Biology and Biotechnology of the Plant Hormone Ethylene

NATO ASI Series Advanced Science Institutes Series

A Series presenting the results of activities sponsored by the NA TO Science Committee, which aims at the dissemination of advanced scientific and technological know/edge, with a view to strengthening links between scientific communities.

The Series is published by an international board of publishers in conjunction with the NATO Scientific Affairs Division

A Life Sciences B Physics

C Mathematical and Physical Sciences D Behavioural and Social Sciences E Applied Sciences

F Computer and Systems Sciences G Ecological Sciences H Cell Biology I Global Environmental Change

PARTNERSHIP SUB-SERIES

1. Disarmament Technologies 2. Environment 3. High Technology 4. Science and Technology Polley 5. Computer Networking

Plenum Publishing Corporation London and New York

Kluwer Academic Publishers Dordrecht, Boston and London

Springer-Verlag Berlin, Heidelberg, New York, London, Paris and Tokyo

Kluwer Academic Publishers Springer-Verlag / Kluwer Academic Publishers Kluwer Academic Publishers Kluwer Academic Publishers Kluwer Academic Publishers

The Partnership Sub-Series incorporates activities undertaken in collaboration with NATO's Cooperation Partners, the countries of the CIS and Central and Eastern Europe, in Priority Areas of concern to those countries.

NATo-PCO-DATA BASE

The electronic index to the NATO ASI Series provides full bibliographical references (with keywords and/or abstracts) to more than 50000 contributions from international scientists published in all sections of the NATO ASI Series. Access to the NATD-PCD-DATA BASE is possible in two ways:

- via online FILE 128 (NATO-PCD-DATA BASE) hosted by ESRIN, Via Galileo Galilei, 1-00044 Frascati, Italy.

- via CD-ROM "NATO-PCD-DAT A BASE" with user-friendly retrieval software in English, French and German (© wrv GmbH and DATAWARE Technologies Inc. 1989).

The CD-ROM can be ordered through any member of the Board of Publishers or through NATO­PCO, OveriJse, Belgium.

3. High Technology - Vol. 34

Biology and Biotechnology of the Plant Hormone Ethylene edited by

A. K. Kanellis Institute of Viticulture Vegetable Crops & Floriculture, National Agricultural Research Foundation, Heraklion, Crete, Greece

Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, Heraklion, Crete, Greece

C. Chang Department of Plant Biology, University of Maryland, College Park, MD, U.S.A.

H. Kende MSU-DOE, Plant Research Laboratory, Michigan State University, East Lansing, MI, U.S.A.

and

D. Grierson BBSRC Research Group in Plant Gene Regulation, Department of Physiology and Environmental Science, University of Nottingham, Loughborough, U.K.

Springer-Science+Business Media, B.V.

Proceedings of the NATO Advanced Research Workshop on Biology and Biotechnology of the Plant Hormone Ethylene Chania, Crete, Greece 9-13 June 1996

A C.I.P. Catalogue record for this book is available from the Library of Congress

ISBN 978-94-010-6336-4 ISBN 978-94-011-5546-5 (eBook) DOI 10.1007/978-94-011-5546-5

Printed on acid-free paper

All Rights Reserved © 1997 Springer Science+Business Media Dordrecht Originally published by Kluwer Academic Publishers in 1997 Softcover reprint of the hardcover 1st edition 1997 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photo­copying, recording or by any information storage and retrieval system, without written permission from the copyright owner.

NATO ASI Series

Series

Biology and Biotechnology of the Plant Hormone Ethylene

A.K. Kanellis C. Chang H. Kende D. Grierson editors

KLUWER ACADEMIC PUBLISHERS DORDRECHT/BOSTON/LONDON PUBLISHED IN COOPERATION WITH NATO SCmNTIFIC AFFAlRS DIVISION

TABLE OF CONTENTS

Prologos xiii Kanellis, A.K, C. Chang, H Kende, and D. Grierson

1. Biochemical and Molecular Mechanisms of Ethylene Synthesis

Structure-function analysis of tomato ACC synthase 1 Tarun, A.S. and A. Theologis

Structure-function analysis of ACC oxidase by site-directed mutagenesis 5 Kadyrzhanova, D.K, TJ McCully, S.A. Jaworski, P. Ververidis, KE. Vlachonasios, KG. Murakami, and D.R. Dilley

l-Aminocyclopropane-l-carboxylate oxidase: molecular structure and catalytic function 15 John, P., Tlturriagagoitia-Bueno, v. Lay, P.G. Thomas, TA. J Hedderson, A. G. Prescott, E.J Gibson, and c.J Schofield,

On l-aminocyclopropane-l-carboxylic acid (ACC) oxidase: Degradation of a-aminoisobutyric acid and structure-function studies on the CO2 binding site 23 Charng, f.-y., f. Liu, JG. Dong" and s.F. Yang

Regulation of auxin-induced ethylene biosynthesis in etiolated pea stems 31 Peck, S. C. and H. Kende

The role of jasmonates in ethylene biosynthesis 39 Saniewski, M

Ethylene biosynthesis and its regulation in ripening "Hayward" kiwifruit 47 Sfakiotakis, E., MD. Antunes, G. Stavroulakis, N. Niklis, P. Ververidis, and D. Gerasopoulos

2. Perception and Signal Transduction Pathways

Two-component regulators and ethylene signal transduction in Arabidopsis Jirage, D. and C. Chang

57

viii

The Ethylene Binding Site of the ETRl Protein Bleecker, A.B.

The ethylene receptor gene family in Arabidopsis Hua, J., H Sakai, and E. M Meyerowitz

Ethylene signal perception and transduction Smith, A.R., A. W Berry N. v.J. Harpham, R.J. Hemsley, M Gholland, 1. Moshkov, G. Novikova and MA. Hall

3. Growth and Development

63

71

77

Ethylene and Arabidopsis rosette development 87 Smalle, J., J. Kurepa, M Haegman, M Van Montagu, and D. Van Der Straeten

Ethylene regulates life-span in cells of reproductive organs in Pisum sativum 93 Granell, A., R. Blay and D. Orzaez

Fluctuations in ethylene formation and flowering in Chenopodium rubrum 99 Machizc1wva, I., N Chauvaux, W. Dewitte and H van Onckelen

Regulation of circadianly rhythmic ethylene production by phytochrome B in Sorghum 105 Morgan, P. W, S.A. Finlayson, I.-J. Lee, KL. Childs, C.-J. He, R.A. Creelman, M C. Drew, and J.E. Mullet

Ethylene involvement in the dormancy of Amaranthus seeds 113 Kr;pczynski, J, M Bihun and E. Kfpczynska

Control of gene transcription by ethylene during tomato fruit ripening 123 Deikman, J., S.A. Coupe, and R. Xu

Molecular genetic analysis of ethylene-regulated and developmental components of tomato fruit ripening: Ethylene and Developmental Signal Transduction in Tomato 133 Giovannoni, J.J.

The role of ethylene in banana fruit ripening 141 Clendennen, S.K., P. B. Kipp, and G. D. May

IX

The modulation of ethylene biosynthesis and ACC oxidase gene expression during peach fruit development and fruitlet abscission 149 Tonutti, P., C. Bonghi, B. Ruperti, and A. Ramina

4. Ethylene and Senescence of Plant Organs

Transcriptional regulation of senescence-related genes in carnation flowers 155 Maxson, JM and W.R. Woodson

Ethylene: interorgan signaling and modeling of binding site structure 163 Woltering, E.J, A. Van der Bent, G.J de Vrije, and A. Van Amerongen

An ethylene-regulated DNA element in abscission-specific gene promoters and the expression of an ETRI homologue in tomato abscission 175 Tucker, ML., G.L Matters, S.M Koehler, D. Zhou, S-B Hong, P. Kalaitzis, A.K. Mattoo, and P. Nath

Spatial and temporal expression of abscission related genes during ethylene-promoted organ shedding 185 Roberts, JA., S.A. Coupe, C.A. Whitelaw, and JE. Taylor

Different endo-J3-1,4-g1ucanases are expressed during abscission and fruit ripening in pepper and peach plants 191 Trainotti, L., L. Ferrarese, and G. Casadoro

The tomato endo-J3-1,4-g1ucanase gene family: regulation by both ethylene and auxin 197 Rose, JK.c., C. Catala, D.A. Brummell, c.c. Lashbrook, C. Gonzalez-Bosch, and A.B. Bennett

5. Stress Ethylene

Ethylene synthesis and a role in plant responses to different stressors 207 Kacperska, A.

Ethylene and the defense against endogenous oxidative stress in higher plants 217 Ievinsh, G. and D. Ozola

x

Potamogeton pectinatus: a vascular plant that makes no ethylene 229 Jackson, MB., J.E. Summers, and L.A.e.J. Voesenek

Hypoxia and fruit ripening 239 Solomos, T. and A.K. Kanellis

Ethylene regulation by the nitric oxide (NO) free radical: a possible mode of action of endogenous NO 253 Haramaty, E. and Y. Y. Leshem

Ethylene syntbesis in tomato plants exposed to ozone: The Role of Ethylene in Ozone Damage 259 Kangasjiirvi, J., J. Tuomainen, e. Betz, D. Ernst, e. Langebartels, H Sandermann Jr

Involvement of ethylene in protein elicitor-induced plant r:esponses 267 Anderson, J.D., F.e. Cardinale, J.e. Jennings, HA. Norman, A. Avni, U. Hanania, and B.A. Bailey

Changes in in vivo and in vitro ACC oxidase activities during chilling and subsequent warming as exemplified by Vigna radiata seedlings 275 Corbineau, F, R. Bogatek, S. Radice, M.A. Picard and D. Come

Impact assessment for ethylene emissions at a petrochemical site 283 Jack, T. R., R. K. McBrien, and B. Dowsley

6. Biotechnological Control of Ethylene

Potential applications of controlling ethylene synthesis and perception in transgenic plants Klee, HJ. and D. Tieman

Regulation of ethylene synthesis and perception in tomato and its control using gene technology Barry, e.s., B. Blume, A. Hamilton, R. Fray, S. Payton, A. Alpuche-Solis, and D. Grierson

Reduced ethylene synthesis and ripening control in tomatoes expressing S-adenosylmethionine hydrolase Kramer, M. G., J. Kellogg, W. Wagoner, W. Matsumura, X Good, S. Peters, G. Clough, and R.K. Bestwick

289

299

307

Interactions of ethylene and polyamines in regulating fruit ripening Mehta, R., A. Handa, andA. Mattoo

Differential expression of ACC oxidase genes in melon and physiological characterization of fruit expressing an antisense ACC oxidase Guis, M, T. Bouquin, H. Zegzouti, R. Ayub, M Ben Amor, E. Lasserre, R. Botondi, J. Raynal, A. Latche, M Bouzayen, C. Balagw!, and J C. Pech

Genetic modification of ethylene biosynthesis and ethylene sensitivity in carnation Van Altvorst, A.C, A.G. Bovy, G.C Angenent, and J.J.M Dons

Modulation of ethylene production in transgenic tobacco Knoester, M, J.F Bol, L.C Van Loon, and H.J.M Linthorst

Index of Authors

Index of Keywords

xi

231

327

339

347

355

359

Prologos

Ethylene is a simple gaseous plant honnone (~I4, the simplest olefin) produced by higher plants and also by bacteria and fungi. Because of its commercial importance and its profound effects on plant growth and development, its biosynthesis, action, and control of its action by chemical, physical and biotechnological means have been intensively investigated. Thanks to new tools available in biochemistIy and molecular genetics, parts of the ethylene biosynthesis, perception and signal transduction reactions have been elucidated. This knowledge has been applied to enhance the quality of a number of agronomically important crops.

The rapid advance in elucidating the mechanisms of ethylene perception and synthesis by plants, the signal transduction pathway, and ethylene control in transgenic plants have made the organization of a series of conferences dedicated to the plant honnone ethylene imperative. It is noted here that studies on ethylene have led the way in advancing our understanding of the biosynthesis of a plant honnone at the biochemical and molecular levels, and future studies should further help in the understanding of the biochemical machinery responsible for the perception and signal transduction of this plant honnone.

The Ethylene Symposia were established two decades ago as important international scientific events. The purpose of the present Symposium was the critical assessment of the existing knowledge and the exchange of new ideas on the mechanisms of ethylene synthesis, perception and signal transduction, its role in pathogenesis and stress, its involvement in plant growth and development and, lastly, the biotechnological control of its function and formation. This book will be of major interest to all academic, industrial and agricultural researchers as well as advanced undergraduate and graduate students in plant biology, biotechnology, biochemistry, genetics, molecular biology and food science.

This volume contains the main lectures presented in the NATO-Advanced Research Workshop and EU-TMR-Euroconference Symposium entitled "Biology and Biotechnology of the Plant Honnone" held in Chania, Crete, Greece, June 9-13, 1996. This international scientific event was organized by the Postharvest Physiology and Biotechnology Group of the Institute of Viticulture, Vegetable Crops and Floriculture­N.AG.RE.F., and the Institute of Molecular Biology and Biotechnology-FO.RT.H., at Heraklion, Crete Greece, and took place on the premises of the Mediterranean Agronomic Institute ofChania (M.A.I.Ch.), Crete, Greece.

We would like to thank the Scientific Affairs Division of NATO for partially funding this event, the European Commission of the European Union and especially the TMR­Euroconference Programme, Cost Action 915, DGXII-SDME 10/47 and DG XIIIE/2. Special thanks go to United States Department of Agriculture, National Agricultural

xiii

xiv

Research Foundation of Greece, General Secretariat of Research & Technology of Greece, Institute of Molecular Biology & Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Crete, Greece, Ministry of Culture of Greece and Hellenic Tourism Organization for their financial support. Appreciation is also extended to a number of private firms which contributed to the success of this important event.

We are particularly indebted to the members of the scientific and local organizing committees as well as to Mr. A Nikolaidis, director, and the personnel of M.AI.Ch., especially Dr. D. Gerasopoulos, Mrs. K. Karapataki and Mrs. A Lioni for their efforts for the success of this Symposium. Lastly, we acknowledge the help of Mrs. A Giannakopoulou for handling secretarial aspects and Mrs. I. Pateraki for the preparation part of the index of keywords.

Heraklion, College Park, East Lansing, Sutton Bonington, 1996

AK. Kanellis C. Chang H. Kende D. Grierson

STRUCTURE-FUNCTION ANALYSIS OF TOMATO ACC SYNTHASE

A. S. T ARUN AND A. THEOLOGIS Department of Plant Biology, UC Berkeley and Plant Gene Expression Center, 800 Buchanan St., Albany CA 94710 USA

1. Introduction

ACC synthase catalyzes the conversion of S-adenosylmethionine to l-amino­cyclypropane-carboxylate (ACC), one of the rate-limiting steps in ethylene biosynthesis. This enzyme has proven to be quite recalcitrant to biochemical characterization because it is labile and in low abundance in plant tissues. The cloning and expression of ACC synthase genes has facilitated more biochemical and structural studies of this enzyme. ACC synthase was first cloned from zucchini [15], and since then a number of ACC synthase genes have been cloned from a number of plants. The emerging picture from the study of these genes is that ACC synthase is encoded by a multi-gene family and that these genes are differentially expressed in response to various internal and external inducers [17]. A comparison of the amino acid sequences encoded by these genes indicates that they are 50-95% identical with the highest variability at the carboxyl end of the protein [17]. ACC synthase also shows homology to another group of pyridoxal-phosphate (PLP) requiring enzymes, the aminotransferases [5,13]. A recent alignment of 14 different kinds of aminotransferases indicates that ACC synthases have the highest homology to sub­group 1 aminotransferases, which includes alanine-, tyrosine-, histidinol-phosphate-, phenylalanine-, and aspartate aminotransferases (AATase), and that they share all of the eleven invariant residues of this subgroup, including four invariant residues present in all aminotransferases [10]. X-ray crystallographic studies of aspartate aminotransferase have indicated that these invariant residues play important roles in binding the PLP co-factor and the substrate a-carboxylate group [9]. This indicates that ACC synthase could be evolutionarily related to the aminotransferases. The homology between these two groups of enzymes also suggests that the structure and co­factor binding sites of these two groups of enzymes may be similar. We have tested this hypothesis by site-directed mutagenesis of some of these conserved residues of tomato LE-ACS2, such as Tyr-92, Lys-278, and Arg-412. We report here that mutagenesis on these residues results in decreased activity or complete inactivation of ACC synthase.

A. K. Kanellis et al. (eds.J, Biology and Biotechnology o/the Plant HOrTn01U! Ethyle1U!, 1-4. © 1997 Kluwer Academic Publishers.

2

2. Results and Discussion

ACC accumulation in the growth medium and ACC synthase activity of crude E. coli protein expressing wild-type or mutant ACC synthase are shown in Table 1.

The residues targeted for mutagenesis, Tyr-92, Lys-278 and Arg-412, were chosen because the corresponding conserved residues in AA Tase have been extensively studied. The data show that mutations at these three conserved residues severely affect the activity of the enzyme. This decrease in activity is not due to a decreased expression or stability of the mutant proteins compared to wild-type because western blots of the wild-type and mutant proteins expressed in E. coli indicate similar levels of expression (data not shown).

The mutation y 92F still retains some activity namely 13% of wild-type ACC accumulation and 33% of wild-type ACC synthase activity. Substituting the Tyr-92 residue with Tryp or Leu reduces the enzyme activity to almost background levels. The corresponding residue in AATase, Tyr-70, is believed to be responsible for anchoring the PLP co-factor to the AA Tase apoenzyme and is one of the residues found at the active site interface of the two subunits of the AATase homodimer [9]. In particular, the Tyr-70 residue of one subunit interacts with the PLP co-factor covalently bound to the active site of the other subunit. A y70p substitution in AATase was found to retain about 8% of wild-type activity and is believed not to be essential for transaminase activity [18]. This similarity in results between the ACC synthase y92F and AATase y70p substitution may indicate similarity in function in binding the PLP co-factor to both enzymes.

The Lys-276 residue in ACC synthase has been previously identified as the covalent binding site of the PLP co-factor [21]. It is thus the only residue in ACC synthase for which a catalytic function has been shown experimentally. A mutation in AATase K258A has been shown to abolish AATase activity [8J. Other researchers have reported similar results when the corresponding active site lysine residue in winters quash [11] and apple [20] has been mutated.

The R412K mutation results in complete inactivation of ACC synthase activity. This is consistent with a recently published report that mutation on the Arg-407 residue to Lys also abolishes enzyme activity in apple ACC synthase [20]. In AATase, Arg-386, the corresponding conserved residue of Arg-412, is believed to form a salt bridge with the a-carboxylate group of its substrate [9J. Substitution of R386y or R38/ip drastically reduces enzyme activity [3]. Thus, it is also possible that the Arg-412 residue of ACC synthase may also form a salt bridge with the a-carboxylate group of its substrate, S­adenosylmethionine.

Our present study indicates that, in the absence of any structural information about ACC synthase, its homology to aminotransferases is useful in determining which residues might be part of its active site. This homology between ACC synthase and aminotransferases may also indicate a similarity in enzyme structure [5, 13]. Aspartate aminotransferase, whose structure is well known, is a homodimer whose functionally independent active sites are formed by the interaction of residues from both subunits and, thus, cannot function as a monomer [4]. It is, thus, possible that ACC synthase

3

may also function as a dimer with shared active sites. Resolving this issue is of particular importance because there are conflicting reports about the subunit structure of ACC synthase. ACC synthase from plant tissues has been reported to be either dimeric, like the zucchini [14], winter squash [12], and mungbean [19] enzymes, or monomeric like the tomato [1, 2] and apple [21] enzymes. There have been reports, however, that heterologously expressed tomato and apple ACC synthases are also dimeric [7, 16, and 20]. The mutants constructed in this study would be used to determine if tomato ACC synthase also functions as a dimer and has shared active sites.

TABLE I. Activity and expression of wild-type and mutantS LE-ACS2 in E. coli.

Sampleb ACC accumulation' % wild-type ACC synthase' % wild-nmoiACC/107 cells nmoIACC.mg·\ protein.hr·\ type

wild-type 1.22 529 Y92F 0.16 13 177 33 Y92W 0.06 5 25 5 Y92L 0.002 4

K278A 0 0 R412K 0 0

SSite-directed mutagenesis ofLE-ACS2 cDNA were carried out using the Kunkel method [6] as well as by PCR. The mutations were carried out to generate the following substitutions: Y92F, Y92W, Y92L, K278A, and R412K. Mutated genes were sequenced to verifY that the desired mutation was the only change from the wild-type sequence. bThe wild-type and mutated ACC synthase cDNA's were subcloned into pKK233-2 expression vector (Pharmacia). Protein expression was verified by fractionating E. coli cell lysate proteins by SDS-PAGE on 10% polyacrylamide gels and blotting onto nytrocellulose membrane. lmmunodetection was done with LE-ACS2 polyclonal antibody using the a1kaline-phosphatase-conjugated anti-rabbit IgG as secondary antibody (Promega). 'ACC accumulation and ACC synthase activity were assayed as described by Sato and Theologis [15]. A unit of ACC synthase activity is defined as the amount of enzyme which catalyzes the formation of 1 nmol of ACC per hour under the stated conditions of the assay, and the specific activity is expressed as units per milligram protein.

3. References

1. Acaster, M. A and Kende, H. (1983) Properties and partial purification of l-aminocyc1opropane-l­carboxylate synthase, Plant Physiol. 72, 139-145.

2. Bleecker, A B., Kenyon, W.H., Somerville, S.C., and Kende, H. (1986) Use of monoclonal antibodies in the purification and characterization of I-aminocyclopropane-I-carboxylate synthase, an enzyme in ethylene biosynthesis, Proc. Natl. Acad. Sci. USA 83, 7755-7759.

3. Danishevsky, AT., Onnufer, J.J., Petsko, G.A and Ringe, D. (1991) Activity and structure of the active-site mutants R386Y and R386F of Escherichia coli aspartate aminotransferase, Biochem. 30, 1980-1985.

4. Ford, G.C., Eichele, G., and Jansonius, J.N. (1980) Three-dimensional structure of a pyridoxal­phosphate-dependent enzyme, mitochondrial aspatate aminotransferase, Proc. Natl. Acad. Sci. USA 77,2559-2563.

4

5. Huang, P.-L., Parks, J.E., Rottmann, W.H., and Theologis, A (1991) Two genes encoding 1-aminocyclopropane-l-carboxylate synthase in zucchini (Cucurbita pepo) are clustered and similar, but differentially expressed,Proc. NatL Acad. Sci. USA 88, 7021-7025.

6.· Kunkel, T. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc. NatL Acad. Sci. USA 82, 488-492.

7. Li, N. and Mattoo, AK. (1994) Deletion of the carboxyl-terminal region of 1-aminocyclopropane-l­carboxylic acid synthase, a key protein in the biosynthesis of ethylene, results in catalytically hyperactive, monomeric enzyme, J. Bioi. Chem. 269, 6908-6917.

8. Malcolm, BA and Kirsch, J.F. (1985) Site-directed mutagenesis of aspartate aminotransferase from E. coli, Biochem. Biophys. Res. Commun. 132,915-921.

9. Mehta, P.K., Hale, T.I., and Christen, P (1989) Evolutionary relationships among aminotransferases, Eur. J. Biochem. 186,295-253.

10. Mehta, P.K., Hale, T.I., and Christen, P. (1993) Aminotransferases: demonstration of homology and division into evolutionary subgroups, Eur. J. Biochem. 214, 549-561.

11. Mori, H., Nakagawa, N., Ono, T., Yamagishi, N., and lmaseki, H.(1993) Structural characteristics of ACC synthase isozymes and differential expression of their genes, in J.C. Pech, A Latche and C. Balague (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, K1uwer Academic Publishers, Dordrecht, pp.I-6.

12. Nakajima, N. and lmaseki, H. (1986) Purification and properties of 1-aminocyclopropane-l­carboxylate synthase of mesocarp of Cucurbita maxima Duch. fruits, Plant Cell Physiol. 27, 969-980.

13. Rottmann, W.H., Peter, G.F., Oeller, P.W., Keller, JA, Shen, N.F., Nagy, B.P., Taylor, L.P., Campbell, AD., and Theologis, A(1991) 1-Aminocyclopropane-l-carboxylate synthase in tomato is encoded by a multigene family whose traoscription is induced during fruit and floral senescence, J. Mol. Bioi. 222, 937-961.

14. Sato, T., Oeller, P.W., and Theologis, A (1990) The 1-aminocyclopropane-l-carboxylate synthase of cucurbita,J. BioL Chem. 266,3752-3759.

15. Sato, T. and Theologis, A (1989) Cloning the mRNA encoding 1-aminocyclopropane-l-carboxylate synthase, the key enzyme for ethylene biosynthesis in plants, Proc. Natl. Acad. Sci. USA 86,6621-6625.

16. Satoh, S., Mori, H., and lmaseki, H. (1993) Monomeric and dimeric forms and the mechanism-based inactivation of 1-aminocyclopropane-l-carboxylate synthase, Plant Cell Physiol. 34, 753-760.

17. Theologis, A (1992) One rotten apple spoils the whole bushel: the role of ethylene in fruit ripening, Cell 70, 181-184.

18. Toney, M.D. and Kirsch, J.F. (1987) Tyr 70 increases the coenzyme affinity of aspartate aminotransferase, J. BioL Chem. 262, 12403-12405.

19. Tsai, D., Arteca, R.N., Bachman, J.M. and Phillips, AT. (1988) Purification and characterization of 1-aminocyclopropane-l-carboxylate synthase from etiolated mung bean hypocotyls, Arch. Biochem. Biophys. 264, 632-640.

20. White, M.F., Vasquez, J., Yang, S.F., and Kirsch, J.F. (1994) Expression of apple 1-aminocyclopropane-l-carboxylate synthase in Escherichia coli: Kinetic characterization of wild-type and active-site mutant forms, Proc. Natl. Acad. Sci. USA 91, 12428-12432.

21. Yip, w.-K., Dong, J-G., Kenny, J.W., Thompson, GA, and Yang, S.F. (1990) Characterization and sequencing of the active site of 1-aminocyclopropane-l-carboxylate synthase, Proc. Natl. Acad. Sci. USA 87,7930-7934.

STRUCTURE-FUNCTION ANALYSIS OF ACC OXIDASE BY SITE­DIRECTED MUTAGENESIS

D.K. KADYRZHANOVA, T.J. McCULLY, S.A. JAWORSKI, P. VERVERIDIS, K.E. VLACHONASIOS, K.G. MURAKAMI AND D.R. DILLEY Postharvest Physiology Laboratory, Department of Horticulture, Plant and Soil Sciences Building, Michigan State University, East Lansing, MJ 48824, USA

1. Introduction

Ethylene is a plant honnone that profoundly influences many diverse aspects of plant growth and development. The ethylene production rate of vegetative tissues and immature flowers and fruits is barely detectable and remains so until the tissue is stressed by abiotic or biotic factors of the environment. Developmentally regulated gene expression accelerates ethylene production in an organ specific and temporal manner as in ripening and senescence. The key enzymes of ethylene biosynthesis are I-aminocyclopropane-l-carboxylate synthase (ACC synthase) and ACC oxidase. Carbon dioxide at concentrations commonly found in the intercellular air space of plant tissues, activates ACC oxidase to produce ethylene. Both the biosynthesis and action of ethylene are modulated by the concentration of carbon dioxide, oxygen, and ethylene in the plant cell. The effect of CO2 on directly modulating ethylene biosynthesis by activating ACC oxidase may have important consequences in affecting plant growth and development by regulating the intercellular ethylene concentration.

The mechanism by which CO2 activates ACC oxidase by 10- to 20-fold is not known. We hypothesize that CO2 activates ACC oxidase in a manner similar to how CO2 activates Rubisco [9], urease [12] and phosphotriesterase [6], i.e., formation of a carbamate that acts as a metallocenter ligand. For Rubisco, this involves carbamylation of the E-amino group oflysine201 in the large subunit [10], which binds magnesium ion. For urease, lysine217 is carbamylated and serves to bridge the two nickel ions at the active site [7]. Similarly, carbamylation of a lysine residue in Pseudomanas diminuata phosphotriesterase is required for the assembly of its binuclear metal center [6]. ACC oxidase is known to require ferrous ion for activity. HiS177, Asp179 and His234 in ACC oxidase are likely ligands for Fe2+ based on similarly placed His and Asp residues in IPNS [15] and other Fe2+/a-ketoglutarate dioxygenases [11, 14]. The core structure of ACC oxidase is thought to be similar to that ofIPNS.

We [3, 13] and others [4, 5] have proposed that the mechanism for ACC oxidase

5

A. K. Kanellis et al. (etis.), Biology and Biotechnology of the Plant Hormone Ethylene, 5-13. © 1997 Kluwer Academic Publishers.

6

activation by CO2 may involve carbamylation of an essential Lys residue at the reaction site of the enzyme. The active species involved in the activation process is CO2 rather than HC03•. The effect of pH on the level of CO2 activation indicates that CO2 reacts with an amino group having an alkaline pK,.. These results strongly suggest, but do not prove, that activation of ACC oxidase involves the formation of a carbamate. Our studies [16] on inactivating ACC oxidase with lysyl specific reagents suggest that a single Lys residue among 29 in the protein may be the target for CO2 carbamylation. Moreover, CO2 pretreatment largely protected the enzyme from inactivation by the lysyl E-NH2 group specific reagent. These results are consistent with CO2

carbamylation of a lysyl residue as the basis for activating the enzyme.

2. Site-directed Mutagenesis

Our strategy to identify the essential lysine and histidine residues was based on important homology found between ACC oxidase and isopenicillin N synthase (IPNS) in primary and secondaty structure by Baldwin's group at Oxford [15]. Their x-ray crystallographic analysis of IPNS has defined the ligands for Fe2+ and other features of the catalytic center. They suggest ACC oxidase and many of the 2-oxoglutarate dependent and Fe2+/ascorbate dioxygenases contain a conserved jelly-roll motif as a new structural family of enzymes. We used IPNS as a model in prioritizing amino acid residues in ACC oxidase for site-directed mutagenesis. We first derived a consensus amino acid sequence for ACC oxidase based on the sequences predicted from 23 cDNAs for ACC oxidase. This is shown in Figure 1. This shows that 7 K residues are completely conserved among the 23 proteins; K 144, K 158, K I72, K 199, K 230, K292 and K296. Since CO2 activation is a feature of all ACC oxidases so far studied, this means that one of the 7 K residues may be the one carbamylated if this is the CO2 activation mechanism. Our strategy was to use arginine, glutamate glutamine and leucine in place of each of the lysine residues. Arginine would provide a positive charge at the respective location but would not be subject to carbamylation so the enzyme should be inactive. Glutamate in place of lysine, although shorter in length, would introduce a negative charge as the carboxylate group and perhaps would mimic a carbamylated E­

NH2 and, if so, the enzyme may be active but without CO2• Alternatively, the glutamate mutant may be inactive. Glutamine is not charged and is not subject to carbamylation and in place of lysine should produce an inactive enzyme. Leucine is hydrophobic and may inhibit enzyme activity by altering protein conformation. According to putative similarity to lPNS, HI77, DI79 and H234 would be expected to be ligands for Fe2+. We employed phenyalanine (F) in place of histidine residues and leucine (L) in place of aspartate. We also prepared a H39F mutation since this H is completely conserved and would serve as a good control for our protocols. We also were interested in determining which of the three cysteine residues is needed for enzyme activity since at least one -SH group is needed. We employed alanine (A) in place ofC28, CI65 and C\33.

Site-directed mutants were made by using the unique restriction site elimination

Figure 1. CONSENSUS SEQUENCE' AND SECONDARY STRUCTURE' FOR 23 ACC OXIDASES

MenFPi geeRaa m I o A C • N .30

p, a, a,

W G F F • I H G i h • mOtVEkmTk H Y k k 60

a,

m E q r F k g • a q a vtDIOWESTF90 -a. p, P.

HIPvSNi o I d • MkdFak 120

a, a, . E k L E • Y L K k F y G s 150

a,

gPn FGTKVSNYPpCPk Pd K G L R A H ," 0 A lBO -a, P,Jp, p, .. .. G G i

p, " L L Q 0 d v s G l Q L l K 0 9 q W I [) V PPm r 210

" p, " p"

s I V v N I G 0 Q I E V I T N G k Y K S" V m H R V i a Q t 240

G ,

P12 " RMSIASF

P ..

a,

Y N P g o a

ekqvYPkFVFdDYMk

E a m k

-P ..

d P I

a"

314

v I -P .. Y a

P a p a k I 270

KFQaKEPR 300

..

7

Figure 1. One letter abbreviations for amino acids: Ala, A; Arg, R; Asn, N; Asp, D; Cys, C; Gln, Q; Glu, E; Gly, G; His, H; De, I; Leu, L; Lys, K; Met, M; Phe, F; Pro, P; Ser, S; Thr, T; Trp, W; Tyr, Y; Val, V. Capital letters represent amino acids completely conserved in all 23 ACC oxidases and lowercase letters represent the most commonly occurring amino acid. Secondary structure as predicted from homology with isopenicillin N synthase and core structure probability score in relation to IPNS crystalline structure by Roach et ai., [15]. The closed triangles ( .. ) are putative ligands for Fe. The open triangles (t.) are residues with side chains within 8°A of the Fe. The amino acids comprising the leucine zipper are shown by an asterisk (*) in a-helix 6. The sequence SI60 NYPpCpI66 with dotted underlining is likely to be in a surface loop near the entrance to the catalytic reaction site. The ppCpI66 sequence is a putative diproline motif implicated in protein/protein interactions with proteins having SH3 homology. Threoninel78 and S231 with open superscript circles (0) are potential phosphorylation sites. KI44, K 158, K172, K199, K230, K292 and K296 are completely conserved in the 23 ACC oxidases and are therefore potential sites for C02 carbamylation and are indicated by closed superscript circles (.).

8

(USE) method of [2] commercially available in kit form. Point mutations were made in the apple ACC-oxidase cDNA [17] using the T7 promoter based expression vector pET-15b with a His-Tag™-tbrombin site. The resulting construct termed as pETAOX x 2a was placed in E. coli strain BL21 (DE3) pLysS (Novagen), which produced authentic and enzymatically active ACC oxidase upon induction with IPTG [16]. Oligonucleotide primers which introduce the desired mutations were synthesized at the Michigan State University Macromolecular Structure Facility. Base changes within the mutagenic oligonucleotides were chosen using codons most frequently used by E. coli. Beside mutagenic primers the USE method involves the primer which mutates a restriction site unique to the plasmid for the purpose of selection. As the selection primer we used the oligonucleotide sequence designed by Pharmacia Biotech which converts a Sca I site to a MIu I site in pET -15b. The Transformer™ site-directed mutagenesis kit from Clontech was employed according to manufacturer's instructions. Mutagenic and selection primers were simultaneously annealed to the denatured double-stranded target plasmid pET AOX x 2a. After DNA elongation and ligation, the heteroduplex DNA was used to transform the repair-deficient E. coli strain BMH71-18 mut S. Plasmids prepared from the pool of transform ants were subjected to selection restriction digestion to enrich for these carrying the selection primer sequence. After the final transformation into the E. coli strain BL21 (DE3) pLysS, plasmids were isolated from individual colonies and analyzed for the presence of the selection and mutagenic primer sequence. The resulting mutations and the integrity of the inserts were confirmed by sequencing (USB Sequence kit).

E. coli BL21 (DE3) pLysS cells transformed with wild-type and mutant forms of pETAOX x 2a wer\! cultured in LB medium, supplemented with antibiotics at 30°C. Protein expression was induced by adding IPTG. The harvested cells were lysed by freeze-thaw. Fusion proteins were purified from E. coli by metal-affinity chromatography and ammonium sulfate precipitation. In general, purification of both wild-type and mutant forms resulted in yields of ~ 0.5 mg of ACC oxidase proteinlliter of induced culture. The homogeneity was tested by SDS-PAGE analysis and immunoblots with ACC oxidase monoclonal antibody. ACC oxidase activity was assayed in E. coli cell lysates and after purification as the fusion protein. The assay conditions were: L-ascorbate (3mM), DTT (1mM), ACC (1 mM), MOPS (50 roM, pH 7.2) in 10% glycerol with 0, 5, 10, and 20 roM HC03- at 20 and 80 J1M Fe2+. Two levels of Fe were employed because some of the mutations introduce ligands which may chelate iron.

The SDS-PAGE analysis indicated that each mutant protein had similar size and mobility as the wild type. Since the expression, immunological and chromatographic properties of the mutants were the same as wild type, we conclude that the structure of the ACC oxidase was not affected by the amino acid changes. Table 1 is a summary of our results for 15 amino acid mutations in ACC oxidase and the consequence of the mutations on enzyme activity and CO2 activation. Our results on site-directed mutagenesis of ACC oxidase are so far confirming the proposition from Baldwin's laboratory at Oxford that ACC oxidase may be a member of a new structural class of enzyme proteins similar to isopenicillin N synthase (IPNS). The crystallographic

Tab

le I

. E

ffec

t of s

ite-

dire

cted

mut

agen

esis

of c

onse

rved

am

ino

acid

s on

act

ivity

and

CO

2 ac

tivat

ion

of A

CC

oxi

dase

as

His

-Tag

enz

yme.

Cel

l Lys

ate

Fusi

on P

rote

in

Am

inoa

cid

z Pu

tati

ve f

unct

ion

mut

ated

A

ctiv

ityY

C

O2

activ

atio

n x

Act

ivit

y Y

C

O2

acti

vati

on x

% o

f nat

ive

nati

ve

mut

ant

% o

f nat

ive

nati

ve

mut

ant

H17

7F

Fe

liga

nd

1.6

9.7

0.9

0.4

10.2

2.

0

H23

4F

Fe

liga

nd

0 9.

8 0

0 11

.7

0

D17

9L

Fe

liga

nd

0 11

.1

0 0

11.7

0

H39

F

Con

serv

ed

86.0

10

.4

12.4

71

.0

7.6

7.1

KI5

8E

CO

z ca

rbam

ylat

ion

32.0

11

.6

12.0

62

.0

6.0

6.0

KI5

8Q

CO

z ca

rbam

ylat

ion

4.7

8.8

10.2

90

.0

7.2

8.4

KI5

8R

CO

2 ca

rbam

ylat

ion

23.0

4.

4 S.

7 30

.0

11.3

16

.3

KI5

8L

CO

2 ca

rbam

ylat

ion

1.4

12.5

37

.8

1.3

10.8

10

.0

K23

0E

CO

2 ca

rbam

ylat

ion

3.1

7.1

5.9

5.1

7.4

8.0

K23

0Q

CO

2 ca

rbam

ylat

ion

22.0

7.

7 8.

5 63

.0

7.0

6.0

K23

0R

CO

z ca

rbllI

l)'y

latio

n 25

.1

12.0

10

.2

38.9

11

.5

11.9

KI9

9E

CO

z ca

rbam

ylat

ion

10.0

6.

5 5.

4 5.

4 6.

5 4.

3

K29

2E

CO

z ca

rbam

ylat

ion

7.3

7.2

7.7

3.7

14.3

14

.0

K29

6E

CO

z ca

rbam

ylat

ion

135.

0 7.

8 7.

8 13

3.0

13.3

14

.6

C16

5A

SH

grou

p 28

.0

7.2

8.0

96.0

9.

8 8.

2

ZSi

ngle

let

ter a

bbre

viat

ions

for

am

ino

acid

s:

Ala

, A; A

sp, D

; Arg

, R;

Cys

, C; G

lu,

E; G

in, Q

; H

is, H

; L

eu, L

; L

ys, K

; Phe

, F.

Y A n

ativ

e en

zym

e w

as p

repa

red

in p

aral

lel a

s th

e co

ntro

l for

eac

h m

utat

ion

prod

uced

in t

he E

. co

li ve

ctor

. A

ctiv

ity

is %

of n

ativ

e en

zym

e ac

tivi

ty.

xCO

z ac

tiva

tion

exp

ress

ed a

s ra

tio

of e

nzym

e ac

tivi

ty a

t CO

z sa

tura

tion

vs.

air

leve

l CO

z.

\0

10

analysis of IPNS has revealed the ligands for Fe2+ at the catalytic center of this dioxygenase.

3. Crucial Amino Acid Residues for ACC Oxidase Activity

3.1. Fe 2+ LIGANDS

We hypothesized that Hl77, D179, H234 and perhaps Q294 of ACC oxidase are the Fe2+

ligands equivalent to H214, D216, H270 and Q330 of IPNS from our secondary structure analysis of ACC oxidase in relation to the structure of IPNS. So far we have shown that Hl77, DI79 and H234 of ACC oxidase are putative Fe2+ ligands. This is based upon loss of ACC oxidase activity by Hl77F, D 179L and H234F mutants (Table 1). The fact that Hl77, DI79 and H234 are essential for ACC oxidase strongly supports their role as ligands for Fe2+ in a catalytic center similar to that of IPNS. We have ruled out H39 as a Fe ligand since the H39F mutant was active. The mutant Hl77F exhibited a reproducible but very low level of enzyme activity that was not activated by CO2. This is in contrast to the H234F and D179L mutants which had no enzyme activity. The Hl77F mutant was examined further. The residual enzyme activity was examined employing 0.25, 0.5 and 1 nmol of enzyme per assay (Table 2). The amount of ethylene produced was proportional to the amount of the mutant enzyme and the kcat was 0.00045 min-I. The native enzyme had a kcat of 0.032±0.004 min-I at air level CO2 and kcat 0.369±0.038 min-I at CO2 saturation. This data strongly suggest that the mechanism of CO2 activation directly involves Hisl77 as an Fe2+ ligand and that Aspl79 and HiS234 are also essential as ligands for Fe2+. The Q 294 mutant remains to be tested as a Fe2+ ligand.

TABLE 2. Histidine 177 is an Fe 2+ ligand essential for ACC oxidase activity and CO 2 activation.

Enzyme Activity ( kcat)'

HC03- Histidine 177 Histidine 177 Phenylalanine

(mM) ±S.D.Y CO2 Activation ' ±S.D.Y CO2

Activation'

0 0.032±0.004 0.00042±0.00008

5 0.316±0.032 9.9 0.00042±0.00004 1.0

10 0.364±0.052 11.4 0.00049±0.00012 1.2

20 0.369±0.038 11.5 0.00046±0.00016 1.1

ZActivity of purified fusion protein for native (HI77) and mutant (HI77F) enzyme expressed as kcat (nmol C2fLJnmoi enzyme/min). YBased on assays of 0.25, 0.5 and 1 nmol of fusion protein (n=9). 'Relative to activity without added HC03' (i.e. air level of CO 2)'

11

3.2. THE ESSENTIAL CYSTEINE RESIDUE

There are three completely conserved cysteine residues; C28, Cl33 and C165. We find that the CI65A mutant enzyme is fully active and CO2 dependent (Table I). CI65 is of particular interest since it is in the polyproline sequence YPPCPKPI68, we suspect is important in enzyme function in protein/protein interactions unrelated to the enzyme reaction (Fig. I). We have shown that ACC oxidase activity is inhibited strongly by sulfhydryl reagents indicating that an SH group is important. Since the CI65A mutant has enzyme activity, CI65 is not essential in the ACC oxidase reaction. Cl33 is in a putative leucine zipper. We hypothesize that C28 exists as SH in the active enzyme and may be the essential cysteine residue. This remains to be proven.

3.3. IS CO 2 ACTIVATION BY CARBAMYLA TION OF A LYSINE RESIDUE?

If CO2 activates ACC oxidase by carbamylating an essential Lys residue, replacing that Lys residue with an amino acid incapable of becoming carbamylated should result in an inactive enzyme. Seven lysine residues are conserved among the 23 ACC oxidases (Fig. I). These are K144, K158, K172, K199, K230, K292 and K296. Our hypothesis that CO2 activates ACC oxidase by carbamylating an essential lysyl residue requires demonstrating that COz-dependent enzyme activity is lost when one of the 7 conserved Lys residues is mutated to another amino acid. We have found that enzyme activity is markedly dependent upon the nature of the amino acid substituted for lysine but, so far in all cases tested, CO2 dependency can be demonstrated in cell lysates and as purified fusion proteins (Table I). We have so far examined K158E, Q, R and L mutants, K230E, Q and R mutants and the K199E, K292E and K296E mutants. All the Lys mutants have variable but significant enzyme activity. The significance of the low activity of the K158L mutant is not clear since the K158E, Q and R mutants were all active. The KI58L mutant is only about 1% as active as the native enzyme but the 1% residual activity is activated by CO2. The K158L mutant may be inactive because of incorrect protein conformation or instability. Substitution of Ala for Lys 158 was found to inactivate ACC oxidase of kiwifruit [8]. This confirms our results with the KI58L mutant. Leu is hydrophobic as is Ala and, when substituted for Lys158, may disrupt protein conformation. We predict that Lysl58 is near the C-terrninus of ~6.

4. Conclusions

CO2 carbamylation of a lysyl residue may not be the mechanism by which CO2 activates ACC oxidase. CO2 dependent enzyme activity was demonstrated by Yang's laboratonT when KI40 KI44 KI58 KI72 KI99 K230 K292 K296 and K304 was replaced b

.&.J ""'" Y arginine leading them to conclude that carbamate formation is not the basis for CO2 activation [I]. So far we have confirmed that K158, K199, K230 and K292 and K296 may be eliminated as carbamylation targets in explaining CO2 activation. CO2-dependent enzyme activity was observed with each of the following mutants: K158E, K158Q,

12

K158R, K158L, K230E, K230Q, K230R, K199E, K292E and K296E. We have yet to confirm essentiality of KI44 and KI72 with respect to COrdependent ACC oxidase activity. These studies are now in progress. Substituting Ala or Cys for K172 in the kiwifruit enzyme yielded an active enzyme [8]. If we confirm CO2-dependent enzyme activity with mutations with all seven of the conserved Lys residues, this will confirm that carbamylation is not the mechanism of CO2 activation of ACC oxidase [1]. It is feasible that CO2 may interact directly with enzyme-bound Fe2+ in forming an Enz­Fe2+-C02 complex at the catalytic site. We have demonstrated that HiS177, HiS234 and Aspl79 are putative ligands for Fe2+. No activity was observed in cell lysates of the H234F and D179L mutants. The purified fusion proteins of these mutants was completely inactive. The low residual activity of the HI77F mutant was not CO2-dependent in the cell lysate assay or as the purified fusion protein. This suggests that CO2 may activate the enzyme as a consequence of interacting directly with HI77 and Fe2+. The H234F and D179L mutants exhibited no enzyme activity as celllysates or as fusion proteins and this may indicate that H234 and DI79 are more critical as ligands for Fe2+ than HiSI77. John et al. [8] observed that the H177Q and D 179E mutants had only about 1 % of the activity of the native kiwifruit enzyme supporting our results.

In summary, our site-directed mutagenesis studies with ACC oxidase strongly indicate that HiS177, Aspl79 and HiS234 are essential for catalytic activity as putative ligands for Fe2+. These Fe2+ ligands are likely to be arranged at the catalytic site similar to that of IPNS. Carbamylation of one of the 7 conserved Lys residues is not likely as the mechanism of CO2 activation. We have ruled out the following Lys residues: K158, K199, K230, K292 and K296. We have yet to test KI44 and K172 which we judge to be unlikely candidates. Moreover, John et. a1. [8] and Charng et. a1. [I] have provided evidence that these are not carbamylated as the CO2 activation mechanism. We have found that the Hisl77F mutant has a low but reproducible enzyme activity that is strictly independent of CO2 suggesting that the CO 2 activation mechanism may involve His 77 at the Fe 2+ center.

5. References

1. Chamg, Y.-Y., Dong, J.-G., and Yang, S.F. (1996) Structure-function studies on the 1-aminocyclopropane-l-carboxylaate acid (ACC) oxidase carbon dioxide binding site, in NATO Advanced Research Workshop, Biology and Biotechnology of the Plant Hormone Ethylene, June 9-13, Chania, Crete, Greece, Poster Abstract No.9.

2. Deng, W.P. and Nickoloff, JA (1992) Site-directed mutagenesis of virtually any plasmid by eliminating a unique site, Anal. Biochem. 200, 81-88.

3. Dilley, D.R., Wilson, I.D., Burmeister, D.M., Kuai, J., Poneleit, L., Zhu, Y., Pekker, Y., Gran, C., and Bower, A (1993) Purification and characterization of ACC oxidase and its expression during ripening in apple fiuit, in J. C. Pech et al. (eds.), Cellular and molecular aspects of the plant hormone ethylene, K1uwer Academic Publishers, Dordrecht, pp. 46-52.

4. Dong, 1.G., Fernandez-Maculet, J.C., and Yang, S.F. (1992) Purification and characterization of 1-aminocyclopropane-l-carboxylate oxidase from ripe apple fiuit, Proc. NatL Acad. Sci. USA 89, 9789-9793. ,

5. Fernandez-Maculet, J.C., and Yang, S.F. (1993) Activation of l-aminocyclopropane-l-carboxylate oxidase by carbon dioxide, Biochem. Biophys. Res. Comm. 193, 1168-1173.

6. Hong, S.-B., Kuo, J.M., Mullins, L.S., and Raushel, F.M. (1995) CO2 is required for the assembly of

13

the binuclear metal center of phosphotries1erase, J. Am. Chem. Soc. 117, 7580-7581. 7. Jahri, E., Carr, M.B., Hausinger, RP., and Karplus, PA (1995) The crystal structure of urease from

Klebsiella aerogenes, Science 268,998-1004. 8. John, P., Iturriagagoitia-Bueno, T., Lay, V., Thomas, P.G., Prescott, AG., Gibson, E.J., and

Scholfield, C.J. (1996) ACC oxidase: molecular structure and catalytic function, NATO Advanced Research Worbhop, Biology and Biotechnology of the Plant Hormone Ethylene, June 9-13, Chania, Crete, Greece, Oral Abstract No.9.

9. Lorimer, G.H. (1983) Carbon dioxide and carbamate formation: the makings of a biochemical control system, Trends Biochem. Sci. 8, 65-68.

10. Lorimer, G.H. and Miziorko, H.M. (1980) Carbamate formation on the e-amino group of Iysyl residue as the basis for the activation of ribulosebisphosphate carboxylase by CO2 and Mi+, Biochemistry 19,5321-5328.

11. McGarvey, J. and Christoffersen, R.E. (1992) Characterization and kinetic parameters of ethylene­forming enzyme from avocado fruit, J. Bioi. Chem. 267, 5964-5967.

12. Park, I.-S. and Hausinger, R.P. (1995) Requirement of carbon dioxide for in vitro assembly of the urease nickel metailocenter, Science 267, 1156-1158.

13. Poneleit, L. and Dilley, D.R (1993) Carbon dioxide activation of l-aminocyclopropane-l­carboxylate (ACC) oxidase in ethylene biosynthesis, Postharvest Bioi. and Technol. 3, 191-199.

14. Prescott, AG. (1993) A dilemma of dioxygenases (or where biochemistry and molecular biology fail to meet), J. Expt. Bioi. 44, 849-861.

15. Roach, P.L., Clifton, I.J., Fulop, V., Harlos, K., Baron, G.F., Hajdu, J., Anderson, I., Schofield, C.J., and Baldwin, J.E. (1995) Crystal structure of isopencillin N synthase is the first from a new structural family of enzymes, Nature 375, 700-704.

16. Ververidis, P. and Dilley, D.R. (1995) Catalytic- and non-catalytic inactivation of 1-aminocyclopropane-l-carboxylate (ACC) oxidase in ethylene biosynthesis: role of cyanide product, Proc. 22nd Ann. Mtg., Plant Growth Regul. Soc. Amer. 1995, pp. 183-199.

17. Wilson, I.D., Zhu, Y., Burmeister, D.M., and Dilley, D.R (1993) The apple ripening-related cDNA clone pAP4 confers ethylene forming ability in transformed Saccharomyces cerevisiae, Plant Physiol. 102,783-788.

1-AMINOCYCLOPROPANE-1-CARBOXYLATE OXIDASE: MOLECULAR STRUCTURE AND CATALYTIC FUNCTION

P. JOHN\ T. ITURRIAGAGOITIA-BUENO\ V. LAy l,2, P.G. THOMAS2, T.A.J. HEDDERSON3, A.G. PRESCOT'll, E.J. GmSON5,

and C.J. SCHOFIELD5

1 Department of Agricultural Botany, School of Plant Sciences, Plant Science Laboratories, The University of Reading, Reading RG6 6AS, UK 2Zeneca Agrochemicals, Jealott's Hill Research Station, Bracknell,

Berkshire, RG42 6ET, UK 3Department of Botany, School of Plant Sciences, Plant Science Laboratories, The University of Reading, Reading RG6 6AS, UK 4Department of Applied Genetics, John Innes Centre, Norwich Research Park, Colney Lane, Norwich NR4 7UH, UK, 5 Dyson Perrins Laboratory and the Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford OX] 3QY, UK

1. Introduction

l-aminocyclopropane-l-carboxylate oxidase (ACO) is the enzyme responsible for the final step in the biosynthesis of ethylene in flowering plants. At the Ethylene Conference that was held at Diepenbeek in 1988, Osborne [1] described the results of a survey of ethylene synthesis among lower plants. It revealed that all representatives of the major groups of non-flowering plants produced ethylene at detectable rates, but many plants were unable to convert l-aminocyclopropane-l-carboxylate (ACC) to ethylene. Figure 1 shows the phylogenetic relationships among the groups of plants studied by Osborne [1] as deduced from analyses [2] of their 18S rRNA sequences, as in [3]. Mapping ACO onto this phylogeny suggests that ACO arose in a common ancestor of the groups represented today by the Cycads, Gymnosperms, Gnetales and Angiosperms, with more basal groups lacking the ability to convert ACC to ethylene. Speculation on the possible evolutionary origin of the ACO raises a host of questions, for which we currently have no answers. For example, the biochemical pathway by which lower plants produce ethylene remains unknown: in the aquatic fern, Regnellidium diphyllum, ACC is clearly not the substrate for ethylene production, and moreover ethylene is not derived from methionine [4].

15 A. K. Kanellis et al. (eds.), Biology and Biotechnology o/the Plant Hormone Ethylene, 15-21. @ 1997 Kluwer Academic Publishers.

16

Chara

Chlorokybus

Coleochaete

Anthoceros

Pallavicinia

Reboulia

Marchantia

Andreaea

Polytrichum

Funaria

Ditrichum

Isoetes

Huperzia

Lycopodium

Lycopodiella

Blechnum

Dicksonia

Adiantum

Hypolepis

Paesia

Histiopteris

Pteridium

Equisetum

Ophioglossum

Botrychium

Psilotum

Tmesipteris

Selaginella krausiana

SelagineUa sp.

Selaginella gaiateaea

Zamia

Gingko

Pinus

Taxus

Gnetum

Oryza

Zea

Glycine

Lycopersicon

Charophycean Green Algae

Bryophytes

Lycophytes

Leptosporangiate Ferns

I Horsetail Ferns

Eusporangiate Ferns

? Lycophytes

I Cycads

I Gymnosperms

I Gnetales

Angiosperms

Figure 1. Tentative phylogeny of land plants based on parsimony and maximum likelihood analyses of 188 rRNA gene sequences, as in [2,3]. Representatives from groups underlined were shown by Osborne [1] to be able to generate ethylene from added ACC. The arrow indicates a possible origin for the ACO.

17

ACOs isolated from a variety of Angiospenns possess strongly conserved motifs that are also found in the sequences of other members of the family of enzymes, the 2-oxoacid dependent dioxygenases (2-0DDs) [5,6J, also known as the Fe(II)-dependent dioxygenases. ACO is unusual among these enzymes, however, in that in vitro activity requires ascorbate and carbon dioxide, and does not require 2-oxoglutarate [7,8]. It has been reported that ascorbate is consumed during the ACO reaction stoichiometrically with the production of ethylene [8J, and consequently it is believed that ascorbate acts as a cosubstrate together with ACC for ACO activity. The role of carbon dioxide is unknown, although some authors [9-12J have suggested, by analogy with the mechanism of carbon dioxide activation of ribulose 1,5-bisphosphate carboxylase/oxygenase, that it may act at a lysine group.

Presumably during the evolution of the land plants, a relatively primitive pathway of ethylene biosynthesis (as yet unidentified) was replaced by the ACC-dependent pathway that now predominates in the Angiosperms and related groups (Fig. 1). The key intermediate of this pathway, ACC, could have been available among the pool of accumulated phytochemicals in the plant group in which the ACO arose. Most likely, the ACO originated with an alteration in substrate specificity in a pre-existing 2-oxoacid-dependent dioxygenase. Since the ancestral enzyme probably used 2-oxoglutarate as a cosubstrate, as do almost all known plant 2-oxoacid-dependent dioxygenases [6J, we might expect to find that ACO has retained a reactivity towards 2-oxoglutarate. The results presented in this paper show that such an expectation is justified, and that 2-oxoacids are potent inhibitors of the ACO. We also show, by site­directed mutagenesis, that ACO is likely to possess structural features present in another 2-oxoacid-dependent dioxygenase [13J.

2. Inhibition by 2-0xoacids

2-0xoglutarate, and a variety of other 2-oxoacids (Table 1) inhibited the ACO activity extracted from ripe pear fruits (Pyrus communis L. cv Conference), showing competitive inhibition kinetics with respect to ascorbate, and non-competitive kinetics with respect to carbon dioxide, ACC and iron(II). Figure 2 show the Lineweaver-Burk plots obtained with 2-oxosuccinate (oxaloacetate), which was the most potent inhibitor examined (Table 1).

TABLE 1. Inhibition constants for the pear ACO [16]

Compounds 2-0xosuccinate 3-0xoglutarate 2-0xoglutarate 2-0xoadipinate

Succinate Pyruvate

Ki(mM) 0.24 0.86 1.35 ",5 >20 >20

K; values were calculated using an Excel curve-fitting program, from data from experiments in which ascorbate and inhibitor concentrations were varied [16].

18

-0.4

12 A

1.5mM 10

! """I

::;;;- 8

~ B 0 ... 6 c::a. lOG e ;::; .s 4 ~ -

2

-0.2 0 0.2 0.4 0.6

1/[A scorbate](mM) -1

14 C

1.5mM

~

1 Q .. "" .. 0.5mM

i ~ ....

OmM

-15 -10 -5 0 5 10 15 20 25

lI[ACC](rnM) .,

-0.4

4 B

"""I 3 1.5mM

:;;--.S B e 2 1:1. ... O.SmM EI ;::; S > -- OmM

-0.2 0 0.2 0.4 0.6

lI[CO .J(" r' 3

D

""I

,-.. ..c:I 1.5n"M

~ 2 0

~ bfl

.@ S 0.5n"M > ---.-<

In"M

.Q4 .Q2 0 0.2 Q.4 0.6

11 [FeSO.]{)lMy>

Figure 2. Lineweaver-Burk plots of the inhibition of pear ACO by 2-oxosuccinate with respect to (a) sodium ascorbate, (b) CO2,(the reaction mixture contained 12 mM sodium ascorbate), (c) ACC, (d) Fe(lI) [16]. ACO was routinely assayed by measuring the ethylene produced after incubation for 15 min at 30°C in a reaction mixture containing 0.2 M MOPS (PH 6.9), 10% (v/v) glycerol, 30 mM sodium ascorbate, 0.1 mM ferrous sulphate and 21 mM NaHC~, with an atmosphere enriched with 20% CO2 [14].

19

3. Site-directed Mutagenesis

In a standard assay of ACO activity, all the mutant proteins fell into one of two categories (Table 2). In one category, the proteins were not greatly affected by mutational change. They included mutant proteins in which KIn was substituted by either alanine or cysteine, and in which G137 was substituted by a proline (Table 2). In the second category were those mutants in which activity was severely reduced so that they showed 1% of the wild-type activity. These included proteins in which substitutions had been made of 0179, HI77, and K158.

TABLE 2. Specific activities of purified recombinant wild-type and mutant kiwifruit ACO.

ACO variant Specific activity Percent (pKatmg protein'l) (%)

Recombinant wild-type 1923 ± 70 100 KI72A 1693 ± 94 85 Kl72C 1223 ± 83 64 Gl37P 1891 ± 179 98 Dl79E 16± 4 H177Q 17± 3 K158A 24± 3 K158C 25± 5

The kiwifruit ACO cDNA clone (PKIWIAOl) was generously donated by Richard Gardner, Auckland, New Zealand, Standard methods were used for sub-cloning, expression and site-directed mutagenesis (Lay et aI., submitted), Mutant proteins were purified using Q-Sepharose chromatography and Mono-Q FPLC, identified by SDS-P AGE, and quantified by western blotting using an Enhanced Laser Densitometer. All determinations were made in triplicate, and the results are expressed as means offour experiments ± S.E.

In ribulose 1,5-bisphosphate carboxylase/oxygenase, carbon dioxide activation occurs as a result of the formation of a carbamate with a lysine residue [17,18]. Sequence alignments of the published ACOs has revealed the presence of 7 conserved lysine residues (Lay et al., submitted), Of these 7 lysines, only one appears to be exclusive to the ACO group and is not shared with any other 2-000s: KIn. The other known 2-0DDs have not been reported to be activated by carbon dioxide, thus conservation of KIn in the ACO family suggested that it may play an important role in ACOs that was not relevant to the activity of the other 2-000s. Of the 7 conserved lysine residues, K158 is the nearest to the KIn in primary sequence and was, therefore, also chosen for substitution to form a comparison with the KIn mutants. The results obtained in the present work indicate that KIn, the putative carbon dioxide activation site appears to be unimportant for activity as it can be substituted by either alanine or cysteine with retention of activity. This suggests that KIn in the kiwifruit enzyme is not the site of carbon dioxide binding, and raises doubts about the

20

concept of a lysine residue being involved in carbon dioxide activation of ACO [11]. An alternative mode of action for carbon dioxide would be to bind at the active-site iron together with ascorbate.

Unexpectedly, K158 appears to be more important than KIn for activity, since substitution of K158 results in a virtually complete loss of activity. Interpretation of these effects is assisted by the crystal structure of isopenicillin N-synthase (IPNS) [13]. Sequence alignment of the tomato ACO and IPNS suggests that the jelly-roll core and

the longest a-helix (00) of the IPNS are also present in the ACO structure [13]. The position of KIn in the kiwifruit ACO sequence is such that it would occur in a short loop, equivalent to ~7 in the IPNS structure, which may be unimportant for ACO activity. By contrast, the position of K158 in the ACO sequence would place this residue, by comparison to the IPNS sequence, at the end of the ~5 strand, which is one of the strands that folds to make the jelly-roll core and is, thus, an essential structural unit of the protein. The loss of activity on mutation of K158 may be explained most readily by a disruption of this structure.

G137 in the kiwifruit ACO occupies a position homologous with G165 in lPNS, a position that is highly conserved among a variety of2-0DDs [13], occurring at a sharp tum at the end of the longest a-helix (00). Proline residues commonly occur at turns in the protein backbone, thus substitution of G137 by a proline is conservative with regard to maintaining flexibility at the end of the 00 helix. Roach et al. [13] have proposed that the function of this 00 helix is to stabilise the distorted jelly-roll core of 2-0DDs; the flexibility at the carboxy-cap of this helix may be essential for permitting conformational changes of the active site on substrate binding. Thus, the conservation of activity on substitution of G 13 7 by proline might be expected.

Both HI77 and D 179 are highly conserved residues in all 2-0DDs, corresponding to the H214 and D216 residues of lPNS, respectively. In lPNS, the side-chains of these residues act as co-ordinators of the iron at the active site [13]. A similar role in ACO would be compatible with the loss of activity observed here on their substitution by other residues. Like histidine residues, glutamine residues are also capable of binding ferrous iron, as described by Roach et al. [13], where a glutamine residue (Q330) in the C-terminal tail of IPNS acts as one of four protein ligands at the metal ion binding site. However, conversion of HI77 to a glutamine in ACO is unable to preserve enzyme activity. Conversion of D 179 to a glutamic residue also resulted in complete loss of enzyme activity. This change was more conservative, involving the introduction of a single methylene group into the side-chain of this putative metal-binding protein ligand. It was unexpected that such a small change would be so deleterious to the enzyme. These findings indicate that space in the active site is very confined and in the case of D 179E, even an additional methylene group cannot be accommodated in the side-chain of this critical residue.

In conclusion, the resUlts presented here indicate that it is unlikely that carbon dioxide activates ACO via carbamylation of lysine residues at positions In or 158 in the kiwifruit enzyme, and may alternatively playa role at the active-site iron.

21

4. Acknowledgements

Work reported here has been supported by the Basque Government, BBSRC, EPSRC, MRC, NERC, and Zeneca.

5. References

1. Osborne, D.J. (1989) The control role of ethylene in plant growth and development., in H. Clijsters et a!. (eds.), Biochemical and Physiological Aspects of Ethylene Production in Lower and Higher Plants, Kluwer Academic Publishers, Dordrecht., pp. 1-11.

2. Swafford, D.L. and Olsen, G.J. (1990) Phylogeny reconstruction, in Hillis, D.M. and Moritz, C. (eds.), Molecular Systematics, Sinauer, Sunderland, pp. 411-501.

3. Hedderson, TA, Chapman, RL., and Rootes, W.L. (1996) Phylogenie relationships of bryophytes inferred from nuclear-encoded rRNA gene sequences, PI. Syst. Evol. 200, 213-224.

4. Osborne, D.l, Walters, l, Milborrow, B.v., Norville, A, and Stange, L.M.C. (1996) Evidence for a non-ACC ethylene biosynthesis pathway in lower plants, PhytochemiStry, 42, 51-60.

5. Prescott, AG.(1993) A dilemma of dioxygenases (or where biochemistry and molecular biology fail to meet),J. Expt. Bot. 44, 849-861.

6. Prescott, AG. and John, P. (1996) Dioxygenases: molecular structure and role in plant metabolism, Annu. Rev. Plant Phys. Plant Mol. BioI. 47,245-271.

7. Smith, J.J., Ververidis, P., and John, P. (1992) Characterisation of the ethylene-forming enzyme activity partially purified from melon, Phytochem. 31, 1485-1494.

8. Dong, J.-G., Fernandez-Maculet., J.C., and Yang, S.F. (1992) Purification and characterization of 1-aminocyclopropane-l-carboxylate from apple fruit., Proc. Natl. Acad. Sci. U.SA. 89,9789-9793.

9. Dilley, D.R, Kuai, J., Poneleit., L., Zhu, Y., Pekker, Y., Wilson, I.D., Burmeister, D.M., Gran, C., and Bowers, A (1993) Purification and characterization of ACC oxidase and its expression during ripening in apple fruit., in lC. Pech, A Latcht and C. Balagut (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, Kluwer Academic Publishers, Dordrecht., pp. 46-52.

10. Yang, S.F., Dong, J.G., Fernandez-Maculet, lC., and Olson, D.C. (1993) Apple ACC oxidase: Purification and characterization of the enzyme and cloning of its eDNA, in J.C. Pech, A Latcht and C. Balague (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, Kluwer Academic Publishers, Dordrecht., pp. 59-64.

11. Fernandez-Maculet., J.C., Dong, J.G., and Yang, S.F. (1993) Activation of l-aminocyclopropane-l­carboxylate oxidase by carbon dioxide, Biochem. Biophys. Res. Commun. 193, 1168-1173.

12. Ververidis, P. and Dilley, D.R (1994) Mechanism studies of CO2 activation of l-aminocyclopropane­I-carboxylate (ACC) oxidase. Evidence for a lysyl residue involvement., Plant Physiol. Supp!. 105, 33.

13. Roach, P.L., Clifton, I.1., Fulop, V., Harlos, K., Barton, G.J., Hajdu, 1., Andersson, I., Schofield, C.l, and Baldwin, J.E. (1995) Crystal structure ofisopenicillin N synthase is the first from a new structural family of enzymes, Nature 375, 700-704.

14. Umbreit., W.W., Burris, RH., and Stauffer, J.F. (1957) Manometric Techniques -a Manual Describing Methods Applicable to the Study of Tissue Metabolism, Burgess Publishing Co., Minneapolis, p. 18.

15. Zhang, Z., Schofield, C.J., Baldwin, J.E., Thomas, P., and John, P. (1995) Expression, purification and characterization of l-aminocyclopropane-l-carboxylate oxidase from tomato in Escherichia coli, Biochem. J. 307, 77-85.

16. lturriagagoitia-Bueno, T., Gibson, E.J., Schofield, C.J., and John, P. (in press) Inhibition of 1-aminocyclopropane-l-carboxylate oxidase by 2-oxoacids, Phytochem.

17. Lorimer, G.H., Badger, M.R, and Andrews, T.J. (1976) The activation ofribulose-l,5-bisphosphate carboxylase by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested mechanism, and physiological implications, Biochem. 15, 529-536.

18. Andrews, T.J. and Lorimer, G.H. (1987) Rubisco: structure, mechanisms, and prospects for improvement., in M.D. Hatch and N.K. Boardman (eds.), The Biochemistry of Plants, A Comprehensive Treatise Vol. 10: Photosynthesis, Academic Press, New York, pp. 131-218.

ON 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID (ACC) OXIDASE Degradation of a-Aminoisobutyric Acid and Structure-Function Studies on the CO2 Binding Site

Y.-y. Chamg1,2, Y. Liu2, lG. Doni, and S.F. Yang1,2 lInstitute of Botany, Academia Sinica, Nankang, Taipei, Taiwan, ROC, 2Department of Vegetable Crops, University of California, Davis, California 95616, USA

1. Introduction

ACC oxidase catalyzes a complex reaction to form the gaseous plant hormone ethylene as shown by the following reaction equation: ACC + O2 + ascorbate 7 C2~ + HCN + CO2 + dehydroascorbate + 2H20 [1]. The reaction requires the presence of the cofactors Fe2+ [16] and CO2 [1,3]. Although the exact reaction mechanism is still unknown, it is believed that the enzymatic reaction proceeds via the intermediate N­hydroxyl-ACC, which is presumably so unstable that it fragments into Cz~ (derived from C-2,3 of ACC) and cyanoformic acid, the latter being further degraded into HCN (derived from C-l of ACC) and CO2 (derived from the carboxyl group of ACC). The notion that ACO is a hydroxylase is supported by the amino acid sequence homology shared between ACO and a number of known hydroxylase [4] as well as by the requirement of the enzyme for molecular oxygen, Fe2+, and ascorbate for catalysis.

a-Aminoisobutyric acid (AIB), a structural analog of ACC, has been shown to inhibit ethylene formation from ACC in plant tissues [13]. It was generally believed that AIB is not metabolized by the tissue to which this compound was fed. However, Liu et al. [8] have demonstrated that AIB is decarboxylated when it was fed to the segments of mungbean hypocotyls. The decarboxylation of AIB was inhibited by ACC and the other inhibitors of ACC conversion to ethylene [8], suggesting that ACO is involved in the degradation of AIB. Here, we tested this hypothesis with the purified recombinant apple ACO. We also examined the oxidative activity of the enzyme on other amino acids.

It is of interest to understand the mechanism by which CO2 as a cofactor activates ACO activity. Preincubation studies suggest that, similar to the case in ribulosebisphosphate carboxylase [9], a carbamate adduct maybe formed between C02 and the e-amino group of a lysine residue of ACO [3]. In an attempt to identify the possible candidate that is involved in the carbamate formation, we have performed site­directed mutagenesis on those lysine residues that are conserved throughout all known ACO sequences. .

23

A. K. Kanellis et aI. (eds.), Biology and Biotechnology o/the Plant Hormone Ethylene, 23-29. © 1997 Kluwer Academic Publishers.

24

2. Degradation of AlB Catalyzed by ACO

If AIB is oxidized by ACO via a similar mechanism as that converting ACC to ethylene, AIB should generate acetone, ammonia, and C02 [8]. We examined first the production of acetone from AIB under the ACO assay condition [1], except that ACC was substituted by AIB in the reaction mixtures. For this purpose, a recombinant apple ACO was purified to homogeneity from E. coli which overexpressed the corresponding gene derived from pAE12 [2]. An enzyme preparation with specific activity of 150 unitlmg was used in this study (one unit of the enzyme activity is defined as the amount of enzyme required to convert one nmol of ACC to ethylene per min at 30°C). After the reaction was initiated by adding an aliquot of the purified recombinant apple ACO to the reaction mixtures, the production of acetone increased as the incubation time progressed. The production rate increased proportionally to the amount of enzyme added (data not shown). Heat treatment of the enzyme preparation at 100°C for 5 min abolished its ability of converting AIB to acetone. Moreover, without the addition of either the enzyme or AIB, no acetone production was observed. These observations support the notion that the conversion of AIB to acetone was catalyzed by ACO.

In order to show that ACO instead of other proteins present in the enzyme preparation catalyzed the conversion of AIB to acetone, the activities in the E. coli crude extracts from cells with or without the transformed apple ACO gene were compared. The crude extract of the E. coli cells without ACO gene did not convert AIB to acetone, while the cells with ACO gene exhibited acetone production activity. Moreover, ascorbate, O2, Fe2+, and CO2 were also required for the activity as is the case for the oxidation of ACC.

Besides acetone, ammonia and CO2 were also produced from AIB catalyzed by ACO as predicted. The stoichiometry of the reaction products (acetone: ammonia: CO2) was determined and was shown to be in 1:1:1 ratio (Table 1). Kinetic studies showed that the Km for AIB was 14.7 mM as compared to 0.23 mM for ACC, while the kcat for AIB was about the same as that for ACC (0.07 sec-I). Similar to the observations of in vivo studies, AIB also competitively inhibited the oxidation of ACC to ethylene using purified ACO, with a Ki of 7.5 mM. The results described above were consistent with the in vivo data reported by Liu et aJ.[9], who predicted that AIB can be oxidized by ACO.

3. Oxidation of Other Amino Acids by ACO

Since ACO can oxidize AIB, we also tested the possibility of ACO oxidizing other common amino acids. First, the inhibitory effect of these amino acids on the conversion of ACC to ethylene was examined. It was observed that the recombinant apple ACO was inhibited more by D-a-aminobutyric acid (D-ABA) and D-alanine than by their L-enantiomers (Table 2), while D,L-valine and D,L-phenylalanine exerted less inhibitory effect, because of due to their bulkier side chains (data not shown). These

25

results are in contrast to the observations of previous in vivo studies, where some L­amino acids were shown to be more potent inhibitor than their D-enantiomers in inhibiting ethylene production [5,6,10,14]. If the effect of D-amino acids on the activity of ACC malonyltransferase was taken into consideration for the in vivo system, it may explain the discrepancy we observed here. Since the D-amino acids are more effective than their L-enantiomers in inhibiting the malonylation of ACC into malonyl­ACC [8], which does not serve as the ethylene precursor, the in vivo concentration of ACC would be expected to be higher in the presence of the D-amino acids, which would indirectly result in higher apparent ACO activity.

TABLE 1. Stoichiometry of AlB oxidation catalyzed by purified recombinant apple ACC oxidase. Assay condition is described in the text. Acetone in the head space was measured with a gas-chromatographic method [17]. Radioactive CO2 liberated from [carboxyl- 14C)AlB was measured with scintillation counter after being absorbed to 2 N KOH. The amount of ammonia was measured by using a Sigma diagnostic kit based on the method of reductive amination of2-oxoglutarate [15).

Products fonned (runo!)

Exp. Acetone co, NH3

202 226

II 90 81

-, not detennined.

