression pattern. Plant Molecular Biology 15, 695-705. Gomez, J., Sanchez-Martinez, D., Stiefel, V., Rigau, J., Puigdomenech, P. & Pages, M. (1988). A gene induced by the plant hormone abscisic acid in response to water stress encodes a glycinerich protein. Nature 334, 262-4. Guerrero, F.D., Jones, J.T. & Mullet, J.E. (1990). Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Molecular Biology 15, 11-26. Guiltinan, M.J., Marcotte, W.R., Jr & Quatrano, R.S. (1990). A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250, 267-71.

150

F . L . O L S E N etal.

Hahn, M. & Walbot, V. (1989). Effects of cold-treatment on protein synthesis and mRNA levels in rice leaves. Plant Physiology 91, 930-8. Hajela, R.K., Horvath, D.P., Gilmour, S.J. & Thomashow, M.F. (1990). Molecular cloning and expression of cor (Cold-/?egulated) genes in Arabidopsis thaliana. Plant Physiology 93, 1246-52. Harada, J.J., De-Lisle, A.J., Bakden, C.S. & Crouch, M.L. (1989). Unusual sequence of an abscisic acid-inducible mRNA which accumulates late in Brassica napus seed development. Plant Molecular Biology 12, 395-401. Hatzopoulos, P., Franz, G., Choy, L. & Sung, R.Z. (1990). Interaction of nuclear factors with upstream sequences of a lipid body membrane protein gene from carrot. The Plant Cell 2, 457-67. Heikkila, J.J., Papp, J.E.T., Schultz, G.A. & Bewley, J.D. (1984). Induction of heat shock protein messenger RNA in maize mesocotyls by water stress, abscisic acid, and wounding. Plant Physiology 76, 270-4. Higgins, C.F., Dorman, C.J., Stirling, D.A., Waddell, L., Booth, I.R., May, G. & Bremer, E. (1988). A physiological role for DNA supercoiling in the osmotic regulation of gene expression in Salmonella typhimurium and Escherichia coli. Cell 52, 569-84. Hong, B., Uknes, S.J. & Ho, T.-H.D. (1988). Cloning and characterization of a cDNA encoding mRNA rapidly induced by ABA in barley aleurone layers. Plant Molecular Biology 11, 495-506. Hooley, R., Beale, M.H. & Smith, S.J. (1991). Gibberellin perception at the plasma membrane of Avena fatua aleurone protoplasts. Planta 183, 274-80. Hornberg, C. & Weiler, E.W. (1984). High-affinity binding sites for abscisic acid on the plasmalemma of Vicia faba guard cells. Nature 310, 321-4. Huang, N., Sutliff, T.D., Litts, J.C. & Rodriguez, R.L. (1990). Classification and characterization of the rice a-amylase multigene family. Plant Molecular Biology 14, 655-68. Huttly, A.K. & Baulcombe, D.C. (1989). A wheat a-Amy2 promoter is regulated by gibberellin in transformed oat aleurone protoplasts. The EMBO Journal 8, 1907-13. Jacobsen, J.V. & Beach, L.R. (1985). Control of transcription of aamylase and rRNA genes in barley aleurone protoplasts by gibberellin and abscisic acid. Nature 316, 275-7'. Khursheed, B. & Rogers, J.C. (1988). Barley a-amylase genes. Quantitative comparison of steady-state mRNA levels from individual members of the two different families expressed in aleurone cells. Journal of Biological Chemistry 263, 18953-60. Khursheed, B. & Rogers, J. (1989). Barley a-amylase genes and the thiol protease gene Aleurain: Use of a single poly (A) addition signal associated with a conserved pentanucleotide at the cleavage site. Proceedings of the National Academy of Sciences (USA) 86, 3987-91.

ABA- and GA-responsive gene expression

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King, G.J., Turner, V.A., Hussey, C.E., Wurtele, E.S. & Lee, S.M. (1988). Isolation and characterization of a tomato cDNA clone which codes for a salt-induced protein. Plant Molecular Biology 10, 401-12. Koornneef, M., Reuling, G. & Karssen, CM. (1984). The isolation and characterization of abscisic acid-insensitive mutants of Arabidopsis thaliana. Physiologia Plantarum 61, 377-83. Kriz, A.R., Wallace, M.S. & Paiva, R. (1990). Globulin gene expression in embryos of maize viviparous mutants. Plant Physiology 92, 538-42. Lanahan, M.B. & Ho, T.-H.D. (1988). Slender barley: A constitutive gibberellin-response mutant. Planta 175, 107-14. Leah, R. & Mundy, J. (1989). The bifunctional a-amylase/subtilisin inhibitor of barley: Nucleotide sequence and patterns of seed-specific expression. Plant Molecular Biology 12, 673-82. Marcotte, W.R., Bayley, C.C. & Quatrano, R.S. (1988). Regulation of a wheat promoter by abscisic acid in rice protoplasts. Nature 335, 454-7. Marcotte, W.R., Jr, Russell, S.H. & Quatrano, R.S. (1989). Abscisic acid-responsive sequences from the Em gene of wheat. The Plant Cell 1, 969-76. Martinez, E., Givel, F. & Wahli, W. (1991). A common ancestor DNA motif for invertebrate and vertebrate hormone response elements. The EMBO Journal 10, 263-8. McCarty, D.R. & Carson, C.B. (1991). The molecular genetics of seed maturation in maize. Physiologia Plantarum 81, 267-72. McCarty, D.R., Carson, C.B., Stinard, P.S. & Robertson, D.S. (1989). Molecular cloning of viviparous-1: An abscisic acid-insensitive mutant in maize. The Plant Cell 1, 523-32. Metzger, J.D. (1988). Hormones and reproductive development. In Plant Hormones and their Role in Plant Growth and Development, ed. P.J. Davies, pp. 431-62. Dordrecht: Kluwer Academic Publishers. Mundy, J. & Chua, N.-H. (1988). Abscisic acid and water-stress induce the expression of a novel rice gene. The EMBO Journal 7, 2279-86. Mundy, J., Hejgaard, J., Hansen, A., Hallgren, L., J0rgensen, K. & Munck, L. (1986). Differential synthesis in vitro of barley aleurone and starchy endosperm proteins. Plant Physiology 81, 630-6. Mundy, J., Yamaguchi-Shinozaki, K. & Chua, N.-H. (1990). Nuclear proteins bind conserved elements in the abscisic acid-responsive promoter of rice rab gene. Proceedings of the National Academy of Sciences (USA) 87, 406-10. Muthukrishnan, S., Chandra, G.R. & Maxwell, E.S. (1979). Hormoneinduced increase in levels of functional mRNA and a-amylase mRNA in barley aleurones. Proceedings of the National Academy of Sciences (USA) 76, 6181-5. Nolan, R.C. & Ho, T.-H.D. (1988). Hormonal regulation of a-amylase expression in barley aleurone layers. Plant Physiology 88, 588-93.

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F . L . O L S E N etal.

Nordin, K., Heino, P. & Plava, E.T. (1991). Separate signal pathways regulate the expression of a low temperature induced gene in Arabidopsis thaliana (L.) Heynh. Plant Molecular Biology 16, 1061-71. Pelham, H.R.B. (1986). Speculations on the functions of the major heat shock and glucose regulated proteins. Cell 46, 959-61. Pena-Cortes, H., Sanchez-Serrano, J.J., Mertens, R. & Willmitzer, L. (1990). Abscisic acid is involved in the wound-induced expression of the proteinase inhibitor II gene in potato and tomato. Proceedings of the National Academy of Sciences (USA) 86, 9851-5. Raikhel, N.V., Bednarek, S.Y. & Leaner, D.R. (1989). In situ RNA hybridization in plant tissues. In Plant Molecular Biology Manual B9, ed. S.B. Gelvin, R.A. Schilperoort & D.P.S. Verma, pp. 1-32. Dordrecht: Kluwer Academic Publishers. Raynal, M., Depigny, D., Cooke, R. & Delseny, M. (1989). Characterization of a radish nuclear gene expressed during late seed maturation. Plant Physiology 91, 829-36. Rogers, J.C. (1988). RNA complementary to a-amylase mRNA in barley. Plant Molecular Biology 11, 125-38. Salmenkallio, M., Hannus, R., Teeri, T.H. & Kauppinen, V. (1990). Regulation of a-amylase promoter by gibberellic acid and abscisic acid in barley protoplasts transformed by electroporation. Plant Cell Reports 9, 352-5. Singh, N.K., Nelson, D.E., Kuhn, D., Hasegawa, P.M. & Bressan, R.A. (1989). Molecular cloning of osmotin and regulation of its expression by ABA and adaptation to low water potential. Plant Physiology 90, 1096-101. Skriver, K. & Mundy, J. (1990). Gene expression in response to abscisic acid and osmotic stress. The Plant Cell 2, 503-12. Smart, C.C. & Trewavas, A.J. (1984). Abscisic-acid-induced turion formation in Spirodela polyrrhiza L. III. Specific changes in protein synthesis and translatable RNA during turion development. Plant, Cell and Environment 7, 121-32. Vilardell, J., Goday, A., Freire, M.A., Torrent, M., Martinez, C , Torne, J.M. & Pages, M. (1990). Gene sequence, developmental expression, and protein phosphorylation of RAB-17 in maize. Plant Molecular Biology 14, 423-32. Vinson, C.R., Sigler, P.B. & McKnight, S.L. (1989). Scissors-grip model for DNA recognition by a family of leucine zipper proteins. Science 246, 911-16. Whittier, R.F., Dean, D.A. & Rogers, J.C. (1987). Nucleotide sequence analysis of a-amylase and thiol protease genes that are hormonally regulated in barley aleurone cells. Nucleic Acids Research 15, 2515-35. Williamson, J.D. & Quatrano, R.S. (1988). ABA regulation of two classes of embryo-specific sequences in mature wheat embryos. Plant Physiology 86, 208-15.

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Wilson, G.F., Rhodes, A.M. & Dickinson, D.B. (1973). Some physiological effects of viviparous genes Vpl and Vp5 on developing maize kernels. Plant Physiology 52, 350-6. Yamaguchi-Shinozaki, K., Mundy, J. & Chua, N.-H. (1990). Four tightly linked rab genes are differentially expressed in rice. Plant Molecular Biology 14, 29-39.

D. GRIERSON, A.J. HAMILTON, M. BOUZAYEN, M. KOCK, G.W. LYCETT and S. BARTON

Regulation of gene expression, ethylene synthesis and ripening in transgenic tomatoes

Introduction The alterations in colour, flavour, texture and aroma that are responsible for transforming an unripe, unpalatable fruit into one that is attractive to a consumer are known collectively as ripening. These ripening changes are catalysed by specific enzymes that cause biochemical modifications required for the ripening process to occur (Tucker & Grierson, 1987). In various fruits, different biochemical pathways have been recruited to the ripening programme during the course of evolution, accounting for the molecular, cellular, and anatomical differences between the many different species that produce fleshy fruits. Thus some fruits undergo no colour change (e.g. kiwi fruit), whereas in others the change in colour may occur in surface layers (e.g. some apples) or throughout the flesh (e.g. tomato). Different mechanisms operate for achieving the same ends, with generation of colour occurring either by the accumulation of carotenoids in chromoplasts (e.g. tomato, banana), anthocyanins in vacuoles (e.g. strawberry, blackcurrant), or the generation of special structures for thin-film interference of light, as in Elaeocarpus (Lee, 1991). During the onset of ripening, in addition to the generation of new structures and compounds, some existing ones such as chloroplast thylakoids and starch grains disappear. These changes are sometimes related to colour production. However, solubilisation of starch and cell walls contributes also to alterations in texture, taste, and juiciness. Further biochemical processes generate a balance of various organic acids and sugars and also produce aromatic compounds which confer a distinctive flavour and aroma to particular fruits. This collection of changes, which together constitute ripening, represents a major transformation in the physiology and biochemistry of Society for Experimental Biology Seminar Series 49: Inducible Plant Proteins, ed. J. L. Wray. © Cambridge University Press, 1992, pp. 155-174.

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the fruit cells. The changes affect all cell compartments (nucleus, vacuole, cytosol, plastids, mitochondria, cell wall, endoplasmic reticulum). The existence of mutations that interfere with specific aspects of ripening, and in extreme cases prevent it from occurring altogether, strengthen the view that specific genes control the ripening process (Grierson et al, 1987). This has been confirmed by biochemical studies which show that during ripening the synthesis of some mRNAs and proteins is dispensed with and the synthesis of new ones begins. The discovery that some fruits undergo a rise in respiration which is associated with the onset of ripening led to the classification of fruits into two groups, those with (e.g. apple, pear, banana, tomato, melon) and those without (e.g. strawberry, grape, citrus) a respiratory climacteric. It is highly probable that alterations in gene expression are responsible for ripening changes that occur in both types of fruits. The main difference appears to be that only the climacteric fruits produce ethylene (ethene) (Fig. 1), which functions as a ripening hormone and stimulates and coordinates various facets of the ripening process (Fig. 2). Ethylene is believed to promote the production of new mRNAs that encode specific enzymes required for ripening of climacteric fruits (Grierson et al., 1986a), whereas some other compound as yet unidentified causes this to occur in non-climacteric fruits. Recent progress in the molecular biology of tomato ripening For various scientific and technical reasons much more research has been carried out on the ripening of tomato than any other fruit. The results of work involving the analysis of proteins generated from the translation of mRNA in vitro indicate that a variety of new mRNAs appear during ripening, others disappear, and the abundance of many, presumably 'housekeeping' mRNAs, remains relatively unchanged (Rattanapanone et al, 1978; Grierson et al, 1985; Biggs et al, 1986). At least 19 different mRNAs that increase in abundance during ripening have been cloned (Slater et al., 1985; Mansson et al., 1985; Lincoln et al, 1987) and cDNAs for the cell wall metabolising enzymes polygalacturonase (Grierson et al, 19866; DellaPenna et al, 1986; Sheehy et al, 1987) and pectinesterase (Ray et al, 1988), and a protease inhibitor (Margossian et al, 1988), have been sequenced. The accumulation of several mRNAs has been shown to be stimulated by ethylene during ripening (Grierson et al, 1986a; Lincoln et al, 1987; Maunders et al., 1987). These results are consistent with the now general view of ripening as a process involving the production of new mRNAs from 'ripening

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Fig. 1. Ethylene biosynthesis. The numbered enzymes are (1) methionine adenosyltransferase, (2) ACC (1-aminocyclopropane-l-carboxylic acid) synthase, (3) ethylene forming enzyme (EFE), (4) 5'-methylthioadenosine nucleosidase, (5) 5'-methylthioribose kinase. Regulation of the synthesis of ACC synthase and EFE are important steps in the control of ethylene production. ACC synthase requires pyridoxal phosphate and is inhibited by aminoethoxy vinyl glycine; EFE requires O2 and is inhibited under anaerobic conditions. Synthesis of both ACC synthase and EFE is stimulated during ripening, senescence, abscission, following mechanical wounding, and treatment with auxins.

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I RIPENING CHANGES colour, flavour, texture, aroma

Fig. 2. Relationship between ethylene synthesis, perception, and gene expression during ripening of climacteric fruits. Enhanced ethylene production occurs following the synthesis of ACC synthase (1) and ethylene forming enzyme (2). Perception of ethylene by the ethylene receptor, which has not been isolated, stimulates an unknown signal transduction chain that results in the expression of 'ripening genes'. These encode enzymes required for catalysing changes in colour, flavour, texture and aroma. In addition to stimulating synthesis of new mRNAs essential for ripening, ethylene also promotes the production of mRNAs for ACC synthase and EFE, leading to autocatalytic ethylene synthesis and the stimulation of ripening. Silver ions and norbornadiene interfere with the perception of ethylene, or a specific component of the signal transduction pathway and thus inhibit ethylene-stimulated processes such as ripening. Aminoethoxy vinyl glycine inhibits ACC synthase and also inhibits ripening. genes', with the expression of at least some of these genes being stimulated by ethylene (Fig. 2; Grierson et al, 1986a). Progress in the molecular analysis of tomato ripening was greatly accelerated by the introduction of plant transformation techniques, utilising gene vectors derived from Agrobacterium tumefaciens. This led to the identification of

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DNA sequences 5' to the polygalacturonase (PG) gene that functioned as cw-acting control regions which could be used to confer a ripening-specific expression pattern on foreign genes transferred to tomato (Bird et al., 1988). Expression of the PG gene in transgenic tobacco also demonstrated that different PG isoenzymes were derived from the same gene (Osteryoung et al., 1990). The development of antisense gene technology enabled the expression of the PG gene to be targeted and inhibited (Smith et al., 1988, 1990a; Sheehy et al., 1988). These experiments showed that antisense genes were stable and inherited (Smith et al., 1990a) and that they achieved a remarkable reduction in the expression of the endogenous target PG gene (Smith et al., 1988, 1990a; Sheehy et al., 1988). The down-regulation of the PG gene by the antisense gene was shown to be caused by the greatly reduced level of accumulation of PG mRNA. Although the precise mechanism of action is not clear, it is likely that sense and antisense transcripts hybridise to form double-stranded RNA in the nucleus and that these hybrids are metabolically unstable and rapidly degraded. Although it has been found that added sense genes can also achieve down-regulation of endogenous genes (Jorgensen, 1990; Smith et al., 19906), a possible explanation is that they function in a way similar to antisense genes, by the unexpected transcription of antisense RNA from the transferred sense gene construct (Grierson et al., 1991). The use of transgenic plants either overexpressing or underexpressing the PG gene in fruit has led to an increased understanding of the function of PG (Smith et al., 1988, 1990a; Giovannoni et al., 1989). It is now recognised that although PG is not the major determinant of softening, it is responsible for the degradation of pectin that occurs during ripening. Inhibiting PG gene expression with antisense genes largely prevents this, generating fruit with increased resistance to mechanical damage and cracking and with improved characteristics for commercial processing (Schuch ef a/., 1991). The control of ethylene production and identification of genes encoding ethylene biosynthesis enzymes Ethylene plays an important role in a number of plant developmental processes, including senescence and abscission of leaves and flowers, responses to wounding, and the ripening of climacteric fruits (Abeles, 1973). In each case ethylene is produced from methionine (Fig. 1). The two enzymes specific to the pathway, ACC synthase and ethylene forming enzyme, increase in activity in response to wounding and during ripening,

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senescence, and abscission, thereby causing enhanced ethylene production. A number of studies have been carried out on ACC synthase, which requires pyridoxal phosphate for the conversion of S-adenosyl methionine to 1-amino-cyclopropane-l-carboxylic acid (ACC). The enzyme has a molecular mass of approximately 55 kDa. Recently, a monoclonal antibody raised against a partially purified ACC synthase preparation was used to screen a cDNA expression library and identify a clone for ACC synthase (Sato & Theologis, 1989). The enzyme has now been purified by a number of laboratories and the sequences of cDNA clones from tomato and squash are now available (van der Straeten et al., 1990; Nakajima et al., 1990; Sato et al., 1991). In contrast, the ethylene forming enzyme has never been purified. Several studies suggested that it was associated with a membrane and activity was lost when attempts were made to solubilise it (Anderson et al., 1979; Mayne & Kende, 1986; McKeon & Yang, 1987; Kende, 1989). All studies on ethylene forming enzyme activity relied on measuring the extent to which the excised plant material converted exogenous ACC to ethylene in the presence of inhibitors such as aminoethoxy vinyl glycine that interfered with the generation of endogenous ACC by ACC synthase (Liirssen et al., 1979; Cameron et al., 1979). The lack of any information about the molecular weight or amino acid sequence of ethylene forming enzyme, and the absence of any antibody, meant that none of the usual methods for identifying cDNA and genomic clones could be used (e.g. screening a cDNA or genomic library with synthetic oligonucleotides derived from the amino acid sequence, or screening expression libraries with antibodies). We adopted a different strategy in an attempt to identify cDNA clones for enzymes involved in ethylene synthesis, by screening a cDNA library from ripening tomatoes (Slater et al., 1985) to identify clones for mRNAs which were also expressed in wounded leaves. The reasoning behind this approach was that both ripening fruit and wounded leaves generate relatively large quantities of ethylene yet there is little other obvious similarity in physiology or biochemistry. This enabled us to identify the clone pTOM 13, previously designated as a ripening-specific cDNA, as being expressed also in wounded leaves (Smith et al., 1986). pTOM 13 was shown to encode a 35 kDa polypeptide, by hybrid-select translation (Slater et al., 1985; Smith et al, 1986) and the sequence of the cDNA clone enabled us to predict the amino acid sequence of the protein (Holdsworth et al., 1987a). At least three genes for proteins related to pTOM 13 were identified in the tomato genome (Holdsworth et al., 1987ft, 1988). Studies on the expression of mRNAs related to pTOM 13 indicated that they accumulated early during ripening (Grierson et al., 1986a; Maunders et al., 1987), within 20 min of wounding (Holdsworth et

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al., 1987a) and in senescing leaves (Davies & Grierson, 1989). We tested our hypothesis that pTOM 13 encoded a protein that was related to ethylene synthesis by generating transgenic tomatoes in which the accumulation of pTOM 13 mRNA was inhibited by antisense genes (Hamilton et al., 1990). Characterisation of these plants revealed that the synthesis of antisense RNA transcribed from the transferred pTOM 13 antisense gene gave rise to a stable transcript in unwounded leaf cells, in which the pTOM 13 gene is not expressed. However, the production of the normal pTOM 13 mRNA in wounded leaves was reduced and its accumulation in ripening fruit was severely inhibited by the antisense transcript (Fig. 3). Inhibition of the accumulation of pTOM 13 mRNAs was greater with two antisense genes compared with one, and there was a corresponding reduction in ethylene synthesis in these transgenic plants (Fig. 4). Interestingly, the antisense transcript and the normal mRNA seemed to interfere with the production of each other (Fig. 3). This provides support for the proposal that antisense genes function at the level of RNA, by generating an unstable RNA-RNA hybrid, causing a reduction in gene expression. The antisense gene was stably inherited, as found also for PG antisense genes (Smith et al., 1990a) and progeny that did not inherit an antisense gene showed a normal pattern of ethylene synthesis. This shows that antisense genes do not permanently alter the target endogenous genes and is also consistent with the idea that antisense genes exert their inhibitory effects at the level of RNA. Since pTOM 13 encoded a 35 kDa protein, whereas ACC synthase was 55 kDa in molecular mass and the DNA sequences were entirely different, we tested whether pTOM 13 encoded the ethylene forming enzyme. Preliminary studies on the transgenic plants in which pTOM 13 mRNA was reduced showed that ethylene forming enzyme activity was inhibited in an antisense-gene-dosage dependent manner (Fig. 5). This suggested that pTOM 13 encoded at least a component of the ethylene forming enzyme, although direct evidence was lacking (Hamilton et al., 1990). Therefore, we sought a direct test of this possibility by expressing the pTOM 13 sequence in Saccharomyces cerevisiae to see whether the protein conferred on yeast cells the ability to convert ACC to ethylene. Before commencing work on the heterologous expression of pTOM 13 in yeast, we examined the DNA sequence to see whether any features were present that might affect its translation. The sequence of the 5' region of the cDNA gave rise to concern, since there were three out of frame ATG codons (protein synthesis initiation codons) upstream from the ATG that began the longest open reading frame (Holdsworth et al., 1987a). According to the ribosome scanning model

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for translation of eukaryotic mRNA (Kozak, 1980), where ribosomes initiate translation at the most 5' ATG sequence, this would give rise to short peptides from the most proximal ATG sequences, with only low initiation of translation from the fourth ATG. Accordingly, the sequence was checked by direct RNA sequencing and it was shown that there were

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two additional bases present in the 5' sequence of pTOM 13 mRNA which were absent from the cDNA, presumably because of a cloning artefact. The addition of these two extra bases brought the first ATG into frame with the fourth ATG adding 60 nucleotides, encoding an additional 20 amino acids, to the 5' end of the longest open reading frame (Hamilton et al., 1991). This sequence was confirmed by isolating and sequencing the corresponding genomic clone (Fig. 6; Kock et al., 1991). This gene was named eth 1 (for ethylene) and the two other relatives of pTOM 13 were designated eth 2 (previously gTOM A: Holdsworth et al., 1987b) and eth 3 (previously gTOM B: Holdsworth et al., 1988). Using this information, a cDNA encoding the complete coding sequence of eth 1 was constructed and transferred to a yeast expression vector, which was then used to transform yeast. Transgenic yeasts synthesised a transcript of the expected size and were capable of converting ACC to ethylene, whereas untransformed yeasts were not (Fig. 7; Hamilton et al., 1991). The ethylene forming enzyme activity in the transgenic yeast showed several characteristics of the authentic activity measured in plant tissues: (i) there was an 80-fold preference for the trans relative to the cis isomer of ACC analogues, (ii) activity was inhibited by cobaltous ions, (iii) an ironchelating compound inhibited activity, suggesting this metal was required by the enzyme, (iv) activity was stimulated by ascorbate. These results provided direct evidence that eth 1 encodes a polypeptide that is capable of catalysing the conversion of ACC to ethylene, confirming the identification made by the antisense approach. Deduced properties of EFE Comparison of the predicted amino acid sequence of the protein encoded by pTOM 13 with the sequence of flavanone 3-hydroxylase showed considerable similarity (A. Prescott and C. Martin, personal communication, cited in Hamilton etal., 1990). This is particularly interesting, since it has been suggested that the conversion of ACC to ethylene might involve a hydroxylation reaction (Yang, 1985). Armed with this information and using conditions developed for extraction and purification of flavanone 3hydroxylase, Ververidis & John (1991) showed that it is possible to solubilise the ethylene forming enzyme from plants and retain full

Fig. 6. Nucleotide sequence of a genomic clone {eth 1) for the pTOM 13 cDNA. The translation start site (V) and termination codon (*) are indicated. Coding sequences are shown in capitals and introns in lower case (after Kock et al., 1991).

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activity. The enzyme requires Fe2+ and is stimulated by ascorbate, which may explain the failure of previous attempts to solubilise and purify it. Inspection of the amino acid sequence of EFE deduced from the eth 1 gene suggested it is not an integral membrane protein and there is no obvious signal peptide for transport through membranes (see also Holdsworth et al., 1987a). Although it is possible that the enzyme may associate with membranes, its expression in yeast shows that it does not require plant membranes or other specific components for activity. Since earlier studies on synthesis, activity and regulation of ethylene forming enzyme were carried out with excised tissue slices or discs, the

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conclusions drawn from these investigations may need to be re-evaluated. The reason for this is that maximum wound induction (caused, for example by cutting plant tissue) of mRNA for the enzyme can occur within 45 min (Holdsworth et al, 1987a), well within the normal time scale for enzyme assays with tissue slices. Effects of reduced ethylene synthesis on tomato ripening The role of ethylene in stimulating processes such as senescence, abscission and ripening has been known for some time (Abeles, 1973). Ethylene is used commercially to ripen unripe bananas and ethylenegenerating compounds are sometimes sprayed onto field-grown tomatoes prior to mechanical harvest. In addition, removal of ethylene from fruit stores is recognised as being crucial for prolonging the storage life of produce such as apples and pears. Similar considerations apply also to retarding processes such as the abscission and senescence of flowers and green leafy vegetables. Although the ethylene receptor has not yet been isolated and its structure is not known, indirect evidence suggests that perception, or a specific aspect of signal transduction leading to the response to ethylene, is inhibited by norbornadiene and silver ions (Fig. 2). Low concentrations of silver, introduced as silver thiosulphate, have been shown to delay ripening, with a corresponding inhibition of accumulation of some ripening-related mRNAs (Davies et al, 1988, 1990). Not all mRNAs that accumulate during ripening are inhibited by silver, however, indicating that only some are controlled by ethylene. The generation of transgenic tomatoes in which ethylene synthesis was inhibited by antisense genes provided an opportunity to test directly the role of ethylene in ripening. Initial experiments indicated that when left attached to the parent plant, these tomatoes ripened more slowly (Hamilton et al., 1990). However, more extreme effects were observed when the fruits were detached from the parent plant at the mature green stage. Normal tomatoes treated in this manner undergo the usual changes in colour, flavour and texture associated with ripening. Those expressing eth 1 antisense genes, and consequently showing greatly reduced ethylene production, turned yellow but failed to turn red. In addition, the softening associated with normal ripening was also reduced. When ethylene was supplied externally to the fruit, normal colour change occurred and the fruits were indistinguishable from controls (Fig. 8). One possible explanation for the difference in ripening behaviour of attached and detached fruit from transgenic plants is that while attached they may receive a ripening stimulus from the parent plant. In this connection, it may be

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Fig. 8. Ripening of normal tomatoes and those expressing pTOM 13 antisense genes. Normal tomatoes (y4) and those homozygous for the pTOM 13 antisense gene (B,C) were grown under similar conditions and fruit were picked when mature green. After 2 weeks tomatoes in (A) ripened normally whereas the antisense fruit (B) turned yellow and failed to ripen further. Supplying ethylene at 20 ul I"1 (Q restored normal ripening to the pTOM 13 antisense fruit. significant that the different eth genes do not have identical DNA sequences and the production of ethylene in fruit was much more severely inhibited than that in wounded leaves (Fig. 4). This is presumably because the eth 1 gene sequences, although efficient at down-regulating the homologous gene expressed in fruit, may be less effective against related but not identical genes expressed in other parts of the plant. Thus, fruits that remain attached may be provided with ethylene from the parent plant. Studies on the expression of the three eth genes will help answer this question, and also enable us to determine the mechanisms involved in regulating ethylene production during ripening, leaf and flower senescence and abscission, and in response to wounding.

References Abeles, F.B. (1973). Ethylene in Plant Biology. New York: Academic Press. Anderson, J.A., Lieberman, M. & Stewart, R.N. (1979). Ethylene production by apple protoplasts. Plant Physiology 63, 931-5. Biggs, M.S., Harriman, R.W. & Handa, A.K. (1986). Changes in gene expressing during tomato fruit ripening. Plant Physiology 81, 395-403. Bird, C.R., Smith, C.J.S., Ray, J.A., Moureau, P., Bevan, M.W., Bird, A.S. Hughes, S., Morris, P.C., Grierson, D. & Schuch, W. (1988). The tomato polygalacturonase gene and ripening-specific expression in transgenic plants. Plant Molecular Biology 11, 651-62. Cameron, A.C., Fenton, C.A.L., Yu, Y.B., Adams, D.O. & Yang, S.F. (1979). Increased production of ethylene by plant tissue treated with 1-aminocyclopropane-l-carboxylic acid. HortScience 14,178-80.

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Davies, K.M. & Grierson, D. (1989). Identification of cDNA clones for tomato (Lycopersicon esculentum) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene. Planta 179, 73-80. Davies, K.M., Hobson, G.E. & Grierson, D. (1988). Silver ions inhibit the ethylene-stimulated production of ripening-related mRNAs in tomato. Plant, Cell and Environment 11, 729-38. Davies, K.M., Hobson, G.E. & Grierson, D. (1990). Differential effect of silver ions on the accumulation of ripening related mRNAs in tomato. Journal of Plant Physiology 135, 708-13. DellaPenna, D., Alexander, D.C. & Bennett, A.B. (1986). Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase levels during ripening. Proceedings of the National Academy of Sciences (USA) 83, 6420-4. Giovannoni, J.J., DellaPenna, D., Bennett, A.B. & Fischer, R.J. (1989). Expression of a climacteric polygalacturonase gene in transgenic rin (ripening-inhibitor) tomato fruit results in polyuronide degration but not fruit softening. The Plant Cell 1, 53-63. Grierson, D., Fray, R.G., Hamilton, A.J., Smith, C.J.S. & Watson, C.F. (1991). Does co-suppression of sense genes in transgenic plants involve antisense RNA? Trends in Biotechnology 9, 122-3. Grierson, D., Maunders, M.J., Slater, A., Ray, J., Bird, C.R., Schuch, W., Holdsworth, M.J., Tucker, G.A. & Knapp, J.E. (1986a). Gene expression during tomato ripening. Philosophical Transactions of the Royal Society, Series B 314, 399^10. Grierson, D., Purton, M.E., Knapp, J.E. & Bathgate, B. (1987). Tomato ripening mutants. In Developmental Mutants in Higher Plants, ed. H. Thomas & D. Grierson, pp. 72-94. Cambridge: Cambridge University Press. Grierson, D., Slater, D., Spiers, J. & Tucker, G.A. (1985). The appearance of polygalacturonase mRNA in tomatoes: one of a series of changes in gene expression during development and ripening. Planta 163, 263-71. Grierson, D., Tucker, G.A., Keen, J., Ray, J., Bird, C.R. & Schuch, W. (1986ft). Sequencing and identification of a cDNA clone for tomato polygalacturonase. Nucleic Acids Research 14, 8595-603. Hamilton, A.J., Bouzayen, M. & Grierson, D. (1991). Identification of a tomato gene for the ethylene forming enzyme by expression in yeast. Proceedings of the National Academy of Sciences (USA) 88, 7334-7. Hamilton, A.J., Lycett, G.W. & Grierson, D. (1990). Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346, 284-7. Holdsworth, M.J., Bird, C.R., Ray, J., Schuch, W. & Grierson, D. (1987a). Structure and expression of an ethylene-related mRNA from tomato. Nucleic Acids Research 15, 731-9. Holdsworth, M.J., Schuch, W. & Grierson, D. (19876). Nucleotide

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D. GRIERSON et al. sequence of an ethylene-related gene from tomato. Nucleic Acids Research 15, 10600. Holdsworth, M.J., Schuch, W. & Grierson, D. (1988). Organisation and expression of a wound/ripening-related small multigene family from tomato. Plant Molecular Biology 11, 81-8. Jorgensen, R. (1990). Altered gene expression in plants due to trans interactions between homologous genes. Trends in Biotechnology 8, 340-4. Kende, H. (1989). Enzymes of ethylene biosynthesis. Plant Physiology 91, 1-4. Kock, M., Hamilton, A.J. & Grierson, D. (1991). eth 1, a gene involved in ethylene synthesis in tomato. Plant Molecular Biology 17, 141-2. Kozak, M. (1980). Evaluation of the "Scanning Model" for initiation of protein synthesis in eucaryotes. Cell 22, 7-8. Lee, D.W. (1991). Ultrastructural basis and function of iridescent blue colour of fruits of Elaeocarpus. Nature 349, 260-1. Lincoln, J.E., Cordes, S., Read, E. & Fischer, R.L. (1987). Regulation of gene expression by ethylene during tomato fruit development. Proceedings of the National Academy of Sciences (USA) 84, 2793-7. Liirssen, K., Naumann, K. & Schroder, R. (1979). 1-Aminocyclopropane-1-carboxylic acid - an intermediate of ethylene biosynthesis in higher plants. Zeitschrift fur Pflanzenphysiologie 92, 285-94. McKeon, T.A. & Yang, S.F. (1987). Biosynthesis and metabolism of ethylene. In Plant Hormones and their Role in Plant Growth and Development, ed. P.J. Davies, pp. 94-112. Dordrecht: Martinus Nijhoff. Mansson, P.-E., Hsu, D. & Stalker, D. (1985). Characterisation of fruit specific cDNAs from tomato. Molecular and General Genetics 200, 356-61. Margossian, L.J., Federman, A.D., Giovannoni, J.J. & Fischer, R.L. (1988). Ethylene-regulated expression of a tomato fruit ripening gene encoding a proteinase inhibitor I with a glutamic acid residue at the reactive site. Proceedings of the National Academy of Sciences (USA) 85, 8012-16. Maunders, M.J., Holdsworth, M.J., Slater, A., Knapp, J.E., Bird, C.R., Schuch, W. & Grierson, D. (1987). Ethylene stimulates the accumulation of ripening-related mRNAs in tomatoes. Plant, Cell and Environment 10, 177-87. Mayne, R.G. & Kende, H. (1986). Ethylene biosynthesis in isolated vacuoles of Vicia faba L. - requirement for membrane integrity. Planta 167, 159-65. Nakajima, N., Mori, H., Yamazaki, K. & Imaseki, H. (1990). Molecular cloning and sequence of a complementary DNA encoding 1aminocyclopropane-1-carboxylic synthase induced by tissue wounding. Plant Cell Physiology 31, 1021-9.

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Osteryoung, K.W., Toenjes, K., Hall, B., Winkler, V. & Bennett, A.B. (1990). Analysis of tomato polygalacturonase expression in transgenic tobacco. The Plant Cell 2, 1239^8. Rattanapanone, N., Speirs, J. & Grierson, D. (1978). Evidence for changes in messenger RNA content related to tomato fruit ripening. Phytochemistry 17, 1485-6. Ray, J., Knapp, J., Grierson, D., Bird, C. & Schuch, W. (1988). Identification and sequence determination of a cDNA clone for tomato pectin esterase. European Journal of Biochemistry 174, 119-24. Sato, T., Oeller, P.W. & Theologis, A. (1991). The 1-aminocyclopropane-1-carboxylate synthase of Cucurbita. Journal of Biological Chemistry 266, 3752-9. Sato, T. & Theologis, A. (1989). Cloning the mRNA encoding 1-aminocyclopropane-1-carboxylate synthase, the key enzyme for ethylene biosynthesis in plants. Proceedings of the National Academy of Sciences (USA) 86, 6621-5. Schuch, W., Kanczler, J., Robertson, D., Hobson, G. Tucker, G., Grierson, D., Bright, S. & Bird, C. (1991). Fruit quality characteristics of transgenic fruit with altered polgalacturonase activity. Hort Science 26, 15-20. Sheehy, R.E., Kramer, M. & Hiatt, W.R. (1988). Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proceedings of the National Academy of Sciences (USA) 85, 8805-9. Sheehy, R.E., Pearson, J., Brady, C.J. & Hiatt, W.R. (1987). Molecular characterisation of tomato fruit polygalacturonase. Molecular and General Genetics 208, 30-6. Slater, A., Maunders, M.J., Edwards, D., Schuch, W. & Grierson, D. (1985). Isolation and characterisation of cDNA clones for tomato polygalacturonase and other ripening-related proteins. Plant Molecular Biology 5, 137-47. Smith, C.J.S., Slater, A. & Grierson, D. (1986). Rapid appearance of an mRNA correlated with ethylene synthesis encoding a protein of molecular weight 35,000. Planta 168, 94-100. Smith, C.J.S., Watson, C.F., Bird, C.R., Ray, J., Schuch, W. & Grierson, D. (19906). Expression of a truncated tomato polygalacturnonase gene inhibits expression of the endogenous gene in transgenic plants. Molecular and General Genetics 224, 477-81. Smith, C.J.S., Watson, C.F., Morris, P.C., Bird, C.R., Seymour, G.B., Gray, J.E., Arnold, C , Tucker, G.A., Schuch, W. & Grierson, D. (1990a). Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes. Plant Molecular Biology 14, 369-79. Smith, C.J.S., Watson, C , Ray, J., Bird, C.R., Morris, P.C., Schuch, W. & Grierson, D. (1988). Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes. Nature 334, 724-6. Tucker, G.A. & Grierson, D. (1987). Fruit Ripening. In The Biochemis-

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D. GRIERSON et al. try of Plants, Vol. 12, ed. D.D. Davies, pp. 265-318. New York: Academic Press, van der Straeten, D., van Wiemeersch, L., Goodman, H.M. & van Montagu, M. (1990). Cloning and sequencing of two different cDNAs encoding 1-aminocyclopropane-l-carboxylate synthase in tomato. Proceedings of the National Academy of Sciences (USA) 87, 4859-63. Ververidis, P. & John, P. (1991). Complete recovery in vitro of ethylene forming enzyme activity. Phytochemistry 30, 725-7. Yang, S.F. (1985). Biosynthesis of ethylene. In Current Topics in Plant Biochemistry and Physiology, Vol. 4, ed. D.D. Randall, D.G. Blevins & R.L. Lasson, pp. 126-38. Columbia: University of Missouri.

