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.

Phosphate starvation inducible enzymes

43

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)

/

(F7kb 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.

Biochemistry and molecular biology of CAM

127

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.

128

H.J. BOHNERT et al.

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.

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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).

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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

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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 # 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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III

NODULE

SYMBIOTIC

EXTERNAL SIGNALS DEVELOPMENT

Early Nodulins

METABOLISM

Late Nodulins

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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MIAO

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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.

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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

•

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?

acidic extra.

PR-Smin acidic extra.

•

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OTHERS- SAR8.2, PR-4.

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•

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•

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PROTEASE INHIBITORS 7

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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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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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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

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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

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PR-l

PR-2

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PR-3

PR-4

iff

PR-5

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PR-1 basic

Basic class HI chitinase

Acidic class H chitinase

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Basic glucanase

1

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

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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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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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Pathway 2 trans-cinnamic acid 4-hydroxy cinnamic acid COOH

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Salicylic Acid (SAR) 4-coumaraldehyde

Naringenin Chalcone

CH

0H Naringenin Flavanone Isoflavonoid Phytoalexins

Flavonoid Pigments

I

?0H

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

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-800

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-600

-500

-400

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-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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etal. STARCH

SUCROSE

GLUCOSE-1-P

(

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

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NAD

CO,

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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

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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

236

E . S . DENNIS

etal. Anaerobic Induction

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competitor DNA

b-Zip proteins and the rbcS-lA promoter

295

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).

296

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

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

b-Zip proteins and the rbcS-lA promoter

297

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)

G-1A

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G-adh

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ACGAATAC

G-chs (P.c.)

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CCATCCGG

G-cabE

CACAATC&G

G-corl80

ACGCTTC&T*****T CCCTTTAT

G-chs (A.m.)-54

CTAATAA ***** *

G-chs (A.m.)-120

ACCCATG T

USF

GTGTAGG

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CTCAGCTA C CAACTGAC SCGGGTGT

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

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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