The inhibition of ACO by D-ABA, D-Ala, and AIB was shown to be competitive (Fig. 1), suggesting that these amino acids also bind to the ACC binding-site. Similar to the case of AlB described above, ACO must also possess capability to oxidize ABA and Ala to yield corresponding aldehydes, CO2, and ammonia. For convenience, the production of ammonia was assayed when these amino acids were employed as substrates. Again, we found that the recombinant apple ACO can oxidatively deaminate D-ABA and D-Ala with appreciable rate as compared to the oxidation of ACC (Table 3). The co-substrates and cofactors required for ACC oxidation activity were also required for the degradation of these amino acids, indicating that ACO was responsible for catalyzing the reaction. The fact that the enzyme exhibits much higher activity with the D-amino acids than with their L-enantiomers at the same substrate concentration (Table 3) is in agreement with the inhibition studies described above.

26

TABLE 2. Effect of various amino acids on the inhibition of the recombinant apple ACC oxidase activity. Ethylene production was assayed with 0.05 mM ACC. In the presence of 10 or 20 mM amino acids as indicated. Each value represents the mean ±SE of two replicates.

Amino acid Relative activity

mM %

None 100 AIB 10 45 ± 5

20 24 ± 2 D -a._aminobutyric acid 10 32 ± 2

20 16 ± I L-a.-1II!1inobutyric acid 10 92 ± 2

20 80 ± 1 D-Ala 10 61 ± 3

20 43 ± 4 L-Ala 10 86 ± 3

20 67 ± 5

TABLE 3. Comparative activity of recombinant apple ACC oxidase towards various amino acid substrates. The concentration of each amino acid substrate was as indicated. For those using ACC as substrate, ethylene production was measured. For the other amino acids, ammonia production was assayed.

Amino acid Product fonned (nmol.min1.mg-1 )

ACC ImM 121.9 lOmM 129.2

AlB lOmM 50.6

D-ABA 10mM 58.5

lrABA lOmM 3.0

D-A1a lOmM 23.3

lrAia lOmM 7.6

The stereospecificity of ACO has been investigated in vivo by measuring the conversion of administered stereoisomers of l-amino-2-ethylcyclopropane-I-carboxylic acid (AEC) into I-butene in plant tissues [5]. It was shown that the most active isomer is (1R,2S)-AEC. We have used the purified enzyme to reexamine the reactivity of ACO toward these compounds. From the kinetic data of these four AECs (Table 4), it was observed that (lR,2S)-AEC was the most active substrate, which is similar to the in vivo result [5]. The Km value for (lR,2S)-AEC was approximately the same as that for ACC, suggesting that the ethyl substituent on the pro-R methylene, trans to the carboxyl group, caused very little or no interference with the enzyme, which supports

27

the model proposed by Hoffman et al. [5]. Moreover, since (1R,2S) and (lR,2R)-AEC have R-configuration as a D-amino acid, the relatively higher apparent affinities and activities of ACO to these two compounds over their S-enantiomers (Table 4) suggest that ACC is recognized by ACO as a D-amino acid.

.-. c: E 0 .-~ 0 E .:. > .....

1.5

• None

/ 0 O-Ala .. AlB

• O-ABA

1.0

~ 0.5

60

lI[ACC] mM -1

Figure 1. Double-reciprocal plots of ACO activity versus concentration of ACC in the absence and presence of 10 mM inhibitor as indicated

The fact that ACO oxidized AIB and other amino acids raises the question, whether the enzymatic reaction really proceeds via the intermediate of N-hydroxyl-amino acid, since it has been reported that several N-hydroxyl-amino acids, such as N-hydroxyl­alanine and N-hydroxyl-aminobutyric acid, are stable compounds [12]. Thus, the stability of the hydroxyl-amino acid under the ACO assay condition needs to be examined. If the corresponding N-hydroxyl-amino acids of AIB, D-ABA, or D-Ala are stable under the assay condition or degrade at slower rate than the oxidation of its corresponding amino acid by ACO, then we have to conclude that ACC is not oxidized to ethylene by ACO via the formation ofN-hydroxyl-ACC.

4. Structure-function Studies on CO2 Binding Site

Carbamate formation between CO2 and the e-amino group of a lysine residue was suggested to be involved in the activation of ACO [3]. If this is true, replacement of this lysine residue with another amino acid should abolish the carbamate formation and, hence, result in an enzyme that can no longer be activated by CO2• We have

28

therefore performed site-directed mutagenesis of the recombinant apple ACO according to the method of Kunkel [7] on lysine residues that are conserved throughout all known ACO sequences individually (residues, 140, 144, 158, 172, 199, 230, 292, 296, and 304 in the apple ACO sequence). In order to introduce as little change as possible in the three dimensional structure of the enzyme, each invariant lysine residue was replaced with an arginine, whose side chain does not form a carbamate adduct with CO2 [11]. We found that all the Lys to Arg mutant enzymes were activated by carbon dioxide and had significant activity compared to that of the wild-type enzyme on the basis of specific activity (Table 5). Therefore, we conclude that the activation of ACO by CO2 does not involve a carbamate formation with a lysine residue of ACO.

TABLE 4. Comparison of the kinetic constants of the recombinant apple ACC oxidase using ACC or stereoisomers of l-amino-2-ethylcyciopropane-l-carboxylic acid (AEC) as substrates

Substrate Structure Km(mM) Relative Vmm: (%)

ACC 0.23 100

(1R,2S)-AEC ~ H009/r. ~H 0.20 91

(IS,2R)-AEC iA H009/r. ~ 5.96 0.6

(IS,2S)-AEC ~ HOO9l, ~Et 9.74 1.6

(lR,2R)-AEC ~ H009/r. ~H 5.18 8.8

TABLE 5. ACO activities of the wild-type and mutant enzymes in the absence and presence of CO2

ACC oxidase activity (nmol.min·l.m!fl) *

Enzyme -CO2 +C02

pET-20(b+) 0.04 0.04 W.T. 0.06 3.2 KI40R 0.04 4.7 KI44R 0.09 29 K158R 0.08 3.8 K172R 0.06 28 KI99R 0.04 1.0 K230R 0.06 1.2 K292R 0.04 28 K296R 0.04 1.6 K304R 0.04 1.9

* Crude extract activites.

29

5. Acknowledgments

This work was supported by a NATO research grant (CGR 930996) awarded to J.C. Pech and S.F. Yang and a Republic of China Research Council grant (NSC85-2321-B-01). We are grateful to Dr. J. C. Pech for his useful discussion.

6. References

1. Dong, 1. G.,Fernandez-Maculet, 1. C., and Yang, s. F. (1992) Purification and characterization of 1-aminocyclopropane-l-carboxylic acid oxidase from ripe apple fruit, Proc. Natl. Acad. Sci. USA 89, 9789-9793.

2. Dong, J. G., Olson, D.,Silverstone, A, and Yang, S. F. (1992) Sequence of a cDNA for a 1-aminocyclopropane-l-carboxylate oxidase homolog from apple fruit, Plant Physiol. 98, 1530-1531.

3. Fernandez-Maculet, J. C., Dong, J. G., and Yang, S. F. (1993) Activation of l-aminocyclopropane-l­carboxylate oxidase by carbon dioxide, Biochem. Biophys. Res. Commun. 193, 1168-1173.

4. Hamilton, A J., Lycett, G. W., and Grierson, D. (1990) Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants, Nature 346,284-287.

5. Hoffinan, N. E., Yang, S. F., Ichiara, A, and Sakamura, S. (1982) Stereospecific conversion of 1-aminocyclopropane-l-carboxylic acid to ethylene by plant tissues. Conversion of stereoisomers of 1-amino-2-ethylcyclopropane-l-carboxylic acid to I-butene, Plant Physiol. 70, 195-199.

6. Hyodo, H. and Nishino, T. (1981) Wound-induced ethylene formation in albedo tissue of citrus fruit, Plant Physiol. 67,421-423.

7. Kunkel, T. A (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection., Proc. Nat!. Acad. Sci. USA 82, 488-492.

8. Liu, Y., Su, L. Y., and Yang, S. F. (1984) Metabolism of a-aminoisobutyric acid in mung bean hypocotyls in relation to metabolism of l-aminocyclopropane-l-carboxylic acid, Planta 161, 439-443.

9. Lorimer, G. H. and Miziorko, H. M. (1980) Carbamate formation on the e-amino group of a lysyl residue as the basis for the activation of ribulosebisphosphate carboxylase by CO2 and Mg2+, Biochemistry 19,5328-5334.

10. LUrssen, K., Naumann, K., and Schroder, R. (1979) l-Aminocyclopropane-l-carboxylic acid. An intermediate of the ethylene biosynthesis in higher plants, Z. Pjlanzenphysiol. 92, 285-294.

11. Morrow, J. S., Keirn, P., and Gurd, F. R. N. (1974) CO2 adducts of certain amino acids, peptides, and sperm whale myoglobin studied by carbon 13 and proton nuclear magnetic resonance, J. BioI. Chem. 249,7484-7494.

12. Neelakantan, L. and Hartung, W.H. (1958) Il-Hydroxylamino nitriles and Il-hydroxylamino acids, J. Org. Chem. 23, 964-967.

13. Satoh, S. and Esashi, Y. (1980) Il-Aminoisobutyric acid: A probable competitive inhibitor of conversion of l-aminocyclopropane- I-carboxylic acid to ethylene, Plant Cell Physiol. 21, 939-949.

14. Satoh, S. and Esashi, Y. (1980) D-Amino acid-stimulated ethylene production in seed tissues, Planta 149,64-68.

15. Van Anken, H. C. and Schiphorst, M. E. (1974) A kinetic determination of ammonia in plasma, Clin. Chim. Acta. 56,151-154.

16. Ververidis, P. and John, P. (1991) Complete recovery in vitro of ethylene-forming enzyme activity, Phytochemistry 30, 725-727.

17. Winterbach, H. E. K. and Apps, P. J. (1991) A gas-chromatographic headspace method for the determination of acetone in bovine milk, blood and urine, Onderstepoort J. Vet. Res. 58, 75-79.

REGULATION OF AUXIN-INDUCED ETHYLENE BIOSYNTHESIS IN ETIOLATED PEA STEMS

1. Introduction

s.C. PECK AND H. KENDE MSU-DOE Plant Research Laboratory Michigan State University, East Lansing, MI 48824, USA

The etiolated pea stem was one of the first model systems for studying auxin-induced ethylene production. Besides being useful for examining the regulation of ethylene biosynthesis, pea seedlings exhibit a number of well-described growth responses mediated, at least in part, by ethylene. This chapter will discuss some of these responses, attempting to integrate earlier observations with more recent information provided by mutants and molecular analysis. It will also summarize our work on the regulation of auxin-induced ethylene biosynthesis and how it may be applicable to the understanding of ethylene-mediated growth responses.

2. Growth responses in etiolated seedlings

2.1. INHIBITION OF GROWTH BY ETHYLENE

Ethylene is generally considered an inhibitor of growth in vegetative tissues. As was first observed by Neljubov (reviewed in [1]), etiolated pea seedlings treated with ethylene are shorter and have an increased diameter. This altered growth pattern is thought to be caused by the reorientation of micro tubules in the elongating cells which leads to a change in deposition of cellulose microfibrils from transverse to longitudinal (reviewed in [6]). Thus, cells are restricted in their vertical expansion but are free to expand radially. Goeschl et al. [8] suggested that this response provided the germinating seedlings with a greater mechanical strength to grow through compacted soil. They showed that when the growth of etiolated seedlings was obstructed by a mechanical barrier, the seedling responded with increased ethylene production and a shortening and swelling of the epicotyl. Because ethylene is implicated in altering the translocation of auxin [4, 19, 20], it is not clear whether the reorientation of microtubules and microfibrils is a direct effect of ethylene or occurs via a redistribution of auxin. Given the inhibitory effect of ethylene on growth, it would appear logical that ethylene could mediate an asymmetric growth response via an asymmetric inhibition of cell elongation.

31

A. K. Kanellis et al. (eds.J, Biology and Biotechnology o/the Plant Hormone Ethylene, 31-38. © 1997 Kluwer Academic Publishers.

32

2.2. FORMATION OF THE APICAL HOOK IN ETIOLATED mCOTS

The apical hook in etiolated dicotyledonous plants is formed by a complex pattern of cell elongation. As the cells move out from the apex, those on the inner side of the hook elongate slower than those on the top, resulting in the curvature of the stem [31]. To fonn a straight stem below the hook, the pattern of elongation must be reversed. The cells on the inner side must rapidly expand to match the length of the outer cells. As long as the seedling remains in its etiolated growth phase, the hook is maintained by new cells passing through this pattern of alternating growth.

Ever since it was demonstrated that auxin causes closure of the apical hook via ethylene [13], it has been postulated that there are gradients of auxin and/or ethylene between the outer and inner portions of the apical hook leading to the asymmetry in growth. While it is difficult to establish the exact auxin concentration at its site of action, experiments with 3H-IAA showed that the IAA level in the inside part of the hook is approximately 4-fold greater than in the outside [30]. Although auxin is normally associated with cell elongation, higher concentrations (3 pM to 1 mM) of IAA inhibit growth by stimulating ethylene production [4]. Thus, a high local concentration of IAA in the inner portion of the hook may be responsible for the higher level of ACC and ethylene production in this region [28] and may, ultimately, lead to inhibition of growth.

While the above supports the assumption that asymmetric ethylene biosynthesis regulates the formation of the hook, there is also evidence that an asymmetric sensitivity or response to ethylene ultimately results in the difference in growth. If the hook is fonned by greater ethylene production on the inner portion, the presence of a saturating amount of ethylene in the atmosphere should abolish any gradient between inner and outer sides. The result would be the loss of the apical hook as both sides would be equally inhibited. However, seedlings grown in the presence of saturating levels of ethylene not only have a closed hook, but the hook is closed even more tightly than nonnal. The etr1 mutant of Arabidopsis, which is defective in a serine/threonine kinase such that it constitutively 'perceives' ethylene, has a similarly exaggerated apical hook [15]. Thus, the inner portion of the hook must be more sensitive or more responsive to ethylene than the outer portion.

Of course, interpretation of cause and effect are complicated by the involvement of both ethylene and auxin in this response. From results with Arabidopsis mutants, it appears that ethylene establishes a gradient in auxin concentration across the hook, not viee versa. Inhibitors of auxin transport abolish the apical hook even in the etr1 mutant [17]. Another mutant, hls1, does not form an apical hook, even in a etr1 background [27]. The HLS1 gene encodes an apparent N-acetyltransferase required to limit the expansion of cells, possibly by affecting auxin transport or auxin metabolism [17]. Because expression of this gene is promoted by ethylene, the gene product of HLS1 may be the intennediate by which ethylene alters the distribution of auxin. Although the HLS1 transcript was expressed equally throughout the hook [17], it was not determined if the protein was differentially localized. Moreover, as will be discussed in section 2.3, expression of genes involved in the formation of the apical hook may undergo rhythmic periodicity, which may cause misleading interpretations of in situ localizations.

33

2.3. NUTATION

Interestingly, the angle of the apical hook does not remain static. During growth, the hook rhythmically opens and closes along with associated stem movements with periods of one to several hours [7]. These nutational oscillations in the stem are primarily in the same plane as the hook, with the greatest bending occurring away from the plumule [2]. Although the response has been suggested to result from gravitropic overcompensation (reviewed in [12]), Heathcote and Aston [9] demonstrated that nutational movements are independent of gravitropic processes. A role for auxin has been implicated because inhibitors of auxin transport prevent nutation [2]. Britz and Galston [3] found that removal of the plumule alone abolishes nutation and that nutation was restored with the application of IAA to the remaining stem. If the hook was also removed, however, IAA could not restore nutation although it could restore growth. Thus, transitory gradients of lAA established by ethylene to form the apical hook may be translated into oscillatory processes of nutation in the stem [3]. The following results may explain the role of ethylene in the rhythmic periodicity observed in nutational movements.

3. Results

3.1 SEQUENTIAL REGULATION OF THE ENZYMES OF ETHYLENE BIOSYNTHESIS.

We have been studying how auxin and ethylene interact to regulate ethylene biosynthesis in etiolated pea seedlings. Because ethylene increases ACC oxidase activity and transcript levels in all portions of the seedling [29, Peck and Kende, unpublished results], we investigated whether IAA, which promotes ethylene synthesis, would also promote the accumulation of ACC oxidase. In the first internode, 100 I'M lAA stimulated ethylene production via an increase in extractable ACC synthase activity [25]. ACC oxidase transcript and activity levels also increased in this tissue between 2 and 4 h after the IAA treatment [25]. This increase was blocked by 2,5-norbornadiene (NBD), a competitive inhibitor of ethylene action, indicating that ethylene mediates the auxin effect via a positive feedback loop.

The increase in ACC synthase transcript and activity levels preceded the increase in ACC oxidase levels and in ethylene production by 1 to 2 h [25]. This observation raised the possibility that ACC oxidase is initially limiting and that the positive feedback loop is necessary for elevated ethylene production. If this assumption is correct, elevating ACC oxidase levels prior to IAA treatment should eliminate the lag time in ethylene production. Seedlings were pretreated with ethylene for 4 h to increase ACC oxidase abundance before the seedlings were sprayed with IAA. As seen in Figure I, pretreatment of the seedlings not only eliminated the lag time but also greatly stimulated ethylene production.

34

3.2 c 0 • . -

/ " 4h C2H4 ~

u :::l 2.4 -c '"" + 0 ~ IAA L. LL a. ....

I CD CI c 1.6 CD .... - I ~

..E: ..E: ~

W C .... ....., 0.8 0

CD ~

c D:: 0.0

0 2 4 6 8 10

Duration of Treatment (h)

Figure 1. Effect of ethylene pretreatment on the rate of auxin-induced ethylene production in the first internode of etiolated pea seedlings. Intact seedlings were placed in desicators with (e) or without (0) 40 p.LlL ethylene for 4 h. The seedlings were then sprayed with 0.1 mM IAA. At the times indicated, 1-cm sections were isolated from the first internode and used for measuring ethylene production. The experiment was performed three times with similar results.

This result, however, is not solely attributable to an increase in ACC oxidase activity. Unexpectedly, ACC synthase transcript abundance and enzyme activity were also higher in tissue pretreated with ethylene (Figure 2). Because ethylene pretreatment alone did not affect ACC synthase levels (Figure 2), the superinduction of ACC synthase must be caused by an altered response to the IAA treatment. Thus, it remains unclear how much the increase in ACC oxidase enzyme activity contributes to the burst of ethylene production after auxin treatment.

+ +

+ +

ACS Activity 2.2 0.6 13.0

PS-ACS2

Figure 2. Effect of ethylene pretreatment on auxin-induced ACC synthase activity and transcript abundance. A portion of the sections used in Figure 1 were used for ACC synthase activity assays and RNA isolation. The experiment was performed twice with similar results.

35

Ethylene negatively regulates its own biosynthesis in vegetative tissue by decreasing the levels of ACC [14]. In etiolated pea stems, ethylene inhibits the auxin­induced accumulation of ACC synthase transcript and activity levels [24]. The lag time of the inhibition of ACC synthase is approximately the same as that for ethylene induction of ACC oxidase.

From our work, we propose a model for the sequential regulation of the ethylene biosynthetic enzymes. IAA causes an increase in ACC synthase transcript abundance leading to an increase in ACC synthase activity. The newly formed ACC is converted to ethylene by a low, constitutive level of ACC oxidase. The ethylene produced then causes an increase in the levels of ACC oxidase via a positive feedback loop. Via a negative feedback loop, ethylene eventually reduces ACC synthase transcript and activity levels, leading to the cessation of IAA-induced ethylene production.

3.2. A SINGLE GENE FOR PS-ACSI ENCODES TWO TRANSCRIPTS

A cDNA clone of one of the IAA-induced ACC synthases, PS-ACSl, was isolated from a cDNA library made from the apical hooks of etiolated pea seedlings treated for 4 h with 0.1 mM IAA. While studying IAA-induced expression of PS-ACSI mRNA, it was observed that the PS-ACSI probe hybridized to two transcripts of 1.6 kband 1.9 kb on RNA blots. Because ACC synthase probes have been shown to hybridize to two transcripts in a variety of species in response to different stimuli [18, 21, 22, 32], we investigated the origin of the two transcripts. The two transcripts accumulated with different time patterns after IAA treatment, indicating that the smaller transcript was not a degredation product of the larger. The full-length PS-ACSI probe hybridized to single

36

bands on a genomic DNA blot. In addition, a probe containing only the 3'-untranslated region hybridized with both transcripts on an RNA blot. These results indicate that both transcripts are encoded by a single gene. Oligonucleotide-based RNase H mapping showed that the transcripts differed in the sequence of their 5' ends. Using 5'-RACE, we determined that the 1.6-kb transcript is lacking the first 385 bases of the 1.9-kb transcript. It is not known whether the putative truncated protein which would be encoded by the 1.6-kb transcript has a regulatory or enzymatic function.

4. Discussion

4.1. THE POSITIVE FEEDBACK OF ETHYLENE ON ACC OXIDASE

Because most ethylene responses are associated with changes in ethylene production [33], the regulation of ethylene biosynthesis is of obvious importance. Thus, it is necessary to determine if stimuli causing ethylene production affect ACC oxidase levels. It has been shown that ethylene treatment increases ACC oxidase activity in citrus leaves [26] and in carnation petals [5] and ACC transcript levels in tomato fruit [10]. NBD inhibits increases in ACC oxidase levels in wounded winter squash mesocarp [11], wounded etiolated mung bean seedlings [16], and in pollinated orchid flowers [23], indicating that these stimuli also increase ACC oxidase levels via ethylene. The presence of the positive feedback loop in a variety of responses emphasizes the importance of performing the proper controls to determine whether an increase in ACC oxidase levels leads to elevated ethylene production or, conversely, an increase in ethylene levels results in increased ACC oxidase activity.

4.2. A POSSIBLE ROLE FOR ETHYLENE IN NUTATION

While ethylene is clearly necessary to maintain the apical hook, differential ethylene biosynthesis per se is probably not responsible for the difference in growth between the inner and outer portions (see section 2.2). The sequential regulation model does, however, provide a possible explanation for the endogenous oscillations of auxin transport implicated by the 'bobbing' motion of the hook and stem of dicots. If local auxin concentrations in the inner side of the hook rose to the levels which induce ethylene biosynthesis, the sequential induction of the biosynthetic enzymes would result in ,a rapid burst of ethylene synthesis. The elevated ethylene production could increase auxin transport to the outer portion of the hook, possibly through the expression of HLSI. The burst of ethylene production simultaneously would begin to inhibit ethylene production via the negative feedback loop on ACC synthase. With the eventual cessation of ethylene production, the active formation qf an auxin gradient would end. Auxin could begin to accumulate in the inner portion of the hook until it reached the threshhold concentration for ethylene production to begin the cycle anew. Therefore, the alternating ethylene production and autoinhibition of ethylene production could establish the periodic oscillations in auxin concentrations resulting in nutational movement.

37

4.3. POSSIBLE CONSEQUENCES OF NUTATIONS ON IN SITU LOCALIZATION RESULTS

While the above is a rather simple model of a complicated growth process, it raises an important consideration. In association with the nutations of the stem, the hook clearly undergoes periodic opening and closing. This observation indicates that the factors which establish the formation of the hook may also undergo similar rhythmic expression. Because in situ localizations only capture the instant at the time of fixation, it may be entirely possible to miss the differential expression of the' genes involved in hook formation. Perhaps it would be preferable to monitor this potentially dynamic system with fusions of the promoters of interest to noninvasive reporter genes such as LUX to visualize the expression patterns of these genes over a period of time.

5. Acknowledgment

This research was supported by the U.S. Department of Energy through grant DE-FG02-9IER20021.

6. References

1. Abeles, F.B, Morgan, P.W., and Saltveit, M.E. (1992) Ethylene in Plant Biology, Academic Press, New York.

2. Britz, S.l. and Galston, A.W. (1982a) Physiology of movements in stems of seedling Pisum sativum L. cv Alaska, I. Experimental separation of nutation from gravitropsism, Plant Physiol. 70, 264-271.

3. Britz, S.l. and Galston, A.W. (1982b) Physiology of movements in stems of seedling Pisum sativum L. cv Alaska, II. The role of the apical hook and of auxin in nutation, Plant Physiol. 70, 1401-1404.

4. Burg S.P. and Burg E.A. (1966) The interaction between auxin and ethylene and its role in plant growth, Proc. Natl. Acad. Sci. USA 55,262-269.

5. Drory A., Mayak S., and Woodson W.R. (1993) Expression of ethylene biosynthetic pathway mRNAs is spatially regulated within carnation flower petals, J. Plant Physiol. 141, 663-667.

6. Eisinger, W. (1983) Regulation of pea internode expansion by ethylene, Annu. Rev. Plant Physiol. 34, 225-240.

7. Galston, A.W., Tuttle A.A., and Penny, P.l. (1964) A kinetic study of growth movements and photomorphogenesis in etiolated pea seedlings, Amer. J. Bot. 51, 853-858.

8. Goeschl, J.D, Rappaport, D.L., and Pratt, H.K. (1966) Ethylene as a factor regUlating the growth of pea epicotyls subjected to physical stress, Plant Physiol. 41, 877-884.

9. Heathcote, D.G. and Aston, T.1. (1970) The Physiology of Plant Nutation, I. Nutation and geotropic response, J. Exp. Bot. 21, 997-1002.

10. Holdsworth M.J., Bird C.R., Ray 1., Schuch W., and Grierson D. (1987) Structure and expression of an ethylene-related mRNA from tomato, Nuc. Acids. Res. 15, 731-739.

11. Hyodo, H., Hashimoto, C., Morozumi, S., Hu, W., and Tanaka, K. (1993) Characterization and induction of the activity of l-aminocylopropane-l-carboxylate oxidase in the wounded mesocarp tissue of Cucurbita TfUlXima, Plant Cell Physiol. 34, 667-671.

12. Jobnsson, A. (1979) Circunmutation, in W. Haupt and M.E. Feinleib (eds.), Encyclopedia of Plant Physiology - New Series, Vol. 7, Springer-Verlag, Berlin.

38

13. Kang, B.G., Yocum, C.S., Burg, S.P., and Ray, P.M. (1967) Ethylene and carbon dioxide, mediation of hypocotyl hook response, Science 156, 958-959.

14. Kende, H. (1993) Ethylene biosynthesis, Annu. Rev. Plant Physiol. Plant Mol. Bioi. 44, 283-307. 15. Kieber, J.J., Rothenburg, M., Roman, G., Feldman, K.A., and Ecker, J.R. (1993) erRl, a

negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases, Cell 72, 427-441.

16. Kim, W.T. and Yang, S.F. (1994) Structure and expression of cDNAs encoding 1-aminocyclo­propane-I-carboxylate oxidase homologs isolated from excised mung bean hypocotyls, Planta 194, 223-229.

17. Lehman, A., Black, R., and Ecker, J.R. (1996) HOOKLESSl, an ethylene responsive gene, is required for differential cell elongation in the Arabidopsis hypocotyl, Cell 85, 183-194.

18. Li, N., Parsons, B.L., Liu, D., and Mattoo, A. (1992) Accumulation of wound-inducible ACC synthase transcript in tomato fruit is inhibited by salicylic acid and polyamines, Plant Mol. Bioi. 18,477-487.

19. Lyon, C.1. (1970) Ethylene inhibition of auxin transport by gravity in leaves, Plant Physiol. 45, 644-646.

20. Morgan, P. W. and Gausman, H. W. (1966) Effects of ethylene on auxin transport, Plant Physiol. 41,45-52.

21. Nakagawa, N., Mori, H., Yamazaki, K., and Imaseki, H. (1991) Cloning of a complementary DNA for auxin-induced 1-aminocyclopropane-l-carboxylate synthase and differential expression of the gene by auxin and wounding, Plant Cell Physiol. 32, 1153-1163.

22. Olson, D.C., Oetiker, J.H., and Yang, S.F. (1995) Analysis of LE-ACS3 , a 1-aminocyclopropane­I-carboxylic acid synthase gene expressed during flooding in the roots of tomato plants, J. Bioi. Chern. 270, 14056-14061.

23. O'Neill, S.D., Nadeau, J.A., Zhang, X.S., Bui, A.Q., and Halevy, A.H. (1993) Interorgan regulation of ethylene biosynthetic genes by pollination, Plant CellS, 419-432.

24. Peck, S.c. (1995) Positive and negative feedback regulation of ethylene biosynthesis induced by indole-3-acetic acid, Ph.D. Dissertation, Michigan State University, East Lansing, Michigan.

25. Peck, S.c. and Kende, H. (1995) Sequential induction of the enzymes of ethylene biosynthesis by indole-3-acetic acid in etiolated peas, Plant Mol. Bioi. 28, 293-301. .

26. Riov, J., and Yang, S.F. (1982) Effects of exogenous ethylene on ethylene production in citrus leaf tissue, Plant Physiol. 70, 136-141.

27. Roman G., Lubarsky, B., Kieber, J.1., Rothenburg, M., and Ecker, J.R. (1985) Genetic analysis of ethylene signal transduction in Arabidopsis thaliana, five novel loci integrated into a stress response pathway, Genetics 139, 1393-1409.

28. Schierle, J. and Schwark, A. (1988) Asymmetric synthesis and concentrations of ethylene in the hypocotyl hook of Phaseolus VUlgaris, 1. Plant Physiol. 133, 325-331.

29. Schierle, J., Rohwer, F., and Bopp, M. (1989) Distribution of ethylene synthesis along the etiolated pea shoot and its regulation by ethylene, 1. Plant Physiol. 134, 331-337.

30. Schwark, A. and Schierle, J. (1992) Interaction of ethylene and auxin in the regulation of hook growth I, The role of auxin in different growing regions of the hypocotyl hook of Phaseolus VUlgaris, 1. Plant Physiol. 140, 562-570.

31. Silk, W.H. and Erickson, R.O. (1978) Kinematics of hypocotyl curvature, Amer. 1. Bot. 65, 310-319.

32. Spanu, P., Boller, T., and Kende, H. (1993) Differential accumulation of transcripts of 1-aminocyclopropane-l-carboxylate synthase genes in tomato plants infected with Phytophthora infestans and in elicitor-treated tomato cell suspension cells, 1. Plant Physiol. 141, 557-562.

33. Yang, S.F. and Hoffman, N.E. (1984) Ethylene biosynthesis and its regulation in higher plants, Annu. Rev. Plant Physiol. 35, 155-189.

34. Yoshi, H. and Imaseki, H. (1982) Regulation of auxin-induced ethylene biosynthesis. Repression of inductive formation of 1-aminocyclopropane-l-carboxylate synthase by ethylene, Plant & Cell Physiol. 23, 639-649.

THE ROLE OF JASMONATES IN ETHYLENE BIOSYNTHESIS

M. SANIEWSKI Research Institute of Pomology and FloriculturePomologiczna 18, 96-100 Skierniewice, Poland

1. Introduction

Jasmonic acid (JA), methyl jasmonate (JA-Me) and some other derivatives are widely distributed in the plant kingdom and play a key role as phytohormones, elicitors and signal transducers [19, 23, 41, 54]. Biosynthesis of (+)-7-iso-jasmonic acid [syn. (+)-2-epi-jasmonic acid») originates from linolenic acid and is easily transformed to (-)­jasmonic acid (Fig. 1). All of different plant responses to jasmonates, wheather applied externally or released internally, appear to be correlated with alterations in gene expression [39).

In this work the role of jasmonates in ethylene biosynthesis is presented. It seems that JA and JA-Me are among the factors controlling the biosynthesis of ethylene through stimulation of ACC synthase and ACC oxidase activities.

epijasmonic acid

spont. -~ o

~ jasmonic acid (JA) R = H

JA-Me, R = CH:s

Figure 1. Structures of two diastereomeric forms ofjasmonic acid [8].

2. Methyl Jasmonate and Ethylene Biosynthesis in Tomatoes

Ripening of tomatoes is associated with the increase of respiration, ethylene production, and synthesis or alteration in the activity of some enzymes, e.g. polygalacturonase. Methyl jasmonate greatly affected many processes in ripening tomatoes.

It has been found that methyl jasmonate inhibits lycopene and stimulates ~-carotene accumulation [11, 42), stimulates chlorophyll degradation [50), stimulates ethylene

39

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 39-45. © 1997 Kluwer Academic Publishers.

40

production, mostly through enhancement of the activity of the ACC oxidase [43, 45, 47] and inhibits polygalacturonase activity [50]. Aminooxyacetic acid (AOA), and inhibitor of ACC synthase, inhibited ethylene production stimulated by methyl jasmonate in normal tomatoes [44]. It is well known that fruits of the non-ripening, nor and rin tomato mutants lack the respiratory climacteric and ethylene evolution characteristics of normal tomato fruit. Methyl jasmonate evidently stimulated ethylene production and ACC oxidase activity in fruits of both mutants, more efficiently in nor than in rin [12].

3. Methyl Jasmonate and Ethylene Production in Apples

Methyl jasmonate stimulated ethylene production, ACC content and ACC oxidase activity in preclimacteric apples cvs. Jonathan, McIntosh and Idared [30, 46, 48, 49]. It inhibited ethylene production in climacteric and postclimacteric apples [30, 46, 48, 49]. Methyl jasmonate did not inhibit ACC oxidase activity in climacteric apples cv. McIntosh, but inhibited the enzyme activity in climacteric apples cv. Jonathan [31].

It is interesting that JA-Me greatly stimulated ACC oxidase activity in preclimacteric apples cvs. Barnack Beauty and Wagner stored in a normal atmosphere as well as in low 02 and CO2 atmospheres in comparison to apples stored under these conditions without additional treatment [26]. Olias et al. [32] found that an application of JA-Me vapors to Golden Delicious apples showed to have significantly stimulated ethylene formation.

Perez et al. [34] found that methyljasmonate vapors (8 ppm) for 4h at 25°C greatly promoted ~-carotene accumulation and chlorophyll degradation in Golden Delicious apple peel. The question is whether the JA-Me directly stimulates carotenoid biosynthesis or indirectly via ethylene stimulation which then affects carotenoid accumulation [34]. Recently, Fan et al. [14] found that in whole apples cv. Golden Delicious JA-Me promoted fruit ripening as indicated by increased ethylene synthesis, accelerated yellowing of surface color and increased loss of flesh firmness. In apple discs, JA-Me modulated ethylene synthesis in a fashion depending on developmental stage and concentration of applied JA-Me. At 100 J.1M or lower concentrations, JA-Me promoted ethylene synthesis while 1000 J.1M JA-Me inhibited ethylene synthesis. It appears JA-Me may have a role in the modulation of apple ripening.

4. The Effect of Methyl Jasmonate on Senescence and Ethylene Production in Flowers

In many flowers the final wilting stage is accompanied by an autocatalytic production of ethylene. Porat et al. [35] showed that methyl jasmonate, applied to Dendrobium and Petunia flowers as an aqueous solution through the cut stem or stigma or as a gas accelerated the senescence and increased ethylene production and ACC content in proportion to the dose of the compound. AOA, inhibitor of ACC synthase, and silver thiosulphate, an inhibitor of ethylene action, completely inhibited the effects caused by

41

JA-Me. Authors concluded that JA-Me enhanced flower senescence via the promotion of ACC and ethylene production.

Application of JA-Me also greatly enhanced Phalaenopsis flower senescence and promoted an increase in ethylene production [36]. Application of linoleic and linolenic acids to Phalaenopsis and Dendrobium flowers enhanced their senescence and promoted ethylene production [36], and this effect was specific to unsaturated fatty acids which serve as substrates for lipoxygenase action, and did not occur following similar treatments with saturated fatty acids.

5. The Effect of Jasmonates on Ethylene Production in Leaves, Cotyledons and other Organs

Ueda and Kato [56] isolated methyl jasmonate from Artemisia absinthium and first found the compound had a strong stimulatory effect on leaf senescence (chlorophyll degradation). Tulip leaf senescence induced by JA-Me did not affect the ethylene production, ACC oxidase activity and ACC content [37, 38], but stimulated ethylene evolution and ACC oxidase activity during gum induction in tulip stem [40, 51, 52]. Also Abeles et aJ. [1] showed that methyl jasmonate induced senescence of excised cucumber cotyledons, however, ethylene did not appear to have been involved in the action of JA-Me. In olive leaf discs methyl jasmonate has a stimulatory action on ethylene production and ACC synthase and oxidase activities [53]. Chou and Kao [9] found that JA-Me evidently stimulated ACC-dependent ethylene production in detached rice leaves. Methyl jasmonate induced tendril coiling of Bryonia dioica and stimulated ethylene production [57]. JA-Me did not stimulate ethylene evolution in seedlings of Chenopodium rubrum [3] and in hypocotyl of soybean seedlings [17].

TABLE 1. Enhancement of biosynthesis ofjasmonic acid as a result of wounding

Species Plant organ References

Bryonia dioica leaves [2] Avena sativa leaves [2] Lycopersicon esculentum leaves [33] Solanum tuberosum leaves [20] Solanum tuberosum tubers [24] Glycine max hypocotyl [10] Nicotiana sylvestris roots and shoots [5] Petunia hybrida corollas [55]

6. Mechanical Wounding in Relation to Jasmonates and Ethylene Biosynthesis

Mechanical wounding is one of the common factors which induce ethylene stress in

42

number of plants [59]. Wounding stimulates the synthesis of ACC synthase. The rise in ACC synthase activity was followed by increases in ACC content and in the rate of ethylene production. Recently, it was shown that mechanical wounding stimulates also ACC oxidase activity [13, 21, 58]. It is well known also that pathogen infection of different organs of plant species is accompanied by an enhancement in ethylene production [6]. In 1992, Czapski and Saniewski [12] suggested that stimulation of ethylene production by wounding and pathogen infection of different organs of various species may be caused by an increased content of endogenous methyl jasmonate or jasmonic acid which control biosynthesis of ethylene. Recently, it was found that jasmonic acid and methyl jasmonate accumulate rapidly and transiently after wounding (Table 1) or pathogen attack [39]. It is well also known thatjasmonic acid and related compounds occur in Botryodiplodia theobromae [4, 28, 29] and Gibberella fUjikuroi [27] and probably in other pathogens. The hypothesis concerning the role of jasmonic acid in control of ethylene biosynthesis in wounded tissue and pathogen infected tissue needs further studies.

1 2

0 0

H\6

! ... ~/ COOH

3

COOH

Figure 2. Structures ofjasmonic acid (1). coronatine (2) and coronafacic acid (3) [25]

7. Similarity of the Phytotoxin Coronatine Activity to Jasmonic Acid Regarding Ethylene Biosynthesis and other Physiological Processes

Coronatine was first isolated from cultures of Pseudomonas syringae pv. atropurpurea and is an amide of coronafacic acid and coronamic acid (I-amino-2-ethylcyclopropane­I-carboxylic acid). Coronatine and jasmonic acid have similar chemical structures (Fig. 2). Ferguson and Mitchell [15] reported that ethylene production increases in Phaseolus vulgaris leaf discs treated with coronatine. The rate of ethylene release from leaves of Nicotiana tabacum was proportional to the concentration of coronatine

43

applied to the leaf surface [22]. The maximum rate of ethylene production occurred 28 to 32 h after application of coronatine. Content of ACC greatly increased in the coronatine-treated tissue of N tabacum. ACC synthase activity increased in Phaseo/us aureus hypocotyls during a 6-h treatment with coronatine [22]. Kenyon and Turner [22] suggest that coronatine induces the synthesis of ethylene from methionine rather than from the breakdown of coronatine itself.

Similarities of the biological activities of coronatine and jasmonic acid were shown in different tests. For example both coronatine and JA-Me inhibited root growth, stimulated anthocyanin accumulation and increased the level of two proteins of approximately 31 and 29 kD in wild-type Arabidopsis [7, 16], induced potato tuberization, inhibited the growth of soybean callus, stimulated senescence of oat leaves [25]. Thus, coronatine acts as a stereospecific analog of jasmonate type signals [18].

8. References

1. Abeles, F.B., Hershberger, W.L., and Dunn, L.J. (1989) Honnonal regulation and intracellular localization of a 33-kD cationic peroxidase in excised cucumber cotyledons, Plant Physiol. 89, 664-668.

2. Albrecht, T., Kehlen, A, Stahl, K., Knofel, M.-D., Sembdner, G., and Weiler, E.W. (1993) Quantification of rapid, transient increases of jasmonic acid in wounded plants using a monoclonal antibody, Planta 191, 86-94.

3. Albrechtova, J.T.P. and Ulhnann, J. (1994) Methyl jasmonate inhibits growth and flowering in Chenopodium rubrum, Bioi. Plant. 36, 317-319.

4. Aldridge, D.C., Galt, S., Giles, D., and Turner, W.W. (1971) Metabolites of Lasiodiplodia theobromae, J. Chem. Soc. (C), 1623-1627.

5. Baldwin, LT., Schmeitz, FA, and Ohnmaiss, T.E. (1994) Wound-induced changes in root and shoot jasmonic acid pools correlate with induced nicotine synthesis in Nicotiana sylvestris Spegazzini and Comes, J. Chem. Ecol. 20, 2139-2157.

6. Barkai-Golan, R, Lavy-Meir, G., and Kopeliovitch, E. (1989) Stimulation of fruit ethylene production by wounding and by Botrytis cinerea and Geotrichum candidum infection in nonnal and non-ripening tomatoes, J.Phytopathol.125, 148-156.

7. Benedetti, C.E., Xie, D., and Turner, J.G. (1995) COil-Dependent expression of an Arabidopsis vegetative storage protein in flowers and siliques and in response to coronatine or methyl jasmonate, Plant Physiol. 109,567-572.

8. Boland, W., Hopke, J., Donath, J., NOske, J., and Bublitz, F. (1995) Jasmonic acid and coronatin induce odor production in plants, Angew. Chem. Int. Ed. Engl. 34, 1600-1602.

9. Chou, C.M. and Kao, C.H. (1992) Stimulation of l-aminocyc1opropane-l-carboxylic-acid dependent ethylene production in detached rice leaves by methyl jasmonate, Plant Sci. 83, 137-141.

10. Creelman, R.A, Tierney, A, and Mullet, J.E. (1992) Jasmonic acidimethyljasmonate accumulate in wounded soybean hypocotyls and modulate wound gene expression, Proc.Natl. Acad. Sci. USA 89, 4938-4941.

11. Czapski, J. and Saniewski, M. (1985) Effect of methyl jasmonate on carotenoids in tomato fruits, Gartenbauwiss. 50,35-37.

12. Czapski, J. and Saniewski, M. (1992) Stimulation of ethylene production and ethylene-forming enzyme activity in fruits of the non-ripening nor and rin tomato mutants by methyl jasmonate, J. Plant Physiol. 139, 265-268.

13. Dunlap, J.R and Robacker, K.M. (1994) Wound induced ethylene production from excised muskmelon fruittissue, 1. Hort. Sci. 69, 189-195.

14. Fan, x., Mattheis, J.P., and Fellman, J.K. (1995) Involvement of methyl jasmonate in fruit ripening, Plant Physiol.. (Suppl.) 108, 80, Abstr.

15. Ferguson, I.B. and Mitchell, RE. (1985) Stimulation of ethylene production in bean leaf discs by the

44

pseudomona phytotoxic coronatine, Plant Physiol. 77,969-973. 16. Feys, B.1F., Benedetti, C.E., Penfold, C.N., and Turner, J.G. (1994) Arabidopsis mutants elected for

resistance to the phytotoxin coronatine are male sterile, insensitive to methyl jasmonate, and resistant to a bacterial pathogen, Plant Cell 6, 751-759.

17. Franceschi, V.R. and Grimes, H.D. (1991) Induction of soybean vegetative storage proteins and anthocyanins by low-level atmospheric methyl jasmonate, Proc. Natl. Acad. Sci. USA 88, 6745-6749.

18. Greulich, F., Yoshihara., T., and Ichihara., A. (1995) Coronatine, a bacterial phytotoxin, acts as a stereospecific analog of jasmonate type signals in tomato cells and potato tissues, J. Plant Physiol. 147,359-366.

19. Hamberg, M. and Gardner, H.G. (1992) Oxylipin pathway to jasmonates: biochemistry and biological significance, Biochim. Biophys. Acta 1165, 1-18.

20. Harms, K., Atzorn, R., Brash, A., Kuhn, H., Wastemack, C., Willmitzer, L., and Pena-Cortes, H. (1995) Expression of a flax allene oxide synthase cDN A leads to increased endogenous jasmonic acid (JA) levels intransgenic potato plants but not to a corresponding activation of JA-responding genes, Plant Cell 7, 1645-1654.

21. Hyodo, H., Hashimoto, C., Morozumi, S., Hu, W., and Tanaka., K. (1993) Characterization and induction of the activity of l-aminocyclopropane-l-carboxylate oxidase in the wounded mesocarp tissue of Cucurbita maxima, Plant Cell Physiol. 34, 667-671.

22. Kenyon, lS. and Turner, 1G. (1992) The stimulation of ethylene synthesis in Nicotiana tabacum leaves by the phytotoxin coronatine, Plant Physiol. 100, 219-224.

23. Koda, Y. (1992) The role of jasmonic acid and related compounds in the regulation of plant development, Inter. Rev. Cytol. 135, 155-198.

24. Koda, Y. and Kikuta, Y. (1994) Wound-induced accumulation ofjasmonic acid in tissues of potato tubers,PlantCellPhysiol. 35, 751-756.

25. Koda, Y., Takahashi, K., Kikuta, Y., Greulich, F., Toshima., H. and Ichihara., A. (1996) Similarities of the biological activities of coronatine and coronafacic acid to those of jasmonic acid, Phytochem. 41, 93-96.

26. Lange, E., Nowacki, J., and Saniewski, M. (1993) The effect of methyl jasmonate on the ethylene producing system in preclimacteric apples stored in low O2 and high CO2 atmospheres, J. Fruit Ornam. Plant Res. 1,9-14.

27. Miersch, 0., Bruckner, B., Schmidt, J., and Sembdner, G. (1992) Cyclopentane fatty acids from Gibberella fojikuroi, Phytochem. 31, 3835-3837.

28. Miersch, 0., Preiss, A., Sembdner, G. , and Schreiber, K. (1987) (+)-Iso-jasmonic acid and related compounds from Botryodiplodia theobromae, Phytochem. 26, 1037-1039.

29. Miersch, 0., Schneider, G., and Sembdner, G. (1991) Hydroxylated jasmonic acid and related compounds from Botryodiplodia theobromae, Phytochem. 30,4049-4051.

30. Miszczak, A., Lange, E., Saniewski, M., and Czapski, J. (1995) The effect of methyl jasmonate on ethylene production and C02 evolution in Jonagold apples, Acta Agrobot. 48, 121-128.

31. Nowacki, 1, Saniewski, M., and Lange, E. (1990) The inhibitory effect of methyl jasmonate on ethylene-forming enzyme activity in apple cultivar Jonathan, Fruit Sci. Rep. 17, 179-186.

32. Olias, 1M., Sanz, L.C., and Perez, AG. (1991) lnfluencia deljasmonato de metilo en la maduracic;n post-cosecha de manzana. In: 1. Recasens., J. Grae1l and M. Vendrell (eds), EI etileno en la maduraci<;n y post recolecci", de frutos y hortalizas. Paper Kite, Lerida, Spain, pp. 60-67.

33. Pena-Cortes, H., Albrecht, T., Prat, S., Weiler, E.W., and Willmitzer, L. (1993) Aspirin prevents wound-induced gene expression in tomato leaves by blockingjasmonic acid biosynthesis, Planta 191, 123-128 ..

34. Perez, AG., Sanz, C., Richardson, D.G., and Olias, J.M. (1993) Methyl jasmonate vapor promotes p­carotene synthesis and chlorophyll degradation in Golden Delicious apple peel, J. Plant Growth Regul. 12, 163-167.

35. Porat, R., Borochov, A, and Halevy, AH. (1993) Enhancement of petunia and dendrobium flower senescence by jasmonic acid methyl ester via the promotion of ethylene production, Plant Growth Regul. 13 , 297-301.

36. Porat, R., Reiss, N., Atzorn, R., Halevy, AH., and Borochov, A (1995) Examination of the possible involvement of lipoxygenase and jasmonates in pollination-induced senescence of Phalaenopsis and Dendrobium orchid flowers, Physiol. Plant. 94, 205-210.

37. Puchalski, J., K1irn, P., Saniewski, M., and Nowacki, J. (1989) Studies of some physiological processes during tulip leaf senescence induced by methyl jasmonate, Acta H ortic. 251, 107-114.

45

38. Puchalski, J., Saniewski, M., and Klim, P. (1985) The effect of methyl jasmonate on tulip leaf senescence and peroxidase patterns, Acta Hortic. 167,247-257.

39. Reinbothe, s., Mollenhauer, B., and Reinbothe, S.C. (1994) J1Ps and RIPs: The regulation of plant gene expression by jasmonates in responses to environmental cues and pathogens, Plant Cell 6, 1197-1209.

40. Saniewski, M. (1989) Relationship between stimulatory effect ofmethyljasmonate on gum fonnation and ethylene production in tulip stem, Bull. Pol. Acad. Sci., BioL Sci. 37, 41-48.

41. Saniewski, M. (1995) Methyl jasmonate in relation to ethylene production and other physiological processes in selected horticultural crops. Acta Hortic. 394, 85-98.

42. Saniewski, M. and Czapski, J. (1983) The effect of methyl jasmonate on Iycopene and p-carotene accumulation in ripening red tomatoes, Experientia 39, 1373-1374.

43. Saniewski, M. and Czapski, J. (1985) Stimulatory effect ofmethyljasmonate on ethylene production in tomato fruits, Experientia 41, 257-257.

44. Saniewski, M. and Czapski, J. (1990) The effect of aminooxyacetic acid on ethylene production induced by methyl jasmonate in tomatoes, Bioi. Plant. 32, 218-222.

45. Saniewski, M., Czapski, J., and Nowacki J. (1987) Relationship between stimulatory effect of methyl jasmonate on ethylene production and l-aminocyclopropane-l-carboxylic acid content in tomatoes, Bioi. Plant. 29,17-21.

46. Saniewski, M., Czapski, J., Nowacki, J., and Lange, E. (1987) The effect of methyl jasmonate on ethylene and l-aminocyclopropane-l-carboxylic acid production in apple fruits, Bioi. Plant. 29, 199-203.

47. Saniewski, M., Nowacki, 1, and Czapski, 1 (1987) The effect of methyl jasmonate on ethylene productiall and ethylene-fonning enzyme activity in tomatoes, J. Plant Physiol. 129, 175-180.

48. Saniewski, M., Nowacki, J., Lange, E., and Czapski, 1 (1986) The effect of methyl jasmonate on ethylene and l-aminocyclopropane-l-carboxylic acid production in preclimacteric and postclimacteric Jonathan apples, Fruit Sci. Rep. 13, 193-200.

49. Saniewski, M., Nowacki, J., Lange, E., and Czapski, J. (1988) The effect of methyl jasmonate on anthocyanin accumulation. and ethylene-fonning enzyme activity in apples, Fruit Sci. Rep.lS, 97-102.

50. Saniewski, M., Urbanek, H., and Czapski, J. (1987) Effects of methyl jasmonate on ethylene production, chlorophyll degradation and polygalacturonase activity in tomatoes, J. Plant Physiol. 127,177-181.

51. Saniewski, M. and W~grzynowicz-Lesiak, E. (1994) Is ethylene responsible for gum fonnation induced by methyl jasmonate in tulip stem ?, J. Fruit Ornam. Plant Res. 2,79-90.

52. Saniewski, M. and W~grzynowicz-Lesiak, E. (1995) The role of ethylene in methyl jasmonate­induced gum fonnation in stem of tulips, Acta Hortic. 394, 305-313.

53. Sanz, L.C., Fernandez-Macule!, IC., Gomez, E., Vioque, 8., and OIias, 1M. (1993) Effect of methyl jasmonate on ethylene biosynthesis and stomatal closure in olive leaves, Phytochem. 33,285-289.

54. Sembdner, G. and Parthier, 8. (1993) The biochemistry and the physiological and molecular actions ofjasmonates, Annu. Rev. Plant Physiol. Plant Mol. BioL 44, 569-589.

55. Tamari, G., Borochov, A, Atzorn, R., and Weiss, D. (1995) Methyljasmonate induces pigmentation and f flavonoid gene expression in petunia corollas: A possible role in wound response, Physiol. Plant. 94, 45-50.

56. Ueda, 1 and Kato, J. (1980) Isolation and identification of a senescence-promoting substances from wormwood (Artemisia absinthium L.), Plant PhysioL 66, 246-249.

57. Weiler, E.W., Albrecht, T., Groth, 8., Xia, Z.-Q., Luxern, M., LiP H., Andert, L., and Spengler, P. (1993) Evidence for the involvement of jasmonates and their octadecanoid precursors in the tendril coiling response of Bryonia dioica, Phytochem. 32, 591-600.

58. W~grzynowicz-Lesiak, E. and Saniewski, M. (1991) The effect of mechanical wounding of different organs of Hip pea strum x hybr. hort. on ethylene-fonning enzyme activity, Bull. Pol. Acad. SCi., Bioi. Sci. 39, 373-377.

59. Yang, S.F. and Hoffinan, N.E. (1984) Ethylene biosynthesis and its regulation in higher plants, Annu. Rev. Plant Physiol. 3S, 155-189.

ETHYLENE BIOSYNTHESIS AND ITS REGULATION IN RIPENING "HAYWARD" KIWIFRUIT

E. SFAKIOTAKIS, M. D. ANTUNES, G. STAVROULAKIS, N. NIKLIS, P. VERVERIDIS and D. GERASOPOULOS Laboratory of Pomology, School of Agriculture, Aristotle University of Thessaloniki, 540 06 Thessaloniki, Greece

1. Introduction

Ethylene plays a crucial role in ripening of kiwifruit, and the elucidation of the controlling factors in ethylene biosynthesis is important in prolonging the storage life and keeping the fruit quality during the handling operations.

Kiwi was believed to be climacteric fruit since its ripening process was mediated by ethylene. Natural ripening showed the fruits to be of climacteric type but, although associated with a peak of ethylene production, the respiratory pattern was somewhat atypical [9]. Reid et al. [10] have suggested that kiwifruit should be classified as climacteric fruit because of the simultaneous increase in respiration rate and C2H4

production. After harvest the kiwifruit rarely produces C2H4 for a long time without exposure to

exogenous C2H4 [18]. Kiwifruit is very sensitive to exogenous C2H4• Very small amount of C2H4, in storage rooms induce excessive fruit softening [6, 3]. Autocatalysis of C2H4 productions plays a key role in ripening of kiwifruit. Many factors can initiate autocatalysis of C2H4 production in the harvested fruit and the control of these factors can be of significance important in prolonging the storage life and keeping the quality of fruit.

2. Internal ethylene concentration of kiwifruit attached to or detached from the vine

Unlike the apple fruit which shows an in increase of internal ethylene concentration (1EC) on the tree [11], kiwifruit attached to the tree showed no increase of C2H4

production during the maturation period as was evaluated by the changes of soluble solids content (SSC) (Fig. 1). The IEC of fruits attached to the tree at ambient orchard temperature was very low during the maturation period. Although the fruit left on the vine for almost 170 days after bloom had a 10.5% SSC (which is above the recommended stage for harvest) and 4.2 kg firmness, there was no remarkable increase of IEC above 0.1-0.5 Ill/I, which is used for apples to determine the optimum maturity stage for harvest [5]. These observations may suggest that kiwi vines produce a "ripening inhibitor" that is translocated to the attached fruits. It has been suggested with other fruits that this factor not only inhibited the accumulation of ACC but also

47

A. K. Kanellis et al. (eds.). Biology and Biotechnology of the Plant Hormone Ethylene. 47-56. © 1997 Kluwer Academic Publishers.

48

suppressed the development of ACC oxidase [17]. Therefore, the IEC in kiwifruit, cannot be used as a maturity index to determine the

harvest period. On the other hand this is an advantage for handling operations to have the harvested kiwifruit with low lEC and if the fruit is placed right after harvest in storage there is no considerable accumulation of C2H4 in the storage room. Kiwifruit can be delayed to be harvested to attain high SSC value and better quality attributes, without the risk of inducing autocatalysis of C2H4 production on the tree.

7

~6 ~ w z ~ g; 5 u.

~ ............ . . . . . .

11 ~ C

10 !z w I-

9 ~ (,)

en 8 g

...J a en 7 w

...J ID

0.023 0.067 0.058 0.086

6 :3 a en ~----~-r----~~--~r-~~5

7u

Figure 1. The effect of harvest (days after bloom) on changes of soluble solids content, firmness and internal ethylene concentration of fruit harvested in different stages. The arrows indicate the sampling days.

3. Climacteric of respiration and ethylene production

Fruit harvested in an early stage of maturity showed a clear climacteric peak of C2H4

production after 20 days shelf life at 20°C (Fig. 2A). The climacteric followed a typical pattern with almost a 20.000 fold increase of the rate of C2H4 production.

After harvest the respiration measurements showed moderate changes with a 2.5 fold increase of the rate of CO2 and this was closely associated with the increase of C2H4

production (Fig. 2A). Treating kiwifruit with C2H4 or propylene it is expected to induce the climacteric of

respiration and autocatalysis of ethylene production with a similar manner to what has been found in apples [12]. In our studies, kiwifruit of the same maturity stage exposed to 130 11111 propylene showed a climacteric peak of respiration without any delay (Fig. 2B) and an increase of the rate of C2H4 production with a typical climacteric with a 5 to 6 days delay (Fig. 2C). This suggests that the two systems of ~H4 production and respiration are different and that the effect of C2H4 or propylene on respiration may be induced by the activation of pre-existed enzymes whereas the induction of C2H4

production may require the de novo synthesis of new enzymes. Fruit of the same maturity stage exposed to propylene at lOoC showed an increase

of respiration (Fig. 2B) without increase in C2H4 production (Fig. 2C) regardless of the storage length at lOoC .Thus, kiwifruit belongs to that group of fruits which shows the respiratory rise before the increase in C2H4 production [4].

100 20"C (air)

• : . i : ,; ~ ! iii ReSPiration. _ •• ~ ~ .~ ..... W 40. f".;

20 " .. ,

Ethylene production

300

~ 2200 ~

~ ~100 Iii

o 2 4

10 15 DAYS

+propylene at 20·C

25

C

14

A 50

i §. ~

300

~ 20z

g a:

10 l§

3u

80 B

~ 1§ 40 15 + propylene at 100C z ......................... ., .............. . i 2 ... air at 2QoC 1S 'll.,~-;." ........... , ......... " ...... -..... .,-........ -.. ~ ....... _"':.~. r:': .. ::,.: ...... ~~ • ..., air at 100 e

°0~------------·8---10----12---1-4-DAYS

30 12.C D

E 25 Pr-> air '--...... "T~h Air+pr

~ ~r: IT , ...... ---. E 20 CIt.... Air->Pr .' Pr->8Ir ... . ~ ", 1 .• ,·- •••••••• ~ 151\Ir->Pr ........ ,

o M

@ ~ 5

~~---50-----1-00-----150~---2~00----~250 HOURS

49

Figure 2. Climacteric of respiration and ethylene production. A: Ethylene production and respiration of kiwifruit after harvest at 20"C ; B: Respiration of kiwifruit at 10° and 20"C treated in air ±13011111 propylene; C: Ethylene production of kiwifruit treated with 130 III II propylene at 20 ° and 10 °C; D: Time course study of respiration induced by propylene-treated (l301111l) kiwifruit: a) kept continuously in air, b) in air + propylene, c) treated with propylene at 20"C then after 24 h transferred to air, then after 48 transferred to air +propylene and finally after 48 h transferred to air.

By conducting a switching experiment, it was found that propylene induced an increase in respiration right after the exposure to propylene, while by removing the propylene, respiration decreased (Fig. 2D). Re-exposing the same fruit to propylene resulted in an increase of respiration. This response is very similar what previous workers reported with non climacteric fruits [4].

Our data support the hypothesis that the kiwifruit stored at room temperatures behaves as a typical climacteric fruit in reference to respiration and C2H4 production, while at low temperature it behaves like a non climacteric fruit with reference to the C2H4 production. This behaviour of the kiwifruit with respect to respiration and C2H4

production offers certain advantages in handling operations. After harvest by keeping the fruit at low temperatures there is no accumulation of C2H4 production in storage rooms, thus the postharvest life of the fruit is prolonged.

50

4. Regulation of ethylene production in kiwifruit

4.1. AUTOCATALYSIS OF ETHYLENE PRODUCTION

Several factors may induce autocatalysis of C2H4 production in the harvested kiwifruit such as exogenous C2H4 or its analogs (propylene) and stresses such as chilling, wounding and fungus infections. Other factors, such as temperature, oxygen and CO2

concentration, may also influence the autocatalytic C2H4 production in kiwifruit. The best method to study autocatalysis of C2H4 production is to apply propylene and

measure C2H4 production and ripening [7, 12]. Treating kiwifruit with 0, 10, 50, 100 and 500 11111 propylene for one week at 20°C stimulated C2H4 production and induced fruit ripening [13]. It was also observed that the propylene-induced ripening was initiated before the onset of C2H4 production. The threshold concentration of propylene for the induction of C2H4 production and initiation of ripening was higher than 10 11111. The concentration of 100 11111 of propylene was found to be the saturation dose for autocatalytic C2H4 production. Propylene applied to kiwifruit at the same concentration for 3 weeks at O°C induced ripening but did not stimulated C2H4 production [13].

4.2. THERMOREGULATION OF THE PROPYLENE INDUCED ETHYLENE PRODUCTION

Kiwifruit is a unique climacteric fruit which lacks the ability for autocatalysis of C2H4

production at low temperature. A study of inducing autocatalysis in different temperatures showed that kiwifruit is not able to produce C2H4 below the temperature range of 11° -14SC [14]. The rate limiting factor is rather the availability of ACC than the activity of ACC oxidase. Further studies showed that optimum rates of C2H4

production occur in the temperature range from 20° to 34°C (Fig. 3A) and this was correlated with accumulation of ACC and high ACC synthase and ACC oxidase activities (Fig. 3B) [1]. The inhibition of C2H4 production at low temperature (lO°C ) and the reduced accumulation of ACC was due to the reduced activity of ACC synthase.

400 + propylene (130 ~VI) .. l\ Ethylene ••••

!li 200

g tu 100

/ ........ .

. .:l ... l

... ... l

TEMPERATURE 'C

A 0.4 + propylene (130 ~VI) B

••••• ACC synthase

'\ 0.00!--~10----:2~0 -~30:---:"!!--~5G

TEMPERATURE 'C

w '" 5 <3 §

o ~

Fig ure 3. The effect of temperature on C;~ production and ACC accumulation (A), and activities of ACC synthase and ACC oxidase (B) of kiwifruit during storage in an atmosphere containing 130 J.!l!l propylene. (ACC synthase unit = nmoles ACC/grl2hrs).

Ethylene production was drastically reduced at temperatures above 38°C and this was

51

attributed rather to the reduced activity of ACC oxidase than to the reduced activity of ACC synthase [1].

4.2.1 Time course study of inhibition of ethylene production by low temperature Measurement of IEC in propylene-treated individual fruit at 0° or 20°C, showed that propylene (130 Illll) induced autocatalytic C2H4 production after a lag period of at least 75-80 h and reaches a peak of C2H4 production 150-160 h of exposure in fruit kept continuously at 20°C [15]. The control fruit at 20°C (without propylene) showed no C2H4 production. However, propylene in another fruit kept continuously at O°C was not able to induce autocatalysis of C2H4 production. Transferring the fruit from 20°C to O°C one day after the commencement of autocatalysis showed strong inhibition of C2H4

production with no delay. Transferring of propylene-treated fruit exposed for 100 h at O°C to 20°C induced autocatalysis of C2H4 production in 80 h and IEC followed the same pattern with fruit kept continuously at 20°C. The inhibition of C2H4 production, caused by low temperature (20° ~ O°C ) was not permanent and with a shift of the temperature again to 20°C the fruit resumed C2H4 production and IEC showed the same pattern with no lag period.

4.3. THE EFFECT OF CARBON DIOXIDE AND OXYGEN CONCENTRATION IN THE PROPYLENE INDUCED ETHYLENE PRODUCTION

High concentration of CO2 or low O2 inhibits autocatalysis of C2H4 production [16]. Increased concentration of CO2 (5-10%) drastically reduced C2H4 production in the propylene treated fruit at 20°e. Previous studies in other fruits showed that the conversion of ACC to C2H4 is O2 dependent [17]. Stavroulakis and Sfakiotakis [16] showed that in kiwifruit, O2 concentration plays a crucial role in the propylene-induced C2H4 production and the accumulation of ACC which is rather the limiting factor than the activity of ACC oxidase.

4.4. THE EFFECT OF CHILLING AND STORAGE IN THE INDUCTION OF ETHYLENE PRODUCTION

Chilling induces the production of C2H4 in kiwifruit upon returning the fruit to room temperature. Pretreated fruit with chilling at 0°,5°, 10°, and 15°C for 5 days showed no C2H4 production during the shelf life at 20°C. The same treatments for 12 days induced C2H4 production at 20°C and the proportional increase at the peak height of the C2H4

production was correlated with the activities of ACC synthase and ACC oxidase (Fig. 4A). Fruit chilled at O°C showed higher rates of C2H4 production and higher activities of ACC synthase and ACC oxidase during the shelf life at 20°C than fruit pretreated at higher temperatures (5°, 10°, 15°C).

Exposure of the fruit to chilling temperature (O°C ) during storage for 12, 18, 60, 120 and 180 days stimulates C2H4 production during the shelf life at 20°C (Fig. 4B). However, following the storage period there was a diminishing capacity for C2H4

production. Fruit exposed for 12 days at O°C produced C2H4 during shelf life with the highest rate (140 Illlkglhr) while the stored fruit for 180 days at O°C produced C2H4

during the shelf life at 20° C with reduced rate (5 Illlkglhr). This diminishing capacity

S2

of the chilled fruit to produce CZH4 with storage period was correlated with reduced activities of ACC synthase and ACC oxidase. In another study [2] exposure of the fruit to such chilling temperatures (O°C ) under controlled atmosphere and ultra low Oz storage (2.5%Oz+S%COz, 1 %Oz+ 1 %COz, 0.7%Oz+0.7%COz) for 60, 120 and 180 days, induced the fruit to produce CZH4 with reduced rate according to the storage period. The fruit removed from Ultra Low Oxygen (ULO) storage showed drastically reduced capacity to produce CZH4, mostly due to the low ACC oxidase activity rather than reduced ACC production or ACC synthase activity [2]. This explains the reduced capacity of kiwifruit to ripen during the shelf life at room temperatures after a prolonged storage in conventional or controlled atmosphere storage.

A Chilling ACS ACO B • 45.06 r ... o•c Gi.1!II 140 .......... days (unltsJmg) (nVglh)

125 5 Days

I - 12 .. 0 2.3 0.2

1100 10"~

_120 ........... 18 ..

f 48.48 1100

'" N ...... 80 .. 1.2 1.5

39.80 120 ..

j --- 180 .. 12 45.7 51.9 ~ 75 "'5°~ w z 80

~ : 26.92 ~ 18 18.7 32.5

.l 15'~ 80 j!: 50 j!: 80 10.1 15.5 w w 40

25 120 14.4 11.8 20

0 180 12.2 13.5

10 12

OO.12O'C

Figure 4. The effect of chilling in ethylene production and the activities of the enzymes Ace synthase and Ace oxidase of kiwifruit. A. Ethylene production at 20"C pretreated at (J' , SO, 19 ° and 15"C for 12 days. B. Ethylene production by kiwifruit at 20"C pretreated with low temperature (O°C) for 5, 12, 18, 60, 120 and 180 days. The table indicates the activities of Ace synthase (ACS) and Ace oxidase (ACO) measured at the end of the experiment. (ACC synthase Unit = pmoles of ACClmg protein/2hrs).

4.S. THE EFFECT OF WOUNDING IN THE INDUCTION OF ETHYLENE PRODUCTION AT LOW TEMPERATURES

Although autocatalysis of CZH4 production ceases at low temperatures, wounding the tissue induced CZH4 production at SoC. The increase in the CZH4 production was noticed after SO h and reached a peak at IS0-200 h, whereas the intact fruit showed no CZH4

e 0.4 A

~ ::1. Z 0.3 o ~ 5 0.2

~ Q.

w ifi 0.1 --' > i!: w o. 0

Wounded fruit + Pr

.......

300

HOURS

Figure 5. Effect of wounding (peeling) on C;~ production in air or air + propylene (130 11111) at5°C

53

production (Fig. 5). Wounding produces considerable amount of C2H4 in low temperatures. The peeled fruit can produce up to 0.35 Jlllkglh at 5°C which is enough to induce ripening of the fruit. Treating the fruit with propylene there was no differences in C2H4 production of the wounded or intact fruit. The wounded fruit incubated at such low temperature showed no fungus growth during the period of experiment.

4.6. THE ROLE OF BOTRYTIS CINEREA IN THE INDUCTION OF ETHYLENE BIOSYNTHESIS

While the kiwifruit does not produce C2H4 during the storage, infected fruit by Botrytis cinerea is producing considerable amount of C2H4 thus causing early softening of the healthy fruit in the storage rooms. Niklis et at. [8] showed that the infected fruits with Botrytis at 10°C produced considerable amount ofC2H4 55 days after inoculation, while the non-inoculated kiwifruit did not produce C2H4 and ACC. Fruit infected with Botrytis cinerea may produce up to 1.5 JlI/kglh C2H4 at O°C and up to 9 Jlllkg/h C2H4 at lOoC at a rate correlated with the infected area. Ethylene production in tissue plungers taken from the infected tissue was high and was associated with the front of fungus growth. Measurements of ACC showed accumulation of ACC at the front area of fungus in tissue plungers taken from infected fruit kept in lOoC. This finding gives evidence that the fungus is inducing C2H4 biosynthesis in the infected kiwifruit through the methionine pathway. Botrytis cinerea incubated in Potato Dextrose Agar (PDA) medium at 10°C produced negligible amounts of C2H4 20 days after inoculation in the growth medium [8]. It was noticed that the fungus is able to induce C2H4 production even at -ISC [8].

5. Discussion

Our results on the autocatalytic C2H4 production in kiwifruit are compatible with the concept that two systems of C2H4 production are involved in the ripening process of climacteric fruits. It has been suggested that System I is the low level of C2H4 present in fruits before the onset of ripening, while system II, represents the autocatalytic burst of C2H4 production which accompanies the ripening ([7], [12]). Furthennore, for System I, Yang et at. [17] suggested that in the preclimacteric fruit the resistance to ripening or the resistance to C2H4 action is mediated through a "ripening inhibitor".

Our results give support to the view that in preclimacteric kiwifruit the System I is responsible to keep low C2H4 production and the application of propylene or C2H4

changes the status of fruit from System I to System II resulting in the autocatalytic burst of C2H4 production. Evidence has been shown that in kiwifruit temperature plays an essential role in the autocatalytic C2H4 production probably suggesting that low temperature (lO°C ) strongly inhibits the conversion from System I to System II (Fig. 6). System I in kiwifruit seems to function in the temperature range from 1.5°C to 11 dc. The rate of C2H4 production can be influenced by several factors and mainly by temperature, wounding and Botrytis infection. The high quality stored fruit at low temperatures produce very small amount of C2H4 that is very low to induce ripening. However, the wounded or infected by Botrytis fruit produces considerable amount of C2H4• The wounded or infected by Botrytis fruit was found to produce C2H4 even a

54

little above the freezing point (-1.5°C). However, high rates of C2H4 production occur at high temperatures (11 °C ) and this points out the importance to store the fruit in the lowest safe temperature. The infected fruit with Botrytis cinerea may produce up to1.5 Illlkglh C2H4 at O°C and up to 9 Illlkg/h C2H4 at 1°C and the rate of C2H4

production is correlated with the infected area.

REGULAlION OF ETHYLENE BIOSYNTHESIS IN KIWIFRUIT

System II

System I

o a: ~ ~ d z o z

F.6CTORS

Propylene (Ethylene)

Temperature

Oxygen

Carbon dioxide

Stress (chHling)

Time of storage

Wounding

Botrytis infections

Fig/R 6. Factors affecting Czl\ biosynthesis within the temperature range from (J' to 40°C and the postulated change from System Ito System II by increasing temperature in the critical range (11 °-14 0C).

System II operates in the temperature range of 14.5° to 40°C and can be induced by propylene (or C2H4) or stresses (chilling, wounding). Propylene (and C2H4) stimulates the fruit to produce C2H4 through the methionine pathway by inducing ACC synthase and ACC oxidase. Several factors may influence the efficiency of System II to operate in the maximum rate such temperature, O2 and CO2 concentrations and duration of storage. Maximum rates of the propylene-induced C2H4 production occur in the temperature range from 20° to 35°C, whereas C2H4 production was drastically reduced at 38° C and above. Exposing the fruit to low temperatures for various period of storage time, C2H4 production was found to occur at room temperatures. The chilled fruit at O°C after 12 days gave the highest rate of C2H4 production. Removing the fruit from the storage, ethylene production occurs during the shelf life of the fruit exposed to the

55

market channels and this is useful to facilitate the softening of the fruit. During prolonged storage there is weathering of the System II and ethylene production was reduced with time of storage. In other studies it was shown that CA or ULO conditions may have strong effect in the efficacy of fruit to produce ethylene during the shelf life. By using ULO treatments during prolonged storage the fruit lost the capacity to produce C2H4•

6. References

1. Antunes, M. D. C. and Sfakiotakis, E. (1996a) Biochemical basis of thennoregulation of ethylene production and ripening of "Hayward" kiwifruit, Acta Hortic. (in press).

2. Antunes, M. D. C. and Sfakiotakis, E. (1996b) Ethylene production of "Hayward" kiwifruit after ultra low oxygen and controlled atmosphere, Acta Hortic. (in press).

3. Arpaia, M. L., Michell, F. G, Kader, A. A., and Mayer, G. (1986) Ethylene and temperature effects on softening and white inclusions of kiwifruit stored in air or controlled atmospheres, J. Amer. Soc. Hort. Sci. 111, 149-153.

4. Biale, J.B. and Yang, S.F. (1981) Respiration and ripening in fruits-retrospect and prospect, in J. Friend and M.J.C. Rhodes (eds.), Recent Advances in the Biochemistry of Fruits and Vegetables, Academic Press, London, pp. 1-39.

5. Dilley, r 1{., (1968) Prediction and verification of proper harvest dates for storage apples, 95th Ann. Rpt. Mich. State Hort. Soc., pp. 45-50.

6. McDonald, B. and Harman, J. E. (1982) Controlled-atmosphere storage of kiwifruit. I. Effect of fruit finnness and storage life, Scientia Hortic. 17, 113-123.

7. McMurchie, E. J., McGlasson, W. B., and Eaks, I. L. (1972) Treatment of fruit with propylene gives infonnation about the biogenesis of ethylene, Nature 237, 235-236.

8. Niklis, N., Sfakiotakis E., and Thanassoulopoulos, C.C.(l993) Ethylene biosynthesis in "Hayward" kiwifruit, in J. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, Kluwer Academic Publishers, Dordrecht, pp. 255-256.

9. Pratt, H. K. and Reid, M. S. (1974) Chinese gooseberry: Seasonal patterns in fruit growth and maturation, ripening, respiration and the role of ethylene, J. Sci. Food Agric. 2 5, 747-757.

10. Reid, M. S., Heatherbell, D. A., and Pratt, H. K. (1982) Seasonal patterns in chemical composition of the fruit of Actinidia chinensis, .J. Amer. Soc. Hort. Sci. 107, 316-319.

11. Sfakiotakis, E. M. and Dilley, D.R. (1973a) Internal ethylene concentrations in apple fruits attached or detached from the tree, J. Amer. Hort. Sci. 98, 501-503.