D.P.S. VERMA AND G.-H. MIAO

Induction of nodulin genes and root nodule symbiosis

Introduction Nitrogen is the most common limiting factor in the growth and productivity of plants. Legume plants, however, have overcome this limitation by developing the ability to harbour a group of soil bacteria (Azorhizobium, Bradyrhizobium, Rhizobiwn and Sinorhizobium spp.) in a symbiotic association. In this mutually beneficial interaction, bacteria invade the root cells of host plants where they become intracellular 'organelles' called bacteroids and are able to fix atmospheric dinitrogen into ammonium for assimilation by the plant. To facilitate this process, the host plant develops an entirely new organ, a root nodule, which houses the nitrogen-fixing bacteria and provides the carbon sources, other nutrients and appropriate environment to support the reduction of dinitrogen (see Verma & Long, 1983; Long, 1989; Verma & Stanley, 1989). The Rhizobium-legume symbiosis, an interaction between a prokaryote (Rhizobium) and a eukaryote (legume), requires a series of sequential induction and function of both bacterium-encoded (bacteroidins) and host-encoded (nodulins) nodule-specific proteins. It has been shown that many plant (Peters et al., 1986; Firmin et al., 1986; Djordjevic et al., 1987; Sadowsky et al., 1988; see also Peters & Verma, 1990) and bacterial (Lerouge et al., 1990; Kondorosi, 1991) signals are involved in the induction of specific genes leading to the development of the root nodule. Many bacterial mutations blocking root hair deformation, induction of cortical cell division, infection thread formation and subsequent events leading to the endocytotic release of bacteria inside the host cell are known (see Rolfe & Shine, 1984). Similarly, mutations affecting nodulation have also been identified in host plants (Carroll et al., 1985; Rolfe & Gresshoff, 1988), suggesting the involvement of many plant genes in this interaction. Society for Experimental Biology Seminar Series49: Inducible Plant Proteins, ed.J. L. Wray. ©Cambridge University Press, 1992, pp. 175-204.

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Communication and titration of plant and bacterial signals The signal for the nodule initiation process comes from the bacterium in response to specific compounds produced by the host plant. It has been found that several leguminous plants are able to synthesise and release into the rhizosphere specific flavonoids which are 3-ring aromatic compounds derived from the normal phenylpropanoid metabolic pathway (Firmin et al., 1986; Peters et al., 1986; Djordjevic et al., 1987; see Peters & Verma, 1990). These low molecular weight compounds induce a set of nodulation (nod) genes in Rhizobium (Mulligan & Long, 1985; Horvath et al., 1987; Spaink et al., 1987). The activation of nodulation genes elicited by flavonoids makes Rhizobium competent to attack the root hair cells of a susceptible host plant. A return signal, which in the case of Rhizobium meliloti has been shown to be a modified oligosaccharide (Lerouge et al., 1990), is produced by the bacterium. This molecule, on its own, is capable of initiating nodule organogenesis by localised application to legume roots (see Truchet et al., 1991). That these signals are finely titrated by bacterium and host is consistent with the observations that overexpression of bacterial nod genes decreases nodulation (Knight et al., 1986) and that nodules are formed only at particular sites on the host root (Bauer, 1981), presumably those sites which are most responsive to the nodule morphogenetic signals. Communication between the two partners is a continuous process from infection to senescence of nodules and appears to involve many discrete signals. This is evident from mutations in both the plant and the bacterium that abort the developmental programme at different stages of nodule morphogenesis and affect symbiotic nitrogen fixation. A generalised scheme of the known and postulated signal molecules affecting plant gene expression is outlined in Fig. 1. It appears that many host genes are affected by bacterial signals either directly or indirectly through a cascade mechanism. Since cortical cell division can be elicited by the purified bacterial signal molecules alone (Lerouge et al., 1990; Truchet et al., 1991), this process is independent of bacterium-plant contact and formation of the infection thread. This confirms earlier observations that nodule morphogenesis can be disassociated from infection and endocytotic release of Rhizobium inside the host cells. Understanding the perception of these bacterial signals by the host and the subsequent expression of host genes encoding nodule-specific proteins will be fundamental to our ability to manipulate the plant-Rhizobium interaction in the future.

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Primary Receptor ? OLIGOSACCHA RIDES v

FLAVONOIDS

t

Inducing early nodulin genes

NODULE MORPHOGENESIS INFECTION & ENDOCYTOSIS i

Other Signal Molecules

i

Late & PBM Nodulin Genes

—

HOST PLANT

Fig. 1. Possible signals and communications in the induction of nodulin genes and the development of root nodule symbiosis.

Nodule ontogeny, metabolism and symbiotic state Successful infection proceeds simultaneously with nodule morphogenesis triggered by the signal compounds produced by Rhizobium in response to its host, and leads to the release of bacteria from the infection threads into the cortical cells. Concomitantly, the bacteria are enveloped in a host-derived plasma membrane called the peribacteroid membrane (PBM) (Verma et at., 1978; Robertson et al., 1978). Both host- and bacterium-derived proteins are specifically targeted to this membrane (Fortin et al., 1985, 1987; Katinakis & Verma, 1985; Katinakis et al., 1988), making it a unique subcellular compartment. During the course of differentiation of bacteria into bacteroids, many bacterial genes are induced and others repressed. One of the induced proteins is the key enzyme, nitrogenase, which catalyses the conversion of dinitrogen to ammonium. The expression of many host genes is essential for the function of this enzyme. The induction of leghaemoglobin to facilitate oxygen diffusion in the host cell is vital for effective symbiosis (Appleby, 1984). The fixed nitrogen (ammonium ions) is transported through the PBM into the cytoplasm of the host cells where it is further assimilated by the host-encoded nitrogen-assimilation enzymes. Most of the enzymes in this pathway are specifically induced or enhanced in the symbiotic state (Verma, 1989). In ureide-producing nodules, the nitrogen assimilation pathways are further compartmentalised in two different cell types (infected and uninfected) in the infection zone. While the early steps of nitrogen assimilation take place in the infected cells, the later steps of nitrogen metabolism are carried out in the uninfected cells. This

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compartmentalisation seems to play a very important role in regulating the expression and function of some key enzymes in this pathway, as well as affecting the efficiency of nitrogen fixation and reallocation of metabolites in the nodule. For an effective symbiotic state, the plant and the microsymbiont must maintain a constant metabolic flow of carbon and nitrogen. While the bacteroids function as an 'engine' for nitrogen fixation the fuel comes from the plant. Dicarboxylic acids are the primary carbon sources fed to the bacteroids by the plant. This unidirectional flow of carbon must be controlled by the PBM. Recently, several specific carbon and amino acid transport systems have been identified in the PBM using isolated peribacteroid units (PBU: Day et al., 1990). Thus, in order for the host plant to house endosymbiotic bacteria and support their metabolic needs, a number of nodulin genes must be induced to support the ontogeny and function of the nodules. Nodulins were first identified in soybean root nodules (Legocki & Verma, 1979, 1980; Fuller et al., 1983). Since then, a large number of nodulins have been isolated and characterised from several legume species (Delauney & Verma, 1988; Verma & Delauney, 1988; Gloudemans & Bisseling, 1989). Based on the differential expression pattern of nodulin genes during the course of nodule development, they have been roughly divided into early and late nodulin genes (Govers et al., 1987). A cascade of expression following a temporal and spatial pattern occurs for many early nodulin genes (Scheres et al., 199Qa,b), and some of these genes can be induced in pseudonodules devoid of any bacteria (Van de Wiel et al., 1990). Early nodulins have been suggested to be involved in the infection processes and root nodule morphogenesis (Nap & Bisseling, 1990) since they are specifically expressed at a distinctive stage of infection and nodule organogenesis. The late nodulin genes are expressed at the later stages of nodule formation, generally following the release of bacteria but prior to the induction of nitrogenase and commencement of nitrogen fixation (Verma et al., 1986).

Structure and function of nodulin genes Early nodulins induced during the infection process and nodule organogenesis Once an appropriate Rhizobium species infects the host plant roots, the plant responds by sequentially turning on a set of specific genes which are required for nodule formation and function. A number of nodule-specific

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genes induced during the early stages of nodule development have been cloned from soybean (Franssen etal., 1987), pea (Scheres etal., 1990a,b; Van de Weil etal., 1990), alfalfa (Dickstein etal., 1988), Sesbania (Strittmatter et ai, 1989) and Vigna (Trese & Pueppke, 1991). Northern blot analysis and in situ hybridisation studies of several early nodulin mRNAs from pea showed that early nodulin genes are expressed in a sequential manner during the course of Rhizobium infection and nodule organogenesis (Scheres et al, 19906; Van de Weil et al., 1990), suggesting that they respond to Rhizobium signals differently or to different signals. This orderly cascade of gene expression further implies that there are multiple regulatory steps involved in the induction of these genes. The expression pattern of the early nodulin genes roughly coincides with the formation of discrete tissue types in the indeterminate root nodules. The mRNA encoding one of the pea early nodulins (PsENOD12) is detected in both infected and uninfected cells of the entire invasion zone (Scheres et al., 1990a). Another early nodulin (PsENOD5) mRNA is primarily expressed in the infected cells of the symbiotic zone, although a low level of expression is also detected in the invasion zone closer to the central tissue of the nodule. The expression of several other early nodulins (Franssen et al., 1987; Scheres et al., 19906) has been detected only in the symbiotic zone and these may be involved in nodule organogenesis rather than in the infection process. More interestingly, ENOD2 is present in the nodule inner cortex (nodule parenchyma) of soybean and pea (Van de Weil et al, 1990) and alfalfa (Allen et al, 1991) and, like PsENOD12, contains two repeating pentapeptides rich in proline, characteristic of cell wall proteins (Cassab & Varner, 1988). Since the nodule inner cortex has been suggested to be an oxygen diffusion barrier (Witty et al, 1986), the presence of ENOD2 in this tissue might indicate the role of nodulespecific cell wall proteins like ENOD2 in establishing and maintaining the oxygen diffusion barrier (Van de Weil et al, 1990). Amino acid sequence analysis of ENOD2 from different plants (Franssen etal, 1987; Dickstein etal, 1988; Van de Weil etal, 1990), revealed that it resembles other plant cell wall proteins (Averyhart-Fullard et al, 1988), containing two repeating pentapeptides, Pro-Pro-Glu-Tyr-Glu and Pro-Pro-His-Glu-Lys. Although there is no direct evidence that the proline residues of ENOD2 are converted into hydroxyproline, or that the protein is further glycosylated, it is likely that ENOD2 is a hydroxyproline-rich glycoprotein (HRGP) since the structural features of ENOD2 are very similar to other well-defined HRGPs (AveryhartFullard et al, 1988; Cassab & Varner, 1988). While PsENOD12 also encodes a putative HRGP (Scheres etal, 1990a), it appears at a different stage and in different cell types of the developing nodules. Thus, ENOD2

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and PsENOD12 might fulfil different roles in nodule formation. Amino acid sequence analysis of other early nodulins (Scheres et al., 19906) has led to the suggestion that PsENOD3 and PsENOD14 genes might encode proteins involved in metal transport, while PsENOD5 might be a membrane protein and is probably targeted to the peribacteroid membrane. Thus early nodulin genes, activated sequentially during nodule development, may perform diverse functions in the formation of this organ. Nodulins involved in oxygen transport and carbon and nitrogen metabolism in root nodules The primary function of root nodules is to allow the reduction of dinitrogen by the Rhizobium and the assimilation of the resultant ammonium by the plant. To accomplish these goals efficiently, the plant has made many necessary adjustments to house the microsymbiont and provide it with the appropriate environment and machinery to sustain the symbiotic state. This is best evidenced by the induction of a number of late nodulin genes prior to the commencement of nitrogen fixation activity. Many late nodulins function as the key enzymes of nitrogen and carbon metabolic pathways in the nodules or as proteins, like leghaemoglobin, that sustain the flux of oxygen under low pO2. The effectiveness of root nodules in fixing nitrogen is directly correlated with the presence of leghaemoglobin (Lb). Lb is the most abundant (up to 20%) protein in root nodules and consists of an apoprotein (a monomeric globin), and its prosthetic group (haem). The globin is encoded by the host plant (Verma et al., 1974), while the haem moiety is primarily synthesised by the bacterium and transported into the host cytoplasm (see Lee & Verma, 1984). This was best demonstrated by the observation that a defect in haem biosynthesis in Rhizobium leads to production of non-functional Lb (Nadler, 1981) and induces Fix" nodules on alfalfa (Leong et al., 1982). However, some plants can synthesise amounts of haem sufficient to maintain the function of Lb in the nodule since haem biosynthesis-deficient Bradyrhizobium japonicum mutants can induce effective nodules on soybean (Guerinot & Chelm, 1985). Recent studies have shown that the haem biosynthetic pathway may be spatially separated between the two partners, since the haem precursor, 6-aminolaevulinic acid, is synthesised in the plant cell while the rest of the haem biosynthetic steps are completed by the bacterium (Sangwan & O'Brian, 1991). Lbs are encoded by a family of genes. In soybean, at least four major Lb species have been characterised: Lba, Lbcl, Lbc2 and Lbc3 (Lee et al., 1983). In addition, truncated genes and pseudogenes of Lb also exist in the soybean genome (Brisson & Verma, 1982). However,

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it is unclear whether these different Lbs play different roles within the nodule. Lb is localised exclusively in the cytoplasm of the infected cells of the nodule (Verma & Bal, 1976; Nguyen et al., 1985), but not inside the PBM. This subcellular location of Lb suggests that it controls the oxygen flux in the cytoplasm of the nodule, but probably not within the PBM. Additional auxiliary oxygen carriers may, therefore, exist for controlling the oxygen level within the PBM. The induction of the oxygen carrier protein Lb in the infected cells of the nodule may help to maintain a flux of oxygen in the cells under conditions where the high demand for oxygen by bacteroids might potentially cause hypoxic conditions. Therefore, Lb may be considered as a 'defence' molecule against hypoxia (Verma et al., 1990a). Lb has also been demonstrated to facilitate respiration in mitochondria (Suganuma et al., 1987) and thus its presence in the host cytoplasm may help oxidative metabolism in the infected cells. Ammonium, the primary product of nitrogen fixation, is transported to the host cell cytoplasm where it is assimilated into amides and, in some cases, further converted into ureides before being transported to the shoot. Since the physiological environment within the nodule is apparently different from the other parts of the plant, nodule-specific or nodule-abundant forms of several enzymes of the nitrogen and carbon assimilation pathways have evolved, and are induced to improve the efficiency of nitrogen and carbon metabolism in nodules. Glutamine synthetase (GS; EC 6.3.1.2) plays a central role in the assimilation of ammonium in nodules. GS is encoded by a small family of genes, the members of which are expressed in different organs of the plant, giving rise to many functional forms of this enzyme (Tingey et al., 1987; Verma, 1989). Nodule-specific GS isoforms have been identified in Phaseolus vulgaris (Lara et al., 1983; Gebhardt et al., 1986; Forde et al., 1989), alfalfa (Dunn et al., 1988), and lupin (Konieczny et al., 1988). In pea, however, it appears that the GS isoform found in nodules is not unique to that organ (Tingey et al., 1987). In soybean, enhanced GS activity in nodules has been reported to be attributable to the synthesis of nodule-specific GS isoforms (Sengupta-Gopalan & Pitas, 1986). However, the results from our laboratory have shown that the increase in soybean nodule GS is caused by ammonium-stimulated expression of GS isoforms existing in the root (Hirel et al., 1987; Miao et al., 1991). All GS> genes so far isolated either through screening a soybean nodule cDNA library (Hirel et al., 1987) or by direct complementation of an Escherichia coli glnA mutant (Miao et al., 1991) seem to be expressed in both roots and nodules. While temperate legumes export symbiotically fixed nitrogen mainly in

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the form of amides, in tropical legumes ureides are the major form of nitrogenous compounds translocated to the shoot (see Schubert, 1986; Verma, 1989). The ureide pathway appears to be superior to the amide pathway since less metabolic energy is used to transport fixed nitrogen via this route. In ureide-producing nodules, nitrogen metabolism is compartmentalised between infected and uninfected cells (Newcomb & Tandon, 1981; Nguyen et al., 1985). Some of the enzymes involved in the early steps of nitrogen assimilation and ureide production (e.g. GS and xanthine dehydrogenase) appear to be located in both infected and uninfected cells (Verma et al., 1986;Branjeonefa/., 1988; Newcomb et al., 1990), whilst others (e.g. uricase II; EC 1.7.3.3) are present primarily in uninfected cells (Nguyen et al., 1985; Kaneko & Newcomb, 1987; Stegink et al., 1987). Nodulin-35 (N-35) represents the second most abundant protein in the soluble fraction of soybean nodules (Legoki & Verma, 1979) and forms a subunit of uricase II which catalyses the conversion of uric acid to allantoin (Bergmann et al., 1983; Suzuki & Verma, 1991). Immunofluorescence localisation and immunogold labelling in soybean nodules showed that N-35 is present in the peroxisomes of the uninfected cells of the nodules (Bergmann et al., 1983; Nguyen et al., 1985). This indicates that the induction of some nodulin genes may not only be under developmental control but also be regulated by cell-specific metabolism. A functional uricase was obtained by expression in E. coli of a soybean N-35 cDNA driven by the bacterial lacZ promoter (Suzuki & Verma, 1991). The uricase activity was mainly found in the cytoplasmic fraction of E. coli and had the same pH optimum and apparent Km values as in the nodules. That N-35 is able to assemble into a functional, tetrameric holoenzyme in E. coli indicates that post-translational modifications, or the presence of peroxisomes, is not essential for its proper assembly and function in this organism. However, N-35 is not active and does not accumulate to any significant levels when it is expressed in transgenic tobacco under the control of the CaMV-35S promoter (our unpublished data). Xanthine dehydrogenase (XDH; EC 1.2.1.37) participates in the ureide biosynthetic pathway and catalyses the oxidation of hypoxanthine to xanthine and the oxidation of xanthine to uric acid (Schubert, 1986). Initially, XDH was localised in the infected cells of soybean nodules (Triplett, 1985), suggesting that the purine biosynthetic pathway occurs there and that uric acid is the most probable intermediate transported from the infected cells. However, a recent study on the localisation of XDH in cowpea nodules (Newcomb et al., 1990) indicated that XDH is located in both infected and uninfected cells and suggests that the plastids

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of the uninfected cells may also carry out purine biosynthesis. The fact that GS is also localised in both cell types (Miao etal., 1991) suggests that the uninfected cells in ureide-producing nodules may be the primary site for assimilation of nitrogen destined for transport while the nitrogen assimilated in the infected cell may be used primarily for maintaining host and bacterial metabolisms. Successful nitrogen fixation depends largely on the rapid flow of carbon from the host plant to the nodules. Sucrose is the main sugar translocated from leaves to nodules. Sucrose synthetase (EC 2.4.1.13) in the nodules appears to be involved in the cleavage of sucrose to support the carbon requirements of the nodules (Reibach & Streeter, 1983). One of the soybean nodulins isolated in our laboratory has been shown to encode a subunit of sucrose synthetase (Thummler & Verma, 1987). The expression of maize sucrose synthetase in transgenic plants showed that it is anaerobically induced in roots and is specifically expressed in the phloem cells (Yang & Russell, 1990). The regulation of soybean sucrose synthetase gene expression in nodules may also be controlled by low levels of oxygen. Moreover, the activity of this enzyme seems to be regulated by the binding of free haem which dissociates this enzyme into subunits (Thummler & Verma, 1987). We have proposed that inhibition of sucrose synthetase by free haem, which may become available during senescence, may restrict the supply of carbon to the bacteroids and thus prevent them from becoming pathogenic to the plant. Besides the abundant nodulins like Lb, GS, uricase and sucrose synthetase, many other enzymes involved in nodule metabolism are induced during the course of nodule development (see Verma & Nadler, 1984). However, owing to the relatively low abundance of these enzymes and their mRNAs in nodules, none of the cognate genes have been cloned and new cloning strategies need to be employed to isolate these relatively less abundantly expressed nodulin sequences. Towards this goal, we have recently taken a direct functional complementation approach and have cloned a soybean cDNA sequence encoding A'-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2) by complementation of an E. coliproC mutant with a soybean nodule cDNA expression library (Delauney & Verma, 1990a,b). Owing to the fact that the activity of P5CR is much higher in soybean nodules than in amideproducing nodules, proline biosynthesis has been postulated to play a role in the flux of carbon for the ureide biosynthetic pathway (Kohl et al., 1988). It has been well established that proline acts as an osmoregulator in many plants (Aspinall & Paleg, 1981). Thus, proline may also be involved in supporting the higher osmoticum found in infected cells compared with that in root cells (Delauney & Verma, 19906; Verma et al.,

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1978). Moreover, treatment of the soybean root with salt (0.4 M NaCl) enhances the expression of this gene (Delauney & Verma, 19906). Recently, we have demonstrated that soybean P5CR is located in the cytoplasm of the root/nodule and in the chloroplast of the leaf (A. Szoke et ah, unpublished data). Localisation of P5CR in different subcellular compartments in root/nodule and leaf suggested that proline is synthesised in different sites in these organs and the genes encoding different isoforms of P5CR may be regulated differently. Many nodulin genes seem to have evolved from genes pre-existing in the legume genome which have been brought under nodule developmental control (Verma et ah, 1990a). The induction of these genes is triggered either by bacterial infection or in some way by the unique nodule environment created during the symbiotic state. Indeed, it has been found that whilst some early nodulin genes (Franssen et ah, 1987; Scheres et ah, 1990a) are highly expressed in nodules, low-level expression of these genes can also be detected in other parts of the plant. However, using the current technologies, no expression of Lb, N-35, nodulin-24 (N-24) nor nodulin-26 (N-26) genes has been found in any other part of the plant. Nodulins of the peribacteroid membrane and the biogenesis of the PBM compartment The release of Rhizobium from the infection thread into the dividing cortical cells follows a well-regulated process resembling endocytosis. Consequently, the invading bacteria do not have any direct contact with the cytoplasm of the infected cells, but instead are enclosed in a hostderived membrane, the peribacteroid membrane (PBM) (Verma et ah, 1978; Robertson et ah, 1978). This membrane keeps the bacteria away from the cytoplasm, rendering the PBM-enclosed bacteria topologically 'outside' the cell. The formation of the PBM is essential for maintaining the bacteria inside the host cells. Failure to develop the PBM may result in triggering of host defence responses and degeneration of the invading bacteria leading to ineffective nodules (Werner et ah, 1984, 1985). The PBM is the primary interface between the host cell cytoplasm and the microsymbiont, and forms the main control point for nutrient and signal exchanges. It has been well documented that the carbon sources required for the function of bacteroids are provided by the host plant primarily in the form of dicarboxylates (see Dilworth & Glenn, 1984). This is strongly supported by the fact that mutations in the bacterial dicarboxylic acid transporter result in ineffective nodules (Birkenhead et ah, 1988; Watson et ah, 1988). In addition, the flow of many other

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compounds essential for nodule function (Streeter, 1987) is also controlled by the PBM and, since these transport functions are unique to the nodule, it is reasonable to assume that there are some nodule-specific transporters present in the PBM. Indeed, a number of transport systems have been identified in the PBM (Day etal., 1990). Among them, the best studied is the dicarboxylic acid transporter (DCT) identified in soybean PBM (Udvardi et al., 1988). This DCT seems to be different from that in bacteria, mitochondria and tonoplasts based on the degree of substrate selectivity and response toward different inhibitors (Udvardi et al., 1988; Day et al., 1990), suggesting the possibility that it may be encoded by a nodule-specific gene. However, none of the putative transporter proteins of the PBM has yet been characterised at the molecular level. Further characterisation of these transporters in the PBM will certainly allow an insight into the nature of the PBM compartment as well as nutrient and signal exchanges between the two partners. It has been determined that the PBM comprises approximately 20 times more membrane than the plasma membrane (Verma et al., 1978). Therefore, massive membrane biosynthesis is required during the course of PBM formation. However, it is not clear what signals are responsible for this highly regulated membrane biosynthesis in the developing nodules. One of the key enzymes in the phospholipid biosynthetic pathway has been shown to be induced in soybean nodules and has been suggested to be a nodulin (Mellor et al., 1986). In addition, a number of nodulins are induced and specifically targeted to the PBM (Verma et al., 19906). Many of the PBM nodulins are glycosylated in the endoplasmic reticulum (ER) and further modified before integration into the membrane (Werner et al., 1988). The induction of different PBM nodulins may require different signals as evidenced by a block in nodule development at a specific stage by a bacterial mutant that results in different patterns of expression of two PBM nodulin genes (Morrison & Verma, 1987). Several PBM nodulins have been identified in soybean (Verma & Fortin, 1989). Among them, N-24 represents a surface PBM protein and N-26 is a major transmembrane protein of the PBM. Structural analysis of N-24 based on amino acid sequence (derived from its cDNA sequence) revealed some very interesting features. The finding that exons 2, 3 and 4 represent the same repeat sequence has led to the suggestion that this repeated domain may have been generated by duplication of an insertion sequence (Katinakis & Verma, 1985). The first and last exons are unique and contain hydrophobic domains. While the first hydrophobic domain at the amino-terminal seems to act as a signal sequence, the hydrophobic domain in the last exon may act as a mem-

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brane anchor allowing the attachment of this protein to the PBM. However, experiments in which the N-24 sequence was fused with the GUS coding region and allowed to translocate into microsomal membranes in vitro (C.-I. Cheon and D. P. S. Verma, unpublished data) showed that no part of this molecule is exposed outside the membrane vesicles and that the entire molecule is protected from trypsin digestion following translation in the presence of microsomal membranes. This suggests that N-24 does not have any membrane anchoring region and that it may be attached to the PBM by some other mechanism. Direct localisation of N-24 in the PBM revealed a cross-reacting band of 32 kDa (Fortin et al., 1987), suggesting that extensive post-translational processing through the Golgi may be responsible for the apparent increase in the size of this protein. Recently, a sequence homologous to N-24, nodulin-16, has been isolated from soybean nodules (Nirunsuksiri & Sengupta-Gopalan, 1990). Like N-24, N-16 also contains N- and Cterminal hydrophobic domains, but is apparently a soluble protein. N-26 shares significant sequence homology with several genes identified in other plants (Guerrero et al., 1990; Johnson et al., 1990; Yamamoto et al., 1990) and other species (Gorin et al., 1984; Rao et al., 1990; Sweet et al., 1990). The striking conservation of these proteins from divergent organisms has led to the proposal that they may be derived from a common ancestor and play a similar role in these different organisms (Baker & Saier, 1990). This reinforces the suggestion that during the course of evolution, legume plants developed the ability to create or modulate a number of genes, which are now induced upon the infection of Rhizobium. Thus, the gene for N-26 has been recruited from a common ancestral gene in the plant to meet the specific need of nodule function. Structural analysis of N-26 predicted that it is a transmembrane protein with six membrane-spanning domains (Miao et al., 1990). Like many transmembrane proteins, this protein lacks a N-terminal signal sequence, but it may have internal signal sequences. In vitro translation experiments suggested that N-26 is co-translationally inserted into microsomal membranes. The translocated protein is further modified by glycosylation as membrane-inserted N-26 binds to Con-A. Chemical cleavage mapping at cysteine residues of trypsinised and untreated N-26 showed that both amino (N) and carboxy (C) termini of the in vitro synthesised N-26 are sensitive to trypsin digestion, indicating that both Nand C-termini of this protein face outside the PBM (i.e. face the cytoplasm) and that the glycosidic residue seems to face the bacteroids. The carboxy end of N-26 contains three potential phosphorylation sites which may interact with acidic lipids present in the PBM. The phosphorylation of nodulins may be regulated by the presence of different

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COOH

Bacteroid

Fig. 2. Proposed topology of soybean nodulin-26 in the peribacteroid membrane: a-f, six membrane-spanning domains. T, Phosphorylation sites; #, potential glycosylation sites that are not glycosylated; 0, glycosylation site facing the bacteroid is in fact glycosylated as shown by Con-A binding; • , trypsin target site closer to the first and last transmembrane domains; ®, phosphate residues possibly interacting with the membrane. protein kinases in root nodules (Suzuki & Verma, 1989). Using a Cterminal synthetic peptide of N-26 as a phosphorylation substrate, Weaver et al. (1991) have shown that the putative phosphorylation sites at the C-terminus of N-26 are indeed phosphorylated by protein kinase(s) present in different parts of plant. However, phosphorylation of native N26 in the PBM is apparently catalysed by a protein kinase located in the PBM (Miao et al., 1992). Based on these studies, a topology of N-26 in the PBM is proposed as shown in Fig. 2. Comparison of the amino acid sequence of N-26 with bovine eye lens major intrinsic membrane protein (MIP-26), a turgor-regulated protein from pea (JM7a), a bean seed tonoplast membrane protein (TIP), a protein coded by the Drosophila neurogenic gene, big brain (bib), and the E. coli glycerol facilitator (GlpF) shows significant homologies. Secondary structure analysis of these proteins also shows extensive similarity. N-26 and the GlpF have a similar isoelectric point while MIP26 and JM7a are similar. It has been shown that the GlpF forms a poretype ion channel in the cytoplasmic membrane of E. coli (Sweet et al., 1990). Based on the proposed secondary structure and its comparison to this conserved group of proteins, it is very likely that N-26 functions as a channel in the PBM. However, the substrate selectivity of these channels probably varies depending on the metabolic needs of each tissue. Since

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the expression of the N-26 gene occurs prior to and independently of nitrogen fixation, N-26 may participate in translocating specific metabolites to support bacteroids. Such a translocation of metabolites through the PBM would allow equilibration of the peribacteroid fluid with the cytoplasmic concentration of those ions and active uptake by bacteroids from peribacteroid fluid follows (Verma et al., 19906). Some of the PBM proteins may be derived from bacteroids (Fortin et al., 1985). It will certainly be interesting to study how these proteins are secreted by the bacteroids and are specifically targeted to the hostderived PBM. Also, in the peribacteroid space (PBS, the space between PBM and bacteroids), several bacteroidins have been detected in pea nodules (Katinakis et al., 1988). Recently, a gene encoding a proteinase inhibitor has also been isolated from senescent nodules (Manen et al., 1991). Proteinase inhibitors are generally present in vacuoles and it has been suggested that PBU shares many properties typical of a lysosomal compartment (Mellor, 1989). This is consistent with observed homology between TIP and N-26 (Johnson et al., 1990), which suggests that the PBM compartment shares some features common to vacuoles. Thus, the PBM may be a mosaic membrane. To determine further the biochemical functions of these important proteins in the PBM, both genetic and biochemical approaches are required. Creating dominant, conditional mutations in these proteins by antisense technology (Delauney et al., 1988) may allow an understanding of the physiological role of these proteins in nodules. One of the most important events in nodule organogenesis is the formation of the PBM, which creates an 'extracellular' compartment within the infected cell. Although the PBM shares many features in common with the plasma membrane, it is extensively modified in the course of nodule development and incorporates many nodulins. The PBM is derived from the plasma membrane surrounding the tip of the infection thread; however, further development and the integrity of the PBM are probably contributed by both ER/Golgi and plasma membrane through endosome routes. Following the targeting routes of different PBM-specific proteins in transgenic legumes may shed some light on the nature of the PBM compartment and the origin of this 'organelle'. How do PBM nodulins distinguish different membranes in the cell and how are they specifically targeted to the PBM? In other systems, it is clear that proteins carry specific targeting signals to guide them to the preprogrammed destination, be it ER, mitochondrion, chloroplast, nucleus, peroxisome, or vacuole (Klausner, 1989). It will be interesting to understand the protein sorting mechanism by which PBM nodulins are targeted to their novel subcellular compartment. Studies from this

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laboratory have shown that the targeting of different PBM nodulins may follow different modes. Targeting of nodulin-24 apparently involves cotranslational cleavage of a signal sequence, while that of N-26 is independent of a N-terminal signal sequence and may use some internal signals (Miao etal., 1992). Regulation of nodulin gene expression during nodule development Developmental control of nodulin genes The activation of early nodulin genes appears to be independent of the. attachment of bacteria and the formation of the infection threads (Scheres et al., 1990a), and consequently the purified nod signal from Rhizobium is able to induce some of these genes along with nodule organogenesis (Denarie & Roche, 1991). These genes seem to be controlled by a number of different signals since they are differentially expressed during nodule organogenesis (Scheres et al., 19906). Expression of early nodulin genes in alfalfa root outgrowths elicited by auxin transport inhibitors (Hirsch et al., 1989) further suggests that the induction of some of the early nodulin genes may not be directly under the control of bacterial signals but under a plant developmental programme or hormonal control. The signal(s) for activating the late nodulin genes is clearly different from that for early nodulin genes since it generally requires the formation of infection threads and release of bacteria into the infected cells (Verma et al., 1988). The expression of different late nodulin genes can be specifically blocked at particular stages of nodule development by Rhizobium mutants (Morrison & Verma, 1987), suggesting that different signals are used to induce these genes. The enhanced nodule expression of some late nodulin genes appears to be under nodule metabolite control (Hirel et al., 1987; Miao et al., 1991) or under the influence of the nodule environment (Larsen & Jochimsen, 1986; Delauney & Verma, 19906). Thus, there are at least three distinct phases in nodule development that temporally control nodulin gene expression, as summarised in Fig. 3. While nodule morphogenesis can be disassociated from infection, endocytosis must proceed via infection and, hence, expression of late nodulin genes does not occur without the formation of the infection thread and release of bacteria. The expression of nodulin genes is mainly controlled at the transcriptional level. The c«-regulatory sequence located in the 5' promoter region of various nodulin genes determines organ specificity and level of expression in nodules by interacting with corresponding trans-acting factors (Verma et al., 1988; Jacobson et al., 1990). DNA sequence com-

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I

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EXTERNAL SIGNALS DEVELOPMENT

Early Nodulins

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Fig. 3. Three distinct groups of signals produced during infection and nodule development inducing early and late nodulin genes. parisons of the 5' flanking region of several nodulin genes revealed the presence of AT-rich consensus sequences among N-23, N-24, Lbc3 (Mauro et al., 1985) and GS (Forde et al., 1990) genes. Using an in vitro transcription assay system, Mauro & Verma (1988) have shown that the induction of N-23, N-24 and Lbc3 genes in soybean nodule nuclei follows similar kinetics. Interestingly, the transcription pattern of N-35, which is expressed in uninfected cells at a later stage of nodule development, compared with the timing of N-23, N-24 and Lbc3 gene expression, appears to be different, suggesting that different factors are involved in activating the N-35 gene. This was further supported by the absence of any consensus sequence motifs between the 5' flanking region of N-35 and those of the N-23, N-24 and Lbc3 genes (Verma et al., 1986). DNAprotein binding experiments have directly demonstrated the presence of trans-diCtmg factors binding to the common AT-rich consensus motifs of 5' flanking regions of N-23 (Jacobsen et al., 1990), Lbc3 (Jensen et al., 1988; Metz etal, 1988) and nodule-specific GS (Forde etal., 1990). Since AT-rich sequence motifs are a common feature of many plant promoters, the factors binding to the nodulin genes may not be directly involved in the control of nodule-specific expression but rather in influencing general transcription. This was evidenced by the recent report (Jacobsen et al., 1990) that some of the factors binding to the AT-rich motifs of nodulin genes are HMG-like proteins which may be involved in changing chromatin conformation rather than controlling organ-specific gene expression. By introducing promoter-reporter gene fusions into transgenic plants, both positive and negative regulatory elements have been located in the promoter regions of Lbc3 and N-23 genes (Stougaard et al., 1987, 1990). A 37 bp sequence between -139 and -102 bp of the Lbc3 gene promoter has been suggested to be a nodule-specific element

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since a -139 deletion fused with the CaMV-35S enhancer resulted in nodule-specific expression (Stougaard et al., 1987). It will be interesting to see how this element interacts with the corresponding fra/is-acting factors and brings about nodule-specific gene expression. Metabolic and environmental regulation of nodulin gene expression While induction of most nodulin genes is under the control of the bacterium, the expression of some nodulin genes involved in nodule metabolism is clearly modulated by nodule metabolites (Verma, 1989; Miao et al., 1991). In soybean, the expression of glutamine synthetase (GS) genes is stimulated following treatment with ammonium ions (Hirel et al., 1987). In order to test this phenomenon directly, we isolated a genomic clone of soybean cytosolic GS and made a reporter gene fusion as shown in Fig. 44 and B (Miao et al, 1991). Introducing this GS-GUS construct into a non-legume (tobacco) and a legume {Lotus corniculatus) plant showed that a soybean GS-GUS gene is strongly expressed in both transgenic tobacco and transgenic L. corniculatus, with activity being primarily localised in the root apices and Lotus nodules. Histochemical localisation of GS-GUS activity in ammonium-treated transgenic L. corniculatus roots showed that GUS activity is increased and is located uniformly in the entire root tissue (Fig. 4). However, this ammonium inducibility of soybean cytosolic GS occurred only in L. corniculatus and not in tobacco. These data suggest that some legume plants have brought the GS gene under the control of ammonium. Elevated GS activity was also observed in ammonium-fed Trifolium roots (Reynolds et al., 1990). The induction of rice GS promoter in transgenic tobacco by ammonium was also reported (Kozaki et al., 1991). The GS genes appear to be regulated in many different ways depending on different isoforms and the physiological status of the plant. In Phaseolus vulgaris, GS genes have been shown to be developmentally controlled and are not responsive to externally supplied ammonium (Cock et al., 1990). In other cases, GS genes are regulated by light (Edwards & Coruzzi, 1989) and by phytochromes (Sakamoto et al., 1990). N-35 is specifically induced in the uninfected cells of nodules in response to nitrogen fixation (Nguyen et al., 1985), suggesting that the expression of this gene is influenced by metabolites produced as a result of nitrogen fixation and assimilation. Larsen & Jochimsen (1986) have hypothesised that low, free oxygen levels induce the expression of N-35, as the activity of uricase II was increased by lowering the oxygen concentration in cultured cells. However, expression of a maize alcohol

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Fig. 4. A, Restriction map of a soybean GS genomic clone (XGS15) which corresponds to cDNA clone pGS20. DNA sequence of the 5' region and the position of Bg/II site, upstream of the initiation codon, used for making the transcription fusion with P-glucuronidase (GUS) gene. B, A portion of the plasmid pBin GS-GUS containing XGS15 promoter (HindlllfBgll) fragment (3.5 kb) and a reporter gene, GUS, is shown. C, D, Root-specific (C) and ammonia-induced (D) expression of soybean GS-GUS in transgenic Lotus corniculatus as revealed by histochemical localisation of GUS activity. For details, see Miao et al., 1991.

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dehydrogenase (ADH) promoter-GUS reporter gene fusion in transgenic Lotus demonstrated that the oxygen level in the uninfected cells is higher than in the infected cells (Hu & Verma, 1990). Therefore, the regulation of N-35 in ureide-producing nodules may not be the same as that observed in root culture or callus tissue (Larsen & Jochimsen, 1986). The expression of some nodulins may be under the control of metabolites produced by bacteria. Lb, a 'mosaic' molecule comprising plant-encoded apoprotein and primarily bacteria-synthesised haem moiety has been suggested to be induced by the availability of haem (Appleby, 1984). Studies on the regulation of Lb by haem in soybean using different haem biosynthesis-deficient mutants have shown that haem biosynthesis is required for the expression of the Lb holoprotein but not the apoprotein in soybean nodules (O'Brian etal., 1987). Haem also has been suggested to regulate soybean sucrose synthetase activity (Thummler & Verma, 1987). Thus, metabolic control of the expression of many nodulin genes seems significant in the light of the fact that nodule tissue has a unique environment, and that many metabolic pathways adjust accordingly. Perspectives The endocytosis of Rhizobium inside the legume plant cell is a highly regulated process mediated by many signals, each at a specific stage of development and finely titrated by each partner to induce a specific set of genes necessary for this interaction. The initial signal(s) produced by bacteria in response to phenolic compounds secreted by the host plant root are transduced via plant hormones responsible for initiation of the nodule meristem. That nodule ontogeny can proceed without infection has been demonstrated by the local application of the return signal (RmR-1) compound from Rhizobium. Development of this pseudonodule structure normally accompanies proliferation of infection and only through fine tuning of events in the infection process leads to the endocytotic release of bacteria inside the host cell. Temporal and spatial expression of specific plant and bacterial genes allows proper development of this unique organ and attainment of a symbiotic state. Root nodules thus provide an ideal developmental system to dissect signal transduction pathways since both micro- and macrosymbiont can be independently genetically modified by mutagenesis or antisense gene control. Elucidation of the mechanisms of communication between the plant and bacteria is essential for our understanding of the evolution of this system and our ability to modify it to establish other useful associations in nature.

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Acknowledgements This study is supported by research grants from the National Science Foundation DCB8904101; DCB8819399 and the USD A GAM8902261. We thank Ashton Delauney for his comments on the manuscript.

References Allen, T., Raja, S. & Dunn, K. (1991). Cells expressing ENOD2 show differential organization during the development of alfalfa root nodules. Molecular Plant-Microbe Interactions 4, 139-46. Appleby, C.A. (1984). Leghemoglobin and Rhizobium respiration. Annual Review of Plant Physiology 35, 443-78. Aspinall, D. & Paleg, L.G. (1981). Proline accumulation: physiological aspects. In The Physiology and Biochemistry of Drought Resistance in Plants, ed. L.G. Paleg & D. Aspinell, pp. 205^41. Sydney: Academic Press. Averyhart-Fullard, V., Datta, K. & Marcus, A. (1988). A hydroxyproline-rich protein in the soybean cell wall. Proceedings of the National Academy of Sciences (USA) 85, 1082-5. Baker, M.E. & Saier, M.H. (1990). A common ancestor for bovine lens fiber major intrinsic protein, soybean nodulin-26 protein, and E. coli glycerol facilitator. Cell 60, 185-6. Bauer, W.D. (1981). Infection of legumes by rhizobia. Annual Review of Plant Physiology 32, 407-49. Bergmann, H., Preddie, E. & Verma, D.P.S. (1983). Nodulin-35: A subunit of specific uricase (uricase II) induced and localized in the uninfected cells of soybean nodules. The EMBO Journal 2, 2333-9. Birkenhead, K., Yang, Y.-P., Noonan, B., Manian S.S. & O'Gara, F. (1988). Characterization of Rhizobium meliloti DCT genes and relationship between utilization of dicarboxylic acids and expression of nitrogen fixation genes. In Molecular Genetics of Plant-Microbe Interactions, ed. R. Palacios & D.P.S. Verma, pp. 137-8. St Paul, MO: APS Press. Branjeon, J., Hirel, B. & Forchioni, A. (1988). Immunogold localization of glutamine synthetase in soybean leaves, roots and nodules. Protoplasma 151, 88-97. Brisson N. & Verma, D.P.S. (1982). Soybean leghemoglobin gene family: Normal, pseudo, and truncated genes. Proceedings of the National Academy of Sciences (USA) 79, 4055-9. Carroll, B.J., McNeil, D.L. & Gresshoff, P.M. (1985). Isolation and properties of soybean (Glycine max (L.) Merr.) mutants that nodulate in the presence of high nitrate concentrations. Proceedings of the National Academy of Sciences (USA) 82, 4162-6.

Induction of nodulin genes and root nodule symbiosis

195

Cassab, G.I. & Varner, J.E. (1988). Cell wall proteins. Annual Review of Plant Physiology and Plant Molecular Biology 39, 321-53. Cock, J.M., Mould, R.M., Bennett, M.J. & Cullimore, J.V. (1990). Expression of glutamine synthetase genes in roots and nodules of Phaseolus vulgaris following changes in the ammonium supply and infection with various Rhizobium mutants. Plant Molecular Biology 14, 549-60. Day, D.A., Ou Yang, L.-J. & Udvardi, M.R. (1990). Nutrient exchange across the peribacteroid membrane of isolated symbiosomes. In Nitrogen Fixation: Achievements and Objectives, ed. P.M. Gresshoff, L.E. Roth, G. Stacey & W.E. Newton, pp. 219-26. New York: Chapman and Hall. Delauney, A.J., Tabaeizadeh, Z. & Verma, D.P.S. (1988). A stable bifunctional antisense transcript inhibiting gene expression in transgenic plants. Proceedings of the National Academy of Sciences (USA) 85, 4300-4. Delauney, A.J. & Verma, D.P.S. (1988). Cloned nodulin genes for symbiotic nitrogen fixation. Plant Molecular Biology Report 6,279-85. Delauney, A.J. & Verma, D.P.S. (1990a). Isolation of plant genes by heterologous complementation in Escherichia coli In Plant Molecular Biology Manual Supplement, ed. S.B. Gelvin, R.A. Schilperoort & D.P.S. Verma, pp. 1-23. Dordrecht: Kluwer Academic Publishers. Delauney, A.J. & Verma, D.P.S. (1990b). A soybean gene encoding A'-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and found to be osmoregulated. Molecular and General Genetics 221, 299-305. Denarie, J. & Roche, P. (1991). Rhizobium nodulation signals. In Molecular Signals in Plant-Microbe Communication, ed. D.P.S. Verma, pp. 295-324. Boca Raton, FL: CRC Press. Dickstein, R., Bisseling, T., Reinhold, V.N. & Ausubel, F.M. (1988). Expression of nodule-specific genes in alfalfa root nodules blocked at an early stage of development. Genes and Development 2, 677-87. Dilworth, M. & Glenn, A. (1984). How does a legume nodule work? Trends in Biochemical Sciences 9, 519-23. Djordjevic, M.A., Redmond, J.W., Batley, M. & Rolfe, B.G. (1987). Clovers secrete specific phenolic compounds which either stimulate or repress nod gene expression in Rhizobium trifolii. The EM BO Journal 6, 1173-9. Dunn, K., Dickstein, R., Feinbaum, R., Burnett, B.K., Peterman, T.K., Thoidis, G., Goodman, H.M. & Ausubel, F.M. (1988). Developmental regulation of nodule-specific genes in alfalfa root nodules. Molecular Plant-Microbe Interactions 1, 66-74. Edwards, J.W. & Coruzzi, G.M. (1989). Photorespiration and light act in concert to regulate the expression of the nuclear gene for chloroplast glutamine synthetase. The Plant Cell 1, 241-8. Ellis, J.G., Llewellyn, D.J., Dennis, E.S. & Peacock, W.J. (1987).

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D . P . S . VERMA AND G . - H . MIAO

Maize ADH-1 promoter sequences control anaerobic regulation addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco. The EMBO Journal 6, 11-16. Firmin, J.L., Wilson, K.E., Rossen, L. & Johnston, A.W.B. (1986). Flavonoid activation of nodulation genes in Rhizobium reversed by other compounds present in plants. Nature 324, 90-2. Forde, B.G., Day, H.M., Turton, J.F., Shen, W.-J. Cullimore, J.V. & Oliver, J.E. (1989). Two glutamine synthetase genes from Phaseolus vulgaris L. display contrasting developmental and spatial patterns of expression in transgenic Lotus corniculatus plants. The Plant Cell 1, 391^01. Forde, B.G., Freeman, J., Oliver, J.E. & Pineda, M. (1990). Nuclear factors interact with conserved A/T-rich elements upstream of a nodule enhanced glutamine synthetase from french bean. The Plant Cell 2, 925-39. Fortin, M.G., Morrison, N.A. & Verma, D.P.S. (1987). Nodulin-26, a peribacteroid membrane nodulin, is expressed independently of the development of the peribacteriod compartment. Nucleic Acids Research 15, 813-24. Fortin, M.G., Zelechowska, M. & Verma, D.P.S. (1985). Specific targeting of the membrane nodulins to the bacteroid enclosing compartment in soybean nodules. The EMBO Journal 4, 3041-6. Franssen, H.J., Nap, J.P., Gloudemans, T., Stiekema, W., van Dam, H., Govers, F., Louwerse, J., van Kammen, A. & Bisseling, T. (1987). Characterization of cDNA for nodulin-75 of soybean: A gene product involved in early stages of root nodule development. Proceedings of the National Academy of Sciences (USA) 84, 4495-9. Fuller, F., Kunstrer, P.W., Nguyen, T. & Verma, D.P.S. (1983). Soybean nodulin genes: Analysis of cDNA clones reveals several major tissue-specific sequences in nitrogen-fixing root nodules. Proceedings of the National Academy of Sciences (USA) 80, 2594-8. Gebhardt, C , Olivier, J.E., Forde, B.G., Saarelainen, R. & Miflin, B.J. (1986). Primary structure and differential expression of glutamine synthetase genes in nodules, roots and leaves of Phaseolus vulgaris. The EMBO Journal 5, 1429-35. Gloudemans, T. & Bisseling, T. (1989). Plant gene expression in early stages of Rhizobium-lcgume symbiosis. Plant Science 65, 1-14. Gorin, M.B., Yancey, S.B., Cline, J., Revel, J.-P. & Horwitz, J. (1984). The major intrinsic protein (MIP) of the bovine lens fiber membrane: Characterization and structure based on cDNA cloning. Cell 39, 49-59. Govers, F., Nap, J.-P., van Kammen, A. & Bisseling, T. (1987). Nodulins in the developing root nodule: An overview. Plant Physiology and Biochemistry 25, 309-22. Guerinot, M.L. & Chelm, B. (1985). Bacterial cAMP and hemes in the

Induction of nodulin genes and root nodule symbiosis

197

Rhizobium-\eg\ime symbiosis. In Nitrogen Fixation Research Progress, ed. H.J. Evans, P.J. Bottomley, W.E. Newton, p. 220. Dordrecht: Martinus Nijhoff. Guerrero, F.D., Jones, J.T. & Mullet, J.E. (1990). Turgor-responsive gene transcription and RNA levels increase rapidly when pea shoots are wilted. Sequence and expression of three inducible genes. Plant Molecular Biology 15, 11-26. Hirel, B., Bouet, C , King, B., Layzell, D., Jacobs, F. & Verma, D.P.S. (1987). Glutamine synthetase genes are regulated by ammonia provided externally or by symbiotic nitrogen fixation. The EM BO Journal 5, 1167-71. Hirsch, A.M., Bhuvaneswari, T.V., Torrey, J.G. & Bisseling, T. (1989). Early nodulin genes are induced in alfalfa root outgrowths elicited by auxin transport inhibitors. Proceedings of the National Academy of Sciences (USA) 86, 1244-8. Horvath B., Bachem, C.W.B., Schell, J. & Kondorosi, A. (1987). Hostspecific regulation of nodulation genes in Rhizobium is mediated by a plant-signal interacting with the nodD gene product. The EM BO Journal 6, 841-8. Hu, C.A. & Verma, D.P.S. (1990). Direct demonstration of the level of intercellular oxygen between infected and uninfected cells of nodules by using an ADH-GUS gene. Plant Physiology 93 (supplement), 83. Jacobsen, K., Laursen, N.B., Jensen, E.O., Marker, A., Poulson, C. & Marcker, K.A. (1990). HMG-like proteins from leaf and nodule nuclei interact with different AT motifs in soybean nodule promoters. The Plant Cell 2, 85-94. Jensen, E.O., Marcker, K.A., Schell, J. & de Bruijn, F. (1988). Interaction of a nodule-specific trans-acting factor with the distinct DNA elements in the soybean LbC3 5' upstream region. The EM BO Journal!, 1265-71. Johnson, K.D., Hofte, H. & Chrispeels, M.J. (1990). An intrinsic tonoplast protein of protein storage vacuoles in seeds is structurally related to a bacterial solute transporter (GlpF). The Plant Cell 2, 525-32. Kaneko, Y. & Newcomb, E.H. (1987). Cytochemical localization of uricase and catalase in developing root nodules of soybean. Protoplasma 140, 1-12. Katinakis, P., Lankhorst, R.M.K., Louwerse, J., van Kammen, A. & van den Bos, R.C. (1988). Bacteroid-encoded proteins are secreted into the peribacteroid space by Rhizobium leguminosarum. Plant Molecular Biology 11, 183-90. Katinakis, P. & Verma, D.P.S. (1985). Nodulin-24 gene of soybean codes for a peptide of the peribacteroid membrane and was generated by tandem duplication of an insertion element. Proceedings of National Academy of Sciences (USA) 82, 4157-61.

198

D . P . S . VERMA AND G . - H . MIAO

Klausner, R. (1989). Sorting and traffic in the central vacuolar system. Cell 57, 703-6. Knight, C D . , Rossen, L., Wells, B. & Downie, J.A. (1986). Nodulation inhibition by Rhizobium multicopy nodABC genes and analysis of early stages of plant infection. Journal of Bacteriology 166, 552-8. Kohl, D.H., Schubert, K.R., Carter, M.B., Hagedorn, C.H. & Shearer, G. (1988). Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis. Proceedings of the National Academy of Sciences (USA) 85, 2036-40. Kondorosi, A. (1991). Regulation of nodulation genes in Rhizobium. In Molecular Signals in Plant-Microbe Communication, ed. D.P.S. Verma pp. 325-40. Boca Raton, FL: CRC Press. Konieczny, A., Szczyglowski, K., Boron, L., Przybylska, M. & Legocki, A.B. (1988). Expression of lupin nodulin genes during root nodule development. Plant Science 55, 145-9. Kozaki, A., Sakamoto, A., Tanaka, K. & Takeba, G. (1991). The promoter of the gene for glutamine synthetase from rice shows organspecific and substrate-induced expression in transgenic tobacco plants. Plant and Cell Physiology 32, 353-8. Lara, M., Cullimore, J.V., Lea, P.J., Miflin, B.J., Johnston, A.W.B. & Lamb, J.W. (1983). Appearance of a novel form of plant glutamine synthetase during nodule development in Phaseolus vulgaris L. Planta 157, 254-8. Larsen, K. & Jochimsen, B.U. (1986). Expression of nodule-specific uncase of soybean callus tissue is regulated by oxygen. The EM BO Journal 5, 15-19. Lee, J.S., Brown, G.G. & Verma, D.P.S. (1983). Chromosomal arrangement of leghemoglobin genes in soybean. Nucleic Acids Research 11, 5541-53. Lee, J.S. & Verma, D.P.S. (1984). Structure and chromosomal arrangement of leghemoglobin genes in kidney bean suggest divergence in soybean leghemoglobin gene loci following tetrapoloidization. The EM BO Journal 3, 2745-52. Legocki, R.P. & Verma, D.P.S. (1979). A nodule-specific plant protein (nodulin-35) from soybean. Science 205, 190-2. Legocki, R.P. & Verma, D.P.S. (1980). Identification of 'nodule-specific' host proteins (nodulins) involved in the development of Rhizobium-legume symbiosis. Cell 20, 153-63. Leong, S.A., Ditta, G.S. & Helinski, D.R. (1982). Heme biosynthesis in Rhizobium. Journal of Biological Chemistry 257, 8724-30. Lerouge, P., Roche, P., Faucher, C , Maillet, F., Truchet, G., Prome, J.C. & Denarie, J. (1990). Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature 344, 781^4. Long, S.R. (1989). Rhizobium-legume nodulation: Life together in the underground. Cell 56, 203-14. Manen, J.-F., Simon, P., Van Slooten, J.-C, Osteras, M., Frutiger, S.

Induction ofnodulin genes and root nodule symbiosis

199

& Hughes, G.J. (1991). A nodulin specifically expressed in senescent nodules of winged bean is a protease inhibitor. The Plant Cell 3, 259-70. Mauro, V.P., Nguyen, T., Katinakis, P. & Verma, D.P.S. (1985). Primary structure of the soybean nodulin-23 gene and potential regulatory elements in the 5'-flanking regions of nodulin and leghemoglobin genes. Nucleic Acids Research 13, 239-49. Mauro, V.P. & Verma, D.P.S. (1988). Transcriptional activation in nuclei from uninfected soybean of a set of genes involved in symbiosis with Rhizobium. Molecular Plant-Microbe Interactions 1, 46-51. Mellor, R. (1989). Bacteroids in the Rhizobium-\egume symbiosis inhabit a plant internal lytic compartment: Implications for other microbial endosymbioses. Journal of Experimental Botany 40, 831-9. Mellor, R.B., Christense, T.M.I.E. & Werner, D. (1986). Choline kinase II is present only in nodules that synthesize stable peribacteroid membranes. Proceedings of the National Academy of Sciences (USA) 83, 659-63. Metz, B.A., Welters, P., Hoffman, H.J., Jensen, E.O., Schell, J. & de Bruijn, F.J. (1988). Primary structure and promoter analysis of leghemoglobin gene of the stem nodulated tropical legume Sesbania rostrata: conserved coding sequences, cis elements and trans-acting factors. Molecular and General Genetics 214, 181-91. Miao, G.-H., Hirel, B., Marsolier, M.C., Ridge, R.W. & Verma, D.P.S. (1991). Ammonia-regulated expression of a soybean gene encoding cytosolic glutamine synthetase in transgenic Lotus corniculatus. The Plant Cell 3, 11-22. Miao, G.-H., Joshi, C.P. & Verma, D.P.S. (1990). Nodulin-26: topology and function in root nodules. In Nitrogen Fixation: Achievements and Objectives, ed. P.M. Gresshoff, L.E. Roth, G. Stacey & W.E. Newton, p. 755. New York: Chapman and Hall. Morrison, N. & Verma, D.P.S. (1987). A block in the endocytotic release of Rhizobium allows cellular differentiation in nodule but affects the expression of some peribacteroid membrane nodulins. Plant Molecular Biology 7, 51-61. Mulligan, J.T. & Long, S.R. (1985). Induction of Rhizobium meliloti nodC expression by plant exudate requires nodD. Proceedings of the National Academy of Sciences (USA) 82, 6609-13. Nadler, K.D. (1981). A mutant strain of Rhizobium leguminosarium with an abnormality in heme biosynthesis. In Current Perspectives in Nitrogen Fixation, ed. A.H. Gibson & W.E. Newton, p. 414. Canberra: Australian Academy of Science. Nap, J.P. & Bisseling, T. (1990). Developmental biology of a plantprokaryote symbiosis: the legume root nodule. Science 250, 948-54. Newcomb, E.H., Datta, D.B. & Triplett, E.W. (1990). Cellular localization of xanthine dehydrogenase (XDH) in root nodules of cowpea (Vigna unguiculata L. Walp). In Nitrogen Fixation: Achieve-

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merits and Objectives, ed. P.M. Gresshoff, L.E. Roth, G.S. Stacey & W.E. Newton, p. 757. New York: Chapman and Hall. Newcomb, E.H. & Tandon, S.R. (1981). Uninfected cells of soybean root nodules: Ultrastructure suggests key role in ureide production. Science 212, 1394-6. Nguyen, T., Zelechowska, M., Foster, V., Bergmann, H. & Verma, D.P.S. (1985). Primary structure of the soybean nodulin-35 gene encoding uricase II localized in the peroxisomes of uninfected cells of nodules. Proceedings of the National Academy of Sciences (USA) 82, 5040-4. Nirunsuksiri, W. & Sengupta-Gopalan, C. (1990). Characterization of a novel nodulin gene in soybean that shares sequence similarity to the gene for nodulin-24. Plant Molecular Biology 15, 835^9. O'Brian, M.R., Kirshbom, P.M. & Majer, R.J. (1987). Bacterial heme synthesis is required for expression of the leghemoglobin but not the apoprotein in soybean nodules. Proceedings of the National Academy of Sciences (USA) 84, 8390-3. Peters, N.K., Frost, J.W. & Long, S.R. (1986). A plant flavone, luteolin, induces expression of Rhizobium meliloti nodulation genes. Science 233, 977-80. Peters, N.K. & Verma, D.P.S. (1990). Phenolic compounds as regulators of gene expression in plant-microbe interactions. Molecular Plant-Microbe Interactions 3, 4-8. Rao, Y., Jan, L.Y. & Jan, Y.N. (1990). Similarity of the product of the Drosophila neurogenic gene big brain to transmembrane channel proteins Nature 345, 163-7. Reibach, P.H. & Streeter, J.G. (1983). Metabolism of I4C-labeled photosynthate and distribution of enzymes of glucose metabolism in soybean nodules. Plant Physiology 72, 634-40. Reynolds, P.H.S., Boland, M.J., McNaughton, G.S., More, R.D. & Jones, W.T. (1990). Induction of ammonium assimilation: leguminous roots compared with nodules using split root system. Physiologia Plantarum 79, 359-67. Robertson, J.G., Lyttleton, P., Bullivant, S. & Grayston, G.F. (1978). Membrane in lupin root nodules I. The role of Golgi bodies in the biogenesis of infection threads and peribacteroid membranes. Journal of Cellular Science 30, 129^*9. Rolfe, B.G. & Gresshoff, P.M. (1988). Genetic analysis of legume nodule initiation. Annual Review of Plant Physiology and Plant Molecular Biology 39, 297-317. Rolfe, B.G. & Shine, J. (1984). Rhizobium-Leguminosae symbiosis: The bacterial point of view. In Genes Involved in Microbe-Plant Interactions, ed. D.P.S. Verma & T. Hohn, pp. 95-128. New York: Springer-Verlag. Sadowsky, M.J., Olson, E.R., Foster, V.E., Kosslak, R.M. & Verma, D.P.S. (1988). Two host-inducible genes of Rhizobium fredii and

Induction of nodulin genes and root nodule symbiosis

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characterization of the inducing compound. Journal of Bacteriology 170, 171-8. Sakamoto, A., Takeba, G., Shibata, D. & Tanaka, K. (1990). Phytochrome-mediated activation of the gene for cytosolic glutamine synthetase (GSO during inhibition of photosensitive lettuce seeds. Plant Molecular Biology 15, 317-23. Sangwan, I. & O'Brian, M.R. (1991). Evidence for an inter-organismic heme biosynthetic pathway in symbiotic soybean root nodules. Science 251, 1220-2. Scheres, B., Van de Wiel, C , Zalensky, A., Horvath, B., Spaink, H., van Eck, H., Zwartkruis, F., Wolters, A., Gloudemans, T., van Kammen, A. & Bisseling, T. (1990a). The ENOD12 gene product is involved in the infection process during the pea-Rhizobium interaction. Cell 60, 281-94. Scheres, B., van Engelen, F., van der Knaap, E., van de Wiel, C , van Kammen, A. & Bisseling, T. (1990b). Sequential induction of nodulin gene expression in the developing pea nodule. The Plant Cell 2,687-700. Schubert, K.R. (1986). Products of biological nitrogen fixation in higher plants: synthesis, transport, and metabolism. Annual Review of Plant Physiology 37, 539-74. Sengupta-Gopalan, C. & Pitas, J.W. (1986). Expression of nodule-specific glutamine synthetase genes during nodule development in soybeans. Plant Molecular Biology 7, 189-99. Spaink, H.P., Wijffelman, C.A., Pees, E., Okker, R.J.H. & Lugtenburg, B.J.J. (1987). Rhizobium nodulation gene nodD as a determinant of host specificity. Nature 328, 337-9. Stegink, S.J., Vaughan, K.C. & Verma, D.P.S. (1987). Antigenic similarity in urate oxidase of major ureide-producing legumes and its correlation with the type of peroxisomes in uninfected cells of nodules. Plant Cell Physiology 28, 387-96. Stougaard, J., Sandal, N.N., Gron, A., Kuhle, A. & Marcker, K.A. (1987). 5' Analysis of the soybean leghaemoglobin Lbc3 gene: regulatory elements required for promoter activity and organ specificity. The EMBO Journal 6, 3565-9. Streeter, J. (1987). Carbohydrate, organic acid, and amino acid composition of bacteroids and cytosol from soybean nodules. Plant Physiology 85, 768-73. Strittmatter, G., Chia, T.-F., Trinh, T.H., Katagiri, F., Kuhlemeier, C. & Chua, N.-H., (1989). Characterization of nodule-specific cDNA clones from Sesbania rostrata and expression of the corresponding genes during the initial stages of stem nodules and root nodules formation. Molecular Plant-Microbe Interactions 2, 122-7. Suganuma, N.M., Kitou, M. & Yamamoto, Y. (1987). Carbon metabolism in relation to cellular organization of soybean root nodules and respiration of mitochrondria aided by leghemoglobin. Plant Cell Physiology 28, 113-22.

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D . P . S . VERMA AND G . - H . MIAO

Suzuki, H. & Verma, D.P.S. (1989). Nodule-specific kinases phosphorylating nuclear factors in isolated nuclei. The Plant Cell 1, 373-9. Suzuki, H. & Verma, D.P.S. (1991). Soybean nodule-specific uricase (nodulin-35) is expressed and assembled into functional tetrameric holoenzyme in Escherichia coli. Plant Physiology 95, 384-9. Sweet, G., Ganor, C , Voeglele, R., Wittekindt, N.A., Beuerle, J., Truniger, V., Lin, E.C.C. & Winfried, B. (1990). Glycerol facilitator of Escherichia coli'. cloning of glpF and identification of the glpF product. Journal of Bacteriology 172, 424-30. Thummler, F. & Verma, D.P.S. (1987). Nodulin 100 of soybean is the subunit of sucrose synthase regulated by the availability of free heme in nodules. Journal of Biological Chemistry 262, 14730-6. Tingey, S.V., Walker, E.L. & Coruzzi, G.M. (1987). Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules. The EMBO Journal 6, 1-9. Trese, A.T. & Pueppke, S.G. (1991). Cloning of cowpea (Vigna unguicutata L.) genes that are regulated during initiation of nodulation. Molecular Plant-Microbe Interactions 4, 46-51. Triplett, E.W. (1985). Intracellular nodule localization and nodule specificity of xanthine dehydrogenase in soybean. Plant Physiology 77, 1004-9. Truchet, G., Roche, P., Lerouge, P., Vasse, J., Camut, S., de Billy, F., Prome, J.-C, & Denarie, J. (1991). Sulfated lipo-oligosaccharide signals of Rhizobium meliloti elicit root nodule organogenesis in alfalfa. Nature 351, 670-3. Udvardi, M.K., Price, G.D., Gresshoff, P.M. & Day, D.A. (1988). A dicarboxylate transporter on the peribacteroid membrane of soybean nodules. FEBS Letters 231, 36-40. Van de Weil, C , Scheres, B., Franssen, H., Van Lierop, M.-J., Van Lammeren, A., Van Kammen, A. & Bisseling, T. (1990). The early nodulin transcript ENOD2 is located in the nodule parenchyma (inner cortex) of pea and soybean root nodules. The EMBO Journal 9, 1-7. Verma, D.P.S. (1989). Plant genes involved in carbon and nitrogen assimilation in root nodules. In Plant Nitrogen Metabolism, ed. J.E. Poulton, T. Romeo & E.E. Conn, pp. 43-63. New York: Plenum Press. Verma, D.P.S. & Bal, A.K. (1976). Intracellular site of synthesis and localization of leghemoglobin in root nodule. Proceedings of the National Academy of Sciences (USA) 73, 3843-7. Verma, D.P.S. & Delauney, A.J. (1988). Root nodule symbiosis: nodulins and nodulin genes. In Temporal and Spatial Regulation of Plant Genes, ed. D.P.S. Verma & R. Goldberg, pp. 169-99. New York: Springer-Verlag. Verma, D.P.S., Delauney, A.J., Guida, M., Hirel, B., Schafer, R. & Koh, S. (1988). Control of expression of nodulin genes. In Molecular

Induction ofnodulin genes and root nodule symbiosis

203

Genetics of Plant-Microbe Interactions, ed. R. Palacios & D.P.S. Verma, pp. 315-20. Minnesota: APS Press. Verma, D.P.S. & Fortin, M.G. (1989). Nodule development and formation of the endosymbiotic compartment. In Cellular and Somatic Cell Genetics of Plants, Vol. 6, The Molecular Biology of Nuclear Genes, ed. J. Schell & l.K. Vasil, pp. 329-53. San Diego, CA: Academic Press. Verma, D.P.S., Fortin, M.G., Stanley, J., Mauro, V., Purohit, S. & Morrison, N. (1986). Nodulins and nodulin genes of Glycine max. Plant Molecular Biology 7, 51-61. Verma, D.P.S., Kazazian, V., Zogbi, V. & Bal, A.K. (1978). Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules. Journal of Cell Biology 78, 919-36. Verma, D.P.S. & Long, S. (1983). The molecular biology of Rhizobium-\egume symbiosis. International Review of Cytology, Supplement 14, pp. 211-45. Verma, D.P.S., Miao, G.-H., Cheon, C.-I. & Suzuki, H. (1990a). Genesis of root nodules and function of nodulins. In Advances in Molecular Genetics of Plant-Microbe Interactions Vol. 1, ed. H. Hennecke & D.P.S. Verma, pp. 291-9. Dordrecht: Kluwer Academic Press. Verma, D.P.S., Miao, G.-H., Joshi, C.P., Cheon, C.-I. & Delauney, A. (19906). Internalization of Rhizobium by plant cells: targeting and role of peribacteroid membrane nodulins. In Plant Molecular Biology 1990, ed. R.G. Herrmann & B.A. Larkins, pp. 121-30. New York: Plenum Press. Verma, D.P.S. & Nadler, K. (1984). Legume-Rhizobium symbiosis: Host's point of view. In Genes Involved in Plant-Microbe Interactions, ed. D.P.S. Verma & T. Hohn, pp. 58-93. New York: Springer-Verlag. Verma, D.P.S., Nash, D.T. & Schulman, H.M. (1974). Isolation and in vitro translation of soybean leghaemoblobin mRNA. Nature 251, 74-7. Verma, D.P.S. & Stanley, J. (1989). The Legume-Rhizobium equiation: A co-evolution of two genomes. In Advances in Legume Biology, ed. C.H. Stirton & J.L. Zarucchi. Monographs in Systematic Botany of the Missouri Botanical Garden 29, 545-57. Watson, R.J. Chan, Y.-K. Wheatcroft, R., Yang, A.-F. & Han, S. (1988). Rhizobium meliloti genes required for C-4-dicarboxylate transport and symbiotic nitrogen fixation are located on a megaplasmid. Journal of Bacteriology 170, 927-34. Weaver, C D . , Crombie, B., Stacey, G. & Roberts, D.M. (1991). Calcium-dependent phosphorylation of symbiosome membrane protein from nitrogen-fixing soybean nodules. Plant Physiology 95, 222-7. Werner, D., Mellor, R.B., Hahn, M.G. & Grisebach, H. (1985). Soybean root response to symbiotic infection: Glyceollin I accumula-

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tion in an ineffective type of soybean nodules with an early loss of the peribacteroid membrane. Zeitschrift fiir Naturforschung 40, 179-81. Werner, D., Morschel, E., Garbers, C , Bassarab, S. & Mellor, R.B. (1988). Particle density and protein composition of the peribacteroid membrane from soybean root nodule is affected by mutations in the microsymbiont Bradyrhizobium japonicum. Planta 174, 263-70. Werner, D., Morschel, E., Kort, R., Mellor, R.B. & Bassarab, S. (1984). Lysis of bacteroids in the vicinity of the host cell nucleus in an ineffective (Fix") root nodule of soybean (Glycine max). Planta 162, 8-16. Witty, J.F., Minchin, F.R., Skot, L. & Sheeky, J.E. (1986). Nitrogen fixation and oxygen in legume root nodules. In Oxford Surveys of Plant Molecular and Cell Biology, Vol. 3, ed. B.J. Miflin, pp. 276314. Oxford: Oxford University Press. Yamamoto, Y.T., Cheng, C.-L. & Conkling, K.A. (1990). Root-specific genes from tobacco and Arabidopsis homologous to an evolutionarily conserved gene family of membrane channel proteins. Nucleic Acids Research 18, 7449. Yang, N.-S. & Russell, D. (1990). Maize sucrose synthase-1 promoter directs phloem cell-specific expression of GUS-gene in transgenic tobacco plants. Proceedings of the National Academy of Sciences (USA) 87, 4144-8.

J. RYALS, E. WARD, P. AHL-GOY and J.P. METRAUX

Systemic acquired resistance: an inducible defence mechanism in plants

Introduction Acquired resistance in plants has long been recognised to play an important role in the preservation of plants against disease (Chester, 1933). While much of this early work focused on viral cross-protection, the plants' ability to induce a defence against disease became a topic of research by the early 1960s (Ross, I961a,b). In these studies tobacco mosaic virus (TMV) was inoculated onto the leaf of a tobacco variety that produced necrotic lesions. Seven days after infection, at a time when lesions had formed on the leaf, both the inoculated leaf and uninfected leaves on the same plant had become resistant to further infection by TMV. The resistance was directed not only against TMV but also other unrelated viruses, as well as certain fungal and bacterial pathogens. Further, the resistance could be induced by other pathogens including viruses, bacteria and fungi, the only common requirement being the development of some necrosis from the infection. Ross referred to the resistance in infected leaves as localised acquired resistance (LAR: Ross, 1961a) and the resistance that developed in the uninfected leaves as systemic acquired resistance (SAR: Ross, 19616). Although resistance to virus was the main topic of these studies, later emphasis has been on the non-specificity and broad spectrum of SAR against various fungal and bacterial diseases (Hecht & Bateman, 1964; Kuc, 1982; Dean & Kuc, 1985). A number of exogenously applied chemicals, including polyacrylic acid, acetylsalicylic acid, salicylic acid and isonicotinic acid (NA) derivatives, have also been shown to induce resistance. Usually the resistance is localised to the treated parts of the plants (Gianinazzi & Kassanis, 1974; White, 1979), but in the INA compounds the resistance can be systemic (Metraux et al., 1991). These observations led to the suggestion that SAR might provide a new strategy for crop protection, either by discovering Society for Experimental Biology Seminar Series49: Inducible Plant Proteins, e.d. J. L. Wray. © Cambridge University Press, 1992, pp. 205-229.

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compounds that stimulate the plants' natural disease resistance mechanisms, or by developing transgenic plants that constitutively express components of the disease resistance mechanism in order to make them more resistant to pathogen attack. One of our research goals over the past few years has been to catalogue the genes involved in maintaining the resistant state (as distinguished from genes involved in inducing the resistant state). These genes can be used in transgenic experiments to elucidate their role in disease resistance and may, in the end, allow for the production of healthier plants. The genes can also be used as tools to elucidate the steps involved in initiating the resistance. Presumably (and possibly naively), Wans-acting factors involved in the induction of a particular maintenance gene will be capable of controlling all of the genes involved in maintenance. A second goal is to identify the endogenous signal compound involved in initiating SAR. This compound could serve as a useful lead toward developing chemical control strategies and might also provide a valuable tool in the identification of cellular receptors involved in signal transduction. A third research goal is to identify the genes involved in initiating the resistance. These genes could also be useful in the development of disease-resistant plants and will lead to a better understanding of the signal transduction pathway that induces this important defence system. This chapter describes the status of experiments conducted in our laboratories directed at understanding the molecular nature of systemic acquired resistance. It is not intended as a comprehensive review of the literature. We have taken the liberty of providing our current working hypotheses in certain areas and in some cases these models may not be supported by a wealth of data.

Genes involved in maintaining the resistant state

Identification and analysis of SAR genes We have identified and characterised many cDNAs that are expressed during the maintenance of systemic acquired resistance. These genes have been isolated using several different strategies including differential cDNA cloning (e.g. SAR8.2: D. Alexander et al., unpublished data), heterologous probing with related sequences (e.g. PR-P and PR-Q: Payne et al., 1990a; PR-Q': Payne et al., 1990b), and probing with degenerate oligonucleotide probes based on the amino acid sequence of purified proteins (e.g. PR-2/N/O: Ward et al., 1991; class HI chitinase: Metraux et al., 1989; Lawton et al., 1992). Throughout this process we have isolated and characterised 29 distinct cDNA species which comprise

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seven families of genes (Fig. 1). Members of six of these families are coordinately induced during SAR. The induction of these genes will be discussed in detail in a later section. The six SAR-related gene families include pathogenesis-related protein 1 (PR-1), (3-1,3-glucanases, chitinases, protease inhibitors, pathogenesis-related protein 4 (PR-4) and SAR8.2. The PR-1 family comprises at least four members that can be grouped into two classes. Class I includes the acidic, extracellular proteins PR-la, PR-lb and PR-lc. Class II includes the basic isoform of these proteins PR-1

CHITINASES

65% PR-1a PR-1b PR-lc

basic form

acidic extracel.

basic ?

Type 65% class 1 basic vac. 3 iso.

•

• function/activity unknown

basic vacuolar

Class II

•

PR-Q'

?

acidic extra.

PR-Smin acidic extra.

•

• B-l ,3-glucans

OTHERS- SAR8.2, PR-4.

6 5% PR-S m ,|

2 iso.

• hydrolyse

•

Class III 56%

PR-2 PR-N PR-0 acidic extra.

•

class IV basic ?

PROTEASE INHIBITORS 7

55% 3 iso.

class III acidic ext. PR-?

hydrolyse chitin fungicidal/n vitro (plus glucanase)

GLUCANASES Class 1

class II acidic ext. PR-P PR-Q •

•

Type II 65%

convergent

Not

Osmolim Osmotin2 basic vacuolar

• Q-amylase/protease

• inhibitors

Related •Peroxidase.

Fig. 1. SAR cDNA families in tobacco. The relationships and functions of the different gene families from tobacco are described. The asterisk below each class denotes that the cDNA has been expressed in transgenic tobacco. The class I chitinase and class I glucanase are not SAR genes as defined in the text.