12. Sfakiotakis, E. M. and Dilley, D.R. (1973b) Induction of autocatalytic ethylene production in apple fruits by propylene in relation to maturity and oxygen, J. Amer. Soc. Hort. Sci. 98, 504-508.

13. Sfakiotakis, E., Stavroulakis, G., Ververides P., and Gerasopoulos, D. (1989) The control of autocatalytic ethylene production and ripening by propylene in "Hayward" kiwifruit, in H. Clijsters et al. (eds.), Biochemical and Physiological Aspects of Ethylene Production in Lower and Higher Plants, Kluwer Academic Publishers, Dordrecht, pp.173-l78.

14. Stavroulakis, G. and Sfakiotakis. E. M. (1993) Regulation by temperature of the propylene induced ethylene biosynthesis and ripening in "Hayward" kiwifruit. in J. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, Kluwer Academic Publishers, Dordrecht, pp. 142-143.

15. Stavroulakis, G. and Sfakiotakis, E. (1995) Time course study of thermolagulation in ethylene biosynthesis and ripening of "Hayward" kiwifruit induced by propylene, Acta Hortic. 397, 429-436.

16. Stavroulakis, G. and Sfakiotakis, E. M. (1996) Regulation of the propylene induced ripening and ethylene biosynthesis by oxygen concentration in "Hayward" kiwifruit, Adv.

56

Hort. Sci. (in press). 17. Yang, S. F., Liu, Y., and Lau, O. L. (1986) Regulation of ethylene biosynthesis in ripening

apple fruits, Acta Hortic. 179, 711-720. 18. Yano, M. and Hasegawa, Y. (1993) Ethylene production in harvested kiwifruit with special

references to ripe rot, J. Japan. Soc. Hort. Sci. 62, 443-449.

TWO-COMPONENT REGULATORS AND ETHYLENE SIGNAL TRANSDUCTION IN ARABIDOPSIS

D. JIRAGE AND C. CHANG Department of Plant Biology, University of Maryland, College Park, MD 20742 USA

1. Two-Component Regulators

The two-component regulator proteins are widely found in prokaryotes where they are involved in the perception and transduction of many different environmental signals [1]. Signal transduction by these regulators leads to numerous adaptive responses such as chemotaxis, phosphate regulation, nitrate regulation, host recognition for pathogen invasion, osmoregulation and stress-induced sporulation. Although two-component regulators are well-characterized in prokaryotes, they are only starting to be found in eukaryotes. In plants, several two-component genes have been cloned and all of them so far appear to be members of a family of ethylene receptors [2-5]. Here, we discuss the two-component features of the Arabidopsis ethylene signal transduction pathway, and examine parallels to a yeast two-component signaling pathway.

®

Signe' -.

~----------- ........ , .... -m ...... , 1,--... , ,

N~ I \ I-c N~C -.output '---1"11 .'gne'

Input histidine receiver output domain autoklnase domain domain

SENSOR RESPONSE REGULATOR

Figure 1. Mechanism of two-component signaling.

The basic units of the two-component system are a "sensor" and a "response regulator" [1] (Fig. 1). In bacteria, these components act together in various combinations to modulate cellular responses to environmental signals. The amino­terminal domain of the sensor is generally located in the periplasmic space of the bacterial cell and is involved in signal perception. The cytoplasmic portion of the sensor is an autophosphorylating histidine protein kinase that acts as a dimer.

57 A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 57-62. @ 1997 Kluwer Academic Publishers.

58

Histidine kinase activity of the sensor is modulated by perception of the signal. A cognate response regulator, which serves as the substrate for the histidine protein kinase, is phosphorylated at a conserved aspartate residue in its "receiver" domain. This phosphorylation controls the response regulator's "output" signaling activity, which could be enzyme activity, DNA binding or protein-protein interactions. [I] Interestingly, none of the ethylene receptors in plants appear to contain such an "output" domain.

The two-component proteins occur in a wide variety of modular arrangements and are readily identified through amino acid sequence homologies. The histidine kinase domain is a module of - 250 amino acids that displays five conserved sequence blocks; one of these is the H box containing the autophosphorylated histidine residue [1]. The receiver domain of the response regulator is characterized by conserved aspartate, lysine and hydrophobic residues in a module of -120 amino acids [I].

2. Ethylene Receptors in Arabidopsis

The isolation of ethylene-response mutants in Arabidopsis have defined a number of loci involved in ethylene signal transduction [6]. One of these, the ETRI locus, was identified through a dominant ethylene insensitive mutation based on the seedling "triple response" to ethylene [7]. In etiolated Arabidopsis seedlings, the triple response consists of inhibition of hypocotyl and root elongation, radial swelling of the hypocotyl and root and exaggeration of the apical hook. The etr 1 mutant lacks the triple response as well as all other measurable responses to ethylene during other stages of growth [7]. In addition, the mutant was shown to saturably bind only one-fifth the amount of ethylene of the wild-type, indicating that ETRI has a role in ethylene perception [7].

When the ETRI gene was cloned, it was found to encode a protein whose carboxyl­terminal region showed striking similarities to the two-component regulators. The predicted ETRl protein consists of 738 amino acids and is a "hybrid histidine kinase" i.e., has a built-in receiver domain as do several bacterial regulators [2]. The amino­terminal region ofETRI is predicted to contain three membrane-spanning regions, and the four known etr 1 mutant alleles each contain a missense mutation in one of these regions. The ETRI protein has been shown to dimerize and was localized to the plasma membrane [8]. The amino-terminal domain of ETRI was recently shown to saturably bind ethylene at physiologically relevant concentrations when expressed in yeast [9]. Furthermore, truncated forms of the protein lacking the amino-terminal domain disrupted the binding, as did the etr 1-1 missense mutation [9]. These experiments suggest that ETRI is indeed an ethylene receptor and that the ethylene binding site is located within the amino-terminal domain of the protein.

Homologs of the ETRI gene have been recently cloned in both Arabidopsis [3] and tomato [4, 5]. All of these homologs possess highly similar amino-terminal domains (which presumably bind ethylene) and a histidine protein kinase domain either with or without an attached receiver domain. The Arabidopsis ERS gene contains a putative histidine kinase domain, but lacks a receiver domain; thus ERS appears to be a typical sensor protein rather than a hybrid sensor [3]. There are no known mutations associated with the ERS gene, but by introducing the same missense mutation iuto ERS

59

as the known etr 1-4 mutation, the altered ERS gene was shown to confer dominant ethylene insensitivity in transformed plants [3]. Two other Arabidopsis genes, ETR2 and EIN4, appear to be hybrid histidine kinases similar to ETR1. For both Em2 and EIN4, there are known mutations that also result in dominant ethylene insensitivity (see Hua et al., in this volume). Based on these sequence similarities and the seemingly identical mutant phenotypes, ETR1, ERS, ETR2 and EIN4 are thought to have similar or overlapping roles in ethylene perception and signal transduction in Arabidopsis.

So far, the only activity that has been demonstrated for this receptor family is ethylene binding by the Eml protein. Based on its sequence similarities with two­component regulators, it seems likely that Eml transduces the ethylene signal through the phosphotransfer mechanisms known in bacterial regulators. In our laboratory, we have performed in vitro assays for both Eml histidine autokinase activity and phosphotransfer to the Eml receiver, but have not yet detected either of these activities [10].

3. The Downstream Raf-Like Kinase CTRI

In contrast to the dominant etr 1 mutants, which are ethylene insensitive, the recessive ctr1 mutants of Arabi do psis exhibit constitutive ethylene responses. [11] That is, ctrJ mutants are phenocopies of wild-type plants exposed to ethylene. The ctr J loss-of­function phenotype suggests that the CTR1 product is a negative regulator of ethylene responses. Double mutant analyses indicate that CTR1 acts at or downstream of the receptors ETR1, ERS, ETR2, and EIN4 [3, 11, 12; A. Bleecker, personal communication] .

The CTR1 gene was cloned and found to encode a protein kinase that is most similar to the Raf family of serine/threonine protein kinases [11]. The regions of similarity include the eleven subdomains that are characteristic of protein kinases. In addition, there is weak homology to Raf family members in the presumed regulatory amino-terminal domain of Cml [11]. In animals, Raf protein kinases phosphorylate MAPK (mitogen activated protein kinase) kinases in signaling cascades for cell differentiation and growth [l3]. These cascades are controlled not by bacterial-like two-component receptors, but by tyrosine protein kinase receptors and seven­transmembrane domain receptors [13].

Recently, our laboratory has obtained preliminary evidence that the putative histidine kinase domains of Eml and ERS can directly interact with the amino­terminal domain of the Cml protein kinase, both in a yeast expression system and in vitro [K. Clark, X. Wang and C. Chang, unpublished]. These interactions might somehow be involved in the regulation of Cml activity. Gain-of-function Raj mutants that can transform cells oncogenically have been found to contain deletions in the amino-terminal portion of the protein [l3]. This region of Raf is thought to exert an autoinhibitory effect on the carboxyl-terminal kinase activity. Phosphorylation of serine-rich regions in this domain result in in vivo regulation of Raf-l [l3]. Raf has also been shown to be activated by recruitment to the plasma membrane [14, 15], and by dimerization [16, 17]. By analogy, Cml activity might similarly be regulated by one or more of the upstream components of the ethylene-response pathway. Gain-of-

60

function mutations in proteins that regulate CTRl (such as the receptors) could conceivably lead to constitutive activation of CTRl; activation of this negative regulator would result in ethylene insensitivity.

4. The Yeast Osmolarity-Response Pathway

The unusual combination of two-component receptors and a putative MAP kinase cascade in the ethylene-response pathway has parallels with the osmolarity-response pathway in the yeast Saccharomyces cerevisiae (Fig. 2).

Arabidopsis Ethylene-Response Pathway Yeast Osmolarity-Response Pathway

+ YPD1 ~

+ SSK1~

+ CTR1 (MAPKKK) SSK2ISSK22 (MAP KKK)

+ "-? PBS2 (MAPKK)

+ + ? HOG1 (MAPK)

+ ? ?

+ + Ethylene Responses Osmolarity Responses

Figure 2. A comparison of the ethylene-response pathway in Arabidopsis and the osmolarity­response pathway in yeast.

61

The two-component system of this yeast pathway is composed of at least three different proteins: SLN1, YPD1 and SSKI. SLN1 is a 1220-amino acid hybrid histidine protein kinase with a novel extracellular amino-terminal domain [IS]. It is thought that this domain perceives osmolarity levels [19]. The small, 167-amino acid YPD 1 protein, which displays weak sequence similarity to histidine protein kinases, comprises a phospho-relay system between SLN1 and SSK1 [20]. SSK1 is a response regulator protein that regulates two MAPKKKs, called SSK2 and SSK22, which in turn activate a MAP kinase cascade that leads to osmolarity responses [19, 21]. There is currently no evidence that the presumed receptor SLN1 physically interacts with the MAPKKKs.

In the model for yeast osmolarity responses, histidine autophosphorylation of SLN1 occurs under normal osmolarity conditions, and is followed by transfer of the phosphate to the conserved aspartate in the SLN1 receiver domain [19, 20]. The phosphate is then transferred to a histidine residue in YPD1, which shuttles the histidine to the conserved aspartate of the SSK1 receiver domain [20]. Phosphorylated SSK1 is incapable of activating the high osmolarity glycerol (HOG) MAP kinase cascade [19]. At high osmolarity, there is no phosphorelay from SLN1 to SSK1, and the unphosphorylated SSKI activates a PBS2-HOG1 MAP kinase cascade via the activation of SSK2 or SSK22 which are MAPKKKs (MAP kinase kinase kinases) [19, 20]. The activated HOG pathway turns on the transcription of appropriate response genes [19].

The yeast osmolarity response also involves a distinct osmosensor called SHO 1 that controls the same MAP kinase pathway by means of its interaction with a proline-rich motif in the MAPKK PBS2 [21]. SHO 1 has no apparent relation to the two­component regulator family, and it apparently interacts with PBS2 through an SH3 domain [21]. At high osmolarity, SH01 is postulated to activate PBS2 either directly or indirectly.

5. Concluding Remarks

Elucidation of the ethylene signaling pathway is emerging as a new and exciting area in plant molecular biology. With the genes in hand, we are in a position to address a number of questions, such as what are the biochemical activities of the ethyl~ne receptors? What are the different roles of the receptors? How do the receptors transduce the signal to CTR1? Working hypotheses are implicated by the bacterial two-component system, the yeast osmolarity-response pathway, as well as the regulation ofRafprotein kinases in metazoans.

6. Acknowledgements

Our research is funded by NRl Competitive Grants Program I USDA grant 95-37304-221S, Maryland Agricultural Experiment Station grant PBIO-97-1S, and a University of Maryland General Research Board Award to C. Chang.

62

7. References

1. Parkinson, J.S. and Kofoid, E.C. (1992) Communication modules in bacterial signaling proteins, Annu. Rev. Genet. 26, 71-112.

2. Chang, C., Kwok, S.F., Bleecker, AB., and Meyerowitz, E.M. (1993) Arabidopsis ethylene response gene ETRI : similarity of product to two-component regulators, Science, 262, 539-544.

3. Hua, J., Chang, C., Sun, Q., and Meyerowitz, E.M. (1995) Ethylene insensitivity conferred by Arabidopsis ERS gene, Science 269, 1712-1714.

4. Wilkinson, J.Q., Lanahan, M.B., Yen, H.-C., Giovannoni, J.J., and Klee, H. (1995) An ethylene inducible component of signal transduction encoded by Never-ripe, Science 270, 1807-1809.

5. Zhou, D., Kalaitzis, P., Mattoo, AK. and Tucker, M.L. (1996) The mRNA for an ETR1 homologue in tomato is constitutively expressed in vegetative and reproductive tissues, Plant Mol. BioL 30, 1331-1338.

6. Ecker, J.R. (1995) The ethylene signal transduction pathway in plants, Science 268, 667-674. 7. Bleecker, AB., Estelle, MA, Somerville, C., and Kende, H. (1988) Insensitivity to ethylene conferred

by a dominant mutation inArabidopsis thaliana, Science 241,1086-1089. 8. Schaller, G.E., Ladd, AN., Lanahan, M.B., Spanbauer, J.M. and Bleecker, AB. (1995) The ethylene

response mediator ETRI from Arabidopsis forms a disulfide-linked dimer, J. Bioi. Chem. 270, 12526-12530.

9. Schaller, E.G. and Bleecker, AB. (1995) Ethylene binding sites generated in yeast expressing the Arabidopsis ETRI gene, Science 270, 1809-1811.

10. Chang C., Clark K., Wang X. and Stewart R (1997) "Two-component" ethylene signaling in Arabidopsis, in SEE Symposium Series, Company of Biologists, London, in press.

11. Kieber, lJ., Rothenberg, M., Roman, G., Feldmann, K.A ,and Ecker, J.R (1993) CTRI, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the raf family of protein kinases, Cell 72, 427-441.

12. Roman, G., Lubarsky, B., Kieber, J.J., Rothenberg, M. and Ecker, J.R (1995) Genetic analysis of ethylene signal transducti<n in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway, Genetics 139, 1393-1409.

13. Daum G., Eisenmann-Tappe, I., Fries, H-W., Troppmair, J. and Rapp, U.R (1994) The ins and outs ofRafkinases, Trends in Biochem Sci 19, 474-480.

14. Leevers, S.J., Paterson, H.F. and Marshal~ C.J. (1994) Requirement for Ras in Raf activation is overcome by targeting Rafto the plasma memrn .!lIe, Nature 369, 411-413.

15. Stokoe, D., Macdonald, S.G., Cadwallader, K., :::ymons, M. and Hancock, J.F. (1994) Activation of Raf as a result of recruitment to the plasma membrane. Science 264, 1463-1465.

16. Farrar, M. A, Alberola-I1a, J. and Perlmutter, R.M. (1996) Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization, Nature 383, 178-181.

17. Luo, Z., Tzivion, G., Belshaw, P.J., Vavvas, D., Marshall, M. and Avruch, J. (1996) Oligomerization activates c-Raf-1 through a Ras-dependent mechanism, Nature 383, 181-185.

18. Ota, I.M. and Varshavsky, A (1993) A yeast protein similar to bacterial two-component regulators, Science 262, 566-569.

19. Maeda, T., Wurgler-Murphy, S.M., and Saito, H. (1994) A two-component system that regulates an osmosensing MAP kinase cascade in yeast, Nature 369,242-245.

20. Posas, F., Wurgler-Murphy, S.M., Maeda, T., Witten, EA, Thai, T.C. and Saito, H. (1996) Yeast HOGl MAP kinase cascade is regulated by a multi-step phosphorelay mechanism in the SLN1-YPD1-SSK1 ''two-component'' osmosensor, Cell 86, 865-875.

21. Maeda, T., Mutsuhiro T. and Saito, H. (1995) Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor, Science 269, 554-558.

THE ETHYLENE BINDING SITE OF THE ETR1 PROTEIN

A. B. BLEECKER Botany Department, University of Wisconsin-Madison, Madison, Wisconsin, WI53706, USA

1. Introductiou

The simple olefin ethylene acts as a hormonal signal in plants. We feel comfortable saying these words because we know that low concentrations of applied ethylene alter plant development, and because endogenously produced ethylene induces these changes in ways that make biological sense to the observer. There is also some basis for considering that ethylene perception conforms to the classic receptor occupancy theory described by Michaelis-Menton kinetics (for a complete discussion see [7]). However, the very simplicity of the ethylene molecule raises questions as to how a proteinaceous receptor might interact with the signal with such a high apparent affinity and specificity. This problem was first addressed by Burg and Burg [5], who suggested that a transition metal at the receptor site could provide the appropriate mechanism for high affinity binding.

The test of Burg's interesting hypothesis has awaited the biochemical characterization of the ethylene receptor. The discovery by two groups in the late 1970's that saturable binding sites for ethylene were present in plant tissues provided a basis for pursuing this goal [10, 17]. However, difficulties in the purification of these binding sites [18] and questions about their physiological relevance have hampered this direct biochemical assault on the problem. With the cloning of the ETRI gene [6] and the demonstration that the product of the gene expressed in yeast is capable of binding ethylene [15], the biochemical characterization of this binding site is close at hand. The connection between the ETRI protein and ethylene physiology is provided by mutations in the ETRI gene that confer insensitivity to ethylene in plants [3]. This combination of binding activity and evidence of a physiological role provide us with the conviction to consider the ETRI protein as a bona fide receptor for ethylene.

In this review we examine some of the emerging biochemical properties of the ETRI receptor and consider the relationship between ETRI and the previously identified ethylene binding sites in plants. We will also consider some of the unresolved inconsistencies between the properties of the ETRI protein and the kinetics of ethylene responses in plants.

63

A. K. Kanellis et al. (eds.), Biology and Biotechnology o/the Plant Hormone Ethylene, 63-70. © 1997 Kluwer Academic Publishers.

64

2. Methodology for Studying Ethylene Binding

Of all known plant hormones, ethylene's property of being a gas that readily diffuses through air spaces, aqueous solutions and across membranes provides unique opportunities for studying binding sites. Given the lack of physiological compartmentation and the minor contribution of metabolic turnover of the signal, one can make reasonable assumptions about the relationship between the concentration of applied ethylene and internal concentration of the hormone when the two are in equilibrium. These properties formed the basis for the method first used to detect saturable binding sites for ethylene in plant tissues.

The discovery of ethylene binding sites resulted from studies of the partitioning of applied ['4C]ethylene into plant tissues [10, 17). Plant tissues equilibrated with 14C2~ can be transferred to a sealed chamber in which the ethylene diffusing from the tissues is absorbed by a solution of mercuric perchlorate and subsequently quantified by liquid scintillation counting [17). A portion of the ethylene recovered represents sample that was dissolved in the aqueous, lipid and air spaces of the tissues. This "background" [14C]ethylene will remain the same if excess [,2C]ethylene is included in the original equilibration, whereas [14C]ethylene will be displaced from specific binding sites by the addition of excess [12C]ethylene. In practice, parallel tissue samples are examined. For example, one sample is equilibrated with [14C]ethylene (0.1 m1Iliter) and one sample is equilibrated with [14C]ethylene (0.1 m1Iliter) plus [12C]ethylene (lOOOm1lliter). The difference in recovered ['4C]ethylene between the two samples is considered to be an estimate of the saturable binding sites for ethylene. Binding sites detected in this way must be high affinity given that O.lm1lliter ethylene in the gas phase is equivalent to about 0.5 nM in the aqueous phase at equilibrium.

There are several caveats to the type of analysis described above. The binding assay requires several hours to complete. Even if problems of compartmentation and metabolism are discounted or accounted for [8), it could be unrealistic to assume that the number of binding sites remains constant over the course of the experiment. In theory, any kinetic data derived from this rather crude type of analysis should be viewed with caution. Some concerns about the in vivo analysis can be addressed by examining binding sites in cell free systems. Surprisingly, at least one class of binding activity characterized in vivo appears to retain similar kinetic properties in membrane preparations, and even after extensive purification of the solublized binding activity (for review see [18)).

3. Properties of Ethylene Binding Sites from Plants

Using the in vivo assay, ethylene binding sites have been detected in a wide range of plant species (for reviews see [8, 18)). Some generalities can be made from the data. Direct measurements of dissociation rates indicate that at least two classes of activities exist in vivo: a fast dissociation class (half life < 30 min.), and a slow dissociation class (half life> 6 h). Measurements of Kd based on dose-binding curves generally range

65

from 0.1 to 5 nM for both classes of sites. The [14C]ethylene is released rapidly from the slow dissociation class if the sample is heated to 60°C. In cell free systems, the slow dissociation class of binding sites is retained, but the fast dissociation class is lost.

In the absence of a direct connection between the ethylene binding activities from plants and any physiological response, a circumstantial case for the relevance of these sites has been made. The effectiveness of a series of antagonists to inhibit ethylene responses is consistent with their abilities to competitively inhibit e4C]ethylene binding (see [8, 18]). It has been pointed out that the Kd's for binding « 1.0 mlIliter), are close to the ethylene concentrations required for half maximal response in a variety of bioassays. However, many ethylene responses operate over concentrations at which these binding components should be saturated (see [7] for further discussion). Attempts have been made to correlate numbers of binding sites with the degree of sensitivity for known responses without success; the most abundant source of binding sites, bean cotyledons, have no identifiable response to the hormone [18]. Finally, response times for growth responses to ethylene can be quite rapid « 1 h) and, thus, are consistent with the fast dissociation class of binding activities, but not the slow dissociation class (see [1]).

Despite the lack of a compelling case for their physiological relevance, the slow dissociation class of binding sites can be assayed in vitro. Consequently, attempts have been made to purify this activity from mung beans [18] and Phaseolis vulgaris [8]. In both cases the activity is initially associated with membranes but can be solubilized with detergent. Purifications ranging up to 600-fold have been achieved, but the specific characteristics of the purified proteins have not been established.

4. Ethylene Binding Activity of the ETRI Protein

The ETRJ gene from Arabidopsis was originally identified by mutations at the locus which render the plant insensitive to ethylene [3]. Based on the evidence that the mutations affected the whole range of ethylene responses and on in vivo binding studies which indicated that the mutant etr J -J showed only 20% of the ethylene binding activity observed in the wild type, we originally speculated that the ETRJ gene could code for an ethylene receptor. However, a definitive interpretation of those original observations was hampered by the fact that only dominant alleles at the ETRJ locus had been identified. The original binding studies were also criticized because the etrJ-J mutant produces more ethylene than wild type, causing possible interference with the binding assay [14]. This issues was addressed with other dominant mutants of Arabidopsis, and it was concluded that some loss of ethylene binding was independent of competition by endogenous ethylene [14].

The cloning of the ETRJ gene and the subsequent discovery that the gene was related to genes coding for large family of receptor-related proteins from bacteria [6], fueled continuing speculations that the ETRJ gene coded for an ethylene receptor. It became clear that the resolution to this question lay in the demonstration that the ETR1 protein could interact directly with ethylene. Consequently, we began

66

experimenting with the expression of the ETRl gene in heterologous systems. We were unable to express the full length ETRI protein in E. coli, but did succeed in expressing portions of the cytoplasmic domain as fusion proteins. These fusion proteins were used to generate polyclonal antibodies. Using these antibodies as reagents, we were able to show that transformation of yeast with the full length ETRl gene resulted in the expression of an immunodetectable protein with the characteristics expected of the full length ETRI protein [16]. For both the ETRI protein expressed in yeast and the native protein in plants, the antibodies detected a 78 kD polypeptide which ran on SDS PAGE as a dimer in the absence of reducing agent.

Yeast turned out to be a suitable system for investigating the ethylene-binding activity of the ETRI protein for several reasons. Yeast does not produce detectable amounts of ethylene so interference form endogenous ethylene in the binding assay would not be expected. We also determined that wild type yeast did not show any detectable ethylene-binding activity. To determine whether yeast expressing the ETRI protein showed binding activity, we developed a variation on the in vivo binding assay developed for ethylene in plant tissues [15]. Yeast cultures were grown to late-log phase and cells were harvested on nitrocellulose membranes. The filtrates collected on membranes were sealed in jars and equilibrated with 0.1 ml/liter [14C]ethylene alone, or in combination with excess [12C]ethylene. After four hours, membranes were removed, aired out for 5 minutes, and placed in fresh sealed chambers along with a mercuric perchlorate solution. The samples were heated to 60°C to release bound ethylene and let stand for 12 hours to allow the released ethylene to be absorbed by the mercuric perchlorate trap. The trapped [14C]ethylene was then quantified by liquid scintillation counting.

The demonstration that yeast expressing the full length ETRI protein contained up to 100-fold higher amounts of ethylene than non-transformed yeast provided the first direct evidence that the ETRI protein was acting as an ethylene receptor. Subsequent experiments indicated that the ethylene binding by the ETRI protein in yeast showed many similarities to the slow-dissociation class of binding sites previously characterized in plants. Several antagonists of ethylene action in planta [15] were effective at blocking the binding of ethylene in yeast. Dose-binding studies indicated a Kd for the ETRI binding site of 0.04 ml/liter (gas phase) and a half life of ethylene release of 12h [15]. Furthermore, the calculated number of binding sites present in yeast was about 100-fold greater than the number found in plants, a value consistent with the immunological data which indicated 100-fold greater amount of immunodetectable protein in yeast than in Arabidopsis on a per gram fresh-weight basis.

The ability to overexpress functional receptor in a heterologous background that is devoid of both endogenous ethylene and ethylene binding activity has obvious advantages. Altered forms of the ETRl gene can be assessed for affects on the binding activity within a matter of days. By expressing truncated forms of the gene, we were able to establish that only the first 165 amino acids of the protein were necessary and sufficient for ethylene binding. This N-terminal domain encompasses three hydrophobic stretches of amino acids that are likely to be membrane spanning

67

sequences [16]. This raises the possibility that the site of ethylene binding is within the membrane. We also established that the etrl-l mutation, representing the conversion of a cysteine in the second hydrophobic stretch, completely eliminated detectable ethylene binding in yeast [15]. This was true whether the conversion was to a tyrosine (etrl-l) or to an serine. This result is particularly intriguing because cysteine is one of a few residues that can act as a ligand for metals. We can speculate that the cysteine in question is a ligand for the metal that binds ethylene and the loss of binding activity when this residue is mutated results from a lack of ability to coordinate the metal.

We have now embarked on a systematic study involving site-directed mutagenesis of residues within the putative ethylene binding domain. The goal of this study is to establish which residues are necessary and sufficient for ethylene binding. Preliminary results indicate that amino acid changes which eliminate binding also confer insensitivity to ethylene in planta. However, we have identified at least one mutant change that affects ethylene sensitivity in plants, but does not affect ethylene binding in the yeast system. We speculate that such mutations may affect the transduction of the signal through the receptor without affecting binding. By using a combination of molecular modeling, in vitro mutagenesis, ethylene binding assays in yeast and physiological assessment of the effects of mutant changes in planta, we hope to define the mechanism of action of this hormone receptor.

5. Evidence for Involvement of a Transition Metal in Ethylene binding

We are currently working on the purification of the ETRI protein from yeast. As an initial step, we examined yeast-membrane preparations for the capacity to bind ethylene. Initial experiments indicated that only a small fraction of the binding detected in vivo it was recovered in extracts. However, addition of copper sulfate to the membrane preparations increased the detectable ethylene binding up to 20-fold (J. Spanbauer, G.E. Schaller and A.B Bleecker, unpublished). No binding was detected in wild type yeast under these conditions. We have tried several other transition metals and they are ineffective with the exception of silver which also promotes an increase in binding activity in isolated membranes (p. Rodriguez, A. Hahr, B. Binder, and A.B. Bleecker, unpublished). These preliminary studies are consistent with the involvement of a copper ion in the interaction of ethylene binding with the ETRI protein. Verification that copper is the active metal awaits the spectroscopic analysis of the purified ETRI protein from yeast. The result with silver is somewhat unexpected since silver inhibits ethylene responses in planta. It has been suggested that silver acts as an inhibitor by displacing the biologically active metal from the receptor binding site [2]. Our results are consistent with this idea, but indicate that the silver may bind ethylene but fail to transduce this binding into a biological response.

68

6. Modeling Signal Transduction by the ETRl Protein

The sequence similarity of the ETRI protein to the two component regulators from bacteria provide for a reasonable model of how the binding of ethylene may be transduced by the ETRI protein into an output signal. In bacteria, the 2 components are refereed to as the sensor and the response regulator. The sensor component transmits the signal through ligand-mediated modulation of protein kinase activity located in the C-terminal kinase domain [13]. In at least some cases, the N-terminal domain of the sensor is integrated in the membrane and is responsible for signal recognition [12]. Bacterial sensors exist as dimers [13]. Phosphotransfer occurs through interactions of the catalytic domain of one monomer and the target histidine residue of the other monomer. Transduction of the signal appears to involve ligand induced conformational changes that are propagated from the membrane associated domains to the kinase domains.

Given the evidence that the ETRI protein exists as a dimer [16], a reasonable model for ETRI function that conforms to the bacterial paradigm can be suggested. We postulate that ethylene interacts with a copper ion that is coordinated in the hydrophobic domain of the ETRI dimer. This interaction induces a conformational change in or between monomers which is propagated through the protein to the kinase domains. Conformational changes between the adjacent kinase domains causes changes in kinase activity and consequently in signal output from the receptor. The validity of this model awaits the measurement of kinase activity by the purified receptor in the presence and absence of ethylene. For a more complete discussion of this issue by the author see Bleecker and Schaller [4].

7. Some Unresolved Questions

While there can be little doubt that the ETRI protein serves a receptor function in Arabidopsis, it is also becoming clear that ETRI does not act as the only ethylene sensing molecule in the plant. Genetic evidence indicates that there is functional redundancy in ethylene perception. The identification of additional genes in Arabidopsis that are functionally and structurally related to ETRI may explain why only dominant mutations in these genes confer ethylene insensitivity [9, Hua et al., this volume). It also provides an explanation for the detection of some ethylene binding in etrl-l mutant plants even though the etrl-l mutation completely eliminated binding to the yeast-expressed protein [3]. Given the existence of functionally redundant isoforms of the receptor, mutant versions of one isoform can override not only the wild-type version of that isoform, but also all other isoforms that may be present. The possible mechanisms for dominant ethylene insensitivity are discussed elsewhere in this volume [Hua et al., this volume]. Briefly, alternative mechanisms depend on ethylene acting to either stimulate or inhibit signal output by the receptor (for further discussion by the author see [4]).

69

Given the similarities between ETRI and the slow-dissociation class of ethylene binding sites previously identified in plant tissues, it is important to consider whether the ethylene binding properties of the ETRI protein in yeast can account for the range of physiological responses observed in planta. The dissociation constant for yeast­expressed ETRI is very similar to the Kd's reported for both the fast- and slow­dissociation sites measured in plant tissues. In both cases, this dose-binding relationship must somehow be reconciled with a number of ethylene responses that operate at ethylene concentrations that are past the saturation point for binding to the measurable sites. Perhaps there are lower affinity sites in plants that are not detected with the crude [14]ethylene assay. If such lower affinity sites are provided by other members of the ETRI gene family, this may be revealed by overexpressing these other isoforms in yeast and examining the binding activities. Receptor isoforms that operate over different ethylene concentrations and feed into a common signal transduction pathway represented by CTRI and EIN2 could explain why so many forms of the receptor seem to be present.

The very slow rate of ethylene dissociation from the yeast-expressed ETRI receptor (I2h) must somehow be reconciled with the much faster recovery time for the return of many ethylene responses to pre-treatment rates once ethylene is withdrawn (e.g. 30 min.; see [I)). These fast recovery times are more consistent with the fast dissociation class of binding sites observed in planta. If the fast and slow dissociations classes represent different receptors, then other isoforms of the ETRI class of receptors could be responsible. Alternatively, the fast dissociation class could represent a completely separate class of receptors unrelated to ETRI. If true, we must explain how mutations in the ETRI receptor class can disrupt the function of this other class.

It is also possible that fast and slow dissociation binding sites observed in planta represent alternate forms of the same receptor. Perhaps there are additional factors in planta which interact with ETRI-like receptors and facilitate fast dissociation. Such factors could be lost when plant tissues are extracted, accounting for the loss of fast­dissociation sites under these conditions (see (18)). These additional factors may not be present in yeast, so only the slow dissociation class is observed with in vivo binding assays in recombinant yeast.

Finally, we should consider the possibility that the slow-dissociation class of receptors are responsible directly for all responses. This possibility can be reconciled with the rapid recovery times of many responses if mechanisms involving receptor sequestration or desensitization are operating in planta. Sequestration can involve selective removal of ligand-receptor complexes from downstream signal transduction components and is known to operate for mammalian receptor kinases [11]. Desensitization often involves covalent modification of ligand-bound receptors which reduces signal output [11]. Interestingly, the bacterial two-component receptor systems that are related to ETRI use a feedback system of receptor methylation to desensitize activated receptors [13 J.

70

8. Conclusion

The identification of ETRl as an ethylene-binding protein that functions in mediating ethylene responses has cracked open the black box of hormone-receptor function in plants. While this work highlights the power of the genetic approach to solving intractable problems, it should be recognized that this success story has been dependent on the convergence of long standing research efforts by many different scientists using physiological, biochemical and molecular approaches. The future of research on the mechanisms of ethylene perception and signal transduction will continue to depend on mutidisciplinaty efforts and on the continued spirit of open communication and cooperation that has characterized this field of research.

9. References

1. Abeles, F.B., Morgan, P.W., and Saltveit, Jr., M.E. (1992) Ethylene in Plant Biology, 2nd edition, Academic Press, San Diego.

2. Beyer Jr., E.M. (1977) A potent inhibitor of ethylene action in plants, Plant Physiol. 58, 268-271. 3. Bleecker, AB., Estelle, MA, Somerville, C., and Kende, H. (1988) insensitivity to ethylene conferred

by a dominant mutation inArabidopsis thaliana, Science 241, 1086-1089. 4. Bleecker, AB. and Schaller, G.E. (1996) The Mechanism of ethylene perception, Plant Physiol. 111,

653-660. 5. Burg, S.P. and Burg, EA (1967) Molecular requirements for the biological activity of ethylene, Plant

Physiol. 42, 144-152. 6. Chang, C., Kwok, S.F., Bleecker, AB., and Meyerowitz, E.M. (1993) Arabidopsis ethylene response

gene ETRI: Similarity of product to two-component regulators, Science 262, 539-544. 7. Chen, Q.G. and Bleecker, AB. (1995) Analysis of ethylene signal transduction kinetics associated

with seedling-growth responses and chitinase induction in wild-type and mutant Arabidopsis, Plant Physiol. 108, 597-607.

8. Hall, MA, Connern, C.P.K., Harpham, M.V.J., Ishizawa, K., Rovada-Hoyos, G., Raskin, I., Sanders, 1.0., Smith, AR., Turner, R., Wood, C.K. (1990) Ethylene receptors and action. in 1. Roberts, C. Kirk, and M. Venis (eds.), Hormone Perception and Signal Transduction in Animals and Plants, The Company of Biologists Limited, Cambridge.

9. Hua, J., Chang, C., Sun, Q., and Meyerowitz, E.M. (1995) Ethylene insensitivity conferred by Arabidopsis ERS gene, Science 269, 1712-1714.

10. Jerie, P.H., Shaari AR., and Hall, MA (1979) The compartmentation of ethylene in developing cotyledons of Ph as eo lis vulgariS, L. Planta 144,503-507.

11. Lauffenburger, DA and Linderman, J.J. (1993) Receptors, Oxford University Press, New York. 12. Milligan, D.L. and Koshland, Jr., D.E. (1991) lntrasubunit signal transduction by the aspartate

chemoreceptor, Science 254,1651-1654. 13. Parkinson, J.S. (1993) Signal transduction schemes of bacteria, Cell 73, 857-871. 14. Sanders, 1.0., Harpham, N.V.J., Raskin, I., Smith, AR., and Hall, MA (1991) Ethylene binding in

wild type and mutant Arabidopsis thaliana (L.) heynh, Annals of Botany 68,97-103. 15. Schaller, G.E. and Bleecker, AB. (1995) Ethylene-binding sites generated in yeast expressing the

Arabidopsis ETRI gene, Science 270, 1809-1811. 16. Schaller, G.E., Ladd, AN., Lanahan, M.B., Spanbauer, J.M., and Bleecker, AB. (1995) The ethylene

response mediator ETRI from Arabidopsis forms a disulfide-linked dimer, J. BioL Chern. 270, 12526-12530.

17. Sisler, E.C. (1979) Measurement of ethylene binding in plant tissue, Plant Physiol. 64, 538-542. 18. Sisler, E.C.(1991) Ethylene-binding components in plants, in AK. Matoo, and J.C. Suttle, (eds.), The

Plant Hormone Ethylene, CRC Press, Boca Raton, pp. 81-99.

THE ETHYLENE RECEPTOR GENE FAMILY IN ARABIDOPSIS

J. HUA, H. SAKAI, AND E. M. MEYEROWITZ Division of Biology, Caltech, Pasadena, CA 91125, USA

1. Introduction

A fundamental question of biology is how a cell senses its environment and responds to it. This process of perceiving and responding is critical to a unicellular organism for its survival in variable conditions. It is equally critical to a multicellular organism for its adaptation. as well as its coordinated development. In plants. some growth regulators have long been shown to act as signals to carry out this process. However. until recently little was known about how a plant cell perceives these signals and how the signals are transduced to cellular responses.

The past several years have seen rapid progress in understanding the signal transduction of the plant growth regulator ethylene. owing to the application of molecular genetics studies on the model plant Arabidopsis thaliana. Genetic screens have identified a dozen or so genes involved in the ethylene response pathway with the 'triple response' assay [1]. The triple response. an ethylene response of etiolated seedlings. consists of the exaggeration of the apical hook. the inhibition of hypocotyl and root elongation. and the radial expansion of the hypocotyl [2]. This simple assay makes it possible to screen thousands of seedlings on a petri dish. Both ethylene insensitive mutants and constitutive ethylene response mutants have been isolated using this screen. The genes involved have been ordered in a genetic pathway by double mutant analysis [1].

The biochemical framework of the signaling pathway has been revealed by the molecular cloning of two important players, coded by the ETR1 and CTR1 genes. The CTR1 gene encodes a serine-threonine protein kinase that is most closely related with manunalian Raf kinases [3]. It is a negative regulator of ethylene responses, as the loss-of-function ctr 1 mutants have constitutive ethylene response. The ETRl gene has been shown to code for an ethylene receptor [4, 5. 6]. The carboxyl-terminal domain of the deduced ETRI protein has homology to the bacterial 'two-component' regulators [5]. A large family of these regulators carry out adaptation responses in bacteria. The amino-terminal domain of the deduced ETRI protein is novel. and it is capable of ethylene binding when expressed in yeast [6]. All known etrl mutants have dominant ethylene insensitivity due to missense mutations in this domain [5]. Therefore. ethylene signaling is probably processed via a phosphorylation cascade. It consists of a receptor homologous to the bacterial 'two-component' regulators and a downstream negative regulator similar to the Raf kinases. Recently. a similar combination of components has been discovered in the osmolarity sensing pathway of yeast [7, 8].

71

A. K. Kanellis et aI. (eils.), Biology and Biotechnology of the Plant Hormone Ethylene, 71-76. © 1997 Kluwer Academic Publishers.

72

Although genetic and biochemical evidence shows that ETRI is an ethylene receptor, it remains an open question how ethylene regulates the activity of ETRI and how ETRI regulates ethylene responses. All available etr 1 mutants to date are dominant, and it is unclear whether they are gain-of-function alleles or loss-of-function alleles. It is therefore difficult to infer the wild-type activity of ETRI from the mutant phenotype. Several possibilities could account for the lack of recessive alleles of ETR1 from genetic screens. These recessive alleles might have a wild-type ethylene response phenotype due to functional redundancy. Alternatively, the ETR1 gene may be essential and etr 1 null mutants thus are lethal. This review on our current research will focus on the discovery of three additional putative ethylene receptors in Arabidopsis and the isolation of etr 1 loss-of-function alleles, which suggests a functional redundancy of ethylene perception at a certain level.

2. ERS

The ERS gene was cloned by its homology to the ETR1 gene [9, Fig 1]. The amino­terminal domain of the encoded ERS protein is 82% similar to that of ETRl. The three putative transmembrane regions where all etr 1 dominant mutations reside are conserved between ETRI and ERS. The carboxyl terminus of ERS is a putative histidine kinase domain with 78% similarity to that of ETRl. It has the five signature motifs of bacterial histidine kinases. The notable difference between ETRI and ERS is that ERS lacks the receiver domain present in ETRl. Nevertheless, the ERS gene is also involved in ethylene perception. A mutant ERS gene engineered in vitro to have the same mutation as etr 1-4 confers ethylene insensitivity when it is transformed into wild-type Arabidopsis. Like etr 1 mutants, the ethylene insensitivity of ers mutants appears to be pleiotropic, including the lack of triple response, lack of inhibition effects of ethylene on the growth of young seedlings, and the delay of leaf senescence when treated with ethylene. ERS does not seem to regulate an independent pathway from that of ETRl. Double mutant analysis showed that ctr 1 mutants are epistatic to ers mutants [9], and thus ETRI and ERS regulate a common downstream player eTRl.

3. ETR2 and EIN4

The identification of ERS as a putative ethylene receptor would seem to complete the picture of ethylene perception. Its potentially redundant function with ETR1 could explain the lack of recessive alleles of etr 1 in the genetic screen. However, ethylene perception in Arabidopsis is in fact more complex. Genetic screens have yielded additional ethylene resistant mutants. Two genes, ETR2 [10] and EIN4 [11], have been identified as dominant mutants in the 'triple response' assay. The ETR2 and E1N4 genes act upstream of CTR1, as shown by double mutant analysis. etr2 and ein4 mutants phenotypically resemble etr 1 and ers mutants. It is thus possible that ETR2 and EIN4 are also ethylene receptors.

The molecular cloning of the ETR2 and E1N4 genes has strengthened this possibility. The ETR2 gene was cloned originally during a chromosome walk to a floral morphology gene [12]. The deduced protein of 773 amino acids exhibits

73

sequence similarity to ETRI [Fig 1]. As it resides close to the genetic map position of the ETR2 locus on chromosome 3, this gene, as derived both from the wild type and from an etr2-1 mutant strain, was sequenced, and a missense mutation was discovered in the mutant strain. The identity of the cloned gene with ETR2 was confirmed by transformation. The amino-terminal portion of ETR2 exhibits a 70% similarity to that of ETRI. The carboxyl domain of ETR2 protein contains a putative histidine kinase domain and a receiver domain, with 57% and 66% similarity to those of ETRI respectively. However, not all of the canonical motifs of bacterial histidine kinases are present in ETR2. Most strikingly, the putative autophosphorylation site corresponding to HiS353 in ETRI and ERS is a glutamic acid instead of a histidine.

1 [ ETRI

ERS

[

ETR2

2 EIN4

amino-terminal putative receiver domain histidine kinase domain

domain

i·Wa2/~

82% 78%

(~:~,~>~. ~_·~<~c~~

67·70% 53·58% 66·70%

(""'t·;;.;,: ::';;.;.';': .. ~ . ..... ·"":···_···-'-...;.·:-·"""':.;')v'lm/~ 77% 69% 81%

( ..... ;.....;..;;;.= ____ .;.;;..: . ..;...J .. >f/'ZW/~

738aa

613aa

773aa

766 aa

Figure 1. Amino acid sequence similarities between ETRl, ERS, ETR2 and EIN4. Plain numbers indicate similarities of the corresponding domains within a subfamily, while numbers in italics indicate similarities between two subfamilies.

The EIN4 gene was cloned by cross-hybridization to ETR2 [Fig. 1]. This homolog of ETR2 maps on top of chromosome 3 where EIN4 resides. That this homolog is indeed the EIN4 gene is indicated by the finding of missense mutations in this gene in three ein4 mutant alleles. The deduced EIN4 protein of 766 amino acids is more closely related to ETR2 than it is to ETRI or ERS. It contains an amino-terminal domain with 77% similarity to that of ETR2. The histidine kinase domain and the receiver domain exhibit 69% and 81% similarity to those ofETR2, respectively. Like the similar regions of ETR2, these two domains are more diverged from the bacterial consensus sequences than the cognate domains ofETRI and ERS. However, a putative autophosphorylation histidine site is present in EIN4.

74

4. The family of Putative Ethylene Receptor Genes

In summary, four homologous genes (ETR1, ERS, ETR2 and EIN4) have been identified in Arabidopsis. They fall into two subfamilies based on sequence similarities. ETRI and ERS are in subfamily 1, while E1R2 and EIN4 are in subfamily 2 [Fig 1]. The carboxyl domains of the four proteins exhibit sequence similarity to the bacterial 'two-component' regulators. ETR1, E1R2 and EIN4 have both the putative histidine kinase domain and the receiver domain, whereas ERS only contains the histidine kinase domain. The most conserved region of the four proteins is the unique amino-terminal domain that has ethylene binding activity in ETRI. All dominant mutants of ETR1, ERS, ETR2, and EIN4 result from missense mutations in this domain of the respective genes.

That each member of this family is involved in ethylene perception is indicated by the ethylene insensitive phenotype of their dominant mutants. They probably all act as ethylene receptors that regulate ethylene responses positively or negatively [Fig. 2]. Then, what specific role might each member play? Firstly, these genes might represent redundant functions to ensure that ethylene perception occurs without failure. Secondly, each member might have different functions in ethylene perception. The difference could occur in their expression pattern or the regulation of their expression. Each could potentially have a different binding affinity to ethylene. Difference between them could also occur at the level of their output activity such as acting as either a kinase or a phosphatase upon ethylene binding. Lastly, some members might sense some molecules other than ethylene [13 ]. As all their dominant mutants have indistinguishable phenotypes except for some difference in severity of different alleles, it is therefore crucial to know the loss-of-function phenotypes of these genes to understand the function of each member, and thus the process of ethylene perception as mediated by these receptors.

a.

b.

ETRI ERS

C2H4 ... ETR2 -; CTRI -; responses

EIN4

ETRI ERS

C2H4 -; ETRl ... CTRI -; responses

EIN4

Figure 2. A family of putative ethylene receptors in Arabidopsis. They could act as positive regulators of ethylene response (a) or negative regulators of ethylene responses (b).

75

5. Loss-of-Function Alleles of ETRI

We plan to address the problem of the function of each member of this family of ethylene-related two-component receptors by isolating loss-of-function alleles of each of these genes. So far, we have obtained several such alleles of etrl. We hypothesized that etr 1 loss-of-function alleles probably would not have a dominant ethylene resistant phenotype because otherwise they should have been isolated from the original genetic screen. It should be possible to recover these alleles as intragenic suppressors of etr 1 dominant mutants if they are not lethal. Through EMS mutagenesis, we have isolated four intragenic suppressors of etr 1 ethylene resistant mutants. Molecular data indicate that these four etr 1 alleles are molecularly null.

These four alleles regain ethylene sensitivity as shown by their close to wild-type 'triple response'. This explains why loss-of-function or recessive alleles of ETRI were not obtained in the original screen for ethylene insensitive mutants. The fact that etr 1 null mutants still retain ethylene sensitivity in the triple response assay indicates that ethylene perception has functional redundancy at a certain level. Detailed analysis of these mutants is under way, to see whether these mutants have subtle ethylene response defects as they might if each receptor has unique functions.

We might be able to answer the question of whether ETRI is a positive or negative regulator of the ethylene response if there are any defects in these putative etr 1 null mutants. In the other scenario, where the other receptor(s) has completely redundant function with ETRl, will not be demonstrable from the phenotype of etr 1 null mutants. However, in principle, it is possible to isolate null mutants of etr2 and ein4 by similar means. By analyzing null mutants of several genes, we may eventually eliminate the activities of several partially redundant family members, and thus see a recessive, loss of function phenotype.

6. Concluding Remarks

Ethylene perception in Arabidopsis probably involves more than one receptor. The four putative receptors, ETRl, ERS, ETR2, and EIN4, encode homologous proteins and have similar dominant phenotypes. Having cloned four of these genes provides more than one way to manipulate ethylene perception in Arabidopsis. Further genetical and biochemical studies should allow the discovery of the role each member plays, and reveal how the ethylene signal is processed by the receptors.

7. Acknowledgements

The isolation of the ETR2 gene is a collaboration between our lab and the labs of Anthony Bleecker and Caren Chang. The isolation of the EIN4 gene is a collaboration between our lab and those of Joe Ecker and Anthony Bleecker. This research is funded by Department of Energy grant FG03-88ER13873.

76

8. References

1. Ecker, J.R. (1995) The ethylene signal transduction pathway in plants, Science 268, 667-675. 2. Guzman, P. and Ecker, 1. R. (1990) Exploiting the triple response of Arabidopsis to identifY ethylene­

related mutants, The Plant Cell 2, 513-523. 3. Kieber, 1.1., Rothenberg, M., Roman, G., Feldmann, K.A and Ecker, J.R. (1993) CTR1, a negative

regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases, Cell 72, 427-441

4. Bleecker, AB., Estelle, MA, Somerville, C., and Kende, H. (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana, Science 241, 1017-1132.

5. Chang, C., Kwok, S.F., Bleecker, AB., and Meyerowitz, E.M. (1993) Arabidopsis ethylene-response geneETRl: similarity of product to two-component regulators, Science 262, 539-544.

6. Schaller, G.E. and Bleecker, AB. (1995) High affinity binding sites for ethylene are generated in yeast expressing the Arabidopsis ETRl gene, Science 270, 1809-1811.

7. Maeda, T., Worgler-Purphy, S.M., and Saito. H. (1994) A two-component system that regulates an osmosensing MAP kinase cascade in yeast, Nature 369, 242-245.

8. Maeda, T., Takekawa, M., and Saito, H. (1995) Activation of yeast PBS2 MAPKK by MAPKKK or by binding of an SH3-containing osmosensor, Science 269,554-558.

9. Hua, 1., Chang, C., Sun. Q., and Meyerowitz, E.M. (1995) Ethyelen insensitivity conferred by Arabidopsis ERS gene, Science 269, 1712-1714.

10. Chen, G. and Bleecker, AB., personal communication. 11. Roman, G., Lubarsky, B., Kieber, J.J., Rothenberg, M., and Ecker, J.R. (1995) Genetic analysis of

ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway, Genetics 139,1393-1409.

12. Sakai, H., Medrano, L.J., and Meyerowitz. E.M. (1995) Role of Superman in maintaining Arabidopsis floral whole boundaries, Nature 378, 199-203.

13. Theologis, A (1996) Plant hormones: more than one way to detect ethylene, Current Biology 6,144-145.

ETHYLENE SIGNAL PERCEPTION AND TRANSDUCTION

AR SMITH1, AW. BERRyl, N.Y.J. HARPHAM\ RJ. HEMSLEY\ M.GHOLLAND1, I. MOSHKOy2, G. NOVlKOYA2 AND M.A HALLI 1 Institute of Biological Sciences, University of Wales, Aberystwyth, U.K 2Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia

1. Introduction

The use of radiolabelled displacement assays has allowed the identification of specific 14C-ethylene binding sites in all tissues so far studied [7, 22]. Yirtually all higher plants investigated contain at least two classes of ethylene-binding site; one of which fully associates and dissociates in about 2 h and a class of sites that takes up to 20 h to become fully saturated and 13 h for half dissociation to occur. Although there appears to be two kinetically distinct populations of binding site, both sites have similar and high affinities for ethylene and its physiologically active analogues [17, 18].

Developing cotyledons of Phaseolus vulgaris contain a higher concentration of ethylene-binding sites than other tissues and only display the slow dissociating component [7]. The affinity and kinetics of this binding site correlate well with those reported for the "ETRl" ethylene receptor gene from Arabidopsis thaliana expressed in yeast [19]. The binding site from Phaseolus which is an integral membrane protein has been purified and resolves into two bands of 26 and 28 kDa on semi-denaturing PAGE and the proteins appear to be single entities on 2-D gels.

Previous work has shown that ethylene promotes protein phosphorylation in membrane fractions from pea epicotyls and that such ethylene-induced phosphorylation can be inhibited by the ethylene antagonist 2,5-NBD [14]. Furthermore, studies on ethylene binding in pea have shown that the binding of ethylene is regulated by phosphorylation. Thus, under conditions which promote phosphorylation ethylene binding is inhibited, whereas the reverse is true under conditions which enhance dephosphorylation. Antibodies raised to the ethylene-binding protein from Phaseolus immunoprecipitate 32P-labelled proteins from membrane preparations from pea [2].

In the remainder of this article, recent progress on the biochemistry of post-receptor ethylene signal transduction processes in pea and Arabidopsis will be described.

77 A. K. Karrellis et al. (eds.), Biology and Biotechnology of the Plant Homwne Ethylene, 77-86. © 1997 Kluwer Academic Publishers.

78

2. Signal Transduction

2.1. PEA SEEDLINGS

The binding of GTP to specific proteins is known to be an important element in signal transduction pathways in animal systems [6]. In contrast, relatively little information is available on the nature and role of GTP binding in higher plant signal transduction although some evidence suggests that GTP-binding proteins are involved in the transduction of light signals [16, 24]) and that of auxin [25]. Experiments are described here which suggest that the transduction of ethylene responses involves the intervention of GTP-binding proteins.

Specific eSS]GTPyS binding and GTPase activity have been recorded in membrane extracts from pea epicotyl tips. Further investigation of Triton X-I00 solubilised membranes which had been pre-washed with 100 and 750mM KCI to remove extrinsic proteins indicated that a Ih treatment with 1 IJ.I r1 ethylene promotes specific GTP­binding (Table 1).

TABLE 1. The effect of in vivo ethylene treatment (1111 rl for Ih) on e5S]GTPyS (0.1 nM; 48.32TBq mmorl) binding and GTPase activity in Triton-solubilised membrane preparations from 5-d-old pea epicotyl tips

Treatment Specific GTP-binding activity Specific GTPase activity finol mg·! % of control nmol mg·! min· % of control

1

Control Ethylene

44.6± 1.1 55.4+1.1

100 124

11.9±O.8 12.2±1.2

100 102

Of the membrane fractions tested, the highest specific activity of GTP binding was in the Triton-solubilised fraction but this fraction contained the lowest level of GTPase activity and the activity was not influenced by ethylene treatment.

In order to confirm the effect of ethylene on GTP binding, another approach using a [a.- 32p]GTP affinity asSay was adopted. This assay relies on covalent binding of periodate-oxidised GTP to the amino group of lysine at the GTP-binding site and depends on the presence of an asparagine-Iysine-X-aspartic acid motif [10]. These conditions are met by few heterotrimeric G-proteins but by most small GTP-binding proteins [4].

The centre panel in Figure 1 shows pronounced specific GTP binding to a Triton­solubilised protein electrophoresing at 28 kDa. Binding in this region is dramatically increased in the presence of ethylene irrespective of whether ethylene was applied for 1 or 20h. Mouse monoclonal antibodies raised to a consensus sequence in small GTP­binding proteins, designated pan-ras, were used for western analysis of the gels (right hand panel). The antibodies cross-reacted with proteins on the gel in the region 20 -30 kDa and six bands with Mr between 22 and 28 kDa. Autoradiographs of the same gel show GTP binding only at ~ 28 kDa where again the promotOIY effect of ethylene

79

was observable. Immunoprecipitation was carried out and the results are shown in the left hand lane. Clearly, the pan-ras antibodies are capable of precipitating a protein of 28 kDa which can be radiolabelled with the [a_32P]GTP probe. The specificity of GTP binding is shown in Figure 2 where it is demonstrated that not only GTP but also GDP and GDP~S eliminate binding whereas CTP and ATP do not.

kOa 1 2 3 4 5 6

94 94 67 67 43 43

30 • • ., • 30

20.1 20.1

14.4 14.4

Figure 1. The effect of ethylene on GTP binding in pea membrane extracts solubilised in Triton X-IOO (lanes 2-5). Lanes 3 and 5; autoradiograph showing the promotion of GTP binding in the presence of ethylene, lanes 2 and 4 representing untreated controls. Lane 6; immunoblot of proteins probed with anti-pan-ras. Lane I; anti-pan-ras immunoprecipitated protein probed with [Cl.- 32p]GTP and electrophoresed.

In addition to the size of this GTP-binding protein, the other characteristics summarised in Table 2 suggest that the protein(s) described here shares properties similar to those of small GTP-binding proteins of the Ras superfamily.

TABLE 2. Characteristics ofGTP binding to membrane protein(s) in pea epicotyls

Ethylene promotes GTP binding to a Triton-solubilised membrane protein Molecular weight of binding protein - 28kDa No activation by Mastoparan which activates heterotrimeric G-proteins Activated by Mg2+ only at fJ.M concentrations Activated by 5fJ.M A1F4' lmmunoprecipitated by anti-pan-ras High and appropriate binding affmity: KD 3.17 x 1O·8M

80

The low GTPase activity associated with the Triton-solubilised fraction most enriched with the small GTP-binding protein activity is consistent with observations that such proteins exhibit low GTPase activity except in the presence of GTPase­activating proteins (GAP) [II).

Previous work has shown that treatment with ethylene affects phosphorylation of a low molecular weight membrane-associated protein in a time-dependent manner [2]. Initially, dephosphorylation of this band occurs in the presence of ethylene but after incubation longer than 30min with radiolabelled ATP, phosphorylation is promoted. This band of Mr 18 kDa cross-reacts with antibodies raised to nucleoside diphosphate kinase (gift from Dr. Paul Millner, Biochemistry Dept., University of Leeds, UK.). Subsequently, the capacity to form radioactive GTP in the presence of unlabelled GDP and radioactive ATP has been shown to occur in solubilised membrane extracts (Figure 3).

c.n C'Q

0. 0. 0. 0. 0. I- 0 0 I- ~ 1mM C " " " u

94- 0/ ..- $" ~, ~ ''* .. ~'" 67-43-

30-

20.1-

14.4-

Figure 2. Autoradiograph illustrating the specificity ofGTP binding for guanine nucleotides.

2.2. ARABIDOPSIS THALIANA

A series of Arabidopsis mutants (designated eti) has been characterised in terms of responsiveness to ethylene [8). One mutant, eti5, proved to be insensitive to ethylene

81

concentrations up to 10,000 f.l.1 rl in seedling triple response screens. The mutation is also reflected in adult plant responses to ethylene. For example, ethylene promotes chlorophyll loss in wild type leaf tissue but this effect is not apparent in eti5 (Table 3).

,.1" ....

"" ", •••• • 11> .......

..O~ -. ... - ... - ... - ...

tt"''' i 1 ,tit : - ... ~ ~ "'-to 10 '0 T ,...;" t 0 ~ 2.0 '\0

•

Figure 3. Autoradiograph of a TLC plate showing the presence of radiolabelled GTP when pea membrane extracts were incubated in the presence of unlabelled GTP and [1'- 32p]ATP.

Compared to wild type, leaf ontogeny in terms of chlorophyll content is delayed by about a week in eti5. However, the data presented in Table 3 are from leaf tissues of the same chronological age. Both wild type and eti5 leaf material are responsive to the cytokinin, benzyladenine, in the delay of chlorophyll loss.

TABLE 3. The effect of ethylene (1 ~l rt) and BA (10.6 M) on total chlorophyll content of detached leaves of wild type and eti5 Arabidopsis incubated in the dark for 72h.

Treatment Control BA Ethylene Ethylene + BA

Chlorophyll (a + b), f1g g.t F.W. wild type eti 5 68.S ± 16.4 171.0 ± 9.8 173.7 ± IS.4 30.9±4.2 143.6 ± IS.8

267.9± 12.9 191.6 ± 6.1 242.3 ± 1l.S

The measurement of ethylene binding in wild type and eti5 has been complicated by the high rates of endogenous ethylene production; especially so in eti5 where ethylene production is released from autoinhibition [17]. Use of inhibitors of ethylene biosynthesis has allowed measurements of ethylene binding to be undertaken and wild type Arabidopsis shows a pattern of fast- and slow-associating sites similar to that found in pea. The slow associating site is present in eti5 in amounts comparable to wild type but the fast associating site is reduced by about 60%.

82

Allowing the effect of ethylene on protein phosphorylation demonstrated in pea seedling extracts, similar experiments were undertaken in leaf extracts from mutant and wild type Arabidopsis. Protein phosphorylation of resuspended membrane preparations from leaves of wild type Arabidopsis is upregulated in response to ethylene treatment (Figure 4). This upregulation is anatagonised when BA was added to the extracts and the phosphorylation profile obtained is similar to that of BA alone. In eti5 extracts, ethylene treatment had little effect on the phosphorylation profile (data not shown) but the addition of BA resulted in a dramatic upregulation of phosphorylation. In addition, the constitutive phosphorylation expressed in eti5 is higher than in wild type.

Eth 1 ",1.1" hot SA S",M

43

30 -

20.1-

14.4-

+ + + +

WT

+

eti 5

Figure 4, The effect of ethylene and BA on in vi1ro phosphorylation of membrane proteins from leaves of wild type and etiS Arabidopsis.

In order to determine whether, at least in part, the effects of ethylene and BA on phosphorylation may be mediated by GTP-binding proteins, the affinity probe was used to measure GTP binding in Triton-solubilised membrane extracts from leaves of wild type and eti5 (Figure 5). As is the case with pea extracts, ethylene promoted GTP binding to protein(s) ofMr -28 kDa in wild type extracts. This promotion is marginal in the presence of BA alone and is decreased when combined application of hormones

83

is made. Contrasting with this, is the lack of effect of ethylene and BA treatment on GTP binding in eti5. The GTP-binding protein(s) at -28 kDa in Arabidopsis membranes immunoprecipitates with anti-pan-ras. It is of interest that the effect of the two hormones on protein phosphorylation and GTP binding correlates with the effects on leaf senescence.

Wild type 130kp etiS 130kp 1mMGTP_+_+_+_+ _+_+_+_+

n-30--..

20.1-,

14.4-

• " • - - .. SA -++--++--++- ++ Eth - - -- -+ + + + - - - -+ + + +

Figure 5. The effect of ethylene and BA on GTP binding in Triton-solubilised membrane pellets from wild type and eti5 leaf extracts of Arabidopsis.

In vitro phosphorylation studies in peas and Arabidopsis have revealed the presence of a common phosphorylated low molecular weight membrane associated protein. As in peas, immunological evidence suggests that this protein in Arabidopsis may be a nucleoside diphosphate kinase and using autoradiography of TLC plates of extracts incubated with GDP and radiolabelled ATP, the presence of active nucleoside diphosphate kinases has been detected.

3. Discussion

The results obtained so far indicate that ethylene responses in pea and in Arabidopsis may be mediated through protein phosphorylation cascades and GTP binding to small GTP-binding proteins. From I-D gels, it is not possible to determine whether more than one small GTP-binding protein is involved in peas but inArabidopsis at least two are activated (Figure 5) and four such proteins have been detected in cotyledon extracts from Phaseolus vulgaris. A link between protein phosphorylation and GTP binding has been established in pea extracts where it is clear that the inhibitor of GTP binding, GDP~S, inhibits ethylene-induced phosphorylation (Figure 6).

84

Although the functionality of the activation by ethylene remains unproven, the work presented here strongly suggests that small GTP-binding proteins are involved in the transduction of the ethylene signal which is consistent with work proposing that GTP-binding proteins are involved in the control of protein kinase activity [11, 12, 15].

Mutants of Arabidopsis have proved of great value in the elucidation of the ethylene signal transduction pathway and Figure 7 presents a model which integrates evidence derived from genetic and biochemical studies. In support of this, biochemical evidence indicates that the phosphorylation status of the receptor affects the capacity to bind ethylene and that ethylene modulates GTP binding to proteins with characteristics similar to those of Ras. The fact that the ethylene-modulated small GTP-binding protein(s) in peas and in Arabidopsis are present in Triton-solubilised membrane fractions is consistent with reports that such proteins are hydrophobic and tightly associated with the inner side of membranes [1].

GDPpS 43

30

20.1

14.4

Control +

Ethylene +

Figure 6. The effect of the inhibitor ofGTP binding, GDPpS, on ethylene-promoted phosphorylation.

Kieber et al. [9] have demonstrated that the protein encoded by the CTRl gene shows homology with Raf type protein kinases which, in animal systems, are known to be regulated by small GTP-binding proteins termed Ras [5, 23]. To what extent other

85

response regulators are involved is unknown, but reports have been made of the importance of guanine nucleotide releasing factors (Sos) and adaptor proteins such as Grb2 in the regulation of the formation of GTP-bound Ras [3, 21].

Deductions from genetic studies in Arabidopsis and from animal cell systems [13] suggest that complexes containing MAPKK activity and Raf-I protein are dependent upon the activity of Ras. Clearly, ethylene influences phosphorylation cascades and recent experiments in our laboratory show that ethylene treatment can promote MAPK activity in supernatants from 130kg centrifugations (unpublished). Determination of the role of kinases and phosphatases in ethylene signal transduction coupled with those of Ras- and Raf-type proteins will be crucial to our understanding of ethylene signalling mechanisms.

EIN!-! }ETRl Histidine

EIN4 + kinase , ~

Response Regulator

+ CTRl MAPKKK

+ + ? MAPKK

+ MAPK

GAP,GDI

Transcription ? EIN3,5,6,7 activators

Figure 7. Model of an ethylene signal transduction pathway based on integration of biochemical and genetic evidence.

4. Acknowledgements

We would like to acknowlegde the support of the BBSRC, INTAS, Royal Society and the EU BRIDGE and BIOTECH Programmes.

5. References

1. Barbacid, M. (1987) ras Genes, Annu. Rev. Biochem. 56,779-827.

86

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21. 22.

23.

24.

25.

Berry, AW., Cowan, D.S.C., Harpham, N.V.1., Hemsley, R.J., Novikova, G.V., Smith, AR., and Hall, MA (1996) Studies on the possible role of protein phosphorylation in the transduction of the ethylene signal, Plant Growth ReguL18, 135-141. Boguski, M.S. and McConnick, F.M. (1993) Proteins regulating Ras and its relatives, Nature 366, 643-654. Bourne, H.R., Sanders, DA, and McConnick, F.M. (1991) The GTPase superfamily: conserved structure and molecular mechanism, Nature 349, 117-127. Daum, G., Eisenmann-Tappe, I., Freiz, H-W., Troppmair, 1., and Rapp, U.R. (1994) The ins and outs ofRafkinases, TIBS 19, 474-480. Gilman, AG. (1987) G-proteins: transducers of receptor-generated signals, Annu. Rev. Biochem. 56, 615-649. Ifarpham, N.V.J., Berry, A W., Holland, M.G., Moshkov, I., Smith, AR., and Hall, MA (1996) Ethylene binding sites in higher plants, Plant Growth ReguL 18, 71-77. Harpham, N.V.1., Berry, A W., Knee, E.M., Roveda-Hoyos, G., Raskin, I., Sanders, 1.0., Smith, AR., Wood, C.K., and Hall, MA (1991) The effect of ethylene on the growth and development of wild-type and mutant Arabidopsis thaliana (L.) Heynh,Ann. Bot. 68, 56-61. Kieber, J.1., Rothenberg, M., Roman, G., Feldmann, K, and Ecker, J.R. (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raffamily of protein kinases, Cell 72, 427-441. Low, A, Faulhammer, H.G., and Sprinze, M. (1992) Affinity labelling of GTP-binding proteins in cellular extracts, FEES Lett. 303, 64-68. McConnick, M. (1989) ras GTPase activating protein: signal transmitter and signalterminator, Cell 56,5-8. Millner, PA (1987) Are guanine nucleotide-binding proteins involved in regulation of thylakoid protein kinase activity, FEES Lett. 226, 155-160. Moodie, SA, Willumsen, B.W., Weber, M.J., and Wolfinan, A (1993) ComplexesofRas.GTP with Raf-1 and Mitogen-Activated Protein Kinase Kinase, Science 260,1658-1661. Novikova, G.V., Moshkov, I.E., Smith, AR., and Hall, MA (1993) Ethylene and phosphorylation of pea epicotyl proteins, in J.C. Pech, A Latche, and C. Balague (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, K1uwer Academic Publishers, Dordrecht, pp.371-372. Porat, R., Borochov, A, and Halevy, AH. (1994) Pollination-induced senescence in Phalaenopsis petals. Relationship of ethylene sensitivity to activity of GTP-binding proteins and protein phosphorylation, PhysioL Plant. 90, 679-684. Romero, L.C., Sommer, D., Gotor, C., and Song, P.S. (1991) G-proteins in etiolated Aveva seedlings. Possible phytochrome regulation, FEES Lett. 282, 341-346. Sanders, 1.0., Harpham, N.V.1., Raskin, I., Smith, AR., and Hall, MA (1991) Ethylene binding in wild-type and mutant Arabidopsis thaliana (L.) Heynh, Ann. Bot. 68,97-103. Sanders, 1.0., Smith, AR., and Hall, MA (1991) Ethylene binding in epicotyls of Pisum sativum L. cv. A1aska,Planta 183, 209-217. Schaller, G.E. and Bleecker, AB. (1995) Ethylene-Binding sites generated in yeast expressing the Arabidopsis ETRI gene, Science 270,1809-1811. Schaller, G.E., Ladd, AN., Lanahan, M.B., Spanbauer, J.M., and Bleecker, AB. (1995) The ethylene response mediator ETRI from Arabidopsis forms a disulfide-linked dimer, J. Bioi. Chem. 270, 12526-12530. Schlessinger, J. (1993) How receptor tyrosine kinases activate Ras, TIBS 18, 273-275. Sisler, E.C. (1990) Ethylene-binding components, in AK Mattoo and J.C. Suttle (eds.), The Plant HormoneEthylene, CRC Press, Baco Raton, pp. 81-100. Warne, P.H., Rodriguez Viciana, P., and Downward, J. (1993) Direct interaction of Ras and the amino-terminal region ofRaf-l in vitro, Nature 364,352-355. Warpeha, KM.F., Hamm, H.E., Rasenick, M.M., and Kaufinan, L.S. (1991) A blue-light-activated GTP-binding protein in the plasma membranes of etiolated peas, Proc. NatL Acad. Sci. USA 88, 8925-8929. Zaina, S., Reggiani, R., and Bertani, A (1990) Preliminary evidence for involvement ofGTP-binding protein(s) in auxin signal transduction in rice (Oryza sativa L.) coleoptiles, J. Plant Physiol. 136, 653-658.

EmYLENE AND ARABIDOPSIS ROSETTE DEVELOPMENT

1. Introduction

J. SMALLE, J. KUREPA, M. HAEGMAN, M. VAN MONTAGU, D. VAN DER STRAETEN Laboratorium voor Genetica, Department of Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

Ethylene is known to affect rosette development in Arabidopsis. By controlling cell size, ethylene exerts a dramatic effect on rosette diameter. Leafblades and petioles are smaller upon ethylene treatment due to an inhibition of cell enlargement rather than cell division [1]. Ethylene is also involved in controlling the onset of leaf senescence [2]. A whole array of Arabidopsis ethylene mutants were isolated based on the triple response [1, 3] and several of these mutants have an altered rosette development. Ethylene-insensitive mutants (etrl, ein2, ainl) have larger rosettes and delayed bolting and flowering, as well as a delay in leaf senescence [3-5]. Ethylene-overproducing mutants (eto) have been identified by screening for seedlings having a constitutive triple response [6]. However, none of these mutants overproduce ethylene in the light, and consequently their rosette phenotype is equal to that of wild-type plants [1]. An Arabidopsis mutant was isolated having a constitutive ethylene response without having higher ethylene biosynthesis [1]. This ctr 1 mutant displays an ethylene phenotype at all stages of Arabidopsis development. At the rosette stage, ctr 1 is severely dwarfed and resembles ethylene-treated wild-type seedlings. Unexpectedly, ctr 1 plants have a delay in bolting, and leaves of intact ctr 1 plants do not senesce faster than do wild-type leaves.

By studying the effects of ethylene on Arabidopsis rosette development, we are trying to address two questions. First, is sensitivity to ethylene differentially regulated in the course of rosette development (from the two-leaf stage to bolting), and in the different parts of the rosette (leaves, petioles, apical meristem)? It is now becoming clear that ethylene sensors are encoded by a multigene family whose members might be differentially expressed during plant development [7]. Although most or all of these putative ethylene receptors are thought to transduce the ethylene signal through a common pathway [3, 8], the possibility that their expression levels and ethylene-binding activities differ, might result in a highly complex and developmentally controlled ethylene-sensing mechanism. Since most of the ethylene mutants have been isolated based on a response of dark-grown seedlings [3], it is

87

A. K. Karrellis et aI. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 87-92. © 1997 Kluwer Academic Publishers.

88

possible that those triple-response mutants that also have an altered rosette development are defining genes occupying key positions in the linear part of the ethylene-sensing pathway [8]. Factors involved in fine tuning the ethylene response or regulating ethylene biosynthesis specifically at the rosette stage might have remained unidentified if not involved in the ethylene response of dark-grown seedlings. In order to test this, we have been using an ethylene response in light-grown Arabidopsis seedlings to isolate novel ethylene mutants.

A second question is whether and to what extent the regulation of ethylene biosynthesis is affecting rosette development. At the molecular level this can be studied by analyzing the regulation of expression of the different ethylene biosynthesis genes. Several of these genes have been cloned and the Arabidopsis l-aminocyclopropane-l-carboxylate (ACC) synthase gene family was shown to contain at least two members that are expressed in rosette leaves [9,10]. Since it is now well established that the regulation of ethylene biosynthesis is predominantly located at the conversion of S-adenosyl- 1 -methionine to ACC by ACC synthase [11], these ACC synthase genes can then be used to determine the factors involved in controlling their expression, and thus the biosynthesis of ethylene, at different stages of rosette development.

2. An Ethylene Response Suitable for Isolating Novel Arabidopsis Ethylene Mutants

Using the triple response for isolating ethylene mutants and subsequent double-mutant analysis, it was convincingly shown that there is only one primary ethylene signal transduction pathway [3]. The cloning of the ETRI gene suggested an explanation for the fact that different ethylene concentrations giving different responses all act through a common transduction pathway. The ETRI gene has significant homology with bacterial two-component regulators [12], and for these kind of receptor systems it is known that they can operate over a wide range of ligand concentrations [13]. Recently, another putative Arabidopsis ethylene receptor was cloned [14], supporting the findings that the Arabidopsis genome encodes a complex family of these receptor-like proteins [12]. If this gene family is differentially expressed throughout development, then screening for ethylene mutants at different stages of development might be required in order to identify all the ethylene-sensing activities. Also, several mutants have been isolated identifying genes that control a subset of the ethylene responses. The hookless 1 (hlsl) mutation results in the loss of apical hook formation in ethylene treated dark grown seedlings [15]. However, both hypocotyl and root respond normally to ethylene. The HLSI gene was shown to act downstream from EIN3 [16]. The eirl (ethylene-insensitive root) mutation affects ethylene sensitivity specifically in the roots. Double-mutant analysis did not allow to position the EIRI gene unequivocally in the ethylene signal transduction pathway [16]. The isolation of these classes of tissue-specific ethylene mutants might indicate that genes acting downstream of the linear part of the ethylene signal transduction pathway will have to

89

be identified by monitoring the ethylene response at different developmental stages, in different plant organs and tissues, and under different growth conditions.

Using the triple response, a number of ethylene-overproducing mutants have been isolated [1, 6]. However, these mutants overproduce ethylene only in the dark [1]. Given the high degree of developmental regulation of the ethylene biosynthesis pathway [11], mutations affecting it will most likely exert their effects at specific stages of development. Thus, screening for mutants having an altered ethylene biosynthesis or response in, for example, the light-grown rosette stage can be complementary to screening based on the triple response.

We have been characterizing an ethylene response in light-grown Arabidopsis seedlings subjected to nutrient stress. This has several advantages compared to screening at the full-grown rosette stage. First, because ethylene is involved in the control of cell enlargement [1], ethylene effects are more pronounced in developing tissues. Therefore, differences in developmental stage, due to variations within a mutagenized population, can be enhanced leading to a high background of ethylene-like phenotypes in screening for ethylene mutants. Seedlings that are grown on low nutrient medium (LNM) are more uniform in size and more synchronized in development. In this case, the growth medium consists of agarose and mineral water (Spa Reine). This medium is deficient in nitrogen and phosphorus, but provides the seedlings with the necessary trace elements. Thus, development of the seedlings is based on the reallocation of nutrients from the cotyledons, in combination with photosynthesis. Growth of seedlings on LNM medium is severely retarded, and the emergence of the true leaves is slow. This leaf emergence can be accelerated by administering the ethylene precursor ACC (50 J,LM). ACC-accelerated leaf emergence is correlated with a faster degreening and senescence of the cotyledons. This response appears to be an ethylene response, since it cannot be induced in the ethylene-resistant mutant etrl, and since it can be blocked by adding Ag+, a known inhibitor of the ethylene response [17]. In theory, this allows the isolation of a set of mutant classes. A first class would consist of seedlings having an accelerated leaf emergence in the absence of ACC. These would be candidates for ethylene overproducers or for mutants showing ethylene responses in the absence of higher ethylene levels. A second class of mutants would, in the absence of ACC, have a slower leaf emergence than wild type. These would be candidates for ethylene underproducers or ethylene insensitives. In a third class, seedlings without accelerated leaf emergence upon ACC treatment would be putative ethylene insensitives. Finally, seedlings having an even faster leaf emergence upon ACC treatment are potentially ethylene-hypersensitive mutants. Some non-ethylene mutants will interfere with this kind of classification. However, using a set of inhibitors of the ethylene biosynthesis and ~ignal transduction pathways in combination with double-mutant analysis should allow to discriminate between ethylene mutants and other non-related classes.

We have been screening Arabidopsis M2 populations after mutagenesis with ethylmethanesulfonate (EMS) or T-DNA insertion [18]. Candidates for all of the suggested mutant classes were obtained. We are now characterizing two classes of

90

mutants, those with a constitutive ACC response and mutants with a hypersensitive response.

The constitutive mutants are named ale (as for ~ccelerated leaf ~mergence). The aiel mutant does not overproduce ethylene. It is an EMS-induced monogenic semi-dominant mutation. When aiel is grown on LNM without ACC, its leaf emergence is markedly faster than that in wild type. The aiel mutant is a dwarf. The rosette is dark green and the inflorescence is more compact than that of wild-type plants. Flowering time in alel is delayed. Etiolated aiel seedlings have a shorter hypocotyl, indicative for a constitutive ethylene response, however the seedlings do not have an exaggerated apical hook. The ale 1 mutation is epistatic to the ethylene-resistant mutation etrl. Double mutants of aiel and etrl still have an accelerated leaf emergence in the absence of ACC, and the hypocotyl length of etiolated double-mutant seedlings remains shorter than that of wild type. This double mutant is ethylene insensitive. Further characterization is needed to position the ALEl gene relative to the other genes in the signal transduction pathway.

A second set of mutants are displaying a hypersensitive response to ACC treatments, i.e., leaf emergence is accelerated even more in response to ACC treatments. In contrast to aiel, these ahs mutants (as for ACC hypersensitive) do not have a constitutive ACC response. Two mutants were isolated from aT-DNA mutagenized population [18]. When grown on Murashige and Skoog medium supplemented with 0.8% sucrose and ACC, both rosette and roots of the ahsl mutant are more severely reduced in size than the wild-type. The ahsl mutant is late flowering. The mutation is monogenic and recessive. As in aiel, ethylene levels in this mutant do not markedly differ from wild type.

3. Transcriptional Regulation oftheAT-ACSI Gene

Like most plant species studied, Arabidopsis was shown to have a complex family of ACC synthase genes [9, 10]. Two of these genes are expressed during rosette development [19, 20]. Since ethylene biosynthesis is predominantly controlled at the conversion of SAM to ACC [11], these genes can be used as starting points for studying developmentally controlled ethylene production in the Arabidopsis rosette. The expression of the AT-ACSl gene was analyzed with RNA gel blot analysis and promoter-~-glucuronidase fusion experiments [10, 19]. It was shown that A T-A CSl is the most abundantly expressed ACC synthase gene in the Arabidopsis rosette, and its expression appeared to be linked to young and developing tissues [10, 19]. The AT-ACS4 gene is also expressed in the rosette and is auxin induced [20].

We have been studying the transcriptional regulation of the AT-ACSl gene by following two strategies. First, a quantitative reverse transcription polymerase chain reaction (PCR) method was designed which allowed detection oftheAT-ACSl message using low amounts of tissue. This method includes the use of an internal RNA control obtained by in vitro transcription from a construct having a small deletion between the primer sites used in the PCR reaction. With this method we could obtain AT-ACSl

91

expression profiles using less than I Ilg of total RNA per sample. To prove that this detection method is reliable, we tested the organ-specific expression of AT-ACSI in flowering Arabidopsis seedlings and indeed obtained the pattern reported by Liang et al. [10]. By determining AT-ACSJ transcript levels in the individual leaves of the Arabidopsis rosette at different time points of development, and in different mutant backgrounds, we are trying to establish a correlation between expression and certain developmental processes.

In a second approach we are attempting to identify factors that control AT-ACSI expression. We have been applying the following strategy. TheAT-ACSI promoter is analyzed for sequences that are bound specifically by proteins from nuclear extracts of Arabidopsis rosettes. Fragments with which one or more protein bands are detected in a gel blotting experiment [21] are then used to isolate the corresponding cDNAs from a cDNA expression library [22]. The proteins encoded by cDNAs isolated in this screen are subsequently tested for their sequence-specific DNA-binding activity using electromobility shift assays [23]. Finally, if specific DNA binding is confirmed, then transgenic plants are made having altered expression levels of the putative A T-ACSI trans-acting regulator. We have isolated a cDNA encoding a DNA-binding protein that interacts specifically with the AT-ACSI promoter. Transgenic Arabidopsis lines overexpressing this putative factor are now being analyzed for their AT-ACSI levels and visible phenotypical alterations.

4. Acknowledgments

We thank Martine De Cock for help in preparing the manuscript. This research was supported by grants from the Belgian Programme on Interuniversity Poles of Attraction (prime Minister's Office, Science Policy Programming, no. 38) and the Vlaams Actieprogramma Biotechnologie (ETC 002). D.V.D.S. is a Senior Research Associate of the National Fund for Scientific Research (Belgium).

5. References

1. Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A, and Ecker, J.R. (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases, Cell 12, 427-441.

2. Grbif, V. and Bleecker, AB. (1995) Ethylene regulates the timing ofleaf senescence in Arabidopsis, Plant J. 8, 595-602.

3. Ecker, J.R. (1995) The ethylene signal transduction pathway in plants, Science 268, 667-675. 4. Van Der Straeten, D., Djudzman, A, Van Caeneghem, W., Smalle, J., and Van Montagu, M. (1993)

Genetic and physiological analysis of a new locus in Arabidopsis that confers resistance to l-aminocyclopropane-l-carboxylic acid and ethylene and specifically affects the ethylene signal transduction pathway, Plant Physiol. 102, 40 1-408.

5. Bleecker, AB., Estelle, M.A, Somerville, C., and Kende, H. (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana, Science 241,1086-1089.

6. Guzman, P. and Ecker, J.R. (1990) Exploiting the triple response of Arabidopsis to identifY ethylene-related mutants, Plant Cell 2, 513-523.

7. Theologis, A (1996) Plant honnones: more than one way to detect ethylene, Curro Bioi. 6, 144-145.

92

8. Chen, Q.G. and Bleecker, AB. (1995) Analysis of ethylene signal-transduction kinetics associated with seedling-growth response and chitinase induction in wild-type and mutant Arabidopsis, Plant Physiol. 108, 597-607.

9. Van Der Straeten, D., Rodrigues-Pousada, RA, Villarroel, R., Hanley, S., Goodman, H.M., and Van Montagu, M. (1992) Cloning, genetic mapping, and expression analysis of an Arabidopsis thaliana gene that encodes l-aminocyclopropane-l-carboxylate synthase, Proc. NatL Acad. Sci. USA 89, 9969-9973.

10. Liang, X, Abel, S., Keller, J.A, Shen, N.F., and Theologis, A (1992) The l-aminocyclopropane-l-carboxylate synthase gene family of Arabidopsis thaliana, Proc. NatL Acad. Sci. USA 89, 11046-11050.

11. Kende, H. (1989) Enzymes of ethylene biosynthesis,PlantPhysiol. 91,1-4. 12. Chang, C., Kwok, S.F., Bleecker, AB., and Meyerowitz, E.M. (1993) Arabidopsis ethylene-response

gene ETRl: similarity of product to two-component regulators, Science 262, 539-544. 13. Parkinson, J.S. (1993) Signal transduction schemes of bacteria, Cell 73, 857-871. 14. Hua, J., Chang, C., Sun, Q., and Meyerowitz, E.M. (1995) Ethylene insensitivity conferred by

Arabidopsis ERS gene, Science 269, 1712-1714. 15. Lehman, A, Black, R., and Ecker, J.R. (1996) HOOKLESSl, an ethylene response gene, is required

for differential cell elongation in the Arabidopsis hypocotyl, Cell 85, 183-194. 16. Roman, G., Lubarsky, B., Kieber, J.J., Rothenberg, M., and Ecker, J.R. (1995) Genetic analysis of

ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway, Genetics 139, 1393-1409.

17. Beyer, E.M. Jr (1976) A potent inhibitor of ethylene action in plants, Plant Physiol. 58, 268-271. 18. Feldmann, KA (1991) T-DNA insertion mutagenesis in Arabidopsis: mutational spectrum, Plant J.

1,71-82. 19. Rodrigues-Pousada, R.A, De Rycke, R., Dedonder, A, Van Caeneghem, W., Engler, G., Van

Montagu, M., and Van Der Straeten, D. (1993) The Arabidopsis l-aminocyclopropane-l-carboxylate synthase gene 1 is expressed during early development, Plant Cell 5, 897-911.

20. Abel, S., Nguyen, M.D., Chow, W., and Theologis, A (1995) ASC4, a primary indoleacetic acid-responsive gene encoding l-aminocyclopropane-l-carboxylate synthase in Arabidopsis thaliana. Structural characterization, expression in Escherichia coli, and expression characteristics in response to auxin, J. BioL Chem.270, 19093-19099.

21. Bowen, B., Steinberg, J., Laemmli, U.K, and Weintraub, H. (1980) The detection of DNA-binding proteins by protein blotting, Nucleic Acids Res. 8, 1-20.

22. Vinson, C.R., LaMarco, KL., Johnson, P.F., Landschulz, W.H., and McKnight, S.L. (1988) In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage, Genes Dev. 2, 801-806.

23. Fried, M. and Crothers, D.M. (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis, Nucleic Acids Res. 9, 6505-6525.

ETHYLENE REGULATES LIFE-SPAN IN CELLS OF REPRODUCTIVE ORGANS IN PISUM SATlVUM

A. GRANELL, R. BLAY AND D. ORZAEZ Instituto de Biologia Molecular y Celular de Plantas. CSIC­Universidad Poliecnica de Valencia, Camino de Vera 14, 46022 Valencia, Spain

1. Introduction

Flowers are terminal structures that evolved to eventually produce a fruit containing the plant offspring. Most of the flower organs are ephemeral and are, therefore, progranuned to die as part of the developmental program of the plant. This program controls the life span of individual cells or organs in response to both endogenous and external factors. Flowers are, therefore, useful organs in the study of factors and genes involved in determining life span and cell death.

Flower senescence has been thoroughly studied in different plants - mainly in morning glory, carnation, petunia, and orchids (see chapters by Woodson, and Woltering on this publication). It has been demonstrated that in the senescence of many flowers (but not all; see [19]) ethylene is a primary coordinating agent. Surprisingly, and despite the widespread use of the pea plant to study the physiological and biochemical effects of ethylene [1], we know very little about the role of ethylene in the senescence of the pea flower. This article summarizes recent progress in characterizing the senescence of flower organs in Pisum sativum, the genes which are differentially regulated during this process, and the role of ethylene in regulating different aspects of flower organ senescence.

The pea flower belongs to the papilionaceous family; it is made up of five petals with the two lower ones joined along their ventral edges and forming the keel which surrounds the stamens and the pistil. The pea plant is entirely autogamic. The anthers dehisce approximately one day before the opening of the flower bud, so that prior to anthesis the anthers and carpel stigma in their growth are withdrawn into the base of the keel and thus pollination and fertilization occurs with high efficiency.

In the pea plant, carpels develop into actively growing fruit upon pollination. As the plant hormone gibberellin can substitute for pollination to produce a seedless but otherwise normal fruit, this hormone appears to be responsible for the induction of fruit development [7,6].

Emasculation prevents pollination, and the carpel, after a quiescent stage of approximately 48h at the end of its growth period, undergoes a senescent process that ends up with the death and abscission of the organ. This program of cell death induced by deprivation of growth factors (gibberellins/pollination) can be delayed or

93

A. K. Ka1U!llis et al. (eds.), Biology and Biotechnnlogy olthe Plant Hormo1U! Ethyle1U!, 93-98. © 1997 Kluwer Academic Publishers.

94

slowed by the application of ethylene action inhibitors (STS or NBD). These results point to the plant honnone ethylene as the inducer of cell death and establish an interesting parallel between senescence of plant organs and programmed cell death in animals, where growth factors acting through a cascade of molecular events can tilt the balance to favor death or to protect against it. [5].

2. Control of Cell Death Process

Cell death in the carpel is a predictable and precise process and is accompanied by the modulation (induction or repression) or specific genes [8,16]. All this suggests that like many other differentiation processes, carpel cell death is just much like any other cell fate. The same occurs in petals: petals grow and develop up to stage dO and then follow a senescence program with specific gene expression.

2.1. SENESCENT ORGANS/CELL SPECIFICATION

In pea, petals seem to function organ-autonomously to initiate death, at least to some extent. Experimehts with flowers progressively deprived of other flower organs and with excised petals show that they wilt and then die at approximately the same time as when attached to the plant. Furthennore pea petals senesce at the same pace in unpollinated or pollinated flowers. The program of petal senescence appears, therefore, to be pre-established (before d-2) and to follow an internal clock rather being than due to organ-organ interaction or in response to a traveling signal (Orzaez, Blay and Granell, submitted).

In the carpel, however, cell fate seems to be more dependent on interactions with other cells, i.e., pollination triggers an increase in gibberellins, and this induces fruit set [6]. Under normal conditions, a signal initiated with pollination induces a rapid development of the fruit. In the absence of such a signal, senescence of the carpel commences at a time that is affected by temperature, daylength, presence or absence of metabolic sinks [3, 15], etc.

Programmed death may therefore occur independently in some flower organs while others develop very rapidly.

2.2. ETIiYLENE IS A SIGNAL THAT TRIGGERS CELL DEAlH

In experiments with STS- and NBD-treated flowers, petals continue growing after anthesis at a time when their untreated counterparts have stopped their developmental program and start to senesce (Orzaez, Blayand Granell, submitted). This suggests that the increase in ethylene production detected at stage dO prematurely aborts the petal's developmental program to induce a program of senescence. Ethylene does not seem to be required during the initial stages of fruit development as STS-treated flowers can develop fruits exactly as the untreated controls dO.

Ethylene action inhibitors, however, prevent or delay the death of susceptible cells in emasculated ovaries as is demonstrated by the extended life span of the ovaries that remain sensitive to gibberellins [14]. The opposite effect is found in flowers treated

95

with ethylene.

NORMAL CELL

SENESCENT CELL

~ DEAD •• 1111,. CELL

Figure 1. Ethylene is a senescence factor inducing the program of cell death in flower organs of Pisum sativum. Life-span of "quiescent cells" is lengthened by ethylene action inhibitors and shortened by ethylene.

Gibberellins can therefore be considered as rescuing or preventing susceptible cells within the carpel from dying whereas ethylene triggers cell death.

The phenotype of STS treated pea flowers is similar to the tomato mutant Never ripe. This tomato mutation is known to be in an ethylene receptor that renders the flower insensitive to ethylene, and thus petals fail to senesce [12] while the fruit continues its growth program. The effect of ethylene on longevity of Arabidopsis leaves has also been demonstrated using a genetic approach [9, 10, 11]. Interestingly, petals and carpels are ontogenically related to leaves.

Ethylene, however, seems not to be involved in the senescence of some other flowers [19]. It is unknown to what extent the death programs in the different types of flowers overlap.

2.3. MECHANISMS BY WHICH CELLS DIE AND DEGRADATION OF DEAD CELLS

Senescent carpels, irreversibly committed to die, show nuclei with increasing

96

intemucleosomal DNA fragmentation, loss of cellular compartmentalization, and loss of protein and fresh weight [14, 18]. DNA fragmentation, one of the hallmarks of programmed cell death in animal systems, seems also to occur in plants during a natural developmental process. Analysis of the DNA extracted from senescent ovaries showed oligomeric bands, multiples of approximately 180 bp (nucleosomal size), which was parallel to the loss of the sensitivity of the ovary to grow in response to gibberellin. While this technique gives us information about the nature of the DNA fragmentation and suggests that it occurs at the unprotected linker regions of the chromatin, it does not provide information about the fate of individual cells in the ovary. The TUNEL reaction enables us to identify which cells in a thin section of tissue are actually undergoing DNA fragmentation. The rationale of this technique is that the nuclei of those cells will contain an increased number of 3'OH termini which can be labeled with terminal transferase and a nucleotide analog. Cells containing an increased number of incorporated analog can be identified with an anti-analog antibody coupled to a enzyme. Using this technique, we were able to identify specific cells with labeled nuclei, mainly located in the outer integument and endothelium of the ovule and in the "a" layer of cells [18] of the carpel endocarp. The nuclei of these cells were in many cases highly condensed, which also occurs in animal systems. At later stages of senescence, a progressive loss of cellular compartmentation was observed [18]. Flowers treated with ethylene action inhibitors showed a delay or inhibition in all the physiological and morphological disorders characteristic of senescent cells, while ethylene applied to presenescent flowers accelerate it [14].

As we mentioned earlier, senescence is an active, programmed cell death process and as such is genetically controlled. By using a molecular approach, particularly by differential screening and differential display techniques, we have isolated a number of cDNAs whose expression is either up- or down-regulated during senescence and by ethylene treatment.

The activity and gene expression levels of a thiolprotease (tpp) related to animal cathepsins increase dramatically with senescence [2, 4, 8], and this enzyme is possibly involved in degradation rather than in the killing of the senescent cells. Cathepsins also increase during animal apoptosis where they have been involved in the execution of the death program, namely contributing to the retrieval of the protein nutrients [17]. Ethylene treatment induced a rapid (less than Ih) increase in the expression of tpp mRNA and protein levels while ethylene action inhibitors STS and NBD blocked its accumulation.

A cDNA with homology to proteins involved in vesicle traffic also accumulates in ethylene-treated and senescent tissues (Orzaez & GranelI, in preparation), suggesting that membrane trafficking may increase to retrieve nutrients from senescent tissues for export to other parts of the plant (or may simply be there to account for the increase in protein trafficking through the secretory pathway in tissues with high levels of ethylene). Interestingly, there is a proliferation of vesicles during the blebling of animal cells during apoptosis.

On the contrary the expression of pdh is down-regulated by ethylene and during flower senescence [13]. Although we do not know the nature of pdh, it is interesting that, as happens with negative regulators of cell death found in animals, the expression of pdh is widespread in young individuals, but in mature individuals pdh appears to be

97

restricted to actively dividing and differentiating tissues. In summary, there are indications that cell death occurring during senescence of

the pea flower organs may share some characteristics with certain apoptotic or programmed cell deaths described in animal systems; interestingly some plants appear to have evolved mechanisms that introduce the plant hormone ethylene as a positive regulator of such a precise and regulated program of cell death. In the absence of other signals, ethylene would limit (shorten) the life-span of developmentally specified cells/organs such as those in petals after anthesis or unpollinated ovaries.

3. Acknowledgments

This research was supported by the Spanish Ministry of Science and Education through Grant No. PB92-0018 and Fundaci6n Caja de Ahorros del Meditemlneo. D.O. is recipient of a Generalitat Valenciana fellowship. Also thanks to F. Barroclough for her help with the English.

4. References

1. Abeles, F.B., Morgan, P.W., and Salveit, Jr. M.E. (1992) Ethylene in Plant Biology, 2nd edition, Academic Press, London.

2. Agiiero. M.S., Granell, A, and Carbonell, J (1996) Fruit set dramatically decreases the levels of thiolproteases in tomato ovaries, Physiol. Plantarum (in press).

3. Carbonell, J. and Garcia-Martinez, J.L. (1980) Fruit set of un pollinated ovaries of Pisum sativum L. Influence of vegetative parts, Planta 147, 444-450.

4. Cercos, M., Carrasco, P., Granell, A, and Carbonell, J. (1992) Biosynthesis and degradation of Rubisco during ovary senescence and fiuit development induced by gibberellic acid, PhysioL Plantarum 8S,476-482.

5. Collins, M.K.L., Perkins, G.R., Rodriguez-Tarduchy, G., Nieto, M.A, and Lopez-Rivas, A (1994) Growth factors as survival factors: regulation of apoptosis, BioEssays 16, 133-138.

6. Eeuwens, C.J. and Schwabe, W.W. (1975) Seed and pod wall development in Pisum sativum L. in relation to extracted and applied hormones, J. Exp. Bot. 26, 1-14.

7. Garcia-Martinez, J.L and Carbonell, J (1980) Fruit set ofunpollina1ed ovaries of Pisum sativum L. Influence of plant growthregulators,Planta 147, 451-456.

8. Grbic, V. and Bleecker, AB. (1995) Ethylene regulates the timing ofleaf senescence in Arabidopsis, Plant J. 8, 595-602.

9. Hensel, L.L., Grbic, V., Baumgarten, D.A and Bleecker, AB. (1993) Developmental and age-related processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis, Plant CellS, 553-564.

10. Hensel, L., Grbic, V., Baumgarten, D., and Bleecker, AB. (1992) Development and age-related processes that influence the longevity and senescence of photosynthetic tissues in Arabidopsis, Plant Cell S, 553-564.

11. Grane1I, A, Harris, N., Pisabarro, AG., and Carbonell, J. (1992) Temporal and spatial expression of a thiolprotease gene during pea ovary senescence, and its regulation by gibberellin, Plant J. 2, 907-915.

12. Lanahan, M.B., Yen, H.C., Giovannoni, 1.J., and Klee, H.J. (1994) The never ripe mutation blocks ethylene perception in tomato, Plant Cell 6, 521-530.

13. Orzaez, D. (1996) Senescence of reproductive organs in Pisum sativum .. Ph.D. Thesis, University of Valencia.

14. Orzaez, D. and Granell, A (1996) DNA fragmentation is regulated by ethylene during carpel senescence inPisum sativum, Plant J. (in press).

15. Peret6, J., Beltran, J.P., and Garcia-Martinez, J.L. (1988) The source of gibberellins in the

98

parthenocarpic development of ovaries on topped pea plants, Planta 175, 493-499. 16. Sanchez- Beltran, MJ, Carbonell, J, Garcia-Martinez, JL, and Lopez-Diaz, I (1992) Gene expression

during two alternative pathways of ovary development in Pisum sativum: fiuit development and ovary senescence,Physiol. Plantarum 85, 69-76.

17. Schwartzman, R.A and Cidlowski, JA (1993) Apoptosis: the biochemistry and molecular biology of programmed cell death, Endoc. Rev. 14, 133-151.

18. Vercher, Y. and Carbonell, J. (1991) Changes in the structure of ovary tissues and in the ultrastructure of mesocarp cells during senescence of fiuit development induced by plants growth substances in P. sativum, Physiol. Plantarum 81, 518-526.

19. Woltering, E.J. and van Doorn, W.G. (1988) Role of ethylene in senescence of petals. Morphological and taxonomical relationships, J. Exp. Bot. 39, 1605-1616.

FLUCTUATIONS IN ETHYLENE FORMATION AND FLOWERING IN CHENOPODIUM RUBRUM

, v , I 2 2 I. MACHACKOV A, N. CHAUV AUX, W. DEWITTE, H. VAN ONCKELEN2

IDe Montfort University, Norman Borlaug Centre for Plant Science, Institute of Experimetal Botany, Academy of Sciences of the Czech Republic, Ke dvoru 15, 166 00 Praha 6, Czech Republic 2Department of Biology, University of Antwerpen, B-2610 Wi/rijk, Antwerpen, Belgium

1. Exogenous Ethylene and Photoperiodic Flower Induction in C. rubrum

In our studies of the possible involvement of ethylene in photoperiodic flower induction in Chenopodium sp. we have shown that ethylene inhibits flower induction in short-day (SD) c. rubrum when applied (in the form of Ethrel) before or during the first half of the inductive darkness [1]. To achieve this inhibition, 100 - 300 J1M Ethrel solution had to be used, after application of which ethylene formation achieved values of 260 -480 nl g-I FW h-I (unpublished results). Moreover, ethylene was shown to mediate auxin (IAA) - induced inhibition of flowering in both SD C. rubrum and long-day (LD) C. murale [2]. In this case, ethylene production was not by far as high as after Ethrel application; 500 J1M IAA induced ethylene formation of no nl.g-1FW h-I at the maximum. If applied 1-2 days after the end of inductive treatment, both Ethrel and IAA at 100-300 J1M stimulated flowering.

Thus, it is clear that high levels of ethylene inhibit flowering during the induction and stimulate flowering after the induction. This raises the question if changes in endogenous ethylene levels could take part in the regulation of flower induction.

2. Endogenous Ethylene and Flowering in C. rubrum

Comparison of ethylene formation rates in both Chenopodium rubrum and Chenopodium murale under continuous light (CL) and 12/12 and 8/16 (light/darkness) photoperiodic regimes, respectively, demonstrated that both species, irrespective of the effect of the photoperiod on flowering, produce more ethylene in CL than under SD (as measured 4 h after the end of the dark period). This difference was shown to be due to an increased capacity to convert l-aminocyclopropane-l-carboxylic acid (ACC) to ethylene in CL [3, 4]. This result suggests that ethylene formation may be controlled by photoperiod. One of the phenomena of photoperiodic control of flowering in SD C.

99

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 99-104. © 1997 Kluwer Academic Publishers.

100

rubrum is endogenous rhythmicity in constant darkness with maxima of flowering at hours 12-13 and 42-44 [5]. The level offree indole-3-acetic acid (IAA) also reveals an endogenous rhythm [6]. Several plants (Vicia, Kalanehoe, Caltha) have been reported to exhibit diurnal variations in ethylene formation [7] and endogenous rhythms in ethylene formation under CL in cotton [8] and under both CL and CD in etiolated barley, wheat and rye seedlings [9] have been described. Our aim was to demonstrate if an endogenous or diurnal rhythmicity in ethylene production exists in C. rubrum and to show to what extent changes in ethylene formation may be correlated to flower induction.

2.l. DIURNAL FLUCTUATIONS IN ETHYLENE FORMATION

Seeds of Chenopodium rubrum, sel. 374 were germinated and cultivated as described by Ullmann et al. (1985) [10], first two days under CL and then under alternating 12 h lightl12h dark regime. All experiments were started at the same circadian time. Nylon nets supporting 5-6 d old plants were transferred onto a layer of small glass beads in glass vessels (free volume 250 mI, total volume 500 mI) containing half­strength Knop's nutrient solution. These vessels were tightly closed and coupled into a flow-through system, as designed and described by de Greef et al. (1976) [11] and de Greef and de Proft (1978) [12]. The experiments lasted for 48 h, measurements in each of running three lines were repeated every 3 h. Plants were kept at 20±I°C, either in constant light, constant darkness or under an alternating (12 h lightl12h darkness) regime. Light was provided by fluorescent tubes at an intensity 20 W m-2•

No regular fluctuations in ethylene production were observed in either constmt light or darkness although ethylene production was, on average, slightly higher in light than in darkness. Under an alternating lightldark regime, however, diurnal variations in ethylene formation with increases occurring in the light and decreases in darkness were observed. However, the range of these fluctuations was relatively narrow: ethylene production ranged between 1-3 nl.g-I FW h-I (Fig. 1). The fluctuations found under this 12/12 photoperiodic regime correspond well to those described by Kapuya and Hall (1977) [7] for Vicia faba and Kalanehoe daigremontiana. The photoperiodic regimes used in their experiments were different, but in most cases a transfer from darkness to light resulted in increased ethylene formation; the effect was especially pronounced in Vieiafaba. Similar results have also been reported for cotton [8]. Dark to light transition resulted in an increased rate of ethylene formation in spite of the known inhibitory effect of light on ethylene production in many plants [13]. This inhibition has been demonstrated to be largely due to the lack of CO2 in the atmosphere of closed vessels kept in the light which affects the conversion rate of ACC to ethylene [14]. In closed vessels, we have observed a similar effect of CO2 free air in Chenopodium rubrum [15]. However, this is excluded in the experiments described here as steady-state conditions were achieved in the flow-through system.

101

4,---------------------

.c 3

~ LL

'": OJ

C 2

Q) c Q)

>-.c .... Q)

ol===~ .... ~~==~ ...... .c==~ o 10 20 30 40 50

time (h)

Figure 1. The effect of photoperiodic regime on ethylene formation by 6-d-old C. rubrum seedlings. Plants on nylon nets were placed on a nutrient solution in special vessels, which were coupled into a flow-through system. Ethylene was trapped for 45 min in 3h intervals. Plants were subjected to continuous darkness (- 0 -), continuous light (- D -) or alternating light/darkness (12 h each) (- II. -) at 20·C. To keep the figure easy to survey, SE values are given only in the case of alternating regime; in CL and CD they did not exceed 12 % of given values.

The results presented above lead to following conclusions: We have shown in the first part of our work that to affect flowering, high levels of

ethylene have to be present. Thus, it is very unlikely that fluctuations of endogenous ethylene production between 1-3 nl.g-1 FW h-1 can influence flowering. Moreover, there is a rhythm in flowering displayed in constant darkness while no fluctuations in ethylene formation were observed in this regime. In addition, application of aminoethoxyvinyl glycine (A VG) did not affect flowering in C rubrum [2]. It is therefore likely that ethylene does not play any important role in regulation of flower induction.

2.2. DIURNAL FLUCTUATIONS IN THE ACC-INDUCED ETHYLENE FORMATION AND IN ACC AND MACC LEVELS

To investigate at which biosynthetic step the diurnal rhythmicity of ethylene formation arises, we studied the time-course of ACC-induced ethylene formation as well as the levels of endogenous ACC and MACC. At saturating concentrations of ACC, the rate of ethylene formation is limited by the activity of ACC oxidase. Intact plants were

102

sprayed every 3h (always a new set of plants) with a 5.10-3 MACC solution (this concentration has been found to be saturating in respect to ethylene fonnation [3]), incubated for 1 h either in light or in darkness according to the phase of light regime and then sealed for 2h into Plexiglas boxes. Incubation and ethylene measurements were perfonned as described by Machackova et al. (1986) [2]. In experiments at lowered CO2 a small vial containing ION NaOH was placed into the box with the plants. Analyses of ACC and MACC were perfonned in the above-ground parts of 6-d-old plants using a method based on the assay of Lizada and Yang (1979) [16]. Alternatively, ACC and MACC levels were determined by TSP LC-MS, using 2H4-ACC as an internal standard [17].

ACC - induced ethylene formation was transiently increased on transfers both from light to darkness and from darkness to light (Fig. 2). The increase always took place during the first few hours after the respective transfer (the maximum was usually attained after 2-3 h) and then ACC- induced ethylene formation decreased again. As these measurements were perfonned in a closed system, we checked the effect of CO2 concentration on the fluctuations (by means of scavenging CO2 in the incubation vessels by NaOH). Lowering the CO2 concentration slightly decreased ethylene fonnation, but to the same extent in darkness and in light, and it had no effect on the fluctuation pattern (Fig. 2).

The level of endogenous ACC in the above-ground parts was constant for the first 3-4 hours of darkness then decreased sharply to a minimum at the end of the dark period before increasing again during the light phase. MACC levels changed in a very similar manner to ACC (Fig. 3).

Comparing the patterns of fluctuation in ethylene fonnation, ACC - induced ethylene fonnation and ACC and MACC levels, we may conclude that the diurnal fluctuations in ethylene production are in greater part a consequence of the changes in the level of ACC with the conversion of ACC to ethylene of lesser significance in this respect. As MACC level changes were similar to those of ACC and ACC decrease was not correlated to MACC increase, it is highly probable that it is ACC synthesis, which is the decisive reaction for the appearance of the fluctuations. Although the mechanism remains to be elucidated, it may be concluded that photoperiodic regime affects ethylene fonnation in C. rub rum plants and that it acts primarily on the rate of ACC synthesis. This finding is in good agreement with recent report by Rodrigues-Pousada et al. (1994) [18] on light and darkness effects on the expression of a construct containing promoter of gene ACS 1 (ACC synthase 1) with gus gene in transfonned Arabidopsis thaliana plants. Presence of the GACGTG hexanucleotide was reported in the ACS promoter, which occurs in light - regulated promoters [19].

01

.s Q)

c: Q)

:;, .s Q)

103

246 10 12 14 16 18 20 22 24 26 28

time (h)

Figure 2. ACC - induced ethylene fonnation by 6-d-old C. rubrum seedlings. Plants on nylon nets were sprayed with ACC solution of saturating concentration (5. 1O-3M) 1 h before beginning of 2 h incubation in sealed boxes at 20°C under alternating light/darkness regime (12 h each) in the absence (- 0 -) and in the presence (- 0 -) ofa small vessel with 10 N NaOH to trap CO2. To keep the figure easy to survey, SE values are given only in the case of CO2 presence, in the other case SE did not exceed 10 % of given values.

60 120

50 100

~ 40 80 ~ LL

LL ';-';- 01

C)

ciJ ciJ 30 60 c: .s u u u U 20 40 « « ::E

10 20

0 0

0 5 10 15 20 25 30

time (h)

Figure 3. Changes during one daily cycle in ACC (- 0 -) and MACC (- 0 -) levels in the above-ground parts of 6-d-old C. rubrum seedlings kept in alternating light/darkness regime (12 h each) at 20°C. Bars represent halfSE.

104

3. References

1. Khatoon, S., Seidlova, F., and Krekule, J. (1973) Time-dependence of auxin and ethrel effects on flowering in Chenopodium rubrumL, Bioi. Plant. 15,361-363.

2. Mach:ickova, I., Krekule, J., Souckova, D., Pfikryl, Z., and Ullmann, J. (1986) Reversa\ of lAA­induced inhibition of flowering by aminoethoxyvinylglycine in Chenopodium, J. Plant Growth Regul. 4,203-209.

3. Mach:ickova, I., Ullmann, 1., Krekule, J., and Stock, M. (1988) Ethylene production and metabolism of l-aminocyclopropane-l-carboxylic acid in Chenopodium rubrum L., as influenced by photoperiodic flower induction, J. Plant Growth Regul. 7, 241-247.

4. Mach:ickova, I., Ullmann, J., Krekule, J., and Stock, M. (1989) Ethylene production and metabolism of l-aminocyclopropane-l-carboxylic acid in a long-day plant Chenopodium murale L., as influenced by photoperiodic flower induction, J. Plant Growth Regul. 8, 175-179.

5. Cumming, B.G. (1969) Circadian rhythms of flower induction and their significance in photoperiodic response, Can. J. Bot. 47, 309-324.

6. Pavlova, L. and Krekule, J. (1984) Fluctuations of free lAA under inductive and non-inductive photoperiods in Chenopodium rubrum, Plant Growth Regul. 2, 91-98.

7. Kapuya, JA and Hall, MA (1977) Diuma\ variations in endogenous ethylene levels in plants, New PhytoL 79, 233-237.

8. Rikin, A, Chalutz, E., and Anderson, J.D. (1984) Rhytlunicity in ethylene production in cotton seedlings, Plant Physiol. 75, 493-495.

9. levinsh, G. and Kreicbergs, O. (1992) Endogenous rhytlunicity of ethylene production in growing intact cereal seedlings, Plant Physiol. 100, 1389-1391.

10. Ullmann, J., Seidlova, F., Krekule, J., Pavlova, L. (1985) Chenopodium rubrum as a model plant for testing the flowering effects ofPGRs, BioI. Plant. 27,367-372.

11. De Greer, J.A, de Proft, M., and de Winter, F. (1976) Gas chromatographic determination of ethylene in large air volumes at the parts-per-billion level, Anal. Chem. 48, 38-41.

12. De Greer, JA and de Proft, M. (1978) Kinetic measurements of small ethylene changes in an open system designed for plant physiological studies, Physiol. Plant. 41, 79-84.

13. Yang, S.F. and Hotlinan, N.E. (1984) Ethylene biosynthesis and its regulation in higher plants, Annu. Rev. PlantPhysiol. 35, 155-189.

14. Kao, C.H. and Yang, S.F. (1982) Light inhibition of the conversion of l-aminocyclopropane-l­carboxylic acid to ethylene in leaves is mediated through carbon dioxide, Planta 155,261 - 266.

15. Ullmann, 1., Pfikryl, Z., Machackova, I., and Krekule, J. (1986) Ethylene production in seedlings of Chenopodium rubrum as influenced by seedling age, light, and CO2 concentration,. Biochem. Physiol. Pjlanz. 181,391-396.

16. Lizada, M.C.C. and Yang, S.F. (1979) A simple and sensitive assay for l-aminocyc\opropane-l­carboxylic acid, AnaL Biochem. 100, 140-145.

17. Chauvaux, N., Van Dongen, W., Esmans, E.L., and Van Onckelen, HA (1993) Liquid chromatographic mass spectrometric quantification of l-aminocyc\opropane-l-carboxylic acid in tobacco, J. Chromatogr. A 657,

18. Rodrigues-Pousada, R.A, De Rycke, R, Dedonder, A, Van Caeneghem, W., Engler, G., Van Montagu, M., and Van Der Straeten, D. (1994) TheArabidopsis l-aminocyclopropane-l-carboxylate synthase gene 1 is expressed during early development, Plant Cell 5, 897-911.

19. Van Der Straeten, D., Rodrigues-Pousada, RA, Villarroel, R, Hanley, S., Goodman, H., and Van Montagu, M. (1992) Cloning, genetic mapping, and expression analysis of an Arabidopsis thaliana gene that encodes l-aminocyclopropane-l-carboxylate synthase, Proc. Natl. Acad. Sci. USA 89, 9969-9973.

REGULATION OF CIRCADIANLY RHYTHMIC ETHYLENE PRODUCTION BY PHYTOCHROME B IN SORGHUM

P.W. MORGAN, S.A. FINLAYSON, I.-I LEE, K.L. CHILDS, C.-I. RE, R.A. CREELMAN, M.C. DREW, IE. MULLET Departments of Biochemistry and Biophysics (KLC, RAC, JEM), Horticulture (CJH, MCD), and Soil and Crop Sciences (PWM, SAF, IJL) Texas A&M University, College Station, Texas 77843, USA

1. Introduction

Sorghum bicolor (L) Moench. is a short-day (SD), quantitatively photoperiodic C-4 monocot. It contains 6 genes which, due to mutations, have been recognized to influence photoperiodic flowering, specifically the delaying effects of long days (LD). These genes have been designated asMa], Ma2, Ma3, Ma4 ,Mas, Ma6 by Quinby [22] and F.R. Miller (unpublished data).

One allele of Ma3, discovered and identified by Quinby and Karper [23] and designated ma/, produced an aberrant, pleiotropic phenotype with long leaf blades and leaf sheaths, reduced leaf blade area, reduced chlorophyll and anthocyanin content, reduced adventitious root development, reduced tiller outgrowth and early flowering [1, 6, 19,23]. This allele was shown by immunological assay to be missing a light stable, 123 kDa, phytochrome B-like phytochrome [3]. Recently Childs et al. [5] sequenced the phytochrome B gene from Mal and from ma/ containing plants and demonstrated that the mutant gene contains a single base pair deletion near the 3' end which results in a frame shift and stop codon in a region shown in phytochrome from other plants to be necessary for dimerization and phytochrome activity [2, 10]. Additionally, Ma3 and PHYB were mapped to the same location on chromosome 1 in sorghum [5, 17]. Thus, Childs et al. [5] concluded that plants containing the allele formerly designated ma/ are nul mutants for phytochrome B. Ma3 was redesignated asPHYB andma3R asphyB-I.

2. Circadian Phenomena in Sorghum

Phytochrome is known to be involved in photoperiodism [28] as is the biological clock [15, 27]. Thus the availability of strongly photoperiodic sorghum genotypes containing a single gene mutation for phytochrome B suggested a means to determine whether or not this phytochrome was involved in the operation of the biological clock. Accordingly, Childs et al. [4] tested the circadian rhythmicity of chlorophyll

105

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene. 105-111. © 1997 Kluwer Academic Publishers.

106

AlB binding protein mRNA and small subunit of RUBIS CO mRNA in sorghum homozygous for genes then designated Ma3, ma3 and mal (now phyB-l [5]). The mRNAs of both genes were expressed in a rhythmic, circadian fashion in sorghum [4] as in other species [21 and reviewed in 4]. In all experiments, however, the amplitude of the mRNA peaks were lower in mal plants than those with ma3 or Ma3 [4]. The latter two genotypes are indistinguishable except for small differences at flowering dates in some locations [19, 22].

Because the genotype designated mal resembled plants treated with GA3 and because GA3 treatments reproduced most aspects of the mal phenotype in genotypes with ma3 and Ma3 [20], we have been interested in the GA economy of genotypes varying at the maturity gene 3 locus. Recently, we asked when during the daily light/dark cycle the GAs are synthesized. Sorghum contains the early-13 hydroxylation pathway (reviewed in [1]), which is summarized as:

In plants homozygous for PHYB (Ma3), GA12, GAS3, GA20 and GAl varied in rhythmic diurnal pulses demonstrated by sampling every 3 hours through a 36 hours cycle [11]. Peaks occurred during the day and were roughly double the concentration of the low points. Whether or not the rhythms persist in constant light, i.e. are driven by the circadian clock, has not been determined. In phyB-l (mal) plants the patterns of GAl 2 and GAS3 peaks were the same, but the timing and shape of peaks of GA20

and GAl were altered [11]. GA20 and GAl were at maximum concentrations near lights on and decreased to minimums near lights off. Thus the phytochrome B deficient, tall genotype experiences its maximum levels of GAl near dawn while plants with the PHYB gene exhibit the same maximum after noon.

3. Circadian Production of Ethylene in Sorghum

Circadian production of ethylene has been noted in several species (see review in [18]). The phenomenon has not been linked to specific physiological processes; however, recently Kathiresan et al. [14] found that ACC oxidase mRNA and enzyme activity exhibited a circadian rhythm in Stellaria longipes. We have examined the production of ethylene by sorghum seedlings growing in test tubes and each three hours the tubes were capped briefly with rubber serum stoppers and sampled. The sorghum genotypes containing mal (58M) and ma3 (90M) exhibited rhythmic variations in ethylene production rates (Fig. 1). 90M contains immunologically detectable phytochrome B and had ethylene peaks with very small amplitudes (data for Ma3-containing 100M were similar). The genotype containing the phyB-l allele behaved as if the regulation of ethylene biosynthesis was de-repressed. It exhibited peaks of ethylene synthesis that were lO-fold higher than the PHYB containing genotypes. The fact that ethylene production follows a diurnal pattern in 58M indicates that of control of timing persists in the absence of phytochrome B.

'"" 40 r-~~-----------....,.

i 30 ~ "2, ~ ;:~ 2' . 2.~ /'" o 20 - ~, ! Q'Q i 10 _0 •••.•• ~·O.O·O •.•.••• ! •.•. . •.•.• ' ~ 0 - •••••••••• I ••

800 2000 800 2000 time

Figure 1. Ethylene production rate of whole sorghum plants of genotypes of 58M (mal) and 90M (ma3). Analysis started 6 days after planting. Dark bar indicates dark period.

107

When 58M plants were moved into continuous light, the rhythmic production of ethylene persisted indicating that the phenomenon is circadian clock driven (data not given). In addition, when sorghum was germinated and maintained in the dark, no rhythm in ethylene production was apparent (data not given). Initial experiments to study rhythmicity in ethylene production employed a 12 hour lightl12 hour dark photoperiod and a synchronous 31122°C thermoperiod. We verified that the day/night ethylene production rhythm persisted at shorter or longer day lengths (data not given). Subsequently we determined that a day/night temperature rhythm as well as a rhythm of light and dark were necessary to establish the pattern of ethylene production illustrated in Figure l. The typical circadian rhythm did not occur if plants were maintained at uniform temperature and subjected to 12 hour photoperiods (data not given).

Because Kathiresan et al. [14] have shown that Stellaria longipes exhibits light and temperature entrainment of ACC oxidase (ACCO) activity and mRNA, we secured a cDNA for ACCO from rice and used it to probe for ACCO mRNA in extracts of 58M and 100M. Shoots of 58M showed an increase of ACCO mRNA during the day (Fig. 2) parallel to the pattern of ethylene production revealed in Figure l. Roots of 58M did not demonstrate significant levels of ACCO mRNA or any detectable diurnal pattern nor did either roots or shoots of 100M.

4. Shade Avoidance Syndrome

The pleiotropic symptoms of the phyB-1 gene in sorghum are parallel with the symptoms of the shade avoidance syndrome studied by Smith [26] and others (Table 1). Previously, ma/·containing 58M was shown to accumulate about 38% more shoot dry weight in 14 days than Ma3-containing 100M (1). Recent studies revealed that the mal mutant genotype has smaller roots and larger shoots (dry weight) than

108

90M and 100M (data not given). Thus in regards to shoot/root ratio as well as other symptoms, phyB-l-containing sorghum duplicates the shade avoidance syndrome. Other phytochrome B mutants (Arabidopsis, cucumber, tomato, and Brassica rapa) exhibit symptoms similar to 5SM sorghum (see review in [5]), and thus they also duplicate the shade avoidance syndrome [26]. Evidence indicates that phytochrome B is the major sensor of shade and regulator of shade avoidance reactions [26], and thus the phytochrome B mutants constitutively over express these reactions.

Roots Shoots

- 1.4 kb

5 8 11 14 17 20 23 2 5 8 11 14 17 20 23 2

1 DDM - 1.4 kb

Figure 2. Diurnal expression of ACCO mRNA in sorghum genotypes S8M and 100M. Total RNA was extracted from sorghum genotypes S8M and 100M (6 days old) harvested every three hours under a 12/12 (31122°C) photoperiod, with lights on at 8:00. The RNA gel blot (10 J!g of total RNA per lane) was hybridized at high stringency with a cDNA encoding ACCO from rice (kindly provided by H. Kende).

TABLE 1. Comparison of the Phenotype of ma3 R -containing, Phytochrome B-deficient Sorghum with the Shade Avoidance Syndrome

mal PHENOTYPE1

longer internodes longer, narrow leaves reduced leaf area reduced tillering early flowering reduced chlorophyll altered root/shoot ratio 2 reduced anthocyanins

SHADE AVOIDANCE3

longer internodes altered leaf shape reduced leaf area increased apical dominance early flowering reduced chlorophyll altered assimilate distribution not documented

lPao and Morgan [19]; Beall et al. [1]; Childs et aL [6] 2Unpublished data, Lee et aL 3Smith [26]

109

5. Aerenchyma Formation, Shade Avoidance and Ethylene

Several root stresses such as reduced O2 levels (hypoxia), physical impedance and nutrient depletion (-N, -P) induce the formation of aerenchyma in roots of maize [8, 9, 12, 24], another tropical C-4 monocot. These air spaces represent areas where cells are digested. Over production of ethylene or increased sensitivity to ethylene has been linked causally to the formation ofaerenchyma in maize roots [8,9, 12, 13], and this suggests that the high ethylene production seen in 58M (Fig.1) and the high shoot/root ratio (data not given) may be related. Although a preliminary experiment indicated higher ethylene production in 58M roots [17], three replicated experiments revealed the following rates (J1lIgFWIh): 58M shoots, 325 ± 20; 58M roots, 69 ± 13; 100M shoots, 34 ± 19; 100M roots, 71 ± 16. Ethylene production rates are similar in roots of 58M and 100M, but the shoots of 58M produce ethylene at rates almost 10-fold higher than 100M. In preliminary experiments, 58M had a higher degree of aerenchyma development than 90M or 100M and hypoxia induced aerenchyma in the later genotypes but not the former (data not given). These observations are being verified, but presently the aberrant distribution of dry matter between shoot and root in 58M compared to 100M appears related to high ethylene production in the 58M shoot. This ethylene may inhibit translocation of assimilates to the roots or it may stimulate shoot growth as known for deep water rice [16]. The apparent slightly higher level of aerenchyma development in 58M may well reflect a change in sensitivity to ethylene as noted in nutrient deficiencies for maize [13], or the effect of shoot-produced ethylene diffusing into the roots. Regardless of the mechanism involved, it seems possible that ethylene may be involved in "normal" shade avoidance behavior occurring in dense stands of monocultures (small grain fields) as implicated here in the "atypical" shade avoidance behavior caused by a mutation of thePHYB gene.

6. Discussion

The recently observed circadian rhythm of ethylene production in phyB-l-containing 58M suggests that some or all of the pleiotropic symptoms of that genotype may be due to ethylene or possibly the timing of ethylene biosynthesis. Current models of biological clocks based on studies with bread mold and fruit fly [7, 25] propose that the clock is composed of one or more genes whose products regulate expression of the clock genes and other genes in a circadian fashion. If this model applies to plants, then it is apparent that expression of certain genes (flowering, shade avoidance, etc.) may be more sensitive to signals at certain times of the day or night. Hormones produced in circadian pulses could be involved in this signaling. We have presented evidence that both GAs and ethylene are produced in rhythmic pulses, that the phyB-1 mutation alters the magnitude or timing of these pulses and therefore these hormone pulses may be involved in the multiple symptoms caused by this single gene mutation.

110

7. Acknowledgments

Supported by USDA-NCRIGP grants #91-31304-6482 (PWM), #94-37300-0331 (JEM), #93-37100-8922 (MCD), #93-37100-8752 (PWM) and Texas Higher Education Board grant #999902-87 (PWM). ACC oxidase eDNA probe supplied by Dr. Hans Kende, Michigan State University.

8. References

1. Beall, F.D., Morgan, P.W., Mander, L.N., Miller, F.R., and Babb, KH. (1991) Genetic regulation of development in Sorghum bieolor. V. The mal allele results in gibberellin enrichment, Plant Physiol. 95, 116-125.

2. Cherry, lR., Hondred, D., Walker, J.M., Keller, J.M., Hershey, H.P., and Vierstra, R.D. (1993) Carboxy-terminal deletion analysis of oat phytochrome A reveals the presence of separate domains required for structure and biological activity, Plant Cell 5, 565-575.

3. Childs, KL., Cordonnier-Pratt, M.-M., Pratt, L.H., and Morgan ,P.W. (1992) Genetic regulation of development in Sorghum bieolor. VII. mal flowering mutant lacks a phytochrome that predominates in green tissue, Plant Physiol. 99, 765-770.

4. Childs, KL., Lu, J.-L., Mullet, lE., and Morgan, P.W. (1995) Genetic regulation of development in Sorghum bieolor. X. Greatly attenuated photoperiod sensitivity in a phytochrome-deficient sorghum possessing a biological clock but lacking a red light-high irradiance response, Plant Physiol. 108,345-351.

5. Childs, KL., Miller, F.R., Cordonnier-Pratt, M.-M., Pratt, L.H., Morgan, P.W., and Mullet, J.E. (1996) The Sorghum bieolor photoperiod sensitive gene, MaJ, encodes a Phytochrome B, Plant Physiol.( in press).

6. Childs, KL., Pratt, L.H., and Morgan, P.W. (1991) Genetic regulation of development in Sorghum bieolor. VI. The mal allele results in abnormal phytochrome physiology, Plant Physiol97, 714-719.

7. Crosthwaite, S.K., Loros, J.l, and Dunlap, J.C. (1995) Light-induced resetting of a circadian clock is mediated by a rapid increase in frequency transcript, Cell 8, 1003-10 12.

8. Drew, M.C., He, C.l, and Morgan, P.W. (1989) Decreased ethylene biosynthesis, and induction of aerenchyma, by nitrogen- or phosphate-starvation in adventitious roots of Zea mays L, Plant Physiol. 91,266-271.

9. Drew, M.C., Jackson, M.B., and Giffard, S. (1979) Ethylene-promoter adventitious rooting and development of cortical air spaces (aerenchyma) in roots may be adaptive responses to flooding in Zea mays L., Planta 147, 83-88.

10. Edgerton, M.D. and Jones, AM. (1993) Subunit interactions in the carboxy-terminal domain of phytochrome, Bioehem. 32, 8239-8245.

11. Foster, KR. and Morgan, P.W. (1995) Genetic regulation of development in Sorghum bieolor. IX. The mal allele disrupts diurnal control of gibberellin biosynthesis, Plant Physiol. 108, 337-343.

12. He, C.-J., Finlayson, SA, Drew, M.C., Jordan, W.R., and Morgan, P.W. (1996) Ethylene biosynthesis during aerenchyma formation in roots of Zea mays L. subjected to mechanical impedance, Plant Physiol. (in press).

13. He, C.-l, Morgan, P.W., and Drew, M.C. (1992) Enhanced sensitivity to ethylene in nitrogen- and phosphate-starved roots of Zea mays L. during aerenchyma formation, Plant Physiol. 98, 137-142.

14. Kathiresan, A, Reid, D.M., and Chinnappa C.C. (1996) Light- and temperature-entrained circadian regulation of activity and mRNA accumulation of l-arninocyclopropane-l-carboxylic acid oxidase in Stellaria longipes, Planta (in press).

15. Lumsden, P.J. (1991) Circadian rhythms and phytochrome, Annu Rev Plant Physiol Plant Mol Bioi 42, 351-371.

16. Metraux, J.P. and Kende, H. (1983) The role of ethylene in the growth response of submerged deep water rice, Plant Physiol. 72,441-446.

17. Morgan, P.W., Childs, KL., Foster, KR., Lee, I.-J., Finlayson ,SA, Ulanch, P.E., Mullet, J.A,

111

Miller, F.R. (1996) Sorghum mutants and photoperiodic flowering, in W.R. Briggs, R.L. Heath, E.M. Tobin (eds.), Regulation of Plant Growth and Development By Light, Amer. Soc. Plant Physiol. Rockville, MD, (in press).

18. Morgan, P.W., He, C.-J., De Greef, JA and De Proft (1990) Does water deficit stress promote ethylene synthesis by intact plants? Plant Physiol. 94, 1616-1624.

19. Pao, C.-I. and Morgan, P.W. (1986) Genetic regulation of development in Sorghum bicolor, I. Role of the maturity genes, Plant Physiol. 82, 575-580.

20. Pao, C.-I. and Morgan, P.W. (1986) Genetic regulation of development in Sorghum bicolor. II. Effect of the mal allele mimicked by G~, Plant Physiol. 82, 581-584.

21. Paulsen, H., Bogorad, L. (1988) Diurnal and circadian rhythms in the accumulation and synthesis of mRNA for the light-harvesting chlorophyll alb-binding protein in tobacco, Plant Physiol. 88, 1104-1109.

22. Quinby, J.R. (1973) The genetic control of flowering and growth in sorghum, in N.C. Brady (ed.). Advances in Agronomy, vol 25, Academic Press inc., New York, pp. 125-162.

23. Quinby, J.R. and Karper, R.E. (1961) Inheritance of duration of growth in the Milo group of sorghum, Crop Sci. 1,8-10.

24. Sarquis, J.I., Jordan, W.R., Morgan, P.W. (1991) Ethylene evolution from maize (Zea mays L.) seedling roots and shoots in response to physical impedance, Plant Physiol. 96, 1171-1177.

25. Sassone-Corsi, P. (1994) Rhythmic transcription and autoregulated loops: Winding up the biological clock, Cell 78, 361-364.

26. Smith, H. (1992) Ecological functions of the phytochrome family. Clues to a transgenic programme of crop improvement, PhotochemPhotobiol56, 815-822.

27. Sweeney, B.M. (1987) RhythmiC Phenomena In Plants, 2nd ed., Academic Press, San Diego. 28. Vince-Prue, D. (1994) The duration of light and photoperiodic responses, in R.E. Kendrick and

G.H.M. Kronenberg, (eds.), Photomorphogenesis in Plants, Kluwer Academic, Boston, pp. 447-490.

ETHYLENE INVOLVEMENT IN THE DORMANCY OF AMARANTHUS SEEDS

J. KEPCZYNSKI,M. BIHUN, E. KEPCmNSKA Department of Plant Physiology, University of Szczecin, Felczaka 3a, 7 J -4 J 2 Szczecin, Poland

1. Introduction

Ethylene stimulation of seed gennination has been known since the 1920s [5]. At present, it is an established fact that the application of ethylene or the ethylene­releasing compound, ethephon, breaks primary dormancy, secondary dormancy in seeds and accelerates the gennination of non-dormant seeds in a large number of species [3,6,12]. Ethylene, ethephon or a precursor of ethylene biosynthesis, 1-aminocyclopropane-l-carboxylic acid (ACC) stimulates the gennination of primary dormant Amaranthus retroflexus [23], [16], secondary dormant Amaranthus paniculatus [18] and non-dormantAmaranthus caudatus [17] seeds. The inhibition of A. caudatus seed germination caused by osmoticum, abscisic acid [ABA] or methyl jasmonate [JA-Me] can be alleviated by ethephon [14,15]. Ethephon or ACC reversed the inhibitory effect of inhibitors of gibberellin biosynthesis, tetcyclacis or paclobutrazol, on A. caudatus seed gennination, suggesting an ethylene-dependent action of gibberellins [13,19]. No or insufficient information on the involvement of endogenous ethylene in dormancy of Amaranthus or other seeds is available.

This paper will consider the participation of exogenous and endogenous ethylene in the regulation primary dormancy, and induction and release of secondary dormancy inAmaranthus seeds.

2. Primary Dormancy

For the studies involving primary dormancy seeds Amaranthus retroflexus weed was used. Freshly harvested seeds genninated partially in darkness at 35°C and were almost unable to genninate at 30°C (Fig. 1 ).

These seeds are regarded as partially dormant at 35°C and dormant at 30°C. Dry storage of seeds for a few months at 25°C breaks dormancy. When seeds were dry stored at 25°C, they genninated almost completely at 30°C or 35°C and began to genninate at 25°C.

113

A. K. Karrellis et al. (etis.), Biology and Biotechnology of the Plant Hormorre Ethylerre, 113-122. © 1997 Kluwer Academic Publishers.

114

~

§

:1 Q)

0

100

80

60

40

20

o +--------'------,--------'----,--25 30

Temperature,OC

35

Figure 1. The effect of temperature on the gennination of freshly harvested

[0] or dry stored [II] A. retroflexus seeds after 7 days. (K.,;pczynski el at.

unpublished data).

Exogenous ethylene, which breaks primary dormancy in, e.g., apple [20], cocklebur [6], subterranean clover [7] and sunflower [2] seeds, strongly stimulated the germination of A. retroflexus dormant seeds at 30°C (Fig. 2). This plant growth regulator, at concentration of 10 ~ allowed the germination of almost all the seeds. This is in agreement with the previous findings of Schonbeck and Egley [23] and K~pczynski et al.[16]. A stimulatory effect of exogenous ethylene suggests a function of endogenous ethylene in the regulation of dormancy release. The reaction of seeds to exogenous ethylene indicates that seeds are equipped with an ethylene response -mechanism.

In order to determine whether ethylene action is physiological, norbornadiene [NED], a competitive inhibitor of ethylene action [22], was used. Previously, it was observed that NBD inhibits the germination of non-dormant A. caudatus and this inhibition was counteracted by ethylene, indicating the requirement of ethylene action for the germination of these seeds [17]. An application of NBD in combination with ethylene partially or almost completely negated the stimulation caused by ethylene (Fig. 3). The counteraction of the effect of ethylene by the inhibitor of ethylene action proves the physiological action of exogenous ethylene and supports the role of endogenous ethylene in breaking of dormancy.

~

d'

:1 G)

0

100

80

60

40

20

0 o 1

Ethylene, ,,1 r1 10

Figure 2. The effect of ethylene on the gennination of A. retroflexus seeds at 30·C after 7 days. (Kcpczynski et al. unpublished data).

100

80

~

i 60

40 G)

0

20

0 0 1 5 10

2,5-norbornadiene, ml r1

Figure 3 The effect of ethylene and 2,5-norbornadiene on the gennination of

A retroflexus seeds at 30·C after 7 days. Ethylene 0 J1I rl [0], ethylene 10

J1I rl [+]. (Kepczynski et al. unpublished data).

115

116

A precursor of ethylene biosynthesis, ACC, did not affect the germination of dormant seeds after 7 days of incubation at the moment when ethylene was active. However, it improved germination after a longer period of incubation, when it was applied at 1O-3M (data not shown).

As in other seeds [1], GA3 also breaks seed dormancy of A. retroflexus seeds [16]. The simultaneous application of norbornadiene with gibberellin partially or completely inhibited seed germination (data not shown). Thus, these data demonstrate that endogenous ethylene action is indispensable for releasing dormacy with GA3.

To develop a study on the involvement of endogenous ethylene in primary dormancy, the content of ACC, ethylene production and ACC oxidase activity in vivo in dormant and non-dormant seeds has been determined. The level of ACC was 2.8 times lower in dormant seeds after 20 h of incubation than in non-dormant ones before radicle protrusion (Table 1). Primary dormant seeds produced less ethylene than non­dormant seeds after 20 and 22 h of incubation (Table 2). Both dormant and non­dormant seeds were able to convert exogenous ACC to ethylene. The rate of ACC conversion was much less in dormant seeds than in non-dormant seeds. The lowered ethylene production by dormant seeds is probably associated with the lack of a sufficient amount of ACC.

TABLE 1. ACC content in donnant and non­

donnant A. retroflexus seeds after 20 hours

imbibition at 30·C. Seeds did not genninate after

20 h. ACC and MACC were detennined as

described by Lizada and Yang [21]. (K~pczynski

et al. unpublished data).

Seeds

D

ND

3. Secondary Dormancy

ACC pmol/g DW

432 ± 150

1209 ± 210

Non-dormant seeds of Amaranthus retroflexus and Amaranthus caudatus cv. atropurpureus did not germinate at 45°C. These seeds lose their ability to germinate when returned to a permissive temperature, thus entering a secondary dormancy termed thermodormancy. Seeds of A. caudatus germinated easily at 25°C when they are non-dormant (Fig. 4). Preincubation for 1 day at 45°C markedly inhibited germination at 25°C.

Ethylene did not affect germination at 45°C, and seeds also did not germinate after a transfer to a lower temperature (data not shown). Likewise, ethephon and ACC did

117

not stimulate gennination at this supraoptimal temperature. This indicates that ethylene did not prevent the establishment of secondary dormancy.

TABLE 2. The effect of ACC on ethylene production by dormant and non-donnant A. retroflexus seeds after 20 and 22 h of imbibition at 30oC. The seeds (200) were incubated

in flasks (4 mJ) 2 h before ethylene detennination. Numbers in brackets are percentages of

seed germination. (K~pczynski et ai. unpublished data).

Ethxlene, 1!1I100 seeds.h

Medium 20h 22h

D ND D ND

Water 6±2 37 ± 11 (1%) 22 ± 6 (0.1%) 63 ± 20 (3%)

ACC1O·3M 46± 20 664 ± 221 ~3%) 129 ± 200.4%) 805 ± 49 ~7%)

The content of ACC and malonyl-ACC [MACC] in seeds preincubated for 1 day at 45°C was similar as dry seeds (Table 3). However, the preincubation at this high temperature lowered the activity of the ACC oxidase in vitro. Previously, Gallardo et al. [11] found that supraoptimal temperatures reduced ACC oxidase activity in vivo in Cicer arietinum seeds. ACC oxidase activity was much lower in seeds incubated at 45°C than in seeds incubated at 25°C (data not shown). ACC oxidase activity in seeds incubated at 25°C rose with increased seed gennination. Thus, incubation at 45°C inhibits both the activity of ACC oxidase and seed gennination.

TABLE 3. Levels of ACC, MACC and ACC oxidase activity inA.caudatus seeds

treated with 45°C for 24 h. ACC oxidase activity in vitro was determined as described

by Fernandez-Maculet and Yang [9]. (K~pczYliski and Bihun, unpublished data).

Treatment ACC MACC nmollg DW nmollg DW

Dry seeds 0.6 ± 0.1 1. 7 ± 0.3

Seeds treated at 45°C 0.6 ± 0.2 1.5 ± 0.6

In vitro ACC oxidase activity: J!!DolIg DW

282 ± 10

127 ± 35

It was demonstrated that ethylene removed thermodormancy in sunflower seeds [3]. Ethylene was also very effective in releasing the secondary dormancy in A. caudatus seeds. Even after 3 days of incubation in the presence of ethylene, complete germination was observed (Fig. 5). This confirms the findings that the ethylene

118

releasing compound, ethephon, caused the total germination of thermodormant A. paniculatus [18] and A. retroflexus [K~pczynski et at. unpubl.] seeds. A requirement of exogenous ethylene for the germination of secondary dormant e.g. cocklebur [8] and lettuce [10] seeds is also well known.

100

80

~

§ 60

:1 40

Q.) e,:,

20

0 0 1 2 3 4 5

Time, days

Figure 4. Gennination of non-donnant (0 - untreated) and thennodonnant

(e - pretreated at 45°C for 1 day) A. caudatus seeds at 25°C. (Kepczynski and Bihun, unpublished data).

Replacing ethylene with a precursor of ethylene biosynthesis caused a similar effect (Fig. 6). However, ACC was less effective in comparison to ethylene. Since ACC could remove secondary dormancy, endogenous ethylene is probably involved in the regulation of the germination of these seeds.

ACC oxidase activity in vivo is probably capable of converting exogenous ACC to ethylene. These data also suggest that the inability of secondary dormant seeds to germinate is related to a lack of sufficient endogenous ACC.

In the following experiment, the content of free ACC in dormant and non-dormant seeds incubated for 10 h at 25°C was measured (Table 4). Dormant seeds contain 3 times more ACC than non-dormant ones. Thus, the higher content of ACC in dormant than in non-dormant seeds can be related to the activation of ACC synthase activity by high temperature. The activation of ACC synthase activity by a high temperature in C. arietinurn seeds has been previously suggested [11]. Dormant and non-dormant seeds showed a similar ability to produce ethylene [Table 5]. Exogenous ACC did not increase ethylene production in dormant seeds but markedly increased it in non-

119

dormant ones. The partial inhibition of exogenous ACC conversion to ethylene due to thermodormancy of sunflower seeds was observed earlier [4]. ACC oxidase activity probably will develop during a later stage of imbibition of dormant A. caudatus seeds since exogenous ACC removed dormancy.

100

80

~

:1 60

40 0 t!)

20

0 ND 0 0,1 1 10

Ethylene, ILl r1

Figure 5. The effect of ethylene on the gennination of secondary donnant A.

caudatus seeds after 3 [0] and 5 days r-:J at 25°C. (K~pczynskiand Bihun,

unpublished data).

TABLE 4. ACC content in secondary donnant

and non-donnantA. caudatus seeds after 10 h of

imbibition at 25°C. Seeds did not genninate after

10 h. (K~pczynski and Bihun, unpublished data).

Seeds ACC pMlg DW

D 1800± 300

ND 600 ± 100

120

100

80

~

§ 60

:1 d)

40 ~

20

0 0 1 2 3 4 5

Time, days

Figure 6. The effect of ACC on the gennination of secondary donnant A. caudatus seeds at 25°C. Non-donnant [0], donnant IACC OM [0], ACC

1O,sM [+], ACC 104 M [*1, ACC 1O·3M [0]/. (K\;pczynski and Bihun,

unpublished data).

TABLE 5. The effect of ACC on ethylene production by secondary

donnant and non-donnant A. caudatus seeds after 10 h of imbibition

at 25°C. The seeds (200) were incubated in flasks [4 mI] 2 h before

ethylene detennination. Numbers in brackets are percentages of seed

gennination. (K\;pczynski and Bihun, unpublished data).

Medium D ND

12 ± 7 14 ± 5

10 ± 4(0.8%) 172 ± 30 (1.4%)

4. Final Remarks

Exogenous ethylene is a limiting factor for the germination of primary dormant A. retroflexus seeds. Primary dormancy is characterized by a lack of a sufficient amount of ACC, as was suggested for sunflower seeds [2], by low ACC oxidase activity and reduced ethylene production. The lower content of ACC in dormant than in non-

121

dormant seeds may suggests lower activity of ACC synthase in dormant seeds. The weak effect of exogenous ACC on primary dormant seed gennination may be associated with a low ability of seeds to convert ACC to ethylene. However, the lower sensitivity of dormant seeds than non-dormant seeds to ethylene could be also considered.

Secondary dormancy induction in A. caudatus seeds cannot be prevented by ethylene; however, exogenous ethylene is a limiting factor for the gennination of these seeds. High temperature, which induces dormancy, decreases the ACC oxidase activity, and presumably, increases ACC synthase activity at 25°C. The inability of secondary dormant seeds to genninate is probably associated with the lack of a sufficient amount of endogenous ACC in order to produce a suitable concentration of ethylene for gennination. Secondary dormant seeds probably require more ethylene than non-dormant seeds.

5. Acknowledgments

This work was partially supported by the U.S. Polish Maria Sklodowska-Curie Joint Fund II, PL-ARS-193 [MRJUSDA-92-92]

6. References

1. Bewley, J.D. and Black, M. (1994) Seeds - Physiology of Development and Germination, Plenum

Press, New York, London.

2. Corbineau, F. and Come, D. (1992) Germination of sunflower seeds and its regulation by ethylene, in

Fu Jiarui and AA Khan (eds.), Advances in the Science and Technology of Seeds, Science Press, Beijing, New York, pp. 277-287.

3. Corbineau, F. and Come, D. (1995) Control of seed germination and dormancy by the gaseous

environment, in J. Kigel and G. Galili (eds.), Seed Development and Germination, Marcel Dekker

Inc, New York, Basel Hong Kong, pp. 397-424.

4. Corbineau, F, Rudnicki, R.M., and Come, D. (1989) ACC conversion to ethylene by sunflower seeds

in relation to maturation, germination and thermodormancy, Plant Growth Regul. 8, 105-115.

5. Egley, G.H. (1982) Ethylene stimulation of weed seed germination, Agriculture and Forestry Bulletin 5, 13-18.

6. Esashi, Y. (1991) Ethylene and seed germination, in AK. Mattoo and IC. Suttle (eds.), The Plant Hormone Ethylene, CRC Press, Boca Raton, Florida, pp. 133-157.

7. Esashi, Y. and Leopold, AC. (1969) Dormancy regulation in subterranean clover seeds by ethylene,

Plant Physiology 44, 1470-1472.

8. Esashi, Y., Okazaki, M., Yanai, N. and Hishinuma, K. (1978) Control of the germination of secondary dormant cocklebur seeds by various germination stimulants, Plant and CellPhysioL 19, 1497-1506.

9. Fernandez-Maculet, J. and Yang, S.F. (1992) Extraction and partial characterization of the ethylene­forming enzyme from apple fruit, Plant Physiol. 99, 751-754.

122

10. Fu, JoR and Yang, S.F. (1983) Release of heat pretreatment-induced donnancy in lettuce seeds by

ethylene or cytokinin in relation to the production of ethylene and the synthesis of ACC during

gennination, J. Plant Growth ReguL 2, 185-192. 11. Gallardo, M., Delgado, M.M., Sanchez-Calle, I.M. and Matilla, AJ. (1991) Ethylene production and

l-aminocyclopropane-l-carboxylic acid conjugation in therm.oinhibited Cicer arietinum L. seeds,

PlantPhysioI97,122-127. 12. K<;pczynski, J. (1985) The role of ethylene in seed germination, Acta Horti. 167,47-56. 13. K<;pczynski, J. (1986) Ethylene-dependent action of gibberellin in seed germination of Amaranthus

caudatus, PhysioL Plant. 67, 584-587. 14. K<;pczynskl, J. (1986) Inhibition of Amaranthus caudatus seed germination by polyethylene g1ycol-

6000 and abscisic acid and its reversal by ethephon or l-aminocyclopropane-l-carboxylic acid,

PhysioL Plant. 67,588-591. 15. K<;pczynski, J. and Bialecka, B. (1994) Stimulatory effect ofethephon, ACC, gibberellin A3 and ~+7

on germination of methyl jasmonate inhibited Amaranthus caudatus L. seeds, Plant Growth Regul.

14,211-216. 16. K<;pczynski, J., Corbineau, F., and Come D. (1996) Responsiveness of Amaranth us retroflexus seeds

to ethephon, l-aminocyclopropane-l-carboxylic acid and gibberellic acid in relation to temperature

and dormancy, Plant Growth Regul. (in press)

17. K<;pczynski, J. and Karssen, C.M. (1985) Requirement for the action of endogenous ethylene during

gennination of non-dormant seeds of Amaranthus caudatus, PhysioL Plant. 63, 49-52. 18. K<;pczynski, J., K<;pczynska, E. (1992) The effect of putrescine, ethephon and ACC on germination of

therm.odormant Amaranthus paniculatus L. seeds. in D. Come and F. Corbineau (eds.), Fourth International Workshop on Seeds. Basis and Applied Aspects of Seed Biology, ASFIS, Paris, pp.-

537-542.

19. Kcpczynski, J., K<;pczynska, E., and Knypl, J.S. (1988) Effects of gibberellic acid and 1-aminocyclopropane-l-carboxylic acid on germination of Amaranthus caudatus seeds inhibited by

paclobutrazol, J. Plant Growth ReguL 7, 59-66. 20. K<;pczynski, J., Rudnicki, R.M., and Khan, AA (1977) Ethylene requirement for gennination of

partly after-ripened apple embryo, Physiol. Plant. 40,292-295. 21. Lizada, M.C.C., and Yang, S.F. (1979) A simple and sensitive assay for l-aminocyclopropane-l­

carboxylic acid, AnaL Biochem. 100, 140-145. 22. Sisler, E. and Yang, S.F. (1984) Ethylene, the gaseous plant hormone, BioScience 34, 234-238. 23. Schonbeck, M.W. and Egley,G.H. (1981) Phase sequence of redroot pigweed seed germination

responses to ethylene and other stimuli, Plant Physiol. 68, 175-179.