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etal.

which has only one species identified so far. The function of the PR-1 family is currently unknown; however, a wealth of data concerning the characterisation and localisation of the protein, as well as studies on the PR-1 gene family and its regulation of expression, have been dealt with in recent reviews (Bol et al., 1990; Carr & Klessig, 1990). A second major family of SAR genes is the chitinases. One function of chitinase is to cleave chitin, a high molecular weight poly[l,4-(N-acetyl-(3D-glucosamine)] - a major cell wall component of fungal pathogens, with the exception of oomycetes. It has been demonstrated that chitinases have an anti-fungal activity in vitro against certain chitin-containing fungi, which is synergistically amplified when the chitinases are combined with P-l,3-glucanases (Mauch et al., 1988). This result suggests that the chitinases could have a direct role in SAR as an anti-fungal enzyme. Two structurally unrelated types of chitinases have been identified in tobacco (Shinshi et al., 1987; Payne et al., 1990a; Lawton et al., 1992). The Type I group consists of two classes, one basic and localised in the vacuole (i.e. class I: Shinshi et al., 1987, 1990; Keefe et al., 1990), the other is acidic and localised in the extracellular spaces of the plant (i.e. class II: Payne et al., 1990a). The class I and class II chitinases share 65% identity at the protein level. The class I chitinase appears to have three structural domains, including a lectin-type domain, a hinge or spacer, and a chitinolytic domain. The class II enzyme has only the chitinolytic domain (Shinshi etal., 1990; Payne etal., 1990a). Interestingly, the class I enzyme has an apparent specific activity at least five times higher than the class II enzyme on a substrate of powdered chitin (Legrand et al., 1987). The Type II chitinases of tobacco also consist of two structurally distinct classes that are related by about 65% amino acid identity (Lawton et al., 1992). This type of chitinase is closely related to the acidic, extracellular chitinase from cucumbers (Metraux etal., 1988, 1989; Boiler & Metraux, 1988). The class III chitinase is acidic and localised in the extracellular space. The protein encoded by the class IV cDNA has not yet been identified in tobacco. However, the putative class IV protein would be somewhat basic in charge. The (5-1,3-glucanases comprise a third family of tobacco SAR genes. At least one enzymatic activity of the (5-1,3-glucanases is to hydrolyse |3-1,3glucan polymers, measured as the amount of reducing sugars released from laminarin, a high molecular weight |3-l,3-glucan. As stated above, the |3-l,3-glucanases have a demonstrated anti-fungal activity in combination with chitinases (Mauch et al., 1988), which may be attributable to the digestion of the |3-l,3-glucan polymer, a cell wall constituent in certain classes of phytopathogenic fungi (Wessels & Sietsma, 1981). At least nine distinct |3-l,3-glucanase cDNA clones, which can be grouped into three

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structural classes (Meins et al., 1991), have been described to date. The classes are interrelated by about 55% identity at the amino acid level. There are at least three species of the class I glucanase, which are basic in charge and localised in the central vacuole (Shinshi et al., 1988). The class II enzymes comprise at least five species which can be further grouped into two subclasses interrelated by about 85% amino acid identity (Ward et al., 1991). One subgroup is comprised of the proteins PR-2, PR-N and PR-O: three acidic, extracellular enzymes. The other subgroup comprises three or four forms, neutral to basic in charge, that have been characterised only at the level of the cDNA and not at the protein level. Only one class III glucanase has been identified thus far in tobacco and this enzyme, PR-Q', is an acidic, extracellular protein (Payne et al., 1990b). The class I glucanases are all relatively high specific activity laminarinases, while only one of the class II enzymes, PR-O, has a high specific activity on laminarin (Kauffmann et al., 1987; T. Gaffney, personal communication). The reason for these differences in activity is not clear. The class III enzyme has a high apparent specific activity on laminarin but it may produce larger oligosaccharides in a limit digest than the other two classes (T. Gaffney, personal communication). The class III enzyme is also a structural heterologue of the soybean elicitor-releasing (51,3-glucanase (Payne etal., 19906; Takeuchi etai, 1990). Pathogenesis-related protein 4 (PR-4) comprises another class of SAR proteins. The PR-4 protein has been recently purified and characterised by partial amino acid sequencing and cDNA clones encoding PR-4 have been isolated (Friedrich et al., 1991). Two species of PR-4, related by about 95% identity, have been found. The proteins are extracellularly localised and slightly acidic with a calculated molecular mass of about 13 500 Da. The function of the protein is unknown, but its primary structure is homologous to both the carboxy-terminal domain of two woundinduced tuber proteins from potato, Win-1 and Win-2 (Stanford et al, 1989), and the C-terminus of hevein, a major protein constituent of rubber latex (Broekaert et al., 1990). Interestingly, Win-1, Win-2 and hevein all contain an amino-terminal lectin domain, as does the class I chitinase from tobacco (Fig. 2). The relationship between PR-4 and Win-l/Win-2/hevein, therefore, is similar to the relationship between the class I and class II chitinases (Shinshi et al., 1990; Friedrich et al., 1991). Based on the structural analysis of tobacco chitinase genes, it has been postulated that DNA encoding the lectin domain from class I chitinase may be capable of transposition to form more complex multi-domain proteins (Shinshi et al., 1990). The occurrence of PR-4 in a form with and without the lectin domain provides further support for this idea. Pathogenesis-related protein 5 (also known as PR-S) represents a fifth

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J. RYALS et al. Leclin domain

Carboxy-tcnniniil domain

Y//////////////////////////////A Hinge

Win2

IHH

V////////////////////////////////A

Hevein

PR-4

Cliilinolylic domain

Class I Chitinase I

Class II Chitinase

L

I

Fig. 2. Comparison of PR-4 to different lectins and chitinases. The dark stippled box represents the encoded protein with structural homology to lectins, such as wheat germ agglutinin. The striped box represents residues with homology to the C-terminal domain from Win-1, whose function is currently unknown. The light stippled box represents residues with homology to the chitinolytic domain of chitinase. Details of the structural alignments can be found elsewhere (Payne et al., 1990a; Friedrich et al., 1991). The sequences represent the following proteins: Winl and Win2, two wound-inducible proteins expressed in potato tubers (Stanford et al., 1989); hevein, a major protein constituent of rubber latex (Broekart et al., 1990); pathogenesis-related protein 4 (Friedrich et al., 1991); class I chitinase (Shinshi et al., 1987); class II chitinase (Payne et al., 1990a).

family of SAR proteins in tobacco. PR-5 is also referred to as the thaumatin-like protein because of a high degree of structural homology that exists between this protein and the sweet-tasting protein, thaumatin, from Thaumatococcus daniellii (Cornelissen et al., 1986). As with several other SAR gene families, there are two structural classes of PR-5 proteins: one class is acidic and extracellularly localised and the other is basic and localised in the vacuole. The two classes are about 65% identical. cDNAs encoding the two forms of the acidic class (PR-5major, also known as PR-S major; and PR-5minor, also known as PR-S minor) have been isolated from tobacco (Cornelissen et al., 1986; Payne et al. 1988a). These cDNAs are about 95% identical to each other and encode the

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major and minor isoforms of PR-5 (Pierpoint et al., 1987). There are two forms of the basic class of protein. These are also known as osmotins, and were characterised as proteins that accumulate as inclusion bodies in the vacuole in response to high salt stress (Singh et al., 1987). The function of the PR-5 family is not known; however, the proteins are 100% homologous and 65% identical to a characterised a-amylase/trypsin inhibitor from maize (Richardson et al., 1987). The mature proteins are about 205 residues long and each contain 16 cysteine residues that are conserved between PR-5 and the trypsin inhibitor. Clearly, the primary structures of PR-5 and the maize protein are closely related, leading to the suggestion that the function of PR-5 may be as some sort of protease or amylase inhibitor. The final SAR gene family, SAR8.2, comprises several highly related cDNAs which were isolated by differential screening of a tobacco cDNA library constructed from RNA of induced resistant leaves (D. Alexander etal., unpublished data). The cDNA encodes a basically-charged protein of about 50 residues, which has not been isolated and whose function is unknown. Gene induction during systematic acquired resistance All of the cDNA clones isolated from tissue in which resistance has been induced encode proteins that can be described as PR-proteins or PR-like proteins (Bol etal, 1990; Carr & Klessig, 1990). The appearance of PRproteins in pathogen-infected tissue and in uninfected portions of the infected plant has been described (Hooft van Huijsduijnen et al., 1986; Metraux et al., 1988; Tuzun et al., 1989). However, a correlation between the induction of mRNA expression and the onset of systemic acquired resistance has not been conclusively demonstrated and the role of the PRproteins in any type of pathogen defence remains questionable (Fraser, 1981; Fraser & Clay, 1983). Our working hypothesis has been that genes responsible for maintaining an induced resistant state would be either expressed at low levels or not expressed at all in healthy, uninduced tissue, and their expression would increase concomitantly with the onset of SAR. We refer to genes that would fulfil these criteria as SAR genes. To determine which of the isolated cDNAs represented SAR genes, their expression was correlated with the onset of SAR. Tobacco plants were inoculated at time zero with a suspension of tobacco mosaic virus on three lower (primary) leaves. At various days after infection, a group of plants was sampled and either used for RNA preparations or challenged on the uninfected (secondary) leaves with a second TMV inoculation. At least three plants were used

212

j . RYALS etal. Table 1. Bioassay for SAR in tobacco. Plants were inoculated with a phosphate buffer solution or with a suspension of TMV (both in carborundum) at day zero. At various times after inoculation, several plants were assayed for SAR by inoculating upper leaves with a suspension of virus. The SAR effect is demonstrated by the reduction in lesion size (in mm) relative to the buffer treated control. Lesions were scored 7 days after the second inoculation Day of challenge inoculation

Buffer TMV

0

3

7

14

3 3

2-3 2-3

2 >0.5

2 >0.5

both for the TMV challenges and for RNA isolations and tissues were pooled prior to RNA isolations. The data from the TMV challenge are shown in Table 1. By day 7 the plants had established SAR, as determined by a dramatic decrease in the size of the TMV-induced lesions, which was maintained for the duration of the experiment. The accumulation of RNA from each of the different cDNA classes is shown in Fig. 3. There are nine classes of cDNAs that are essentially co-induced Fig. 3. Expression of SAR genes in response to TMV infection. The autoradiographs are from northern blot experiments using RNA extracted from leaves of tobacco at various times after injection with TMV. The RNA was extracted from either inoculated (primary) leaves or from uninoculated (secondary) leaves of the same plant. The numbers represent days after inoculation. The northern blots were probed with the following clones: PR-la cDNA (T. Parks, N. Desai and J. Ryals, unpublished data); PR-2 (class II glucanase: Ward et al., 1991); PR-3 (class II chitinase: Payne etal., 1990a); PR-4 (Friedrich etal., 1991); PR5cDNA(Paynee/a/., 1988a); PR-1 basic (Payne etal., 1989); basic class III cDNA (Lawton et al., 1992); acidic class III chitinase (Lawton et al., 1992); PR-Q' (class III glucanase: Payne et al, 1990b); basic chitinase cDNA (class I chitinase: Shinshi et al., 1987); basic glucanase cDNA (Shinshi et al., 1988); SAR8.2 cDNA (D. Alexander et al., unpublished data); acidic peroxidase (Lagrimini etal., 1987). Relative levels of each mRNA cannot be compared between blots owing to different specific activities of the probes and different autoradiographic exposure times.

Primary Leaf 0 1 2 3 4 5 6 7 9

12 14 C

Secondary Leaf 0 1 2 3 4 5 6 7 9

12 14

t—n|

PR-l

PR-2

f|

PHIf

—M

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PR-3

PR-4

iff

PR-5

••fpi

PR-1 basic

Basic class HI chitinase

Acidic class H chitinase

PR-Q'

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1

^ B ?W ^B '"1^ Wfe Basic chitinase

SAR 8.2

tmffi.'i

Acidic peroxidase

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in the uninfected (secondary) leaves. A large increase in RNA accumulation beginning at day 6 correlates well with the onset of SAR as determined by the bioassay. Thus, the PR-1 acidic, PR-1 basic, PR-2 (class II glucanase; PR-2, PR-N and PR-O), PR-3 (class II chitinase; PRP and PR-Q), PR-4, PR-5, PR-Q' (class III glucanase), class III chitinase acidic and class III chitinase basic cDNAs fulfil the criteria set for SAR genes. These RNAs are also co-induced in the infected leaves of the plant with a peak of accumulation at days 5-7. The basic, class I chitinase and basic, class I f5-l,3-glucanase show patterns of induction similar to one another: they do not appear to be consistently induced in the secondary tissue but are induced to high levels in the primary tissue. A third pattern of expression is illustrated by the SAR8.2 gene, which is induced in primary tissue similarly to the other genes but is induced in secondary tissue starting at day 4 or 5 with levels of mRNA slowly increasing to reach maximal expression by day 12. The acidic, lignin-forming peroxidase (Lagrimi et al., 1987) is somewhat induced by primary tissue but is not induced in the secondary tissue. Expression in transgenic tobacco A direct role for the SAR genes in effecting SAR cannot be inferred from the data presented above. However, it is plausible that any of the SAR genes may encode proteins with antibiotic activity which could play a direct role in maintaining the disease-resistant state. To address this issue homozygotic, transgenic tobacco lines producing high levels of each of these proteins were developed by expressing the corresponding cDNAs from a strong constitutive promoter. Using these lines we have asked whether the expression of a single SAR protein is capable of conferring a disease resistant phenotype. Our goal was to develop a high-level expression system capable of routinely producing the transgenic protein at a level of at least 0.1% total protein. In order to achieve that level of expression, an expression cassette, pCGN1761, based on the double 35S promoter was constructed (Kay et al., 1987). The full-length SAR cDNAs were subcloned into pCGN1761 and the resulting expression cassette was subcloned into the Agrobacterium binary vector pCGN1541 (McBride & Summerfelt, 1990). Agrobacteria containing the different binary constructs were used to transform tobacco leaf discs, transformed tissues were selected and plants were regenerated. About 20 independent transformants were selected from each transformation experiment and homozygous lines were developed by sequential selfing of the plants. The lines were evaluated for expression of the transgenic protein, which in many cases was present at

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levels exceeding 1% of the total protein. From one to several lines expressing high levels of each SAR protein were selected for disease resistance evaluation. Several lines have now been evaluated for resistance to bacterial (Pseudomonas syringe pv. tabaci, Pseudomonas syringae pv. syringae), fungal (Cercospora nicotianae, Phytophthora parasitica, Rhizoctonia solani, Peronspora tabacina) and viral (TMV, PvY) pathogens. In the first round of analysis, positive disease resistance results were referred to as indications. An indication is promoted to a bona fide resistance once the resistance has been demonstrated repeatedly in several independent transformants. Among the more interesting indications thus far are lowlevel resistance to P. tabacina conferred by PR-la, significant resistance to Rhizoctonia solani conferred by the class I, class II and class III chitinases, and a high level of resistance to Phytophthora parasitica conferred by SAR8.2. Using the homozygous lines, Fl plants have been developed by sexual crosses that express pairs of SAR gene products. The evaluation of these lines is currently under way. Homozygous lines expressing high levels of the antisense of each of the SAR cDNAs have also been developed. In these plants expression of the encoded protein target is strongly depressed. At this point, no indications that the absence of a single SAR protein results in increased disease susceptibility have been found. Identification of the biochemical signal for SAR Identification and analysis of a candidate signal from cucumber The work described to this point has involved experiments in tobacco. Experiments designed to identify a biochemical signal that would mediate the SAR response were carried out, however, in cucumber. The cucumber system is attractive because phloem exudate can be collected from decapitated plants with relative ease and the SAR system is well characterised in these plants. It had been proposed that a biochemical signal is produced in the infected leaf and transmitted through the phloem (Ross, 1966; Guedes et al., 1980). Therefore, we decided to analyse phloem exudate for newly synthesised metabolites that would appear somewhat before the onset of SAR. If candidate metabolities were found it might be possible to purify the compounds, determine their chemical structure, and test the ability of synthesised compounds to induce SAR. This type of experimental approach could provide compelling evidence for the identity of the signal molecule.

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etal.

To begin the experiment a number of cucumber plants were inoculated with either a suspension of Colletotrichum lagenarium spores or a suspension of tobacco necrosis virus (TNV). At various times after infection, a set of five plants was selected: two were used for the preparation of phloem exudate and three were used to bioassay for SAR using a C. lagenarium challenge (Metraux et al, 1990). Analysis by HPLC of methanol extracts from phloem exudate showed that a fluorescing metabolite appeared in the phloem exudate and accumulated dramatically, reaching a peak about one day before resistance could be detected in the upper leaves of the plant. In further experiments, the presence of this fluorescent metabolite was shown to be tightly correlated with SAR and thus the metabolite was a candidate for a biochemical signal. The metabolite was purifed from the exudate and the structure was determined by gas chromatography-mass spectrometry to be 2-hydroxybenzoic acid or salicylic acid (SA) (Metraux et al., 1990). It has already been shown that SA can induce resistance to C. lagenarium when applied exogenously to cucumber leaves and that this resistance is not caused by fungitoxic metabolites of SA (Mills & Woods, 1984). Also, the acidic chitinase from cucumbers has been shown to serve as a good biochemical marker for SAR (Metraux et al., 1988) and it is clear that this gene is induced by exogenous applications of SA (Metraux et al., 1989). These results argue for a causal role of SA in the induction of SAR. Further support of SA as a possible signal in SAR was provided by Malamy et al. (1990), who demonstrated that the accumulation of SA was induced by TMV infection of tobacco which reacted hypersensitively to the virus but not in tobacco that was susceptible to the virus (i.e. did not react hypersensitively). Furthermore, accumulation of SA preceded the accumulation of PR-1 mRNA, which has been shown here to be a biochemical marker for SAR. These results provide evidence that SA could act as the biochemical signal in tobacco as well. Interestingly, a similar hypothesis had been advanced earlier to explain the induction of PRproteins after virus infection or ethylene treatment (Van Loon & Antoniw, 1982). Ironically, it took almost a decade for researchers to determine experimentally that SA is actually synthesised by the plant, and that its appearance is correlated with the induction of PR-protein accumulation. Two observations, however, do not neatly fit a model of SA as the endogenous signal. First, in order to induce resistance, the compound must be applied in high concentrations, typically in the order of 10 ITIM (Mills & Woods, 1984; J.P. Metraux etal., unpublished data). Secondly, the exogenous application of SA will induce resistance only in leaves that are treated with the compound: it does not act systematically. The

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requirement of high concentrations of exogenously applied SA for the induction of resistance may be caused by the rapid conversion of SA to a non-active form. For example, it has been demonstrated that exogenously applied SA is rapidly metabolised (Metraux et al., 1990), possibly by glucosylation (Balke et al., 1987). It is not clear why exogenously applied SA does not induce resistance systemically, but this could also be due partly to the high rate of metabolism. In summary, we have identified salicylic acid as a candidate for the endogenous signal that mediates SAR. The addition of this compound to leaves induces the biological response and also induces the synthesis of marker genes that have been correlated with the onset of SAR. However, it has not been possible to induce the phenomenon systematically by the addition of exogenous SA and this is apparently due either to the rapid metabolism of the compound or to its inability to gain access to the vascular system. Gene induction by salicylic acid If SA is indeed the biochemical signal that mediates SAR in cucumber and tobacco then exogenous application of the compound should induce the same types of genes induced during the pathogen-induced response. Indeed, this was found for the induction of the acidic, cucumber chitinase. However, the pattern of gene induction in cucumbers is less well understood than in tobacco. As previously described, nine classes of mRNAs that accumulate to high levels in uninfected leaves during the induction of SAR in tobacco have been identified. In order to test whether SA would induce the same set of genes, the expression of the mRNA classes in tobacco in response to exogenously applied SA was catalogued. Leaves of 6-week-old tobacco plants were treated with 50 mM sodium salicylate solution and groups of plants were harvested at 0, 2,4,8,12,24 and 48 h after treatment. This concentration of salicylate allows very strong expression of mRNAs but is not so high as to produce phytotoxicity within the time frame of the experiment. The leaves of several plants were pooled and RNA was extracted. The accumulation of mRNA for PR-1 acidic, PR-1 basic, class I, II and III glucanase, class I, II, III and IV chitinase, PR-4, PR-5, SAR8.2 and the lignin-forming peroxidase was determined in these samples by northern blot analysis. Within 2-4 h after SA treatment RNA accumulation was dramatically increased for PR-1 acidic, PR-1 basic, class II and III glucanase, class II, III and IV chitinase. PR-4, PR-5 and SAR8.2. There was not a consistent increase in the mRNA for class I glucanase, class I chitinase or the lignin-forming peroxi-

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dase. Therefore, the results showed that, at least qualitatively, the same set of genes is induced by SA as is induced during the onset of pathogeninduced SAR. Biosynthesis of salicylic acid The biosynthetic pathway for salicylic acid is not clear. At present, at least two pathways have been proposed. Each branches from phenylpropanoid biosynthesis after phenylalanine has been converted to transcinnamic acid by phenylalanine ammonium lyase (PAL). In one scheme (Pathway 1: Fig. 4), frans-cinnamic acid would be converted to 2-hydroxy cinnamic acid (or 2-coumaric acid) by a cinnamate 2-hydroxylase. This compound could then be converted to salicylic acid via |3-oxidation possibly through an acetyl coenzyme A (CoA) intermediate. Alternatively, rrarcs-cinnamic acid could be oxidized to benzoic acid and then hydroxylated via a postulated o-hydroxylase activity. The details of this pathway, particularly in tobacco and cucumber, deserve further study. In either of the proposed pathways, salicylic acid is synthesised from frarcs-cinnamic acid. This is an intriguing observation and may provide a clue as to how and why the induction of SAR is tightly linked to the formation of a necrotic lesion. When plants react hypersensitively to pathogen attack, many biochemical changes occur, including the induction of phenylpropanoid biosynthesis. In bean, as well as other plants, this induction seems to be at least partly caused by an increase in the synthesis of phenylalanine ammonium lyase and other enzymes involved in the biosynthesis of isoflavonoid phytoalexins, flavonoid pigments and \

Pathway 1

VCHJ-C-COOH

phenylalanine

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CH

0H Naringenin Flavanone Isoflavonoid Phytoalexins

Flavonoid Pigments

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4-coumaryl alcohol

lignin precursor lignin (physical barrier)

Fig. 4. Possible biosynthetic pathway for salicylic acid in plants. Structural names are given in bold and enzymes in small letters.

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lignin. The result is an increased flux through this pathway. Since SA is probably synthesised from this pathway, downstream of ?ra/M-cinnamic acid, it is reasonable that an increase in fraAis-cinnamic acid production could lead to an increase in the accumulation of SA. Clearly, this model for the biosynthesis of SA is somewhat speculative and needs further experimentation, but it is attractive in that the linkage of the two biosynthetic pathways may explain the biological observation that SAR is induced when the plant reacts to a pathogen by the formation of a necrotic lesion. Identification of SA dependent trans-acting factors

Analysis of the PR-la promoter Another principal research objective, as a possible approach to understanding the signal transduction mechanism, has been to identify the transcriptional factors involved in inducing the expression of the SAR gene family. A step toward achieving that goal is to identify ds-acting DNA elements that bind these factors. We refer to these putative binding sites as Salicylic Acid Responsive Elements or SAREs. We have isolated a genomic clone encoding PR-la, which is the most highly salicylateinducible SAR gene in tobacco (Payne et al., 1988; J. Ryals, unpublished data). The transcriptional start site of the mRNA was mapped by primer extension analysis and SI nuclease protection (Payne et al., 19886) and 900 base pairs of the 5' flanking sequence including the 31 bp untranslated leader was fused to a |3-glucuronidase (GUS) reporter gene (Jefferson et al., 1987). The 900bp 'full-length' construct and a series of deletions were transformed into tobacco via Agrobacterium leaf disc transformation. The expression of GUS enzyme activity from either healthy plants or plants treated with salicylic acid or tobacco mosaic virus was determined. In healthy plants, the basal level of GUS expression remained unaffected when the promoter was deleted from -900 to —318. However, GUS activity decreased dramatically when the promoter was deleted to —222. GUS activity decreased to virtually undetectable levels when the promoter was deleted to -150. Further deletion to -75 slightly increased the level of constitutive expression to about the level of the —222 deletion. These results suggest that there may be an element in the —318 to —222 region which is important for non-specific transcription. Further, there may be a negative element that suppresses transcription around the -150 region that is lost when the promoter is deleted to -75. When the plants were induced by infecting either with TMV or treating

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with salicylic acid, GUS expression was induced by about 50 to 100-fold in constructs containing 900, 825 or 660 bases of the 5' flanking region. However, in constructs containing 318 bases of the 5' DNA, the gene cannot be substantially induced by either SA or TMV. In similar experiments, Van de Rhee et al. (1990) have shown that deletions containing 680 bases of the 5' flanking region of PR-la can induce GUS activity but promoter constructs containing 640 bp of PR-la DNA cannot. Therefore, it appears that the 5' border of the SARE lies between —660 and —640. It also appears that the architecture of the PR-la promoter is somewhat complex with at least three elements that may be important for the control of transcription.

Dependence of SAR RNA accumulation on cycloheximide An important aspect of the regulation of SAR induction is to understand whether the gene is regulated by factors that are synthesised de novo after the signal has been transduced or whether transcription is regulated by factors already present in uninduced cells. A metholodogy frequently used to address this issue is to determine the effects of protein synthesis inhibitors on RNA accumulation. Therefore, leaf tissue was treated with cycloheximide (CHX), salicylic acid, or both salicylic acid and CHX and the accumulation of SAR gene mRNA after 4 and 24 h was determined. The results of these experiments indicated that treatment of the tissue with CHX alone induces the accumulation of mRNA encoding all of the SAR genes. SA treatment and treatment with both SA and CHX also induces mRNA accumulation to high levels (data not shown). These results are somewhat surprising and suggest either that SAR gene expression is under negative regulation by a labile repressor or that the SAR system is also sensitive to the interruption of protein synthesis. In any case, the results do not favour a model of a positive regulator which is synthesised de novo and which acts to stimulate RNA transcription. The results are compatible with either a model where a negative regulatory factor is released from the DNA and thus allows increased transcription or a model where a positive factor is modified (e.g. phosphorylated) and then binds to the promoter causing increased transcription. However, considering the complexity of the deletion analysis results, a model that combines negative regulation and positive regulation by a modified factor is also likely. The promoter deletion analysis data and the CHX data are summarised in a model of the PR-la promoter shown in Fig. 5. The promoter appears to have a complex architecture comprising several cw-acting domains

Systemic acquired resistance

900

-800

-700

-600

-500

-400

221

-300

-200

-100

Fig. 5. Hypothetical model of the PR-la promoter. The numbers under the drawing indicate base pairs relative to the start site of transcription.

which may bind positive or negative regulatory factors. Some of these factors may be induction-specific and others important for non-specific transcription regulation. The induction of the promoter is apparently not dependent on de novo protein synthesis of a transcription factor, so promoter activation could occur via the turnover of a labile repressor or the activation/inactivation (e.g. phosphorylation) of a positive/negative transcription factor. Cell types that respond to salicylic acid Using the transgenic tobacco plants, the cell types that respond to the action of salicylate could also be determined. Nine independent PR-la/ GUS transgenic lines were developed and analysed for the cell types responding to SA by an in situ enzyme activity stain (Jefferson et al., 1987) followed by histological analysis. The expression pattern of six of these lines was qualitatively similar. The strongest expression was in the spongy mesophyll; there was less in the palisade cells and little expression in other cell types. In one strongly expressing line there was also detectable expression in the epidermal cells and the trichomes. In the other three lines the expression of GUS was not inducible by either TMV or salicylate. In two of these lines there was a constitutive, high level of expression in most of the plant tissues and in the third there was no observed staining of the cells. We conclude that the constitutive, noninducible expression patterns are aberrant and represent ectopic gene expression. While it seems clear that the cell types that respond to SA using the PR-la promoter are the mesophyll cells of the leaf, it may be premature to generalise this response to all of the SAR genes. However, it is interesting that the observed expression pattern is consistent with the observation that SAR protects best against foliar pathogens.

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Development of a chemical induction system Early in our experiments with PR-la gene expression it became clear that the PR-la promoter was very dynamic in transgenic plants and in certain cases after TMV or chemical treatment the expression of GUS was driven to high levels. We have used this property of the PR-1A promoter to develop a chemically regulated chimaeric gene expression system for use in transgenic plants. In these experiments the PR-la promoter and leader sequence from +31 to —903 was fused to either the BtK gene, a gene that encodes the insecticidal delta endotoxin protein from Bacillus thuringensis subsp. kurstaki (Geiser et al., 1986), or a mutant acetohydroxyacid synthase (AHAS) gene obtained from Arabidopsis thaliana by selecting for resistance to sulphonylurea herbicides (Haughn et al., 1988). In the first case, we would expect to develop transgenic plants that become resistant to insect damage after chemical treatment; in the second, we would expect to develop plants that become tolerant to herbicides upon induction. Although salicylic acid could be used to induce the transgenes, the exogenous application of high concentrations of this compound resulted in unacceptable levels of phytotoxicity. Therefore, we also began to search for compounds that would serve as better inducers. One compound, currently called 'Inducer X', was found that could induce strong expression from the transgenic PR-la promoter but did not present phytotoxicity problems. BT-13 is a homozygous transgenic seed line that carries the PR-la promoter fused to the BtK coding sequence. Six-week-old plants were treated with a foliar application of either water or Inducer X and the plants were allowed to grow for another week. At that time 10 first instar larvae of Manduca sexta (tobacco hornworm) were applied to each plant and the insects allowed to feed for 10 days. The most heavily damaged plant from each treatment is shown in Fig. 6. It is clear that the insects stripped virtually all of the leaf material from the water-treated plant while the Inducer X treated plant received little damage. Control, nontransgenic plants did not show an insecticidal effect. In similar experiments using the PR-la promoter to control AHAS gene expression, the mRNA was strongly induced by the addition of Inducer X. In addition, a marked decrease in the herbicidal effect of sulphonylurea application could be observed in the chemically treated plants. Thus, the expression of a chimaeric gene can be controlled in transgenic plants by the addition of an exogenous compound and this gene induction can result in dramatic phenotypes. This type of regulated chimaeric gene expression may have applications

Fig. 6. Induction of insect control in transgenic tobacco. The PR-l promoter was fused to the Btk gene and the construct was used to transform tobacco. Both plants are from seeds of a transformed line Bt13. One plant was sprayed with water and the other with an inducing agent. The plants were challenged after 7 days with Manduca sexta (tobacco hornworm). Control, untransformed plants treated with the inducer do not show insect tolerance.

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j . RYALS etal. 4 . SAR genes are induced to high levels and expressed for many days. Large amounts of proteins are synthesized . which maintains the resistant state.

3 . SA is bound by a receptor and the signal is transduced independent of protein synthesis (ie. phosphorylationJu

2 . Salicylic acid is released and translocated systemically.

Pathogen induces necrotic response, -phenylprop biosyn. pathway is induced. -salicylic acid is synthesized off phenylpropanoid pathway.

Fig. 7. Current model for SAR in tobacco.

both in agriculture and in the laboratory. Regulated expression of traits such as insect tolerance may lessen the natural selection for resistance in natural populations. In laboratory applications, regulated induction or repression of gene expression can be studied. For instance, sense or antisense versions of genes of interest could be activated by chemical induction at specific times in plant development.

Summary The goal of our research over the past few years has been to understand better the molecular events involved in systemic acquired resistance. Our current hypothesis of the phenomenon is detailed in Fig. 7. This is a speculative model at this stage but it serves as our working hypothesis on which to plan future experiments. When pathogen infection induces a necrotic lesion many biochemical changes take place. Among these are

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the induction of the phenylpropanoid pathway which leads to the synthesis of flavonoids and lignins and to the synthesis of SA. The SA is released into the phloem, where it is translocated throughout the plant and is eventually perceived by its target cells, which comprise the leaf mesophyll cells and possibly other cell types. Presumably, SA binds a receptor which transduces the signal, by a process that is apparently independent of protein synthesis, leading to the induction of a number of genes to very high levels in the target cells. The proteins synthesized from these genes then act cooperatively to protect the plant from further infection by other pathogens.

Acknowledgements This paper is dedicated to Dr Hans Geissbuehler on his retirement as Head of R & D of the Agricultural Division of CIBA-GEIGY Limited. We thought about your vision and impact on this project many times while writing this manuscript. 'Doc would listen to any kind of nonsense and change it for you to a kind of wisdom. His mind had no horizon . . . and his sympathy had no warp' (Steinbeck, 1945). We thank Drs Kay Lawton, Scott Uknes, Tom Gaffney and Mary-Dell Chilton for critical review of the manuscript, and Dr Ray Hammerschmidt for useful discussions concerning the biosynthetic pathway for the salicylic acid. We would especially like to thank the members of our laboratories who have supplied the data and to our colleagues who have helped with various phases of this work over the years.

References Balke, N., Davies, M. & Lee, C. (1987). Conjugation of allelochemicals by plants. Enzymatic glucosylation of salicylic acid by A vena sativa. In Allelochemicals: Role in Agriculture and Forestry, ed. G.R. Walker, pp. 214-27. Washington, DC: American Chemical Society. Bol, J.F., Linthorst, H. & Cornelissen, B. (1990). Pathogenesis-related proteins induced by virus infection. Annual Review of Phytopathology 28, 113-38. Boiler, T. & Metraux, J.P. (1988). Extracellular localization of chitinase in cucumber. Physiological and Molecular Plant Pathology 33, 11-16. Broekaert, W., Lee, H., Kush, A., Chua, N. & Raikhel, N. (1990). Wound-induced accumulation of mRNA containing a hevein sequence in lactifers of rubber tree (Hevea brasiliensis). Proceedings of the National Academy of Sciences (USA) 87, 7633-7. Carr, J. & Klessig, D. (1990). The pathogensis-related proteins of

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J. RYALS etal. plants. In Genetic Engineering, Principles and Methods, Vol. 11, ed. J. Setlow, pp. 65-109. New York: Plenum Press. Chester, K. (1933). The problem of acquired physiological immunity in plants. Quarterly Review of Biology 8, 275-324. Cornelissen, B., Hooft van Huijsduijnen, R. & Bol, J. (1986). A tobacco mosaic virus-induced tobacco protein is homologous to the sweet-tasting protein thaumatin. Nature 321, 531-2. Dean, R. & Kuc, J. (1985). Induced systemic protection in plants. Trends in Biotechnology 3, 125-9. Fraser, R. (1981). Are "pathogenesis-related" proteins involved in acquired systemic resistance of tobacco plants to tobacco mosaic virus? Journal of General Virology 58, 305-13. Fraser, R. & Clay, C. (1983). Pathogenesis-related proteins and acquired resistance: causal relationship or separate effects. Netherlands Journal of Plant Pathology 89, 283-92. Friedrich, L., Moyer, M., Ward, E. & Ryals, J. (1991). Isolation and characterization of cDNA clones encoding pathogenesis-related protein 4 from tobacco. Molecular and General Genetics 230, 113-19. Geiser, M., Schweitzer, S. & Grimm, C. (1986). The hypervaiable region in the genes coding for entomopathogenic crystal proteins of Bacillus thuringiensis: nucleotide sequence of the kurhdl gene of subsp. kurstaki HD1. Gene 48, 109-18. Gianinazzi, S. & Kassanis, B. (1974). Virus resistance induced in plants by poly acrylic acid. Journal of General Virology 23, 1-9. Guedes, E., Richmond, S. & Kuc, J. (1980). Induced systemic resistance to anthracnose in cucumber as influenced by the location of the inducer inoculation with C. lagenarium and the onset of flowering and fruiting. Physiological and Plant Pathology 17, 229-33. Haughn, G., Smith, J., Mazur, B. & Sommerville, C. (1988). Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides. Molecular and General Genetics 211, 266-71. Hecht, E. & Bateman, D. (1964). Non-specific acquired resistance to pathogens resulting from localized infection by Thielaviopsis basicola or virus in tobacco leaves. Phytopathology 54, 523-39. Hooft van Huijsduijnen, R., van Loon, L. & Bol, J. (1986). cDNA cloning of six mRNA's induced by TMV infection of tobacco and a characterization of their translation products. The EMBO Journal 5, 2057-61. Jefferson, R., Kavanagh, T. & Bevan, M. (1987). GUS fusions: |3glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal 6, 3901-7. Kauffmann, S., Legrand, M., Geoffrey, P. & Fritig, B. (1987). Biological function of "pathogenesis-related" protein: four PR proteins of tobacco have l,3p* glucanase activity. The EMBO Journal 11, 3209-12. Kay, R., Chan, A., Daly, M. & McPherson, J. (1987). Duplication of

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CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236, 1299-1302. Keefe, D., Hinz, U. & Meins, F. (1990). The effect of ethylene on the cell-type-specific and intracellular localization of |3-l,3-glucanase and chitinase in tobacco leaves. Planta 182, 43-51. Kuc, J. (1982). Induced immunity to plant disease. Bioscience 32, 854-9. Lagrimini, M., Burkhart, W., Moyer, M. & Rothstein, S. (1987). Molecular cloning of complementary DNA encoding the lignin-forming peroxidase from tobacco: Molecular analysis and tissue specific expression. Proceedings of the National Academy of Sciences (USA) 84, 7542-6. Lawton, K., Ward., Payne, G., Moyer, M. & Ryals, J. (1992). Acidic and basic class III chitinase mRNA accumulation in response to TMV infection of tobacco. Molecular and General Genetics (in press). Legrand, M., Kauffmann, S., Geoffrey, P. & Fritig, B. (1987). Biological function of pathogenesis-related proteins: four tobacco pathogenesis-related proteins are chitinases. Proceedings of the National Academy of Sciences (USA) 84, 6750-4. McBride, K. & Summerfelt, K. (1990). Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Molecular Biology 14, 269-76. Malamy, J., Carr, J., Klessig, D. & Raskin, I. (1990). Salicylic acid: a likely endogenous signal in the resistance response of tobacco to viral infection. Science 250, 1002^. Mauch, F., Mauch-Mani, B. & Boiler, T. (1988). Antifungal hydrolases II. Inhibition of fungal growth by combinations of chitinase and (3-1,3glucanase. Plant Physiology 88, 936-42. Meins, F., Neuhaus, J.-M., Sperisen, C. & Ryals, J. (1992). The primary structure of plant-pathogenesis related glucanohydrolases and their genes. In Genes Involved in Plant Defense, Plant Gene Research. Vol. 8, ed. F. Meins & T. Boiler. New York: Springer-Verlag (in press). Metraux, J.P., Ahl-Goy, P., Staub, Th., Speich, J., Steinemann, A.. Ryals, J. & Ward, E. (1991). Induced systemic resistance in cucumber in response to 2,6-dichloro-isonicotinic acid. In Advances in Molecular Genetics of Plant-Microbe Interactions, Vol. 1, ed. H. Hennecke & D.P.S. Verma, pp. 432-9. Dordrecht: Kluwer Academic Publishers. Metraux, J.P., Burkhart, W., Moyer, M., Dincher, S., Middlesteadt, W., Williams, S., Payne, G. & Ryals, J. (1989). Isolation of a complementary DNA encoding a chitinase with structural homology to a bifunctional lysozyme/chitinase. Proceedings of the National Academy of Sciences (USA) 86, 896-900. Metraux, J., Signer, H., Ryals, J., Ward, E., Wyss-Benz, M., Gaudin, J., Raschdorf, K., Schmid, E., Blum, W. & Inverardi, B. (1990). Increase in salicylic acid at the onset of systemic acquired resistance in cucumber. Science 250, 1004-6.

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Metraux, J.P., Streit, L. & Staub, T. (1988). A pathogenesis-related protein in cucumber is a chitinase. Physiological and Molecular Plant Pathology 33, 1-9. Mills, P. & Woods, R. (1984). The effect of polyacrylic acid, acetyl salicylic acid and salicylic acid on resistance of cucumber to Colletotrichum lagenarium. Phytopathologische Zeitschrift 111, 209-16. Payne, G., Ahl, P., Moyer, M., Harper, A., Beck, L., Meins, F. & Ryals, J. (1990a). Isolation of complementary DNA clones encoding pathogenesis-related proteins P and Q, two acidic chitinases from tobacco. Proceedings of the National Academy of Sciences (USA) 87, 98-102. Payne, G., Middlesteadt, W., Desai, N., Williams, S., Dincher, S., Carnes, M. & Ryals, J. (1989). Isolation and sequence of a genomic clone encoding the basic form of pathogenesis-related protein 1 from Nicotiana tabacum. Plant Molecular Biology 12, 595-6. Payne, G., Middlesteadt, W., Williams, S., Desai, N., Parks, T., Dincher, S., Carnes, C. & Ryals, J. (1988a). Isolation and nucleotide sequence of a novel cDNA clone encoding the major form of pathogenesis-related protein R. Plant Molecular Biology 11, 223-4. Payne, G., Parks, T., Burkhart, W., Dincher, S., Ahl, P., Metraux, J. & Ryals, J. (19886). Isolation of the genomic clone for pathogenesisrelated protein la from Nicotiana tabacum cv. Xanthi. Plant Molecular Biology 11, 89-94. Payne, G., Ward, E., Gaffney, T., Ahl-Goy, P., Moyer, M., Harper, A., Meins, F. & Ryals, J. (19906). Evidence for a third structural class of P-l,3-glucanase in tobacco. Plant Molecular Biology 15, 797-808. Pierpoint, W., Tatham, A. & Pappin, D. (1987). Identification of the virus-induced protein of tobacco leaves that resembles the sweetprotein thaumatin. Physiological and Molecular Plant Pathology 31, 291-8. Richardson, M., Valdes-Rodriguez, S. & Blanco-Labra, A. (1987). A possible function for thaumatin and a TMV-induced protein suggested by homology to a maize inhibitor. Nature 327, 432-4. Ross, A.F. (1961a). Localized acquired resistance to plant virus infections in hypersensitive hosts. Virology 14, 329-39. Ross, A.F. (19616). Systemic acquired resistance induced by localized virus infections in plants. Virology 14, 340-58. Ross, A. (1966). Systemic effects of local lesion formation. In Viruses of Plants, ed. A. Beemster & J. Dijkstra, pp. 127-50. Amsterdam: North Holland Press. Shinshi, H., Mohnen, D. & Meins, F. (1987). Regulation of a plant pathogenesis-related enzyme: Inhibition of chitinase and chitinase mRNA accumulation in cultured tobacco tissues by auxin and cytokinin. Proceedings of the National Academy of Sciences (USA) 84, 89-93.

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Shinshi, H., Neuhaus, J.M., Ryals, J. & Meins, F. (1990). Structure of a tobacco endochitinase gene: evidence that different chitinase genes can arise by transposition of sequences encoding a cysteine-rich domain. Plant Molecular Biology 14, 357-68. Shinshi, H., Wenzler, H., Neuhaus, J.M., Felix, G., Hofsteenge, J. & Meins, F. (1988). Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro-P1,3-glucanase. Proceedings of the National Academy of Sciences (USA) 85, 5541-5. Singh, S., Bracker, C , Hasegawa, P., Handa, A., Buckel, S., Hermondson, M., Pfankoch, E., Regnier, F. & Bressan, R. (1987). Characterization of osmotin, a thaumatin-like protein associated with osmotic adaptation in plant cells. Plant Physiology 85, 529-36. Stanford, A., Bevan, M. & Northcote, D. (1989). Differential expression within a family of novel wound-induced genes in potato. Molecular and General Genetics 215, 200-8. Steinbeck, J. (1945). Cannery Row, 5th edn, p. 24. New York: Viking Press. Takeuchi, Y., Yoshikawa, M., Takeba, G., Kunisuke, T., Shibata, D. & Horino, O. (1990). Molecular cloning and ethylene induction of mRNA encoding a phytoalexin elicitor-releasing factor P-l,3-glucanase, in soybean. Plant Physiology 93, 673-82. Tuzun, S., Rao, N., Voegli, U., Schardl, C. & Kuc, J. (1989). Induced systemic resistance to blue mold: early induction and accumulation of P-l,3-glucanases, chitinases, and other pathogenesis-related proteins (b-proteins) in immunized tobacco. Phytopathology 79, 979-83. Van de Rhee, M., Van Kan, J., Gonzalez-Jean, M. & Bol, J. (1990). Analysis of regulatory elements involved in the induction of two tobacco genes by salicylate treatment and virus infection. The Plant Cell 2, 357-66. Van Loon, L. & Antoniw, J. (1982). Comparison of the effects of salicylic acid and of ethephon with virus-induced hypersensitivity and acquired resistance in tobacco. Netherlands Journal of Plant Pathology 88, 237-56. Ward, E., Payne, G., Moyer, M., Williams, S., Dincher, S., Sharkey, K., Beck, J., Taylor, H., Ahl-Goy, P., Meins, F. & Ryals, J. (1991). Differential regulation of P-l,3-glucanase mRNAs in response to pathogen infection. Plant Physiology 96, 390-7. Wessels, J. & Sietsma, J. (1981). Fungal cell walls: a survey. In Encyclopedia of Plant Physiology, Vol. 13B. Plant Carbohydrates 11, ed. W. Tanner & F. Loewus, pp. 352-94. Berlin: Springer. White, R.F. (1979). Acetylsalicylic acid (aspirin) induces resistance to tobacco mosaic virus in tobacco. Virology 99, 410-12.

E. S. DENNIS, M. OLIVE, R. DOLFERUS, A. MILLAR, W.J. PEACOCK and T.L. SETTER

Biochemistry and molecular biology of the anaerobic response The anaerobic response When plants experience anoxic conditions there is a shift in carbohydrate metabolism from an oxidative to a fermentative pathway (Fig. 1). In the absence of oxygen, ATP is generated not by the Krebs cycle but by alcoholic fermentation, i.e. glycolysis and ethanol synthesis. As well as the change in carbohydrate metabolism there is a change in the pattern of polypeptide synthesis under anoxia (Sachs et al., 1980; Bailey-Serres et al., 1988). Synthesis of polypeptides normally present under aerobic conditions stops and synthesis of a number of specific polypeptides - the anaerobic polypeptides (ANPs) - commences. In maize there are about 20 ANPs which have been identified chiefly as enzymes associated with the flow of carbon into and through glycolysis and through alcoholic fermentation; in particular UDP-sucrose synthetase, pyruvate decarboxylase and alcohol dehydrogenase (ADH) are induced approximately 10-fold (Lazlo & St Lawrence, 1983; Springer et al., 1986). Glucose phosphate isomerase (Kelley & Freeling, 1984a), one of the isozymes of glyceraldehyde 3-phosphate dehydrogenase, and cytoplasmic aldolase (Kelley & Freeling, 19846) have also been shown to be induced to a lesser degree. The levels of two enzymes which are thought to be responsible for regulating the glycolytic pathway, phosphofructokinase and pyruvate kinase, do not change significantly during anaerobiosis (Bailey-Serres et al., 1988). It is assumed that the enzymes of glycolysis are induced by anaerobiosis to allow a greater flux of carbohydrate through the pathway because only 2 molecules of ATP are produced per molecule of glucose under anaerobic conditions whereas 36 molecules of ATP are produced under oxidative conditions. Nuclear runoff experiments show that there is transcriptional control of ANP synthesis (at least for Adhl and Adh2, and sucrose synthetase: Society for Experimental Biology Seminar Series 49: Inducible Plant Proteins, ed. J. L. Wray. ©Cambridge University Press, 1992, pp. 231-245.

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SUCROSE

GLUCOSE-1-P

(

glucose phosphate ] isomerase J

I

glyceraldehyde-3-phosphate dehydrogenase

GLUCOSE —•PYRUVATE USES NAD PRODUCES NET: 2 moles ATP/mol glucose ETHANOL (

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NAD

CO,

ACETALDEHYDE-"

PRODUCES NAD

•PYRUVATE pyruvate| decarboxylasej

'LACTATE

( lactate | Idehydrogenasel

Fig. 1. The pathway of carbon in alcohol fermentation. Enzymes which have been identified as being anaerobically induced are boxed. Rowland & Strommer, 1986; Dennis et al., 19886). At the same time the synthesis of aerobically expressed proteins is repressed at the level of translation, suggesting that an anaerobic specific translation factor functions under anaerobic conditions. The Anaerobic Response Element (ARE) regulates anaerobic induction of the maize Adh gene In order to investigate the DNA sequences responsible for the anaerobic induction of ANPs we cloned and sequenced the Adhl and 2 genes of

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maize (Dennis et al., 1984, 1985) and pea (Llewellyn et al., 1987) and the aldolase gene of maize. These were analysed at a functional level. Subsequently, a number of other anaerobically induced genes (strawberry Adh, maize sucrose synthetase, Arabidopsis Adh) have also been cloned and sequenced. The maize Adhl gene is regulated at the level of transcription. In order to identify the sequences responsible for this anaerobic regulation of transcription we analysed the promoter region by both 5' and 3' deletions and by linker scan mutations which replace bases within the Adhl promoter eight at a time (Walker et al., 1987). The Adhl promoter and mutant derivatives of it were linked to a reporter gene (either CAT or GUS) and introduced into maize protoplasts by electroporation. Expression of the introduced reporter gene paralleled that of the endogenous gene under different conditions of aeration, giving confidence that any change in activity of the promoter-reporter gene construct reflected that of the endogenous gene (Howard et al., 1987). This deletion and mutation analysis of the promoter resulted in the. identification of a region located between —140 and —100, relative to the transcription start, the Anaerobic Responsive Element (ARE), which was critical for anaerobic gene expression. Two subregions exist within this promoter, both of which are necessary for activity, and each of these two subregions contained a core sequence T/CGGTTT. The same core sequence T/CGGTTT was identified in a number of other anaerobically inducible genes (Dennis et al., 1987). In the aldolase (Dennis et al., 1988a) and pea Adh genes (Llewellyn et al., 1987) these same sequences were shown to lie in a region which was functionally important as determined by deletion analysis. All Adh genes contained multiple copies of this core anaerobic sequence, lying both outside the functionally important regions and within them. We suggested that a common regulatory sequence controls all anaerobically induced genes by binding a common transcription factor and that the multiple copies of the core anaerobic sequence are responsible for the different rates and different levels of anaerobic induction seen with the different genes. A more detailed analysis of the ARE showed that when more copies of the ARE were linked to the reporter gene an increased level of anaerobic induction was obtained, with six copies giving 16-fold induction (Olive et al., 1990). The ARE alone was sufficient for anaerobic induction. The element gave anaerobic induction when placed in either orientation, showing the ARE has enhancer-like qualities. While the two subregions within the ARE were both critical for anaerobic induction the region between could be mutated without loss of activity, and the spacing between the two subregions of the ARE could be increased by up to 60 bp

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with little change in activity. The necessity for both subregions to be present suggested that they are both needed simultaneously to bind a regulatory protein. Proteins bind to the ARE Transcription is mediated through proteins binding to specific c/s-acting regulatory sequences. This DNA-protein complex then interacts with RNA polymerase to form an effective transcription complex which results in gene expression. Having identified the region of the maize Adhl promoter critical for anaerobic gene expression, we could look for evidence of protein interactions in this region. Ferl & Nick (1987) used in vivo dimethyl sulphate (DMS) protection to identify putative protein binding sites in the maize Adhl promoter. Two of the sites they footprinted coincided with the two subregions of the ARE. These footprints are present constitutively but the signal appeared to intensify under anaerobic conditions. Two other sites outside the ARE show protection only under anaerobic conditions. We have used gel retardation assays to study proteins binding to the ARE. We used a probe consisting of the 42 base ARE. When the probe was multimerised in four or six tandem copies, protein, AREF (ARE Factor) from nuclear extracts of anaerobically induced suspension cultures was bound to the probe and showed a ladder of retarded bands (Fig. 2). This ladder of bands was completed specifically by the 4x ARE probe but not by a probe containing four copies of the octopine synthase enhancer element or pUC DNA even in 100-fold excess. Probes which contained one or two copies of the ARE did not show any binding. The need to multimerise the probe presumably results either from low affinity of the protein for the DNA or low abundance of AREF in nuclear extracts. Functional structure of the ARE In order to define the limits of the ARE and the bases critical for binding of the protein and for transcriptional activity, the ARE sequence was subjected to a mutational analysis. Mutations were introduced into the ARE using mutant oligonucleotides and these mutant sequences were multimerised to give a four copy ARE promoter. These 4x mutant ARE constructs were assayed for their ability to bind the protein factor or to compete with binding of the wild-type ARE element to the protein. The 4x mutant ARE elements were also linked to the GUS reporter gene and introduced into protoplasts by electroporation to determine their activity as transcriptional effectors under aerobic and anaerobic conditions.

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EXTRACT +/SPECIFIC NONSPECIFIC FREE PROBE rEXCESS COMPETITOR 1 DNA

10 NIL

50

4ARE

Fig. 2. Binding of a nuclear extract to the ARE probe. A four copy radioactively labelled ARE probe was incubated with a nuclear extract from maize suspension culture cells. From the left the lanes represent: free probe; probe plus extract; probe plus extract competed with a 10fold excess of unlabelled ARE and, lastly, with a 50-fold excess of unlabelled ARE. The top bands are competed by unlabelled ARE but not by other unlabelled DNAs.

These studies showed that the bases critical for the anaerobic induction of the Adh promoter extended beyond the T/CGGTTT motif. While mutations of this motif certainly eliminated expression, mutations outside this region also had a marked effect on expression. The effect on binding was even more dramatic: regions outside this motif were critical for binding, while alterations in the T/CGGTTT motif had virtually no effect on binding. A summary of the results is shown in Fig. 3. Mutations of both GGTTT sequences within the T/CGGTTT motifs (Mut4) affects expression but not binding; mutation of nucleotides including the TGGTTT sequence (Mut2) also affects expression but not binding. Mut6 shows that mutation of the GC-rich region immediately

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-,« ARE MUT4

-,o,

CTGCAGCCCCGGTTTCGCAAGCCGCGCCGTGGTTTGCTTG r—i

MUT6

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Binding Activity

W->

+++

+++

-

+++

1

cm MUT3 MUT2

Fig. 3. The ARE sequence and mutated versions. The regions altered in the mutated AREs are indicated by blocks. The mutated AREs were multimerised and binding activity assayed by gel retardation. Each multimerised ARE was linked to a GUS reporter gene, electroporated into maize protoplasts and assayed for anaerobically induced expression of GUS. Binding Activity ARE

CTGCAGCCCCGGTTTCGCAAGCCGCGCCGTGGTTTGCTTG

AI All

1 I

MUT4C MUTGT-1 MUT7 MUTGC-1

MUTGC-2 MUTGT-2

+++

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+++ +++

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+++

Fig. 4. Protein binding domains of the ARE. Legend is as in Fig. 3. AI and All show the deleted portion of the ARE. Only binding activity has been assayed so far.

upstream of the TGGTTT sequence abolishes both binding and transcription. In Mut3, loss of the 4C region also abolishes both binding and expression (this result is confirmed by Mut4C: Fig. 4). In Mut 7 both binding and anaerobic induction are retained. Anaerobic expression can be lost while binding is retained, but in no case is there expression without binding of AREF. This suggests that binding of AREF is a necessary prerequisite for expression but binding alone is not sufficient for anaerobically induced expression to occur. The more detailed analysis of binding of AREF to the ARE is shown in

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-140

-99 'V*

*

CH1GCAGCCCCGGTTT CGCAAGCCGCGCCIGTGGTTT GCTTGCC GiACGTCGGGGCCAAA GCGTT CGGCGCGGCACCAAACGAACGG */

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*

In vivo dimethylsulfate protected residues (Ferl & Nick, 1987)

D

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D

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!..! Transcription not yet tested

Fig. 5. Important regions of the ARE. A summary of the results obtained with all mutant promoters.

Fig. 4 where it is clear that the GC-rich regions are critical for binding (GC-1, GC-2 and 4C) but the GGTTT sequence is not (MutGT-1, GT-2). The TGGTTT sequence (All) can be deleted with no loss of binding. Both the 4C box and the GC-1 and GC-2 sequences must be present for binding to occur. The regions we had identified as critical for binding and transcription are shown in Fig. 5. A much larger proportion of the ARE than we previously believed is important for both transcription and binding, and both subregions I and II must be present. The induction of the Adhl gene by anaerobic conditions is not caused by a greatly increased level of binding activity during anaerobic conditions. The binding activity is present at similar levels in the extract under both aerobic and anaerobic conditions. This suggests that when specific transcription occurs under anaerobic conditions a secondary modification of the ARE binding factor occurs (such as phosphorylation) and the T/ CGGTTT motif must also play a role. Alternatively, a second molecule could bind to AREF to convert the binding protein complex from an ineffective to an effective state. In vivo footprinting results of Ferl & Nick (1987) also suggest that proteins are associated with the ARE under both aerobic and anaerobic conditions. A possible oxygen sensor We have previously suggested that haemoglobin may act as an oxygen sensor in roots (Appleby et al., 1988). In plants, haemoglobin was orig-

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inally thought to be associated only with nodules of nitrogen-fixing plants where it transports oxygen at a suitable level to the terminal oxidases of the symbiotic bacteria. We have recently shown that haemoglobin is also present in the roots of the non-nodulating, non-nitrogen-fixing Trema and in the roots of Parasponia. Haemoglobin genes in the different species which maintain nitrogen-fixing symbioses have a common evolutionary origin and probably also share a common ancestry with animal haemoglobin genes. We suggested that haemoglobin has been maintained through evolution because of a function alternative to its nitrogen-fixing role. This alternative function may be to sense the level of oxygen in cells and, when it becomes too low, to invoke the anaerobic response. There is some precedent for this suggestion as in several cases a haem protein can act as an oxygen sensor and effect gene induction. In the erythropoietin system, hypoxia induces the expression of erythropoietin mRNA and a haem protein is integrally involved in the oxygen-sensing mechanism (Goldberg et al., 1988). A model in which a ligand-dependent conformational change regulates gene expression was proposed. Another example of a haem protein being postulated to play a role in hypoxic gene regulation is the expression of the nitrogen-fixation genes of Rhizobium meliloti which are induced under conditions of low oxygen tension. Two regulator proteins, FixJ and FixL, initiate the oxygen response cascade; FixL appears to be a transmembrane sensor that modulates the activity of the FixJ product. FixL is an oxygen-binding haemoprotein and it is proposed that FixL senses oxygen through its haem moiety and transduces the signal by phosphorylation of another protein (Gilles-Gonzales et al., 1991). Thus, in the anaerobic response the plant haemoglobin protein present in the roots could assume different conformations depending upon the oxygen concentration. This conformational change could activate another molecule which in turn could alter transcription factor binding and induce the anaerobic response (Fig. 6). This remains a most fascinating question in the anaerobic response: how does the plant sense that it is experiencing anaerobic conditions and mount a gene response? The anaerobic response is conserved Many plant species synthesise ANPs in response to anaerobic conditions. The most easily assayed feature, the induction of ADH by low oxygen tension, has been widely demonstrated. Induction of the anaerobic polypeptides and the cessation of aerobic protein synthesis has been shown to occur in maize, cotton and, to a lesser extent, soybean (Russell et al., 1990). That the DNA signals specifying anaerobic induction are

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Anoxia Haemoglobin Oxyhaemoglobin Modifies transcription factor Induces Anaerobic Polypeptides Fig. 6. Root haemoglobin as an oxygen sensor. Model for root haemoglobin sensing oxygen levels and inducing the anaerobic response. There could be many more steps in this pathway than shown in the figure.

conserved between species is shown by the fact that the maize Adhl promoter is anaerobically induced when introduced into the cereals (wheat, barley and rice) or even when linked to a suitable enhancer element and introduced into the dicot tobacco (Ellis et al., 1987). In the fermentative mode, lactate dehydrogenase is initially induced, but as lactate is produced, the pH of the cell decreases. At this point pyruvate decarboxylase (PDC) and ADH are induced. PDC produces acetaldehyde and ADH reduces this to ethanol. Acetaldehyde is a toxic compound and must be removed rapidly following its production by PDC. Once the switch to ethanolic fermentation occurs the pH of the cell is stabilised and plants such as maize can survive up to 72 h of anoxia. This ethanolic fermentation does not occur in vertebrates. The ability of plants to use this metabolic pathway for the regeneration of NAD used in glycolysis (Fig. 1) ensures that plants can maintain high levels of carbon flux through glycolysis while minimising acidosis which may be the result of lactate synthesis. This capacity explains why plants can survive anoxic conditions far better than vertebrates. Importance of the anaerobic response to survival during anoxia Seedlings which are mutant for the Adhl allele and do not show any ADH activity have a lower survival under anoxic conditions (Schwartz,

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1966; Jacobs et al., 1988). Under anoxic conditions alcoholic fermentation occurs and in the absence of this fermentation the pH continues to fall, and cytoplasmic acidosis and cell death occur (Roberts et al., 1984a,b). Mutants of maize containing only low levels of ADH activity survived but were unable to germinate when submerged under airsaturated water, while seeds containing wild-type ADH activity showed normal germination. Maize Adh] mutants with null or low ADH activity can be selected by germination under air-saturated water (Chen et al., 1986). We have been studying the anaerobic response in cotton, a crop which experiences a reduction in growth rate during irrigation or waterlogging. Cotton shows a level of anaerobically inducible ADH activity comparable with that of maize, a plant which is relatively resistant to anoxia (A. Millar, unpublished data; T.L. Setter, unpublished data). However, in cotton the level of the enzyme catalysing the preceding step in the fermentation pathway, PDC, is relatively low and this may lead to low rates of ethanol synthesis and hence low tolerance to anoxia. We are at present using molecular biology techniques in cotton to test the effects of variation in the levels of ADH and PDC on rates of ethanol synthesis and survival during anoxia (Fig. 7). cDNA clones containing the complete coding region of an anaerobically induced Adh gene have been isolated from cotton (A. Millar, unpublished data). A cDNA clone of maize PDC has been isolated using the polymerase chain reaction. Both coding regions are being cloned behind each of two promoters, the constitutive 35S promoter of cauliflower mosaic virus and the anaerobically inducible Adh promoter of pea. The clones are being introduced in the positive orientation to evaluate the effects of increasing the amount of ADH and PDC enzymes on survival under anoxia. The Adh constructs are also being introduced with the Adh open reading frame in the opposite orientation so as to produce antisense RNA and decrease the level of ADH protein. The constructs are being mobilised into Agrobacterium vectors, introduced into cotton hypocotyls or cotyledons, and callus generated. The levels of ADH and PDC in the callus will then be assayed together with the ability of the callus to grow and to survive during anoxic conditions. In this way we should be able to test the importance of ADH and PDC in the anaerobic response. A benefit of this methodology is that because individual transformed plants show variation in the levels of expression of introduced genes, transformed callus containing a range of levels of both enzymes will be obtained. Clearly, the relative activity levels of ADH, PDC and lactate dehydrogenase will be important and it may be too simplistic to envisage altering the level of one enzyme alone. A combination of overproducers achieved by extra copies of the Adh, Pdc or Ldh coding sequences, or underpro-

35S

Adh

Nos

peaAdh

Adh

Nos

35S

hdA

Nos

peaAdh

hdA

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Agrobacterium

Cotton callus

Anoxia survival ?

35S

Pdc

Nos

Pea Adh

Pdc

Nos

Agrobacterium

I I

Cotton callus

B

Anoxia survival ?

Fig. 7. Diagrams of the schemes for modifying levels of A, alcohol dehydrogenase and B, pyruvate decarboxylase activity and testing for survival of anoxia. In A, constructs contain the 35S promoter of the cauliflower mosaic virus (35S) driving expression of the cotton Adh cDNA in either the sense (Adh) or antisense (hdA) orientation, linked to the 3' termination signal of the nopaline synthase gene (Nos). Alternatively, the expression of cotton Adh cDNA is under control of the pea Adh promoter sequence (pea Adh). In B, either the 35S promoter or the pea Adh promoter is used to drive expression of the maize pyruvate decarboxylase cDNA (Pdc), linked to a Nos 3' termination sequence. Constructs are introduced into cotton via Agrobacterium tumefaciensmediated infection of cotton. Transformed cotton callus is then assayed for its ability to survive anoxia.

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ducers achieved by the use of antisense RNA or ribozymes against the appropriate mRNAs, may be necessary. The exciting thought is that it is possible to use molecular techniques to test these various hypotheses for survival during anoxia. Signal perception in the anaerobic response The Adh gene is induced by anaerobiosis and the response has been extensively studied. In order to study the conservation of the signals for anaerobic induction across the plant kingdom, and because of the power of molecular genetics and the ease of transformation of Arabidopsis, we have commenced analysis of the Arabidopsis Adh promoter. Sequences similar to the ARE core motif are present in the Arabidopsis promoter, and we are presently carrying out a deletion and specific mutagenesis analysis to delineate the sequences critical for anaerobic expression. The Arabidopsis Adh promoter is also induced by the auxin 2,4-D. Recently we have found that it is, in addition, inducible by low temperature (4 °C), 10~4M ABA and wilting. The induction by ABA is inhibited by GA3. It is also known that other Adh genes are induced by elicitors such as arachidonic acid. The multiplicity of stimuli capable of Adh induction raises the question of whether each inducer has a specific cw-regulatory sequence in the Adh promoter which binds a specific transcription factor, or whether each stimulus is working through a single promoter element interacting with a common signal transduction pathway which branches earlier in the pathway. An example of the latter model would be if mitochondrial function was inhibited by all these stimuli and induction of Adhl was mediated by a signal which indicates that energy supply is too low and fermentative metabolism should be activated. This possibility was supported when recent experiments with cotton showed that even moderate damage of roots by agitation for 5-10 min, with associated mitochondrial damage, showed a five-fold increase in root ADH activity similar to the five-fold induction seen during anoxia treatments. We intend to test the role of mitochondrial dysfunction by determining the effect of TCA cycle inhibitors or uncouplers on ADH induction. Since the Adh promoter is affected by cold, anoxia, wounding, etc. our name for the promoter regulatory region, the Anaerobic Responsive Element (ARE) may be too restrictive and perhaps it should more correctly have been called the Energy Regulatory Element or Energy Deficiency Regulatory Element in recognition of its more central role in plant metabolism.

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Acknowledgements This work was supported by the Cotton Research Council and Agrigenetics Company. References Appleby, C.A., Bogusz, D., Dennis, E.S. & Peacock, W.J. (1988). A role for haemoglobin in all plant roots? Plant, Cell and Environment 11, 359-67. Bailey-Serres, J., Kloeckner-Gruissem, B. & Freeling, M. (1988). Genetic and molecular approaches to the study of the anaerobic response and tissue specific gene expression in maize. Plant, Cell and Environment 11, 351-7. Chen, C.-H., Freeling, M. & Merckelbach, A. (1986). Enzymatic and morphological consequences of Ds excisions from maize Adhl. Maydica 31, 93-108. Dennis, E.S., Gerlach, W.L., Pryor, A.J., Bennetzen, J.L., Inglis, A., Llewellyn, D., Sachs, M.M., Fed, R.J. & Peacock, W.J. (1984). Molecular analysis of the alcohol dehydrogenase {Adhl) gene of maize. Nucleic Acids Research 12, 3983^000. Dennis, E.S., Gerlach, W.L., Walker, J.C., Lavin, M. & Peacock, W.J. (1988a). Anaerobically regulated aldolase gene of maize. A chimeric origin? Journal of Molecular Biology 202, 759-67. Dennis, E.S., Sachs, M.M., Gerlach, W.L., Beach, L. & Peacock, W.J. (19886). The Dsl transposable element acts as an intron in the mutant allele Adhl-Fm335 and is spliced from the message. Nucleic Acids Research 16, 3815-28. Dennis, E.S., Sachs, M.M., Gerlach, W.L., Finnegan, E.J. & Peacock, W.J. (1985). Molecular analysis of the alcohol dehydrogenase-2 (Adh2) gene of maize. Nucleic Acids Research 13, 727-43. Dennis, E.S., Walker, J.C., Llewellyn, D.J., Ellis, J.G., Singh, K., Tokuhisa, J.G., Wolstenholme, D.R. & Peacock, W.J. (1987). The response to anaerobic stress: Transcriptional regulation of genes for anaerobically induced proteins. In Plant Molecular Biology, ed. D. Von Wettstein & N.H. Chua, pp. 407-17. New York: Plenum Press. Ellis, J.G., Llewellyn, D.J., Dennis, E.S. & Peacock, W.J. (1987). Maize Adhl promoter sequences control anaerobic regulation: Addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco. The EMBO Journal 6, 11-16. Ferl, R.J. & Nick, H.S. (1987). In vivo detection of regulatory factor binding sites in the 5' flanking region of maize Adhl. Journal of Biological Chemistry 262, 7947-50. Gilles-Gonzales, M.A., Ditta, D.S. & Helinski, D.R. (1991). A haemoprotein with kinase activity encoded by the oxygen sensor of Rhizobium meliloti. Nature 350, 170-2.

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Goldberg, M.A., Dunning, S.P. & Bunn, H.F. (1988). Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 242, 1412-15. Howard, E.A., Walker, J.C., Dennis, E.S. & Peacock, W.J. (1987). Regulated expression of an alcohol dehydrogenase-1 chimeric gene introduced into maize protoplasts. Planta 170, 535-40. Jacobs, M., Dolferus, R. & Van Den Bossche (1988). Isolation and biochemical analysis of ethyl methane sulfonate induced alcohol dehydrogenase null mutants of Arabidopsis thaliana (L.) Heynth. Biochemical Genetics 26, 105-12. Kelley, P.M. & Freeling, M. (1984a). Anaerobic expression of maize glucose phosphate isomerase 1. Journal of Biological Chemistry 259, 673-7. Kelley, P.M. & Freeling, M. (1984b). Anaerobic expression of maize fructose-l,6-diphosphate aldolase. Journal of Biological Chemistry 259, 14180-3. Lazlo, A. & St Lawrence, P. (1983). Parallel induction and synthesis of PDC and ADH in anoxic maize roots. Molecular and General Genetics 192, 110-17. Llewellyn, D.J., Finnegan, E.J., Ellis, J.G., Dennis, E.S. & Peacock, W.J. (1987). Structure and expression of an alcohol dehydrogenase I gene from Pisum sativum (cv. Greenfeast). Journal of Molecular Biology 95, 115-23. Olive, M.R., Walker, J.C., Singh, K., Dennis, E.S. & Peacock, W.J. (1990). Functional properties of the anaerobic responsive element of the maize Adhl gene. Plant Molecular Biology 15, 593-604. Roberts, J.K.M., Callis, J., Jardetzky, O., Walbot, V. & Freeling, M. (1984a). Cytoplasmic acidosis as a determinant of flooding intolerance in plants. Proceedings of the National Academy of Sciences (USA) 81, 6029-33. Roberts, J.K.M., Callis, J., Wemmer, D., Walbot, V. & Jardetzky, O. (1984b). Mechanism of cytoplasmic pH regulation in hypoxic maize root tips and its role in survival under hypoxia. Proceedings of the National Academy of Sciences (USA) 81, 3379-83. Rowland, L.J. & Strommer, J.N. (1986). Anaerobic treatment of maize roots affects transcription of Adhl and transcript stability. Molecular and Cellular Biology 6, 3368-72. Russell, D.A., Wong, D.M.L. & Sachs, M.M. (1990). The anaerobic response of soybean. Plant Physiology 92, 401-7. Sachs, M.M., Freeling, M. & Okimoto, R. (1980). The anaerobic proteins of maize. Cell 20, 761-7. Schwartz, D. (1966). An example of gene fixation resulting from selective advantage in suboptimal conditions. American Naturalist 103, 479-81. Springer, B., Werr, W., Starlinger, P., Clark Bennett, D., Zokolica, M.

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& Freeling, M. (1986). The Shrunken gene on chromosome 9 of Zea mays L. is expressed in various plant tissue and encodes an anaerobic protein. Molecular and General Genetics 205, 461-8. Walker, J.C., Howard, E.A., Dennis, E.S. & Peacock, W.J. (1987). DNA sequences required for anaerobic expression of the maize alcohol dehydrogenase 1 gene. Proceedings of the National Academy of Sciences (USA) 84, 6624-8.

F. SCHOFFL, V. DIEDRING, M. KLIEM, M. RIEPING, G. SCHRODER and K. SEVERIN

The heat shock response in transgenic plants: the use of chimaeric heat shock genes

Introduction The heat shock (hs) response in living cells is a fascinating subject for studying molecular mechanisms of stress-dependent regulation of gene expression and its phenotypic consequences (for reviews see Schofrl, 1988; Neumann etai, 1989; Morimoto etal., 1990; Nover, 1990). Particularly important is the transient acquisition of increased thermotolerance following a sublethal heat stress. Although exceptions occur, a positive correlation exists between the amount of hs proteins (hsps) synthesised in response to hyperthermia and the degree of tolerance. The synthesis of hsps seems to be necessary but may not be sufficient for thermotolerance. In different organisms different hsps are important for thermotolerance; the inability to synthesise hsps is usually correlated with thermosensitivity and an inability to acquire thermotolerance. An example of a naturally occurring inability to synthesise hsp60, the sole hsp of the genus Hydra, is found in the thermosensitive species H. oligactis which is restricted to an ecological niche where it rarely encounters increasing water temperatures (Bosch etal., 1988). Mutations negatively affecting the hs response and thermotolerance properties have been described for Dictyostelium, Tetrahymena, Escherichia coli and yeast (for reviews see Neidhardt etal., 1984; Lindquist, 1986; Lindquist & Craig, 1988). A contrasting phenotype is exhibited by double mutations in two ubiquitin-conjugating enzymes of yeast which lead to the constitutive expression of hsps (Jentsch et al., 1990). The expression of hs genes is primarily regulated at the level of transcription. The coordinate transcription is initiated by the binding of the activated hs factor (HSF) to conserved promoter elements (HSE) present in multiple copies in the promoter regions of hs genes. During high Society for Experimental Biology Seminar Series 49: Inducible Plant Proteins, ed. J. L. Wray. © Cambridge University Press, 1992, pp. 247-266.

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temperature stress the hs-induced transcripts are preferentially translated into hsps which belong to different molecular mass classes. These classes are grouped into high molecular mass (hmm) hsps comprising hsp60, hsp70, hsp80-90 classes and low molecular mass (lmm) hsps comprising hsps below 30 kDa. Hmm hsps are generally considered as molecular chaparonins which are involved in transport, maturation and assembly of temporarily associated intracellular proteins. The functional properties of lmm hsps are less well investigated. However, hsps belonging to this group are structurally related, most prevalent in plants, and certain members are transported into chloroplasts. Most investigations of hs-regulated gene expression in plants have focused on genes of this group, particularly from soybean. The use of transgenic plants has become increasingly important for the analysis of transcriptional regulation of hs genes. The scrutiny of sequences which are required for heat-inducible, high levels of expression is highly relevant for the identification of target sites for tarns-acting regulatory factors. The delimitation and use of such sequences in chimaeric gene constructions have great potential for hs-regulated expression of genes that may alter genetic traits, e.g. pathogen resistance, transposable elements, plant growth regulators, and for the isolation of genes and mutants that affect the hs response, e.g. HSF and other components of the signal transduction pathway. Sequences involved in transcriptional regulation can be subdivided into target sites for regulation of heat inducibility and sequences affecting the amplitude of the transcription. Both categories of sequences are located predominantly in the 5' flanking region of the soybean hs genes (Baumann etal., 1987; Schoffl etal., 1989; Czarnecka etal., 1989, 1990). Heat inducibility is mediated by HSEs with the general consensus sequence of alternating units of -GAA- and -TTC- (Schoffl et al., 1991). At a minimum, three units are required for in vitro binding of multimeric HSF complexes isolated from yeast and Drosophila (Sorger & Nelson, 1989; Perisic et al., 1989). The multiple binding sites which occur in most natural hs-promoters seem to be particularly important for cooperative binding of HSF and perhaps for differential expression of hs genes (Xiao etal., 1991). Accessory sequences, upstream from HSEs containing soybean hs promoter regions, have a significant influence on the amplitude of gene expression (Baumann etal., 1987; Czarnecka et al., 1989). Little is known about the functional structures within these regions and the molecular mechanism that enhances hs gene expression. Other sequences, e.g. hs gene mRNA leader and trailer sequences, are also very important for

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faithful regulation of chimaeric gene expression in plants (Schoffl et al., 1989, 1991) but are not considered further here. Several strategies have been outlined for manipulation of the hs response in transgenic plants using chimaeric hs genes (Schoffl, 1988). These manipulations can be focused on either alterations of the expression of single hs genes and gene families or on changes of the expression of the whole set of hs genes and proteins. Pleiotropic, negative regulatory mutations that alter the expression of all hs genes would presumably have the most pronounced impact on thermotolerance. However, they must be the most difficult to isolate. A prerequisite to mutant isolation is the development of suitable selection schemes applicable to plants. The use of chimaeric hs genes as possible selection markers for mutations in the hs response will be discussed. Manipulation of individual hs genes or gene families has a lower priority. However, considering the detrimental effects that pleiotropic regulatory mutations could cause (for example HSF~ mutants of yeast: Sorger & Pelham, 1988), it also seems rational to follow strategies that would affect only parts of the hs response system, for example constitutive expression of hs sense and antisense RNA. Here we describe the constitutive, heat-stable transcription of a soybean hs gene and its antisense variant in transgenic tobacco. The homology between tobacco and soybean hsp gene families is evident but it is not yet known whether the similarities between their nucleotide and protein sequences are sufficient to result in functional modifications with respect to the heat shock response. Functional analysis of heat shock promoter elements The soybean hs gene Gmhspl7.3-B is one of the primary targets for functional analysis of a plant hs promoter. 5' deletions of the native gene (Baumann et al., 1987) and 3' promoter deletions fused to the chloramphenicol acetyltransferase (CAT) reporter gene were analysed in transgenic tobacco plants (Schoffl et al., 1989). Overlapping HSEs were identified as functionally significant sequences for heat inducibility of gene expression. Multiple HSEs seem to modulate the amplitude of transcription. Other elements, e.g. CCAAT box sequences which are present in three copies immediately upstream from the most distal HSE of Gmhspl7.3-B, also exert a stimulatory influence on promoter activity. One single upstream HSE, which is usually not sufficient for heat inducibility of transcription, is active in chimaeric genes when flanked by its native CCAAT box sequences (Rieping & Schoffl, 1992). Other upstream sequences, including a run of adenine/thymine bases, stimulate

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hs-induced transcription of the Gmhspl7.3-B promoter synergistically, as demonstrated by chimaeric constructs with the P-glucuronidase (GUS) reporter gene. However, elevated levels of gene expression of both heatinduced and basal GUS activity are observed (Schoffl et al., 1991). The leakiness of hs promoter activity is a phenomenon common to all plant systems investigated to date. Traces of native Gmhspl7.3-B message were detected by Sl-mapping in 25 °C control mRNA isolated from soybean seedlings (Schoffl & Baumann, 1985). Higher levels of promoter leakage, apparently indicated by reporter gene activity, may result from higher stability of the chimaeric mRNA especially when terminated by the commonly used 3' NOS-ter sequence (Schoffl et al., 1991). Replacement of NOS-ter by the native hs-ter region has contrasting effects on mRNA levels during hs and recovery, probably because of differences in RNA stability (M. Rieping and F. Schoffl, unpublished data). Very little is known about developmental regulation of plant hs genes. In pea, small hsps are synthesised during embryogenesis and early germination (Vierling & Sun, 1987). Developmentally dependent hsps and their mRNAs were found in dry, and early imbibing, embryos of wheat (Helm & Abernethy, 1990). Similar, as yet unconfirmed, observations in other plants suggest a common concept of developmental regulation of hsps during embryogenesis. Since nothing is known about this type of temperature-independent regulation of hs gene expression in tobacco, we considered it worthwhile to test the soybean hs promoter for developmental induction of the CAT reporter gene in seeds derived from selfed plants. Seeds containing a hs promoter —321/—152 CaMV-CATter gene (Schoffl et al., 1989) or a similar construct with a synthetic hs promoter, 5XHSE2 (Schoffl et al., 1990), were tested for CAT activity. Neither imbibed nor dry seeds showed significant levels of CAT activity without hs (Fig. 1). This result suggests that a putative developmental regulation of this soybean hs gene in tobacco requires additional ris-active sequences which are not present in the region spanning HSE and CCAAT box elements. However, we cannot exclude the possibility that post-transcriptional insufficiencies, e.g. adverse effects on CAT protein deposition in tobacco seeds, or tissue specificity of developmental regulation, interfere with the detection of developmentally regulated reporter gene activity. On the other hand, heat inducibility of these gene constructs (Fig. L4) seems to be drastically impaired in imbibed seeds. Seeds of 5xHSE2 CaMV-CATter derived plants show a small but insignificant increase of the otherwise basal levels of CAT activity after hs (Fig. 1). However, this maximum level of CAT activity is much lower than the basal levels measured in protein extracts of leaves from the non-induced parental plants (Fig. 1). Two to 20 hours of imbibition prior to the heat treatment

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A Gmhsp17.3-B -'"

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B 5 x HSE2

Gmhsp17.3-B

5 x HSE2

seed

leaf

acm

cm

ds

is

is hs

ds

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is

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Fig. 1. CAT activities in seeds of transgenic tobacco plants containing hs promoter-CAT constructs. A, Schematic structure of chimaeric genes introduced into tobacco. Details of the construction are described by Schoffl et al. (1989, 1991). HSE sequences with the consensus C-GAATTC-G are symbolised by boxes; the synthetic HSE2 is represented by two overlapping soybean HSEs. The CaMV promoter is a truncated silent version of the 35S promoter, providing only the TATA box and the transcription start site. B, CAT assays were performed as described by Schoffl et al. (1989), using 50 u.g protein from seed extracts and 10 ng from leaf extracts. Dry seeds (ds) without imbibition and 20 h imbibed seeds (is), derived from the same plant, were used. Heat treatment (hs) was carried out for 2 h at 40 °C prior to protein extraction. Control extracts (c) were prepared from leaves incubated at 25 °C. cm, l4Cchloramphenicol; acm, acetylated form of cm.

were not sufficient to increase the CAT activities in seeds significantly. Lethal effects can be excluded as a possible cause of this inability since the rate of germination was not adversely affected by the heat treatment. However, the inability of seeds to respond to heat stress during early

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Gmhsp 17.3-B

Gmhsp 18.5-C

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00

•

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germination is in accordance with similar observations in other systems. Lack of heat inducibility of hs genes at certain developmental stages has been observed during embryogenesis in Drosophila (Zimmerman et al., 1983) and embryogenesis/early germination of wheat (Helm & Abernethy, 1990). It is still conceivable that the ability to elicit the hs response is not completely absent in germinating plant seeds. However, it might be confined to certain tissues of the embryo and/or seed and therefore escape detection in total extracts of seed proteins. Impaired translation of CATmRNA during hs can be largely excluded since it is known that 35SmRNA initiated transcripts are efficiently translated in plants (Schoffl et al., 1989). Gene fusion constructs with the extended Gmhspl7.3-B promoter containing a large part of the 5' non-translated leader region showed the same result (data not shown). Cis-active SAR sequences and heat shock gene expression It is a general observation that the expression of introduced genes varies between individual transgenic plants and does not appear to be correlated with differences in gene dosage. This shortcoming is inconvenient for a quantitative, functional analysis of hs genes (Baumann et al., 1987; Schoffl et al., 1989). Position-independent, high levels of hs-regulated gene expression are a prerequisite for many applications involving regulatory hs promoter sequences (Schoffl, 1988). Native 5' upstream sequences flanking soybean hs promoter regions have pronounced effects on the level of transcription of Gmhspl7.3-B (Baumann et al., 1987) and Gmhspl7.5-E (Czarnecka et al., 1989) in transgenic plants. One common structural feature of these regions is the occurrence of A- and T-rich sequences which seem to interact with Fig. 2. Scaffold attachment regions (SAR) flanking soybean hs genes. The sequences of the small soybean hs genes were screened for the presence of scaffold binding sequences: • , Drosophila A-box consensus AATAAA(TC)AAA; • , T-box consensus TT(AT)T(TA)TT(TA)1T (Gasser & Laemmli, 1986); T, topoisomerase II consensus GTN(AT)A(TC)ATTNATNN(GA) (Sander & Hsieh, 1985); • , mouse MAR consensus AATATTTTT; O, reduced MAR consensus ATATTT (Cockerill & Garrard, 1986). The hs genes were aligned with respect to their coding sequence (bold); the start site of transcription is indicated by an arrow. Fragments identified by in vitro binding to isolated nuclear scaffolds (SAR), their position and that of non-binding flanking fragments, are indicated.