CONTROL OF GENE TRANSCRIPTION BY ETHYLENE DURING TOMATO FRUIT RIPENING

J. DEIKMAN, S.A. COUPE, AND R. XU Department of Biology, 208 Mueller Laboratory, Pennsylvania State University, University Park, PA 16802, USA

1. Introduction

The effect of ethylene on fruit ripening has been exploited since biblical times, when gashing of figs was practiced to enhance ripening [1]. It is now known that this treatment would result in the production of wound ethylene that would promote ripening. In climacteric fruit such as tomato, the onset of fruit ripening is normally accompanied by a large increase in ethylene biosynthesis, which is autocatalytic [2]. Tomato fruit ripening involves the autolysis of cell wall pectins, the synthesis of lycopene and other carotenoid pigments, and changes in the acid and sugar content associated with taste [3]. The role of ethylene in controlling fruit ripening is supported by several decades of physiological experiments, and has recently been confirmed by analysis of transgenic tomato plants that are impaired in ethylene production [3]. Fruit with greatly reduced levels of endogenous ethylene either failed to ripen or had significantly retarded ripening. Also, a tomato mutant, Never-ripe, which is impaired in fruit ripening, has recently been shown to be defective in an ethylene receptor [4].

Ethylene controls ripening processes at least in part by activation of gene transcription. In tomato, a set of genes, which includes E4 and E8, was shown to be transcriptionally activated by ethylene when unripe fruit were treated with ethylene [5]. The mRNA levels for E4 and E8 increase within 30 minutes of exposure of unripe fruit to ethylene [5]. Considering that it takes approximately 15 minutes for ethylene gas to achieve 50% of its final concentration in fruit [6], this response is quite rapid and may represent a primary genetic response to ethylene. Although its function during fruit ripening is not yet known, the predicted polypeptide encoded by E4 has significant sequence identity with a peptide methionine sulfoxide reductase protein from Escherichia coli [7]. The amino acid sequence of the E8 gene indicates that it is a dioxygenase, and it has significant similarity to ACC oxidase [8]. Analysis of antisense suppression of E8 in transgenic plants has shown that E8 negatively regulates ethylene biosynthesis in fruit [9].

The regulation of the E4 and E8 genes by ethylene has been extensively characterized. Transcription of E4 is rapidly activated by ethylene in both leaves and fruit, and E4 is not expressed in mutant or transgenic fruit which do not produce

123

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 123-131. © 1997 Kluwer Academic Publishers.

124

ethylene and do not ripen [10-13]. Treatment of ripening inhibitor (rin) mutant fruit with ethylene results in normal levels of E4 mRNA accumulation [11]. Thus, the expression of E4 appears to have an absolute requirement for elevated levels of ethylene, and its expression is not tissue-specific. In contrast, expression of the E8 gene is more fruit-specific. E8 is highly induced by ethylene in tomato fruit but is not induced in response to ethylene in leaves [10). In addition, E8 can be expressed in ripening fruit in the absence of ethylene. For example, in fruit from the ripening mutant rin, E8 is expressed at approximately 30% of its normal level [11]. The level of E8 expression increases to that of wild-type fruit if the rin fruit are treated with ethylene. E8 is also expressed in transgenic tomato fruit with greatly reduced ethylene levels [13]. Thus, although E4 and E8 expression during fruit ripening is coordinate, E8 is controlled both by ethylene and by separate developmental signals, while E4 expression is strictly dependent on ethylene. Comparison of the molecular mechanism of E4 and E8 gene activation should be valuable for discovering the essential elements of ethylene responsive gene transcription, as well as the basis for tissue-specific ethylene responses.

2. cis-active DNA sequences required for ethylene responsive expression of E4 andES

DNA sequences required for ethylene-responsive transcription of both E4 and E8 were initially defined by 5' deletion analyses in stably transformed plants [14, 7). This work indicated that sequences required for E8 response to ethylene are located between -2181 and -1088 bp relative to the transcription start site, but an E4 deletion to -193 was still ethylene responsive (Fig. 1). It is important to note that a 5' deletion mutation identifies the 5' boundary for an ethylene-response element, but it does not indicate the 3' boundary. For example, it is possible that sequences 3' to -1088 are also required for ethylene responsiveness of E8.

Additional promoter deletions of an E4-1uciferase (LUC) gene were tested in a transient assay system in which DNA was delivered to pericarp pieces by particle bombardment [7]. These studies demonstrated that sequences required for both ethylene responsiveness and fruit ripening regulation of the E4 gene are contained within 161 bp of the E4 transcription start site. Furthermore, an internal deletion of sequences from -193 to -85 within the 1421 bp promoter region almost completely eliminated expression of the E4-LUC gene in both ethylene-treated unripe fruit and in ripening fruit [7].

Figure 1 compares the locations of DNA sequences required for ethylene responsiveness of the E4 and E8 genes. It also identifies regions of the E8 promoter that are important in expression during fruit ripening, in response to ethylene­independent signals. Interestingly, the differences in the E4 and E8 expression patterns are reflected in these data. That is, E8 is expressed both in response to ethylene and in response to ethylene-independent fruit ripening signals, and promoter elements involved in regulation of the E8 gene in response to each of these signals

125

were identified. In contrast, the E4 gene requires ethylene for its expression, and sequences required for ethylene responsiveness of E4 were not separated from sequences required for expression during fruit ripening.

-1421

-409 -263

-161 to -85 I

-34 to -18

Figure 1. Promoter regions required for ethylene-regulated and ethylene-independent expression of E4 and E8, and selected DNA-binding proteins that interact with these regions. Hatched boxes, sequences required for ethylene response; R, sequences required for expression in response to ethylene-independent ripening signals; white boxes, transcribed regions of E8 and E4 genes; *, TAT A box; hatched ball, DNA-binding protein that interacts with sequences from -142 to -110 ofE4 promoter.

3. DNA-binding Proteins that Interact with E4 and E8 5' Flanking Sequences

A DNA-binding protein that interacts with 5' flanking sequences of both the E4 and E8 genes was identified, and its recognition sequences were determined by methylation interference assay [8, 15]. The binding site for this protein spans the E4 TATA box but is located from position -936 to -920 upstream of the E8 transcription start, and sequences in common between the two sites are those that flank the E4 TAT A sequences [15]. We have named this protein the E41E8 Binding Protein (E41E8BP). Although the E41E8BP does not interact directly with the sequences shown to be required for ethylene responsiveness, in the case of both the E4 and E8 genes it interacts with sequences 3' to those necessary for ethylene response (Fig. 1). This similarity in promoter structure is intriguing and suggests that the E41E8BP may be involved in ethylene response.

The binding activity ofE41E8BP correlates with the expression of E4 and E8. That is, it is greater in extracts from ripening fruit than in extracts from unripe fruit [15]. However, the binding activity does not increase significantly in response to ethylene [8]. Although the DNA-binding activity is not ethylene-dependent, this protein may still participate in ethylene-responsive gene transcription. In some cases of inducible

126

gene transcription, transcription factor binding is constitutive, but the factor does not activate transcription until modified by other proteins such as kinases [16].

A second nuclear protein was shown to interact with E4 sequences from -142 to -DO, within the region required for E4 response to ethylene [7]. However, this DNA­binding activity did not correlate with E4 gene transcription. Instead, it had greatest activity in nuclear extracts from unripe fruit, its activity was reduced in extracts from ethylene-treated unripe fruit, and was absent in extracts from ripening fruit [7]. This protein could either be a repressor that interacts with sequences close to those of the binding site of a positive regulator of E4 transcription, or its affinity for the DNA could be reduced in its activated state [7]. In either case, the ethylene-responsiveness of this DNA-binding activity suggest that it is involved in controlling expression of the E4 gene in response to ethylene.

4. Detailed Analysis of E4 Promoter Structure

Because the regulation of the E4 gene in response to ethylene appears to be simpler than that of E8, we have focused on it, and have carried out a more detailed study of the sequences necessary for ethylene responsive expression during fruit ripening. For this analysis, we have continued to use the transient assay system shown by Montgomery et al. [7] to reproduce the in vivo pattern of E4 expression.

Our results indicated that E4 transcription in response to ethylene requires the interaction of two cooperative cis-elements [17]. When sequences from -193 to -85, or from -193 to -40 that are required for ethylene response were fused to a minimal (46 bp) Cauliflower Mosaic Virus (CaMV) 35S promoter-LUe-NOS gene, the resulting constructs were not ethylene responsive. As a complementary experiment, the 35S enhancer was fused to the E4 promoter truncated to -85, and this construct was also not ethylene responsive, indicating that sequences downstream from -85 bp do not contain sufficient information for ethylene-responsive transcription. Some possibility remained that spacing of the upstream element is extremely critical with respect to the TATA box, so that when the upstream ethylene-responsive sequences were fused to the minimal 35S TAT A the construct was not ethylene responsive. However, two pieces of evidence make this possibility unlikely. The first is that when we introduced a 2 bp insertion between the upstream and the downstream element, we found that there was a large negative effect on the level of expression of the construct [17]. However, the mutated construct was still ethylene responsive. In contrast, our constructs with the upstream element fused to the minimal 35S promoter have greater activity in ripening fruit than the minimal 35S promoter alone, but they are not ethylene responsive. Secondly, in our constructs with the upstream element fused to the minimal 35S promoter, the spacing between the upstream element and the TAT A box is altered from wild type by 10 bp. A 10 bp difference represents approximately one turn of the DNA double helix, and should have a minimal effect on protein/protein interactions. We concluded that the region from -193 to -85 must act cooperatively with a specific cis-element in the region of the E4 TATA box to produce an ethylene responsive

127

promoter, and refer to these two elements as the upstream and downstream elements (Fig. 2).

To further define the upstream element, a linker scan analysis was carried out in which a series of 10 bp substitutions was made each 10 bp from -160 to -91 [17]. This experiment indicated that sequences from -150 to -121 are most important for ethylene responsiveness. Interestingly, these sequences overlap, but do not perfectly coincide, with the binding site for the protein identified by Montgomery et al. [7], which extended from -142 to -110. The failure of these two sequences to align perfectly suggests that they represent interaction of different proteins. For example, perhaps the identified DNA-binding protein that has greater activity in extracts of unripe fruit than in ethylene-treated unripe fruit or ripening fruit is a repressor which is displaced by a positive regulator that interacts with the sequences identified by the linker scan analysis.

-150 ACAAGTTTG'I'l'TTTG'l'TTTTACTACCAACA -121

/ -34 ATTTCTATATAAAGAAA -18

/ URE

-150 to -121 ORE

-40 to +65

Figure 2. Summary of DNA sequences important for expression of the E4 gene in response to ethylene. URE, upstream regulatory element; DRE, downstream regulatory element. Sequences from -150 to -121 were shown by linker scan mutation to be important, and the sequences in bold were most critical. The E41E8BP recognition sequence (-34 to -18) is shown, with the TAT A box underlined. Arrow, start of transcription.

We tested the role ofE41E8BP in regulation of the E4 gene in transient assay. We created site-specific mutations of its binding site within the E4 promoter, but our mutations were limited to those that did not disrupt the TAT A sequences. Mutations which reduced affinity for the E41E8BP also reduced the expression of the gene in transient assay [17]. These mutations had reduced expression in every stage of fruit ripening, but since we had only affected protein binding affinity and not the activation function of the putative transcription factor, the remaining activity was ethylene responsive. Our data support a role for the E41E8BP in normal regulation ~f the E4 gene, and it may participate as part of an ethylene-responsive transcription factor complex.

12S

5. Cloning the E41E8BP Gene

In order to make further progress in understanding the molecular mechanisms of ethylene control of gene transcription during fruit ripening, it is necessruy to clone the genes that encode the DNA-binding proteins involved in this control. We decided to begin by cloning the gene that encodes the E4IESBP, so we screened a cDNA expression libTal)' with the E8 recognition sequence for the E4IESBP under conditions that would allow DNA-protein interactions [IS]. Approximately one million plaques were screened, and three plaques expressing putative DNA-binding proteins were identified. Proteins produced by purified phage were then interacted with both the E8 probe and with a control probe that consisted of sequences from the 3' end of the gene encoding polygalacturonase [19]. We found that the protein produced by two of these phage interacted equally with both probes, but proteins from one of the phage (clone 7-3) interacted only with the E8 probe and not with the control probe (Fig. 3). We are now in the process of further characterizing this cDNA, and testing whether the protein it encodes functions in control of E4 in response to ethylene.

Probe:

Non-specific DNA-binding

protein

E4/E8BPa clone 7-3

E8 PG

Figure 3. Proteins from purified phage interacted with radiolabeled double-stranded DNA probes. The probes are the E41E8BP recognition sequence from E8, or a fragment from the 3' end of the PG gene.

6. Discussion

Our results indicate that two cis-elements are required for ethylene responsiveness of E4. The upstream element is located between -150 and -121, and the downstream element is located between -40 and +65. The recognition site for the E4IESBP is necesSal)' for normal E4 expression. and may be the downstream element.

129

The requirement for two cis-elements for ethylene-responsive transcription of E4 is different from what has been found for control of other genes by ethylene. For genes encoding pathogenesis-related proteins such as chitinase [24], glucanase [21], and basic PR-l type proteins [23], a similar cis-element, which contains a Gee motif, is present in the 5' flanking region of the gene. A 47 bp fragment containing two copies of this motif was shown to be sufficient to confer ethylene responsiveness to a neutral promoter [22]. A family of genes encoding DNA-binding proteins that interact with the Gee box were cloned, and their products were called the Ethylene-Responsive Element Binding Proteins [22). Interestingly, the accumulation of mRNAs for these genes is induced by ethylene. No Gee box motif is present within the 5' flanking regions of the E4 or E8 genes. Therefore, different proteins are likely to be involved in ethylene activation of genes during fruit ripening and in response to pathogens. The study of genes induced by ethylene during carnation flower petal senescence has focused on the regulation of the glutathione-S-transferase (GSTJ) gene [20). A 126 bp sequence from the 5' flanking region of the GSTJ gene was shown to be both necessary and sufficient for ethylene regulation in carnation petals in transient assay. A protein that interacts within this sequence was identified, and part of its binding site resembles part of the binding site for the protein that interacts with E4 sequences from -142 to -110 [20). It is possible that a related protein could be involved in regulating gene transcription in response to ethylene during fruit ripening and flower petal senescence.

The cooperation of two or more cis-elements for regulation of gene transcription is not unusual. It appears to be required for the regulation of a-amylase gene transcription by gibberellic acid in barley aleurone [25], auxin-responsive transcription of the PS-JAA 4/5 gene [26], and for the regulation of gene transcription in response to light [27].

Figure 4 depicts a model for how the two cis-elements we have identified interact with previously studied DNA-binding proteins to activate gene transcription in response to ethylene. It is likely that the E4IE8BP could interact with sequences spanning the E4 TAT A box simultaneously with the binding of TAT A binding protein (TBP), and the associated general transcription factors. Recent crystallographic structures of TBPfIFIIAfTFIm interactions with DNA indicate adequate room remains for the interaction of additional proteins in this region [28]. Our model proposes that a protein which acts as a positive regulator binds at the upstream element and interacts with the E4IE8BP when the E4 gene is actively transcribed.

Two alternative mechanisms have been proposed for how ethylene stimulates this proposed transcription factor complex to activate E4 gene transcription [17]. The first hypothesis suggests that the DNA-binding protein described by Montgomery et al. [7] is a repressor, and that it binds to the upstream element in the absence of ethylene, preventing the binding of a positive regulator. The binding activity of the repressor would be altered after ethylene treatment, so that it would no longer bind the upstream element, and the positive regulator could then bind. Although our linker scan analysis did not provide evidence for a negative regulatory element, it is possible that these putative positive and negative regulatory proteins interact with overlapping sequences,

130

so that mutation of the site would destroy binding of both negative and positive regulatory proteins, and only elimination of the positive element would be detected.

The second mechanism that was proposed is that the synthesis or activity of one of the positive regulators shown in Figure 4 that interact with either the upstream or the downstream element is stimulated by ethylene treatment. These two mechanisms are not mutually exclusive.

Figure. 4. Model of interaction ofE41E8BP with protein bound at upstream element to activate E4 transcription. TBP, TATA binding protein; TAPs, TBP associated factors; DRE, downstream regulatory element; URE, upstream regulatory element. From Xu et al. [17]

The model shown in Figure 4 is undoubtedly oversimplified, and additional proteins are most likely involved. Our future studies are aimed at identifying other components of this complex, and in determining how it is activated in response to ethylene.

7. References

1. Galil, J. (1968) An ancient technique for ripening sycomore fiuit in east-Mediterranean countries, Econ. Bot. 22,178-190.

2. Yang, S.F. and Hoffinan, N.E. (1984) Ethylene biosynthesis and its regulation in higher plants, Annu. Rev. Plant Physiol. 35,155-189.

3. Gray, J.E., Picton, S., Giovannoni, J.J., and Grierson, D. (1994) The use of transgenic and naturally occurring mutants to understand and manipulate tomato fiuit ripening, Plant, Cell and Environ. 17, 557-571.

4. Wilkinson, J.Q., Lanahan, M.B., Yen, H.-C., Giovannoni, J.J., and Klee, H.J. (1995) An ethylene­inducible component of signal transduction encoded by Never-ripe, Science 270, 1807-1809.

5. Lincoln, J.E., Cordes, S., Read, E., and Fischer, R.L. (1987) Regulation of gene expression by ethylene during Lycopersicon esculentum (tomato) fiuit ripening, Proc. Natl. Acad. Sci. USA 84, 2793-2797.

6. Cameron, C.C. and Yang, S.F. (1982) A simple method for the determination of resistance to gas diffusion in plant organs, Plant Physiol. 70, 21-33.

131

7. Montgomery, J., Goldman, S., Deikman, J., Margossian, L., and Fischer, R.L. (1993) Identification of an ethylene-responsive region in the promoter of a fiuit ripening gene, Proc. Natl. Acad. Sci. USA 90, 5939-5943.

8. Deikman, J. and Fischer, R.L. (1988) Interaction of a DNA binding factor with the 5'-flanking region of an ethylene-responsive fiuit ripening gene from tomato, EMBO J. 7,3315-3320.

9. Penarrubia, L., Aguilar, M., Margossian, L., and Fischer, R.L. (1992) An antisense gene stimulates ethylene hormone production during tomato fiuit ripening, Plant Cell 4, 681-687.

10. Lincoln, J.E. and Fischer, R.L. (1988) Diverse mechanisms for the regulation of ethylene-inducible gene expression, Mol. Gen. Genet. 212, 71-75.

11. Lincoln, J.E. and Fischer, R.L. (1988) Regulation of gene expression by ethylene in wild-type and rin tomato (Lycopersicon esculentum) fiuit, Plant Physiol. 88, 370-374.

12. DellaPenna, D., Lincoln, J.E., Fischer, R.L., and Bennett, AB. (1989) Transcriptional analysis of polygalacturonase and other ripening associated genes in Rugters, rin, nor, and Nr tomato fiuit, Plant Physiol.90, 1372-1377.

13. Theologis, A, Oeller, P.W., Wong, L.-M., Rottmann, W.H., and Gantz, D.M. (1993) Use ofa tomato mutant constructed with reverse genetics to study fiuit ripening, a complex developmental process, DeveL Genet. 14, 282-295.

14. Deikman, J., Kline, R., and Fischer, R.L. (1992) Organization of ripening and ethylene regulatory regions in a fiuit-specific promoter from tomato (Lycopersicon esculentum), Plant Physiol. 100, 2013-2017.

15. Cordes, S., Deikman, J., Margossian, L.J., and Fischer, R.L. (1989) Interaction ofa developmentally regulated DNA-binding factor with sites flanking two different fiuit-ripening genes from tomato, Plant CellI, 1025-1034.

16. Hunter, T. and Karin, M. (1992) The regulation of transcription by phosphorylation, Cell 70, 375-387.

17. Xu, R., Goldman, S., Coupe, S., and Deikman, J. (1996) Ethylene control ofE4 transcription during tomato fiuit ripening involves two cooperative cis-elements, Plant Mol. Bioi., (in press).

18. Singh, H., Clerc, R.C., and LeBowitz, J.H. (1989) Molecular cloning of sequence-specific DNA binding proteins using recognition site probes, Biotechniques 7, 252-261.

19. Bird, C.R., Smith, C.J.S., Ray, J.A, Moureau, P., Bevan, M.W., Bird, AS., Hughes, S., Morris, P.C., Grierson, D., and Schuch, W. (1988) The tomato polygalacturonase gene and ripening-specific expression in transgenic plants, Plant Mol. BioI. 11, 651-662.

20. Itzhaki, H., Maxson, J.M., and Woodson, W.R. (1994) An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GSTl) gene, Proc. Natl. Acad. Sci. USA 91, 8925-8929.

21. Vogeli-Lange, R., Frondt, C., Hart, C.M., Nagy, F., and Meins, J., F. (1994) Developmental, hormonal, and pathogenesis-related regulation of the tobacco class I b-l,3-glucanase B promoter, PlantMol. BioI. 25, 299-311.

22. Ohme-Takagi, M. and Shinshi, H. (1995) Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element, Plant Cell 7 , 173-182.

23. Sessa, G., Meller, Y., and Fluhr, R. (1995) A GeC element and a G-box motif participate in ethylene­induced expression of the PRB-l b gene, Plant Mol. Bioi. 28, 145-153.

24. Shinshi, H., Usami, S., and Ohme-Takagi, M. (1995) Identification of an ethylene-responsive region in the promoter of a tobacco class I chitinase gene, Plant Mol. Bioi. 27, 923-932.

25. Rogers, J.C., Lanahan, M.B., and Rogers, S.W. (1994) The cis-acting gibberellin response complex in high-pi alpha-amylase gene promoters, Plant Physiol. 105, 151-158.

26. Ballas, N., Wong, L.-M., Ke, M., and Theologis, A (1995) Two auxin-responsive domains interact positively to induce expression of the early indoleacetic acid-inducible gene PS-lAA 4/5, Proc. Natl. Acad. Sci. USA 92, 3483-3487.

27. Terzaghi, W.B. and Cashmore, AR. (1995) Light-regulated transcription, Annu. Rev. Plant Phyiol. Plant Mol. Bioi. 46,445-474.

28. Burley, S.K. (1996) Picking up the TAB, Nature 381,112-113.

MOLECULAR GENETIC ANALYSIS OF ETHYLENE-REGULATED AND DEVELOPMENTAL COMPONENTS OF TOMATO FRUIT RIPENING

Ethylene and Developmental Signal Transduction in Tomato

J. J. GIOVANNONI Department of Horticultural Sciences, Crop Biotechnology Center, Texas A&M University, College Station, TX 77843-2133

1. Introduction

Fruit ripening represents a biological process unique to plant species in which developmental and hormonal signaling systems orchestrate a variety of biochemical and physiological changes which, in summation, result in the "ripe" stage of fruit maturation. In so called "climacteric" fruits such as tomato, cucurbits, banana, apple, and many others, the initiation of ripening is characterized by a dramatic increase in respiration and biosynthesis of the gaseous hormone ethylene [I]. Inhibition of ethylene biosynthesis or ethylene perception via application of inhibitors, or endogenous expression of transgenes, has been shown to have profound inhibitory effects on ethylene-mediated plant processes, including climacteric fruit ripening [reviewed in 1, 2, 3, 4, 5, 6, 7]. As a result, the majority of scientific effort devoted to ripening research has been in the areas of ethylene biosynthesis, ethylene responses, and, more recently, ethylene perception and signal transduction [8, 9]. Nevertheless, careful examination of the physiological, biochemical, genetic, and molecular data collected in recent decades (particularly from analysis of ripening tomato fruit) suggests a significant developmental component of fruit ripening which interacts with and modulates ethylene biosynthesis and signaling during ripening, and has been largely overlooked [8, 10].

l.l. TOMATO RIPENING MOLECULAR BIOLOGY AND ETHYLENE SIGNALING

The critical role of ethylene in coordinating climacteric ripening at the molecular level was first observed via analysis of ethylene inducible ripening-related gene expression [reviewed in 4]. Ripening-related genes have been isolated via differential gene expression patterns [11, 12] and biochemical function [13, 14, 15, 16, 17, 18]. Promoter analysis of ripening genes has been performed via examination of promoter/reporter construct activities in transient assays and transgenic plants. The result has been the identification of cis-acting promoter elements and trans-acting

133

A. K. Kanellis et al. (ells.), Biology and Biotechrwlogy of the Plant Hormone Ethylene, 133-140. © 1997 Kluwer Academic Publishers.

134

factors which are responsible for both ethylene and non-ethylene regulated aspects of ripening [19, 20].

The in vivo functions of several ripening-related genes, including PG, PME, ACC synthase, ACC oxidase, and phyfoene synthase have been tested via antisense gene repression and/or mutant complementation in tomato. For example, the cell wall pectinase, polygalacturonase, was shown to be necessary for ripening-related pectin depolymerization and pathogen susceptibility, though with little effect on fruit softening [21, 22, 23]. Inhibition ofphytoene synthase resulted in reduced carotenoid biosynthesis and reduction in fruit and flower pigmentation [24]. Reduced ethylene evolution resulting in ripening inhibition occurred with ACC synthase and ACC oxidase antisense [17, 25].

1.2. DEVELOPMENTAL REGULATION

Further analysis of transgenic tomatoes inhibited in ethylene biosynthesis demonstrates that climacteric ripening responds to a combination of ethylene regulation and developmental control. Although antisense ACC synthase tomatoes which failed to produce ethylene did not ripen, several ripening-related genes were still expressed. This observation confirms the presence of a developmental (non-ethylene regulated) component of ripening. Indeed, the gene encoding the rate-limiting activity in ethylene biosynthesis, ACC synthase, is itself initially induced during ripening by a developmental signal [10].

Gene expression in the non-ripening rin and nor tomato mutants is impaired for most ripening-related genes examined [26, 27]. In addition to demonstrating a lack of ethylene inducible gene expression (due to the lack of climacteric ethylene), similar to that observed in transgenic ethylene reduced fruit, the rin and nor mutants are also deficient in expression of developmentally regulated genes such as PG, E8, and ACC synthase [10,26]. This fact, in combination with the observations that 1) rin and nor fruit ripening inhibition cannot be reversed with exogenous ethylene, and 2) many ethylene regulated genes can be induced in mutant fruit with the application of ethylene (demonstrating rin and nor are ethylene responsive), strongly supports the hypothesis that rin and nor represent lesions in the developmentally regulated component of climacteric ripening. Alternatively, rin and nor may represent steps downstream of primary ethylene signaling as does the Arabidopsis hookless (HLS) gene [28]. However, the lack of ethylene biosynthesis and inhibited expression of developmentally regulated genes such as PG, argue in favor of the former hypothesis.

1.3. ETHYLENE SIGNAL TRANSDUCTION IN TOMATO

Lanahan [29] observed that the Nr mutation is manifested as a block in a host of ethylene responses including inhibition of the seedling triple response and incomplete fruit ripening. The Nr gene was cloned, sequenced and shown to have high homology to the ETRl and ERS genes of Arabidopsis [8]. All three genes have significant homology to members of the "two component" class of protein kinases [30]. The reduction of ethylene binding capacity in (etrl) mutants [31] and yeast expressing a

135

mutant ETRl cDNA [32], in combination with the fact that the ETRl gene product is apparently involved in protein phosphorylation, suggests that ETRl (and presumably Nr and ERS) is likely to encode an ethylene receptor which initiates signaling via modification of protein phosphorylation.

Nr gene expression is distinct from the constitutive expression of ETRl and ERS in that Nr expression is itself ethylene inducible, and the ethylene responsiveness of Nr is additionally dependent on the developmental stage of the fruit [8]. This observation provides additional evidence for a significant role of non-ethylene developmental signals in coordinating climacteric fruit ripening and demonstrates that such signals are important for the regulation of ethylene signaling.

The tomato Epi mutant likely represents an additional component of ethylene signal transduction [3]. The Epi mutation was originally characterized as a semi­dominant, single locus mutation resulting in leaf epinasty, vertical growth, minimal branching, and highly branched root structure. These effects are consistent with ethylene over-production or constitutive ethylene signaling [7]. Although elevated ethylene biosynthesis has been reported in some tissues of the Epi mutant, treatment with inhibitors of ethylene biosynthesis or action had little effect on mutant phenotype, suggesting that Epi represents a lesion in ethylene signal [33]. The Arabidopsis ctrl mutant is also characterized by constitutive ethylene signal transduction, and the corresponding CTRl gene has been isolated and shown to have homology to the Raj family of protein kinases [34]. It is possible that Epi may represent a tomato homologue of CTR1. Alternatively, Epi may represent a unique ethylene signal transduction component.

In summary, analysis of ripening-related gene expression, ripening mutants, and ripening impaired transgenic plants points to a significant role for developmental cues in coordinating ethylene biosynthesis, ethylene perception, and additional non-ethylene regulated components of climacteric fruit ripening.

2. Results and Discussion

2.1. ISOLATION AND EXPRESSION OF A PARTIAL TCTR eDNA

DNA gel blot hybridization of the Arabidopsis CTRl full-length cDNA to tomato genomic DNA (at high stringency) indicates the presence of one to three related sequences in the tomato genome. In addition, one band appeared predominantly with each of the 5 restriction enzymes tested, suggestive of one highly homologous locus. 100,000 clones from a breaker stage tomato fruit cDNA library were screened using the CTRl cDNA as probe, and resulted in the isolation ofa 1.3 kb cDNA which shares 68% DNA sequence identity and 95% amino acid identity with CTR1. The tomato cDNA corresponds to approximately the middle third of the 3 kb CTRl sequence. We have tentatively named this cDNA TCTR [So Lee and J. Giovannoni, unpublished].

Preliminary gene expression analysis indicates that the TCTR transcript is approximately 3 kb in length, which is similar to that reported for Arabidopsis [34]. Of particular interest was the observation that TCTR is induced during ripening, and

136

by exogenous ethylene in mature green fruit (Table 1). TCTR is additionally ethylene inducible in the rin and nor mutants, and expressed at very low levels in both ripening and ethylene treated Nr fruit. This pattern of expression in fruit is similar to that observed for the Nr ethylene receptor [8] and distinct from the constitutive expression pattern reported for CTRI [34]. Nevertheless, TCTR is apparently constitutively expressed and considerably more abundant in total RNA derived from leaves and 9-day-old seedlings (Table 1). Together, these results suggest that TCTR is constitutively expressed in some tissues or during particular stages of development and modulated by developmental and/or hormonal signals in other tissues or developmental stages. Additional regulation of putative (TCTR) and known (Nr) ethylene signal transduction components during tomato fruit development suggests that ethylene signal transduction is more tightly regulated in tissues whose proper development is largely dependent on effects resulting from ethylene hormone action.

Table 1. Expression ofTCTR in tomato fruit, seedlings and leaves.

Ripe Breaker Green Leaf Seedling

Yes Yes No Yes Yes Yes

2.2. GENETIC CHARACTERIZATION OF THE Epi MUTATION

The tomato Epi (Epinastic) mutant was characterized as a partially dominant single locus trait by Ursin [35]. Preliminary analysis of segregation of the epinastic phenotype in an F2 population suggests a single-locus recessive mutation. Specifically, we have crossed L. esculentum (cv. VFN8) homozygous for the Epi mutation to L. cheesmannii (LA483) and generated an F2 population of over 1000 plants from which 123 mutant individuals have been identified (or approximately half the number expected for a recessive mutation). In this regard, it is noteworthy that poor transmission of the recessive Arabidopsis ctrl mutant allele has also been reported [34]. We are currently considering the mutant epi allele to be essentially recessive. Accurate assessment of the dominance of the mutant epi allele is an important aspect of our future efforts.

To date, only one ethylene signal transduction mutant resulting in constitutive ethylene signaling has been identified [ctrl; 34]. TCTR was utilized as an RFLP probe on the subset of 123 mutant F2 progeny described above, in order to test the possibility that the epi mutation may represent a defect in the TCTR gene. The TCTR RFLPs segregated in a 1:2: 1 ratio within the subpopulation of mutant F2s, indicating that the TCTR locus is not linked to Epi [So Lee and J. Giovannoni, unpublished]. This result suggests that Epi represents an ethylene signaling component whose Arabidopsis counterpart has either not been identified, or does not exist.

137

2.3. EFFECTS OF Epi ON ETHYLENE-REGULATED GENE EXPRESSION AND PLANT MORPHOLOGY

Tomato plants homozygous for the mutant Epi allele are characterized by vertical growth, minimal lateral branching, and leaf epinasty as described by Ursin [35]. In addition, Epi seedlings demonstrate a constitutive triple-response phenotype as was previously shown by Fujino [33]. Treatment of seedlings with ethylene enhanced the triple-response phenotype, suggesting that the constitutive ethylene response displayed by Epi is not saturated [So Lee and J. Giovannoni, unpublished].

Based on the constitutive ethylene response phenotype of Epi seedlings and leaves, we predicted that fruit ripening, pedicel abscission, leaf senescence, and additional ethylene-mediated developmental processes may be accelerated or exaggerated in the epi mutant. In a preliminary test of 10 normal and 10 mutant fruit, tagged at anthesis, we did not observe any significant change in time to onset or completion of ripening. One explanation for this result is that developmental regulators required for ripening control this process via modulation of ethylene signal transduction during fruit development. Consistent with this hypothesis is the observation that all ripening­related and ethylene-inducible genes examined were normally regulated during Epi fruit ripening [So Lee and J. Giovannoni, unpublished]. In contrast, the ethylene inducible chitinase 9 gene [CH9; 36] was constitutively expressed in both leaves and mature green fruit of the Epi mutant and ethylene inducible in the corresponding non­mutant [So Lee and J. Giovannoni, unpublished]. This result is consistent with the observation of constitutive chitinase expression in the Arabidopsis ctrJ mutant [34], and provides additional support for the hypothesis according to which ethylene signal transduction is developmentally regulated during certain critical times of development.

2.4. ISOLATION OF DEVELOPMENTALLY REGULATED RIPENING GENES

A total of 57 ripening-related display PCR products were identified and eluted from acrylamide gels as described in Oh et al. [37]. Following re-amplification, each display-cDNA was radiolabeled and hybridized to an RNA gel-blot containing RR, BR, MG, and MG+ethylene total RNA (RR red ripe; BR breaker; MG mature green). Twenty two display-cDNAs demonstrating fruit RNA gel-blot patterns identical to those observed in the corresponding differential display acrylamide gels were recovered and hybridized to whole seedling and leaf total RNA to verify fruit specificity. Several ripening-related display-PCR products showing ethylene inducibility were also included among the original 57, and four were confirmed via RNA gel-blot analysis [B. Oh and J. Giovannoni, unpublished]. Thus, we have isolated a total of 22 ripening-related sequences of which 18 are not inducible with 8 h of 20 ppm ethylene. We refer to these display-cDNAs with the acronym DDTFR (differential display tomato fruit ripening).

To date, we have completely sequenced 21 of the 22 ripening-related display­cDNAs (ranging from 124 - 600 bp). GENBANK database analysis of 20 of theses sequences resulted in the identification of five display-cDNAs which corresponded to previously characterized tomato genes. For example, DDTFR-2 and DDTFR-3 show

138

perfect homology with the 3' end of tomato fruit polygalacturonase, while DDTFR-9 and DDTFR-15 are identical to 3' sequences found within the heat shock-like cDNA, pTOM66 [38J. Both DDTFR-ll and DDTFR-14 showed greater than 70% sequence identity with two different Arabidopsis STS (sequence tagged site) clones of unknown function. The remaining DDTFR clones showed no significant homologies with any sequences deposited in GENBANK, nor with each other, suggesting that they represent at least 15 novel ripening-related genes, 10 of which are fruit-specific and not induced by 8 h of 20 ppm ethylene treatment.

3. Summary

Research in the area of fruit ripening molecular biology has been primarily focused on the genetic basis of ethylene biosynthesis and resulting terminal. Genes encoding the regulated steps in ethylene biosynthesis have been isolated. as have numerous genes which respond to ethylene during ripening. Promoter sequences from a number of these ethylene-inducible genes have additionally been isolated and characterized. Although great strides have been made toward understanding ethylene signal transduction in the model system of Arabidopsis, little has been done to characterize corresponding genes in additional species. The need for such an effort is exemplified by the fact that the one tomato ethylene signaling gene reported to date, Nr, is characterized by unique developmental and ethylene mediated regulation [8J. This observation suggests that ethylene signal transduction may be modulated. under certain circumstances, by additional layers of developmental or hormonal regulation to ensure optimal ethylene responses during plant growth and development

4. References

1. Abeles, F., Morgan, P., and Saltveit M. (1992) Ethylene in Plant Biology, Academic Press, San Diego.

2. Mattoo, AK. and Suttle, J.C. (1991) The Plant Hormone Ethylene, CRC Press, Boca Raton. 3. Theologis, A (1992) One rotten apple spoils the whole bushel: the role of ethylene in fiuit ripening,

Cell 70, 181-184. 4. Giovannoni, J. (1993) Molecular and genetic analysis of tomato fiuit development and ripening,

Methods in Plant Biochemistry 10, 251-285. 5. Hobson, G. and Grierson, D. (1993) Tomato, in Seymour GB, Taylor JE, Tucker GA (eds.)

Biochemistry of Fruit Ripening, Chapman and Hall, London pp. 405-442 6. Gray, lE., Picton, S., Giovannoni, J.l, and Grierson, D. (1994) The use of transgenic and naturally

occurring mutants to understand and manipUlate tomato fiuit ripening, Plant Cell Env.17, 557-571 7. Ecker, lR. (1995) The ethylene signal transduction pathway in plants, Science 268, 667-675. 8. Wilkinson, J., Lanahan, M., Yen, H., Giovannoni, J., and Klee, H. (1995) An ethylene-inducuble

component of signal transduction encoded by Never-ripe, Science 270, 1807-1809. 9. Yen, H., Lee, S., Tanksley, S., Lanahan, M., Klee, H., and Giovannoni, J. (1995) The tomato

Never-ripe locus regulates ethylene-inducible gene expression and is linked to a homologue of the Arabidopsis ETR1 gene, Plant Physiol. 107, 1343-1353.

10. Theologis, A, Oeller, P., Wong, L., Rothmann, W., and Gantz. D. (1993) Use of a tomato mutant constructed with reverse genetics to study fiuit ripening, a complex developmental process, Dev Gen. 14,282-259

139

11. Slater, A, Maunders, M., Edwards, K., Schuch, W., and Grierson, D. (1985) Isolation and characterization of cDNA clones for tomato polygalacturonase and other ripening-related proteins, Plant Mol. Bioi. 5, 137-147.

12. Lincoln, J., Cordes, S., Read, E., and Fischer, R. (1987) Regulation of gene expression by ethylene during Lycopersicon esculentum (tomato) fiuit development, Proc. NatL Acad. Sci. USA 84, 2793-2797.

13. DellaPenna, D., Alexander, D., and Bennett, A (1986) Molecular cloning of tomato fiuit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening, Proc. NatL Acad. Sci. USA 83, 6420-6424.

14. Sheehy, R., Pearson, J., Brady, C., and Hiatt, W. (1987) Molecular charictarization of tomato fiuit polygalacturonase, Mol. Gen. Genet. 208, 30-36.

15. Biggs, M. and Handa, A (1989) Temporal regulation of polygalacturonase gene expression in fiuits of nonnal, mutant, and heterozygous tomato genotypes, Plant Physiol. 89, 117-125.

16. Harriman, R., Tieman, D., and Handa, A (1991) Molecular cloning of tomato pectin methylesterase gene and its expression in Rutgers, ripening inhibitor, nonripening and Never ripe tomato fiuits, Plant PhysioI97,80-87.

17. Oeller, P.W., Wong, L,M., Taylor, L.P., Pike, D.A, and Theologis, A (1991) Reversible inhibition of tomato fiuit senescense by antisense l-aminocyclopropane-l-carboxylate synthase, Science 254,427-439.

18. Yelle, S., Chetelat, R., Dorais, M., DeVema, J., and Bennett, AB. (1991) Sink metabolism in tomato fiuit. IV. Genetic and biochemical analysis ofsuCTose accumulation, Plant Physiol95, 1026-1035.

19. Deikman, J., Kline, R., and Fischer, R. (1992) Organization of ripening and ethylene regulatory regions in a fiuit-specific promoter from tomato (Lycopersicon esculentum), Plant Physiol. 100, 2013-2017.

20. Montgomery, J., Pollard, V., Deikman, J., and Fischer, R. (1993) Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fiuit pericarl, Plant Cell 5,1049-1062.

21. Smith, C., Watson, C., Ray, J., Bird, C., Morris, P., Schuch, W., and Grierson, D. (1988) Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes, Nature 334, 724-726.

22. Giovannoni, J., DellaPenna, D., Bennett, AB., and Fischer, R. (1989) Expression of a chimeric polygalacturonase gene in transgenic rin (ripening inhibitor) tomato fiuit results in polyuronide degradation but not fiuit softening, Plant CellI, 53-63.

23. Kramer, M., Sanders, R., Sheehy, R., Melis, M., Kuehn, M., and Hiatt, W. (1990) Field evaluation of tomatoes with reduced polygalacturonase by antisense RNA, in AB. Bennett, and S. O'Neill, (eds.), HorticulturalBiotechnology, Alan R. Liss. pp347-355.

24. Fray, R. and Grierson, D. (1993) Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencning, complementation, and co-suppression, Plant Mol. Bioi. 22, 589-602.

25. Hamilton, A, Lycett, G., and Grierson, D. (1990) Antisense gene that inhibits synthesis of the honnone ethylene in transgenic plants, Nature 346, 284-287.

26. DellaPenna, D., Lincoln, J.E., Fischer, R.L., and Bennett, AB. (1989) Transcriptional analysis of polygalacturonase and other ripening associated genes in Rutgers, rin, nor, and Nr tomato fiuit, Plant Physio[ 90,1372-1377.

27. Picton, S., Gray, J.E., Barton, S.L., AbuBaker, u., Lowe, A, and Grierson, D. (1993) cDNA cloning and characterisation of novel ripening-related mRNAs with altered patterns of accumulation in the ripening inhibitor (rin) tomato ripening mutant, Plant Mol. Bioi. 23, 193-207

28. Lehman, A, Black, R., and Ecker, J. (1996) HOOKLESSJ, an ethylene response gene, is required for differential cell elongation and de-etiolation in the Arabidopsis hypocotyl, Cell 85,183-194.

29. Lanahan, M,B" Y enm H,C" Giovannoni, J.J., and Klee, H.J. (1994) The Never Ripe mutation blocks ethylene perception in tomato, Plant Cell 6, 521-530.

30. Koshland, D. (1995) The two-component pathway comes to eukaryotes, Science 262, 532. 31. Bleeker, A, Estelle, M., Somerville, C., and Kende, H. (1988) Insensitivity to ethylene conferred by a

dominant mutation inArabidopsis thaliana, Science 241,086-1089. 32. Schaller, G. and Bleeker, A (1995) Ethylene-binding sites generated in yeast expressing the

ArabidopsisETRJ gene. Science 270,1809-1811. 33. Fujino, D., Burger, D., and Bradford, K. (1989) Ineffectiveness of ethylene biosynthetic and action

inhibitors in phenotypically reverting the Epinastic mutant of tomato (Lycopersicon esculentum Mill.). J. Plant Growth Regul. 8, 53-61.

140

34. Kieber, J., Rothenberg, M., Roman, G., Feldman, K., and Ecker, J. (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases, Cell 72, 427-441

35. Ursin, V. (1987) Morphogenetic and physiological analyses of two developmental mutants of tomato, Epinastic and diageotropica, Ph.D. Dissertation, University of California, Davis.

36. Danbash, N., Wagemakers, C., van Kan, J., and de Wit, P. (1993) Molecular characterization offour chitinase cDNAs obtained from Cladisporiumfolvum-infected tomato, Plant Mol. Bioi. 22, 1017-1029.

37. Oh, B. J., Balint, D., and Giovannoni, J. (1995) A modified procedure for PCR-based differential display and demonstration of use in plants for isolation offruit ripening-related genes, Plant Mol. BioL Reporter 13, 70-81.

38. Gray, J.E., Picton, S., Shabbeer, J., Schuch, W., and Grierson, D. (1992) Molecular biology of fruit ripening and its manipulation with antisense genes, Plant Mol. Bioi. 19, 69-87

THE ROLE OF EmYLENE IN BANANA FRUIT RIPENING

s. K. CLENDENNEN, P. B. KlPP, AND G. D. MAY Boyce Thompson Institute for Plant Research, Cornell University, Tower Road, Ithaca, NY 14853

1. Introduction

Bananas, plantains, and cooking bananas are crops of vital importance to the food security of hundreds of millions of people in developing countries. Nearly all inhabitants in the tropics of all continents benefit directly or indirectly from Musa crops as a source of food or cash export. The food value of bananas and plantains is widely recognized. They are high in carbohydrates (about 35%) and fiber (6-7%) [1], and are an important source of major elements, such as potassium, magnesium, phosphorus, calcium. and iron as well as vitamins A and C [2].

Fleshy fruit characteristics are considered to be relatively new in an evolutionary context. These fall into two groups: fruits with a rind (citrus, banana, and fruits of the Cucurbitaceae) and fruits without a rind (berries [tomato], drupes [peach, plums], and pomes [apple and pear)). While fruits can be compared or contrasted on the basis of their anatomy, two classes can also be defined based upon physiological processes that occur during ripening [3]. This is related to the absence or presence of a large increase in ethylene synthesis at the onset of ripening. In non-climacteric fruit (such as citrus and strawberries), ripening is not dependent on an increase in ethylene synthesis. Climacteric fruit produce basal levels of ethylene during early fruit development followed by dramatic ethylene synthesis increases during ripening.

Bananas are a climacteric fruit and ripen in response to ethylene. Banana fruit ripening is characterized by a number of biochemical and physiological changes, including fruit softening, changes in peel color, and an increase in respiratory activity [reviewed in 4]. Although ethylene is produced by the fruit, ripening can also be stimulated by the application of ethylene. The physiological changes that occur during ripening are not altered considerably by the application of ethylene. For example, starch and sugar content, acidity and concentration of pectic substances are approximately equivalent at the same stage of ripening in ethylene-treated and untreated control fruit [5-7]. Primarily, the post-harvest application of ethylene influences physiology by hastening the changes that are associated with the ripening process. Application of exogenous ethylene results in an increase in ethylene production and decreases the pre-climacteric period in fruit of a Cavendish banana variety by approximately seven days [6,7].

141

A. K. Karrellis et al. (eds.), Biology and Biotechnology of the Plant HOrTnOrre Ethylerre, 141-148. © 1997 Kluwer Academic Publishers.

142

2. Controlling Exposure to Ethylene

Exposure to ethylene (either applied or produced by the fruit) accelerates ripening and senescence of banana fruit. To prolong the pre-climacteric storage life (or green-life) of fruit, great efforts are currently being made to control exposure/response to ethylene. Studies on the storage of harvested banana fruit under modified atmospheric conditions have indicated that low concentrations of oxygen (10%) delay the onset of ethylene production and the rise in respiration, but only under very low concentrations of exogenous ethylene [8]. Very low concentrations of CO2 decrease the rate of respiration in ripening bananas [9] and very high CO2 concentrations (60%) reduce the progress of the respiratory climacteric and delay degreening of the peel even in the presence of exogenous ethylene. However, these storage conditions do not delay the initiation of ripening [10]. High CO2 concentrations can also result in injury to the ripening fruit [9,11].

Ethylene absorbents have been successfully used to remove exogenous ethylene from the storage atmosphere. Potassium permanganate-based "scrubbers" and combinations of charcoal and palladium chloride have been shown to prolong the storage life and decrease softening of banana fruit [12,13]. Exclusion of ethylene by packing fruit in sealed polyethylene bags can prolong storage life, and sealed polyethylene bags when used in combination with ethylene absorbents prolonged the pre-climacteric or storage phase of bananas even further [11,14-17]. The use of waxy coatings on the fruit can achieve similar results, especially in combination with a fungicide treatment, and may also reduce water loss from ripening fruit [18,19]. It has been shown that water loss and low relative humidity shorten the pre-climacteric period and hasten the ripening process, likely as a result of increased ethylene production in the peel [20-22]. Treatment of the fruit with low intensity gamma radiation also delays ripening [23], presumably by decreasing the sensitivity of the fruit to endogenous ethylene, since the subsequent response to exogenous ethylene is unaffected [24].

3. Ethylene Production in Banana Fruit

Ethylene is formed from the precursor l-aminocyclopropane-l-carboxylic acid (ACC) by the activity of ACC oxidase (ACO), also referred to as the ethylene-forming enzyme (EFE). In bananas, ethylene production is low or undetectable during fruit development and is only observed during fruit ripening [25]. After harvest of the fruit, ACC and ethylene production are low in banana fruit during the pre-climacteric phase. At the onset of ripening, ethylene production and ACC levels increase markedly, with ethylene production reaching a peak before the respiratory climacteric [26]. Treatment with a competitive inhibitor of ethylene has been shown to prolong the pre-climacteric period [27].

It is generally agreed that under normal ripening conditions, ACO activity in the pulp is a key factor leading to ethylene production, and that banana ripening is

143

coordinated primarily by pulp ethylene production [20,28]. Trace amounts of endogenous ethylene may be responsible for the gradual elevation of ACO activity during the pre-climacteric stage [27]. Ethylene produced in the pulp is essential for both ethylene production in the peel [28] and for de-greening of the peel [29] since neither event occurs in peel samples that have been separated from the pulp. Although ethylene production is higher in the pulp than in the peel during the pre-climacteric phase, ACO activity in the peel (measured as the rate of ethylene evolution) can actually exceed that in the pulp during the later stages of ripening [28,30]. However, the increase in ACO activity in the peel does not occur until after, and is dependent upon, the peak in ethylene biosynthesis in the pulp [28]. Localization of the ACO transcript in banana fruit was determined on whole-fruit tissue blots by northern analysis (Fig. 1). In post-climacteric fruit that had been treated with ethylene, the A CO transcript was more abundant in the pulp than in the peel and was most prevalent near the pulp-peel interface.

Figure 1. Tissue northern blot demonstrating the distribution pattern of the ACO transcript in an ethylene-treated ripening banana (peel color index 3).

ACO has been purified and characterized from banana [31], and genes encoding ACO have been isolated in our laboratory. Six different genomic isolates ofACO have been characterized from the banana variety Grand Nain. Restriction enzyme mapping and sequence analysis indicate that ACO is encoded by a small gene family in banana (Fig. 2).

144

I I " 'II I

II' " , I

Figure 2. Schematic diagram of banana ACO genomic isolates representing two putative subclasses of the ACO gene family based on patterns of restriction sites and relative homologies of 5' proximal sequence. Heavy lines represent regions of the lambda isolates that hybridize with the ACO cDNA Light lines represent 5' and 3' proximal regions. Short vertical lines indicate the position of restriction sites within the clones.

The banana A CO gene family is apparently comprised of at least two subclasses based on differences in restriction enzyme patterns and homology comparisons between the various genomic isolates. Possibly, genes of one subclass are responsible for wound- or pathogen-inducible ethylene production while the genes of the other subclass are responsible for ripening-related ethylene production. Sequence analysis of the 5' flanking region reveals the presence of a putative TATA-box at position -81 to -77. One genomic isolate contains four exons separated by three small introns. Electrophoretic mobility shift assays indicate that protein(s) from banana pulp whole cell nuclear extracts bind specifically to the 5' flanking region of the A CO gene (Figure 3).

+ S NS

Figure 3. Electrophoretic mobility shift assays of the banana ACO 5' proximal element using banana pulp whole nuclear extracts. -, +, S, and NS represent free-probe, probe plus nuclear extract, specific competitor, and non­specific competitor, respectively.

145

Ethylene production not associated with the normal ripening process can be stimulated in banana fruit by a number of biotic and abiotic factors. Irradiation of banana fruit with high tluence uve increases ethylene production by an unknown mechanism [32]. Ethylene production in banana pulp is also stimulated in the presence of the protein synthesis inhibitor cyclohexamide and the RNA synthesis inhibitor Actinomycin D [33]. Infection by fungal pathogens results in an increase in ethylene production in banana fruit and in a similar acceleration of changes associated with fruit ripening, such as increased respiration [34]. Ironically, ethylene has further been shown to induce germination and appressorium formation in fungal spores, suggesting that fungal pathogens may have co-opted the host's own hormone signal to trigger the infection process [35].

4. Ethylene Perception in Bananas

When applied ethylene is used to accelerate the ripening process, there is a minimum treatment time required to achieve the desired response. The treatment time is approximately 24 h for most commercial varieties, but is variable between cultivars [36]. Longer or continuous exposure to ethylene has no further effect [37], and fruit subjected to shorter exposure times may return to a pre-climacteric state after re­exposure to air [36,37].

Attempts to characterize the ethylene binding site in bananas have been few and have focused on identifying compounds that interfere with the ethylene response in fruit. The application of ethanol, acetylene, and allene induce the ethylene response in banana [6,7,38]. 2,5-Norbomadiene (NBD) and other competitive inhibitors of ethylene suppress the ethylene response as evidenced by an inhibition of peel de­greening [39] and prolongation of the pre-climacteric phase [27]. Most of the compounds that affect the ethylene response in banana diffuse away from the binding site upon exposure to air [38,39]. An exception is diazocyclopentadiene (DAPC), which has been shown to inhibit banana ripening and exhibits permanent inactivation of the ethylene receptor in the presence of light [40]. Treatment with the short-chain fatty acid octanoic acid during the pre-climacteric suppresses ethylene synthesis, and may be interacting with ethylene binding as well [36].

The current understanding of ethylene perception and signal transduction in plants has been recently reviewed [41,42]. Much is being discovered about the ethylene signal transduction pathway using ethylene-response mutants of Arabidopsis [43]. Genes encoding a putative ethylene receptor and other elements of the ethylene response pathway have been isolated from Arabidopsis and tomato. Virtually nothing is known about the ethylene receptor in bananas or about the signal transduction pathway leading to the various ethylene responses associated with fruit ripening. However, there is some evidence to suggest that the signal transduction pathway in banana may share some similarity to the pathway as it is understood inArabidopsis. A gene fragment with sequence homology to CTR1, which encodes a negative regulator of the ethylene perception pathway in Arabidopsis [44], was recently amplified from

146

banana leaf cDNAs in our laboratory. Further characterization of this gene in banana is now underway.

5. Conclusion

An understanding of ethylene-induced changes in fruit ripening and other aspects of post-harvest physiology has allowed major advances in the production and distribution of fruit products over the last three decades. In the near future, we can anticipate increased interest in using the tools of agricultural biotechnology to "design" genetic traits related to the ripening process. These advances will be dependent upon our total information base related to cellular and molecular events associated with fruit development.

While many genes and their respective proteins involved in the process of fruit ripening have been characterized, much less is known about their regulatory mechanisms. Typical plant gene expression studies have involved the generation of transgenic plants. Some species, such as tobacco, tomato, and potato, are readily transformed and regenerated. These traits allow for the testing of numerous promoter/reporter gene constructs. However, in fruit-bearing species such as citrus, apple or banana, the time required for transformation/regeneration limits the testing of large numbers of expression vectors. Information about the patterns of fruit-specific gene expression is essential to experiments directed toward improvement of value­added traits in banana.

6. Acknowledgments

This work was supported in part by Zeneca Plant Science and, in part, by the Banana Improvement Program (sponsored by the Common Fund for Commodities, the F AO Inter-Governmental Group on Bananas (FAOIIGB) and the World Bank), and by the Boyce Thompson Institute.

7. References

1. Forsyth, W.G.C. (1980) Banana and plantain, in S. Nagy and P.E. Shaw (eds.), Tropical and Subtropical Fruits, A VI Publishing, Westport, Conn., pp. 258-278.

2. Anon. (1959) Bananas: Versatile in health or illness, United Fruit Co. 3. Brady, C.J. (1987) Fruit ripening, Ann. Rev.PlantPhysioL, 38,155-178. 4. John, P. and Marchal, J. (1995) Ripening and biochemistry of the fiuit, in S. Gowen (ed.), Bananas

and Plantains, Chapman and Hall, New York, pp. 434-466. 5. Agravante, J.u., Matsui, T., and Kitagawa, H. (1990) Starch breakdown in ethylene-treated and

ethanol-treated bananas: changes in phosphorylase and invertase activities during ripening, J. Jpn. Soc. Food Sci. Technol. 37, 911-915.

6. Agravante, J.U., Matsui, T., and Kitagawa, H. (1991) Changes in pectimnethylesterase, polygalacturonase, lind pectic substances of ethanol-treated and ethylene-treated bananas during ripening, J. Jpn. Soc. Food Sci. Technol. 38, 527-532.

147

7. Agravante, 1.u., Matsui, T., and Kitagawa, H. (1991) Sugars and organic acids in ethanol-treated and ethylene-treated banana fruits, J. Jpn. Soc. Food Sci. Technol. 38, 441-444.

8. Acedo, AL. Jr. and Bautista, OK (1993) Banana fruit response to ethylene at different concentrations of oxygen and carbon dioxide, Asean Food J. 8, 54-60.

9. Pal, RK and Buescher, R W. (1993) Respiration and ethylene evolution of certain fruits and vegetables in response to carbon dioxide in controlled atmosphere storage, J. Food Sci. Technol. 30, 29-32.

10. Kubo, Y., Tsuji, H., Inaba, A, and Nakamura, R (1993) Effects of elevated carbon dioxide concentrations on the ripening in banana fruit by exogenous ethylene, J. Jpn. Soc. Hortic. Sci. 62, 451-455.

11. Abdullah, H., Rohaya, MA, and Yunus, J.M. (1993) Irnprovement on storage of banana (!vfusa sp. cv. Mas) under modified atmosphere, MARDI Research J. 21, 163-169.

12. Jayaraman, KS. and Raju, P.S. (1992) Development and evaluation of a permanganate based ethylene scrubber for extending the shelf life of fresh fruits and vegetables, J. Food Sci. Technol. 29, 77-83.

13. Abe, K and Watada, AE. (1991) Ethylene absorbent to maintain quality of lightly processed fruits and vegetables,J. Food Sci. 56,1589-1592.

14. Abdullah, H., Rohaya, MA, and Yunus-J-M. (1993) Effects of precooling, ethylene absorbent and partial evacuation of air on storage of banana (Musa sp. cv. Berangan) under modified atmosphere, MARDI ResearchJ. 21,171-177.

15. Bai, J.H., Ueda, Y., and Iwata, T. (1990) Effect of packaging with polyethylene bags on shelf life and volatiles production of ripening-initiated bananas, J. Jpn. Soc. Food Sci. Technol. 37, 971-977.

16. Satyan, S., Scott, KJ., and Grabam, D. (1992) Storage of banana bunches in sealed polyethylene tubes,J. Hortic. Sci. 67, 283-287.

17. Krishnamurthy, S. and Kushalappa, C.G. (1985) Studies on the shelf life and quality of robusta bananas as affected by post-harvest treatments, J. Hortic. Sci. 60, 549-556.

18. Desai, B.B., Shukla, D.V., and Chouqule, BA (1989) Biochemical changes during storage of chemical treated banana fruits, J. Maharashtra Agric. Univ. 14, 44-47.

19. Rao, D.V.R and Chundawat, B.S. (1986) Effect of certain chemical retardants on ripening changes of banana cultivar lacatan at ambient temperatures, Prog. Hortic. 18, 189-195.

20. Burdon, J.N., Dori, S., Lomaniec, E., Marinansky, R, and Pesis, E. (1994) The post-harvest ripening of water stressed banana fruits, J. Hortic. Sci. 69,799-804.

21. Xue, Y., Kubo, Y., Inaba, A, and Nakamura, R (1995) Effects of humidity on ripening and texture in banana fruit, J. Jap. Soc. Hortic. Sci. 64, 657-664.

22. Finger, F.L., Puschmann, R, and Barros, RS. (1995) Effects of water loss on respiration, ethylene production and ripening of banana fruit, Revista Brasileira de Fisiologia Vegetal. 7, 115-118.

23. Strydom, G.1., VanStaden, 1., and Smith, M.T. (1991) The effect of gamma radiation on the ultrastructure of the peel of banana fruits, Environ. Exp. Bot. 31, 43-50.

24. Strydom, G.J. and Whitehead, C.S. (1990) The effect of ionizing radiation on ethylene sensitivity and postharvest ripening of banana fruit, Sci. Hortic. (Arnst.) 41: 293-304.

25. Munasque, V.S. and Mendoza, D.B., Jr. (1990) Developmental physiology and ripening behavior of senorita banana (Musa-sp L.)fruits,AseanFoodJ. 5,152-157.

26. Li, W. and Huang, B. (1988) Studies on ethylene production and respiration rate in relation to other ripening changes of three banana cultivars, Acta Hortic. Sin. 15, 18-22.

27. Gao, J.P., Kubo, Y., Nakamura, R, and Inaba, A (1990) Induction of ethylene biosynthesis in banana fruit under different ripening conditions, J. Jpn. Soc. Hortic. Sci. 59,665-672.

28. Dorniniguez, M. and Vendrell, M. (1993) Ethylene biosynthesis in banana fruit: evolution of EFE activity and ACC levels in peel and pulp during ripening, J. Hortic. Sci. 68, 63-70.

29. Ke, L.S. and Tsai, P.L. (1988) Changes of ACC content and EFE activity in peel and pulp of banana fruit during ripening in relation to ethylene production. J. Agric. Assoc. China New. Ser. 143, 48-60.

30. Xie, H.H., Wang, Y.R, and Liu, H.x. (1993) Chilling-induced ethylene production in the peel and pulp of banana, Acta Bot. Sin. 35, 526-532.

31. Moya-Leon, MA and John, P. (1994) ACC oxidase from banana fruit, purification and biochemical characterization, Biologia Plantarum (Prague) 36, S32.

32. Wade, N.L., Tan, S.C., and Kavanagh, E.E. (1993) White light prevents increased catechin synthesis by ultraviolet irradiation in banana fruits, J. Hortic. Sci. 68, 637-644.

33. Areas, JAG., Garcia, E., and Lajolo, F.M. (1988) Effect of protein synthesis inhibitors on the climacteric of banana Mus a acuminata, J. Food Biochem. 12, 51-60.

148

34. Schiffinann-Nadel, M., Michaely, H., Zaubennan, G., and Chet, I. (1985) Physiological changes occurring in picked climacteric fruit infected with different pathogenic fungi, Phytopathol. J. 113, 277-284.

35. Flaishman, MA and Kolattukudy, P.E. (1994) Timing of fungal invasion using host's ripening honnone as a signal, Proc. NatL Acad. Sci. USA 91, 6579-6583.

36. Whitehead, C.S. and Bosse, CA (1991) The effect of ethylene and short-chain saturated fatty acids on ethylene sensitivity and binding in ripening bananas, J. Plant Physiol. 137, 358-362.

37. Inaba, A and Nakamura, R. (1986) Effect of exogenous ethylene concentration and fruit temperature on the minimum treatment time necessary to induce ripening in banana fruit, J. Jpn. Soc. Hortic. Sci. 55, 348-354.

38. Sisler, E.C. and Wood, C. (1988) Competition of unsaturated compounds with ethylene for binding and action in plants, Plant Growth Regul. 7, 181-192.

39. Sisler, E.C., Blankenship, S.M., and Guest, M. (1990) Competition of cyclooctenes and cyclooctadienes for ethylene binding and activity in plants, Plant Growth Regul. 9, 157-164.

40. Sisler, E.C. and Blankenship, S.M (1993) Diazocyclopentadiene (DACP), a light- sensitive reagent for the ethylene receptor in plants, Plant Growth Regulation 12, 125-132.

41. Chang, C. and Meyerowitz, E.M. (1995) The ethylene hormone response in Arabidopsis: a eukaryotic two-component signaling system, Proc. Natl. Acad. Sci. USA 92, 4129-4133.

42. Bleecker, AB. and Schaller, G.E. (1996) The mechanism of ethylene perception, Plant Physiol. 111, 653-660.

43. Chang, C., Kwok, S.F., Bleecker, AB., and Meyerowitz, E.M. (1993) Arabidopsis ethylene-response gene ETR1: Similarity of product to two-component regulators. Science 262, 539-544.

44. Kieber, J.J., Rothenberg, M., Roman, G., Feldmann, K.A, and Ecker, J.R. (1993) CTR1, a negative regulator of the ethylene response pathway in Arabidopsis, encodes a member of the Raf family of protein kinases. Cell7Z, 427-441.

THE MODULATION OF ETHYLENE BIOSYNTHESIS AND ACC OXIDASE GENE EXPRESSION DURING PEACH FRUIT DEVELOPMENT AND FRUITLET ABSCISSION

P. TONUTTI, C. BONGlll, B. RUPERTI AND A. RAMINA Department of Environmental Agronomy and Crop Science, University of Pad ova, Agripolis, 35020 Legnaro (padova), Italy

1. Introduction

It has been shown that an increase in ethylene biosynthesis is associated with specific stages of peach fruit development [11]. Besides accelerating ripening, exogenous ethylene induces the activation of the fruitlet abscission zone (AZ3) located between the pericarp and the receptacle, leading to fruitlet shedding, both under field and laboratory conditions [1, 8]. As far as fruit development is concerned, the highest rates of ethylene evolution are detectable during early peach fruit development and when fruit have softened to about 20 N [4, 1]. At ripening, the appearance of 1-arninocyclopropane-l-carboxylate oxidase (ACO) mRNA precedes the ethylene burst [2] and the highest transcript accumulation is coincident with the climacteric [3]. No information on ACO gene expression pattern during early fruit growth exist.

Considering peach fruitlet abscission, the postulated regulatory role of ethylene is based on the promoting effect of the applied hormone exogenous application on cell wall hydrolases (polygalacturonase, PG, and endo-~-1,4 glucanase, EG) responsible for cell separation at AZ3 level [I]. No knowledge exists on the relationship between fruitlet abscission and endogenous ethylene biosynthesis as well as ACO gene expression at AZ3 level and surrounding tissues.

2. Peach Fruit Development

During the four stages (81, 82, 83 and 84) of peach fruit development, ethylene levels rise above basal levels during the early fruit growth phase and at ripening. In fruit of cv. "Springcrest", an early ripening variety, ethylene evolution was 1.2 n1 h-1g-1fw at the first sampling date (27 days after full bloom, AFB), and it declined through SI reaching an almost undetectable level that was maintained through 82, 83 and part of 84. The climacteric rise started 83 days AFB and, at the last sampling date (88 days AFB), ethylene evolution reached the value of 3 .03 n1 h-1 g-lfw. ACC content and ACO

149

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 149-153. © 1997 Kluwer Academic Publishers.

150

activity measured at 27 (SI), 70 (S3), and 88 (S4) days AFB showed that the amount of ethylene detected at 27 days AFB is associated to a low ACC content (0.15 nmol/gfw) and a high ACO activity (28.2 nl h-1 g-lfw). The undetectable level of ethylene at 70 days AFB is concurrent with the lowest values of ACC concentration (0.11 nmol/gfw) and ACO activity (15 nl h-1g-1fw). The ethylene climacteric parallels a dramatic increase of ACC (2.18 nmol h-1g-1fw) and ACO activity (54 nl h-1g-1fw). When total RNA, extracted from fruit collected at 27, 70 and 88 days AFB, was probed with an ACO cDNA clone, a weak hybridization signal (l.4 kb) was detected at 27 days AFB, whereas transcripts were undetectable at 75 days AFB. The high levels of ethylene evolution and ACO activity detected at 88 days AFB were concurrent with a dramatic accumulation of ACO mRNA.

When propylene (500 ppm) treatment was performed for 48 h on fruitlets collected at 27 days AFB, a 2-fold increase of ethylene biosynthesis was observed if compared with fruitlets maintained in air for the same period. ACC content was unaffected by propylene. ACO activity and gene expression were greatly stimulated by the ethylene analogue.

Detailed observations of ethylene physiology performed on a single fruit basis during the ripening stage (from 75 to 90 days AFB) reconfirmed that the measurable ethylene climacteric rise occurred only after the fruits have already started to soften, as previously reported [10]. Nevertheless, significant increases in mesocarp ACC content and ACO activity were measured at 80 days AFB when the whole fruit ethylene evolution was still undetectable and before any change in fruit firmness. The highest value of ACC content (2.4 nmol/gfw) was observed 85 days AFB, then it declined; ACO activity reached the highest value (190 nl/h/gfw) at the last sampling date (90 days AFB), concurrently with the ethylene climacteric. ACO transcripts were undetectable at the first sampling date (75 days AFB), whereas a hybridization signal appeared 80 days AFB and increased in intensity through ripening.

3. Peach Fruitlet Abscission

Abscission, although considered part of the senescence program, is a highly co­ordinated, active metabolic process. In fact, activation of peach fruitlet AZ3 was paralleled by an increase, in the same region, of total protein: this is due to a stimulated synthesis of specific polypeptides (Fig. 1).

Enhanced ethylene biosynthesis in explants consisting of AZ3 and surrounding tissue (non-zone, NZ), was already observed 20 min after embrioctomy, although it reached significant levels (3.1 nl h-1g-1fw) only 40 min later. Ethylene evolution remained high 12 (6.3 nl h-1g-1fw) and 24h (7.2 nl h-1g-1fw) following activation. Considering the tissue specificity, it appeared that a decreasing gradient of ethylene evolution from the distal NZ (pericarp side), through AZ3, to the proximal NZ (pedicel side) exists. The highest values of ethylene were detected in AZ3 (7.8 nl/h/gfw) and distal NZ (8.2 nl h-1g-1fw) after 48h of incubation in air. The same gradient was observed in propylene-flushed explants, although the ethylene burst was anticipated:

151

ethylene evolution reached the highest values of 6.1, 8.8 and 10.2 nl h-1g-1fw in the proximal NZ, AZ3 and the distal NZ, respectively, after 12 h of treatment. ACO activity paralleled the kinetics of ethylene evolution both in air and propylene-flushed explants. ACO mRNA was undetectable in the proximal NZ and AZ3 at the beginning of experiment, while a hybridization signal was present in the distal NZ (Fig. 2).

, -+-"-__ -J...--.-.!---i •• --I...........~l..-•• -~~.,-'----'--... I._._ • ...Jiij..! _

_ 1111 prupyl"II"4f'Himtlllt

011 _wI i ~--------------------------------~i

-0. ;<:IZl~--r--.-_.---"_' ~,-...,...-Ir.--";--"r"-.+ 97 66 45 31 21 14

MolBCUIllr Weig/W ( Jr.D)

Figure 1. Densitometric scan along SDS-PAGE separation of the in vitro translation products derived from fruitlet abscission zone RNA The RNA was extracted before (control) and after 12 h of propylene (500 ppm) treatment. The arrows indicate polypeptides showing marked differences after propylene treatment.

After 12 h of incubation, both in air and propylene, ACO transcripts appeared in the proximal NZ and the AZ3, and strongly accumulated in the distal NZ. At 48 h in air, a decrease of signal intensity was registered in both the proximal and the distal NZ, but not in the AZ3. After 48h of propylene treatment, transcript accumulation was almost undetectable in the proximal NZ (5% of maximum), whereas the signal intensity remained clearly detectable in both the AZ3 and the distal NZ. When the tissue specificity was considered in terms of cell wall hydrolases, it appeared that dramatic increases of enzyme activities were confined to the AZ3 region and that propylene exerted a major stimulatory effect on EG.

152

.1 ~ s .... Q

'1e 0

100

50

0 0

Hours of treatment

• Proximal NZ OAZ3 mDistalNZ

Figure 2. Effect of air and propylene treatments (12 and 48 h) on the accumulation ACO mRNA in the proximal NZ, AZ3, and the distal NZ. The intensity of hybridization bands was measured by laser densitometry. The strongest hybridization signal, detected after 12 h of propylene treatment in distal NZ, represents the 100% value.

4. Conclusions

Results demonstrated that both in developing fruit and activated AZ3, increases of ethylene evolution are concurrent with high ACO activity levels and accumulation of related transcripts. Peculiar to fruit in early S I appears the enhancement of ethylene evolution, which takes place without any detectable change in ACC content; this may suggest a regulatory role of ACO in ethylene biosynthesis.

Cell separation, occurring as a consequence of AZ3 activation, is preceded by a burst of ethylene evolution that takes place within 60 min both in the AZ3 and the NZs. However, a decreasing gradient of ethylene evolution, as well as of ACO activity and related transcripts, has been observed. These data indicate that a lack of specificity of AZ3 and surrounding tissues in relation to induced ethylene biosynthesis exists. In contrast, a marked specificity has been shown for cell wall hydrolases whose stimulated activities appeared to be always confined to the AZ3. A rise in activity of EG and PG restricted to the site of cell separation has been reported by Roberts et al. [9] in Sambucus nigra leaflets. As reported for other species [5, 6, 7, 12], propylene exerts a stimulatory role on ethylene evolution, ACO activity, and mRNA accumulation, both in developing peach fruits and AZ3. It is interesting to note that, in SI, propylene stimulates ethylene biosynthesis through an enhancement of ACO gene expression without affecting ACC content.

153

5. Acknowledgments

Research reported in this paper was supported by the Research National Council (CNR, special project RAISA) and by the Ministry of Agriculture and Forestry (project "Frutticoltura") of Italy.

6. References

1. Bonghi, C., Rascio, N., Ramina A, and Casadoro, G. (1992) Cellulase and polygalacturonase involvement in the abscission ofleaf and fruit explants of peach, Plant Mol. BioI. 20, 839-848.

2. Callahan, AM., Morgens, P.H., Wright, P., and Nichols, K.E. Jr. (1992) Comparison of Pch313 (pTOM13 homolog) RNA accumulation during fruit softening and wounding of two phenotipically different peach cultivars, Plant Physiol1 00, 482-488.

3. Lester, D.R., Speirs, J., Orr, G., and Brady, C.l (1994). Peach (Prunus persica) endopolygalacturonase cDNA isolation and mRNA analysis in melting and nonmelting peach cultivars, Plant Physiol105, 225-231.

4. Miller, AN., Krizek, BA, and Walsh, C.S. (1988). Whole fruit ethylene evolution and ACC content. J. Amer. Soc. Hort. Sci. 113, 119-124.

5. O'Neill, S.D., Nadeau, lA, Zhang, X.S., Bui, AQ., and Halevy, AH. (1993), Interorgan regulation of ethylene biosynthetic genes by pollination. Plant Cell 5, 419-432.

6. Peck, S.C. and Kende, H. (1995) Sequential induction of the ethylene biosynthetic enzymes by indole-3-acetic acid in etiolated peas, Plant Mol. BioI. 28, 293-301.

7. Picton, S., Barton, M., Bouzayen, M., Hamilton, AJ., and Grierson D. (1993) Altered fruit ripening and leaf senescence in tomatoes expressing an antisense ethylene-forming enzyme transgene Plant J. 3,469-481.

8. Ramina, A, Rascio, N., and Masia, A (1989) The abscission process in peach: structural, biochemical and hormonal aspects, in D.l Osborne and M.B. Jackson (eds.), Cell Separation in Plants, NATO ASI Series Vol 35H, Springer-Verlag Berlin Heidelberg, pp 233-238.

9. Roberts, JA, Taylor, J.E., Coupe, SA, Harris, N., and Webb S.T.l (1993) Changes in gene expression during leaf abscission, in J.C. Pech, A Latche, and C. Balague (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, Kluwer Academic Publishers, pp 272-277.