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nuclear proteins (Schoffl et al., 1990; Czarnecka et al., 1990). The motifs for topoisomerase II binding, and scaffold or matrix attachments, are very similar to these sequences. Scaffold attachment regions (SARs) were shown to cohabit with enhancer-like sequences in Drosophila (for review see Jackson, 1986; Gasser & Laemmli, 1987). The proximity of such sequences to the lysozyme gene in chimaeric constructions introduced into animal cells results in elevated and gene dosage-dependent gene activity (Stief et al., 1989). A computerised search for potential SARs, matrix attachment regions (MAR) and topoisomerase II binding sites in soybean hs genes revealed a surprisingly frequent occurrence of such sequences. They are located at a relatively short distance upstream and/or downstream from most of the genes (Fig. 2). A number of fragments containing SAR and MAR sequences become selectively associated with nuclear scaffolds in vitro (M. Kliem and F. Schoffl, unpublished data). Highest binding affinity was shown for a 395 bp fragment of the 3' flanking region of Gmhspl7.6-L. This fragment contains at least two MAR and one topoisomerase II consensus sequences, and was chosen for testing its functional properties with respect to heat-inducible expression of a chimaeric Gmhspl7.3l-12 hs promoter-GUS reporter gene (Schoffl et al., 1991) in transgenic tobacco plants. Constructions were made that contained this fragment either at one side (5' or 3') or at both sides of the test gene. Several plants were analysed for each individual type of chimaeric construction by determination of GUS activity and gene copy number (data not shown). A 4 to 10-fold increase in the average heat-inducible GUS activity was observed in plants containing constructs with SAR fragments at both sides. A 3 to 4-fold elevation of the levels of GUS activity was determined for constructs containing the SAR fragment only at the 5' flanking location, but no stimulation was observed when it was located in the 3' flanking region (F. Schoffl etal., unpublished data). These results suggest that SAR sequences have a similar stimulatory effect on expression of closely linked genes in plants as they do in animal cells. The molecular mechanism by which this enhancement of gene expression is brought about is still obscure. We have not yet tested whether SAR sequences from the flanking regions of hs genes are also effective for enhanced expression of non-hs test genes. It may be that position effects on gene expression are caused to some extent by the presence or absence of endogenous SAR sequences in proximity to integrated transgenes. Position effects may become less pronounced if SARs are co-introduced as flanking sequences, since a correlation between gene activity and copy number can be deduced (F. Schoffl et al., unpublished data). Scaffold attachment may influence adjacent genes either by torsional changes of

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DNA conformation, allowing access of trans-iactors to regulatory regions, or alternatively by maintaining close contact between genes and the transcriptional machinery, which is presumed to be located at the basis of chromosomal scaffolds (Jackson, 1986). Constitutive expression of heat shock genes Constitutive expression of an otherwise tightly regulated gene is one possible means for changing the functional and phenotypic properties of a genetic trait. The properties of thermal tolerance may also be affected by constitutive expression of individual hs genes as shown for Hsp70, which accelerates the recovery of nucleolar morphology following hs (Pelham, 1984). Constitutive transcription of the soybean Gmhspl7.6-L gene driven by the CaMV-35S promoter yielded high levels of mRNA at 25 °C but only reduced levels following hs (Schoffl et al., 1987). This reduction was thought to be a consequence of decreased promoter activity at high temperature. Improvement of the expression properties might be expected by the use of dual promoters which are active at both normal and heat shock temperatures. Two types of design were chosen for generating a 'constitutive hs-promoter'. One type of construction contains the constitutive CaMV-35S promoter together with the synthetic HSE2 promoter elements, whilst the other is based on the native hs promoter but is flanked by the constitutive enhancer of the 35S promoter (Fig. 3). The native CaMV-35S promoter contains all sequences necessary for high levels of constitutive transcription (Schoffl et al., 1987). The synthetic HSE2 sequences (Schoffl et al., 1989) were inserted into the EcoRV site, located 59 bp upstream from the native TATA box sequence of the 35S promoter. The promoter was linked within the 5' leader sequences via suitable restriction sites to the Gmhspl7.6-L protein-coding sequence. The transcript levels derived from this gene in transgenic tobacco plants indicate that the properties of constitutive and heat-stable transcription are exhibited (Fig. 4). This finding suggests that HSE2 sequences confer heat inducibility to the constitutive and otherwise heat stress-impaired promoter. The interruption of the 35S promoter by the two HSE2 sequences has no detectable effect on constitutive transcription. The addition of the 35S enhancer region, spanning the 336 bp Accll Hgal fragment (positions — 55/-392 in the CaMV promoter), to a location upstream of the hsL gene promoter, yields only heat-inducible but no constitutive transcripts of Gmhspl7.6-L (Fig. 4). This unexpected result suggests an incompatibility between the basal hs promoter and the constitutive enhancer complexes. Other genes, for example the maize

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etal. Gmhsp17.6-L !Si»T

HSE

TATA

CaMV-Enhancer

B

CaMV-P

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TATA

Fig. 3. Schematic diagrams of chimaeric Gmhspl7.6-L genes transcribed constitutively in sense and antisense orientation. A, The native soybean hs gene (Schoffl et al., 1990, 1991). The protein-coding region is indicated by dark boxes; the orientation is marked by an arrow. B, The same gene as in A except for the replacement of the 5' promoter upstream region by the CaMV-35S enhancer. C, The hs promoter of Gmhspl7.6L is replaced by the complete CaMV-35S promoter including also the TATA box and transcriptional start site. Two synthetic HSE2 were inserted upstream from the TATA box sequence. This design adds heat inducibility of transcription to the constitutive promoter. D, Same construct as C but with an internal inversion of the coding region.

A hs

B c I hs

Ci c I hs

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I hs

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c I hs c | hs

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Adh-1 and a truncated wheat Cab-1 gene promoter, are activated in transgenic tobacco by this enhancer region (Ellis et al., 1987; Nagy et al., 1987). We cannot exclude the possibility that the enhancer has a stimulatory effect on heat-activated transcription, but it is difficult to discriminate between enhancer function and position effects. A larger sample of independent transgenic plants has to be tested to determine a possible enhancement of hs-induced transcription.

The generation of antisense hsRNA The strategy of antisense RNA transcription has been successfully applied to inhibit gene expression specifically and hence generate a mutant phenotype. Complementary sequences between sense (mRNA) and antisense RNA are prerequisite for this effect, and one criterion for effective antisense RNA function has been the concomitant reduction within the cells of the target mRNA level and enzyme activity, for example as shown for the suppression of polygalacturonase in tomato (Sheehy et al., 1988). Antisense hs gene constructions were made using the dual 'constitutive hs promoter' described above. This promoter was linked with the Gmhspl7.6-L gene coding region containing a large inversion (Fig. 3). The antisense character of this gene was generated by an internal inversion of a 324 bp fragment, which was generated by Bsml/Styl digestion followed by reinsertion into the same site of the gene after blunt end formation. Tobacco plants transformed with this antisense gene yielded different levels of antisense RNA. One class of transgenic plants showed reduced levels of antisense RNA after hs (Fig. 5A), but no reduction of endogenous levels of tobacco hs-mRNAs was detectable (Fig. 5B). The lack of transgenic plants with both reduced levels of antisense RNA and endogenous hs-mRNAs suggests that the homology between soybean and tobacco small hsp genes is not sufficient to exert the expected negative effect. It is, however, conceivable that the antisense RNA has effectively

Fig. 4. Constitutive transcription of Gmhspl7.6-L mRNA in transgenic tobacco plants. The constructs A, B and C shown in Fig. 3 have been used for transformation of tobacco. Total RNA was isolated from leaves of individual plants which had been subjected to heat stress (hs) for 2 h at 40 °C, or incubated only at 25 °C (c). 30 ug RNA per lane were separated by electrophoresis and blots were hybridised with a genespecific DNA probe (Schoffl et al., 1987). A,B,C refer to transgenic plants transformed with the chimaeric genes depicted in Fig. 3. C, and Q represent two individual plants. RNAs from soybean (sb) and untransformed tobacco (tob) were used as references.

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tob hs

. sense • C c i hs

c ' hs

antisense D, D2 c

hs

D3

c > hs

c

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*

Fig. 5. Gmhspl7.6-L antisense RNA in transgenic tobacco and levels of endogenous hs mRNAs. Sense (C) and antisense constructs (D) as depicted in Fig. 3 were used for transformation of tobacco. Total RNA was isolated from leaves of individual plants which had been subjected to heat stress (hs) for 2 h at 40 °C, or incubated at 25 °C (c). 30 u.g RNA per lane were separated by electrophoresis and blots were hybridised (A) with the same gene-specific DNA probe as used in Fig. 4. This hybridisation identifies the levels of antisense RNA in individual plants D 1; D2 and D3. Hybridisation (B) of an identical blot with a single stranded antisense RNA probe representing only the inverted region of construct D (Fig. 3), shows the levels of hs-induced endogenous tobacco hsp-mRNAs. tob, untransformed tobacco plants used as a control.

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eliminated one highly homologous (complementary) tobacco hs mRNA species but failed to reduce the other, less conserved hs mRNAs which still cross-hybridise. Only if the thermotolerance properties of transgenic plants were changed dramatically could one conclude that soybean antisense RNA is effective in other plant species. To date there is no evidence for either a change of hsp synthesis patterns or altered thermotolerance properties. The heat shock transcription factor, HSF HSF is the central target for manipulation of the hs response. The conservation of its DNA-binding site in virtually all hs genes underscores its importance in the transduction of stress signals into transcriptional activation of hs genes. The activity of HSF is heat stress-dependent; the nature of this activation and the mechanism by which it activates transcription of hs genes is still unknown. In plants, genes encoding HSF have been isolated from cDNA expression libraries of tomato by DNA ligand screening using a synthetic oligonucleotide (Scharf et al., 1990). In contrast to yeast and Drosophila, HSF of plants appears to be encoded by more than one gene. At least three different types of cDNAs have been isolated by South-Western screening. These tomato Lp-HSF genes are divergent; sequence similarity is confined only to the putative DNAbinding domain, spanning approximately 90 amino acid residues near the N-terminus, and a nearby leucine zipper motif. These common structural motifs are also found in yeast HSF (Sorger & Pelham, 1988; Wiederrecht et al., 1988) and Drosophila HSF genes (Clos et al., 1990). The small size of the tomato HSF Lp-HSF24 of approximately 33 kDa, deduced from a nearly full-length cDNA clone, and the heat inducibility of its mRNA, are surprising features of the plant system. Southern blot analyses showed that Lp-HSF24 cross-hybridises with different but distinct DNA fragments of different plant species including soybean, tobacco and Arabidopsis (data not shown). This is the first clue to a perhaps common feature that the hs response is mediated by heterooligomeric HSF complexes. It is also conceivable that different types of HSF regulate different classes of hs genes or are involved in autoregulation and suppression of active multimeric forms of HSF (Clos et al., 1990). With the availability of gene probes for plant HSF it seems possible to manipulate the hs response in transgenic plants. However, the possibility that multiple and different HSF genes are involved in mediating the hs response in plants may complicate the analysis.

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HSF-dependent chimaeric hs genes as selection markers If the model of multiple, different and alternative HSF molecules holds true for plants, it will be difficult to isolate mutations in HSF genes. However, if there is a common path of signal transduction that leads to the activation of HSF and subsequently to the transcription of hs genes, then there is a possibility that mutants can be generated and hopefully selected by a suitable phenotype. Different hs promoter/reporter gene constructions have been employed to reach this goal. A heat-inducible hygromycin resistance gene, driven by a soybean Gm.hspl7.6-L hs promoter, was used for generating heat-inducible levels of resistance to the drug in tobacco (Severin & Schoffl, 1990). This same chimaeric hpt gene can also be induced by repeated application of hs to transgenic Arabidopsis thaliana plants, as shown in Fig. 6. The faithful regulation of this gene and the high levels of antibiotic resistance generated allow the possibility of selecting mutants of the transgenic host plants that survive on hygromycin at room temperature. Such frans-acting mutations should cause a constitutive activation of HSF and thus elicit a constitutive hs

Gmhsp17.6-L

hpt

nos

Fig. 6. Heat-inducible hygromycin resistance in transgenic Arabidopsis seedlings containing the chimaeric hs promoter-hpt gene. F2 seeds from a transgenic plant containing the chimaeric construct shown were germinated on MS medium containing 30mg/l hygromycin. Plants were grown for 4 weeks, A, continuously at 25 °C, or B, at 25 °C but interrupted for 1 h incubation at 37 °C every second day.

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response. This selection scheme would allow the isolation of mutations either in HSF or in any component of the signal pathway leading to the activation of HSF. The reverse type of mutations, that is those exhibiting a HSF null phenotype, have an even lower probability of being selected. A defect in one HSF gene might be compensated by other HSFs, and a mutation that negatively affects the activation of all the different HSFs might be lethal. The yeast HSF gene is essential for growth (Sorger & Pelham, 1988), but nothing is known about the activation and function of HSF in other systems. One possible strategy to select a HSF" phenotype is based on the use of a heat-inducible adh gene which has been placed in an Adh null background. Adh null lines of Arabidopsis are available (Jacobs et al., 1988) and have been transformed with an Arabidopsis adh gene that is under the control of a soybean hs promoter (K. Severin and F. Schoffl, unpublished data). Transgenic lines containing the Adhf* gene express significant levels of Adh activity only after heat stress. These levels are sufficient to cause susceptibility to allyl alcohol (data not shown) and thus it might be possible to use this approach for the isolation of HSF" mutations. Perspectives The manipulation of the hs response in plants by genetic engineering will contribute significantly to our understanding of the molecular mechanisms underlying stress-related control of gene expression. Moreover, it may also provide deeper insights into stress physiology and initiate the novel design of agronomically important genetic traits. The studies presented in this paper are the first attempts to generate mutants and other heritable phenotypic changes of the hs response by reverse genetics. The use of chimaeric hs genes has been rewarding with respect to the identification and functional characterisation of cisregulatory sequences, e.g. hs promoter elements, SAR- and enhancerlike sequences (Schoffl et al., 1989, 1990, 1991). Future subjects for this type of investigation will be the as yet largely neglected field of developmental regulation of hs genes. The results of our preliminary study indicate that a developmental stimulus requires additional sequences which are not present in a typical, HSE-containing region of a soybean hs promoter. It will also be important to know why certain, but not all, hsps are developmentally regulated and expressed during embryogenesis and whether this 'developmental response' is related to heat stress resistance, water deficiency or developmental processes (Vierling & Sun, 1987; Helm & Abernethy, 1990).

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F. SCHOFFL

etal.

Not directly related to the hs response may be the influence of SAR sequences on the level of chimaeric gene expression in transgenic plants. The proximity of SAR sequences to soybean hs genes implies a correlation between scaffold attachment and the amplitude of gene expression, as suggested by Gasser & Laemmli (1987) and Jackson (1986). Many applications involving chimaeric genes will benefit if this stimulatory effect is not restricted to hs promoters but can be extended to the expression of other transgenes in plants. So far transcriptional regulation has been the primary target for investigations of hs-regulated gene expression, but the focus of research will undoubtedly shift towards the secondary levels of regulation, that is the processes of post-transcriptional control, for example of mRNA stability and translatability. 5' and 3' nontranslated regions of mRNAs seem to be involved in these processes (Schoffl et al, 1989; M. Rieping and F. Schoffl, unpublished data). These may not only contribute to differential expression of hs genes, since translational control also seems to dominate the expression of hsps in certain developmental stages (Helm & Abernethy, 1990). A new field in the investigation of the hs response became accessible when the hs transcription factor and its genes were isolated from yeast (Sorger & Pelham, 1988; Perisic et al, 1989). Although this factor recognises the same type of target sequences, the HSF and its genes isolated from yeast, Drosophila, mammals and plants, differ extensively in their primary nucleotide and deduced amino acid sequences (Clos et al, 1990; Scharf et al, 1990). The most conserved regions are the DNAbinding domain (approximately 50% identity) including also a short helixturn-helix motif present in E. coli sigma-factors (Clos et al, 1990), and an array of heptad repeats of hydrophobic residues representing a leucine zipper motif which seems to be important for multimerisation of HSF by coiled-coil interaction. It is still a matter for discussion as to whether the activation of HSF by hs is intrinsic to its metastable structure or due to covalent modifications, for example phosphorylation, or to its release from repressing components (Clos et al, 1990; Jakobsen & Pelham, 1991). The isolation of several different cDNA sequences for HSF from tomato adds an as yet unknown complexity to the hs response system (Scharf et al, 1990) and renders it more difficult to make predictions about manipulations concerning the hs response via HSF. The generation and selection of mutants affecting the signal transduction pathway and consequently the hs response will inevitably depend on the molecular mechanism by which HSF is activated. The generation of Arabidopsis lines, containing either hs-inducible hygromycin resistance or Adh genes, provides suitable genetic backgrounds for selection of possible mutants. If such pleiotropic positive or negative mutations of the hs response cannot be obtained because they may be lethal, other strategies for

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manipulation, e.g. overexpression of distinct hsps and blocking of gene expression by antisense RNAs, become more important.

Acknowledgements We thank Ute Lutterschmid and Petra Heller for their help in preparing the manuscript. The research was supported by grants from the Deutsche Forschungsgemeinschaft (Scho 242/5-1) and Stiftung Volkswagenwerk (1/ 65 985).

References Baumann, G., Raschke, E., Bevan, M. & Schoffl, F. (1987). Functional analysis of sequences required for transcriptional activation of a soybean heat shock gene in transgenic tobacco plants. The EMBO Journal 8, 2195-202. Bosch, T.C.G., Krylow, S.M., Bode, H.R. & Steele, R.E. (1988). Thermotolerance and synthesis of heat shock proteins: these responses are present in Hydra attenuata but absent in Hydra oligactis. Proceedings of the National Academy of Sciences (USA) 85, 7927-31. Clos, J., Westwood, J.T., Becker, P.B., Wilson, S., Lambert, K. & Wu, C. (1990). Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation. Cell 63, 1085-97. Cockerill, P.N. & Garrard, W.T. (1986). Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44, 273-82. Czarnecka, E., Fox, P.C. & Gurley, W.B. (1990). In vitro interaction of nuclear proteins with the promoter of soybean heat shock gene Gmhspl7.5-E. Plant Physiology 94, 935-43. Czarnecka, E., Key, J.L. & Gurley, W.B. (1989). Regulatory domains of the Gmhspl7.5-E heat shock promoter of soybean. Molecular and Cellular Biology 9, 3457-63. Ellis, J.G., Llewellyn, D.J., Dennis, E.S. & Peacock, W.J. (1987). Maize Adh-1 promoter sequences control anaerobic regulation: addition of upstream promoter elements from constitutive genes is necessary for expression in tobacco. The EMBO Journal 6, 11-16. Gasser, S.M. & Laemmli, U.K. (1986). Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. Cell 46, 521-30. Gasser, S.M. & Laemmli, U.K. (1987). A glimpse at chromosomal order. Trends in Genetics 3, 16-22. Helm, K.W. & Abernethy, R.H. (1990). Heat shock proteins and their mRNAs in dry and early imbibing embryos of wheat. Plant Physiology 93, 1626-33.

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Jackson, D.A. (1986). Organization beyond the gene. Trends in Biochemical Sciences 11, 249-52. Jacobs, M., Dolferus, R. & Van Den Bossche, D. (1988). Isolation and biochemical analysis of ethyl methanesulfonate-induced alcohol dehydrogenase null mutants of Arabidopsis thaliana (L.) Heynh. Biochemical Genetics 26, 105-22. Jakobsen, B.K. & Pelham, H.R.B. (1991). A conserved hepta peptide restrains the activity of the yeast heat shock transcription factor. The EMBO Journal 10, 369-75. Jentsch, S., Seufert, W., Sommer, T. & Reins, H.-A. (1990). Ubiquitinconjugating enzymes: novel regulators of eukaryotic cells. Trends in Biochemical Sciences 15, 195-8. Lindquist, S. (1986). The heat-shock response. Annual Reviews of Biochemistry 55, 1151-91. Lindquist, S. & Craig, E.A. (1988). The heat-shock proteins. Annual Review of Genetics 22, 631-77. Morimoto, R.I., Tissieres, A. & Georgopoulos, C. (ed.) (1990). Stress Proteins in Biology and Medicine. Cold Spring Harbor: Cold Spring Harbor Laboratory Press. Nagy, F., Boutry, M., Hsu, M.Y., Wong, M. & Chua, N.-H. (1987). The 5'-proximal region of the wheat Cab-1 gene contains a 268 bp enhancer-like sequence for phytochrome response. The EM BO Journal 6, 2537^2. Neidhardt, F.C., VanBogelen, R.A. & Vaughn, V. (1984). The genetics and regulation of heat-shock proteins. Annual Review of Genetics 18, 295-329. Neumann, D., Nover, L., Parthier, B., Rieger, R., Scharf, K.-D., Wollgiehn, R. & zur Nieden, U. (1989). Heat shock and other stress response systems of plants. Biologisches Zentralblatt 108, 1-156. Nover, L. (ed.) (1990). Heat Shock Response. Boca Raton, FL: CRC Press. Pelham, H.R.B. (1984). Hsp70 accelerates the recovery of nucleolar morphology after heat shock. The EMBO Journal 3, 3095-100. Perisic, O., Xiao, H. & Lis, J.T. (1989). Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit. Cell 59, 797-806. Perisic, O., Xiao, H. & Lis, J.T. (1989). Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit. Cell 59, 797-806. Rieping, M. & Schoffl, F. (1992). Synergistic effect of upstream sequences, CAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobacco. Molecular and General Genetics 231, 226-32. Sander, M. & Hsieh, T.S. (1985). Drosophila topoisomerase II doublestrand DNA cleavage: analysis of DNA sequence homology at the cleavage site. Nucleic Acids Research 13, 1057-72.

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Scharf, K.-D., Rose, S., Zott, W., Schoffl, F. & Nover, L. (1990). Three tomato genes code for heat stress transcription factors with a region of remarkable homology to the DNA-binding domain of the yeast HSF. The EM BO Journal 9, 4495-501. Schoffl, F. (1988). Genetic engineering strategies for manipulation of the heat shock response. Plant, Cell and Environment 11, 339-43. Schoffl, F. & Baumann, G. (1985). Thermo-induced transcripts of a soybean heat shock gene after transfer into sunflower using a Ti plasmid vector. The EMBO Journal 4, 1119-24. Schoffl, F., Rieping, M. & Baumann, G. (1987). Constitutive transcription of a soybean heat-shock gene by a cauliflower mosaic virus promoter in transgenic tobacco plants. Developmental Genetics 8, 365-74. Schoffl, F., Rieping, M., Baumann, G., Bevan, M.W. & Angermiiller, S. (1989). The function of plant heat shock promoter elements in the regulated expression of chimaeric genes in transgenic tobacco. Molecular and General Genetics 217, 246-53. Schoffl, F., Rieping, M. & Raschke, E. (1990). Functional analysis of sequences regulating the expression of heat shock genes in transgenic plants. In Genetic Engineering of Crop Plants, ed. G.W. Lycett & D. Grierson, pp. 79-84. London: Butterworth. Schoffl, F., Rieping, M. & Severin, K. (1991). The induction of the heat shock response: activation and expression of chimaeric heat shock genes in transgenic plants. In Plant Molecular Biology, NATO-ASI Series, ed. R.G. Herrmann & B. Larkins, pp. 685-94. New York: Plenum Press. Severin, K. & Schoffl, F. (1990). Heat-inducible hygromycin resistance in transgenic tobacco. Plant Molecular Biology 15, 827-33. Sheehy, R.E., Kramer, M. & Hiatt, W.R. (1988). Reduction of polygalacturonase activity in tomato fruit by antisense RNA. Proceedings of the National Academy of Sciences (USA) 85, 8805-9. Sorger, P.K. & Nelson, H.C.M. (1989). Trimerization of a yeast transcriptional activator via a coiled-coil motif. Cell 59, 807-13. Sorger, P.K. & Pelham, H.R.B. (1988). Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell 54, 855-64. Stief, A., Winter, D.M., Stratling, W.H. & Sippel, A.E. (1989). A nuclear DNA attachment element mediates elevated and positionindependent gene activity. Nature 341, 343-5. Vierling, E. & Sun, A. (1987). Developmental expression of heat shock proteins in higher plants. In Environmental Stress in Plants. Biochemical and Physiological Mechanisms Associated with Environmental Stress Tolerance in Plants, ed. J. Cherry, pp. 343-54. New York: Springer-Verlag. Wiederrecht, G., Seto, D. & Parker, C.S. (1988). Isolation of the gene encoding the S. cerevisiae heat shock transcription factor (1988). Cell 54, 841-53.

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Xiao, H., Perisic, O. & Lis, J.T. (1991). Cooperative binding of Drosophila heat shock factor to arrays of a conserved 5 bp unit. Cell 64, 585-93. Zimmerman, J.C., Petri, W. & Meselson, M. (1983). Accumulation of a specific subset of Drosophila melanogaster heat shock mRNAs in normal development without heat shock. Cell 32, 1161-70.

L. CATTIVELLI and D. BARTELS

Biochemistry and molecular biology of cold-inducible enzymes and proteins in higher plants Introduction Plants growing in temperate climates are often exposed to cold stress which can kill the plants. The cold or low temperature stress can be subdivided into chilling (temperatures above 0 °C) and freezing (subzero temperatures) stress. The absolute temperatures which lead to damage in plants vary markedly between different plant species. Cold resistance measured as the frost killing point is not a constant for a genetically pure variety but is strongly influenced by environmental factors and the developmental stage of the plants. Two basic mechanisms to survive a low temperature stress have evolved in plants: either to avoid the fatal temperature or to adapt to the low temperature by developing tolerance towards the stress. Although the plant is essentially unable to avoid the freezing temperature of its environment, it is suggested by Levitt that some protection can be obtained by supercooling or accumulation or antifreeze as a mechanism for avoidance (Levitt, 1980). On the other hand, many temperate plant species have the capability of cold acclimation: exposure to a period of low, non-freezing temperatures can lead to a significant increase of cold tolerance (Levitt, 1980). This process is termed cold acclimation or cold hardening. The interrelationship of these different processes is shown in Fig. 1. The subject of this chapter will be to summarise the biochemical and molecular changes which take place during the process of cold acclimation and the acquisition of freezing stress tolerance. We will discuss how polypeptides correlated with the acclimation process might play a role in increased cold tolerance and we will focus on recent results emerging from molecular studies. For further treatment of the subject the reader is referred to a number of recent review articles (Steponkus, 1984; Guy, 1990; Thomashow, 1990). The biochemistry and physiology of cold acclimation is described in detail by Levitt (1980). Society for Experimental Biology Seminar Series 49: Inducible Plant Proteins, ed. J. L. Wray. © Cambridge University Press, 1992, pp. 267-288.

268

L. CATTIVELLI AND D. BARTELS Optimal growth temperature

I

\

Lower temperature

INJURY RESPONSE Chilling injury (tropical and subtropical plants)

ADAPTIVE RESPONSE ;lin Cold acclimation or rder Cold hardening

T

Freezing injury (all plants)

T increased frost tolerance

Fig. 1. Cold-stress responses in higher plants. Cold acclimation is a very complex process and genetic experiments suggest that the inheritance of the capacity for cold acclimation-induced freezing tolerance is a quantitative character controlled by a number of additive genes (Guy, 1990; Thomashow, 1990). However, in wheat genetic analysis indicates that some genes with major effects on cold tolerance are located on chromosome 5A, closely linked with the locus Vrnl controlling vernalisation requirements (Sutka & Snape, 1989). The problem of isolating components involved in cold stress and cold acclimation can be approached in two ways: one strategy is to investigate known physiological pathways under low temperature conditions, and alterations in enzyme activities or quantitative changes of metabolites will be noticed. Secondly, the strategy adopted by molecular biology to reveal cold-stress-related components is to discover differential gene activity by comparing a stress and non-stress situation and then to isolate stressrelated genes by differential hybridisation. Most of these genes will encode unknown products and therefore the next step will be to prove the direct role of the identified genes in the stress phenomenon by testing their effect in transgenic plants. To date differential gene activity has been shown for many cold-stressed plants and a number of genes have been isolated (see below), but no reports of transgenic plants are yet available. Before discussing the role of proteins in cold acclimation the physical effects caused by low temperature are briefly considered. A decrease of temperature leads to altered rates of enzymatic catalysis. Formation of hydrogen bonds and electrostatic interactions are thermodynamically more stable at a lower temperature whereas hydrophobic interactions are

Cold-inducible enzymes and proteins

269

energetically favoured at elevated temperatures. So even modest changes in temperature should effect the kinetics of the different metabolic reactions. However, when temperatures decrease below 0°C, the extracellular water starts to freeze and the solute concentration increases, as a result of which the intracellular water leaves the cell causing cellular dehydration. Hence freezing stress also imposes an osmotic stress on the cell. Since protoplast dehydration occurs during water, salt or low temperature stress it is likely that common components are involved in the reactions of plants to these different stresses (see below). When the temperature is raised and the extracellular ice crystals melt, the cells rehydrate. Only plasma membranes of cold-hardened cells are able to withstand the water efflux and influx. This observation suggests that membranes must be one of the major focal points in cold stress research (see Steponkus, 1984). Biochemical responses to low temperature The exposure of plants to low temperatures induces many changes in physiological and biochemical parameters. Many studies have attempted to investigate the relationship between low temperature and enzyme activities. The effects of low temperature treatments on the protein levels and on activity of some plant cell enzymes are reported in Table 1. As a response to cold treatment there is a modification both in the level and in the activity of many important enzymes. In rice the mRNA coding for the Rubisco small subunit decreases drastically at low temperature, as do the levels of both the small and large Rubisco subunits (Hahn & Walbot, 1989). In rye Huner & Macdowall (1979) also observed structural and functional changes in Rubisco during growth at low temperature. As a consequence of modifications in enzyme activities several soluble components can change their levels during the cold response. For instance an increase or decrease of components like soluble carbohydrates, proline and polyamines has been correlated with the acquisition of cold tolerance. There are many cases where sugars have been shown to increase in plants during cold-hardening and to decrease as they deharden (Levitt, 1980). In leaves of Dactylis, for instance, low temperature induces synthesis and accumulation of fructosan (Pollock & Ruggles, 1976). The role of carbohydrates in the development of chilling tolerance has been demonstrated in tomato seedlings where the chilling sensitivity changes with a diurnal cycle according to the contents of carbohydrates. Maximum sensitivity is at the end of the dark period probably resulting from

Table 1. Changes in protein levels and enzyme activities observed at low temperature Enzyme

Plant

Observation

Enzymes involved in zarbohydrate t metabolism Rubisco Decrease of both Rice Rubisco subunits Rapeseed Rubisco Wheat Sucrose synthase Increase of activity Changing ratios of Wheat Invertase isozymes Increase of activity Potato tubers Invertase Glucose 6-phosphate Poplar twigs Decrease of activity 6-phosphoglucanate Poplar twigs Decrease of activity 'i dehydrogenase Enzymes involved in proline metabolism Wheat Increase of activity Glutamine synthetase Wheat Increase of activity Ornithine transaminase Proline dehydroWheat Increase of activity genase Increase of activity Glutamate dehydro- Wheat genase Other enzymes Catalase IAA oxidase RNA

polymerase Phenylalanine ammonia lyase Hydroxycinnamyltransferase Galactolipase

Glutathione reductase Glutathione reductase Ferridoxine NADP reductase

Reference

Hahn & Walbot, 1989 Meza-Basso et al., 1986 Calderon & Pontis, 1985 Roberts, 1975 Sasaki et al., 1971 Sagisaka, 1985 Sagisaka, 1985

Charest & Phan, 1990 Charest & Phan, 1990 Charest & Phan, 1990 Charest & Phan, 1990

Cucumber Cucumber Wheat

Decrease of activity Increase of activity Increase of activity

Omran, 1980 Omran, 1980 Sarhan & Chevrier, 1985

Tomato fruits

Increase of activity

Rhodes & Wooltorton, 1977

Tomato fruits

Increase of activity

Rhodes & Wooltorton, 1977

Wild tomato species

Gemel etal., 1988

Poplar twigs

Higher activity in chilling-sensitive species Increase of activity

Spinach

Increase of activity

Guy & Carter, 1984

Wheat

Increase of activity

Riov & Brown, 1976

Sagisaka, 1985

Cold-inducible enzymes and proteins

271

carbohydrate depletion, while the highest chilling tolerance is found during the light period (King et al., 1988). Potato tubers stored at low temperature accumulate fructose and glucose owing to the high level of induction of invertase activity (Sasaki et al., 1971). During cold acclimation in wheat, the ratio between the different forms of invertase changes, probably because the isoenzymes found in cold-hardened plants enable them to survive better at low temperatures (Roberts, 1975). In wheat the increase in sucrose synthase activity is associated with an increased level of sucrose (Calderon & Pontis, 1985), which leads to an improved cold tolerance. The accumulation of soluble carbohydrates represents a general mechanism to increase the osmotic concentration of a cell. However, increases in the levels of other metabolites can lead to the same result. Proline levels, for instance, increase in response to many environmental stresses including drought stress and low temperature (Hanson et al., 1979; Swaaij et al., 1985; Duncan & Widholm, 1987; Songstad et al., 1990). Increasing activity of the enzymes involved in the proline pathways has been demonstrated under low temperature conditions (Charest & Phan, 1990). Polyamines have also been implicated in the plant's response to cold, since both spermidine and proline accumulate in leaves of Citrus species during hardening (Kushad & Yelenosky, 1987). Exposure to low temperature not only induces modifications in enzyme activities but also alters the lipid composition of the cell membranes. A general observation is that the percentage of disaturated phosphatidylglycerol (PG) of thylakoid membranes goes up concomitantly with increasing cold sensitivity of plant species within a genus (Roughan, 1985). Coldtolerant plants contain less than 2% of disaturated PG in thylakoid membrane lipids, while in most chilling-sensitive plants the disaturated PG represents about 4-5% of thylakoid membrane lipids. High contents of disaturated PG result in a phase separation of membrane lipids at low temperature. Many changes in the lipid composition of the plasma membrane have been associated with hardening (Lynch & Steponkus, 1987). In rye (cv. Puma) an increase of the total lipid content has been measured during hardening (Cloutier, 1987). Free sterols increased while steryl-glycoside and acylated steryl-glycoside decreased. In addition the phospholipid contents of the plasma membrane increased. The important role of sterol components in the acquisition of freezing tolerance has recently been demonstrated using an Arabidopsis mutant with altered steryl-ester metabolism. Compared with the wild-type the mutant has no visible phenotype at standard growth temperature but exhibits clear symptoms when exposed to low temperatures (Hugly et al.,

272

L. CATTIVELLI AND D. BARTELS

1990). In cereals the role of unsaturated fatty acids in cold acclimation has been demonstrated using a chemical (BASF 13-338) that reduces the linolenic acid content in polar lipids (St John et al, 1979). Molecular analysis of cold-responsive genes and proteins Cold-induced changes in polypeptides and translatable mRNAs As long ago as 1970 Weiser proposed that transcriptional activation of genes normally not expressed under non-acclimated conditions, and the synthesis of new proteins, are involved in the cold acclimation process (Weiser, 1970). Weiser's hypothesis was substantiated by the observation that, with a few exceptions, the soluble protein content increased during cold acclimation (Levitt, 1980; Sakai & Larcher, 1987). Consequently a number of groups investigated qualitative and quantitative changes in the protein patterns during low temperature treatment. The experimental approach used was as follows. Total soluble proteins or in vivo radiolabelled proteins were fractionated by 1- and 2-dimensional SDS-PAGE and detected by either staining or autoradiography. The protein patterns obtained from cold-stressed plants were compared with the patterns from plants grown at normal control temperatures. These types of experiments were carried out with plant species ranging from chilling-sensitive to frost-resistant plants employing a series of low temperature treatments for different lengths of time. The results clearly established that low temperature treatment leads to the synthesis of new proteins, referred to here as cold-regulated proteins. A decrease in the level of some proteins was also observed. Lists of these protein changes have been compiled by Guy (1990) and Thomashow (1990). In those cases for which it was examined it was found that cold-induced changes in the polypeptide pattern can be accounted for by changes in translatable mRNA populations. Low temperature-induced changes in mRNA populations have been reported for a number of plant species using in vitro translation assays in combination with protein electrophoresis (Meza-Basso et al, 1986; Mohapatra et al, 1987a,b\ Gilmour et al, 1988; Cattivelli & Bartels, 1989; Lang et al, 1989). The analysis of the in vivo and in vitro protein data indicates that during cold treatment new proteins and mRNAs can be detected, but the overall protein pattern is not dramatically different from the one observed at normal control temperatures, unlike the situation for heat shock or anaerobiosis (Sachs & Ho, 1986). At low temperature the majority of proteins and mRNAs continue to be synthesised. This would

Cold-inducible enzymes and proteins

273

be consistent with the need to maintain basic cellular reactions, although at a reduced rate. It is therefore likely that the set of mRNAs common to low and control temperature probably includes most of the housekeeping genes. This supports the view that cold acclimation is an adaptive response during which polypeptides and other metabolites accumulate. The induction kinetics of cold-regulated proteins can vary between a few hours and several days. The fastest accumulation of cold-regulated proteins was seen in the case of a 75 kDa barley protein (Cattivelli & Bartels, 1989) and a group of four 47 kDa Arabidopsis proteins (Gilmour et al., 1988). A comparison between different plant species does not point to the appearance or disappearance of a specific set of proteins; the size of the inducible polypeptides ranges from 10 to 200 kDa (Mohapatra et al., 1987a,b; Perras & Sarhan, 1989). It is, however, remarkable that in several species in vitro- and in vivo- synthesised polypeptides of around 160 kDa have been found (Arabidopsis, spinach, citrus, wheat: Guy & Haskell, 1987, 1988; Gilmour et al., 1988; Perras & Sarhan, 1989; Lin et al., 1990). It will be interesting to determine whether they are homologous. Up to now little or no information is available concerning the identity or function of the cold-regulated proteins and mRNAs. The co-identity of different proteins can be confirmed only by comparisons of amino acid sequences or by immunological cross-reactions. That cold-regulated polypeptides have a role in the acquisition of cold tolerance is supported by the general observation that the appearance of most proteins is temporally correlated with the onset of freezing tolerance. The polypeptides are detected as long as the plants are kept at low temperatures but decline when the temperature is raised as, for example, demonstrated by Guy & Haskell (1987, 1988) for spinach. Similar associations of appearance and decline of polypeptides have been reported for alfalfa (Mohapatra et al., 1987a,b).

Isolation and characterisation of cold-regulated genes and encoded polypeptides Analysis of changes in in-vivo- and /n-vi7ro-synthesised proteins is a very descriptive approach to understanding the phenomenon of cold acclimation and cold tolerance. For a functional analysis a molecular dissection of the process is required. This will allow investigators to determine the level (transcriptional/translational) at which the appearance of the new mRNAs is regulated, and the sequence information of the cold-regulated genes should point to the biochemical features of the encoded proteins.

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To date several mRNAs which accumulate in response to low temperature have been cloned as cDNAs. The experimental route taken was to construct a cDNA library of RNA isolated from cold-acclimated plants and to screen it by differential hybridisation with probes from RNA extracted from acclimated and non-acclimated plants. By this method several cDNA clones encoding cold-regulated transcripts were isolated from alfalfa, Arabidopsis, barley, spinach, tomato and wheat (see Table 2 for a summary of the data and the corresponding references). RNA hybridisation analysis demonstrated that all the cDNA clones contain inserts that hybridise strongly to RNA from cold-stressed plants. The nucleotide sequences of several cDNA clones have been determined (Table 2). These data show that most of the isolated genes encode novel polypeptides with unknown function. An exception to this is that a cloned mRNA from tomato, designated C14, encodes a polypeptide closely related to thiol proteases (Schaffer & Fischer, 1988, 1990). By using the maize cDNA clone encoding alcohol dehydrogenase (Adhl), Christie et al. (1991) showed for maize and rice seedlings that Adhl mRNA accumulated when the plan tie ts were exposed to low temperature (10 °C) but the mRNA levels declined when the stress was relieved. The mRNA levels coincide with altered enzymatic activity of alcohol dehydrogenase. The authors propose that during the stress period the energy metabolism is shifted from aerobic to anaerobic respiration to compensate for impaired mitochondrial function. This could be considered as an adjustment to a changed metabolism during low temperature. Interestingly, several of the cold- and freezing-labile enzymes belong to the respiratory pathways (Guy, 1990). Although no functions could be assigned to any of the other coldregulated genes, some remarkable features will be pointed out which may hint at a role in cold acclimation and/or osmoprotection. Sequence comparisons showed that the KM gene product from Arabidopsis has similarities (41% on the amino acid level) to fish antifreeze proteins, and therefore it is suggested that the predicted KM protein may serve as an antifreeze protein (Kurkela & Franck, 1990). The fish antifreeze proteins are able to depress the freezing temperature of the aqueous body fluids by inhibiting the growth of ice crystals (Franks et al., 1987). The characteristic features of the deduced KM polypeptide are its hydrophilicity, alanine-rich sequence-repeat motifs and an unusual amino acid composition with alanine, glycine and lysine being the prevailing amino acids whilst arginine, cysteine, histidine, isoleucine, proline, tryptophan and tyrosine are lacking. A different class of hydrophilic proteins is encoded by the rab (ABA responsive) genes. These genes were first discovered as ABA-induced

Cold-inducible enzymes and proteins

275

genes abundantly expressed during late embryogenesis, but subsequently they have also been related to drought stress (Mundy & Chua, 1988; Close et al, 1989; Piatkowski et al., 1990; Skriver & Mundy, 1990). Several examples have emerged where these genes are also induced by cold stress: in rice, barley and spinach (Hahn & Walbot, 1989; L. Cattivelli, unpublished data; C.L. Guy, personal communication). The characteristic features of the rab gene products are a contiguous stretch of 8 or 9 serine residues and the conservation of a lysine-rich motif which occurs repeatedly in the proteins. It has been hypothesised (Dure et al., 1989) that the function of the rab gene-encoded proteins is to stabilise cellular proteins during dehydration. The evolutionary conservation of characteristic motifs in the gene structure must be related to functional constraints among diverse plant species and supports a general osmoprotective role for these proteins. Based on biochemical properties some protein products of the coldregulated genes from Arabidopsis and wheat {cor genes) are closely related to the hydrophilic proteins described above. The cor gene products are distinguished by an unusual biochemical property: they are 'boiling stable', that is, they stay in solution after they have been denatured by boiling (Lin et al., 1990). This feature has been reported for the dehydrins (Close et al., 1989), a group of proteins found in dehydrated barley and maize seedlings whose encoding genes display the characteristic structure described above for the rab genes. Some of the antifreeze proteins of fish do not precipitate after boiling (DeVries, 1983). Although no protein sequence data are available and the corresponding genes have not been isolated, cryoprotective proteins from coldhardened leaves of spinach and cabbage exhibit biochemical properties related to the rab and cor gene products (Volger & Heber, 1975; Hincha et al., 1989, 1990). These cryoprotective proteins protected thylakoid membranes from freezing damage by increasing their resistance against the osmotic stress which they experience during a freeze/thaw cycle. Hydrophilic properties are also evident for two cold-regulated proteins predicted from isolated barley genes (Cattivelli & Bartels, 1990). These sequences appear to be different from the ones described above and the most remarkable feature is the presence of several arginine residues in close proximity. Arginine-rich motifs have been shown to be important in protein-RNA interactions in the case of viral proteins (Veidt et al., 1988; Lazinski et al., 1989). Further investigations are needed to reveal whether the cold-regulated barley proteins could have a similar function. There is no homology between these cold-regulated barley genes and that reported by Dunn et al. (1990). However, when the cold-regulated barley cDNA clones were used as probes cross-hybridisation was found to

Table 2. Characteristics of cloned cold-regulated transcripts Plant

Clone name

Alfalfa Alfalfa

pSM1409 pSM784 pSM2201 pSM2358

Observations

Inducibility by other factors

References

ABA

Mohapatra et al., 1988 Mohapatra et al., 1989

Arabidopsis

Kinl*

Similarities to a fish antifreeze protein

ABA, water stress

Kurkela & Franck, 1990

Arabidopsis

pHH7.2(cor 47) pHH28 pHH29 pHH67

Encoding a 'boiling-stable' polypetide

ABA, ABA, ABA, ABA,

Hajela et al., 1990

Barley

pT59" pV60 pAO29 PAO86" pAF93

Barley

pBLT14fl

Barley

Rab21*

Maize

Adhl"

Rice

Rab21*

Spinach

CAP79 CAP85 CAP160

Related to 70 kDa heat shock protein Related to LEA and dehydrins

Tomato

C14" C17 C19

Homology to thiol protease

Wheat

pWGl

Encoding a 'boiling-stable' polypetide, homologous to pHH7.2

water water water water

stress stress stress stress

Cattivelli & Bartels, 1990

ABA, water stress

L. Cattivelli, unpublished Dunned/., 1990 L. Cattivelli, unpublished Christie etal., 1991

Alcohol dehydrogenase ABA, water stress

Genes down-regulated by low temperature Rice Rubisco ssu Cab Chlorophyll binding protein

Hahn & Walbot, 1989 C. L. Guy, personal communication

Heat shock

Schaffer & Fischer, 1988, 1990 Lin etal., 1990

Hahn & Walbot, 1989

Notes:

°Nucleotide sequence published. fc The Rab21 rice cDNA probe detects three different transcripts. Two of them (1.5 and 0.9 kb) are cold-induced. The smallest of these transcripts corresponds in size to the Rab21 transcript described (Mundy & Chua, 1988). In barley the same cDNA clone detects a cold induced transcript of 2.4 kb.

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closely related cereals, suggesting that the genes have been conserved (Cattivelli & Bartels, 1990; Dunn etal, 1990).

Induction kinetics of cold-regulated transcripts The availability of the cDNA clones allowed a study of the time course of their induction and their tissue-specific expression. The overall picture is that transcripts start to accumulate within the first 24 h after the plants have been exposed to low temperature, they remain at a steady-state level and decline when the temperature is raised. During cold acclimation oi Arabidopsis the mRNA detected by the Kinl cDNA clone (homology to fish antifreeze protein) started to increase during the first 6 h of cold treatment and remained high during the whole acclimation period (up to 7 days) (Kurkela & Franck, 1990). This observation is in agreement with RNA hybridisation data obtained for four other cold-regulated transcripts from Arabidopsis: the transcripts started to accumulate between 1 and 4 h after the onset of the cold treatment, reached a maximum after about 12 h and declined rapidly when the plants were returned to normal growth temperature (Hajela et al., 1990). The two tomato transcripts C14 and C17 were detected at significant levels after 8 h of cold treatment and their steady-state levels persisted during prolonged exposure to cold for several days (Schaffer & Fischer, 1988). The cold-regulated transcripts characterised in alfalfa accumulated rapidly during the first day and at a slow rate during the following 7 days of cold stress (Mohapatra et al., 1988). A comparable situation was found in barley: after one day of exposure to low temperature five different transcripts were detected and persisted during the stress but declined when the temperature was elevated (Cattivelli & Bartels, 1990). An interesting finding of the expression studies in barley was that the transcripts showed a tissue-specific accumulation when young and mature leaves as well as root tissues were examined. Correlation between freezing tolerance and coldregulated products Several groups have reported a correlation between cold-regulated gene expression and freezing tolerance when steady-state mRNA levels are measured with probes for cold-regulated genes. Mohapatra et al. (1989) found that the expression of mRNAs is correlated with the level of freezing tolerance displayed by certain alfalfa cultivars. Schaffer & Fischer (1988) showed that the level of the cold-regulated tomato transcripts C14 and C17 is different in cold sensitive and cold tolerant varieties; they

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concluded that genetically determined cold tolerance influences coldinducible gene expression. The expression of cold-regulated transcripts from barley was tested in spring and winter varieties but no clear relationship could be established (Cattivelli & Bartels, 1990; Dunn etal, 1990). However, conclusions from a comparison of two different varieties have to be interpreted with caution, because these lines most likely differ at many genetic loci not related to cold tolerance behaviour. Involvement of ABA in the low temperature response Many physiological studies established a correlation between cold acclimation and increasing ABA levels in plant tissues (Chen et al., 1983). Furthermore, exogenous ABA application can induce cold-hardening to the same degree as a low temperature treatment does (Chen & Gusta, 1983) while plant species which do not show a hardening capacity at low temperature do not harden even after ABA treatment. Arabidopsis plants grown on medium containing 15 mg/1 of ABA show the same freezing tolerance as plants hardened during a low temperature exposure (Lang et al., 1989). A mutation affecting ABA biosynthesis prevents the development of cold tolerance in Arabidopsis (Heino et al., 1990). The mutant has lost the ability to cold-acclimate when subjected to low temperature. However, an addition of exogenous ABA to the growth medium complements almost fully the mutational defect. These observations support the hypothesis that ABA has a key role in the acquisition of cold-hardening. Despite many studies on the effect of ABA on plant tissues there is no demonstration that ABA can regulate the level of enzyme activities and specially of those involved in cold acclimation. On the other hand, ABA certainly induces modifications in gene expression and many genes are modulated by ABA (Skriver & Mundy, 1990). Indeed several in vivo protein-labelling and in vitro translation experiments have demonstrated that the level of certain proteins or mRNAs increased in response to both low temperature and exogenous ABA applications (Robertson et al., 1987; Mohapatra et al., 1988; Lang et al, 1989). Several of the cold-regulated genes isolated so far in Arabidopsis (Kurkela & Franck, 1990; Hajela et al., 1990), alfalfa (Mohapatra et al., 1988) and barley (L. Cattivelli, unpublished data), show ABA-inducibility, but, at least in alfalfa and in barley, not all (Mohapatra et al., 1989; L. Cattivelli, unpublished data). These results support the hypothesis that although ABA is probably essential for hardening (in fact an ABA-deficient mutant does not cold-acclimate), it is not responsible for all the modifications induced during exposure of plants to low

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temperatures. This suggests that the plant response to cold could be split in two pathways: an ABA-regulated pathway and another, not ABArelated, which might also include modifications in enzyme activities. Further studies in this direction will contribute to a better understanding of the relation between ABA and hardening. Because ABA is also involved in drought stress, several investigations have been carried out to check whether the cold-regulated and ABAinduced genes are expressed under drought conditions. Indeed in Arabidopsis the cold-regulated transcript homologous to the clone KM (Kurkela & Franck, 1990) and, in barley, the pAF93-rt\&t&& transcript (L. Cattivelli, unpublished data) are also induced under water stress. As mentioned before it has been shown that transcripts homologous to the rab gene family (Mundy & Chua, 1988; Skriver & Mundy, 1990) are induced in response to cold in rice (Hahn & Walbot, 1989) and barley (L. Cattivelli, unpublished data). Therefore the molecular responses to drought and cold share some common features probably mediated through ABA. This finding is not surprising as drought as well as cold stress imposes an osmotic strain upon the cell. Relationship between low temperature stress and heat shock response Several experiments have been performed to check whether coldregulated proteins and/or genes are induced also under heat shock conditions. The majority of the isolated cold-regulated genes do not show any relation to heat shock transcripts (see also Table 2) (Mohapatra et al., 1989; Cattivelli & Bartels, 1990). However, an interesting exception is represented by the cDNA C14 corresponding to a thiol protease gene of tomato. Exposure for 8 h or longer of tomato fruits to 40 °C results in the accumulation of thiol protease mRNA. This response is more rapid than that at 4 °C but slower than the induction of many heat shock mRNAs (Schaffer & Fischer, 1990). Therefore this response cannot be included in the typical heat shock response. As suggested by Schaffer and Fischer, the heat induction of C14 mRNA may reflect a need to degrade polypeptides damaged during temperature stress. In spinach exposed to 5 °C a protein named CAP79 was detected which turns out to be a complex of 70 kDa heat shock cognates (C.L. Guy, personal communication). However, the general conclusion still remains that cold stress and heat shock activate different sets of genes.

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Organisation of the cold-related genes When genomic Southern analyses were performed, the data showed that the cold-regulated genes are low or single-copy number genes; this was found for the Arabidopsis genes (Hajela et al., 1990; Kurkela & Franck, 1990) and the barley genes (Dunn etai, 1990; L. Cattivelli, unpublished data). Only for alfalfa cold-regulated genes did it turn out that at least one is present as a multigene family (Mohapatra et al., 1989).

Subcellular localisation As it is thought that modifications of membranes are important in the cold stress response, a question which has not been addressed yet is the subcellular localisation of the cold-regulated gene products. No clear experimental data are available. However, from the cDNA sequences determined so far it can be deduced that the predicted proteins lack obvious signal sequences and are therefore very likely to be localised in the cytoplasm (Schaffer & Fischer, 1988; Cattivelli & Bartels, 1990; Kurkela & Franck, 1990). These data are in agreement with comparisons of in vivo and in vitro synthesised protein patterns, because for many polypeptides no post-translational modifications were predicted from 2dimensional electrophoresis analyses (Cattivelli & Bartels, 1989). With the availability of cloned probes this problem can be analysed directly by the functional expression of the cDNA clones and the production of antibodies which can be used for immunolocalisation of the corresponding proteins. Regulation of gene induction One important objective is to determine the molecular mechanism responsible for the accumulation of cold-temperature-induced gene transcripts. The increased levels of specific transcripts could either reflect transcriptional activation of a cold-regulated set of genes or post-transcriptional control mechanisms, like preferential translation or selective thermostability of specific mRNAs. This question has begun to be addressed by performing nuclear run-on transcription experiments. The tomato C14 transcript encoding a thiol protease-homologous-protein is induced through transcriptional induction (Schaffer & Fischer, 1990). In contrast to this, the cold-regulated expression of three Arabidopsis cor genes was controlled primarily at the post-transcriptional level, while, for a fourth cor gene, regulation was found at both transcriptional and post-transcriptional levels, with transcriptional control having the major role (Hajela

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et al., 1990). More experiments, and in particular promoter analysis of genomic clones, are required to analyse in detail the transcriptional and post-transcriptional regulatory mechanisms responsible for expression of cold-regulated genes.

Future perspectives The property of cold acclimation resulting in freezing tolerance has evolved in many temperate plants. The metabolic pathways of this complex process are mostly unknown. However, from recent work it is clear that gene activation is involved. By means of molecular biology some cold-regulated transcripts have been isolated and have become amenable to further analysis. The cDNA clones so far available probably correspond to only a small number of the transcripts which are affected by low temperature. One may therefore expect that the number of cDNA clones and genomic clones available for characterisation will increase in the near future. Two major areas of research on gene expression in response to low temperature require extensive studies: to determine the function of the cold-regulated transcripts and to understand the regulation of gene expression. Much effort needs to be put into experiments to define functions for the proteins encoded by the isolated genes. As the capacity for cold tolerance cannot be defined in a Mendelian fashion, but is likely to be determined by the interplay of several genes, no defined mutants are available. This fact complicates the task of assigning functions. However, the effect of single genes can be tested by overexpression in transgenic plants. Gene isolation also opens the way to isolate the corresponding proteins and to test their cryoprotective behaviour in assays like those performed by Hincha et al. (1990). In vitro mutagenesis together with structural, biochemical and biophysical studies on the novel cold-regulated proteins may reveal how they act as cold-protective molecules. There are only a few studies on the correlation between the expression of specific transcripts and freezing tolerance (see earlier), but nucleotide probes for these genes may contribute to selection schemes in breeding programmes. Since from physiological studies it was evident that increased carbohydrate levels are able to confer resistance to cold, it is surprising that none of the cloned transcripts can so far be related to carbohydrate metabolism. One explanation for this may be that low temperature activates or modifies enzymes which are involved in metabolism at normal temperatures, and therefore transcripts for these enzymes would not

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be detected by differential screening procedures. An integrative physiological and molecular approach is necessary to analyse particular metabolic routes. As pointed out in the introduction the cold acclimation capacity is a quantitative trait and work is just beginning to link such complex traits to molecular markers using restriction fragment length polymorphism (RFLP). Eventually, also, information about genes involved in environmental stress can be expected from these experiments. The second area essential to study is the mechanism by which plant gene expression is controlled by low temperature. These studies will reveal whether common elements are responsible for activating a set of cold-regulated genes. It should further address the question of how other factors, in particular the plant hormone ABA, are involved in the expression of cold-regulated genes and whether there is a common mechanism for osmotic stress response in drought, cold and salt stress. Recently much progress has been made by cloning a transcriptional activator gene related to ABA induction (Guiltinan et al., 1990). A genetic dissection of gene activation should be facilitated by exploiting mutants in the ABA pathway which are available for Arabidopsis and barley (Raskin & Ladyman, 1988; Walker-Simmons et al, 1989; Heino et al, 1990). For Arabidopsis the involvement of ABA in cold acclimation has already been shown, and powerful genetic systems like Arabidopsis should provide new insights into the old physiological problem of cold-hardening and freezing tolerance. Acknowledgements Work partially supported by the National Research Council of Italy, special project RAISA, subproject N. 2 paper N. 47. References Calderon, P. & Pontis, H.G. (1985). Increase of sucrose synthase activity in wheat plants after a chilling shock. Plant Science 42,173-6. Cattivelli, L. & Bartels, D. (1989). Cold-induced mRNAs accumulate with different kinetics in barley coleoptiles. Planta 178, 184-8. Cattivelli, L. & Bartels, D. (1990). Molecular cloning and characterization of cold-regulated genes in barley. Plant Physiology 93, 1504-10. Charest, C. & Phan, C.T. (1990). Cold acclimation of wheat (Triticum aestivum): properties of enzymes involved in proline metabolism. Physiologia Plantamm 80, 159-68. Chen, H.-H., Li, P.H. & Brenner, M.L. (1983). Involvement of abscisic acid in potato cold acclimation. Plant Physiology 71, 362-5.

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Chen, T.H.H. & Gusta, L.V. (1983). Abscisic acid-induced freezing resistance in cultured plant cells. Plant Physiology 73, 71-5. Christie, P.J., Hahn, M. & Walbot, W. (1991). Low temperature accumulation of alcohol dehydrogenase-1 mRNA and protein activity in maize and rice seedlings. Plant Physiology 95, 699-706. Close, T.J., Kortt, A.A. & Chandler, P.M. (1989). A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn. Plant Molecular Biology 13, 95-108. Cloutier, Y. (1987). Lipid and protein changes in cold- and droughthardened cereals. Phytoprotection 68, 87-96. DeVries, A.L. (1983). Antifreeze peptides and glycopeptides in cold water fishes. Annual Review of Physiology 45, 245-60. Duncan, D.R. & Widholm, J.M. (1987). Proline accumulation and its implication in cold tolerance of regenerable maize callus. Plant Physiology 83, 703-8. Dunn, M.A., Hughes, M.A., Pearce, R.S. & Jack, P.L. (1990). Molecular characterization of a barley gene induced by cold treatment. Journal of Experimental Botany 41, 1405-13. Dure, L. Ill, Crouch, M., Harada, J., Ho., T.-H.D., Mundy, J., Quatrano, R., Thomas, T. & Sungh, Z.R. (1989). Common amino acid sequence domains among the LEA proteins of higher plants. Plant Molecular Biology 12, 475-86. Franks, F., Darlington, J., Schenz, T., Mathias, S.F., Slade, L. & LeVine, H. (1987). Antifreeze activity of antarctic fish glycoprotein and a synthetic polymer. Nature 325, 146-7. Gemel, J., Saczynska, V. & Kaniuga, Z. (1988). Galactolipase activity and free fatty acid levels in chloroplast of domestic and wild tomatoes with different chilling tolerance. Physiologia Plantarum 74, 509-14. Gilmour, S.J., Hajela, R.K. & Thomashow, M.F. (1988). Cold acclimation in Arabidopsis thaliana. Plant Physiology 87, 745-50. Guiltinan, M.J., Marcotte, W.R., Jr & Quatrano, R.S. (1990). A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250, 267-71. Guy, C.L. (1990). Cold acclimation and freezing stress tolerance: role of protein metabolism. Annual Review of Plant Physiology and Plant Molecular Biology 41, 187-223. Guy, C.L. & Carter, J.V. (1984). Characterization of partially purified glutathione reductase from cold-hardened and nonhardened spinach leaf tissue. Cryobiology 21, 454-64. Guy, C.L. & Haskell, D. (1987). Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins. Plant Physiology 84, 872-8. Guy, C.L. & Haskell, D. (1988). Detection of polypeptides associated with the cold acclimation process in spinach. Electrophoresis 9, 787-96.

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Hahn, M. & Walbot, V. (1989). Effects of cold-treatment on protein synthesis and mRNA levels in rice leaves. Plant Physiology 91, 930-8. Hanson, A.D., Nelsen, C.E., Pedersen, A.R. & Everson, E.H. (1979). Capacity for proline accumulation during water stress in barley and its implications for breeding for drought resistance. Crop Science 19, 489-93. Hajela, R.K., Horvath, D.P., Gilmour, S.J. & Thomashow, M.F. (1990). Molecular cloning and expression of cor (cold-regulated) genes in Arabidopsis thaliana. Plant Physiology 93, 1246-52. Heino, P., Sandman, G., Lang, V., Nordin, K. & Palva, E.T. (1990). Abscisic acid deficiency prevents development of freezing tolerance in Arabidopsis thaliana (L.) Heynh. Theoretical and Applied Genetics 79, 801-6. Hincha, D.K., Heber, U. & Schmitt, J.M. (1989). Freezing ruptures thylakoid membranes in leaves, and rupture can be prevented in vitro by cryoprotective proteins. Plant Physiology and Biochemistry 27, 795-801. Hincha, D.K., Heber, U. & Schmitt, J.M. (1990). Proteins from frosthardy leaves protect thylakoids against mechanical freeze-thaw damage in vitro. Planta 180, 416-19. Hugly, S., McCourt, P., Browse, J., Patterson, G.W. & Somerville, C. (1990). A chilling sensitive mutant of Arabidopsis with altered sterylester metabolism. Plant Physiology 93, 1053-62. Huner, N.P.A. & Macdowall, F.D.H. (1979). The effects of low temperature acclimation on the catalytic properties of its ribulose bisphosphate carboxylase-oxygenase. Canadian Journal of Biochemistry 57, 1036-41. King, A.I., Joyce, D.C. & Reid, M.S. (1988). Role of carbohydrates in diurnal chilling sensitivity of tomato seedlings. Plant Physiology 86, 764-8. Kurkela, S. & Franck, M. (1990). Cloning and characterization of a cold- and ABA-inducible Arabidopsis gene. Plant Molecular Biology 15, 137-44. Kushad, M.M. & Yelenosky, G. (1987). Evaluation of polyamine and proline levels during low temperature acclimation of citrus. Plant Physiology 84, 692-5. Lang, V., Heino, P. & Palva, E.T. (1989). Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh. Theoretical and Applied Genetics 77, 729-34. Lazinski, D., Grzadzielska, E. & Das, A. (1989). Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif. Cell 59, 207-18. Levitt, J. (1980). Responses of Plants to Environmental Stresses, 2nd edn, Vol. 1. New York: Academic Press.

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L. CATTIVELLI AND D. BARTELS Lin, C , Guo, W.W., Everson, E. & Thomashow, M.F. (1990). Cold acclimation in Arabidopsis and wheat. Plant Physiology 94, 1078-83. Lynch, D.V. & Steponkus, P.L. (1987). Plasma membrane lipid alterations associated with cold acclimation of winter rye seedlings (Secale cereale L. cv. Puma). Plant Physiology 83, 761-7. Meza-Basso, L., Alberdi, M., Raynal, M., Ferrero-Cadinanos, M.-L. & Delseny, M. (1986). Changes in protein synthesis in rapeseed (Brassica napus) seedlings during a low temperature treatment. Plant Physiology 82, 733-8. Mohapatra, S.S., Poole, R.J. & Dhindsa, R.S. (1987a). Changes in protein patterns and translatable messenger RNA populations during cold acclimation of alfalfa. Plant Physiology 84, 1172-6. Mohapatra, S.S., Poole, R.J. & Dhindsa, R.S. (1987b). Cold acclimation, freezing resistance, and protein synthesis in alfalfa (Medicago sativa L. cv. Saranac). Journal of Experimental Botany 38, 1697-703. Mohapatra, S.S., Poole, R.J. & Dhindsa, R.S. (1988). Abscisic acid regulated gene expression in relation to freezing tolerance in alfalfa. Plant Physiology 87, 468-73. Mohapatra, S.S., Wolfraim, L., Poole, R.J. & Dhindsa, R.S. (1989). Molecular cloning and relationship to freezing tolerance of coldacclimation-specific gene of alfalfa. Plant Physiology 89, 375-80. Mundy, J. & Chua, N.-H. (1988). Abscisic acid and water-stress induce the expression of a novel rice gene. The EMBO Journal 7, 2279-86. Omran, R.G. (1980). Peroxide levels and the activities of catalase, peroxidase, and indoleacetic acid oxidase during and after chilling cucumber seedlings. Plant Physiology 65, 407-8. Perras, M. & Sarhan, F. (1989). Synthesis of freezing tolerance proteins in leaves, crown, and roots during cold acclimation of wheat. Plant Physiology 89, 577-85. Piatkowski, D., Schneider, K., Salamini, F. & Bartels, D. (1990). Characterization of five abscisic acid-responsive cDNA clones isolated from the desiccation-tolerant plant Craterostigma plantagineum and their relationship to other water-stress genes. Plant Physiology 94, 1682-8. Pollock, C.J. & Ruggles, P.A. (1976). Cold-induced fructosan synthesis in leaves of Dactylis glomerata. Phytochemistry 15, 1643-6. Raskin, I. & Ladyman, J.A.R. (1988). Isolation and characterization of a barley mutant with abscisic-acid-insensitive stomata. Planta 173, 73-8. Rhodes, M.J. & Wooltorton, L.S. (1977). Changes in the activity of enzymes of phenylpropanoid metabolism in tomatoes stored at low temperatures. Phytochemistry 16, 655-9. Riov, J. & Brown, G.N. (1976). Comparative studies of activity and properties of ferrodoxin-NADP+ reductase during cold hardening of wheat. Candian Journal of Botany 54, 1896-902.

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Roberts, D.W.A. (1975). The invertase complement of cold-hardy and cold-sensitive wheat leaves. Canadian Journal of Botany 53, 1333-7. Robertson, A.J., Gusta, L.V., Reaney, M.J.T. & Ishikawa, M. (1987). Protein synthesis in bromegrass (Bromus inermis Leyss) cultured cells during the induction of frost tolerance by abscisic acid or low temperature. Plant Physiology 84, 1331-6. Roughan, P.G. (1985). Phosphatidylglycerol and chilling sensitivity in plants. Plant Physiology 77, 740-6. Sachs, M.M. & Ho, T.-H.D. (1986). Alterations of gene expression during environmental stress in plants. Annual Review of Plant Physiology 37, 363-76. Sagisaka, S. (1985). Injuries of cold acclimatized poplar twigs resulting from enzyme inactivation and substrate depression during frozen storage at ambient temperatures for a long period. Plant and Cell Physiology 26, 1135-45. Sakai, A. & Larcher, W. (1987). Frost Survival of Plants: Responses and Adaptations to Freezing Stress. New York: Springer-Verlag. Sarhan, F. & Chevrier, N. (1985). Regulation of RNA synthesis by DNA-dependent RNA polymerases and RNases during cold acclimation in winter and spring wheat. Plant Physiology 78, 250-5. Sasaki, T., Tadokoro, K. & Suzuki, S. (1971). Multiple forms of invertase of potato tuber stored at low temperature. Phytochemistry 10, 2047-50. Schaffer, M.A. & Fischer, R.L. (1988). Analysis of mRNAs that accumulate in response to low temperature identifies a thiol protease gene in tomato. Plant Physiology 87, 431-6. Schaffer, M.A. & Fischer, R.L. (1990). Transcriptional activation by heat and cold of a thiol protease gene in tomato. Plant Physiology 93, 1486-91. Skriver, K. & Mundy, J. (1990). Gene expression in response to abscisic acid in osmotic stress. The Plant Cell 2, 503-12. Songstad, D.D., Duncan, D.R. & Widholm, J.M. (1990). Proline and polyamine involvement in chilling tolerance of maize suspension cultures. Journal of Experimental Botany 41, 289-94. Steponkus, P.L. (1984). Role of the plasma membrane in freezing injury and cold acclimation. Annual Review of Plant Physiology 35, 543-84. St John, J.B., Christiansen, M.N., Ashworth, E.N. & Gentner, W.A. (1979). Effect of BASF 13-338, a substituted pyridazinone, on linolenic acid levels and winterhardiness of cereals. Crop Science 19, 65-9. Sutka, J. & Snape, J.W. (1989). Location of a gene for frost resistance on chromosome 5A of wheat. Euphytica 42, 41-4. Swaaij, van A.C., Jacobsen, E. & Feenstra, W.J. (1985). Effect of cold hardening, wilting and exogenously applied proline on leaf proline content and frost tolerance of several genotypes of Solanum. Physiologia Plantarum 64, 230-6.

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Thomashow, M.F. (1990). Molecular genetics of cold acclimation in higher plants. Advances in Genetics 28, 99-131. Veidt, I., Lot, H., Leiser, M., Scheidecker, D., Guilley, H., Richards, K. & Jonard, G. (1988). Nucleotide sequence of beet western yellow virus RNA. Nucleic Acids Research 16, 9917-32. Volger, H.G. & Heber, U. (1975). Cryoprotective leaf proteins. Biochimica et Biophysica Acta 412, 335-49. Walker-Simmons, M., Kudrna, D.A. & Warner, R.L. (1989). Reduced accumulation of ABA during water stress in a molybdenum cofactor mutant of barley. Plant Physiology 90, 728-33. Weiser, C.J. (1970). Cold resistance and injury in woody plants. Science 169, 1269-78.

U. SCHINDLER, A.E. MENKENS and A.R. CASHMORE

GBF-1, GBF-2 and GBF-3: three Arabidopsis b-Zip proteins that interact with the light-regulated rbcS-lA promoter Introduction Gene transcription requires the interplay between transcription factors with their cognate promoter elements. Structural and functional analyses of many of these transcription factors revealed a modular protein structure, composed of a DNA-binding domain and a transcriptional activation domain (Johnson & McKnight, 1989; Mitchell & Tjian, 1989). The DNA-binding domain of the b-Zip proteins is characterised by the presence of a basic region with an adjacent leucine zipper (Landschulz et al., 1988). Whereas the basic region is required for specific protein-DNA interaction and directly contacts the DNA, the leucine zipper facilitates homodimer and heterodimer formation (Hu et al., 1990 and references therein). Transcriptional activation domains are often enriched in acidic amino acids (Hope & Struhl, 1987; Ptashne, 1988), glutamines (Courey et al., 1989) or prolines (Mermod et al., 1989). Although many DNA-binding proteins have been identified in plant nuclear extracts, only a few cDNA sequences encoding these proteins have been cloned. Plant b-Zip proteins for which cDNAs have been isolated include the wheat proteins, EmBP-1 (Guiltinan et al., 1990) and HBP-1 (Tabata et al., 1989), the maize proteins OCSBF-1 (Singh et a!.., 1990) and Opaque2 (Schmidt et al., 1990) and the tobacco proteins TGAla and TGAlb (Katagiri et al., 1989). HBP-1, as originally identified in crude nuclear extract, was shown to interact with the hexamer (Hex) motif TGACGT found in several histone promoters (Mikami et al., 1987, 1989a,b,c). The cDNA identified as encoding HBP-1 was isolated by screening an expression library for specific DNA binding to an oligonucleotide derived from the wheat histone 3 promoter and containing the conserved Hex motif (Tabata et al., 1989). Sequences similar to the Society for Experimental Biology Seminar Series49: Inducible Plant Proteins, ed. J.L.Wray. ©Cambridge University Press, 1992, pp. 289-304.

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Hex motif are found in the octopine synthase (ocs) and nopaline synthase (nos) promoters of the Ti-plasmids (Bouchez et al., 1989), as well as in the cauliflower mosaic virus 35S promoter (as-1 motif) (Katagiri et al., 1989). The proteins OCSBF-1 and TGAla recognise the nos, as-1 and Hex motif. On the other hand, TGAlb is specific for the Hex sequence (Katagiri et al., 1989). Previously, we have shown that the promoters of the genes encoding the small subunits of the ribulose 1,5-bisphosphate carboxylase/ oxygenase (RBCS) are often characterised by conserved DNA sequences, designated L-, I- and G-boxes (Giuliano et al., 1988). In the case of the Arabidopsis thaliana rbcS-lA promoter, we demonstrated that both the G-box, as defined by the palindromic sequence CCACGTGG, and the I-boxes are required for expression of the GUS reporter gene in both transgenic tobacco and Arabidopsis plants (Donald & Cashmore, 1990). We identified a protein, designated GBF, in plant nuclear extracts, which specifically interacts with the G-box motif (Giuliano et al., 1988; Donald era/., 1990). G-box-like elements are also found in other plant promoters, including the cab-E promoter of N. plumbaginifolia (Castresana et al., 1988; Schindler & Cashmore, 1990) where a mutation of this sequence results in substantial loss of expression (P. Bringmann and A.R. Cashmore, unpublished data). Similarly, G-box-like sequences are present in chalcone synthase promoters (Schulze-Lefert et al., 1989; Staiger et al., 1989). In the case of the parsley chalcone synthase promoter, this element was shown to be required for UV light induced expression and in vivo footprinting studies demonstrated that the binding of a GBF-like factor was altered upon UV light induction (Block et al., 1990; Schulze-Lefert et al., 1989). The Arabidopsis alcohol dehydrogenase promoter, which mediates anaerobically enhanced root expression, also contains a G-boxlike sequence which is bound by a GBF-like factor, both in vivo (Ferl & Laughner, 1989) and in vitro (DeLisle & Ferl, 1990; McKendree et al., 1990). ABA-induced genes have also been shown to contain functional G-box-like sequences within their promoters (Marcotte et al., 1989; Mundy et al., 1990). From the preceding discussion it is apparent that G-box-like elements are located in a variety of plant promoters where they have been demonstrated to be either essential for expression and/or bound by a GBF-like factor. These G-box-containing promoters are induced by a variety of stimuli including red and UV light, ABA and anaerobiosis. These observations prompt the following questions: is there a single or multiple G-box-binding factor mediating these distinct expression characteristics? How does a single promoter element mediate these quite diverse expression characteristics?