10. Tonutti, P., Bonghi, C., and Ramina, A (1996) Fruit fmnness and ethylene biosynthesis in three cultivars of peach (Prunus persica L. Batsch),J. Hort. Sci. 71,141-147.

11. Tonutti, P., Casson, P., and Ramina, A (1991) Ethylene biosynthesis during peach fruit development, J. Amer. Soc. Hort. Sci. 116,274-279.

12. Woodson, W.R., Park, K.Y., Drory, A, Larsen, P.B., and Wang, H (1992). Expression of ethylene biosynthetic pathway transcripts in senescing carnation flowers, Plant Physiol. 99, 526-532.

TRANSCRIPTIONAL REGULATION OF SENESCENCE-RELATED GENES IN CARNATION FLOWERS

J.M. MAXSON AND W.R. WOODSON Department of Horticulture, Purdue University, West Lafayette, IN 47907-1165, USA

1. Introduction

The senescence of flower petals in several plant species is associated with a dramatic increase in the production of ethylene. There is considerable evidence pointing to a role for this increased ethylene in regulating the processes of programmed cell death leading to senescence [13]. A number of years ago we reported that the senescence of carnation flower petals was associated with the expression of new genes, which led us to isolate a number of senescence-related cDNA clones that have been useful in elucidating the mechanism by which ethylene regulates their transcription [13]. In addition to its influence on the cell death program leading to floral senescence [27] ethylene controls the expression of genes during tomato fruit riperung [3,15] and pathogen attack [9,18,23]. Ethylene-respousive elements in the promoter regions of several genes have been identified [2,5,11,20]. While nuclear factors have been shown to specifically interact with some of these regulatory regions [5,11,20], little is known about the nature of these DNA binding proteins. Recently, Ohme-Takagi and Shinshi [23] have demonstrated that an 11-bp Gee box, conserved in the promoter regions of ethylene-regulated pathogenesis-related proteins, is sufficient for conferring ethylene­responsiveness to a heterologous promoter. Four different cDNAs encoding DNA binding proteins capable of interacting with the Gee box were cloned (EREBP-1 to 4). The DNA binding domain of the EREBPs does not share homology with other known DNA binding proteins.

2. Identification of an Ethylene-Responsive Enhancer Element in the Carnation GSTI Gene

One of the flower senescence-related genes from carnation was previously shown to encode a glutathione S-transferase (GST) [19]. This gene was shown to be transcriptionally activated by ethylene specifically during flower petal senescence [10,14] and has been used as a model to study the mechanism by which ethylene regulates transcription [10,11]. To investigate the sequences responsible for ethylene­responsive expression of the carnation GST 1 gene, a series of 5' deletions of the GST 1

155

A. K. Kanellis et al. (eds.). Biology and Biotechrwlogy olthe Plant Hormone Ethylene. 155-162. © 1997 Kluwer Academic Publishers.

156

promoter fused to the reporter gene GUS were introduced into petals by particle bombardment. Subsequent analysis of GUS activity in the presence or absence of ethylene, or during senescence revealed that sequences present between -667 and -470 bp 5' of the transcriptional start site were necessary for transcriptional activation of the chimeric gene (Fig. 1). Sequences within this region were further characterized by placing them in the context of a minimal CaMV-35S promoter (-46) containing only a TAT A-box fused to GUS. This analysis revealed that the sequences responsible for ethylene-regulated expression resided between -596 and -470 (Fig. 2). Furthermore, delivery of these constructs into petals showed that these sequences conferred ethylene­responsive expression upon the minimal promoter regardless of orientation, suggesting that and ethylene-responsive enhancer (ERE) element was present. Taken together, these results indicate that the ERE present in the GST 1 promoter is both necessary and sufficient for ethylene-responsive expression.

3. Identification of Nuclear Proteins that Interact with the GSTI ERE

To identify nuclear proteins from carnation petals that interact specifically with the ERE element, gel shift assays were performed [17]. These results are shown in Figure 3. DNA binding activity was present in both fresh (non-senescing) and senescing petals, and interacted -specifically with sequences present between -596 to -470 as evidenced by the successful competition for binding activity with the full length probe (-667 to -470). DNase I footprinting was performed to more precisely localize the sequences involved in this binding [17]. The interaction of nuclear protein with DNA was localized to a 22 bp element with the nucleotide sequence 5' -GTGATTTACCACCTATTTCAAAG-3'. When taken together, these data show that a DNA binding factor is present in nuclear extracts of carnation petals that interacts specifically with a region of the GSTl promoter containing the ERE and may represent trans-acting factors involved in the ethylene-regulated expression of GST 1.

4. Cloning of an ERE-Binding Protein

To clone the gene encoding the carnation ERE DNA binding protein, a cDNA expression library was screened with the double-stranded ERE oligo concatamer. From 600,000 recombinant phage representing an amplified senescing petal cDNA library, two identical clones (1-1 and 5-1) were isolated to purity based on the ability of the expressed b-Galactosidase fusion protein to recognize the ERE oligonucleotide concatamers. This gene product was designated Carnation ERE Binding Protein 1 (CEBP-l). The specificity of the interaction was tested during purification steps by screening with an oligonucleotide concatamer corresponding to another region of the GST1 promoter that was not involved in ethylene responsiveness [-597 to -580; 12]. This promoter region was not recognized by the CEBP-l fusion protein indicating there is some specificity in the interaction between CEBP-l and the ERE oligo.

-1415~7 ________________________ -1~~E2~ L I GUS-NOS I

250

~ 200 a. C 'e 150 ""-=> ~ Q) (5 100 E a.

50

0

-956

LI --------------1�JG~U~S~-N~O~sl

D • ~

> ~ IV

(,)

-667 I

Air

-470 I

Ethylene

Senescing

,.. co It) It) v (7) ..- . .

I GUS-NOS I

I GUS-NOS I

-195

Y GUS-NOS I

,.. ~ It)

~ (7) ..,. ..-. .

Figure 1. Deletion analysis of the GSTl 5' flanking DNA GSTl promoter deletion endpoints relative to the start of transcription are indicated in bp shown on the abscissa. Chimeric genes were delivered into carnation petals by particle bombardment. Petals were incubated in ethylene-free air or ethylene (10 ppm) for 16 hr following bombardment and then assayed for GUS activity. Senescing petals were bombarded 5 days after harvest and incubated in air as they entered into programmed senescence.

157

158

~ -46 I- +8

I C~V I GUS-rileS 3' I A «

-667 ~

+8 -470 -461-

I GSTl vi I

B I CaMV : GUS-IbcS 3' I «

-667 ~

-580-461- +8

I GSTl ~~V I GUS-rIleS3'1 C «

-596 ~

-470-461- +8

I GSTl ¥ claMV I GUS-rIleS 3' I 0 « ~

-470 -596-461- +8

I GSTl ~.l:1 I I CaMV: GUS-rbcS 3' I E

« -596 -470 -596 -470 -596

~ -470-461- +8

I GSTl ~.! GSTl ~ GSTl ~ baMV I GUS-rileS 3' I F

30

25

5 CD

20 .e-c:: 'E :5 15 ~ CD 15

10 E Q.

5

0 A B C 0 E F

Construct

Figure 2, Effects of 5' flanking sequences of GSTl on the ethylene-responsiveness of a minimal CaMV-35S promoter fused to GUS, Constructs are indicated in (A) and activity of GUS following bombardment and treatment with ethylene is indicated in (B),

159

-667 A

-470

C B

o day 6 day

Compo (20x) A B C A B C

Protein + + + + + + + + , , Bound 'I ':~

Free

Figure 3. Gel mobility-shift analysis of the ERE-containing GST 1 promoter region using crude nuclear extracts from carnation petals at anthesis (0 day) or senescence (6 day). Binding of the end-labeled GSTl promoter fragment from -667 to -470 with petal nuclear protein competed with a 20x molar excess of unlabeled DNA fragments: A, -667 to -470; B, -596 to -470; C, -667 to -580. Products analyzed on a 4% nondenaturing polyacrylamide gel.

,"" ~!~,

Nucleotide sequence analysis of the cDNA representing CEBP-I revealed a 292 amino acid open reading frame encoding a 32 kDa polypeptide (GenBank accession number U38483). Comparison of the deduced polypeptide with protein sequences in the GenBanklEMBL database identified significant homology with nucleic acid binding proteins from maize [Nucleic Acid Binding Protein (NBP)-72% similarity; 4] and Arabidopsis [FMV3bp-79% similarity; 8]. The conserved amino acid regions have been implicated as the nucleic acid binding domain [16]. Within this region are two highly conserved motifs, designated RNP-I and RNP-2. The RNP (ribonucleoprotein)

160

motif is the only RNA-binding sequence for which structural information is available. Three-dimensional structures of the RNA binding proteins UI snRP has been determined and the interaction of the RNP consensus sequence with RNA has been verified [21]. However, the RNP motif is not unique to RNA binding proteins. An RNP sequence in several transcription factors has also been recognized. These include the human Enhancer Factor I [BFI; 24] and Y-box I [Yb-I; 7] proteins, the Xenopus laevis FRG Y2 factor [I] and the E. coli cold shock protein CspA [12]. Interestingly, the ERE oligonucleotide sequence used to isolate CEBP-I (5'­ATTTACCACCTATTTCAAA-3') is similar to the recognition sequences for other RNP-I-containing proteins including maize NBP (5'-... TTTCGCAAGCCG .. 3'), Arabidopsis FMV3bp (5'-... CAAAAAAG ... -3') and E.coli CspA (5'­... AATTTA. .. ACCACCC ... -3'). The mRNAs of the recently cloned ethylene­responsive element binding proteins (EREBPs) from tobacco have been shown to accumulate in the presence of ethylene [23]. However, these DNA binding proteins have been isolated based on their interaction with the GCC box motif. The carnation ERE of GST J does not contain a GCC box element and is therefore likely to associate with a distinct DNA binding protein. An octameric motif within the carnation ERE (5'-ATTTCAAA-3') is also present in the ethylene-controlling region of genes from tomato and bean [2,5,20]. Both ES and E4 genes are upregulated during tomato fruit ripening in response to ethylene. The 5' flanking sequences reported to be necessary for ethylene-inducibility contain the sequence 5'-AATTCAAA-3'. Footprinting of the E4 promoter has determined that this sequence is protected from DNaseI digestion. Similarly, the pathogen-induced chitinase gene (CH5B) from bean possesses an ethylene-regulatory region containing this motif. The PRB-J b gene is a component of the pathogenesis-related basic PR-I protein family from tobacco and is transcriptionally activated by ethylene [IS]. Two separate regions of DNA-protein interactions have been identified within the 94 bp ethylene-responsive region. One region contains a GCC box motif while the other contains a core sequence (5'­... ATTGAAA .. 3') that is similar to the ERE oligonucleotide. Two distinct nuclear factors were shown to interact with these elements. Perhaps transcription of ethylene­regulated genes can be directed by individual or combinations of cis-elements, including the GCC box and the AT -rich element reported here.

In addition to its affect on transcription, ethylene has also been shown to exert translational control. Overexpression of the tomato ACC synthase cDNA LE-ACS2 in its antisense orientation prevents the climacteric rise in ethylene production characteristic of the ripening stage of development [22]. The plants only ripen when treated with ethylene for six days. The expression of polygalacturonase (pG) mRNA is observed in the antisense plants, but the PG protein does not accumulate until the fruit has been exposed to ethylene. The presence of RNP motifs in CEBP-I provides an intriguing possibility for its involvement in ethylene-regulated transcription and translation. Conceivably, CEBP-I may be involved not only in the activation of GSTI transcription, but in the processing, stability or transport of the RNA. Such a precedent has been set by the TFIIIA transcription factor from Xenopus laevis that activates the transcription of the 5S RNA gene [25]. The RNA made then binds to the same protein. Accumulation of 5S RNA occurs as long as there is free protein

161

available. This has been suggested to provide a mechanism for the accumulation of massive amounts of 5S RNA before the 18S and 28S rRNA synthesis begins and ribosome assembly occurs. The WIllA protein interacts with DNA and RNA through zinc finger motifs. A similar function has also been ascribed to the FRG Y2 Y -box transcription factor from Xenopus [6]. As a DNA -binding protein it stimulates transcription from a variety ofY-box promoters. As an RNA-binding protein, it serves to protect mRNA against degradation. Perhaps the same is true of CEBP-l. Because the activation of GST 1 expression occurs at a stage in development in which the cells are undergoing a programmed death, it may be necessary to protect the transcripts encoding the proteins that are part of the cell death regime. Future experiments will address the ability of CEBP-l to interact with RNA, especially GSTl RNA.

5. Acknowledgments

This research was supported by the National Science Foundation (ffiN-9206729). J.M. Maxson was supported by a Purdue University Plant Physiology Program Fellowship.

6. References

1. Bienz, M. (1986) A CCAAT box confers cell-type-specific regulation on the Xenopus hsp70 gene in oocytes, Cell 46,1037-1042.

2. Broglie, KE., Biddle, P., Cressman, R and Broglie, R (1989) Functional analysis of DNA sequences responsible for ethylene regulation of a bean chitinase gene in transgenic tobacco, Plant Cell 1, 599-607.

3. Christofferson, RE., Warm, E. and Laites, G.G. (1982) Gene expression during fruit ripening in avocado, Planta 155, 52-56.

4. Cook, W.B. and Walker, J.C. (1992) Identification of a maize nucleic acid-binding protein (NBP) belonging to a family of nuclear-encoded chloroplast proteins, NucL Acids Res. 20, 359-364.

5. Deikman, J. and Fischer, R.L. (1988) Interaction of a DNA binding factor with the 5' -flanking region of an ethylene-responsive fruit ripening gene from tomato, EMBO J. 7,3315-3320.

6. Deschamps, S., Viel, A, Garrigos, M., Denis, H. and Ie Maire, M. (1992) mRNP4, a major mRNA­binding protein from Xenopus oocytes is identical to transcription factor FRGY2, J. BioI. Chem . 267, 13799-13802.

7. Didier, D.K, Schiffenbauer, 1., Woulfe, S.L., Zaceis, M. and Schwartz, B.D. (1988) Characterization of the cDNA encoding a protein binding to the major histocompatibility complex class II Y box, Proc. Natl. Acad. Sci. USA 85, 7322-7326.

8. Didier, D.K and K1ee, H.J. (1992) Identification of an Arabidopsis DNA-binding protein with homology to nucleolin, Plant Mol. BioI. 18,977-979.

9. Ecker, J.R and Davis, R W. (1987) Plant defense genes are regulated by ethylene, Proc. Natl. Acad. Sci. USA 84, 5202-5206.

10. Itzhaki, H. and Woodson, W.R (1993) Characterization of an ethylene-responsive glutathione s­transferase gene cluster in carnation, Plant Mol. BioI. 22, 43-58.

11. Itzhaki, H., Maxson, J.M. and Woodson, W.R (1994) An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione s-transferase (GSTJ) gene, Proc. Natl. Acad. Sci. USA 91, 8925-8929.

12. Landsman, D. (1992) RNP-l, an RNA-binding motif is conserved in the DNA-binding cold shock domain, Nucl. Acids Res. 20, 2861-2864.

13. Lawton, KA, Huang, B., Goldsbrough, P.B. and Woodson, W.R (1989) Molecular cloning and characterization of senescence-related genes from carnation flower petals, Plant PhysioL 90, 690-696.

14. Lawton, KA, Raghothama, KG., Goldsbrough, P.B. and Woodson, W.R (1990) Regulation of senescence-related gene expression in carnation flowers by ethylene, Plant Physiol. 93, 1370-1375.

162

15. Lincoln, J.E., Cordes, S., Read, E. and Fischer, R.L. (1987) Regulation of gene expression by ethylene during Lycopersicon esulentum (tomato) fruit development, Proc. Natl. Acad. Sci. USA 84, 2793-2796.

16. Mattaj, I.W. (1989) A binding consensus: RNA-Protein interactions in splicing, snRNPs and sex, Cell 57,1-3.

17. Maxson, J.M. and Woodson, W.R (1996) Cloning of a DNA-binding protein that interacts with the ethylene-responsive enhancer element of the carnation GSTJ gene, Plant MoL Bioi. 31, 751-759.

18. Meller, Y., Sessa., G., Eyal, Y. and Fluhr, R (1993) DNA-protein interactions on a cis-DNA element essential for ethylene-regulation, Plant Mol. Bioi. 23, 453-463.

19. Meyer, RC., Goldsbrough, P.B. and Woodson, W.R (1991) An ethylene-responsive flower senescence-related gene :from carnation encodes a protein homologous to glutathione S-transferases, Plant MoL Bioi. 17,277-281.

20. Montgomery, 1., Goldman, S., Deikman, 1., Margossian, L. and Fischer, RL. (1993) Identification of an ethylene-responsive region in the promoter of a fruit ripening gene, Proc. NatL Acad. Sci. USA 90, 5939-5943.

21. Nagai, K, Oubridge, C., Jessen, T.H., Li, J. and Evans, P.R (1990) Crystal structure of the RNA­binding domain of the Ul small nuclear ribonucleoprotein A, Nature 348, 515-520.

22. Oeller, P.W., Wong, L.M., Taylor, L.P., Pike, D.A and Theologis, A (1991) Reversible inhibition of tomato fruit senescence by antisense RNA, Science 254, 437-439.

23. Ohme-Takagi, M. and Shinshi, H. (1995) Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element, Plant Cell 7, 173-182.

24. Ozer, 1., Fabers, M., Chalkey, R and Sealy, L. (1990) Isolation and characterization of a cDNA clone for the CCAAT transcription factor EFIA reveals a novel structural motif, J. BioL Chem. 265, 22143-22152.

25. Pelham, H.RB. and Brown, D.D. (1980) A specific transcription factor that can bind either the 5S RNA gene or 5S RNA,Proc. Natl. Acad. Sci. USA 77, 4170-4174.

26. Raghothama., KG., Lawton, KA, Goldsbrough, P.B. and Woodson, W.R (1991) Characterization of an ethylene-regulated flower senescence-related gene from carnation, Plant Mol. Bioi. 17, 61-71.

27. Woodson, W.R (1987) Changes in protein and mRNA populations during the senescence of carnation petals, Physiol. Plant 71, 495-502.

EmYLENE: INTERORGAN SIGNALING AND MODELING OF BINDING SITE STRUCTURE

E.J. WOLTERING, A. VAN DER BENT, G.J. DE VRIJE, and A. VAN AMERONGEN Agrotechnological Research Institute (ATO-DLO), PO Box 17, 6700 AA Wageningen, The Netherlands.

1. Introduction

Ethylene is involved in many developmental processes including the senescence of petals in ethylene sensitive flower species such as carnation, orchids and Petunia. The mode of action of ethylene in petal senescence may be its effect on the expression of numourous genes, among them genes coding for enzymes directly involved in cell death and genes encoding ethylene biosynthetic enzymes or components of the ethylene perception and signal transduction route(s). In carnation, a range of different senescence-related (SR) genes were isolated by Woodson and co-workers. Expression of most of these genes increased during the rise in ethylene production while treatment with the ethylene inhibitor 2,5-norbornadiene generally was inhibitory, indicating that expression is controlled by ethylene [I]. Based on homology studies with other known proteins, putative roles of some of the proteins in petal senescence is expected. Among the carnation SR genes, besides from the ethylene biosynthetic genes ACC synthase and ACC oxidase, the following activities have been reported: beta-glucosidase, beta-galactosidase, glutathione-S-transferase, carboxyphosphonoenolpyruvate mutase and thiol protease [I and references therein]. Although some of these enzymes may be directly or indirectly involved in the processes leading to petal senescence, definite proof of their function e.g in transgenic plants, is lacking.

Apart from being a direct inducer of cell death and senescence of plant organs, ethylene and its amino acid precursor, I-aminocyclopropane-I-carboxylic acid (ACC), have been implicated as molecules involved in interorgan communication. In waterlogged tomato plants, ACC produced in the roots was shown to be translocated, through the xylem, to the aerial parts of the plant where conversion to ethylene occurs. The ethylene produced causes epinastic curvature of the leaves [2]. Also in other plant species, waterlogging was shown to elicit ethylene symptoms in the aerial parts of the plant which may be ascribed to the root-to-shoot translocation of ACC [e.g. 3, 4]. Root-to-shoot translocation of ACC was also suggested to be involved in pathogenic symptom expression upon root knot nematode infection of tomato plants [5] and in leaf abscission in water stressed citrus plants following dehydration [6]. Additional evidence for root-to-shoot ACC transport was provided by experiments with radiolabeled ACC [7].

163

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 163-173. © 1997 Kluwer Academic Publishers.

164

Besides xylem translocation of ACC it has been demonstrated that ACC could also be translocated through the phloem. In tomato, application of radiolabeled ACC to a single leaflet led to accumulation of radioactivity in other leaves and in roots [8, 9].

Also within flowers, ACC translocation has been implicated in interorgan communication. Sequential increases in ACC concentrations in different flower parts following pollination or stigma wounding indicated ACC translocation [10, 11]. More direct evidence was obtained in experiments where flowers were treated with radiolabeled ACC. In carnation, Reid et al. [12] showed that petals produced radiolabeled ethylene following treatment of the stigma with radiolabeled ACC. Similarly, in orchid (Cymbi­dium) flowers, petals produced radiolabeled ethylene following application of radiolabeled ACC to the rostellum (modified lobe of the stigma) [13]. The above data suggest that ACC may act as a mobile senescence factor in flowers.

Ethylene gas has also been considered as a translocatable factor in plants but received much less attention than ACC. It was shown that ethylene, applied to the roots of tomato plants, diffused to the aerial parts and it was argued that ethylene translocation was one of the factors contributing to symptom expression in waterlogged plants [14]. In Vicia faba, however, ethylene was relatively immobile [15]. Also in gravistimulated flower stems ethylene seems rather immobile as relatively steep gradients in internal concentrations were detected between upper and lower parts of gravistimulated stems [16, 17].

Ethylene is thought to interact with a receptor molecule. Ethylene binding in plant tissues has been a subject of research for many years and, based on dissociation curves, it has been established that at least 2 binding activities are present [18]; a slow binding activity (in the order of hours) and a fast binding activity (in the order of minutes). Until recently no clear picture of the possible structure of such binding proteins existed.

In the last couple of years, components of the ethylene response pathway have been identified by isolating ethylene-response mutants in Arabidopsis using the triple response as a screenable marker. The first mutant identified was etr, an ethylene-insensitive, dominant mutation [19]. The protein encoded by the ETRl gene displays extensive homology to the hybrid bacterial histidine kinases of the two-component signal sensing systems. In the ETRI protein a histidine kinase domain is joined to a response-regulator domain present in the carboxy terminus. The sensor domain is probably located at the amino terminus which contains hydrophobic sequences, suggesting that it is an integral membrane protein with the kinase and regulator domains located cytoplasmically [20]. Mutations in the hydrophobic region lead to ethylene insensitivity and alter binding characteristics. Currently another putative receptor protein has been identified (ERS) which resembles the ETRI protein except that it lacks the response regulator domain [21].

ETRI acts upstream of a second identified mutant, etrl (constitutive triple response), suggesting that this mutant affects ethylene signal transduction. The CTRl gene codes for a putative serine/threonine protein kinase most similar to the Raf family of protein kinases [22]. This suggests that ETRIIERS is complexed to a MAP-kinase like cascade of protein kinases. Activation of such protein kinase cascades by extracellular signals affects many cellular processes such as gene transcription, cell cycle progression, differentiation and cell death in mammals, insects and nematodes and most likely also in yeast. In plants, a similar pathway may regulate the responses to ethylene.

165

The cloning and characterization of ethylene receptor genes allows the study of ethylene binding at the molecular level. Sufficient recombinant protein may be produced to facilitate elucidation of the 3-dimensional structure of ethylene receptors with X-ray crystallography or NMR techniques. Unfortunately, the membrane embedded nature of the sensor domain makes it very difficult or even impossible to study its structure with these analytical techniques. Furthermore, except for the kinase domain, there is no structural information available of homologous proteins. The construction of models with well established homology modelling techniques is therefore not possible. However, the sequence information presented by Chang et al. [20] suggests the existence of three hydrophobic membrane spanning stretches at the amino terminus of ETRI which may form the basis for a de novo modelling approach. The three hydrophobic stretches likely transverse the membrane as alpha-helices. The residual N-terminus presumably resides in the apoplasm, whereas the large C-terminal moiety (which includes effector functions) is probably located in the cytosol. Expression of truncated forms of ETRI in yeast demonstrated that ethylene binding indeed takes place in the N-terminal, membrane embedded domain [23]. All known ethylene-insensitive mutants are mutated in the putative transmembrane (TM) helices, indicating that the ethylene binding site is located within the membrane embedded domain.

This paper describes experiments aiming at examining the relative contributions of ACC and ethylene translocation in interorgan communication during senescence of flowers. In addition, using the available sequence information of the membrane embedded ethylene binding domain of ETR1, molecular models have been developed. Though preliminary, these models are useful tools that may enhance our understanding of the molecular basis of ethylene perception.

2. Results

2.1. REDISTRIBUTION OF RADIOACTIVITY

Translocation studies were done in cut Cymbidium and Petunia flowers. Radiolabeled ACC was pipeted onto the stigma of a flower, previously treated with a mixture of aminoethoxyvinylglycine (A VG) and cobalt chloride to block ACC synthase and ACC oxidase activities. Similarly, the ACC analog, a-aminoisobutyric acid (AlB), was applied to non-pretreated flowers. In Petunia, stigmas were either intact or wounded by sqeezing between forcep-tips prior to treatment with the radiolabeled chemicals. Twenty (Cymbidium) or 10 h (Petunia) following application of the radiolabeled chemicals, the amount of radio-activity in extracts from different flower parts was measured. Flower parts were frozen in liquid N2 and extracted by immersion in an 80% methanol solution at 60°C for 24 h. Recovery of radioactivity was generally over 95%.

In addition to the translocation studies in whole flowers, exudation of radioactive materials from excised central columns, being a fusion of styles and stamens, (Cymbidium) and styles (Petunia) was studied. Following treatment of the stigma with radiolabeled chemicals, these flower parts were placed with their cut base in either water or solutions with different EDTA concentrations to prevent blockage of phloem cells [24]. Accumulation of radioactivity in the holding solutions was measured.

166

Both in Cymbidium and Petunia flowers, most of the radioactivity, either applied as ACC or AlB, remained at the site of application while generally less than 1 % was recovered from the remaining tissues (Tables 1 and 2). No ethylene was produced during the experimental period. This lack of redistribution of ACC and AlB was confIrmed in experiments where the translocation of radiolabeled compounds was studied in isolated central columns and styles. During the experimental period less than 0.5 % of the applied radioactivity was recovered from the holding solutions (data not shown).

2.2. ETHYLENE DIFFUSION IN CYMBIDIUM

The transport of gases was studied in Cymbidium flowers. 30 mL tubes were mounted over the stigmatic region of the central column and over one of the petals. The tube that was positioned over the central column was flushed with a mixture of 10 J.illL of either ethylene or propylene in air with a flow rate of 5 mUmin. Air samples (1.5 mL) were regularly withdrawn from the tube holding the petal using a Photovac gas chromatograph equiped with a photo ionization detector and a sample pump. During sampling, the volume withdrawn was replenished with air. A fan was located in front of the experimental setup to prevent any exchange of gases through the air between the two tubes. For more details on experimental procedures see [13, 25, 26].

Following ethylene treatment, ethylene was released by the petals within approximately 0.5 h (Figure lA). Similar experiments done with propylene yielded comparable results (Figure lB). Air samples taken from locations between the column and the petals only yielded background levels of ethylene/propylene (data not shown).

2.0

-I 1.5 ..5 ~ c: 1.0 G)

>-.c ... 0.5 G)

0.0

2.0

::i 1.5 ..5

G) c 1.0 G)

>-D-o li 0.5

0.0

A. Ethylene

B. Propylene

0.0 0.5 1.0

time (hI

1.5 2.0

Figure 1. Ethylene emission by an individual petal following treatment of the central column with a mixture of either 10 IIllL ethylene (A) or propylene (B) in air.

167

TABLE 1. Redistribution of stigma-applied radiolabeled ACC and AlB in Cymbidium. Ten IJL of an aqueous I'C-ACC solution (5 nmol, 0.7 * 106 dpm) was applied to the stigma of a previously

AVG/CoCl2-treated flower; I'C-Am (5 nmol, 0.6 * 106 dpm) was applied to a non-pretreated flower. Treatment with an A VG/CoCI2 mixture was done by pipetting 10 IJL of a solution containing 10 mM A VG and 10 mM CoCl2 onto the stigma approximately 20 h before treatment with radio labeled chemicals. Means (n=2) are given for tissues other than the upper part of the central column (containing the stigma). Values for radioactivity in this part of the flower were calculated assuming that the remaining part of the applied activity was located here.

Flower part

Upper part column Lower part column Ovary Pedicel Petals + sepals Labellum Holding solution

Radioactivity (% of applied)

AlB

99.2 0.24 0.20 0.01 0.10 0.25 0.04

TABLE 2. Redistribution of stigma-applied radiolabeled ACC and AlB in Petunia.

ACC

99.1 0.11 0.23 0.05 0.10 0.33 0.05

Two IJL of an aqueous I'C-ACC solution (10 nmol, 2.2 * 10" dpm) was applied to the stigma of a previously AVG/CoCl2-treated flower; I'C-AlB (10 nmol, 0.11 * 10· dpm) was applied to a non-pretreated flower. Treatment with an A VG/CoCI2 mixture was done by pipetting 2 IJL of a solution containing 5mM A VG and 5 mM CoCl2 onto the stigma and placing the flower with its cut base in an 0.1 mM CoCI2 solution approximately 16 h before treatment with radiolabeled chemicals. Means (n=3) are given for tissues other than the stigma/style. Values for radioactivity in the stigma/style were calculated assuming that the remaining part of the applied activity was located here.

Treatment Flower part

Intact

Wounded

Style + stigma Corolla Ovary Calyx Pedicel Holding solution

Style + stigma Corolla Ovary Calyx Pedicel Holding solution

Radioactivity (% of applied)

AlB

99.S 0.06 0.07 0.04 0.06 0.002

99.2 0.10 0.24 0.19 0.29 0.002

ACC

99.9 0.03 0.002 0.004 0.004 < 0.001

99.6 0.26 0.13 0.003 0.009 < 0.001

168

2.3. MOLECULAR MODELLING OF ETRI MEMBRANE EMBEDDED DOMAIN

All modelling studies were performed on a Silicon Graphics Indigo workstation with the Sybyl 6.2 package from Tripos Inc., USA. The Kollman universal force field was used in conformational searching procedures. The neural network system PHDhtm [27], accessible at the EMBL Internet site, was employed to predict putative transmembrane domains in the ETRI sequence.

With high probability, and in accordance with Chang et al. [20], PHDhtm predicts the presence of three TM helices. The (less certain) boundaries for these helices are: HI: 26-43; H2: 53-77; H3: 82-106 (Figure 2) .

.... / .... 1 .... / .... 2 .... / .... 3 .... / .... 4 .... / .... 5 ...• / .... 60 MEVCNCIEPQWPADELLMKYQYISDFFIAIAYFSIPLELIYFVKKSAVFPYRWVLVQFGA! aaaaaaaaaaaaaaaaaaaaaaaaaTTTTTTTTTTTTTTTTTTcccccccccTTTTTTTT

.... / .... 7 .... / .... 8 .... / .... 9 .... / .... 10 ... / .... 11 ... / .... 120 FIVLCGATHLINLWTFTTHSRTVALVMTTAKVLTAVVSCATALMLVHIIPDLLSVKTREL! TTTTTTTTTTTTTTTTTaaaaTTTTTTTTTTTTTTTTTTTTTTTTTcccccccccccccc

Figure 2. Deduced amino acid sequence ofETRl protein and putative transmembrane stretches (T), apoplasrnic (a) and cytoplasmic (c) amino acids.

Similar to other known helical TM receptor domains, the helices of ETRI are likely to be closely packed. Bundles of 3 (anti-)parallel helices either contain a triangular arrangement of "straight" helices or pack in a coiled coil motif. From the specifically packed hydrophobic interior (heptad repeat) and polar exterior of the latter, only straight helices were considered for ETRI. Standard alpha-helices and side chain conformations were used to construct an initial model of the ETRI sensor domain. At this stage, the kink that Pro36 may induce in TMHI was neglected.

TMH 2 & 3 both contain a cysteine (C65 & 99) which are positioned in the membrane at a similar "depth". These residues may therefore form an (intermolecular) cysteine bridge. Assuming this, we performed a systematic search of the conformational space that is accessible from the 5 rotatable dihedral angles in the connected side chains (Experimental details: esse dihedral +/- 900, all others full grid search with 100 increments; Force Field + charges: Koll-Uni; Vanderwaals scaling 0.75; distance constraints on termini: 13 A maximum).

The produced low energy conformations of the covalently bonded TMH2 & 3 helices show interhelical angles in the range 160-180° and a fairly dense packing at their interface. The two histidines at position 69 and 107 consistently appear on the same face of the bundle.

Figure 3. Apoplasmic view on anticlockwise and clockwise topologies for ETRI monomers.

169

As shown in Figure 3, two topologies have to be considered for the 3 helices in ETRI. A preference for either of these should arise from their compatibility with biological data and the quality of the resulting model with respect to hydrogen bonding, salt bridges, spatial packing etcetera. This also holds for the degrees of freedom in combining HI with H2&3 (relative height, tilt angle, rotation angle). Systematic searches aimed at the optimal packing of TMHI against 2&3 are not possible without the input of additional constraints. Therefore, TMHI was added "by eye". The manual addition ofTMHl to the lowest energy conformation from this search has resulted in preliminary models like the one depicted in Figure 4.

Figure 4. anticlockwise topology model for a three helix bundle of the membrane embedded domain of ETR 1.

170

3. Discussion

The redistribution studies in Cymbidium revealed that applied ACC and AlB are largely immobile both in intact flowers and in isolated central columns. Earlier it was found (in non-cobalt treated Cymbidium flowers) that petals produced radiolabeled ethylene following treatment of the stigma with radiolabeled ACC. To explain the amount of radiolabeled ethylene produced by the petals in terms of ACC translocation, approximately 25% of the applied ACC must have been translocated to the petals [13]. In A VG/CoCI2-treated flowers, that did not produce ethylene in response to ACC treatment, we would therefore expect significant accumulation of radioactivity in the petals. Similarly, following treatment of the stigma with the ACC analog, AlB, we would expect accumulation in the petals as AlB is not actively metabolized. As in none of the experiments more than 1 to 2% of the applied radioactivity was recovered from the petals and, in addition, these chemicals were also immobile in isolated central columns, it is concluded that ACC is not likely to playa role in interorgan signaling in Cymbidium. The lack of redistribution of stigma-applied ACC and AlB in Petunia flowers and isolated Petunia styles, confirms the view that ACC does not playa significant role in interorgan communication in flowers.

It was shown earlier that, during senescence, orchid petals do not accumulate any ACC and, in accordance with this, have no ACC synthase activity and show no expression of ACC synthase genes [26, 28]. Senescence of the petals, being an ethylene-regulated event, is therefore totally dependent on processes in other flower parts. As ACC was found to be immobile in these flowers, the observed production of ethylene by the petals and subsequent petal senescence may be from translocated column-produced ethylene.

The rate of ethylene and propylene translocation was therefore studied by direct meas­urement of ethylene/propylene release by the petals following treatment of the stigmatic region with ethylene or propylene. These experiments were only carried out in Cymbidium flowers because, contrary to Petunia, the different flower parts in Cymbidium are spatially seperated from each other allowing measurements of ethylene emission of individual flower parts. Ethylene and propylene were found to be translocated from the site of application to the petals within less than 0.5 and 1.0 h, respectively. Considering that the applied gases must first diffuse into the tissue, thereafter over a distance of several cm through the internal tissues of the central column to the petal and, subsequently, out of the petal to allow detection, these results show that ethylene diffusion is fast enough for a possible role role in interorgan communication in these flowers. In addition, it shows that ethylene translocation may account for the rapid ethylene production by petals following application of ACC to the stigma [13]. In should be noted here that the central column does not contain any air channels; on average less than 10 J.JL air is recovered from 1 g tissue by vacuum extraction. Apparently ethylene rapidly diffuses through the intercellular spaces due to the existence of a concentration gradient. During senescence of these flowers, concentrations of up to 15 J.JLIL ethylene were found in the internal air spaces of the central column [26], indicating that the applied concentrations may be regarded physiologically relevant. Altogether our results show that ethylene, apart from its direct role in cell death and organ senescence, plays an important role in interorgan signaling.

171

The ethylene binding protein ETRI most likely plays a major role in ethylene signal perception and transduction. The various assumptions that were made in the construction of 3-D models of the N-terminal domain of ETRI as shown in Figure 4 require that the results are interpreted with great care. In fact, after the development of these models, Schaller and Bleecker [29] reported novel data from ethylene binding studies in yeast­expressed mutated ETRI proteins that are in conflict with one of our assumptions. Mutation of Cys65 to serine was found to completely abolish specific ethylene binding, whereas mutation of Cys99 to serine did not. This questions the validity of the cysteine bridge that we built into our models. When the cysteine bridge is omitted, systematic packing of TMH2 & 3 is no longer possible and the three transmembrane helices may be combined in numerous additional relative orientations. Despite the obvious limitations of the current models, some interesting conclusions appear to have general validity. In support of the occurrence of ETRI as a dimer [23], the models suggest that monomers are not suited for ethylene binding, because: i) Competitive antagonists like 2,5-norbornadiene are too large to be accommodated in the core of the 3-helix bundle models, ii) A receptor-bound Cu ion is thought to intermediate in ethylene binding, thus necessitating an even larger binding site and, iii) Not all hydrophilic residues can be packed in the core between the three helices. As the membrane is highly hydrophobic, these residues likely reside at the dimer interface. We therefore suggest that the interface area between the monomers in a dimer is the site where Cu coordination, and therefore ethylene binding, occurs.

Of the polar residues that may serve as ligands in Cu coordination, the histidines in H2 (His69) and/or H3 (HisI07) are preferred candidates. These large polar residues are among the most common in Cu coordination. Also, they very rarely occur in TM regions, suggesting a special role in ETRI. In this case, dimer topologies with H2 and/or H3 at the interface are clearly the most likely. From the 2-fold axial symmetry that the homodimer will likely possess, the actual coordination of Cu is likely to involve pairs of equivalent histidines, one from each monomer. Depending on the coordination of the Cu ion (e.g. tetrahedral coordination for Cu(I) or square planar coordination for Cu(ll», additional electron donors in the form of proximate polar residues may be localized with the aid of the monomer models. Like the two histidines, these are interesting candidates for mutation studies that may shed light on the structural requirements for the coordination of Cu. Vice versa, the qualitily of the current models of both monomer and dimer topologies may be much improved once that more data from site-directed mutagenesis become available.

Currently we are incorporating the latest experimental data of the site-specific mutagenesis approach applied by Bleecker and co-workers and the sequence information of ETRI and ERS genes from other species. The prediction of the boundaries of the transmembrane helices now becomes more accurate. By including the Arabidopsis ERS and geranium ETR sequences as input for the PHDhtm program, Hisl07 is shifted to a position just outside TMH3 and, therefore, to the cytoplasmic domain, whereas His69 remains in the membrane. Thus, only the latter remains a likely candidate for the coordination of Cu. Interestingly, His69 is located exactly one turn "up" from Cys65. It is therefore very close to, and at the same face of the helix as, the cysteine residue whose mutation to serine completely abolishes specific ethylene binding. It is therefore proposed that Cys65 and His69 are jointly involved in the coordination of Cu.

172

4. Conclusion

The presumed role of ACC as a mobile senescence factor in flowers could not be established. On the contrary, ACC was largely immobile in Cymbidium and Petunia flowers. Ethylene translocation was found to be very fast and may account for interorgan communication following e.g. pollination. This emphasizes a dual role for ethylene in flower senescence: a direct role in death of individual cells and tissue senescence and a signaling role to coordinate and integrate the senescence process.

In the monomer models, the mutant positions ofthe ethylene-insensitive etr 1-1 to etr 1-4 alleles all align about half-way the membrane. This supports the proposed role of the transmembrane domain as the key region in ethylene binding. The monomer models suggest that ethylene binding requires the formation of dimers. This combines well with the general view that Cu ions play a role in ethylene binding. Here, a symmetrical coordination of the Cu ion(s) by two cysteines (C65) and two histidines (H69) at the dimer interface of two TMH2 helices is proposed. Updated models that are based on recent experimental data are under construction. We hope these will contribute to a better understanding of the structure of the binding site in the N-terminal transmembrane domain of ethylene receptors.

5. Acknowledgement

The authors are grateful to A.B. Bleecker for stimulating discussions and providing unpublished results and to D.G. Clark for providing the geranium ETR sequences.

6. References

1. Woodson, W.R., Brandt, A.S., Itzhaki, H., Maxon, J.M., Wang, H., Park, K.Y., and Larsen, P.B. (1993) Ethylene regulation and function of flower senescence-related genes, In: J.C. Pech et al. (eds), Cellular and molecular aspects of the plant Iwrmone ethylene, Kluwer Academic Publishers, The Netherlands, pp. 291-297.

2. Bradford, K.J. and Yang, S.F. (1980) Xylem transport of 1-aminocyclopropane-l-carboxylic acid, an ethylene precursor, in waterlogged tomato plants, Plant Physiol. 65, 322-326.

3. Seliskar, D.M. (1988) Waterlogging stress and ethylene production in the dune slack plant, Scirpus americanis, J. Exp. Bot. 39, 1639-1648.

4. Voesenek, L.A.C.J., Harren. F.lM., Bogemann, G.M., Blom, C.w.P.M., and Reuss, J. (1990) Ethylene production and petiole growth in Rumex plants induced by soil waterlogging, Plant Physiol. 94, 1071-1077.

5. Glazer, J., Apelbaum, A., and Orion, D. (1984). Reversal of nematode-induced growth retardation in tomato plants by inhibition of ethylene action, J. Amer. Soc. Hort. Sci. 109, 886-889.

6. Tudela, D. and Primo-Millo, E. (1992) l-Aminocyciopropane-l-carboxylic acid transported from roots to shoots promotes leaf abscission in Cleopatra mandarin (Citrus reshni Hort. ex Tan) seedlings rehydrated after water stress, Plant Physiol. 100, 131-137.

7. Finlayson, S.A., Foster, K.R., and Reid, D.M. (1991) Transport and metabolism of I-aminocyciopropane­I-carboxylic acid in sunflower (Helianthus annuus L.) seedlings, Plant Physiol. 96, 1360-1367.

8. Amrhein, N., Breuing, F., Eberle, J., Skorupka, H., and Tophof, S. (1982) The metabolism of I-am­inocyciopropane-l-carboxylic acid, In: P.F. Waering (ed), Plant Growth Substances. Academic Press. LondonlNew York. pp. 249-258.

9. Morris, D.A. and Larcombe, N.J. (1995) Phloem transport and conjugation of foliar-applied 1-aminocyciopropane-l-carboxyJic acid in cotton (Gossypium hirsutum L.), J. Plant Physiol. 146,429-436.

173

10. Nichols, R., Bufler, G., Mor, Y., Fujino, D.W., and Reid, M.S. (1983) Changes in ethylene production and l-aminocyclopropane-l-carboxylic acid content of pollinated carnation flowers, J. Plant Growth Regul. 2, 1-8.

11. Nichols, R. and Frost, C.E. (1985) Wound-induced production of l-aminocyclopropane-l-carboxylic acid and accelerated sl(nescence of Petunia corollas, Sci. Hortic. 26, 47-55.

12. Reid, M.S., Fujino, D.W., Hoffman, N.E., and Whitehead, C.S. (1984) l-aminocyclopropane-l-carboxylic acid (ACC) - The transmitted stimulus in pollinated flowers, J. Plant Growth Regul. 3, 189-196.

13. Woltering, E.J. (1990) Interorgan translocation of l-aminocyclopropane-l-carboxylic acid and ethylene coordinates senescence in emasculated Cymbidium flowers, Plant Physiol. 91, 837-845.

14 Jackson, M.B. and Campbell, D.J. (1975) Movement of ethylene from roots to shoots, a factor in the responses of tomato plants to waterlogged soil conditions, New Phytol 74, 397-406.

15. Zeroni, M., Jerry, P.H., and Hall, M.A. (1977) Studies on the movement and distribution of ethylene in Vicia Faba L., Planta 134, 119-125.

16. Woltering, E.J. (1991) Regulation of ethylene biosynthesis in gravistimulated Kniphojia flower stalks, J. Plant Physiol. 138, 443-449.

17. Philosoph-Hadas, S., Meir, S., Rosenberger, I., and Halevy, A.H. (1996) Regulation of the gravitropic response and ethylene biosynthesis in gravistimulated snapdragon spikes by calcium chelators and ethylene inhibitors, Plant Physiol. 110,301-310.

18. Sisler, E.C. (1991) Ethylene binding components in plants, In: A.K. Mattoo and J.C. Suttle (eds), The plant hormone ethylene, CRC press, Boston/London, pp. 81-99.

19. Bleecker, A.B., Estelle, M.A., Somerville, C., and Kende, H. (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana, Science 241, 1086-1089.

20. Chang, C., Kwok, S.F., Bleecker, A.B., and Meyerowitz, E.M. (1993) Arabidopsis ethylene-response gene ETRI: Similarity of product to two-component regulators, Science 262, 539-599.

21. Hua, I., Chang, C., Sun, Q., and Meyerowitz, E.M. (1995) Ethylene insensitivity conferred by Arabidopsis ERS gene, Science 269, 1712-1714.

22. Kieber, U., Rothenberg M., Roman, G., Feldmann, K.A., and Ecker, I.R. (1993) CTRl, a negative regulator of the ethylene response pathway in Arabidopsis encodes a member of the Raf family of protein kinases, Cell 72, 427-441.

23. Schaller, G.E., Ladd, A.N., Lanahan, M.B., Spanbauer, I.M., and Bleecker, A.B. (1995) The ethylene response mediator ETRI from Arabidopsis forms a disulfide-linked dimer, Journal of Bioi. Chern. 270, 12526-12530.

24. King, R.W. and Zeevaart, I.A.D. (1974) Enhancement of phloem exudation from cut petioles by chelating agents, Plant Physiol. 53, 96-103.

25. Woltering, E.I., Somhorst, D., and Van der Veer, P. (1995) The role of ethylene in interorgan signaling during flower senescence, Plant Physiol. 109, 1219-1225.

26. Woltering, E.J. (1990) Interrelationship between the different flower parts during emasculation-induced senescence in Cymbidium flowers, J. Exp. Bot. 41, 1021-1029.

27. Rost, B., Casidio, R., Fariselli, P., and Sander, C. (1995) Prediction of helical transmembrane segments at 95% accuracy, Prot. Science 4, 521-533.

28. O'Neill, S.D., Nadeau, I.A., Zhang, X.S., Bui, A.Q., and Halevy, A.H. (1993) Interorgan regulation of ethylene biosynthetic genes by pollination, Plant Cell 5, 419-432.

29. Schaller, G.E. and Bleecker, A.B. (1995) Ethylene-binding sites generated in yeast expressing the Arabidopsis ETRI gene, Science 270, 1809-1811.

AN ETHYLENE·REGULATED DNA ELEMENT IN ABSCISSION· SPECIFIC GENE PROMOTERS AND THE EXPRESSION OF AN ETRI HOMOLOGUE IN TOMATO ABSCISSION

M.L. TUCKER, G.L. MATTERS, S.M. KOEHLER, D. ZHOU, S-B HONG, P. KALAITZIS, A.K. MATTOO, P. NATH USDA, ARS, Plant Molecular Biology Lab, Bldg 006, Beltsville, MD 20705, USA

1. Introduction

Abscission is evoked in response to both environmental and developmental cues, e.g., fruit ripening, senescence, disease, insect damage, fertilization, drought, heat, etc. Both the quality and yield of a crop can be affected by the number of flowers and young fruit that abscise prematurely. An understanding of the biology of abscission is of both commercial and academic interest. The intent of this article is to review progress in my laboratory towards an understanding of the hormonal and cell-specific regulation of abscission.

A common feature in the abscission of plant organs is the synthesis or activation of hydrolases that degrade the cell wall and middle lamella to form the fracture plane between the two separating parts. Bean has for the past few decades been the model most often used to study abscission. It was from bean that the first cell wall hydrolase, cellulase (endo-I ,4-p-D-glucanase), was identified [14], purified [13], antiserum prepared [5], and a cDNA clone characterized [21]. Immunolocalization [20] and in situ hybridization with RNA probes [22] confirmed that this cellulase is directly associated with the separation layer cells in the abscission zone. Outside the vascular bundle in the cortical cells of the bean abscission zone, cellulase protein and mRNA were restricted to 1 to 2 cell layers on either side of the fracture plane. However, within the vascular bundle, cellulase was synthesized in cells up to -4 mm distance from the separation layer. Synthesis of cellulase in the vascular bundles relatively far from the separation layer may be important in the sealing off of the vascular system to reduce water loss and pathogen infection.

In addition to the cell-specific expression of the cloned cellulase mRNA, the hormonal regulation controlling the accumulation of cellulase mRNA during abscission is consistent with its potential role in the formation of the separation layer. Exogenous ethylene initiates and enhances the rate of abscission in most plants. In bean, accumulation of cellulase mRNA in

175

A. K. Kanellis et al. (eds.), Biology and Biotechnology o/the Plant Hormone Ethylene, 175-183. © 1997 Kluwer Academic Publishers.

176

abscission zones is dependent upon the continuous presence of ethylene' [21]. In addition, auxin, an inhibitor of abscission, prevents the accumulation of cellulase mRNA in abscission zones even in the presence of high concentrations of ethylene [21].

Several years ago we reported on the cloning and sequencing of the bean abscission cellulase (BAC) gene [23]. In addition, we reported preliminary results with the transient expression of a GUS reporter gene fused to the BAC gene promoter [23]. Here we describe recent work on the BAC gene promoter and introduce studies on the abscission-specific expression of polygalacturonases [9] and an ETRI homologue (putative ethylene receptor) [25] in tomato.

2. Analysis of the BAC gene promoter

In addition to bean, we have studied abscission in soybean. As a part of the soybean project, we identified a cDNA and genomic clone for an abscission cellulase from soybean [12]. The expression of the soybean abscission cellulase (SAC) gene is very similar ~o that of the BAC gene [11]. To aid in the analysis of the bean cellulasl'l gene promoter, we compared the 5' upstream DNA seqbences for the BAC gene with the comparable region of the SAC gene (Fig. 1). Two regions of high sequence identity are noteworthy and are circled in Figure 1. The encircled region labeled A between -1 and -200 is of particular interest here and will be discussed in greater detail below.

Bean promoter

-1200

, .

-800 -400 I

, B ,. '!to' '~I

..... . . , . ,.

J.,. , . ' .. . ,

, , .. .,. ... . ,.! .' J! • , , .

-1 ~ - (-1)

A I ,.~I

.'-" (-3'J1)

• .. , (-801)

.

r (-1301)

III

S j ~ en

Figure 1. Dot matrix comparison of the BAC promoter sequence 'with the comparable region of the SAC gene sequence. Regions of high sequence similarity are circled with a dashed line. The numbering for nucleotide position in the SAC sequence is in parentheses to emphasize that these are relative to a predicted start of transcription based solely on sequence similarity with the BAC gene sequence .

Initially, we prepared several BAC 5' promoter constructs fused to a GUS reporter gene [23]. Transient expression from these constructs after particle gun bombardment of bean abscission zones gave disappointingly low levels of in vitro GUS activity. To improve the quantitative assay for transient expression driven by the BAC promoter we incorporated a luciferase reporter gene [18] (Fig. 2). In addition, we used a double 35S promoter [10] fused to a GUS gene as an internal control to normalize expression from various BAC-Iuciferase constructs (Fig. 2).

177

Figure 2. Gene construct design and preparation of explants for transient expression assay. A. Chimeric gene constructs used to obtain transient expression results shown in Figure 3" The BKm3 construct contains 2.8 kbp of 5' BAC sequence which includes 37 bp of 5'

A 5' cellulase sequence luc

-2800 +1 +47

3'cellulase term. seq. (880bp)

BKm3 • :; "m';"

Ill"

2-9m -21_~'N=" double 35-S

promoter

-343 -90-343 +1 035S'5 '

GUS

50% ()

AO% \

NOS voId particle (1.8 micron) -

petiole leaf abscission zone (lAZ)

untranslated sequence and 10 bp of translated sequence fused to the luciferase (luc) open reading frame. In addition, after the stop codon for luciferase, BKm3 is terminated by 680 bp of 3' BAC sequence which includes 168 bp of 3' untranslated BAC sequence. The 2-9m construct includes 210 bp of BAC sequence 5' to the start of transcription and 47 bp of BAC sequence down from the start of transcription fused to luciferase which is terminated with the nopaline synthetase (NOS) termination sequence. The 035S construct includes a double CaMV 35S promoter fused to a GUS reporter gene and NOS termination sequence. The 035S plasmid and either BKm3 or 2-9m were co­precipitated at equal molar concentration onto 1.6 micron gold particles. 035S acts as an internal control. B. Oiagram showing how explants were prepared for particle gun bombardment.

Figure 3 Cellulase activity and transient expression of chimeric BAC-Iuciferase constructs in bean. Constructs were prepared and co-precipitated onto gold particles as shown in Figure 2A.

~10000 .;t g 7500

j5000 ~ 2500

~ 5000 3 .1:

~ 4000 .;t

~ 3000

J2000 ~ 1000

A

c

~ 100

~ 75 <.'J g 50

c: .0 100

~ en 75

i5 50 g

8

BKm3 2-9m

BKm3 2-9m

B

o

Leaf abscission zones (LAZ), stems and petioles were prepared as shown in Figure 2B and bombarded with gold particles. A and B. Explants were exposed to 1.0 JlL/L ethylene for 48 h and the bombarded surface (1 mm) harvested and assayed for cellulase, luciferase and GUS activity. A. Cellulase activity per gram of tissue. B. The ratios of luciferase to GUS activity normalized to the mean ratio for BKm3 in this series of experiments. C and D. All abscission zone explants received 2 IlL of a 50 mg/mL mannitol solution on the bombarded surfaces before exposure to 1.0 J1llL ethylene. Samples labeled 2,4-0 received 100 JlM 2,4-dichlorophenoxy-acetic acid in the mannitol solution. Samples labeled NBO were exposed to 5,000 JlllL 2,5-norbornadiene. C. Cellulase activity per gram of tissue. D. The ratio of luciferase to GUS activity was normalized to the mean ratio for BKm3 in this series of experiments. Numbers above standard error bars indicate number of replicates (9 explants per replicate) for each type of explant or treatment.

178

To date, two constructs have been fully tested for transient expression in bean using particle gun bombardment. The larger construct, BKm3, includes both a 2.8 kb 5' BAC promoter and a 680 bp 3' BAC gene fragment (Fig. 2). The smaller construct, 2-9m, includes 210 bp 5' BAC sequence (Fig. 2). These two BAC gene constructs were tested for their relative expression in stems, abscission zones and petioles (Figs. 3A & 3B). In addition, the constructs were tested for their expression in the presence and absence of the ethylene inhibitor 2,5-norbornadiene (NBO) and the auxin analog 2,4-dichlorophenoxyacetic acid (2,4-0) (Figs. 3C & 3~).

Both BKm3 and 2-9m were expressed more abundantly in abscission zones than stems or petioles (Fig. 3B), and both were inhibited by NBO and 2,4-0 (Fig. 3~). These results suggest that the shorter 210 bp promoter fragment includes enough sequence information to confer tissue-specific and hormonal regulation. Nevertheless, the larger construct includes information that can enhance thr. level of this expression (for more details see ref. 12).

Figure 4. Expression 3f the -2W BAC promoter in 2-9-7 transgenic R! plants. RNA was isolated from 0, 24, 48 and 72 h ethylene-induced leaf abscission zones (LAZ) and adjacent stem (S) and petiole (Pt) l'Iections. IAA in la::tolin paste was applied to the petiolar stump of

..:....!... .:.. ...:. 1M - "+ STS --=---

ex?lants at concentrations of 0 (- IAA) or 50 ~M e- iAA) 4 h prior to a 61 h exposure to ethylene. J~xplants were treated with sooium thiosulfate

-!!!-~ ...!!.!!.......!!.!!.. ~H.t{_ STS) or silver thiosulfate (+ S1"S) and exposed

• GUS

t-:> ethylene for 88 h. After 61 or 88 h of 25 ~1JL ethylene exposure for IAA or STS treated explants, respectively, RNA was isolated from leaf

• TAPO abscission zones (LAZ). Ten ~g of total RNA were .:oaded per lane. The RNA blot was sequentially

• TAE1 ill'Obed for mRNA encoding GUS, T APG and T AE 1.

In addition to analysis by transient expression in bean, we used Agrobacterium tumefaciens to stably transform tomato with the BAC 5' sequence fused to the GUS reporter gene. Unfortunately, we were unable to get significant GUS activity from any of our transgenic plants. However, one construct (2-9, 210 bp BAC promoter) expressed a low level of GUS transcript. The accumulation of the GUS transcript was examined for tissue­specificity and hormonal regulation (Fig. 4). GUS transcript did not accumulate in ripening fruit (data not shown) nor stems, petioles or abscission zones of transgenic tomato prior to exposure to ethylene (Fig. 4); however, GUS transcript did accumulate in ethylene-induced abscission zones and petioles but not stems (Fig. 4). Silver thiosulfate (STS), an inhibitor of ethylene action, fully inhibited abscission and accumulation of GUS transcript in abscission zones of transgenic tomato (Fig. 4). However, IAA, which also fully inhibited abscission, only partially inhibited GUS transcript accumulation (Fig. 4). This is in contrast to PG mRNA accumulation which was completely blocked by the IAA treatment (Fig. 4). The observation that IAA only partially inhibited GUS mRNA expression suggests that sequences are missing from the 210 bp BAC construct that confer IAA sensitivity or

179

that tomato is somehow different than bean in this regard (for more detail and discussion see ref. 12).

3. Polygalacturonase expression in tomato abscission

In part, because of some of the anomalies we observed for expression of the 2-9 construct in stably transformed tomato and transient expression in bean, we decided to examine hydrolase expression in tomato abscission. We initially identified a single PO clone, T APO 1, that was specific for tomato abscission zones [9J. T APO 1 mRNA did not accumulate in stems, petioles or ripe fruit. On rescreening the cDNA library for full-length clones, we identified two more POs expressed in abscission. The three POs are closely related, each having between 85 to 93% amino-acid sequence identity with the others; however, their sequence identity with the ~omato fruit PO [4J is approximately 45% for each. In order to avoid potential cros~-hybridization problems on northern blots, we used an RNaSe protection protocol to determine change~ 10 the accumulation of gene-specific PG transcripts (Fig. 5). Although th( sequences for the three PG& m-e very similar, the temporal accumulation of the respective mRNAs are not identical (Fig" 5), TAPO 1 and 2 have fairly similar accumulation rates while T AP04 accumulates much earlier in both flower and leaf abscission zones (Fig. 5). After 24 h of ethylene exposure, the l!lRNAs for all three PGs accumulate in distal flower pedicles. The function of PO in distal flower pedicles is unknown.

Figure 5. Expression patterns for three different PO genes expressed in tomato abscission. Polysomal RNA was extracted from abscission zones (A) and tissue proximal (Pr) and distal (0) to the abscission zones of flower pedicles, and from abscission zones (A) of leaf explants exposed to 25 IllJL ethylene for the indicated intervals of time. RNA was also extracted from anthers and pistils (stigma and styles only) of fully open tomato flowers. Labeled RNA probes were prepared for each of the three PO clones (TAPGt, TAPG2, TAPG4) and hybridized with 51lg of the RNA samples. After hybridization, samples were incubated with RNase ONE, separated on acrylamide gels, and exposed to x-ray film. Undigested probe (P) was loaded onto each gel to show the size of probe that escaped RNase digestion in treated samples.

Anthers and pistils (stigmas and styles only) were also examined for PO expression. Several reports have shown hydrolase activity a:;sociated with

180

these reproductive tissues [1,15,16]. All three PGs show a low level of transcript accumulation in pistils, but not anthers (Fig. 5). At this time, we do not know if the PG transcript in the pistil samples are associated with the pollen or the maternal pistil tissue.

gP03 gPG2 gPG1 - ~ ..... ~ 1kb SaE E Sm HHSmEK E EK E

", "" " ' " n

, ... >..TAPG2-12a (14 kb) ., ~ APG1-2(14kb) .1

Chromosomal DNA

gPG4 gPGS ,. APG11-1 (13 kb). __ I

....... ~ E E HBB X E E , ',,'" ,

1-4 >..pG3-3 (13 kb) .r~romoscmBI DNA

~e~I'.1 (~4Jc!JJ .~

Figure 6. Chromosomal linkage of PG genes. Overlapping A phage genomic clones have been identified for each of the three PG cDNAs expressed in abscission. Genomic clones have been partially mapped by restriction endonucleases and sequencing. Sin, Sal I; E, EcoR I; Sm, Sma I; H, H:/td III; K, Kpn I; B, BamH I· \ ,:c 1.

In addition to the identification of three PG cDNA clones, we have identified several ~enomic clones that nybridize to these cDNAs. Endonuclease restriction maps and seque~cing of overla9ping genomic clones show that TAPGl and 2 (gPGI and g!>C'l,) are closely linked (Fig. 6). TAPG4 (gPG4), however is not as closely lioke'! (Fig 6). Tw,,: ~dditional PG genes were discovered in the genomic clonee, gPG3 and 5 (F!g, 6). RNase protection studies show that gPG3 is not expressed in leaf or flower abscission zones. We have not yet tested for expression of gPG5.

4. Expression of ETRI homologues (ethylene receptors) in abscission

We routinely use ethylene to initiate and synchronize abscission. It has been proposed that ethylene is the natural regulator of abscission in plants [7]. We decided to examine the expression of putative ethylene receptors during abscission. Chang et a1. [3] identified a clone for the gene that is responsible for the ethylene insensitivity of the Arabidopsis etrl mutant. Recently, Schaller and Bleecker [19] showed that the in vitro expressed protein for ETRI binds ethylene. Using the Arabidopsis ETRI cDNA as a probe [3], we identified two different homologues from tomato, TAEI [25] and TFE27 [26]. The expression of TAEI is shown in Figures 4, 7 and 8. TAEI mRNA level (based on equal loading of RNA) changes by only a few-fold in all the tissues examined, including leaf and flower abscission zones, stems, petioles, pedicles and fruit (Fig. 7). Moreover, neither auxin (IAA) nor silver thiosulfate (STS) affected TAEI transcript levels (Fig. 8). Transcript levels for TFE27 are very similar to TAEI (data not shown). These results indicate that the regulation of abscission by ethylene is not regulated by the presence or absence of mRNA for the putative ethylene receptor. Nevertheless, control

181

of ethylene perception by genetic manipulation of the ethylene receptor is viable mechanism for regulating abscission in agronomic plants.

Flower 1Ibec. Fruit Figure 7. RNA expression patterns for two ethylene receptor genes, TAB and NR, in tomato. RNA was extracted from ethylene treated and untreated stems (S), leaf abscission zones (A), petioles (Pt), proximal pedicles (P) pedicle abscission zones and green (0), breaker (B) and ripe (R) fruit. The same northern blot was first probed with 32p labeled T ABl and then several months later with NR

TAE1-

HR-

STS

1M

o 24 024h

- + - +

C2H4 .!.. ~ ~ !!. h ....!!.!!.... 48h

TAE1-

HR-

Figure 8. Effect of silver thiosulfate (STS) and auxin (JAA) on RNA accumulation of TABl and NR mRNA in leaf abscission zones from ethylene­treated tomatc ~xp'ant!L S'f~ :4_: .. 1AA treatments were the same as that des<:ribcl L,:: ,4lgure 4.

Recently, Wilkinson et al. [24] cloned the Never-ripe (NR) gene that confers ethylene insensitivity to tomato. The NR gene has very high sequence identity with the ERS gene from A.rabidopsis [6] which is also closely related tc ETR1. Based on the published sequence for the NR gene [24], we used peR to clone the NR mRNA. The NR gene has approximately 68% nucleic-acid sequence identity with the TAEI sequence. We probed the same blots that were probed with TAEI with the NR probe (Figs. 7 and 8). The hybridization conditions were such that the two probes did not cross­hybridize with the other gene's transcript. The mRNA levels for the NR gene showed greater variability than the TAEI mRNA levels (Fig. 7). Particularly noteworthy are the changes in the mRNA levels in fruit (Fig. 7). NR transcript increases markedly during fruit maturation while that for TAEI is fairly constant. Auxin treatments which fully inhibited leaf abscission had little effect on NR mRNA levels in leaf abscission zones (Fig. 8). However, STS inhibited NR transcript levels several-fold in abscission zones without changing the mRNA levels for TAEI (Fig. 8).

5. Concluding remarks

An applied goal of our project is to modify abscission through genetic engineering to benefit agricultural productivity. One approach for delaying abscission to increase the yield of selected crops is to express in an abscission-specific manner a mutant form of the ethylene receptor that confers dominant ethylene insensitivity. For example, a chimeric gene could be constructed that uses an abscission-specific PG gene promoter fused to a mutant ethylene-receptor gene. In addition, we are interested in (1) using

182

cis-acting elements in the gene promoters to identify trans-acting factors that might have interesting abscission regulating properties and (2) establishing visual abscission-specific markers that can be used to study cell-to-cell signaling and cell differentiation in abscission. To create a visual marker for abscission, we will fuse an abscission-specific promoter (e.g., TAPGl or 4) to a GUS [8] or GFP (green fluorescent protein) [2] reporter gene. A good abscission-specific marker would allow us not only to study cell-to-cell signaling but also provide a tool to study the cell differentiation events that occur in adventitious abscission. Adventitious abscission is the formation of a separation layer at positions along the stem or petiole where abscission would not normally occur. It is assumed that the abscission zone for normal abscission at the base of an organ (Le., leaf, fruit, flower, etc.) is determined early in development [17]. However, cell commitment in adventitious abscission must be induced either by unique environmental conditions or hormone treatment~ In addition to the importance of ab&cission to agriculture productivity, abscission provides the opportunity ~c study many interesting facets of cell biology, development and hormonal regulation.

6. References

1. Brown, S.M. and Crouch, M.L. (1990) Characterization ~i a family abundantly expressed in Oenothera organesis pollen that shows sequenu d.milarity to polygalacturonase. Plant Cell 2, 263-274.

2. Chalfie, M., Tu Y., Euskirchen G., Ward W.W., and Prasher, D.C. (1994) Green Fluorescent Protein as a marker for gene expression. Science 263, 802-805.

3. Chang, c., Kwok, S.F., Bleecker, A.B., and Meyerowitz, E.M. (1993) Arabidopsis ethylene-response gene ETRl: Similarity of product to two-component regulators. Science 262, 539-544.

4. Della Penna, D., Alexander, D.C., and Bennett, A.B. (1986) Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening. Proc Natl Acad Sci USA 83, 6420-6424.

5. Durbin, M.L., Sexton, R., and Lewis, L.N. (1981) The use of immunological methods to study the activity of cellulase isozymes (/J-l,4-g1ucan 4-g1ucan hydrolase) in bean leaf abscission. Plant Cell Environ 4, 67-73.

6. Hua, J., Chang, c., Sun, Q., and Meyerowitz, E.M. (1995) Ethylene insensitivity conferred by Arabidopsis ERS gene. Science 269, 1712-1714.

7. Jackson, M.B. and Osborne, D.J. (1970) Ethylene, the natural regulator of leaf abscission. Nature 22S, 1019-1022.

8. Jefferson, R.A., Kavanagh, T.A., and Bevan MW (1987) GUS fusions: /J-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO 6, 3901-3907.

9. Kalaitzis, P., Koehler, S.M., and Tucker, M.L. (1995) Cloning of a tomato polygalacturonase expressed in abscission. Plant Mol Bioi 28, 647-656.

10. Kay, R., Chan, A., Daly, M., and McPherson, J. (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236, 1299-1302.

11. Kemmerer, E.C. and Tucker, M.L. (1994) Comparative study of cellulases associated with adventitious root initiation, apical buds, and leaf, flower, and pod abscission zones in soybean. Plant Physiol 104, 557-562.

12. Koehler, S.M.; Matters, G.L., Nath, P., Kemmerer, E.C., and Tucker, M.L. (1996) The gene promoter for a bean abscission cellulase is ethylene-induced in transgenic tomato and shows high sequence conservation with a soybean abscission cellulase. Plant Mol Bioi (in press)

183

13. Koehler, D.E., Lewis, L.N., Shannon, L.M., and Durbin, M.L. (1981) Purification of a cellulase from Kidney bean abscission zones. Phytochemistry 10, 409-412.

14. Lewis, L.N. and Varner, J.E. (1970) Synthesis of cellulase during abscission of Phaseolus vulgaris leaf explants. Plant Physiol46, 194-199.

15. Milligan, S.B. and Gasser, C.S. (1995) Nature and regulation of pistil-expressed genes in tomato. Plant Mol BiollB, 691-711.

16. Neelam, A. and Sexton, R. (1995) Cellulase (endo IJ-I,4 glucanase) and cell wall breakdown during anther development in the Sweet Pea (Lathyrus odoratus L.): Isolation and characterization of partial cDNA clones. J Plant Physiol 146, 622-628.

17. Osborne, D.J. (1989) Abscission. Crit Rev Plant Sci B, 103-129. 18. Ow, D.W., Wood; KV., DeLuca, M., DeWet, J.R., Helinski, D.R., and Howell, S.H.

(1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 134, 856-859.

19. Schaller, G.E. and Bleecker, A.B. (1995) Ethylene binding sites generated in yeast expressing the Arabidopsis ETRI gene. Science 170, 1809-1811.

20. Sexton, R., Durbin, M.L., Lewis, L.N., and Thomson, W.W. (1981) The immunocytochemical localization of 9.5 cellulase in abscission zones of bean (Phaseolus vulgaris cv. Red Kidney). Protoplasma 109, 335-347.

21. Tucker, M.L., Sexton, R., del Campillo, E., and Lewis, L.N. (1988) Bean abscission cellulase: Characterization of a eDNA clone and regulation of gene expression by ethylene and auxin. Plant PhysiolBB, 1257-1262.

22. Tucker, M.L., Baird, S.L., and Sexton, R. (1991) Bean leaf abscission: Tissue specific accumulation of a cellulase mRNA. Planta 186,52-57.

23. Tucker, M.L., Matters G.L., Koehler, S.M., Kemmerer, E.C., and Baird S.L. (1993) Hormonal and tissue-specific regulation of cellulase gene expression in abscission. In: Pech JC, Latche A, Balague C (eds), Cellular and Molecular Aspects of the Plant Hormone Ethylene, Current Plant Science and Biotechnology in Agriculture, Vol 16 Kluwer Academic Publishers, Boston, pp. 265-271.

24. Wilkinson, J.Q., Lanahan, M.B., Yen, H-C., Giovannoni, U., and Klee, H.J. (1995) An ethylene-inducible component of signal transduction encoded by Never-ripe. Science 170, 1807-1809.

25. Zhou, D., Kalaitzis, P., Mattoo, A.K., and Tucker ML (1996) The mRNA for an ETRI homologue in tomato is constitutively expressed in vegetative and reproductive tissues. Plant Mol Bioi 30, 1331-1338.

26. Zhou, D., Mattoo, A.K., and Tucker, M.L. (1996) Molecular cloning of a tomato cDNA (accession no. U47279) encoding an ethylene receptor. Plant PhysiolllO, 1435-1436.

SPATIAL AND TEMPORAL EXPRESSION OF ABSCISSION-RELATED GENES DURING ETHYLENE-PROMOTED ORGAN SHEDDING

IA. ROBERTS, S.A. COUPE, C.A. WHITELAW and IE. TAYLOR Department of Physiology and Environmental Science, Faculty of Agricultural and Food Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LEi2 5RD, UK

1. Introduction

During the course of development a spectrum of organs are shed from a plant [1]. Although the cellular and molecular mechanisms by which the abscission process is brought about have yet to be ascertained, the final event is the dissolution of the cell wall at the site where shedding takes place. A major difficulty encountered when studying abscission is that only a few rows of cells may undergo separation. In bean, there is convincing evidence that only one or two rows of cells may contribute to the loss of the primary leaf [2] while in tomato flowers there may be 5-10 rows of cells involved in the process [3]. In an attempt to examine the molecular changes which lead to abscission we have chosen to study the shedding of leaflets in Sambucus nigra [4,5,6]. This is a well characterised system and as the leaflet abscission zone is a composite of three sites where cell separation takes place, as many as 50 rows of cells may be involved in the process [7]. Moreover, leaflet abscission can be induced in approximately 24 hours by exposure to ethylene (10 III r1) and the use of explants leads to a close degree of synchronisation of the process [5].

2. Regulation of Cell Wall Degradation

Ultrastructural analysis of leaf abscission has revealed that one of the first visible signs of cell separation is the breakdown of the middle lamella [7]. This observation indicates that the pectin-rich material of the wall matrix is degraded and a role for such enzymes as polygalacturonase (pG) and pectin esterases in the process has been proposed. Evidence that leaf abscission is associated with an increase in the activity of PG has been shown in a number of systems including S. nigra [5] and in this species an analysis of the cell wall material has shown that cell separation is accompanied by an increase in the solubility of polyuronides within the zone tissue. An increase in the activity of ~ 1,4 glucanase is commonly associated with the time course of abscission and although it has been hypothesised that this enzyme may contribute to the

185

A. K. Kanellis et al. (etis.), Biology and Biotechnology of the Plant Hormone Ethylene, 185-190. © 1997 Kluwer Academic Publishers.

186

dissolution of the primary cell wall there is, as yet, no definitive role for this enzyme in the cell separation process. Other enzymes that could contribute to the cell wall loosening process include the recently characterised enzyme xyloglucan endotransglycosylase (XET) [8], however, there is no evidence that the activity of this enzyme changes in either the zone or adjacent non-zone tissue during abscission in S. nigra [Webb, Fry and Roberts unpublished].

3. Changes in Gene Expression during Leaflet Abscission in Sambucus nigra

3.1 CELL WALL DEGRADING ENzyMES

As the leaflet abscission zone of S. nigra is clearly distinguishable prior to cell separation and is composed of many layers of cells, it is possible to extract mRNA from tissue which is relatively uncontaminated with "non-zone" material. This facility has enabled us to generate an abscission zone cDNA library that can be screened using both heterologous and homologous probes. Furthermore, by adopting a differential screening strategy additional abscission-related clones may be identified.

Using a cDNA encoding a 13 1,4 glucanase from abscission zones of P. vulgaris [9] as a heterologous probe, we have isolated a hybridising clone from the S. nigra library [10]. This cDNA, named pJETl, encodes an mRNA with 67% sequence identity to the bean mRNA and 48% identity to a 13 1,4 glucanase mRNA expressed during ripening of avocado fruit. Northern analysis of the expression of JETl has shown that the mRNA accumulates specifically in the abscission zone tissue 18 hours after exposure to ethylene [10]. Interestingly, the 13 1,4 glucanase mRNA seems to show signs of degradation within the abscission zone tissue and this is not a consequence of the extraction process as other abscission-related mRNAs remain intact [10].

A similar heterologous screening strategy for the isolation of an abscission zone PG has proved unsuccessful. However, a degenerate PCR approach has resulted in the isolation of two PG cDNAs within the abscission zone library. These clones, termed pJET37 and pJET39, show close homology to other PGs within the database (Figure 2) although JET39 has features which distinguish it from the other groups. An abscission­related PG has been cloned from tomato [11] and as predicted the peptides differs markedly from the fruit enzyme [12]. The time course of expression of JET37 and JET39 mRNA shows that both PG genes are up-regulated during abscission within 18 hours of ethylene treatment and that expression is restricted to the zone tissue (Figure 1). The level of expression of JET37 is much greater than that of JET39 and their is evidence that both mRNAs are unstable, a feature that they share with the mRNA encoding 13 1,4 glucanase.

JET37

JET39

Non-zone

o 12 18 24 24 -~~

•• y ... ,'~' ~ ::~ .{.

, t" ~~~:it i'~,'" ~, . ,,,!~,, t",~, :.7~,'·

Zone

o 12 18 24 24 -~~

"j', J ; ~: 't~:: ~

187

Figure 1, Northern blot of total RNA (10 1Jg) from S. nigra leaflet abscission zone (Zone) and non abscission zone (Non-zone) tissues that have been exposed to different durations of ethylene or for 24 h in the absence of the gas (~I4). The Northern blot was hybridized to a radiolabelled JET37 or JET39 cDNA insert.

~ PGLRACT.PRO PJET37.PRO PGLRL YC.PRO PGLRPER.PRO 9A1A.PRO

i PJET39.PRO PGTOM.PRO PGLRMA.PRO

61.3 I

60 50 40 30 20 10 0

Figure 2. Phylogenetic tree of JET37 (PJET37.PRO) and JET39 (PJET39.PRO) to abscission-related (PGTOM.PRO - accession number U23053). fruit-related - tomato; (PGLRLYC.PRO - accession number P05117). - kiwifruit, (PGLRACT.PRO - accession number p35336). avocado; (pGLPER.PRO - accession number - L06094). pod dehiscence-related -Brassica napus; (9AIAPRO - accession number Z49971). and pollen-related polygalacturonases - Zea mays; (pGLRMAPRO - accession number S66022). Generated using DNAStar Clustal method (PAM250 residue weight table). The scale beneath the tree measures the distance between sequences. Units indicate the number of substitution events.

188

3.2 PATHOGENESIS-RELATED PROTEINS

With the aid of a differential screening strategy using mRNA extracted from ethylene treated non abscission zone tissue we have isolated a number of abscission-related clones from the S. nigra library. One of these encodes a Type 2 metallothionein [13] and the expression of the gene is closely correlated with abscission at both a temporal and spatial level. The role of a metallothionein-like protein during abscission has yet to be determined, however, a number of groups have recently demonstrated that such peptides may be up-regulated during developmental events such as ripening and senescence where ethylene may have a co-ordinating role [14,15]. Moreover, the promoter of an MT -like protein from pea has been sequenced and a putative ethylene domain identified (Fordham-Skelton unpublished). An MT -like protein is also expressed during senescence of S. nigra leaflets although the size of the mRNA encoding the peptide is larger that the abscission zone protein when analysed on a northern gel [13].

Six additional abscission-related gene products have been isolated using the differential screening approach. Northern analysis has shown that their patterns of expression have subtle differences both in the time course of their expression and the capacity to be up-regulated by ethylene. By comparing the sequences of the cDNAs with others in the database an identity for each of them has been ascribed. They are a polyphenoloxidase, a pathogenesis-related (PR-l) type protein, a win (PR-4) type protein, a protease inhibitor (PR-6) type protein, an acidic (PR-3) type and a basic (PR-8) type chitinase.

The cDNAs can be grouped into two distinct classes. Expression of the first group is specifically related to both the abscission zone tissue and the presence of ethylene. These include polyphenoloxidase, protease inhibitor and the basic chitinase. The expression of the other group is also abscission-related but is apparent both in the presence and absence of ethylene. This observation suggests that wounding associated with explant generation could be a sufficient signal to promote their up-regulation. Some differences are also seen in their time course of expression with the acidic chitinase peaking at ISh of ethylene treatment while the basic enzyme does not reach a maximum until 6 hours later. These observation suggest that the regulatory elements dictating the expression of these abscission-related gene products may differ.

3.3 OTHER ABSCISSION-RELATED PEPTIDES

In an effort to identify other abscission-related proteins we have carried out differential display on total RNA extracted from ethylene-treated abscission zone and non zone tissue. First strand copies of both RNAs were made by reverse transcription using an oligo-dT primer that has a specific dinucleotide at its 3' end. This anchor primer and an arbitrary lO-mer were used to amplify cDNAs to which both primers hybridize. A number of PCR products have been identified that appear to represent mRNAs that are differentially expressed. Some of these are up-regulated within the zone tissue while others appear to be down-regulated (Figure 3a). These have been reamplified using

189

appropriate primers (Figure 3b), cloned and sequenced and are currently under further analysis.

a Zone Non-zone b

Figure 3. a) Differential display autoradiogram of S. nigra abscission zone and non-zone RNA showing putative differentially expressed mRNAs. b) A 2.0% agarose gel containing one of tbe putative differential (~200bp) which has been eluted and reamplified using tbe same primers as in tbe differential display peR.

4. Conclusions

Leaflets of S. nigra have proved to be an excellent model system on which to study abscission. Using a range of techniques a spectrum of abscission-related clones have been isolated and these encode a mixture of cell wall degrading enzymes and pathogenesis-related proteins. The latter group of peptides would appear to be expressed in the abscission zone to protect the fracture surface from invading pathogens at the time of cell separation. The induction of PR-proteins in bean abscission zones has been previously reported [16]. The role of MT-like proteins during abscission has yet to be elucidated but it is unlikely to contribute to sequestration of heavy metals at this site. More likely is the proposed role of plant MT -like proteins in the binding of free radicals as these potentially harmful components have been hypothsised to playa role in the induction ofPR-proteins [17].

It is evident from northern analyses of the abscission-related mRNAs that the expression of these genes varies both temporally and spatially. Moreover some of them seem to be less dependent on ethylene for their up-regulation than others. This raises

190

the possibility that the promoters of these genes have different controlling elements and possibly that their expression is restricted to different cells within the abscission zone complex. Thus the assumption that an abscission zone is composed of only a single type of ethylene responsive cell may prove to be an over-simplification of the situation. Work is underway to examine this hypothesis further.

5. References

1. Sexton, R. and Roberts, JA (1982) Cell biology of abscission, Annual Review of Plant Physiology 33, 133-162.

2. Wright, M. and Osborne, D.l (1974) Abscission in Phaseolus vulgaris. The positional differentiation and ethylene-induced expansion growth of specialized cells, Planta 120, 163-170.

3. Roberts, JA, Schindler, C.B. and Tucker, GA (1984) Ethylene-promoted tomato flower abscission and the possible involvement ofan inhibitor, Planta 160,159-163.

4. Roberts, JA, Taylor, J.E., Coupe, S.A, Harris, N. and Webb, S.T.J. (1993) Changes in gene expression during leaf abscission, in lC. Peche (ed.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, pp.272-277.

5. Taylor, J.E., Webb, S.T.J., Coupe, SA, Tucker, GA and Roberts, JA (1993) Changes in polygalacturonase activity and solubility of polyuronides during ethylene-stimulated leaf abscission in Sambucus nigra, Journal of Experimental Botany 258, 93-98.

6. Webb, S.T.J., Taylor, lE., Coupe, SA, Ferrarese, L. and Roberts, JA (1993) Purification of J31,4 g1ucanase from ethylene-treated abscission zones of Sambucus nigra, Plant Cell and Environment 16, 329-333.

7. Osborne, D.l and Sargent, J.A (1976) The positional differentiation of ethylene-responsive cells in rachis abscission zones in leaves of Sambucus nigra and their growth and ultrastructural changes at senescence and separation, Planta 130,203-210.

8. Fry, S.C., Smith, R.C., Renwick, K.F., Martin, D.J., Hodge, S.K. and Matthews, K.G. (1992) Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants, Biochemical Journal 282, 821-828.

9. Tucker, M.L. and Milligan, S.B. (1991) Sequence analysis and comparison of avocado fruit and bean abscission cellulases, Plant Physiology 95, 928-933.

10. Taylor, J.E., Coupe, S.A, Picton, S.J. and Roberts, J.A (1994) Isolation and expression of a mRNA encoding an abscission-related ~ 1,4 g1ucanase from Sambucus nigra, Plant Mol. Bio!. 24, 961-964.

11. Kalaitzis, P., Koehler, S.M. and Tucker, M.L. (1995) Cloning of a tomato polygalacturonase expressed in abscission, Plant Molecular Biology 28, 647-656.

12. Taylor, lE., Tucker, GA, Lasslett, Y., Smith, C.J.S., Arnold, C.M., Watson, C.F., Schuch, W., Grierson, D. and Roberts, JA (1991) Polygalacturonase expression during leaf abscission of normal and transgenic tomato, Planta 183, 133-138.

13. Coupe, S.A, Taylor, J.E. and Roberts, JA (1995) Characterisation of an mRNA encoding a metallothionein-like protein that accumulates during ethylene-promoted abscission of Sambucus nigra L. leaflets, Planta 197, 442-447.

14. Ledger, S.E. and Gardner, R.C. (1994) Cloning and characterization of five cDNAs for genes differentially expressed during fruit development of kiwifruit (Atinidia deliciosa var. deliciosa), Plant Molecular Biology 25,877-886.

15. Buchanan-Wollaston, V. (1994) Isolation of eDNA clones for genes that are expressed during leaf senescence in Brassica napus, Plant Physiology 105,839-846.

16. del Campillo, E. and Lewis, L.N. (1991) Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones, Plant Physiology 98, 955-961.

17. Lamb, C.l (1994) Plant disease resistance genes in signal perception and transduction, Cell 76, 419-422.

DIFFERENT ENDO-~-1,4-GLUCANASES ARE EXPRESSED DURING ABSCISSION AND FRUIT RIPENING IN PEPPER AND PEACH PLANTS

L. TRAINOTTI, L. FERRARESE, G. CASADORO Dipartimento di Biologia, Universita di Padova,via Trieste 75, 1-35121 Padova, Italy

1. Introduction

Senescence of leaves, flowers and fruits usually ends with the formation of an abscission zone at the base of the organ involved. The abscission zone consists of a few layers of cells whose walls undergo extensive digestion processes leading to loss of adhesion between cells [1]. Also ripening of fleshy fruits includes an aging requirement which involves similar cell wall changes during the softening of the tissues. Though the rate of senescence can be either delayed or increased by plant hormones, in many cases ethylene has been found to have promotive effects on the senescence phenomena. In particular, this hormone accelerates abscission and ripening, although, in term of regulation, different responses are shown by climacteric and nonclimacteric fruits. These ethylene-mediated physiological processes involve synthesis of different mRNAs which include those encoding cell wall hydrolases [2].

The participation of endo-~-1,4-g1ucanase (EGase; EC 3.2.1.4) in the hydrolysis of the cell wall during softening offruits and abscission has long since been known [3,4]. Subsequent research has shown that this enzyme is present in plants in different forms as, for instance, in bean where two isoforms with different isoelectric points (PI 4.5 and 9.5, respectively) have been demonstrated in the leaf abscission zones. The 9.5 form is the abscission EGase and is highly induced by ethylene, while the 4.5 form shows a decreased activity during leaf abscission [5].

In peach two different isoforms (PI 6.5 and 9.5, respectively) are responsible for the EGase activity observed during the abscission of both leaves and fruits. However, in peach ethylene is able to increase the activity of both enzymes, but it is the 6.5 form which shows the highest levels in leaf abscission zones [6]. Exogenously applied ethylene also causes a significant increase of a 2.2 kb transcript, recognized by the avocado ripening EGase cDNA, in the abscission zones of both organs, although the amount in leaves is much higher than that observed in fruit abscission zones [5]. Also in pepper two isoenzymes (PI 7.2 and 8.5) have been characterized and their different involvement in abscission of leaves and ripening of fruits examined [7].

191

A. K. Kanellis et al. (eds.), Biology and Biotechrwlogy of the Plant Hormone Ethylene, 191-196. © 1997 Kluwer Academic Publishers.

192

In this study we report the presence of three different EGase genes which show different responses to ethylene in the cell separation events occurring during fruit ripening and abscission in both peach and pepper plants.

2. Results

2.1. PEACH ENDO-~-1,4-GLUCANASES

Plants of Prunus persica (L.) Batsch cv. Redhaven were used for this study. Explants of both leaf and fruit abscission zones were prepared and treated with ethylene according to Bonghi et al. [6]. Fruits at various stages of development (SI, S2, S3, S4 as described in Zanchin et al. [7]) were collected and used without further treatment.

A number of primers constructed on the basis of known higher plant EGases [8] were used in RT -PCR experiments with total RNA. The result of these experiments consisted of three cDNA fragments named pCellO (788 bp), pCel20 (239 bp; accession no. X96854) and pCel30 (224 bp; accession no. X96855), respectively. Sequencing of the fragments revealed that they encode different EGases whose similarities range between 51.4% and 62%, although highly conserved regions are present in all of the three sequences.

In order to better assess their respective roles in the cell separation events which characterized the abscission of leaves and fruits and the fruit development, the three cDNA fragments were used as probes in northern analyses. These experiments yielded positive results only with pCellO which hybridized to a 2.2 kb transcript when used with RNA from ethylene-activated abscission zones. In accordance with previous data [6], obtained by using a ripening avocado EGase cDNA [9], also the pCellO probe gave a much stronger hybridization signal with the leaf RNA than with that of fruit (Table I). These results indicate that the gene coding for the pCellO-related mRNA can be regarded as the "abscission" EGase gene in peach.

Table 1. Expression analysis of the three peach EGase cDNAs. The table was built by using the result of northern (asterisks) and RT-PCR (crosses) experiments. In the northern analysis the pCellO probe recognized an mRNA of2.2 kb in ethylene-treated abscission zones ofleaves and fruits. The expression of the pCel20 and pCel30 related mRNAs was detected by RT-PCR. The amplification fragments were separated in agarose gels and blotted onto nylon membranes. The blots were probed either with pCel20 or with pCe130. While pCel20 seemed to be constitutively expressed in abscission zones and in fruits, the pCel30-related mRNA showed increased amounts in fruits at stage 3 (83) and in ethylene-activated abscission zones offruits.

Peach EGases: leaf fruit fruit abscission zone abscission zone Air C2iL Air C2iL 81 82 83 84

pCellO ***** ** pCel20 + + + + + + + + pCel30 + + + ++ + + ++ +

193

The more sensitive RT -PCR method was then used to determine the expression pattern of the two other EGases. While the pCel20-related mRNA seemed to be constitutively expressed, a different expression pattern was found for the pCe130-related mRNA whose amount was extremely low in the leaf zones but showed an increase in the ethylene-activated fruit abscission zones (Table 1). Interestingly, also fruits at stage 3 (S3) of development had increased amounts of this transcript (Table 1). On the basis of its expression pattern, pCellO was used as a probe to screen a genomic library. This yielded a 5445 bp region containing its cognate gene ppEGl (accession no. X96856). The 2650 bp fragment shown in Figure 1 contains the ORF, plus two flanking region at the 5' and 3' termini, respectively. Compared to the other known EGase genes in higher plants, ppEG 1 appears to be much shorter due to the reduced size of its intron regions (Fig. 1). This might be a consequence of the smaller size of the peach genome (265 Mbplhaploid genome) compared to the avocado (883 Mbp/h.g.), bean (637 Mbp/h.g.) and pea (-4000 Mbp/h.g.) genomes [12]. From the comparison shown in Figure 1 it is also evident that the four EGase genes are structurally divergent as regards both the size and the number of the exonlintron regions. However, if we disregard the intron sizes and consider that exon 7 in the peach and bean genes roughly corresponds to the sum of the exons 7 and 8 in avocado, it appears that these three genes, which are ethylene-inducible, share some structural similarity and are quite different from the pea one, which is auxin-inducible [13].

avocado

1II-~IIJ--(!]-.H--~l!H!I-I 7 •

bean

peach

pea 00...-____ 1_000 bp

Figure 1. Comparison of the structure offour EGase genes: avocado [10], bean [11], peach [this paper] and pea [13]. White blocks represent exons, while black bars represent introns. The gray areas represent 5' and 3' untranslated regions of the genes.

194

2.2. PEPPER ENDO-~-1,4-GLUCANASES

Experiments were carried out with plants of Capsicum annuum var. longum (DC) Sendt. Abscission of leaves and ripening of fruits were induced by treatments with ethylene as described in Ferrarese et al. [8]. Three cDNA fragments encoding different EGases in pepper [8] were used as probes to isolate the relative full length clones (cCell: accession no. X97188; cCel3: accession no. X97189; cCel2: accession no. X97190) from cDNA libraries. Both northern and RT-PCR analyses were performed in order to examine the expression pattern of their cognate genes (Table 2). This study revealed a tissue specific expression of the three EGase genes. cCell was solely expressed at increasing amounts during the late stage of fruit ripening, while high amounts of cCel2-related mRNA were only detected in ethylene-treated abscission zones of both leaves and flowers. Expression of cCe13-related mRNA could be detected by RT-PCR in leaf abscission zones and the amount of the transcript was increased by ethylene treatments.

Table 2. Expression analysis of the three pepper EGase cDNAs. The table was built by using the result of Northern (asterisks) and RT-PCR (crosses) experiments. The cCell probe recognized an mRNA of 2.4 kb during fruit ripening. The cCel2 probe recognized an mRNA of 2.2 kb in ethylene-1reated abscission zones of leaves and flowers. Expression of the cCel3-related mRNAs was revealed by RT-PCR The amplification fragments were separated in agarose gels and blotted onto a nylon membrane which was subsequently probed with cCel3. cCel3 transcripts were only detected in leaf abscission zones and their amount was increased by ethylene.

Pepper leaf Dower fmit EGases: abscission zone abscission zone

Air CzRt Air CzRt Green Orange Red cCell ** ***** cCe12 **** ***** cCel3 + ++ n.d. n.d.

Salicylic acid has recently been shown to counteract the promotive effects of ethylene on the abscission of pepper leaves [14]. By means ofRT-PCR using specific primers for each of the three cDNAs, we could show that the observed quenching effect was due to a reduction in the level of expression of the cCel2 transcript, while cCe13 seemed insensitive to the chemical.

3. Discussion

A phylogenetic tree was recently proposed, based on the deduced amino acid sequences, in order to correlate a number of higher plant EGases on the basis of their physiological role [13]. Unfortunately, this kind of comparison did not seem to give coherent results due to the fact that very similar EGases could be expressed either during ripening of fruits or during abscission of organs. Figure 2 shows a phylogenetic

195

tree constructed on the basis of the deduced amino acid sequences of 13 higher plant EGases. A number of groups of similar enzymes are quite evident besides the fact that the pea EGase (a growth enzyme) is set apart from the others. Moreover, one of the clusters groups the "ripening" avocado enzyme with the abscission enzymes of both peach (pCell) and pepper (cCeI2) [15]. Another cluster groups the pepper (cCell) "ripening" EGase with the tomato (tomcell) [16], the elder [17] and bean [18] "abscission" enzymes. These data clearly demonstrate that is impossible to predict in which physiological role a certain EGase is involved on the sole basis of its primary structure.

39.4 I

35 30

r ____ c==~cCell r tomCell a (r)

.... ------elder a I ... -------bean a ..... -----pCe130

.... ____ pCeI20

cCe13 a tomCel2 r (a)

----------TPPI8 pistil .... ------avoCell r .... ----- pCell a

--------cCeI2 a EGL 1 cell extension

I I I 25 20 15 10 5 o

Figure 2. Phylogenetic tree built with the neighbor joining method using the alignment of the deduced amino acid sequences obtained by the Clustal method. The lengths of the branches are proportional to the distances among the sequences. The scale beneath the tree measures the distance between sequences. In uppercase the sequence names, in lowercase the phenomena in which their are expressed: "r"= ripening; "a"= abscission (between brackets stands for low expression).

The finding that in very close species, like tomato and pepper (they are both Solanaceae), highly similar EGases are expressed in different physiological process [8,16] suggests that the higher plants EGases may have evolved from an ancestral gene before the actual evolution of the plant species. This might explain why similar EGases have evolved different physiological roles within different plant species. Moreover, the presence of different EGase forms might supply the biochemical variability required to bring about different cell separation events in different organs.

196

4. Acknowledgments

This research was supported by grants from C.N.R. and M.U.R.S.T.

5. References

1. Sexton, R., Roberts, JA (1982) Cell biology of abscission, Annu. Rev. Plant Physioz. 33, 133-162. 2. Abeles, F.B., Morgan, P.W., and Saltveit, M.E. Jr. (1992) Ethylene in plant biology (2nd edition),

Academic Press, Inc., San Diego. 3. Pesis, E., Fuchs, Y., and Zaubennan, G. (1978) Cellulase activity and fruit softening in avocado,

PlantPhysioz. 61,416-419. 4. Horton, R.F. and Osborne 0.1 (1967) Senescence, abscission and cellulase activity in Phaseolus

vulgaris, Nature 214,1086-1088. 5. del Campillo, E., Durbin, M., and Lewis, L.N. (1988) Changes in two fonns of membrane-associated

cellulase during ethylene induced abscission, Plant Physiol. 88, 904-909. 6. Bonghi, C., Rascio, N., Ramina, A, and Casadoro, G. (1992) Cellulase and polygalacturonase

involvement in the abscission ofleaf and fruit explants of peach, Plant Mol. BioI. 20, 839-848. 7. Zanchin, A, Bonghi, C., Casadoro, G., Ramina, A, and Rascio, N. (1994) Cell enlargement and cell

separation during peach fruit development, Int. J. Plant Sci. 155,49-56. 8. Ferrarese, L., Trainotti L., Moretto, P., Polverino de Laureto, P., Rascio, N., and Casadoro, G. (1995)

Differential ethylene-inducible expression of cellulase in pepper plants, Plant Mol. BioI. 29,735-747. 9. Tucker, M.L., Durbin, M.L., Clegg, M.T., and Lewis, L.N. (1987) Avocado cellulase: nucleotide

sequence of a full length cDNA clone and evidence for a small multi-gene family, Plant Mol. BioI. 9, 197-203.

10. Cass, L.G., Kirven, KA, and Christoffersen, R.E. (1990) Isolation and Characterization ofa cellulase gene family member expressed during avocado fruit ripening, Mol.Gen. Gen. 223,76-86.

11. Koehler, S.M., Matters, G.L., Nath, P., Kemmerer, E.C., and Tucker,M.L. (1995) The gene promoter for a bean abscission cellulase is ethylene-induced in transgenic tomato and shows high sequence conservation with a soybean abscission cellulase, Unpublished Accession no. U34754.

12. Arumuganathan, K. and Earle, E.D. (1991) Nuclear DNA content of some important plant species, PlantMol. BioI. Rep. 9,208-218.

13. Wu, S.C., Blumer, 1M., Darvill, AG., and A1bersheim, P. (1996) Characterization of an endo-j3-1,4-g1ucanase gene induced by auxin in elongating pea epicotils, Plant Physiol. 110, 163-170.

14. Ferrarese, L., Moretto, P., Trainotti, L., Rascio, N., and Casadoro, G. (1996) Cellulase involvement in the abscission of peach and pepper leaves is affected by salycilic acid,J. Exp. Bot. 47, 251-257.

15. Trainotti et az. (Unpublished): accession numbers: cCel1: X97188; cCel3: X97189; cCel2: X97190. 16. Lashbrook, C.C., Gonzales-Bosh, C, and Bennet, AB. (1994) Two divergent endo-j3-1,4-g1ucanase

genes exhibit overlapping expression in ripening fruits and abscission flowers, Plant Cell 6, 1485-1493.

17. Taylor, J.E., Coupe, SA, Picton, S., and Roberts, JA (1994) Characterization and accumulation pattern of an mRNA encoding an abscission-related j3-1,4-g1ucanase from leaflets of Sambucus nigra, Plant Mol. BioI. 24,961-964.

18. Tucker, M.L., Sexton, R., del Campillo, E., and Lewis, L.N. (1988) Bean abscission cellulase: characterization of a cDNA clone and regulation of gene expression by ethylene and auxin, Plant Physioz. 88, 1257-1262.

THE TOMATO ENDO-P-l,4-GLUCANASE GENE FAMILY: REGULATION BYBOTHETHYLENEANDA~

J.K.C. ROSE, C. CATALA, D.A. BRUMMELL, C.C. LASHBROOK, C. GONZALEZ-BOSCH AND A.B. BENNETT Mann Laboratory, Department of Vegetable Crops, University of California Davis, California 95616 USA.

1. Introduction

The primary cell wall of plants is a complex polymeric network that provides a rigid constraint to cell turgor, a resistant barrier to pathogen attack and yet be sufficiently compliant to allow controlled cell elongation. The cell wall has been described as a network of cellulose microfibrils embedded in a hemicellulosic polysaccharide matrix, consisting primarily of the polymer xyloglucan in dicots. The hemicellulose component interacts to some degree with an additional co-extensive matrix of pectin and some less abundant components including structural proteins [4]. It is thought that the cellulose microfibrils are tethered by xyloglucan polymers [22, 34] and in a turgid cell, cleavage of these potentially load-bearing xyloglucan chains could provide a rate limiting step to cell wall loosening during expansive growth. Several studies linking xyloglucan metabolism with cell expansion include the release of xyloglucan fragments from the wall during elongation growth induced by the plant hormone auxin [16,10], a decrease in the molecular weight of the remaining polymeric xyloglucan [2, 1,9] and partial dissolution of the wall-bound xyloglucan [30]. Metabolism ofxyloglucan has also been strongly correlated with fruit ripening in a wide range of species [8, 17]. Furthermore, fruit of the rin (ripening inhibitor) tomato mutant, which soften extremely slowly, do not exhibit any decrease in xyloglucan molecular weight during ripening, unlike wild type where substantial depolymerization occurs [20]. Modification of xyloglucan structure, abundance, distribution and interaction with other cell wall components may therefore be integral to both cell expansion and fruit ripening.

One group of enzymes that may play an important role in xyloglucan modification is the endo-~-1,4-glucanase (EGase) family, which catalyse the endo-hydrolysis of ~-1,4 glucan linkages. Plant EGases have also been termed 'cellulases' despite a lack of evidence that they are able to hydrolyze crystalline cellulose, or 'carboxymethylcellulases' (CMCases) based on their ability to hydrolyze carboxymethylcellulose, an artificial cellulose derivative [28]. Although the in vivo substrate(s) for EGases is unknown, one likely candidate is xyloglucan which has a

197

A. K. Kanellis et al. (etis.), Biology and Biotechrwlogy o/the Plant Hormone Ethylene, 197-205. @ 1997 Kluwer Academic Publishers.

198

~-1,4 linked glucan backbone. There is however some evidence to suggest that some EGases may also act on other hemicellulose substrates [13, 6]. Such an activity could contribute to cell wall loosening during elongation growth and to cell wall degradation associated with physiological events such as organ abscission and fruit ripening. EGases are encoded by a multigene family in tomato with divergent members exhibiting highly regulated developmental and tissue-specific patterns of expression. For example, an EGase gene family member has recently been described with specific expression in abscission zones [5]. However, the specific function of different EGase isoforms remains relatively unexplored beyond localization to particular tissues or organs.

Plant hormones regulate a variety of physiological processes, for example ethylene is intimately associated with climacteric fruit ripening and organ abscission and auxin has been shown to regulate elongation growth. Characterization of the hormonal regulation of EGases may therefore provide insight into their function in vivo and we provide evidence here for the regulation of divergent members of the EGase gene family by both ethylene and auxin. Several additional enzymes exist in the apoplast that may interact directly or indirectly with either EGases or their proposed substrate in muro, xyloglucan. For example, xyloglucan endotransglycosylases (XETs) are xyloglucan specific transglycosylases that have been associated with rapidly expanding tissues and ripening fruit [1,9,20]. XETs cut the xyloglucan backbone and religate the newly generated non-reducing end to an adjacent xyloglucan polymer or oligomer. The generation of such an oligomer could result from EGase activity. Since certain EGases and XETs potentially act on the same substrate, they may therefore play a cooperative role in the cell wall disassembly that accompanies both expansion growth of tissues and fruit ripening. Analysis of the co-expression of both classes of genes and the potential for hormonal co-regulation represents a new approach to characterizing the role of EGases, and in the long term to understanding xyloglucan metabolism in the cell wall.

2. Results and Discussion

Tomato EGases comprise a multigene family of at least seven divergent members. The full length cDNAs derived from five of these genes have been cloned and sequenced and Figure 1 shows their deduced amino acid homology to EGases from a variety of other plant species.

2.l. CELl AND CEL2: EXPRESSION AND DIFFERENTIAL REGULATION BY ETHYLENE

Cell and Cel2 cDNAs were both cloned from a ripening tomato fruit cDNA libraty [18]. They showed only 50% deduced amino acid identity and Cell was most homologous to EGases from bean and elder abscission zones whereas Cel2 was more

199

similar to EGases from pepper and avocado fruit (Fig. 1). In addition to transient expression during tomato fruit expansion, accumulation of Cell and Cel2 mRNA occurred coincident with the burst of ethylene production at the onset of ripening [18]. A large increase in extractable CMCase activity has also previously been reported at this developmental stage [12, 14]. Levels of Cell mRNA declined as ripening progressed whereas Ce12 expression increased throughout ripening [18]. A degree of differential spatial distribution was apparent in fruit as Cell showed minimal levels of expression in locules, whereas Cel2 abundance increased in locular tissue coincident with the initiation of ripening, and may therefore contribute to locule liquefaction [11].

I I

I I

I L

Bean BAC1

Elder JET1

Tomato Cel1

Tomato Cel2

Pepper Ccx3

Avocado Cel1

Poplar Cel1

Tomato Cal4

Pea EGL 1

Tomato Cal7

Tomato Cel3

Figure 1. Clustai analysis using of deduced amino acid sequences of a variety of plant EGases using PAUP software. Gene references: Bean [33]; Elder [29]; Pepper [7]; AVOcado [32]; Poplar [26]; Pea [35]

Both Cell and Cel2 appeared to be regulated by ethylene during ripening and expression was of each gene was induced following post-harvest ethylene treatment and inhibited by application of the ethylene action inhibitor norbornadiene (NBD).

The regulatory pathways were dissected further using the rin (ripening inhibitor) mutant. Wild type (Fig. 2A) or rin fruit (Fig. 2B) were held in either air or the ethylene analogue propylene and Cell and Cel2 mRNA abundance quantified over a 20 day period. Both genes were barely detectable in rin fruit when held in air, however Cell, was strongly induced 7 days after propylene treatment. No significant effect of propylene on Cel2 expression was observed. The differential effect of the rin mutation on Cell and Cel2 expression suggests that although both genes are ethylene regulated, they may represent end points of two distinct ethylene regulatory pathways.

200

Furthermore, since rin fruit do not soften to any great extent, we can conclude that Cell is insufficient to cause fruit softening.

A. Wild Type

Cell

Cel2

~ ~ ~ " ~ " ~ " ~ ... Col) ... ... ... Air Propylene

B. Tin

Cell

Cel2

~ ~ ~ " ~ " ~ " ~ ... Col) ... ... ... Air Propylene

Figure 2. Expression of Cell and Cel2 mRNA in (A) wild type and (B) rin mutant tomato fruit pericarp tissue (cv. Ailsa Craig). Fruit were harvested at the Mature Green stage (TO) and held in air or propylene.

2.2. CEL3: GENE STRUCTURE AND EXPRESSION

Ce13 mRNA is relatively more abundant in vegetative tissues than in fruit or floral organs and is expressed at higher levels in rapidly growing tissues, such as the zone of elongation in growing hypocotyls (unpublished data). This suggests a role for Ce13 in cell wall modification during elongation growth although application of a variety of growth related plant hormones including ethylene, auxin and gibberellins appeared to have no regulatoty effect. Analysis of the deduced structure of Ce13 suggests that it represents a novel type of EGase. Plant EGases generally possess a typical eukaryotic hydrophobic signal sequence at their N-terminus which would target them to the ER for processing and secretion into the apoplast. However, the deduced amino acid sequence of tomato Ce13 is highly divergent from that of other plant full-length

201

deduced EGase proteins, is approximately 20% longer and consists of three domains (Fig. 3).

Membrane Spanning Domain

__ W///////////#/~ Highly Charged Domain

Catalytic Core

Figure 3. Schematic representation of tomato EGase Cel3 deduced protein structure. Cel3 consists of three domains, an N-tenninal highly-charged region (stippled box), a membrane­spanning domain (black box), and the catalytic core (striped box).

The N-terminal region of the deduced sequence consists of a highly charged, hydrophilic region, followed by a hydrophobic domain possessing high homology to membrane-spanning regions of other genes. The catalytic core of Ce13 possesses all the conserved amino acid domains characteristic of plant EGases, together with seven potential sites for N-linked glycosylation. Antibodies raised to a portion of Cel3 expressed in E. coli detected proteins in plasma membrane and Golgi-rich membrane fractions. The mature Ce13 protein thus appears to be an integral membrane protein. Interestingly, a membrane-anchored EGase (celC) has also been described in the cellulose-synthesizing bacterium Agrobacterium tumefaciens, where it is part of the operon responsible for cellulose biosynthesis [21]. Disruption of this EGase by transposon insertion abolished cellulose biosynthesis, showing that the gene is required for this process [21]. Possible roles for the Ce13 protein may include the cleavage of newly synthlsised cellulose microfibrils to appropriate lengths prior to incorporation in the cell wall, or metabolism of xyloglucan during its synthesis, or secretion into the apoplast. The functions of tomato Ce13 remain to be established and are currently under investigation.

2.3. CEU: PATTERNS OF GENE EXPRESSION AND ETHYLENE INDUCTION

This EGase was originally identified as a pistil-predominant gene and termed TPPI8, with highest levels of expression early in pistil development during a phase of rapid expansion, and barely detectable levels in roots and young seedlings [25]. The same gene was isolated separately from a ripe tomato fruit cDNA library and for consistency of nomenclature has been referred to as Ce14. Further analysis revealed that the gene was also expressed at relatively high levels in the elongation zones of etiolated

202

hypocotyls and in young expanding leaves [3J. Low levels of expression were also detected in young expanding fruit but were negligible in fully expanded or ripening fruit. Treatment of hypocotyls with either ethylene or higher concentrations of auxin caused an approximately two-fold increase in mRNA abundance. It is known however that the application of high concentrations of auxin induces the production of ethylene [15J. Since both ethylene and auxin treatments caused a similar increase in Cel4 mRNA abundance, and also similar phenotype in the form of a lateral swelling below the apical hook, it is probable that the response in both cases was directly or indirectly ethylene-mediated.

2.4. CEL7: CO-EXPRESSION WITH AN XET GENE (LEEXT) AND REGULATION BY AUXIN

Since auxin is known to promote xyloglucan metabolism in elongating tissues, we initiated a search for novel auxin-induced tomato EGases. Cel7 was identified as an auxin-induced EGase and the full length cDNA cloned from a tomato root library. Northern blot analysis revealed that Cel7 was a relatively abundant EGase with the highest levels of expression in rapidly expanding vegetative tissues and in developing fruit, where mRNA accumulation was maximal at the immature and mature green stages (unpublished data). Interestingly, the gene was not detectable at the breaker stage of fruit development, suggesting that gene expression was effectively down­regulated by ethylene. This was confirmed in subsequent experiments using ethylene treated etiolated hypocotyls. Since both EGases and XETs may utilize xyloglucan as a substrate, and have both been implicated as playing a role in cell wall loosening that accompanies elongation growth, they may act coordinately in elongating tissues. This was addressed by examining the auxin-induced co-expression of Ce17, and an auxin­induced XET that shares 100% identity with the tomato LeEXT cDNA isolated by Okazawa et al. [27J, in etiolated tomato hypocotyls. Both hypocotyl segment elongation and enhanced Cel7 and LeEXT gene expression were induced after incubation with 2,4-D (an auxin analogue) and showed the same concentration dependency over a range of 0.1-10 J1M 2,4-D (unpublished data).

Similarly, the temporal relationship between auxin-induced Cel7 and LeEXT gene expression was examined. Both Cel7 and LeEXT mRNA accumulation showed a only transient increase in control segments. In the presence of auxin however, Cel7 and LeEXT mRNA accumulation was more prolonged, peaking at 12 h. Cel7 mRNA continued to accumulate up to 24 h, a period of sustained hypocotyl elongation (Fig. 4) whereas LeEXT expression decreased considerably at the 24 h time point. LeEXT levels at this point were however greater than in the control segments. To address whether the observed increase in Cel7 and LeEXT abundance was an auxin-specific effect or the indirect result of ethylene production following auxin application, two mutant varieties of tomato Nr and dgt were used that are insensitive to ethylene and auxin respectively [31, 15J. Induction by auxin of both genes was apparent in the Nr mutant to a similar degree as wild type, however no increase was detectable in the dgt

203

mutant, suggesting that the dgt but not the Nr mutation lies on a pathway regulating expression of both Cel7 and LeEXT.

Incubation Time (b)

0 2 6 12 24

+2,4-D

ee17 ' .. ........ Control

+2,4-D

LeEXT --- Control

Figure 4. Time course of Cel7 and LeEXT mRNA expression in 5mM 2,4-0 treated, or control (buffer) treated etiolated tomato hypocotyl segments

An additional approach to the question of cooperativity between these two genes that is currently being pursued, is the determination their spatial distribution within specific regions of elongating tissues using in situ hybridization. These studies may provide valuable information about the individual and potentially cooperative roles of these genes in cell expansion.

The complexity of expression and regulation of the tomato EGase gene family illustrates the corresponding complexity of the regulation of xyloglucan metabolism during plant growth and development. Certain EGases are expressed in a variety of tissues and may have parallel functions in seemingly different developmental processes. For example, a particular EGase may play an important role during both hypocotyl elongation and fruit ripening; apparently dissimilar processes yet possibly linked by disassembly of the same substrate. Conversely, the overlapping expression of multiple EGases in the same tissue or organ suggests a specificity of function. This may relate to several factors including substrate micro-heterogeneity where distinct

204

structural domains within the xyloglucan polymer act as active sites for divergent enzymes, or it may reflect the regulation by a range of both environmental stimuli and plant hormones that are certainly involved in an elaborate crosstalk. An additional consideration is the range of enzymes that interact with each other, and with their potential common substrate in the apoplast. A current model of xyloglucan metabolism and cell wall disassembly during cell expansion and fruit ripening might include not only isoforms of EGases and XETs, but also glycosidases and expansins, a group of enzymes that induce loosening in isolated plant cell walls [23, 24]. All these enzymes appear to interact with xyloglucan to some degree, and an examination of the cooperativity between them may be an effective way to address the complexity of xyloglucan metabolism and the role ofEGases in plant growth and development.

3. References

1. Arrowsmith, DA and de Silva, J. (1995) Characterisation of two tomato fiuit-expressed cDNAs encoding xyloglucan endo-transglycosylses, Plant Mol. BioI. 28, 391-403.

2. Bret-Harte, M.S. and Talbott, L.D. (1993) Changes in the composition of the outer epidermal cell wall of pea stems during auxin-induced growth,Planta 190, 369-378.

3. Brummell, DA, Bird, C.R, Schuch, W., and Bennett, AB. (in press) An endo-l,4-f3-g1ucanase expressed at high levels in rapidly expanding tissue, Plant Mol. BioI.

4. Carpita, N.C. and Gibeaut, D.M. (1993) Structural models of primary cell walls in flowering plants; consistency of molecular structure with the physical properties of the the walls during growth, Plant Journal 3, 1-30.

5. del Campillo, E. and Bennett, AB. (1996) Pedicel breakstrength and cellulase gene expression during tomato flower abscission, Plant Physiol. 111,813-820.

6. Durbin, M.L. and Lewis, L.N. (1988) Cellulases inPhaseolus vulgaris, Methods Enzymol. 160,342-351.

7. Ferrarese, L., Trainotti, L., Moretti, P., Polverino de Loretto, P., 7. Rascio, N., and Casadoro, G. (1995) Differential ethylene-inducible expression of cellulase in pepper plants, Plant Mol. BioI. 29, 735-747.

8. Fischer, RL. and Bennett, AB. (1991) Role of wall hydrolases in fiuit ripening, Annu. Rev. Plant Physiol. Plant Mol. BioI. 42,675-703.

9. Fry, S. (1995) Polysaccharide-modifYing enzymes in the plant cell wall, Annu. Rev. Plant Physiol. Plant Mol. BioI. 46,497-520.

10. Gilkes, N.R and Hall, MA (1977) The hormonal control of cell wall turnover in Pisum sativum L., NewPhytoi. 78, 1-5.

11. Gonzalez-Bosch, C., Brummell, DA, and Bennett, AB. (1996) Differential expression of two endo-1,4-f3-glucanase genes in pericarp and locules of wild-type and mutant fiuit, Plant Physiol. 111, 1313-1319.

12. Hall, C.B. (1964) Cellulase activity in tomato fiuits according to portion and maturity, Bot. Gaz. 125, 156-157.

13. Hatfield, R and Nevins, D.J. (1986) Characterization of the hydrolytic activity of avocado cellulase, PlantCellPhysiol. 27, 541-552.

14. Hobson, G.E. (1968) Cellulase activity during the maturation and ripening of tomato fiuit, J. Food Sci. 33, 588-592.

15. Kelly, M.O. and Bradford, K.J. (1986) Insensitivity of the diageotropica tomato mutant to auxin, PlantPhysioi. 82, 713-717.

16. Labavitch, J.M. and Ray, P.M. (1974) Turnover of cell wall polysaccharides in elongating pea stem segments, Plant Physiol. 53, 669-673.

17. Lashbrook, C.C., Brummell, DA, Rose, J.K.C., and Bennett, AB. Non-pectolytic cell wall metabolism during fiuit ripening, in J. Giovannoni, (eds.), Fruit Ripening Molecular Biology, Gordon

205

and Breach Publishing, (in press) 18. Lashbrook, C.C., Gonzalez-Bosch, C., and Bennett, AB. (1994) Two divergent endo-fJ-1,4-giucanase

genes exhibit overlapping expression in ripening fruit and abscising flowers, Plant Cell 6, 1485-1493. 19. Lorences, E.P. and Zarra, I. (1987) Auxin-induced growth in hypocotyl segments of Pinus pinaster

Aiton: changes in molecular weight distribution of hemicellulosic polysaccharides, J. Exp. Bot. 38, 960-967.

20. Maclachlan, G. and Brady, C. (1994) Endo-1,4-~-giucanase, xylogiucanase and xylogiucan endo­transgiycosylase activities versus potential substrates in ripening tomatoes, Plant PhysioL 105, 965-974.

21. Matthysse, AG., White, S., and Lightfoot, R. (1995) Genes required for cellulose synthesis in Agrobacterium tumefaciens, J. BacterioL 177, 1069-1075.

22. McCann, M.C., Wells, B., and Roberts. K. (1990) Direct visualization of cross-links in the primary cell wall, J. Cell Sci. 96, 323-334.

23. McQueen-Mason, S.J., Durachko, D.M. and Cosgrove, D.J. (1992) Endogenous proteins that induce cell wall expansion in plants, Plant Cell 4, 1425-1433.

24. McQueen-Mason, S.J. and Cosgrove, D.J. (1995) Expansin mode of action on cell walls, Plant PhysioL 107, 87-100.

25. Milligan, S.B. and Gasser, C.S. (1995) Nature and regulation of pistil-expressed genes in tomato, Plant MoL BioL 28, 691-711.

26. Nakamura, S., Moo, H., Sakai, F., and Hayashi, T. (1995) Cloning and sequencing of a cDNA for poplar endo-l,4-fJ-giucanase, Plant CellPhysiol. 36, 1229-1235.

27. Okazawa, K., Sato, Y., Nakagawa, T., Asada, K., Kato, I., Tomita, E. and Nishitani, K. (1993) Molecular cloning and cDNA sequencing of endoxylogiucan transferase, a novel class of giycosyltransferase that mediates molecular grafting between matrix polysaccharides in plant cell walls, J. BioL Chem. 268, 25364-8.

28. Reese, E.T., Siu, R.G.H., and Levinson, H.S. (1950) The biological degradation of soluble cellulose and its relationship to the mechanism of cellulose hydrolysis, J. BacterioL 59,485-497.

29. Taylor, J.E., Coupe, SA, Picton, S., and Roberts, JA (1994) Characterization and accumulation of an mRNA encoding an abscission-related ~-1,4-giucanase from leaflets of Sambucus nigra, Plant MoL BioL 24,961-964.

30. Terry, M.E., Jones, R.L., and Bonner, BA (1981) Soluble cell wall polysaccharides released from pea stems by centrifugation. I. Effect of auxin, Plant Physiol. 68, 531-537.

31. Tigchelaar, E., McGlasson, W., and Buescher, R. (1978) Genetic regulation of tomato fruit ripening, Hortic. Sci. 13,508-513.

32. Tucker, M.L., Durbin, M.L., Clegg, M.T., and Lewis, L.N. (1987) Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene family, Plant Mol. BioL 9,197-203.

33. Tucker, M.L. and Milligan, S.B. (1991) Sequence analysis and comparison of avocado fruit and bean abscission cellulases, Plant PhysioL 95, 928-933.

34. Whitney, S.E.C., Brigham, J.E., Darke, AH., Reid, J.S.G., and Gidley, M.J. (1995) In vitro assembly of cellulose/xylogiucan networks: ultrastructural and molecular aspects, Plant J. 8, 491-504.

35. Wu, S.-C., Blumer, J.M., Darvill, AG., and Albersheim, P. (1996) Characterization of an endo-fJ-l,4-Glucanase gene induced by auxin in elongating pea epicotyls, Plant Physiol. 110, 163-170.

ETHYLENE SYNTHESIS AND A ROLE IN PLANT RESPONSES TO DIFFERENT STRESSORS

A. KACPERSKA Department of Plant Resistance, Institute of Experimental Plant Biology, Warsaw University, Pawinskiego 5, PL-02-106 Warsaw, Poland

1. Introduction

Most of current knowledge concerning ethylene synthesis and action comes from studies on ethylene involvement in control of plant growth and development. Within this context, phenomena such as embryogenesis, gennination, hypocotyl hook opening, leaf abscission and senescence and most of all - fruit ripening have been extensively studied. On the other hand, there is an ample evidence that different stress-evoking factors (stressors), such as pathogen attack, wounding, mechanical pertwbations or impedance, anaerobiosis, waterlogging and submergence, desiccation, chilling, freezing, salt stress, heavy metals, ozone, electric currents, certain herbicides (Table 1) induce the burst of ethylene, which could be observed in a relatively short time, depending on the stress factor and the way of its application.

TABLE 1. Stresses which increase ethylene formation in plant tissue

Stress-evoking factors

Mechanical wounding and cutting Pathogens infection Drought Chilling Freezing Desiccation Waterlodging and submergence Ozone Anaerobiosis Mechanical impedance Salinity (NaCI) Heavy metals: mercuric ion:

copper zinc

Electric current The auxin-type herbicides

207

References

as cited in [83] [68] and as cited in [83] as cited in [83] as cited in [83] [20,47,48] [48] as cited in [42, 43] [27,34] [56] [63] [13,14] [30] [29,60] [29] [40] [32]

A. K. Kanellis et al. (eds.). Biology and Biotechnology o/the Plant Hormone Ethylene. 207-216. © 1997 Kluwer Academic Publishers.

208

The following questions can be asked: l. What are the implications of the stress-induced perturbations in cell functioning

and structure for ethylene biosynthesis? 2. What are the implications of the increased ethylene production for plant resistance

to stress-inducing factors?

2. The Stress-induced Alterations in Cell Functions and Ethylene Synthesis

According to the current knowledge on mechanisms of action of different stressors, the oxidative stress seems to be the primary stress that plant cells have to cope with. The oxidative burst has been observed in plants exposed to chilling, freezing, desiccation, ozone and paraquat treatments (as cited in [58]), osmotic and mechanical pressure [81], copper treatment [80], as well as to pathogen attacks [59, 72]. In relation to ethylene production it seems necessary to differentiate between effects of a mild stress and a heavy and/or rapidly acting factor (shocks) [46]. Under moderate (mild) stress conditions formation of hydrogen peroxide and of oxygen radicals (active oxygen species, AOS: singlet oxygen, superoxides, hydroxyl and organic radicals) remains under control of enzymatic (superoxide dismutase, catalase) or non-enzymatic (ascorbic acid, tocopherol, glutathione, carotenoids and others) free radical scavenging systems (as cited in [58]). However, heavy or rapidly acting, sublethal stressors induce a reversible destabilization of membranes which leads to overproduction of AOS in chloroplasts [23], mitochondria, endoplasmic reticulum, microbodies (peroxisomes and glyoxysomes), plasma membrane and cell wall [58]. The freezing- or desiccation­induced reversible membrane alterations has been shown to be a necessary requirement for stress ethylene production in vegetative tissues [2, 20, 47, 48]. Enhancement of ethylene synthesis in plant tissues by a free radical-generating system, such as exogenous hydrogen peroxide, has been also reported [38]. Therefore, the involvement of active oxygen species in the formation of ethylene in the stress-affected plant tissues ought to be taken into consideration. The question is which of the ethylene biosynthetic steps can be affected by the oxidative stress.

There is no doubt that biosynthesis of stress ethylene occurs via the same pathway as that described for synthesis of ethylene in non-stressed tissues [34, 49, 82]. The increased availability of ACC seems to be the key regulatory step in biosynthesis of stress ethylene: increased activity of ACC synthase was observed in plants subjected to different environmental stressors (Table 2). Wounding or fungal elicitors have been reported to affect synthesis of ACC synthase at transcriptional or postransriptional level (Table 2). It is also of interest that hydrogen peroxide increases the enzyme activity [38]. The question what are the primary plant cell responses which result in differential expression of ACC synthase genes remains to be answered in a future.

Unlike ACC synthase, ACC oxidase is a constitutively expressed in most vegetative tissues and its activity does not seem to be the rate limiting factor in ethylene synthesis [49, 83]. However, there is mounting evidence that the activity of the enzyme and/or the amount of ACC oxidase transcripts increase in plants in response to internal or

209

external factors (Table 2). It has been also proved that the amount of ethylene produced by stressed plants can be decreased by inhibitors of ACC oxidase or by introduction of antisense ACC oxidase constructs into a plant (Table 2). Therefore, the involvement of ACC oxidase in control of stress ethylene ought to be taken into consideration.

TABLE 2. Stress-induced modifications in ethylene biosynthesis

Type of modification Type of a stressor

ACC accumulation: All the stressors

- increased activity of ACC synthase

- increased expression of ACC synthase genes wounding

- regulation at the post- transcriptional level fungal elicitors

Enzymatic oxidation of ACC:

- ACC oxidase activity increased

- enhanced ACC oxidase gene expression

- antisense ACC oxidase construct

decreases ethylene production

- ACC oxidase inhibitors* prevent or decrease the stress-induced burst of ethylene production

water stress

flooding

electric current

pathogens

wounding flooding

flooding

ozone copper rubbing

electric current

mechanical impedance

References

[27, 29, 34, 38, 47, 48]

and as cited in [83]

[29], as cited in [49]

as cited in [49]

[57]

[21]

[40]

[17] and as cited in [49]

[18] [44]

[21]

[34] [60] [4,5]

[40]

[63]

* the ACC-dependent "residual actvity" (insensitive to ACC oxidase inhibitors) is frequently observed

There is experimental evidence that active oxygen species may be involved in the non-enzymatic conversion of ACC to ethylene, both in vitro and in vivo (Table 3).

It seems that defense mechanisms, involved in scavenging of oxygen and organic radicals and operating in tissues affected with moderate stress, are very likely to inhibit ACC oxidase in such tissues (Table 4), even as the stress-induced accumulation of ACC takes place [78]. On the other hand, in tissues responding to a sudden and acute sublethal stresses an overproduction of active oxygen species is likely to result in the non-enzymatic oxidation of ACC molecules localized at the sites of free radicals generation (e.g. in cell wall). However, there is a lack of direct experimental evidence

210

which can confirm such a possibility. Usually, the ACC oxidase activity is evaluated by determinations of ethylene amount produced by the ACC-supplied samples (tissues or a cell-free system). This approach does not allow for a discrimination between ACC oxidase-dependent and free radical-induced, non-enzymatic conversion of ACC to ethylene.

TABLE 3. The involvement of oxidative stress in non-ezymatic conversion of ACC to ethylene

system studied

A in vitro (promotion) superoxide radicals H20 2 generating system + Mn 2+

lipid peroxidation

B. in vivo (promotion) Oxidative free radicals LOX-dependent lipid peroxidation due to freezing, desiccation, wounding pulse heat treatment

C. Free radical scavengers (inhibition)

References

as cited in [48, 82] [70] as cited in [48]

[26,27]

[47,48] [37] [69]

as cited in [48, 83]

It is known that ethylene synthesis is promoted by jasmonate, the product of lipoxygenase-mediated lipid peroxidation (for a review see [62]. Therefore, synthesis of stress ethylene may be affected by the oxidative stress in a indirect, JA-dependent way, as well.

3. Possible Role of Ethylene in Plant Responses to Stress-inducing Factors

Diverse effects of ethylene, reported in the literature (Table 4), can be brought to two types of the hormone action: 1) modification of tissue growth and development 2) induction or promotion of defense mechanisms (Table 5).

It seems that stress ethylene may be involved in control of two types of plant resistance to stress factors: 1) a plant ability to avoid stress by an exclusion of a stressor from cells and tissues (changes in symmetty of cell growth, promotion of aerenchyma formation, increased rooting activity, promotion of organ abscission and tissues senescence, etc., which allow plants to cope with mechanical stress, oxygen depletion and water deficits; in the case of the pathogen attack this type of responses is known as "a hypersensitive reaction" which limits spreading of pathogens within a plant, 2) an ability to tolerate the stress-evoking agent within a cell or a tissue by an induction or activation of certain proteins (Table 5), responsible for osmotic or metabolic

211

adjustments, modification of cell wall structure, protection of other macromolecules, wound healing, or for defense reactions against pathogens etc. Possibility that ethylene is involved in control of antioxidative mechanisms [38] calls for further studies.

TABLE 4. Consequences of oxidative stress for ethylene biosynthesis

Mechanisms Consequences for ethylene References production

Superoxide dismutase (SOD) - H20 2 inhibits ACC oxidase - (dismutation of superoxide to H20 2) in vitro and in vivo [67] Catalase - salicylic acid-binding protein - (dismutation ofH20 2 to shares high sequence identity with water and oxygen) catalase and allows for an increase

ofH202and AOS in pathogen-affected cells [11,12]

- salicylic acid inhibits ACC oxidase and [53] ACC synthase [54]

Endogenous free radical scavengers: - ascorbic acid - necessary requirement for ACC ]

oxidase [66,67] - dehydroascorbate (product of the scavenging reaction) - inhibits purified ACC oxidase [67]

- polyamines [16] - inhibit synthesis of ACC synthase and ACC conversion to ethylene [1, 24, 25, 71]

Compatible solutes: sorbitol, - proline inhibits ACC to mannitol, myo-inositol, proline [65] ethylene conversion in the halophyte [14]

Currently, ethylene signal transduction pathway has become the object of intensive studies [6, 15]. It has been proposed [10] that the primary steps in ethylene-signal perception and processing are mediated by the products of ETRl, CTRl, and EIN2 genes, which act in combination to produce a secondary signal that mediates the divergent responses, listed in Table 5. The nature of the output signal from the primary pathway is unknown but could be a second messenger, e.g. ea2+ or an enzyme, e.g. protein kinase [10]. Calcium requirement for a variety of ethylene-dependent processes has been indicated [8, 22, 61]. Calcium was found to be involved in control of seedling responses to mechanical and cold stress [45, 50]. Obviously, further studies on ethylene transduction pathway and calcium involvement in plant responses to different stressors are needed.

212

TABLE 5. Ethylene-controlled plant responses to different stressors.

Phenomenon

Inhibition of stem elongation promotion oflateral expansion - orientation of cortical MT

longitudinally to cell axis

Promotion of root and stem elongation in submerged plants

Promotion of aerenchyma formation Stimulation of rooting activity]

Promotion of leaf abscission and a tissue senescence,

Cell wall modifications: - stimulation of cell wall loosening - increased lignification accumulation ofhydroxyproline­rich glycoproteins (HPRG]

Stimulation of alternative electron transfer in plant mitochondria [77]

Modification ofphenylpropanoid synthesis

References

[4,5] [73]

as cited in [64]

as cited in [43]

as cited in [43] [3]

[75] [17,52]

[36] [39]

[74]

[56]

as cited in [33]

Synthesis of pathogen-related [18, 19, 55] (PR) proteins(also in UV- and wound-affected cells - basic (extracellular) PR-l, PR-2, desiccation [7]

PR-3, PR-5 (homologous to osmotin) basic chitinase [10, 41]

- osmotin (induced also by NaCl, wounding, UV, desiccation) [9]

- HPRG proteins [19]

4. References

Implications for stress resistance

coping with mechanical stress

coping with flooding

coping with flooding recovery from wounding

coping with water stress defense against spreading of pathogens

coping with flooding increased tissue resistance to mechanical impedance desiccation, freezing and pathogens

coping with anaerobiosis protection against oxidative stress

different defence mechanisms in stressors

defense mechanism against pathogens, plant adjustment to certain abiotic stresses such as, salt or freezing [35]

1. Apelbaum, A, Burgoon, AC., Anderson, J.D., Lieberman, M., Ben-Arie R. and Mattoo, AK. (1981) Polyamines inhibit biosynthesis of ethylene in higher plant tissues and fruit protoplast, Plant Physiol. 68, 453-456.

2. Bailey, B.A, Dean, J.F.D. and Anderson, J.A (1990) An ethylene biosynthesis-inducing endoxylanase elicits electrolyte leakage and necrosis in Nicotiana tabacum cv Xanthi leaves, Plant Physiol. 94, 1849-1854.

3. Bollmark, M. and Elliason, L. (1990) Ethylene accelerates the breakdown of cytokinins and thereby stimulates rooting in Norway spruce hypocotyl cuttings, Physiol. Plant. 80, 534-540.

213

4. Boyer, N., Desbiez, M-O., Hofmger, M. and Gaspar, Th. (1983) Effect of lithium on thigmomorphogenesis in Bryonia dioica. Ethylene production and sensitivity, Plant PhysioL 72, 522-525.

5. Boyer, N., De Jaeger, G., Bon, M.-C. and Gaspar, Th. (1986) Cobalt inhibition of thigmomorphogenesis in Bryonia dioica: possible role and mechanism of ethylene production, Physiol. Plant. 67, 552-556.

6. Bowler, C. and Chua, N.-H. (1994) Emerging themes of plant signal transduction, The Plant Cell 4, 1529-1541.

7. Brederode, F.Th., Linthorst, H.J.M. and Bol, J.F. (1991) Differential induction of acquired resistance and PR gene expression in tobacco by virus infection, ethephon treatment, UV light and wounding, Plant Mol. Bioi. 17, 1117-1125.

8. Ceverry, J.L., Pouliquen, J., Le Guyader, H. and Marcellin, P. (1988) Calcium regulation of exogenous and endogenous 1-aminocyclopropane acid bioconversion to ethylene, Physiol. Plant. 74, 53-57.

9. Chang, P-F.L., Cheah, K, Narasimhan, M.L., Hasegawa, P. M. and Bressan, RA (1995) Osmotin gene expression is controlled by elicitor synergism, PhysioL Plant. 95, 620-626.

10. Chen, Q. G. and Bleecker, A (1995) Analysis of ethylene signal transduction kinetics associated with seedling-growth response and chitinase induction in wild-type and mutant Arabidopsis, Plant Physiol. 108, 597-607.

11. Chen, Z. and Klessig, D.F. (1991) Identification of a soluble salicylic binding protein that may function in signal transduction in the plant disease resistance response, Proc. Natl. Acad. Sci. USA 88, 8179-8183.

12. Chen, Z., Silva, H. and Klessig, D.F. (1993) Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid, Science 262, 1883-1886.

13. Chrominski, A, Bhat, RB., Weber, D.J. and Smith, B.N. (1988) Osmotic stress-dependent conversion of 1-aminocyclopropane-1-carboxylic acid in the halophyte Allenrolfea occidentalis, Envir. Exp. Bot.U8, 171-174.

14. Chrominski, A, Halls, S., Weber, D.J. and Smith, B. (1989) Proline affects ACC to ethylene conversion under salt and water stresses in the halophyte Allenrolfea occidentalis, Envir. Exp. Bot .. 29,359-363.

15. Clark, K, Jirage, D., Wang. x.. and Chang, C. (1996) Two-component regulators in ethylene perception and signal transduction, NATO-ARW, Biology and Biotechnology of the Plant Hormone Ethylene, June 9-13, Chania, Crete, Greece

16. Drolet, G. F., Dumbroff , E.B., Legge, RL. and Thompson, J. E. (1986) Radical scavenging properties ofpolyamines,Phytochemisfry 25,367-371.

17. Dutta, S. and Biggs, R H. (1991) Regulation of ethylene biosynthesis in citrus leaves infected with Xanthomonas campestris pv. citri, PhysioL Plant. 82, 225-230.

18. Ecker, J.R and Davis, R W. (1987) Plant defense genes are regulated by ethylene, Proc. Natl. Acad. Sci. USA 86,1539-1542.

19. Ecker, J., Roman, G., Rothenberg" M., Lehman, A, Lubarsky, B., Chao, Q., Raz, V., Alonso, J.M., Nourizadech, S.D. and Solano, R (1996) Genes and gene interactions controlling ethylene signal transduction, NATO-ARW, Biology and Biotechnology of the Plant Hormone Ethylene, June 9-13, Chania, Crete, Greece

20. Elstner, E.F. and Konze, J.R (1976) Effect of point freezing on ethylene and ethane production by sugar leaf disks, Narure 263, 351-352.

21. English, Ph. J., Lycett, G. W., Roberts, J. A and Jackson, M. B. (1995) Increased 1-aminocyclopropane-l-carboxylic acid oxidase activity in shoots of flooded tomato plants raises ethylene production to physiologically active level, Plant Physiol. 109, 1435-1440.

22. Evenson, K B. (1984) Calcium effects on ethylene and ethane production and 1-aminocyclopropane­I-carboxylic acid content in potato disks,PhysioL Plant. 60, 125-128.

23. Foyer, C.H., Lelandais, M. and Kunert, K (1994) Photooxidative stress in plants, Physiol. Plant. 92, 696-717.

24. Fuhrer, J., Kaur-Sawhney, R, Shih, L.M. and Galston, W. (1982) Effects of exogenous 1,3-diaminopropane and spermidine on senescence of oat leaves. II. Inhibition of ethylene biosynthesis and possible mode of action, Plant PhysioL 70, 1597-1600.

25. Gallardo, M., Matilla, A and Sanchez-Calle, I.M. (1992) Effects of spermine, abscisic acid and temperature upon ethylene production in Cicer arietinum seeds, Plant Physiol. Biochem. 30, 19-27.

214

26. Gaspar, Th., Penel, C., Castillo, F.J. and Greppin H. (1985) A two step control of basic and acidic peroxidases and its significance for growth and development, PhysioL Plant. 64,418-423.

27. Glick, RE., Scblagnhaufer, C.D., Arteca, R and Pell, E.J. (1995) Ozone-induced ethylene emission accelerates the loss of ribulose-l,5-bisphosphate carboxylase/oxygenase and nuclear-encoded mRNAs in senescing potato leaves, Plant PhysioL 109,891-898.

28. Gomez-Lim M., Vald-Lopez, V., Cruz-Hernandez, A and Saucedo-Arias, L.J. (1993) Isolation and characterization of a gene involved in ethylene biosynthesis from Arabidopsis thaliana, Gene 134, 217-221.

29. Gora, L. and Clijsters, H. (1989) Effect of copper and zinc on the ethylene metabolism in Phaseolus vulgariS L., in: H. Clijstres et al. (eds), Biochemical and Physiological Aspects of Ethylene Production in Lower and Higher Plants,. K1uwer Academic Publishers, Dordrecht, pp. 219-228.

30. Goren, R and Siegel, S.M. (1976) Mercury-induced ethylene formation and abscission in citrus and coleus plants, Plant Physiol. 57, 628-631.

31. Gude, H. and van der Plas, L.H.W. (1985) Endogenous ethylene formation and the development of the aIternative pathway in potato tuber discs, Physiol. Plant. 65, 57-62.

32. Hall, lCh., Bassi, P. K., Spencer, M. S. and Van den Born, W.H. (1985) An evaluation of the role of ethylene in herbicidal injury induced by picloram or c10pyralid in rape seed and sunflower plants, Plant Physiol. 79, 18-23.

33. Harber, RM. and Fuchigami, L.H. (1989) Ethylene-induced stress resistance, in: P.H. Li (ed.), Low Temperature Stress Physiology in Crops, CRC Press Inc., Boca Raton, Florida, pp. 81-90.

34. Hogsett, W.E., Raba, RM. and Tingey, D.T. (1981) Biosynthesis of stress ethylene in soybean seedlings: Similarities to endogenous ethylene biosynthesis, Physiol. Plant. 53, 307-314.

35. Hon, W-C., Griffith, M., Mlynarz, A, Kwok, Y.C. and Yang, D.S.C. (1995) Antifreeze proteins in winter rye are similar to pathogenesis-related proteins, Plant Physiol. 109, 879-889.

36. Hoson, T., Maeda, S., Sakaguchi, K., Onishi, H. and Ohta, H. (1990) Changes in osmotic pressure and cell wall properties during auxin- and ethylene-induced growth of intact coleoptiles of rice, Physiol. Plant. 78,277-284.

37. Ievinsh, G. (1992) Soluble lipoxygenase activity in rye seedlings as related to endogenous and exogenous ethylene and wounding, Plant Sci. 82, 155-159.

38. Ievinsh, G. (1996) Ethylene synthesis and the defense against endogenous oxidative stress, in AK. Kanellis, C. Chang, H. Kende' D. Grierson (ed.), Biology and Biotechnology of the Plant Hormone Ethylene, K1uwer Academic Publishers, Dordrecht, ( in press).

39. Ievinsh, G. and Romanovskaya, 0.1. (1991) Accelerated Iignificationn as a possible mechanism of growth inhibition in winter rye seedlings caused by ethefon and l-aminocyclopropane-l-carboxylic acid, Plant PhysioL Biochem. 29,327-331.

40. Inaba, A, Gao, 1 P. and Nakamura, R. (1991) Induction by electric currents of ethylene biosynthesis in cucumber (Cucumis sativus L.) fruit, Plant Physiol. 97, 1161-1165.

41. Ishige, F., Yamazaki, K., Mori, H. and Imaseki, H. (1991) The effects of ethylene on the coordinated synthesis of multiple proteins: accumulation of an acidic chitinase and a basic glycoprotein induced by ethylene in leaves of Azuki bean, Vigna angularis, Plant CellPhysiol. 32, 681-690.

42. Jackson, M. B. (1993) Are plant hormones involved in root to shoot communication?, Adv. Bot. Res. 19,103-187

43. Jackson, M.B. (1985) Ethylene and responses of plants to soil waterlogging and submergence, Annu. Rev.PlantPhysiol. 36,145-174.

44. Jackson, M.B. (1996) Regulation of ethylene production by ACC oxidase during adaptive responses to flooding and submergence, NATO-ARW, Biology and Biotechnology of the Plant Hormone Ethylene, June 9-13, Chania, Crete, Greece

45. Jones, R and Mitchell, C.A (1989) Calcium ion involvernent in growth inhibition of mechanically stressed soybean (Glycine max) seedlings, Physiol. Plant. 76, 598-602.

46. Kacperska, A (1996) Are there common bases for plant responses to different stress factors?, in: F. Dubert and st. Grzesiak (eds.), Ecophysiological Aspects of Plant Responses to Stress Factors, (in Polish), Zaklad Fizjologii Roslin PAN, Krakow, Poland, in press.

47. Kacperska, A and Kubacka-Zebalska, M. (1985) Is lipoxygenase involved in the formation of ethylene from ACC?, Physiol. Plant. 63,399-405.

48. Kacperska, A and Kubacka-Zebalska, M. (1989) Formation of stress ethylene depends both on ACC synthesis and on the activity free radical-generating system, PhysioL Plant. 77, 231-237.

49. Kende' H. (1993) Ethylene biosynthesis, Annu. Rev. Plant Physiol. Plant Mol. BioI. 44, 283-307.

215

50. Knight, H., Trewavas, AJ. and Knight, M. (1996) Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation, The Plant Cell 8, 489-503.

51. Reference omitted. 52. Lawton, KA, Raghothama, KG. Goldsbrough, P.B. and Woodson, W. (1990) Regulation of

senescence-related gene expression in carnation flower petals by ethylene, Plant Physiol. 93, 1370-1375.

53. Leslie CA and Romani RJ. (1988) Inhibition of ethylene biosynthesis by salicylic acid, Plant Physiol. 88, 833-837.

54. Li, N., Parsons, B.L., Liu, D. and Mattoo, AK (1992) Accumulation of wound-inducible ACC synthase transcript in tomato filIit is inhibited by salicylic acid and polyarnines, Plant Mol. Bioi. 18, 477-487.

55. Lincoln, J.E. and Fischer, RL. (1988) Diverse mechanisms for the regulation of ethylene-inducible gene expression,Moi. Gen. Genet. 212, 71-75.

56. Marissen, N., Kanneworlf, WA and van der Plas, L.H.W. (1991). Effect of anareobiosis on ethylene production, respiration and flowering in iris bulbs, Physiol. Plant. 82, 465-473.

57. McKeon, T. A, Hoffinan, N.E. and Yang, S.F. (1982) The effect of plant-hormone pretreatments on ethylene production and synthesis of l-aminocyclopropane-l-carboxylic acid in water stressed wheat leaves, Planta 1255, 437-443.

58. McKersie, B.D. and Leshem, Y. Y. (1994) Stress and Stress Coping in Cultivated Plants. Kluwer Academic Publishers, Dordrecht

59. Mehdy, M.C. (1994) Active oxygen species in plant defense against pathogens, Plant Physioi. 105, 467-472.

60. Pennazio, S. and Roggero, P. (1991) Rapid ethylene production in soybean in response to the cupric ion, Annals of Botany 67,247-249.

61. Raz, V. and Fluhr, R. (1992) Calcium requirement for ethylene-dependent responses, The Plant Cell 4, 1123-1130.

62. Saniewski, M. (1996) The role of jasmonates in ethylene biosynthesis, NATO-ARW, Biology and Biotechnology of the Plant Hormone Ethylene, June 9-13, Chania, Crete, Greece

63. Sarquis, J. I., Jordan, W.R and Morgan, P.W. (1991) Ethylene evolution from maize (Zea mays L.) seedling roots and shoots in response to mechanical impedance, Plant Physioi. 96, 1171-1177.

64. Shibaoka, H. (1994) Plant hormone-induced changes in the orientation of cortical microtubules. Alterations in the cross-linking between microtubules and the plasma membrane, Annu. Rev. Plant Physioi. Plant Mol. Bioi. 45, 527-544.

65. Smirnoff, N. and Cumbes, Q.J. (1989) Hydroxyl radical scavenging activity of compatible solutes, Phytochemistry 28, 1057-1060.

66. Smith, J. T., Ververidis, P. and John, P. (1992) Characterization of ethylene-forming enzyme partially purified from melon, Phytochemistry 31, 1485-1494.

67. Smith, J.J., Zhang, Z.H., Schofield, C., John, P. and Baldwin, J.E. (1994) Inactivation of 1-arninocyclopropane-l-carboxylate (ACC) oxidase. J. Experimental Botany 45,521-527.

68. Spanu, P. and Boller, P. (1989) Ethylene biosynthesis in tomato plant affected by Phytophtora infestans, J.Plant Physiol. 134,533-537.

69. Steed, C.1. and Harrison, MA (1993) Regulation of ethylene synthesis after short-termed heat treatment in etiolated pea sterns,Physioi. Plant. 87,103-107.

70. Stegink, S.J. and Siedow J.N. (1986) Ethylene production from l-arninocyclopropane-l-carboxylic acid in vitro: A mechanism for explaining ethylene production by a cell-free preparation from pea epicotyls,Physioi. Plant. 66,625-631.

71. Suttle, J.C. (1981) Effects ofpolyarnines on ethylene production., Phytochemistry 20, 1477-1480. 72. Sutherland, M.W. (1991) The generation of oxygen radicals during host plant responses to infection,

Physioi. Mol. Plant Pathoi. 38,79-93. 73. Taiz, L. Rayle, D.L. and Eisinger, W. (1983). Ethylene-induced lateral expansion in etiolated pea

sterns. The role of acid secretion, Plant Physiol. 73, 413-417. 74. Toppan, A, Roby, D. and Esqurre-Tugaye, M.T. (1982) Cell surfaces in plant-microorganism

interactions. II In vivo effect of ethylene on hydroxyproline- rich glycoprotein accumulation in the cell wall of diseased plants. Plant. Physiol. 70, 82-86.

75. Tudela, D. and Primo-Millo, E. (1992) l-Aminocyclopropane-l-carboxylic acid transported from roots to shoots promotes leaf abscission in Cleopatra mandarin (Citrus reshni Hort., e. Tan.) seedlings rehydrated after water stress, Plant Physiol. 100, 131-137.

216

76. Ververidis, P. and John, P. (1991) Complete recovery in vitro of ethylene-forming enzyme activity, Phytochemistry 30,725-727.

77. Wagner, AM. and Krab, K. (1995) The alternative respiration pathway in plants: Role and regulation, Physiol. Plant. 95,318-325.

78. Wang, C. Y. and Adams, D. O. (1980) Ethylene production by chilled cucumbers (Cucumis sativus L.),PlantPhysiol. 66,841-843.

79. Weiss, C. and Bevan, M. (1991) Ethylene and a wound signal modulate local and systemic transcription ofwin2 genes in transgenic potato plants, PlantPhysiol. 96,943-951.

80. Weckx, J.E.J. and Clijsters, H.M.M. (1996) Oxidative damage and defense mechanisms in primary leaves of Phaseolus vulgaris as a result of root assimilation of toxic amounts of copper, Physiol. Plant. 96, 506-512.

81. Yahraus, T., Chandra, S., Legendre, L. and Low, P.S. (1995) Evidence for a mechanically induced oxidative burst, Plant Physiol., 109, 1259-1266.

82. Yang, S.F. and Dong, G. (1993) Recent progress in research of ethylene biosynthesis. Bot. Bull. S. Acad. Sin. 34,89-101.

83. Yang, S.F. and Hoffinan, N. E. (1984). Ethylene biosynthesis and its regulation in higher plants, Annu. Rev. PlantPhysiol. 35,155-189.

ETHYLENE AND THE DEFENSE AGAINST ENDOGENOUS OXIDATIVE STRESS IN HIGHER PLANTS

G. IEVINSH AND D. OZOLA Department of Plant Physiology, Institute of Biology, Latvian Academy of Sciences, 3 Miera Str., LV-2169 Salaspils, Latvia

1. Introduction

In higher plants, activation of ethylene biosynthesis is a common and well-known stress-related response. Depending on plant species and tissues studied, the stress­related increase in ethylene production may vary from several-fold to many hundred­fold [1]. Both endogenously produced and exogenous ethylene is known to regulate the accumulation of specific plant mRNAs, as well as control the rate of transcription of specific plant genes [2,3]. Therefore, it is believed that the stress-related ethylene is a signal for plants to activate defense mechanisms. Although endogenously produced stress ethylene has been postulated to have a role in the regulation of stress adaptation reactions, it is not known to what extent a higher capacity to produce ethylene in stress conditions reflects a better chance to cope with a stress. No data has been presented yet to show the range of endogenous ethylene production intensity over which response reactions are mediated.