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Relationship between GBF-1 and other plant b-Zip proteins To identify cDNAs encoding G-box-binding proteins, we screened a XZAPII cDNA expression library for DNA-binding activity specific to a synthetic oligonucleotide bearing the tomato rbcS-3A G-box-like sequence (aCACGTGG). Two positive phage were detected in the initial screen. Subsequent DNA sequence analysis revealed that both cDNAs were derived from the same mRNA species (data not shown). The longest cDNA, GBF-1, encoded a 34 kDa protein that was characterised by a N-terminal proline-rich region and a C-terminal putative DNAbinding domain composed of a basic region and a leucine zipper. A comparison between the putative DNA-binding domain of GBF-1 with those of other known plant b-Zip proteins is illustrated in Fig. 1 A. The 31 amino acids (219-249) encompassing the basic region of GBF-1 showed some similarity (45 and 39%, respectively) to the two tobacco proteins, TGAla and TGAlb (Katagiri et al., 1989). An even greater similarity (55% in both cases) was observed when GBF-1 was compared with the two maize proteins, O2 (Schmidt et al, 1990) and OCSBF-1 (Singh et al., 1990). TGAla, TGAlb and OCSBF-1 have all been shown to interact with TGACG-related motifs present in several plant promoters (Katagiri et al, 1989; Singh et al, 1990). The most striking similarity to the basic region of GBF-1 was noticed for two wheat proteins, EmBP-1 (87%) (Guiltinan et al, 1990) and the protein identified as HBP-1 (90%) (Tabata et al, 1989). EMBP-1 was shown to interact with a G-box-like motif found in the ABA response element of the wheat Em promoter (Guiltinan et al, 1990). Even though GBF-1 and EmBP-1 interact with similar DNA recognition sites, they are quite divergent outside the basic motif (R.S. Quatrano, unpublished data). By contrast, GBF-1 and the protein identified as HBP-1 showed striking sequence similarity not only within the basic region, but also within parts of the N-terminal proline-rich region (69% similarity within a stretch of 45 amino acids, Fig. IB). This sequence conservation, especially within the basic region, was unexpected since HBP-1, as identified in nuclear extracts, was shown to interact with the conserved hexamer motif TGACGT found in several histone promoters (Mikami et al, 1987, 1989a,b,c). The cDNA encoding a protein identified as HBP-1 was isolated by screening an expression library with an oligonucleotide containing the conserved hexamer motif of the wheat histone 3 promoter.

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b-Zip proteins and the rbcS-lA promoter

293

GBF-1 interacts with the G-box but not with the TGACGT motif unless present in the context of a G-boxlike sequence In view of the observed sequence similarity between the basic region of GBF-1 and HBP-1, we considered it imperative to determine whether GBF-1 was a G-box, or a hexamer-binding protein. Sequences similar to the histone hexamer motif TGACGT are also found in the CaMV-35S promoter (as-1 element). Interestingly, the TGACGT hexamer motif contains the core ACGT sequence of the G-box (CCACGTGG). Furthermore, the oligonucleotide used to screen for the wheat protein HBP1 contained an overlapping G-box-like sequence (Hex, TGACGTGG, Fig. 2A). In the mutant oligonucleotide (Hexml, TtACtTGG, Fig. 2A) used in the screen for HBP-1, both the hexamer motif and the G-box-like sequence were altered and thus these experiments did not distinguish binding of the two sequences (Tabata et al., 1989). To investigate further whether GBF-1 would interact with the TGACGT motif, a competition assay with several synthetic oligonucleotides was performed. The results shown in Fig. 2B demonstrated that binding of the in vitro translated protein, GBF-1, to the G-3A oligonucleotide was specifically competed by the oligonucleotide itself (lane 4), but not by the mutant oligonucleotide (G-3Am, lane 5). This results was in agreement with what we expected from the nature of the oligonucleotides used in the initial screen. Specific competition was also obtained when the Hex oligonucleotide (lane 6) was included in the

Fig. 1. GBF-1 is similar to other plant DNA-binding proteins. A, above: Schematic presentation of GBF-1, indicating the location of the prolinerich region (Pro), the basic region (BR) and the leucine zipper domain (LZ); below: amino acid comparison between the DNA-binding domain of GBF-1 and other plant DNA-binding proteins (Katagiri et al., 1989; Tabata et al., 1989; Guiltinan et al., 1990; Schmidt et al., 1990; Singh et al., 1990). Identical amino acids are indicated by an asterisk, BR-1 and BR-2 designate the two clusters of basic amino acids, the leucine residues within the leucine zipper are highlighted and numbered. B, Dot matrix analysis of amino acid similarities between GBF-1 and the protein identified as HBP-1. The location of the proline-rich region and the basic region is indicated. The sequence alignment below illustrates the similarity between the two proteins within the proline-rich region; one gap was inserted within HBP-1 to optimise the alignment. The numbers indicate the amino acid positions. Identical amino acids are indicated by an asterisk.

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b-Zip proteins and the rbcS-lA promoter

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binding reaction. Furthermore, no competition was observed with the Hexml oligonucleotide (lane 7); this result was similar to the result obtained by Tabata et al. (1989) for the protein identified as HBP-1. To distinguish whether binding of GBF-1 required the G-box-like sequence or simply the overlapping TGACGT motif present within the Hex oligonucleotide, the Hexm2 mutant was designed (Fig. 2A). This oligonucleotide contained two base pair substitutions within the G-boxlike sequence, but it retained the integrity of the TGACGT motif. GBF-1 did not interact with this sequence (Fig. 2B, lane 8). Furthermore, the oligonucleotide as-1, derived from the 35S promoter and containing one TGACGT and one TGACGC motif (Fig. 2A), was also not recognised by GBF-1 (Fig. 2B, lane 9). These results clearly demonstrated that GBF-1 is a G-box-binding protein and that it does not recognise the TGACGT motif unless present in the context of a G-box-like sequence (tgACGTGG). GBF-1 mRNA is expressed in both roots and leaves and its transcription does not appear to be regulated by light In view of the possibility that GBF-1 may play a role in mediating the expression of genes such as rbcS-lA that are light-regulated and selectively expressed in leaf tissue, we determined the expression characteristics of GBF-1. RNA isolated from Arabidopsis leaves and roots was subjected to analyses by northern blot (Fig. 3). The leaf RNA was prepared from 6-day-old seedlings which were grown either under standard daylight conditions or in constant darkness. A single mRNA band of almost equal intensity was detected under all conditions, indicat-

Fig. 2. GBF-1 does not interact with TGACG related motifs. A, DNA sequence of the oligonucleotides used to determine the binding specificity of GBF-1. The G-3A oligonucleotide is derived from the tomato rbcS-3A promoter; the mutant, G-3Am, carries 4 base pair substitutions within the core sequence. The Hex motif is derived from the wheat histone 3 promoter, Hexml and Hexm2 are mutant derivatives carrying the indicated base pair substitutions, as-1 contains the TGA-la binding sites of the CaMV-35S promoter. The arrows designate the location of the G-box motif. The highlighted base pairs indicate the TGACG related motifs. B, Competition assay using in vitro translated GBF-1 protein. The labelled G-3A oligonucleotide was incubated with either no extract (lane 1), rabbit reticulolysate without RNA (lane 2) or in vitro translated GBF-1 (lanes 3-9). Different oligonucleotides were induced in the binding reactions as indicated above each lane (lanes 4-9).

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L

D

R

— 28S

— 18S

Fig. 3. Expression characteristics of GBF-1. Total RNA (40 |xg per lane) was isolated from Arabidopsis leaves, grown under standard daylight (L) conditions or in complete darkness (D), and from roots (R). The RNA was electrophoretically separated, transferred to nitrocellulose and hybridised to the radiolabelled cDNA encoding GBF-1. The position of the two ribosomal RNAs, 28S (3.5 kbp) and 18S (1.8 kbp), is indicated. ing that the gene encoding GBF-1 is transcribed in both photosynthetically active and inactive tissue. This result is in agreement with our previous findings that GBF activity is present in extracts prepared from both light- and dark-grown tissue (Giuliano et al., 1988; Schindler & Cashmore, 1990).

GBF-1 does not bind to all G-box-like promoter sequences Several genes, which are induced by many different stimuli and are expressed in a variety of tissues, contain G-box-like promoter sequences. In many instances this element has been shown to be bound by nuclear proteins and/or has been demonstrated to be important for expression. In attempting to evaluate whether GBF-1 - or related factors - may be involved in mediating the expression of these diverse genes, we considered it important to investigate the binding properties of GBF-1 for some of these naturally occurring G-box-like promoter sequences. GBF-1 binds to the promoters of a number of light-regulated genes. In addition to binding to the perfectly palindromic G-box sequence of the Arabidopsis rbcS-lA promoter, we also observed binding to the nonpalindromic tomato rbcSSA sequence (aCACGTGG; it was this sequence that was used for the initial screen) and the N. plumbaginifolia cab-E promoter sequence (agACGTGG). One example of a non-lightregulated Arabidopsis gene containing a 'perfect' G-box promoter

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sequence is the Adh gene, which is induced by a variety of different stimuli. In binding studies employing an oligonucleotide derived from the Adh promoter (G-adh, Fig. 4), this element was bound by GBF-1 with a level of efficiency similar to that observed for the rbcS-lA G-box. In contrast, only weak binding was obtained with the element derived from the cold-induced Arabidopsis Corl80 gene promoter (G-corl80, Fig. 4). This element differs by three base pair substitutions (atCAGTGt) from the perfectly-palindromic G-box motif. GBF-1 bound very strongly to the palindromic G-box (box II) found in the parsley (Petroselinum crispum)

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core sequence Fig. 4. GBF-1 interacts with some, but not all, G-box-like elements identified in plant promoters. DNA sequence of oligonucleotides derived from different plant promoters containing G-box-like elements. G-1A represents the motif found in the Arabidopsis rbcS-lA promoter (Donald & Cashmore, 1990). G-adh is derived from the Arabidopsis alcohol dehydrogenase promoter (DeLisle & Ferl, 1990), G-chs (P.c.) from the P. crispum chalcone synthase promoter (Schulze-Lefert et al., 1989), G-cabE from the N. plumbaginifolia cab-E promoter (Schindler & Cashmore, 1990) and G-corl80 represents the motif located in the cold-induced Arabidopsis corl80 promoter (M. Thomashow, personal communication). The two elements G-chs (A.m.) —54 and —120 are found in the A. majus chalcone synthase promoter (Staiger et al., 1989) and the USF element is derived from the adenovirus major late promoter (Gregor et al., 1990). The arrows indicate the location of the Gbox, the asterisks mark the base pairs which are identical to the palindromic G-box motif; ' + + + ' indicates high affinity, ' + + ' medium affinity, ' + ' low affinity, ' - / + ' DNA binding only at high protein concentrations, '—' no binding.

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chalcone synthase promoter (Schulze-Lefert et al., 1989; G-chs (P.a), Fig. 4). In contrast, neither of the two G-box-like elements in the promoter of A. majus chalcone synthase gene (Staiger et al., 1989) were efficiently bound by GBF-1 (G-chs (A.m.) -54 and -120, Fig. 4). A similar negative result was obtained with an oligonucleotide containing the binding site for the mammalian transcription factor USF (Sawadogo & Roeder, 1985), which differs by only one base pair (CCACGTGa, Fig. 4) from the perfectly-palindromic G-box motif. Similar results were obtained with GBF in crude nuclear extracts. These data indicate that GBF and GBF-1 have similar DNA-binding properties and that they are distinct from the binding characteristics of USF, a helix-loop-helix/ leucine zipper protein. The fact that GBF-1 bound to the cab-E G-box-like sequence, but bound only weakly to the USF recognition sequence and the G-box-like elements of the A. majus chalcone synthase promoter, contrasted with results reported for the plant nuclear factor CG-1 (Staiger et al., 1989). This latter factor was shown to interact with both the USF recognition sequence and one of the G-box-like elements of the A. majus chalcone synthase promoter; it did not interact with the cab-E motif. These cumulative DNA-binding studies indicate that GBF-1 and CG-1 have different DNA-binding properties. How do we interpret these somewhat conflicting binding studies involving the 'perfect' G-box sequence of the parsley chalcone synthase promoter (that binds to GBF-1) and the divergent G-box-like sequence of the Antirrhinum promoter (that does not bind to GBF-1 - indeed, it binds in vitro to a factor that is clearly distinct from GBF-1)? One interpretation is that the expression of RBCS genes may involve GBF-1-like leucine zipper proteins whereas, as was suggested (Staiger et al., 1989), the expression of chalcone synthase genes may involve a USF-(helix-loophelix)like protein. The difficulty with this interpretation is that mutation of the parsley G-box sequence to a sequence corresponding to the USF binding site results in loss in expression (Block et al., 1990). Clearly, additional studies are needed to clarify these points. GBF-1 interacts as a dimer with the G-box Leucine zipper proteins have been shown to interact with their cognate recognition sites as dimers (Landschulz et al., 1988). This dimer formation is mediated by the leucine zipper domain and is absolutely required for DNA binding (Hu et al., 1990 and references therein). We employed the method of Hope & Struhl (1987) to investigate the ability of GBF-1 to form dimers in the presence of DNA. Different portions of the cDNA

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encoding GBF-1 were translated in vitro and employed in DNA binding studies. As expected, when the full-length protein GBF-1 and the truncated version GBF-1 (199-288), containing amino acids 199-288, were individually assayed for DNA binding only one major protein-DNA complex was observed in each case (Fig. 5, lanes 3 and 4, complex I and III). When both proteins were synthesised separately and mixed in the presence of DNA, again the two corresponding protein-DNA complexes were formed (Fig. 5, lane 5). However, when mRNAs corresponding to G-IA

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Fig. 5. GBF-1 binds as a homodimer. The labelled G-1A (lanes 1-6) or Hex (lanes 7-12) oligonucleotides were incubated with either no protein (lanes 1 and 7), rabbit reticulolysate (RRL) without RNA (lanes 2 and 8), the in w?ro-translated full-length protein GBF-1 (lanes 3 and 9) or the truncated version GBF-1 (199-288) (lanes 4 and 10). For the binding reactions shown in the last two lanes of each set, the two proteins were translated separately and mixed in the binding assay (lanes 5 and 11) or were cotranslated (lanes 6 and 12). I, II and III indicate the predicted dimers.

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the two proteins were cotranslated and the product assayed for DNA binding, a complex of intermediate mobility appeared (complex II, lane 6). A similar result was obtained when both proteins were translated separately and incubated together prior to the addition of the DNA (data not shown). We interpret the formation of complex II as corresponding to a heterodimer formed between the full-length and the truncated versions of the protein. It is interesting to note that the rbcS-lA G-box used as a probe in the left panel (Fig. 5, lanes 1-6) represented a perfect palindrome (Fig. 4). In contrast, the Hex oligonucleotide (Fig. 2A) which was also used in these experiments (Fig. 5, lanes 7-12) contained two mismatches in the 5' half of the recognition site. Binding of GBF-1 to this Hex oligonucleotide also indicated dimerisation. These findings suggested that GBF-1 binds as a dimer, even in those cases where the DNA-binding sequence is quite asymmetric. Arabidopsis thaliana contains multiple genes encoding GBF-like proteins Given that the G-box motif mediates the expression of a diverse array of plant genes, it was clearly of interest to know how may G-box-binding proteins existed in a simple diploid plant such as Arabidopsis. To explore this question, we used a DNA probe corresponding to the conserved basic DNA-binding domain of GBF-1 to screen an Arabidopsis cDNA library by low stringency DNA hybridisation. Two positively hybridising phage were isolated and shown to contain cDNA inserts of approximately 1.4 and 1.5 kb. DNA sequencing studies showed that these cDNAs encoded proteins (GBF-2 and GBF-3) with putative DNA-binding domains exceedingly similar to the basic region of GBF-1 (Fig. 6). Within 219 GBF-1

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Fig. 6. Amino acid sequence similarity between the three G-box-binding proteins isolated from Arabidopsis. The location of basic region 1 (BR1) and 2 (BR-2) is indicated. The leucine residues within the leucine zipper are highlighted and numbered. The hydrophobic amino acids interdigitating the leucine repeats are designated a-d. 219 and 249 denote amino acid positions in GBF-1; asterisks indicate identical amino acids.

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this region of 31 amino acids, GBF-2 and GBF-3 share 84% amino acid similarity when compared with GBF-1. The amino acid similarity was not restricted to the basic region but extended into the leucine zipper domain (Fig. 6). In addition to the conservation of each leucine residue (L1-L5), position a, b and c, interdigitating the heptad repeats, were also conserved. These amino acids are believed to be involved in the hydrophobic interactions between two leucine zipper domains (Hu et al., 1990). Binding studies employing GBF-2 and GBF-3 indicated that the two proteins are, like GBF-1, G-box-specific DNA-binding proteins (data not shown). Furthermore, our sequence data for GBF-2 and GBF-3 (U. Schindler et al., unpublished data) indicate that, whereas these proteins do contain proline-rich regions, neither of these sequences is as similar in this region to GBF-1 as is the protein identified as HBP-1. This sequence conservation within parts of the proline-rich regions of GBF-1 and HBP-1 suggests that these proteins might fulfil a common function in both plant: species. In contrast, GBF-2 and GBF-3 might be involved in other signal transduction pathways. What are the functions of the multiple GBF-like proteins in Arabidopsis! G-box-like binding sites are found in a variety of different plant promoters. In several cases these DNA sequences either have been shown to be important for the expression of the corresponding gene (SchulzeLefert et al., 1989; Block et al, 1990; Donald & Cashmore 1990; Guiltinan et al., 1990) and/or they have been bound (in vivo or in vitro) by a GBF-like protein (Giuliano et al., 1988; Ferl & Laughner, 1989; Marcotte et al., 1989; Staiger et al., 1989; DeLisle & Ferl, 1990). Most of these promoters are activated under different developmental or environmental conditions. These observations raise some obvious questions. How many different G-box-binding proteins exist in Arabidopsis and what is the role of these multiple GBF-like proteins? Is there a distinct GBF-like protein that mediates the expression of photoregulated genes or are there multiple GBF-like proteins that mediate this process? Are there additional classes of GBF-like proteins that mediate, for example, ABA-induced or ADH gene expression and/or in some cases does a single GBF-like protein mediate the expression of genes with quite distinct properties? Having demonstrated that there are multiple Arabidopsis GBF proteins, a difficult task now will be to determine the function of these individual proteins. In order to address this point directly, clearly a genetic, or reverse genetic approach will be necessary.

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References Block, A., Dangl, J.L., Hahlbrock, K. & Schulze, L.P. (1990). Functional borders, genetic fine structure, and distance rquirements of cis elements mediating light responsiveness of the parsley chalcone synthase promoter. Proceedings of the National Academy of Sciences (USA) 87, 5387-91. Bouchez, D., Tokuhisa, J.G., Llewellyn, D.J., Dennis, E.S. & Ellis, J.G. (1989). The ocs-element is a component of the promoters of several T-DNA and plant viral genes. The EMBO Journal 8, 4197-204. Castresana, C , Garcia-Luque, I., Alonso, E., Malik, V.S. & Cashmore, A.R. (1988). Both positive and negative regulatory elements mediate expression of a photoregulated CAB gene from Nicotiana plumbaginifolia. The EMBO Journal 7, 1929-36. Courey, A.J., Holtzman, D.A., Jackson, S.P. & Tjian, R. (1989). Synergistic activation by the glutamine-rich domains of human transcription factor Spl. Cell 59, 827-36. DeLisle, A.J. & Ferl, R.J. (1990). Characterization of the Arabidopsis Adh G-box binding factor. The Plant Cell 2, 547-57. Donald, R.G.K. & Cashmore, A.R. (1990). Mutation of either G-box or I-box sequences profoundly affects expression from the Arabidopsis rbcS-lA promoter. The EMBO Journal 9, 1717-26. Donald, R.G.K., Schindler, U., Batschauer, A. & Cashmore, A.R. (1990). The plant G-box promoter sequence activates transcription in Saccharomyces cerevisiae and is bound in vitro by a yeast activity similar to GBF, the plant G-box binding factor. The EMBO Journal 9, 1727-35. Ferl, R.J. & Laughner, B.H. (1989). In vivo detection of regulatory factor binding sites of Arabidopsis thaliana Adh. Plant Molecular Biology 12, 357-66. Giuliano, G., Pichersky, E., Malik, V.S., Timko, M.P., Scolnik, P.A. & Cashmore, A.R. (1988). An evolutionarily conserved protein binding sequence upstream of a plant light-regulated gene. Proceedings of the National Academy of Sciences (USA) 85, 7089-93. Guiltinan, M.J., Marcotte, W.R. & Quatrano, R.S. (1990). A plant leucine zipper protein that recognizes an abscisic acid response element. Science 250, 267-71. Hope, I.A. & Struhl, K. (1987). GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA. The EMBO Journal 6, 2781-4. Hu, J.C., O'Shea, E.K., Kim, P.S. & Sauer, R.T. (1990). Sequence requirements of coiled-coils: analysis with X repressor-GCN4 leucine zipper fusions. Science 250, 1400-3. Johnson, P.F. & McKnight, S.L. (1989). Eukaryotic transcriptional regulatory proteins. Annual Review of Biochemistry 58, 799-839.

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Katagiri, F., Lam, E. & Chua, N.-H. (1989). Two tobacco DNA-binding proteins with homology to the nuclear factor CREB. Nature 340, 727-30. Landschulz, W.H., Johnson, P.F. & McKnight, S.L. (1988). The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240, 1759-64. Marcotte, W.R., Russell, S.H. & Quatrano, R.S. (1989). Abscisic acidresponsive sequences from the Em gene of wheat. The Plant Cell 1, 969-76. McKendree, W.L., Paul, A.-L., DeLisle, A.J. & Ferl, R J . (1990). In. vivo and in vitro characterization of protein interactions with the dyad G-box of the Arabidopsis Adh gene. The Plant Cell 2, 207-14. Mermod, N., O'Neill, E.A., Kelley, T.J. & Tjian, R. (1989). The proline-rich transcriptional activator of CTF/NF-1 is distinct from the replication and DNA binding domain. Cell 58, 741-53. Mikami, K., Nakayama, T., Kawata, T., Tabata, T. & Iwabuchi, M. (1989a). Specific interaction of nuclear protein HBP-1 with the conserved hexameric sequence ACGTCA in the regulatory region of wheat histone genes. Plant and Cell Physiology 30, 107-19. Mikami, K., Tabata, T., Kawata, T., Nakayama, T. & Iwabuchi, M. (1987). Nuclear protein(s) binding to the conserved DNA hexameric sequence postulated to regulate transcription of wheat histone genes. FEBS Letters 223, 273-8. Mikami, K., Takase, H., Tabata, T. & Iwabuchi, M. (19896). Multiplicity of the DNA-binding protein HBP-1 specific to the conserved hexameric sequence ACGTCA in various plant gene promoters. FEBS Letters 256, 67-70. Mikami, K., Sakamoto, A., Takase, H., Tabata, T. & Iwabuchi, M. (1989c). Wheat nuclear protein HBP-1 binds to the hexameric sequence in the promoter of various plant genes. Nucleic Acids Research 17, 9707-17. Mitchell, P.J. & Tjian, R. (1989). Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245, 371-8. Mundy, J., Yamaguchi, K.S. & Chua, N.H. (1990). Nuclear proteins bind conserved elements in the abscisic acid-responsive promoter of a rice rab gene. Proceedings of the National Academy of Sciences (USA) 87, 1406-10. Ptashne, M. (1988). How eukaryotic transcriptional activators work. Nature 335, 683-9. Sawadogo, M. & Roeder, R.G. (1985). Interaction of a gene-specific transcription factor with the adenovirus major late promoter upstream of the TATA box region. Cell 43, 165-75. Schindler, U. & Cashmore, A.R. (1990). Photoregulated gene expression may involve ubiquitous DNA binding proteins. The EM BO Journal 9, 3415-27.

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Schmidt, R.J., Burr, F.A., Aukerman, M.J. & Burr, B. (1990). Maize regulatory gene opaque-2 encodes a protein with a "leucine-zipper" motif that binds to zein DNA. Proceedings of the National Academy of Sciences (USA) 87, 46-50. Schulze-Lefert, P., Dangl, J.L., Becker-Andre, M , Hahlbrock, K. & Schulz, W. (1989). Inducible in vivo DNA footprints define sequences necessary for UV light activation of the parsley chalcone synthase gene. The EMBO Journal 8, 651-6. Singh, K., Dennis, E.S., Ellis, J.G., Llewellyn, D.J., Tokuhisa, J.G., Wahleithner, J.A. & Peacock, W.J. (1990). OCSBF-1, a maize Ocs enhancer binding factor: isolation and expression during development. The Plant Cell 2, 891-903. Staiger, D., Kaulen, H. & Schell, J. (1989). A CACGTG motif of the Antirrhinum majus chalcone synthase promoter is recognized by an evolutionarily conserved nuclear protein. Proceedings of the National Academy of Sciences (USA) 86, 6930-4. Tabata, T., Takase, H., Takayama, S., Mikami, K., Nakatsuka, A., Kawata, T., Nakayama, T. & Iwabuchi, M. (1989). A protein that binds to a cw-acting element of wheat histone genes has a leucine zipper motif. Science 245, 965-9.

Index

Page references in italics refer to figures or tables. ABA (abscisic acid) alcohol dehydrogenase gene Adhl 242 antagonism with gibberellic acid 1414, 145 CAM induction 128 cold acclimation 279-80 cold-regulated proteins 274-80, 276, 277 DNA response element 144-7 dormancy 139 gene expression 139-47, 142, 143, 144, 145, 274-80, 276, 277, 283 osmotic stress 139-41, 140 ACC (1-aminocyclopropane-l-carboxylic acid) synthase amino acid sequence 166 cDNA clone 160-7 ethylene biosynthesis 157, 158, 158-60, 164 expression in yeast 161, 165-7, 168 properties 167-9 sense and antisense RNA expression 161-7, 169-70, 762, 163, 164, 165, 170 transgenic plants 161-7, 162, 163, 164, 165 acid phosphatase (epsi) excreted phosphate starvation inducible 25, 27-37, 30, 31, 32, 34, 38 alcohol dehydrogenase abscisic acid 242 Adhl promoter 232-7, 235, 236, 237 antisense constructs 240-2, 241 mutant 239-40 transgenic plants 240-2, 241 alcohol fermentation pathway 232 a-amylase DNA response element 144-7

gibberellic acid 141, 144, 146 anaerobic polypeptides 231-2, 238 anaerobic response alcohol dehydrogenase 231-42 conservation 238-9 gene expression 231-3 oxygen sensor 237-8 pyruvate decarboxylase 231, 239, 2401,241 signal perception 242 survival during anoxia 239-42 antisense mRNA ACC synthase 161-7, 162, 163, 164, 165 effect on fruit ripening 169-70, 170 alcohol dehydrogenase 240-2, 241 chalcone synthase 97 cinnamyl alcohol dehydrogenase 107 heat shock protein 256, 257-9, 258 nitrite reductase 65 polygalacturonase 159 pyruvate decarboxylase 240-2, 241 ARE (anaerobic response element) Adhl gene 232-7 protein binding 234-7, 235, 236, 237 structure 233-7, 236, 237 b-Zip proteins 289-304 see also GBF-1, G-box factors CAT (chloramphenicol acetyltransferase), see reporter gene fusions CAM (crassulacean acid metabolism) abscisic acid 128 biochemistry and molecular biology 113-31 gene expression in the ice plant 123-8, 126 gene promoters 128-31

306

Index

CAM (cont.) induction 113-15, 127-8 pathway 113-16, 775 phosphoeno/pyruvate decarboxylase 115-31 chalcone synthase 97 chitinase systemic acquired resistance 206-8, 209, 270, 212, 213, 214, 215, 216, 217 cinnamyl alcohol dehydrogenase antisense mRNA 107 biochemistry 105-6 cDNA clones 105-6 purification 106 cold-stress responses 267-88, 268 abscisic acid 274-80, 276, 277, 283 biochemistry 267-72, 270, 281 molecular analysis 272-82, 276, 277 cold-regulated genes 272-81, 276, 277, 281

polypeptides and translatable mRNA 272-8, 281 relationship to heat shock response 280 4-coumarate: CoA ligase phenylpropanoid pathway 99, 99 DNA-binding proteins Adhl promoter 234-7, 235, 236, 237 b-Zip proteins 289-304 G-box factors 289-304 heat shock transcription factor 259 nodulin gene promoter 190 ppcl promoter 128-30 rbcS-lA promoter 289-304 DNA response elements abscisic acid response element (ABRE) 144-7 anaerobic response element (ARE) 232-7, 236, 237 gibberellic acid response element (GARE) 144-7 heat shock element (HSE) 249-55 metal response element (MRE) 17 salicyclic acid responsive element (SARE) 219-21, 221 ethylene ACC synthase 161-8, 162, 163, 164, 165, 168 biosynthetic pathway 157, 158, 158-160 control 159-67 genes 159-67 fruit ripening 158, 169-70, 770

gene expression 158, 159-67 perception 158 production in transgenic plants 161, 164 reduced synthesis 161, 164, 169-70, 170 -stimulated mRNA 156 ethylene forming enzyme, (see ACC synthase fruit ripening ethylene perception 158 ethylene synthesis 158 gene expression 156-9, 158, 169-70, 170 in transgenic tomato plants 169-70, 770 reduced ethylene synthesis 169-70, 170 GA (gibberellic acid) a-amylase 141, 144, 146 antagonism with ABA 141-4, 145 DNA response element 144-7 gene expression 139, 141-7 GBF-1 (G-box factor 1) interaction with G-box 293-5, 294, 296-300, 297, 299 mRNA expression 295-6, 296 relationship to other b-Zip proteins 291-2, 292 G-box factors (GBF-1, GBF-2 and GBF3) 289-304 function 301 multiple genes 300-1, 300 gene expression ABA-responsive 139-47, 742, 143, 145, 274-80, 276, 277, 283 anaerobic response 231-3 CAM 123-38, 726 cold-inducible 272-81, 276, 277 ethylene biosynthesis 159-67, 158, 162, 163, 164, 165, 168 fruit-ripening 156-9, 158, 169-70, 770 GA-responsive 139, 141-7 G-box factors 289-301 glutamine synthetase 81-92, 181, 191, 792 heat shock 249-59, 257, 252, 258 light-regulation 62-4, 88-90, 289-90, 295-6 metal-regulated 12-18 nitrate reductase 62-7 nitrite reductase 62-7 nodule development 178-93, 790, 192 phenylpropanoid biosynthesis 106

307

Index phosphate starvation inducible metabolism 34-6, 35, 36, 39-40 systemic acquired resistance 206-215, 217-18, 207, 212, 213 p"-l,3-glucanase systemic acquired resistance 207-9, 214, 217, 207, 212, 213 glutamine synthetase ammonium regulation 84, 86, 88-9, 91, 181 genes 81-9,52, 181, 191,792 gln-a 82, 85, 86-8, 87 gln-0 82, 85, 86, 87 gln-y 82-6, 82, 85, 91 gln-6 82, 88-9, 85, 87, 88-9 inducibility of gene family 79-95 isoenzymes 81-2, 82, 83, 85, 87, 91, 181-3 light regulation 88-90 root nodules 82, 82-6, 89-92, 91, 181, 183, 191, 192 transgenic plants 84, 86, 191, 192 glutamate synthetase/glutamate synthase pathway 79-80, 79 GUS (|3-glucuronidase) see reporter gene fusions haemoglobin as an oxygen sensor 237-8, 239 heat shock 247-63 antisense RNA 256, 257-9, 258 chimaeric genes as selection markers 260-1, 260 constitutive gene expression 255-7, 256, 257 gene expression and cis-active SAR (scaffold attachment region) sequences 252, 253-5 promoter elements 249-53, 251 relationship to cold-stress response 280 signal transduction pathway mutants 260-1 transcription factor, HSF 259 transgenic plants 248-63 light regulation glutamine synthetase 88-90 nitrate reductase 62-4 nitrite reductase 62-4 rbcS-lA promoter 289-304 lignin biosynthesis 105-6, 105 metal-binding polypeptides 2-12, 7-9 metal-binding proteins 1—24

metal homeostasis and metal tolerance 18-19 metallothionein amino acid sequence 15-18, 16, 17 animal 3-4,//, 12-13 fungal 4, 11, 13 genes 12-18 plant 6, , 11, 15-19, 16, 17 role 18-19 Mesembryanthemum crystallinum (ice plant) biochemistry and molecular biology of CAM 113-37 Mimulus gultatus metal-binding polypeptides 6-12, 7-9 metal-regulated genes 14-18 molybdenum cofactor domain 52-3, 59-61 structure 59, 60 synthesis 59-61 mutants alcohol dehydrogenase 239-240 heat shock signal transduction pathway 260-1 nitrate reductase cnx 60 intragenic complementation 61 nia 50, 50-1, 61 phosphate starvation inducible metabolism 35-6, 37 nitrate assimilation pathway 45-7, 46 nitrate reductase amino acid sequences 51-61, 52-6 catalytic activities 49-51 cnx mutants 60 domains 49-61, 52-6 FAD 55, 57-9 haem 54, 56-7 MoCo 52-3, 59-61, 60 gene expression 62-67 intracellular location 48 isoforms 47-8 nia mutants 50, 50-1, 61 intragenic complementation 61 reducing substrates 47-8 regulation circadian rhythm 64-7, 66 light 62^* nitrate 6 2 ^ nitrogen metabolites 64-7, 66 structure-function model 48-61 subunits 49-51 transgenic plants 64-5

308

Index

nitrite reductase antisense mRNA 65 gene expression 62-7 regulation circadian rhythm 64-7 light 62-4 nitrate 6 2 ^ nitrogen metabolites 64—7 transgenic plants 65 nodulins carbon metabolism 183-4 early nodulins induced during the infection process and nodule organogenesis 178-80 gene expression during nodule development 189-93 developmental control 189, 190, 191 metabolic control 191-3 nitrogen metabolism 181-3, 191, 192 oxygen transport 180-1 peribacteroid membrane 184-9, 187 transgenic plants 189-93 osmotic stress 139-41, 140 ABA-responsive genes 140-1 PEPCase (phospheno/pyruvate carboxylase) dendrogram of C3, C4 and CAM sequences 124 expression in E. coli 118-19, 118 expression in insect cells 118-19, 118 genes 123-5,126 GUS fusions 129 isoforms 117-19, 118 ppcl and ppc2 gene promoters 128-31 regulation 119-23 transgenic plants 128-9 peribacteroid membrane biogenesis 185—9 nodulins 184-9, 187 phenylalanine ammonia-lyase biochemistry 100-1 cDNA clones 99, 102 deletion analysis of gPAL2 promoter 104 environmental regulation 101 genes gPAL2 expression 102-3, 103 gPAL3 expression 103-4, 103 transgenic plants 102-4 phenylpropanoid biosynthesis biochemistry 100-1, 105-6 cinnamyl alcohol dehydrogenase 105-7 4-coumarate: CoA ligase 99, 99

genes 99, 102^1, 106 pathway 97-9, 95 phenylalanine ammonia-lyase 99-104, 99, 103

phosphate starvation inducible (psi) metabolism 25^14 cDNA 34-5, 35 epsi acid phosphatase 25, 27-37, 30, 31,32,34,38 epsi ribonuclease 37-40 mRNA 34-6, 55, 36 pho stimulation 40-1 protein phosphorylation 40-1, 41 resistant cell lines 35-6, 37 phosphate starvation resistant cell lines 35-6, 37 pho stimulon 40-1 phytochelatins fungal 4 metal regulation of synthesis 13-14 plant 5 role 18-19 phytochelatin synthase 13-14 polygalacturonase antisense RNA 159 expression in transgenic tobacco 159 promoter deletion analysis a-Amy 116-4 gene 144-6 Adhl gene 232-7, 235, 236, 237 CAM genes 128-30 gPAL2 gene 104 heat shock genes 249-55, 251, 252 light-regulated genes 289-90 nodulin genes 189-91 PR-la gene 219-20, 221 rabl6 gene 144-6 protein phosphorylation phosphate starvation inducible metabolism 40-1, 41 PR (pathogenesis-related) proteins systemic acquired resistance 206-7, 207, 209-11, 210, 212, 213, 214, 215, 216, 217, 219-20, 221, 222 pyruvate decarboxylase anaerobic response 231, 239, 240-1, 241 transgenic plants 240-2, 241 reporter gene fusions GUS (fS-glucuronidase) alcohol dehydrogenase gene Adhl 233-36, 236 glutamine synthetase genes, 84, 86, 191-3, 192 heat shock genes 250, 254

Index phenylalanine ammonia-lyase genes, 102-4, 103 phospheno/pyruvatc carboxylase genes 128-30 PR-la gene 219-21, 221 CAT (chloramphenicol acetyltransferase) a-amylase gene a-Amy 1/6-4 144-6 alcohol dehydrogenase gene Adhl 233 heat shock gene Gmhsp 17.3-B 24953, 251 rabl6 gene 144-6 ribonuclease (epsi) excreted phosphate starvation inducible 37-40 root nodule symbiosis communication and titration of plant and bacterial signals 176, 177 nodulin gene expression 178-93, 190 ontogeny 177-8 symbiotic state 177-8 salicyclic acid biochemical signal for SAR 215-17 biosynthesis 218-9, 218 gene induction 217-18, 221-2 responsive elements (SARE) 219-21 SAR (systemic acquired resistance) biochemical signal 215-17 chemical induction system 222—4, 223 chitinasc 206-8, 209, 210, 212, 213, 214, 215, 216, 217 dependence of RNA accumulation on cycloheximide 220 genes 206-11,207, 210 gene induction 211-14, 212, 213, 21718

309 P-l, 3-glucanase 207-9, 214, 217, 207, 212, 213 model in tobacco 224-5, 224 PR (pathogenesis-related) proteins 206-7, 207, 209-11, 210, 212, 213, 214, 215, 216, 217, 21920, 221, 222 PR-la promoter 219-20, 221 salicyclic acid 215-19, 221-2, 225 TMV infection 205, 211-14, 212, 213, 216 transgenic plants 214-15, 219-22, 223 TMV infection systemic acquired resistance 205, 211— 14, 212, 213, 216 transgenic plants ACC synthase gene 161-7, 162, 163, 164, 165 alcohol dehydrogenase gene 240-2, chemical induction system 222-24, 223 glutamine synthetase gene 84, 86, 191, 192 heat shock response genes 248-63 insect control 222^1, 223 nitrate reductase gene 64-5 nitrite reductase gene 65 phenylalanine ammonia-lyase gene 102^1 phosphoeno/pyruvate carboxylase gene 128-9 polygalacturonase gene 159 nodulin genes 189-93 pyruvate decarboxylase gene 240-2, 241 SAR genes 214-15, 219-22, 223