Several lines of evidence associate ethylene as a regulative factor with endogenous oxidative stress: (i) a parallelity between active biosynthesis of ethylene, intensive formation of H2(h, high activity of ascorbate peroxidase and lipoxygenase in meristematic tissues; (ii) induction of ethylene biosynthesis by H20 2; (iii) increase of ethylene biosynthesis in parallel with enhanced level of H20 2 and activation of ascorbate peroxidase and lipoxygenase in the stressed tissues.

Consequently, if the induction of intensive biosynthesis of ethylene is necessary for initiation of adaptation reactions, it is possible to assume that plants with higher capacity to produce ethylene in response to stress treatment may have higher tolerance against stress. In this report, we have specifically focused on a possible relationship between the ethylene biosynthesis as a potent regulative factor and production and degradation of activated oxygen in different physiological situations. An extreme diversity of ethylene production in response to stress conditions revealed in a variety of objects will be analyzed as well.

217

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 217-228. © 1997 Kluwer Academic Publishers.

218

2. Relationship between ethylene and oxidative stress

2.1. HYDROGEN PEROXIDE

In the same way as ethylene is constitutively produced in higher plants, the formation of active oxygen species, i.e. singlet oxygen, superoxide and hydroxyl radicals, as well as H20 2 within plant tissues is a fundamental consequence of aerobic metabolism [4]. These reactive oxygen species are highly toxic and may lead to severe damage of cell structures [5]. The oxidative metabolism is based on the scavenging of superoxide by superoxide dismutase, leading to formation of H20 2, which in turn is removed by catalase and ascorbate peroxidase.

Oxygen-derived free radicals are involved in plant metabolic processes, e.g. photosynthesis, and therefore are constitutively formed in plants. However in normal conditions the balance between formation and consumption of active oxygen species is tightly controlled [6]. The set of intracellular or extracellular conditions that lead to an increase in the steady-state concentration of reactive oxygen species and lipid hydroperoxides beyond the capacity of the tissue to scavenge them is termed oxidative stress [7]. Increase in tissular concentrations of H20 2 has been shown to occur in non­stressed meristematic tissues [7,8], in senescent tissues [9,10], and in response to different kinds of stresses, including pathogen attack [11,12], cold treatment [13,14], and mechanical stimulation [15]. All the above mentioned situations are characterized by intensive biosynthesis of ethylene as well [1]. A close relationship between rise of ethylene c biosynthesis and the level of peroxides has been shown to occur during senescence of plant tissues [16].

There is a body of data indicating the role of H20 2 in signal transduction during stress response functioning as a diffusible signal for selective induction of defense genes [6,12,17]. Recent studies have shown that oxidative burst is an early event in a pathogen-induced response pathway [18]. The increase in ethylene production after a pathogen attack is a latter event, suggesting the role of ethylene in amplification of defense responses [19]. It should be noted, that the generation of hydrogen peroxide (oxidative burst) in plant tissues can occur within minutes after applying of stimulus [15], thus appearing to be the earliest defense mechanism. The concentration of H20 2

in plant tissues due to stress treatment can increase several-fold from 0.5 J.IlIlol per g fresh weight up to 1.6 J.IlIlol within 5 min [13].

Ethylene biosynthesis can be induced by H20 2 treatment in variety of plant systems both in intact and detached plant tissues. In detached Scots pine needles, H20 2 resulted in more than a 600-fold increase of ACC level accompanied by more than a 200-fold enhancement of ethylene evolution [20]. Protein synthesis de novo was essential for H20 r dependent ethylene evolution from the needles. Numerous experiments in our laboratory have shown that it is possible to induce in vivo ethylene production in Solanum tuberosum stem explant culture, as well as in leaves of cereal seedlings by H20 2 [21]. In pear fruits, H20 2 accelerated ripening with a concomitant increase of ethylene production [9]. On the other hand, limited amount of data suggests a possibility to induce formation of peroxides by treatment with exogenous ethylene [22]. However, the latter effect seems to be associated with general physiological changes due to ethylene rather than with a direct effect of ethylene.

219

Inhibition of several enzyme activities by H20 2, including ACC oxidase [23], ascorbate peroxidase [24] has been documented. Therefore, the dual role of H20 2 in regulation of physiological responses should be assumed. As it has been noted that H20 2 may act as a chemical signal only within an extremely narrow range of concentration [13] it is evident, that quantitative and spatial aspects of peroxide production must be taken into the account.

2.2. LIPOXYGENASE

Lipoxygenase, catalyzing the incorporation of molecular oxygen into fatty acids with formation of an unsaturated fatty acid hydroperoxides, is the major cause of lipid peroxidation in plant tissues [25]. Fatty acid hydroperoxides, which are the primary products of lipoxygenase catalyzed reaction, are metabolized into molecules with regulatory activities, e.g., jasmonic acid [26]. In higher plants, lipoxygenase activity is under control of both endogenous and exogenous ethylene [27] and can be induced in stress situations [28]. In particular, lipoxygenase activity has been shown to increase as a result of pathogen attack with a putative role of the subsequent products as antibacterial agents or signaling molecules [29]. High lipoxygenase activity has been monitored in actively growing vegetative tissues in parallel with intensive ethylene formation [27].

Due to close association between ethylene biosynthesis and lipoxygenase activity as well as because of the fact that ACC can be converted into ethylene in various chemical systems containing lipoxygenase it was suggested previously that lipoxygenase may participate in the formation of ethylene in vivo [30,31]. However, recent studies have shown that native ACC oxidase is not identical with lipoxygenase [32]. Yet, several higher plant species have been reported to produce ethylene through peroxidation of membrane lipids in conditions of severe stress. These include an aquatic plant Spirodela oligorrhiza [33], spruce needles [34], and Scots pine needles [20]. Based on selenomethionine experiments, it was concluded, that in Scots pine needle system only ethylene evolution induced by Na2S20S at concentrations above 8 mM may be derived from lipid peroxidation [20].

Taken together, the data indicate that besides the direct effect of ethylene on lipoxygenase expression there should be a common regulative pathway for both ethylene biosynthesis and lipoxygenase activity.

2.3. ASCORBATE PEROXIDASE

Ascorbate peroxidase is one of the enzymes central to antioxidant responses, found in the cytoplasm and stroma and thylakoid compartments of the chloroplast [24]. As catalase is localized only in peroxisomes, ascorbate peroxidase is most important H20 2 eliminating factor both in the cytosol and chloroplasts. A high activity of ascorbate peroxidase has been shown to occur during early phases of vegetative growth [ai,ak,al] as well as in stressed plant tissues [38,39,40].

220

Ethylene as well as ACC has been shown to increase the activity of ascorbate peroxidase in pine and potato [21,41] and the cytoplasmic ascorbate peroxidase transcript levels were found to increase in response to application of ethylene generating chemical, ethephon in peas [42], although additional controls in gene expression were observed at the translational level. On the other hand, treatment with exogenous H20 2 induced ascorbate peroxidase activity in different plant objects [38,43,44], in particular, both in intact needles of pine seedlings as well as in detached needles [41]. Inhibitor studies demonstrated that protein synthesis de novo was necessary for peroxide-dependent activation of ascorbate peroxidase. However, it was difficult to judge if RNA synthesis was also required for the rise in ascorbate peroxidase activity. In addition, H20 r associated increase in ascorbate peroxidase activity was abolished by inhibitors of both ethylene biosynthesis (aminooxyacetic acid) and ethylene action (silver thiosulphate) [41]. In potato stem explant culture increase in ethylene production due to H20 2 treatment was accompanied by activation of ascorbate peroxidase [21].

It should be noted, that ascorbic acid, a substrate for ascorbate peroxidase catalyzed reaction of H20 2 destruction, is required for a full activity of ACC oxidase [32]. Ascorbate is known to be presented in the chloroplasts, cytosol, vacuole and apoplastic space of plant cells at high concentrations [45]. However, a general availability of ascorbate within plant tissues does not exclude a possibility that a certain kind of competition may occur between different ascorbate-utilizing enzymes.

3. Diversity of Ethylene Production

It is broadly accepted that ethylene production rates differ greatly between plant species and tissues analyzed [1]. However, usually different units of measurement used for expression of ethylene production rates as well as various experimental setups make direct comparison extremely difficult. Here we have tried to use an another approach for characterization of diversity in ethylene production between plant species, namely, to search for a minimum vs. maximum possible rates of ethylene production both for basal ethylene biosynthesis as well as for that induced by stress conditions. Figure 1 summarizes the results from a broad spectrum of plant species. It is evident, that if basal rates of ethylene production were concerned, the maximum rate was l20-fold the minimum rate. Among stress-induced ethylene production intensity, the difference was 72-fold. If the increase in ethylene production was taken into the account, the maximum relative increase was 86-fold the minimum increase.

Actually, the data summarized in Figure 1 do not reveal any physiological nor phylogenetic relationship between ethylene production rates within species and tissues analyzed. In general, there was no causal relationship between the basal rates of ethylene production and those induced by different stress treatments, which is not surprising, as ethylene production rate for a particular plant may be related to the actual degree of stress-related injury, at least, in part [63,64]. In the same way, ACC levels both in intact as well as in stressed plant tissues differed significantly, however, in a lesser extent than in the case of ethylene production rates (Fig. 2).

Cuc

umbe

r M

ung

Pin

e co

tyle

dons

[47

) b

ean

[49)

ne

edle

s [5

0)

Bar

ley

Mai

Ze

Bea

ns

Bea

n O

at

Bar

ley

[47]

co

leop

tile

[47

) [5

4)

leav

es [

55)

cole

optl

le (

47]c

oleo

ptil

e (4

7]

Cuc

umbe

r S

oybe

an

Cuc

umbe

r P

eppe

r fr

uit

[46)

[4

8)

hypo

coty

l [4

7]

leaf

di

scs

[51)

Tob

acco

A

rabi

dops

is

Tom

ato

Cit

rus

App

le

leaf

[5

3)

segm

ents

[47

) le

af

shoo

ts [

57)

disc

s [5

2)

disc

s [5

6)

Whe

at

cole

opti

le [

47)

o o

o C

ontr

ol

00

0

Eth

ylen

e (n

mol

g-l

FW

h-l

)

o 0

0 0

00

o

0.0

07

0

.02

0

.02

4

0.0

24

0.0

36

0

.04

50

.04

5

0.0

48

0

.05

0

.05

40

.08

9

0.1

0

.11

0

.12

o~

09

2 Q

QQ

S

tres

sed

o o

Oo

oQ

D

0.2

4

1.12

0

.36

0.

67

0.7

1.

32

1.3

4

A

~!:"':;Y

';""':''

'' ()

o ®

";!::

>{',

: ':

,-:>

-0

'"

< i,

:/~>::

~.'::-

'~~"

,. ":'

34

8

12

0

78

8

25

o e

.'"

,,:.

1',

,:.

0, 0

0

8 14

1

4

25

1

5

e 5

Incr

ease

(ti

mes

)

@

2 1

03

31

Figu

re 1

, D

iver

sity

of b

asal

rat

es o

f eth

ylen

e pr

oduc

tion

vs.

str

ess-

indu

ced

ethy

lene

pro

duct

ion,

o G

e

o

6 7

1.4

1.6

~ ....

222

Tomato roots 1581

0.1

®

Mung bean

hypocotyl1571 Soybean 1591

Control 0

0.2 0.4

Stre •• ed

@ G ®

Beans 1601

ACC (nmol ,-1 FW)

0 0 0.4 1.6

(0 0 @

Increase (times)

Soybean leaves 1611

0 1.6

G (])18

Figure 2. Diversity of ACC content in intact vs. stressed plant tissues.

Ethylene ACC

Parsley cell

culture 1621

a 8

o 4

nmol h- I g-IFW Increase.

nmolg-IFW Increase,

Time, h times times

Control 0 0.02 0.04

Detachment- 12 0 0.37 GD 19 0 0.13 3 induced

24 0 0.15 • 8 0 0.22 6

H20 2- 12 0 4.40 .220 01.50 .. 38 induced

G 24 0 1.30 • 65 625

Figure 3. Comparative increase in ethylene production intensity and ACC content in stressed Scots pine (Pinus sylvestris L.) needles. Data from ref. [20].

1.4

1.2 c 0 ;i "II. l~ 0.8 "':!: • Ci 0.6 c e .. - c !- 0.4

0.2

0 o m m ~ ~ ~ • ~ N ~ 0 m m ~ ~ ~ ~ ~ N

N ~ ~ ~ ~ ~ m moo _ N ~ ~ ~ ~ ~ m

Individual ne.dle.

Figure 4. Distribution of ethylene production intensity by individual pine needles along the stern of individual seedling.

14 6

12 .. .!! 10 ... I c 8 '0 ~ 6 .. ... e 4 " z

2

0 ., It) ., ~

... .... It) ., co '" ., ... '" '" ... '" "! ~ ~

.., "': ... "! It) It) co "! co .,; ., "! "! "": ~

0 0 0 0 0 0 .,; 0 .,; .,; .,; 0 0 0 0 0

Ethylene production (nmol/h/gFW)

Figure 5. The distribution of pine needles in groups according to their ethylene production rates.

i II.

':' ... 0.8

':' c E C 0.6 S ~ i 0.4 • .. .. ~ 0.2

I! .. II.

00 2 4 6 7

Neadle group

223

Figure 6. Peroxidase activity in the respective pine needle groups differing in ethylene production rates (according to Fig. 5).

224

Scots pine needles treated with H20 2 were among the plant objects producing ethylene at extremely high rates [20]. However, the basal rate of ethylene production from the needles was relatively small. It is evident, that the proportional increase in ACC level in stressed pine needles is considerably more expressed than the increase in ethylene production (Fig. 3). Consequently, to our knowledge, Scots pine needles represent plant tissues exhibiting the highest relative increase of ACC content as a result of stress treatment. One may ask, if there is a real physiological necessity for accumulation of ACC 600-fold above normal level and for evolution of ethylene 200-times above basal rates?

Variation in ethylene production between individual plants of a particular species has been noted for conifers [65,66] and cereal seedlings [67]. Less commonly studied phenomenon is the diversity of ethylene production between repetitive subunits, e.g. leaves, within an individual plant. In physiological studies it has been assumed mostly that the leaves of one age from a particular plant represent identical objects both physiologically and biochemically. However, the coefficient of variance between the intensity of ethylene production by individual needles within an individual Scots pine seedling was up to 60 % [21]. At the same time, the coefficient of variance for needle weight within an individual seedling was between 9 and 15 %.

In Scots pine, needles are arranged around the stem in a Fibonacci spiral with a 3/8 phyllotactic arrangement [68]. Respective individual needles from individual seedlings were collected sequentially according to the spiral and their ethylene production rates were determined (Fig. 4). There were no pronounced regularities in distribution of ethylene production rates from individual needles along the stem. In order to relate ethylene production rates by individual needles to their capacity to destroy endogenous hydrogen peroxide, respective needles were grouped according to ethylene production intensities and peroxidase activity was determined. The distribution of needles in groups according to their ethylene production rates is shown in Figure 5. The data in Figure 6 presents an evidence that there is a correlation between the intensity of ethylene production by individual needles and their peroxidase activity. Consequently, it is possible to conclude that ethylene production intensity by individual needles is related to the capacity to destroy endogenous hydrogen peroxide.

In spite to the well established role of ethylene in regulation of plant defense responses, a causal relationship between endogenous ethylene production in stress situations and a particular response reactions against endogenous oxidative stress has not yet been demonstrated. Our data represents the first evidence for a direct link between ethylene production and antioxidative stress responses in repetitive subunits within an individual plant.

4. Cooperative Interactions between Ethylene and Oxidative Stress

In general, it is possible to suggest that there is no threshold between normal metabolism and stress metabolism. On the other hand, there is no threshold between the basal rate of ethylene biosynthesis and that induced by a sudden change of internal or external factors which may be called "stress". It is well accepted, that ethylene biosynthesis requires almost constitutive renewal of ACC synthase protein level due to

225

extremely short life of the enzymatic protein molecule [69]. Consequently, this provide an excellent point for nearly immediate regulation of ethylene production, both positive and negative, in response to any changes in internal and external environment.

The main signaling pathway of stress responses evidently goes through peroxide/hydroperoxide metabolism where tightly controlled balance between formation and degradation of peroxide molecules occurs. According to Eistner et al. [70], regulation of metabolic responses differs significantly before and after the "stress point" that characterizes transition from heterolytic to homolytic reactions. In a situation before the "stress point" the increase in H20 2 content is limited in time and the peroxide itself may be suggested as a signaling factor. In addition, H20 2 may act as a chemical signal only within an extremely narrow range of concentration [13]. This may serve as an inducer of ethylene biosynthesis at the level of gene expression. As a result, the produced ethylene may induce expression of several other genes of defense proteins, including those for ascorbate peroxidase. After the "stress point" when free radical mechanisms associated with membrane degradation are initiated the inhibitory activity of peroxides may prevail leading to self-destruction of cells or tissues.

As there seems to be no absolute requirement of activation of ethylene biosynthesis for induction of oxidative stress responses, it may be suggested that other, non-ethylene pathway for induction of defense genes should exist. This fact is obvious also from the studies of the role of ethylene during wound response. It was demonstrated, that only less than 15% of wound-induced gene expression is dependent on ethylene [71].

The intermediate role of H20 2during juvenile growth and senescence, as well as in stress situations may be concerned. Ethylene, in turn, may be assumed to playa role in maintaining a high levels of expression for enzymes related to antioxidative metabolism, e.a., ascorbate peroxidase, as well as lipoxygenase. On the other hand, lipoxygenase activity may be directly regulated by the formation of H20 2. It is possible to conclude, that ethylene production in higher plants is casually related to both formation and degradation of reactive oxygen species that emphasizes the role of ethylene as an intermediate molecule during adaptation reactions.

5. References

1. Abeles, F.B., Morgan, P.W., and Saltveit, M.E. (1992) Ethylene in Plant Biology, Academic Press, San Diego.

2. Ecker, J.R. and Davis, R.W. (1987) Plant defense genes are regulated by ethylene, Proc. Natl. Acad. Sci. USA 84, 5202-5206.

3. Boller, T. (1991) Ethylene in pathogenesis and disease resistance, in AK Mattoo and lC. Suttle (eds.), The Plant Hormone Ethylene, CRC Press, Boca Raton, pp. 293-314.

4. Hendry, GAF. and Crawford, R.M.M. (1994) Oxygen and envirorunental stress in plants - an overview,Proc. Royal Soc. Edinburgh 1028, 1-10.

5. E1stner, E.F. and Osswald, W. (1994) Mechanisms of oxygen activation during plant stress, Proc. Royal Soc. Edinburgh 1028,131-154.

6. Inze, D. and Van Montagu, M. (1995) Oxidative stress in plants, Curro Opin. BioL 6, 153-158. 7. Puntarulo, S. (1994) Effect of oxidative stress during imbibition of soybean embryonic axes, Proc.

Royal Soc. Edinburgh 1028, 279-286. 8. Puntarulo, S., Sanchez, RA and Boveris, A (1988) Hydrogen peroxide metabolism in soybean

embryonic axes at the onset of germination, Plant Physiol. 86, 626-630.

226

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

Brennan, T. and Frenkel, C. (1977) Involvement of hydrogen peroxide in the regulation of senescence in pear, Plant Physiol. 59,411-416. Irigoyen, J.J., Emerich, D.W. and Sanchez-Diaz, M. (1992) Alfalfa leaf senescence induced by drought stress: photosynthesis, hydrogen peroxide metabolism, lipid peroxidation, and ethylene evolution,Physiol. Plant. 84,67-72. Apostol, I., Heinstein, P.F. and Low, P.S. (1989) Rapid stimulation of an oxidative burst during elicitation of cultured plant cells: role in defense and signal transduction, Plant Physiol. 90, 109-116. Levine, A, Tenhaken, R., Dixon, R. and Lamb, C. (1994) H202 from the oxidative burst orchestrates the plant hypersensitive disease resistance response, Cell 79, 583-593. Prasad, T.K, Anderson, M.D., Martin, BA and Stewart, C.R. (1994) Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide, Plant Cell 6, 65-74. Okuda, T., Matsuda, Y., Yamanaka, A and Sagisaka, S. (1991) Abrupt increase in the level of hydrogen peroxide in leaves of winter wheat is caused by cold treatment, Plant Physiol. 97, 1265-1267. Yahraus, T., Chandra, S., Legendre, L. and Low, P.S. (1995) Evidence for a mechanically induced oxidative burst.,PlantPhysioZ. 109, 1259-1266. Sylvestre, I., Droillard, M.-J., Bureau, J.M. and Paulin, A (1989) Effects of the ethylene rise on the peroxidation of membrane lipids during the senescence of cut carnations, Plant Physiol. Biochem. 27, 407-413. Chen, Z., Silva, H. and K1essig, D.F. (1993) Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid, Science 262, 1883-1886. May, M.J., Hammond-Kosack, KE. and Jones, J.D.G. (1996) Involvement of reactive oxygen species, glutathione metabolism, and lipid peroxidation in the Cfgene-dependent defense response of tomato cotyledons induced by race-specific elicitors of Cladosporium folvum, Plant Physiol. 110, 1367-1379.

19. Hammond-Kosack, KE., Silverman, P., Raskin, I. and Jones, J.D.G. (1996) Race-specific elicitors of Cladosporium folvum induce changes in cell morphology and the synthesis of ethylene and salicylic acid in tomato plants carrying the corresponding Cf disease resistance gene, Plant Physiol. 110, 1381-1394.

20. Ievinsh, G. and Tillberg, E. (1995) Stress-induced ethylene biosynthesis in pine needles: a search for the putative l-aminocyclopropane-l-carboxylic acid-independent pathway, J. Plant Physiol. 145, 308-314.

21. Ievinsh, G. (1996) Ethylene and Peroxidases in the Control of Plant Development and Adaptation, Habilitation Theses, University of Latvia, Riga.

22. Frenkel, C. and Brennan, T. (1977) Upsurge in respiration and peroxide formation in potato tubers as influenced by ethylene, propylene, and cyanide, Plant PhysioZ. 59, 411-416.

23. Smith, J.J., Zhang, Z.H., Schofield, C.J., John, P. and Baldwin, J.E. (1994) Inactivation of 1-aminocyclopropane-l-carboxylate (ACC) oxidase,J. Exp. Bot. 45, 521-527.

24. Asada, K (1992) Ascorbate peroxidase - a hydrogen peroxide-scavenging enzyme in plants, Physiol. Plant. 85,235-241.

25. Droillard, M.-J., Roudet-Mayer, M.A, Bureau, J.-M. and Lauriere, C. (1993) Membrane-associated and soluble lipoxygenase isoforms in tomato pericarp. Characterization and involvement in membrane alterations, Plant Physiol. 103, 1211-1219.

26. Creelman, R.A and Mullet, J.E. (1995) Jasmonic acid distribution and action in plants: regulation during development and response to biotic and abiotic stress, Proc. Natl. Acad. Sci. USA 92,4114-4119.

27. Ievinsh, G. (1992) Soluble lipoxygenase activity in rye seedlings as related to endogenous and exogenous ethylene and wounding, Plant Sci. 82, 155-159.

28. Hildebrand, D.F. (1989) Lipoxygenases, Physiol. Plant. 76,249-253. 29. Bell, E. and Mullet, J.E. (1993) Characterization of an Arabidopsis lipoxygeanse gene responsive to

methyljasmonate and wounding, Plant PhysioZ. 103, 1133-1137. 30. Bosquet, J.F. and Thirnann, KV. (1984) Lipid peroxidation forms ethylene from 1-

aminocyc1opropane-l-carboxylic acid and may operate in leaf senescence, Proc. Natl. Acad. Sci. USA 81,1724-1727.

31. Kacperska, A and Kubacka-Zebalska, M. (1989) Formation ofstress ethylene depends both on ACC synthesis and on the activity offree radical-generating system, Physiol. Plant. 77, 231-237.

32. Kende, H (1993) Ethylene biosynthesis, Annu. Rev. Plant Physiol. Plant Mol. Bioi. 44,283-307.

227

33. Mattoo, AK, Baker, J.E. and Moline, H.E. (1986) Induction by copper ions of ethylene production in Spirodela oligorrhiza: evidence for a pathway independent of l-aminocyclopropane-l-carboxylic acid, J. Plant PhysioL 123, 193-202.

34. Chen, Y.-M., Tabner, B.J. and Wellburn, AR. (1990) ACC-independent ethylene fonnation in brown Norway spruce needles involves organic peroxides rather than hydroperoxides as possible precursors, PhysioL MoL Plant Pathol. 37,323-337.

35. Ievinsh, G. (1992) Characterization of the peroxidase system in winter rye seedlings: compartmentation and dependence on leaf development and hydrogen donors used, J. Plant Physiol. 140,257-263.

36. Cakmak, I., StrbaC, D. and Marscbner, H. (1993) Activities of hydrogen peroxide-scavenging enzymes in germinating wheat seeds, J. Exp. Bot. 44, 127-132.

37. De Gara, L., De Tullio, M., Paciolla, C., Liso, R. and Arrigoni, O. (1993) Cytosolic ascorbate peroxidase in angiosperms and the different expressions of its isoforms in maize embryos during germination, in KG. Welinder, S.K Rasmussen, C, Penel and H. Greppin (eds.), Plant Petroxidases: Biochemistry and Physiology, University of Copenhagen and University of Geneva, pp. 251-255.

38. Mehlhorn, H. (1990) Ethylene-promoted ascorbate peroxidase activity protects plants against hydrogen peroxide, ozone and paraquat, Plant Cell Environ. 13, 971-976.

39. Irigoyen, J.J., Emerich, D.W. and Sanchez-Diaz, M. (1992) Alfalfa leaf senescence induced by draught stress: photosynthesis, hydrogen peroxide metabolism, lipid peroxidation and ethylene evolution, Physiol. Plant. 84,67-72.

40. Mittler, R., Pitcher, L.H. and Zilinskas, BA (1993) Molecular biology of pea cytosolic ascorbate peroxidase and its response to oxidative stress, in KG. Welinder, S.K Rasmussen, C, Penel and H. Greppin (eds.), Plant Petroxidases: Biochemistry and PhYSiology. University of Copenhagen and University of Geneva, pp. 263-270.

41. Ievinsh, G., Valcina A and Ozola, D. (1995) Induction of ascorbate peroxidase activity in stressed pine (Pinus sylvestris L.) needles: a putative role for ethylene, Plant Sci. 112, 167-173.

42. Mittler, R. and Zi1inskas, BA (1992) Molecular cloning and characterization of a gene encoding pea cytosolic ascorbate peroxidase,J. BioI. Chem.267, 21802-21807.

43. Mittler, R. and Tel-Or, E. (1991) Oxidative stress response and shock proteins in the unicellular Cyanobacterium synechococcus R2 (PCC-7942), Arch. Microbiol. 155, 125-130.

44. Zheng, X. and van Huystee, R.B. (1992) Anionic peroxidase cataiysed ascorbic acid and IAA oxidation in the presence of hydrogen peroxide: a defence system against peroxidative stress in peanut plant, PhytochemiStry 31, 1895-1898.

45. Arrigoni, O. (1994) Ascorbate system in plant development, J. Bioenerget. Biomembr. 26,407-419. 46. Inaba, A, Gao, J.P. and Nakamura, R. (1991) Induction by electric currents of ethylene biosynthesis

in cucumber (Cucumis sativus L.)fruit,PlantPhysiol. 97, 1161-1165. 47. Saltveit, M.E. and Dilley, D.R. (1978) Rapidly induced wound ethylene from excised segments of

etiolated Pisum sativum L., cv. Alaska. I. Characterization of the response, Plant Physiol. 61, 447-450.

48. Tittle, F.L., Goudey, J.S. and Spencer, M.S. (1990) Effect of 2,4-dichlorophenoxyacetic acid on endogenous cyanide, beta-cyanoalanine synthase activity, and ethylene evolution in seedlings of soybean and barley, Plant Physiol. 94, 1143-1148.

49. Yu, Y.B. and Yang, S.F. (1980) Biosynthesis of wound ethylene, Plant Physiol. 66,281-285. 50. Bucher, lB. (1981) S02-induced ethylene evolution of forest tree foliage, and its potential use as

stress-indicator, Eur. J. For. Path. 11,369-373. 51. Nanaiah, G.K. and Anderson, JA (1991) Electrolyte leakage and evolution of ethylene and ethane

from pepper leaf disks following temperature stress and fatty acid infiltration, J. Amer. Soc. Hort. Sci. 117,846-851.

52. LangebarteIs, C., Kerner, K, Leonardi, S., Schraudner, M., Trost, M., Heller, W. and Sandermann, H. (1991) Biochemical plant responses to ozone. I. Differential induction of polyamine and ethylene biosynthesis in tobacco, Plant Physiol. 95, 882-889.

53. Rakitina, T.Y., Vlasov, P.V., Jalilova, F.K and Kefeli, V.I. (1994) Abscisic acid and ethylene in mutants of Arabidopsis thaliana differing in their resistance to ultraviolet (UV-B) radiation stress, Russ. J. Plant Physiol. 41, 682-686.

54. Morgan, P.W., He, C.l, De Greer, lA and De Proft, M.P. (1990) Does water deficit stress promote ethylene synthesis by intact plants? Plant Physiol. 94, 1616-1624.

55. Arteca, R.N. and Schlagnhaufer, C. (1984) The effect of brassinosteroid and 2,4-D-L-amino acid conjugates on ethylene production by etiolated mung bean segments, Physiol. Plant. 62,445-447.

228

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

Riov, jJ and Yang, S.F. (1982) Autoinhibition of ethylene production in citrus peel discs. Suppression of l-aminocyclopropane-l-carboxylic acid synthesis, Plant Physiol. 69, 687-690. Kim, W.T., Silverstone, A, Yip, W.K, Dong, J.G. and Yang, S.F. (1992) Induction of 1-aminocyclopropane-l-carboxylate synthase mRNA by auxin in mung bean hypocotyls and cultured apple shoots, Plant Physiol. 98,465-471. Wang, T.-W. and Arteca, R.N. (1992) Effects oflow 02 root stress on ethylene biosynthesis in tomato plants (Lycopersicon esculentum Mill cv Heinz 1350), Plant Physiol. 98,97-100. Rodecap, KD. and Tingey, D.T. (1983) The influence of light on ozone-induced 1-aminocyclopropane-l-carboxylic acid and ethylene production from intact plants, Z. PjlanzenphysioL 110,419-427. Fuhrer, J. (1985) Ethylene production and premature senescence of needles from fir trees (Abies alba), Eur. J. For. Path. 15,227-236. Pennazio, S. and Roggero, P. (1992) Effects of free radical scavengers on stress ethylene in soybean leaves hypersensitively reacting to tobacco necrosis virus, Ann. Bot. 69,437-439. Chappell, J., Hahlbrock, K and Boller, T. (1984) Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase, Planta 161, 475-480. Bressan, R.A, LeCureux, L., Wilson, L.G. and Filner, P. (1979) Emission of ethylene and ethane by leaf tissue exposed to ijurious concentrations of sulfur dioxide or bisulfite ion, Plant Physiol. 63, 924-930. Kimmerer, T.W. and Kozlowski, T.T. (1982) Ethylene, ethane, acetaldehyde, and ethanol production by plants under stress, Plant Physiol. 69, 840-847. Telewski, F.W. and Jaffe, M.J. (1986) Thigmomorphogenesis: the role of ethylene in the response of Pinus taeda andAbiesfraserito mechanical perturbation,Physiol. Plant. 66,211-218. Telewski, F.W. (1990) Growth, wood density, and ethylene production in response to mechanical perturbation inPinus taeda, Can. J. Forest Res. 20, 1277-1282. Ievinsh, G. and Kreicbergs, 0. (1992) Endogenous rhythmicity of ethylene production in growing intact cereal seedlings, Plant Physiol. 100,1389-1391. Namboodiri, KK and Beck, C.B. (1968) A comparative study of the primary vascular system of conifers. I. Genera with helical phyllotaxis, Amer. J. Bot. 55,447-457. Acaster, M.A and Kende, H. (1983) Properties and partial purification of l-aminocyclopropane-l­carboxylate synthase, Plant Physiol. 72, 139-145. Eistner, E.F., Wagner, G.A and Schutz, W. (1988) Activated oxygen in green plants in relation to stress situations, Curro Top. Plant Biochem. Physiol. 7, 159-187. Henstrand, J.M. and Handa, AK (1989) Effect of ethylene action inhibitors upon wound-induced gene expression in tomato pericarp, Plant Physiol. 91, 157-162.

POTAMOGETON PECTINATUS: A VASCULAR PLANT THAT MAKES NO ETHYLENE

B. JACKSON, lE. SUMMERS andL.A.C.l VOESENEK1

lAeR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol BS18 9AF, United Kingdom, I Department of Ecology, University of Nijmegen, Toernooiveld 1,NL-6525 ED, Nijmegen, The Netherlands

1. Introduction

The advent of inexpensive and sensitive gas chromatographs made highly replicated trace analyses of low molecular mass hydrocarbons routine widely available. Since 1959 when HueHn and Kennet [1] used gas chromatography to detect ethylene

from apples, a wealth of ethylene emission data helped dispelled misgivings about categorising ethylene as one of the principal endogenous plant hormones. Implicit in this view of ethylene as a major hormone is the assumption that ethylene production is an inextricable feature of aerobic plant metabolism. A second widespread belief is that the penultimate step in ethylene biosynthesis, catalyzed by the enzyme 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase) determines the rate of ethylene production rather than the final step in which ACC is converted to ethylene by the enzyme ACC oxidase. This view is based on the realization that while ACC oxidase is a relatively stable enzyme and seemingly present in amounts that exceed those needed to oxidise available endogenous ACC, ACC synthase transcripts, and enzyme, are unstable and thus require continued re-synthesis [2]. A third, widely-held view is that ethylene-promoted underwater elongation of submerged shoots is an obligate adaptive feature that allows aquatic and amphibious species to regain their position at or near-to the water surface and, thus, to survive submergence [3, 4, 5].

Each of the above generalizations is shown not to apply to Potamogeton pectinatus, a vigorous and widespread temperate aquatic monocot. that frequents lakes, rivers and drainage channels of several continents [6]. We show that the shoots of light or dark grown plants are unable to synthesise ethylene in well-aerated conditions even when challenged with a range of treatments that commonly promote ethylene biosynthesis in other species. Thus, P. pectinatus appears to be the first reported example of a vascular plant whose growing shoots are constitutively incapable of synthesising ethylene. Although P. pectinatus does not form ethylene, it does synthesise ACC. We show it is the absence of discernible ACC oxidase activity rather than deletion of ACC synthesis that is responsible for the lack of ethylene production. Despite the absence of ethylene, submergence in water still promotes elongation by shoots of P. pectinatus. This faster growth is explained, in part, by the promoting effects of both oxygen shortage (especially the complete absence of oxygen) and of carbon dioxide.

229 A. K. Karrellis et al. (eds.), Biology and Biotechnology o/the Plant Hormone Ethylene, 229-237. @ 1997 Kluwer Academic Publishers.

230

Pisum Potamogeton Potamogeton

~

2,000 (A) 2,000 (8) (C) 20

U) Scale expanded x100

Cl 1,500 1,500 ..lI:: 15

IISd

(5

IISd E ..e, 1,000 1,000 10 c: 0

13 ::J 500 500 5 -0 e 0. CD 0 c: 0 0 CD >. ~

W 0 10 25 0 10 25 0 10 25

Concentration of ACC applied (mmol m-3)

Figure 1. Ethylene production rates by growing shoots of (A) Pisum satiwm and (B) Potamogeton pectinatus over 15 h starting 4 h after submerging in 0, 10 or 25 mol m-3 ACC for 2 h. In the right-hand graph (C), the resuh for P. pectinatus is redrawn with the ethylene production scale expanded 100 times.

2. Dark-grown Shoots

2.1. ETHYLENE PRODUCTION (GAS CHROMATOGRAPHY)

Ethylene production by approximately 0.5 g fresh weight of tubers of P_ pee/ina/us each with a vigorously elongating shoot was indistinguishable from background levels of the gas after 4 h enclosure in 23 x 10-6 m-3 test tubes following 2.5 h prior submergence in well-aerated water. A longer accumulation time of 15 h also failed to give a clear ethylene signal (Fig- lB). When 10 mol m-3 ACC was included in the submerging solution, FID-GC again failed to detect an ethylene signal significantly above background. Only when the plants were immersed in 25 mol m-3 ACC was ethylene production detected (Fig. lB). However, the production rate was extremely slow compared to that of pea plants (Fig. lA); the scale of the graphs for P_ pee/inatus require a 100-fold expansion to reveal an effect of the highest ACC concentration (Fig­lC). The rate of production induced by 25 mol m-3 was less than that of pea plants treated with water_ When similar experiments were repeated with 0, 0.1, 1.0 or 10 mmol m-3 aminoethoxyvinylglycine (A VG), ethylene formation in peas was strongly inhibited. But, in P_ pee/inatus, there was no discernible difference between plants treated only with water and those supplied with A VG.

231

2.2. ETHYLENE PRODUCTION (LASER PHOTOACOUSTICS)

The sensitivity of gas chromatography is limited to approximately 0.01 ppm in 1 x 10-3

m-3. Sometimes, this made it difficult to discriminate with certainty between background ethylene and possible small emissions of ethylene from P. pectinatus even when the instrument when set to run at maximum sensitivity. To improve the situation we turned to laser-driven photoacoustics [7]. When tuned to the most actively absorbing laser line, this provided a working sensitivity of 50 parts per trillion in an injected volume of 1 x 10-6 m3• This is 200 times more sensitive than FID-GC. A valve switching device allowed cuvettes containing the plants (Fig. 2) to be sampled .on-line every 20 min for up to 40 h. De-rooted pea plants were handled similarly. Where required, tubers or seedlings were treated with chemicals by injecting 20 x 10-6 m3 of treatment solution through a needle fitted to each cuvette (Fig. 2). After 4 h, the solution was withdrawn.

When using the photoacoustic detector, no discernible ethylene production was seen when up to six tubers with growing shoots in glass cuvettes (Fig. 2) were connected, in line to the detector and production monitored every 20 min for 25 h (Fig.3B).

Air out

./

Needle for adding/removing solutions

Figure 2. Diagram of 30 x 10.6 m3 glass cuvette in which plant material held in place with glass ballotini, was subjected to 4-h-long submergence treatments and connected to a laser photoacoustic detector sensitive to 50 parts per trillion, v/v (equivalent to 2 nmol m·3).

In contrast, similar amounts of pea shoot tissue produced considerable quantities of ethylene especially during the first few hours following transfer to cuvettes (Fig. 3A). These initially fast rates of evolution are attributable to the minor physical damage caused by handling. A range of treatments that usually enhance ethylene production in

232

plants were applied to both species. These included submergence in water for 4 h to entrap any ethylene produced, followed by de-submergence to release the accumulated ethylene in a few minutes. A clear peak of released ethylene was seen for pea shoots (Fig. 3C) but no ethylene was released by P. pectinatus. (Fig 3D). Physical wounding by lightly crushing with a small roller, submergence in 0.1 mol m-3 IAA and increasing carbon dioxide concentrations to 10 % (vlv) all promoted ethylene formation strongly in peas but were without effect on P. pectinatus. Submerging the tissues for 4 h in 10 mol m-3 ACC raised ethylene production rates by peas from approximately 1 pmol kg-! sol to almost 2000 pmol kg-! sol 40 h after treatment. In contrast, production by P. pectinatus was raised only slightly to 10 pmol kg-! sol 40 h after treatment [9].

-":" en

":" 0) ~

'0 E Q.

... :::I c.. ... :::I 0 CI) c CI)

>. .c ... w

10 [A] 10 [B]

8 8

6 6 4 4 2 2 0 ........... _ ..... _ .......................... 0 -.~

0 5 10 15 20 0 5 10 15 20 15 [C] 15 [0]

10 10

5 5

0 ··· __ ·_··_-··'-·f·_·····_·····_·_··· __ ·· 0 .. ~ .....

0 10 20 30 40 0 10 20 30 40

Time (h)

Figure 3. Ethylene production by shoots of Pisum sativum (A & C) and Potamogeton pectinatus (B & D) measured every 20 min by a laser-driven photoacoustic detector. (A,B) Production during the first 25 h following transfer of plants to the detection system. (C,D) Production before during and after a 4-h-long submergence in water. The time axes in C & D begin 15 h after transfer of plants to the cuvettes. Arrows show the submergence period. Modified from Summers et al. [9].

233

2.3. ACC AND ACC OXIDASE

Although P. peetinatus made no ethylene except in extremely small amounts when given high concentrations of ACC, shoot tissues contained considerable quantities of endogenous ACC (25.6 mmol kg-I fresh weight). ACC was five times more concentrated in P. peetinatus than in dark-grown pea shoots (5.2 mmol kg-I). Submerging the tissue in 0.1 mol m-3 IAA for 4 h increased ACC levels in P. peetinatus four-fold.

Extracts of P. peetinatus shoots contained no detectable ACC oxidase activity even when the amount of extract included in the reaction mixture was increased four-fold (Fig. 4). In contrast, extracts from ~ shoots with a similar protein content to extracts of P. peetinatus (1-2 mg in 10-6 m-3), contained high ACC oxidase activity which increased linearly and in strict proportion as the amount of extract tested was increased (Fig. 4).

20 >

Pea r2 = 0.99 b = 3.35 ... ·s

.';::; u 11:1

CP II) 11:1 -c .;: 0 (J (J

«

~ 15 ,

II)

o:t :::t: 10 N (J

'0 E 5 :!:.

0

0

Potamogeton

Isd

r2 = 0.23 b = 0.018

... _-_ .... _ ......•.. _ ....... _._ .... _ ........... _ ...... _ ................................ .

0.2 0.4 0.6 0.8 -6 3 Volume of extract (10 m )

Figure 4. ACC oxidase activity in extracts of dark-grown shoots of Potamogeton pectinatus and Pisum sativum. The results demonstrate the absence of ACC oxidase activity in P. pectinatus. Increasing volumes of extract were incubated in the reaction mixture and the response analyzed by linear regression. Points are means of 5 replicates. Assay method based on Smith and John [11]. Figure modified from Summers et al. [9].

2.4. GROW1H RESPONSES TO ETIIYLENE, A VG AND ACC

The shoot of P. peetinatus tubers initially comprises a stem and one visible leaf. Both the stem and leaf actively elongate in moist air with the leaf being the more vigorous. Despite the absence of ethylene production, responsiveness to the gas has not been lost entirely. Five days in a flow of moist air containing 10 ppm ethylene (0.41 mmol m-3)

promoted leaf extension. Growth in air was 18.6 mm and 48.5 mm in 10 p8,m ethylene (lsd 1.35, p = 0.05). The stems did not respond. A VG (0.1 - 10 mmol m- ) and ACC (1.0 - 25 mol m-3) supplied to submerged plants for 5 d failed to influence elongation

234

rate. This is consistent with the inability of A VG to influence ethylene production and with the extremely small amounts of additional ethylene generated in response to exogenous ACC.

140

--- 120 I Isd

E E 100 --c 0 80 en c Q) 60 X (iii) Aerated Q) .... 40 0 0 ..c 20 en

0 0 2 4 6

Time (d)

Figure 5. Time course over 6 d of cumulative extension by shoots (leaf + stem) of Potamogeton pectinatus tubers submerged in the dark in water that was (i) anaerobic and unsparged, (ii) unsparged and partially depleted of oxygen by respiration, (iii) sparged with air. Modified from Summers and Jackson [12].

2 . .5. GROWTH RESPONSES TO OXYGEN DEFICIENCY AND CARBON DIOXIDE

A lack of ethylene did not prevent P. peetinatus shoots from responding positively to two other changes in the internal gaseous environment that submergence brings about. In the dark, the submerging water impedes the entry of oxygen from the atmosphere and itself becomes depleted of some or all of its dissolved oxygen. The stems of P. peetinatus respond to this deprivation by accelerating their rate of extension growth strongly. The overall effect is a lengthening of the shoot. The most vigorous growth was seen in the complete absence of oxygen, achieved using an anaerobic work station in which traces of oxygen in an the atmosphere of nitrogen and hydrogen are removed catalytically. The slowest growth was in water sparged with air. Intermediate rates of elongation were seen in water that was not sparged with air but became partially depleted of oxygen by respiration over the 6 d of the experiment ( see [12] and Fig. 5).

The stems of P. peetinatus also responded positively to elevated concentrations of carbon dioxide. This gas can be expected to accumulate in submerged tissues in the dark and also dissolve readily in the surrounding water under unsparged conditions.

235

Once more, the growth promotion centred on the stem rather than the leaf After 5 d, stem lengths in water sparged with air containing 10 % (vlv) carbon dioxide extended 21.7 mm, while those grown in water sparged with carbon dioxide-free air extended only 6.8 mm [13].

3. Light-grown Shoots

3.1. ETHYLENE PRODUCTION

Exposing plants of P. peetinatus to 24 h of light in their cuvettes did not induce ethylene production as measured by photoacoustics. When gas chromatography and head space analysis were used to examine production by excised shoots of plants grown in the glasshouse in full light for several weeks, no ethylene production above background was detected. When these shoots were treated with 1 mol m-3 ACC there was no statistically significant increase in ethylene production. Only when 10 mol m-3

ACC was given was some ethylene production recorded. However, the rate was vel)' slow (50.9 pmol kg-I S-I~ which was less than that of green pea shoots treated only with water (71.5 pmol kg-I s- ).

Ethylene production was also examined in shoots taken from mature plants growing in natural river conditions. This was the only occasion when ethylene production by untreated P. peetinatus was detected. However, rates were vel)' slow, highly variable and thus of questionable statistical significance (p = 0.05), and restricted to the leaves; stem material forming no ethylene (leaves 4.39 pmol kg-I S-I (lsd 3.46), stems 0.95 pmol kg-I S-I (lsd 0.74).

3.2. ACC AND ACC OXIDASE

Shoots of P. peetinatus taken from plants grown in the glasshouse contained ACC. Concentrations (mmol kg-I) were 2.926 ± 0.44 in leaves and 2.4 ± 0.46 (n = 6) in stems. These values were appreciably smaller than those of dark-grown plants (25.5 mmol kg-I) and half those of shoots from light-grown peas seedlings (4.8 ± 0.58). ACC oxidase activity in extracts of P. peetinatus shoots containing approximately 2.4 mg in 1 x 10-6 m3 was undetectable when compared with heat-denatured counterparts. Extracts of light-grown pea shoots were highly active and gave dose dependent release of ethylene from ACC similar to that shown previously for dark-grown plants in Figure 3.

4. Concluding Remarks

Analyses by FID-GC and laser photoacoustics failed to detect ethylene evolution from dark-grown and vigorously elongating shoots borne on overwintering tubers of P. peetinatus. This was the case even when 4 h worth of possible ethylene production was concentrated within the shoot by submergence in water and then released within a few minutes by de-submergence. Thus, at this stage in development, P. peetinatus appears to be a vascular plant without ethylene. Equally surprising was the failure of several treatments to elicit a response from Potamogeton that invariably stimulate ethylene biosynthesis when applied to other species. Physical wounding, IAA and carbon dioxide were each without effect. Similarly, treatments with A VG or reduced

236

concentrations of oxygen (5 %) that slow ethylene fonnation in pea shoots did not change the strength of the ethylene signal from P. pectinatus, which remained indistinguishable from background. Treatment with ACC did result in some ethylene fonnation but the amounts were extremely small compared to those released by pea shoots supplied with ACC. At no time did ACC treatment to Potamogeton, even at the highest concentration (25 mol m-3) increase rates ethylene production above the level of peas given only water. This barely detectable response to ACC and the presence of large amounts of endogenous ACC that are naturally present in dark-grown P. pectinatus suggest that the block to ethylene fonnation is at the ACC oxidase step. This was continned by the absence of any ACC oxidase activity in extracts with a wide range of protein content. That the earlier steps in the conventional biosynthetic pathway still operate in this species seems clear from the presence of so much natural ACC and from the large increase in ACC levels seen in IAA-treated plants. IAA is known to stimulate the ACC synthase step in other species and it also appears to have this effect in P. pectinatus, although, of course, without an accompanying production of ethylene.

The possibility that the absence of ethylene production is limited to dark-grown plants arising from tubers was eliminated by results showing that illumination for 24 h, which results in some greening of the shoots, did not induce ethylene fonnation. Shoots from more mature plants grown for several weeks also made no ethylene. Furthennore, extracts from these plants were without any ACC oxidase activity when tested in a system where high activity was found in comparable extracts from light­grown peas. Only in shoots from mature plants taken from the river was evidence of an extremely small amount of natural ethylene production obtained. This was by leaf rather than stem tissue but was highly variable between samples and not statistically significant. Thus, we doubt this was true plant-produced ethylene. P. pectinatus tissue in the wild is known to be heavily contaminated by endophytic bacteria [14] and this may have been the source of the trace amounts of ethylene we sometimes detected from our unsterilized material.

Although P. pectinatus does not make ethylene it can respond to the gas. Leaves in the dark elongated more quickly when given ethylene, although the stems were unresponsive. This might conceivably have some adaptive value in sustaining fast underwater elongation in water containing significant amounts of dissolved ethylene generated by other sources such as other macrophytes, bacteria etc.

The above findings not only mark-out P. pectinatus as being highly unusual because it lacks a major plant honnone but because unlike other water plants it does not place reliance on ethylene-mediated fast underwater shoot extension for survival [5, 15]. Clearly, ethylene-mediated depth accommodation is not available to P. pectinatus. We have identified two features that may compensate for this. Firstly, the stem of the shoot responds to partial oxygen shortage by elongating more rapidly. Since submergence can restrict the availability of oxygen [16]), this deficiency probably accelerates shoot extension in natural situations. The response to partial oxygen shortage is an extension of the effect of complete anoxia, which promotes stem elongation to an even faster rate [l2]. This has relevance to the early growth in spring when the tubers begin to elongate whilst buried in the substrate of lakes, pools and rivers which is often anaerobic. A second feature that may enhance underwater shoot elongation in the absence of ethylene is carbon dioxide. This gas can accumulate in submerged tissues and in the water, especially in the dark [17]. Our results indicate that stem extension by P. pectinatus can be substantially enhanced by supplying carbon dioxide and slowed by sparging the water with air from which carbon dioxide has been removed.

237

5. References

1. Huelin, F.E. and Kennet, B.H. (1959) Nature of olefines produced by apples, Nature 184, 996. 2. Nakagawa, N., Mori, H., Yamazaki, K. and Imaseki, H. (1991) Cloning ofa complimentary DNA for

auxin-induced 1-aminocyclopropane-l-carboxylate synthase and differential expression of genes by auxin and wounding, Plant and Cell PhysioL, 32, 1291-1298.

3. Musgrave, A, Iackson, M.B., and Ling, E. (1972) CaIlitriche stem elongation is controlled by ethylene and gibberellin, Nature New Biology, 238, 93-96.

4. Ridge, I. (1987) Ethylene and growth control in amphibious plants, in RM.M. Crawford (ed.), Plant Lifo in Aquatic and Amphibious Habitats, Blackwell, Oxford, pp. 53-77.

5. Voesenek, LAC.J., Van Der Sman, AI.M., Harren, F.H., and Blom, C.W.P.M. (1992) An amalgamation between honnone physiology and plant ecology: a review on flooding resistance and ethylene, J. Plant Growth Regul., 11, 171-178.

6. Yeo, RR (1965) Life history of sago pondweed, Weeds, 13, 314-321. 7. Harren, F.I.M., Bijnen, F.C.G., Reuss, I., Voesenek, LAC.I., and Blom C.W.P.M. (1990) lntercavity

photoacoustic measurements with a CO2 wave guide laser; detection of C2Rt as a trace gas at ppt level, Applied Physics, 850,137-144.

8. Lizada, M.C.C. and Yang, S.F. (1979) A simple and sensitive assay for l-aminocyclopropane-l­carboxylic acid, Annals Biochem., 100, 140-145.

9. Sununers, I.E., Voesenek, L.AC.J., Blom, C.W.P.M., Lewis, M.I., and Iackson, M.B. (1996) Potamogeton pectinatus is constitutively incapable of synthesizing ethylene and lacks 1-aminocyclopropane-l-carboxylic acid oxidase, PlantPhysioL 111 (in press).

10. Hall, K.C., Pearce, D.M.E., and Iackson, M.B. (1989) A simplified method for detennining 1-aminocyc\opropane-l-carboxylic acid (ACC) in plant tissue using a mass selective detector, Plant Growth ReguL 8, 297-307.

11. Smith, I.J. and Iohn, P. (1993). Maximising the activity of the ethylene forming enzyme, in J.C. Pech, A Latcht! and C. Balage (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, Kluwer Academic Publishers, Dordrecht, pp. 33-38.

12. Sununers, I.E. and Jackson, M.B. (1994) Anaerobic conditions strongly promoteextension by an aquatic monocot (Potamogeton pectinatus L.), J. Exp. Bot. 45, 1309-1318.

13. Sununers, J.E. and Jackson, M.B. (1996) Anaerobic promotion of stem extension in Potamogeton pectinatus. Roles for carbon dioxide, acidification and hormones, Physiol. Plantar. 96, 615-622.

14. Ailstock, D.O., Fleming, W.I., and Cooke, T.I. (1991) The characterization of axenic culture systems suitable for plant propagation and experimental studies of the submersed aquatic angiosperm Potamogeton pectinatus (sago pondweed), Estuaries, 14, 57-64.

15. Jackson, M.B. (1982) Ethylene as a growth promoting hormone, in P.F. Wareing (ed), Plant Growth Substances 1982, Academic Press, London, pp. 291-301.

16. Waters, I., Annstrong, W., Thompson, C.J., Setter, T.L., Adkins, S., Gibbs, I., and Greenway, H. (1989) Diurnal changes in oxygen transport and ethanol metabolism in roots of submerged and non­submerged rice seedlings, New Phytologist, 113, 439-451.

17. Rose-John, S., and Kende, H. (1985) Short-tenn growth responses of deep-water rice to submergence and ethylene, Plant Science, 38, 129-134.

HYPOXIA AND FRUIT RIPENING

T. SOLOMOS i AND A.K. KANELLIS2 IDepartment of Horticulture & Landscape Architecture, University of Maryland, College Park, MD 20741-5611 USA, 2Institute of Viticulture Vegetable Crops and Floriculture, Agricultural Research Foundation, PO Box 1841, GR-711 10 Heraklion, Crete, Greece, 3Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology - Hellas, PO Box 1527, GR-711 10 Heraklion, Crete, Greece

1. Introduction

Storage of detached horticultural crops under low O2 and/or high CO2 greatly extends their commercial life. Although the commercial application of controlled atmosphere (CA) storage began some 60 years ago, the biochemical and molecular aspects underlying the action of low O2 on the senescence of detached plant organs remain a mystery [11, 17, 29]. There is compelling experimental evidence indicating that the retarding effects of hypoxia on fruit ripening involve the inhibition of ~Rt action because the inclusion of relatively high levels of the gas in low O2 treatments fails to substantially alleviate the retarding effects oflow O2 on fruit ripening [11, 12-14, 19]. The retardation of plant senescence, however, is not the sole metabolic manifestation of hypoxia. For instance, hypoxic preconditioning enhances both the synthesis of anoxic proteins and the ability of tissues to survive the subsequent imposition of anoxia [I, 3, 6, 7, 26, El-Mir, Gerasopoulos, Metzidakis, Kanellis, unpublished results). In potato tubers, hypoxia greatly inhibits the accumulation of sugars during storage at 1°C and suppresses the induction of acid invertase [35]. In addition, low O2 inhibits respiration in such tissues as potato tubers and sweet potato roots, where ethylene is not involved [21, 35). The overt effects of hypoxia on climacteric-type fruits include a diminution in respiration, a delay in the onset of the climacteric rise in C2Rt evolution and a decrease in the rate of ripening of fruits whose ripening has been initiated either naturally or by brief exposure to exogenous ~Rt [8, 9, 12-17,29]. In this presentation we shall attempt to address the question of the action of low ~ on fruit ripening and on metabolic processes where the inhibition of ~Rt action is not at issue.

2. Effect of Low 0 1 on Respiration

In the past, the effects of low O2 and/or high CO2 were mainly restricted to establishing appropriate CA environments for individual crops. Since CA atmospheres always reduced respiration. it was assumed that this physiological response played a crucial role in extending the storage life offruits [see 29].

Previous work has shown that the decrease in respiration with decreasing external O2 concentration is usually biphasic in nature, in that it includes an initial gradual

239

A. K. Kanellis et al. (etis.), Biology and Biotechnology o/the Plant Hormone Ethylene, 239-252. © 1997 Kluwer Academic Publishers.

240

decrease at relatively high O2 concentrations, followed by a rapid decline as the oxygen concentration approaches zero [2, 21, 22, 27, 33]. This has been attributed in turn to the existence of a "regulatory" protein which exerts a feedback inhibitory effect on the initial stages of glucose oxidation (2, 27, 33], to the existence of two terminal oxidases with different affinities for O2 [21], and to resistance to O2 diffusion [4].

Table 1. Preclimacteric CO2 minimum of "Gala" apples

Oxygen %

21 8 6 4 3 2

Rate of respiration J.lIC02 g.t h·t

1.83 1.41 1.36 1.05 0.85 0.52

% of Air

22.95 25.68 42.62 53.55 71.58

In the case of preciimacteric "Gala" apples, the rate of respiration at the preciimacteric minimum decreases when the external O2 concentration falls below the range of between 8% and 6% (Table 1). This concentration of O2 is not expected to restrict any of the mitochondrial terminal oxidases, since the calculated internal O2

concentration is too high to inhibit either of the mitochondrial terminal oxidases [28]. Because preciimacteric fruits evolve small amounts of C2~' and because low O2

inhibits C2~ action, we used peeled sweet potato roots to calculate an apparent Km for O2 of respiration. To this end, the diffusivity of O2 through the flesh was first determined and the root was assumed to consist of a solid cylinder at the center, having a radius of 0.1 cm, surrounded by 29 layers of hollow cylinders, each having a thickness of 0.1 cm [34]. Fick's diffusion equation was solved for solid and hollow cylinders [5], and an apparent Km for O2 of 1.31% was calculated. The value of the resultant Km for O2, along with the observed V max, was used in a nonlinear regression analysis [23], to calculate the rate of CO2 evolution as a function of O2 concentration. It may be seen from the data presented in Figure 1 that the calculated and observed rates are very similar. Furthermore, the model predicted that the apparent Km for O2 of an enzyme with an affinity for O2 similar to that of cytochrome oxidase (0.0037%) would be 0.144%, which is much lower than 1.31%. It is obvious that the diminution of respiration of intact roots with decreasing O2 concentration may not be ascribed to the restriction of cytochrome oxidase. Moreover, a restriction of cytochrome oxidase may be expected to result in the accumulation of glycolytic products, i.e. ethanol and lactic acid. In "Gala" apples kept under 1.5% O2 for extensive periods of time, there is no accumulation of ethanol [28]. In fresh sweet potato slices, where cytochrome oxidase is the main terminal electron acceptor, a 30% decrease in the rate of O2 uptake leads to an increase in pyruvate and lactate (Table 2), whereas a similar decrease in the rate of CO2 output in intact roots subjected to 10% O2 does not produce an increase in lactate (27). In addition, the treatment of sweet potato roots with 10 ,.u rl C2~ in air

241

and 3% O2 results in a 40% decrease by low O2 of the respiratory peak observed in air (Fig. 2a). This is also reflected in changes in pyruvate concentration (Fig. 2b). In short, the available experimental data indicate that the restriction of the low affinity "oxidase" leads to a feedback inhibition of the rate of pyruvate synthesis, implying a decrease in the glycolytic flux. In animal tissues subjected to frequent changes from normoxia to anoxia, there is a decrease through phosphorylation in the activities of key glycolytic regulatory enzymes [32]. In carnation flowers, hypoxia decreases the activity of pyruvate kinase [3]. Since a decrease in respiration is expected to decrease the rate of ATP biosynthesis, and since its steady-state level does not decrease appreciably in either hypoxic carnation flowers or corn seedlings [26, 30], its turnover should be restricted in hypoxic tissues. It thus appears that hypoxia induces a metabolic depression, thereby decreasing the demand for biological energy.

25 .------------------,

21

OBSERVED

~ .. --... ---.-. ---------

~ 17 (!J -N

o o ~ 13

9

5 2 4 6 8 1 0 12 14 16 18 20 22

% OXYGEN

Figure 1. Six sweet potato roots were used for each oxygen concentration (1, 4, 6, 8, 10, and 21%). The rate ofCOz output was monitored in a flow-through system every two hours. A non-linear regression analysis was used for the calculated values.

242

165

132

~ 99 '" 0

U 66 :t

33

0 0

Table 2. Effect of oxygen concentration on pyruvate and lactate accumulation

No. of O2 concentration Time (min) Pyruvate Lactate experiments (JIM) (nmolesJg) (nmolesJg)

2

250.0 10.5

250.0 10.5

60 60

90 90

350 522

380 578

47 356

58 642

The slices were kept under the indicated O2 concentration in ImM phosphate buffer pH 6.5

6

165

132 0 ~ 99 2 [ 66

33

0

10 14 20 24 28 32 0 16

Hoursin~~ HoursinC2~

Figure 2. Rate of CO2 output (A) and pyruvate content (B). The experimental set-up is similar to that of Figure 1. Two treatments were used: air plus ethylene and 3% O2 plus ethylene. At the times indicated tissue plugs were removed from each individual root for pyruvate determination.

AIR

34

In order to gain a better insight into the regulation of respiratory metabolism during hypoxia, we have recently utilized the well characterized anaerobic adaptation system found in yeast (Saccharomyces serevisiae), in order to isolate putative anaerobic plant genes. [36]. Part of the response of yeast to anaerobiosis is regulated by heme and mediated by protein factors that can induce or repress transcription [36]. The hap locus has been identified as the main regulatory complex of that response [20]. We recently isolated a plant gene from A. thaliana that shows 80% identity with the yeast gene and can compliment hap5 deficient yeast strains (Makris, Aggelis, Kanellis, unpublished results). The HAP5 protein is a recently characterized component of a conserved trimeric complex (HAP2/3/5). This complex is heme regulated, binds to

243

"Upstream Activated Sequence 2 (UAS2)" on the CCAAT motif and is responsible for the induction of respiratory genes in yeast [20].

3. Effect of Hypoxia on the Onset of the Climacteric Rise in C1H4 Evolution

It is well known that the effectiveness of low O2 in delaying fruit ripening increases with decreasing O2 concentration as long as the O2 partial pressure does not drop below the level which engenders anaerobic fermentation [8, 29]. In addition, the rapid establishment of an appropriate CA environment is much more effective in retarding fruit ripening than it is when it is delayed.

Table 3. Concentration of ACC in "Gala" apples after four months storage at the indicated partial pressures

Oxygen % ACC content Ethylene (nJ g.1 h· l )

21 18.64 9.34 8 17.90 9.94 6 8.53 8.25 4 3.75 4.36 3 0.36 0.21 2 0.24 0.10

The data concerning the range of O2 pressure that can delay the onset of the climacteric rise in C2~ evolution are very limited. In the case of "Gala" apples, for O2 to retard the onset of the C2~ increase its partial pressure must be decreased below 8% [28]. The results also show, as expected, that the retarding effect of low O2 on the initiation of fruit ripening is inversely related to the partial pressure of O2. In short, the retardation of the onset of the climacteric rise in C2~ evolution exhibits saturation kinetics. Similar results were obtained with carnation flowers [30). Since preclimacteric apples produce small amounts of C2~, it may be suggested that in preclimacteric fruits low O2 inhibits the action of C2~, thereby retarding the onset of the autocatalytic rise in C2~ evolution. A number of observations indicate, however, that the retarding effects of low O2 on the onset of ripening may not be ascribed to its inhibitory effects on C2~ action alone. In "Gala" apples kept under 3% O2 the onset of the climacteric rise in 1987 and 1988 differed by more than 100 days. Yet the rate of preclimacteric Cz~ evolution was similar in both years [28]. In carnation flowers, hypoxia extends their vase life by 2-3 fold over those treated with inhibitors of C2~ action [30]. Neither can the delaying effects of hypoxia be ascribed to the inhibition of the induction of ACC-oxidase because the rate of C2~ evolution is closely related to the internal ACC concentration (Table 3), indicating a suppression of ACC-synthase. Furthermore, once the induction of C2~ has been initiated, imposition of hypoxia does

244

not inhibit its eventual increase. In contrast, the application of low O2 at the preclimacteric stage severely suppresses the climacteric rise in C2~. In "Gala" apples, the imposition of 1.5% O2 immediately after harvest totally inhibits the rise in C2~ evolution for 215 days (Fig. 3). On the other hand, if the fruits are transferred to 1.5% O2 after 37 or 72 days, the rate of ~~ evolution decreases initially but with time it increases substantially (Fig. 3). This is also reflected in the accumulation of ACC­oxidase protein (Fig. 4). It thus appears that in preclimacteric fruits the retardation of the onset of the induction of C2~ biosynthesis may not be ascribed to the inhibition of C2~ action alone but rather to the suppression of developmentally regulated genes which precede the induction of ~~ biosynthesis and whose expression is necessary for the induction of ACC- synthase and oxidase, hence ripening.

5

4

~ 3

u 2 'i:l

1

o

.,... AIR

~

I 39 Days inAIR·1.5% 0,

I 75 Days in AIR·1.5% 0, !'~ \ 4;: ttl of;:

.Jl ,:

,f *,/ .

." : ~ , , , , .. :. : ,', . ., : ,

... 1, . , \,

3 12 29 44 60 86 109 137 171 209 Days in Storage

Figure 3. Ethylene evolution of "Gala" apples stored at 1°C in a f1ow­through system. One group of apples was kept in air continuously. The second and third groups were transferred to 1.5% O2 after 39 and 75 days postharvest. The last group was kept under 1.5% ~ continuously.

4. Effect of Hypoxia on the Rate of Ripening

As has already been pointed out, hypoxia decreases the rate of ripening of fruits where ripening has been initiated either naturally or by brief exposure to ~~. In these fruits a range of O2 concentrations between 2.5 - 5.5% prevents the accumulation of cellulase and polygalacturonase proteins and the rise in activity of cellulase and PG isoforms [15]. The suppressive effects of O2 on the cellulase protein are also reflected in the accumulation of its mRNA [15]. Concentrations of O2 above 7.5% exert no inhibitory

245

effect on the protein, activity and mRNA accumulation of cellulase and PG [15]. In short, the retarding effects of hypoxia on the rate of ripening are also saturable. Moreover, it should also be noted that the magnitude of the suppressive effects of hypoxia on the above cell wall hydro lases are inversely related to the concentration of O2• In addition, it was found that the same range of O2 concentrations which inhibited the synthesis of enzymes attending fruit ripening also induced the synthesis of the anoxic isoenzymes of alcohol dehydrogenase (ADH) [9, 15, 16]. Furthennore in carnation flowers and avocado fruits, hypoxia enhances the activity of ADH [3, 20] and increases the both the rate of ethanol synthesis and longevity over those of the control flowers and avocados when the flowers and fruits are transferred to N2 [3, 20].

1 2 '3 4 Figure 4. Protein blot of ACC-oxidase of "Gala" apples after 120 days post harvest. Lanes: I-air, 2-fruits were transferred to 1.5% ~ after 7S days in air, 3-ftuits were transferred to 1.5% O2 after 39 days in air, 4-ftuits were transferred to 1.5% O2 after 2 days post harvest.

Transferring bananas to 2.5% O2 after they had been kept in 10 1.1.1.1-1 C2lit for 24 hours at 18°C decreased the sugar accumulation by about 40%, coincident with an inhibition of the increase in sucrose phosphate synthase and acid invertase (Fig. 5a,c).

246

5

o

CzH.-A1R SPS

I rn 10 9 INVERTASE CzH.-A1R

f 8 [[J j 7 6 CzH.-LOWo.

CzH.-LOWo. ~ 5 \ \ o

CC LOW 0.

I III \ 1 4 6

Days 8 20

40 SSACTIVITY

(]] ~ 30 fil CzH.-A1R

-a 20 §. t

10

o _ L-

o 4

! 4 3

~ 2 1 0

C,H.-LOWo.

6 Days

CONT LOW 0,

0 1 4 6 Days

CONT LOW 0.

\

8 20

Figure 5. Effect of 2.5% O2 on the activities of sucrose phosphate synthase [AJ, invertase [BJ and sucrose synthase [C]. Preclimacteric banana fruits were kept in ethylene (10111.1-1) for 24h in air, then transferred either to 2.5% O2 or air. A third group of fruits was kept continuously under 2.5% O2

without ethylene.

8 20

On the other hand, 2_5% O2 induced a 2.5-fold increase in sucrose synthase, an anoxic protein (Fig- 5c). In preclimacteric bananas, low O2 strongly inhibits the increases in sugars, sucrose phosphate synthase and acid invertase, while increasing the activity of extractable sucrose synthase (Figs- 5a,b). In short, in bananas, as in avocados, hypoxia suppresses the induction of enzymes associated with normal ripening while inducing the synthesis of anoxic proteins.

In low O2 stressed fruits, induction of specific protein synthesis and gene expression also takes place. Alcohol dehydrogenase, lactate dehydrogenase (LDll) and glucose phosphate isomerase (GPI) isoenzymes are expressed in low O2 in both preclimacteric and initiated avocado fruit [8, 15]. This increase in ADH protein is reflected in its mRNA levels [8, 15]_ Recently, we have concentrated on isolating low 02-regulated genes from tomato fruits. We have applied differential display on RNA

247

isolated from tomato fruits subjected to 0%, 3% and 21% O2 for various periods of time. We have isolated some clones, which are expressed in both hypoxia and anoxia, only in hypoxia or anoxia or air only. The analysis and further characterization of these clones are under investigation (Aggelis and Kanellis, unpublished data).

5. Effects of Hypoxia on the Responses of Cold Stress in Potato Tubers

Storage of potatoes at chilling temperature is attended by extensive physiological and biochemical changes [9, 31]. In addition, cold stress does not engender ~~ biosynthesis in tubers. It was, thus, considered a convenient experimental material for the study of the effects of hypoxia on a tissue where the inhibition of ~~ action is not at issue. We observed that 1.5% O2 strongly inhibited the increase in respiration, sugar and chiorogenic acid concentrations. More importantly, 1.5% O2 suppress the cold-induced acid invertase, alternative oxidase, and three isoforms of amylase (data not shown). The suppression of invertase was also reflected in the accumulation of its mRNA (Fig. 6). On the other hand. low O2 has no effect on starch phosphorylase, whose activity is not influenced by temperature [17]. It thus appears that hypoxia inhibits the expression of new gene products but has no effect on pre-existing proteins.

6. Effects of Hypoxia on Gene Expression

Early work with com seedlings showed that anoxia induces dramatic changes in protein synthesis, in that the synthesis of pre-existing proteins is drastically reduced and a number of new polypeptides, anoxic proteins, are induced [25]. In vitro translation of the mRNA showed that the mRNA of pre-existing proteins were not destroyed, although their translation had ceased [24, 25]. Anoxic com roots, however, die within about 90h, whereas the life of hypoxic fruits can be extended for up to a year or even longer.

It has already been pointed out that in avocado and banana fruit and cut carnation flowers, hypoxia on the one hand suppresses the induction of enzymes associated with normal ripening and on the other induces the synthesis of anoxic enzymes such as ADH and sucrose synthase [3, 11-16], (Fig. 5). The effects of hypoxia on the synthesis of pre-existing proteins have not been investigated in fruits. Does hypoxia inhibit the translation of pre-existing mRNA, as does anoxia in com seedlings [25]? In order to investigate this aspect, we carried out a two-dimensional analysis of total proteins of both preclimacteric and propylene-treated avocado fruits which had subsequently been transferred to 0%, 1%, 3%, 5%, 10% and 21% O2• It was shown that in both preclimacteric and propylene-initiated avocado fruits, low O2 regimes induced the appearance of new in addition to increasing the staining intensity of certain pre­existing polypeptides in air [15]. In contrast, suppression of polypeptides was noticed only in avocado whose ripening had been initiated and held in low O2• Thus, it appears that low O2 induces the appearance of new polypeptides in both preclimacteric

248

and initiated avocado fruits, whereas it suppresses only the synthesis of de novo synthesized ripening polypeptides [15].

lIa." II T 1°(,) IU 0: 1(; ) air

IU air

7

I air

II .'41 \U I \0 I

I.S air air 1.5 air air 1.5

Figure 6. RNA blot of potato acid invertase. The tubers were kept in air at 10°C, and 1°C in 1.5% O2 at 1 °C.

Similar results were obtained at the RNA expression level. One- and two­dimensional gel electrophoresis of in vitro translation products of poly (At RNA isolated from both preclimacteric and initiated avocado fruits subjected to low O2 stress revealed that low O2 causes no elimination of pre-existing mRNA in air in both type fruits, whereas it exhibits strong suppressive effects on the synthesis of mRNA associated with avocado ripening. On the other hand, the inductive effect of low O2

was observed in both types offruits held in 0-5% O2 [15, 16, Loulakakis and Kanellis; manuscript in preparation]. In order to characterize further protein synthesis and gene expression of specific proteins synthesized during ripening under low O2 environment, we have performed protein and RNA blots using total protein and RNA extracts isolated from preclimacteric and propylene-initiated avocado fruits which were held in zero, 1%,3%,5%, 10% and 21% O2. The results showed that the accumulation of the

249

cellulase protein and its mRNA in preclimacteric fruit was irrespective of O2 tensions [15, 16]. Thus, a 48h treatment with low O2 did not prevent the accumulation of pre­existing proteins and mRNA, which is consistent with the steady-state amount of poly (A) RNA. In initiated fruits, however, low O2 (0-5%) prevented the accumulation of cellulase protein and its poly (A) RNA. Therefore, it seems that low O2 suppresses a further accumulation of cellulase protein and poly (A) RNA which is associated with ripening and is C2~ regulated [8,15].

The data presented earlier indicate that the effects of low O2 on respiration, induction of the onset of the climacteric rise, and on the rate of ripening show saturation kinetics, indicating that there may be a mechanism that senses the level of O2, (an "oxygen sensor?") which in turn initiates the induction, or suppression, of a number of genes.

It is perplexing that the extension of the storage life of fruits and vegetables under a CA environment is always associated with a decrease in respiration entailing a decrease in the rate of A TP biosynthesis. A limited number of A TP determinations indicates that its steady-state concentration in hypoxic tissues may not be very different from that in the normoxic ones [26, 30]. If this is the case, then the rate of ATP utilization must decrease appreciably in hypoxic detached plant organs. Since protein biosynthesis is considered an extensive sink of A TP utilization, it is tempting to suggest that hypoxia decreases the rate of protein synthesis. The fact that the profiles of soluble proteins do not show a great many changes in apples which were kept under 1.5% O2 for four months indicates that the turnover of the most predominant proteins that are visualized with the usual staining techniques may be decreased by hypoxia. Does, then, hypoxia induce a metabolic depression akin to that observed in animal tissues and yeast [32, 36], thereby decreasing the rate of fruit development, ultimately resulting in delayed ripening?

7. Effects of Hypoxia on Post CA Fruit Ripening

The results with avocado fruits show that the suppressive effects of low O2 on the level of protein of cellulase, polygalacturonase and cellulase mRNA are inversely related to the O2 concentration: the lower the O2 pressure, the higher its suppressive effects [14, 15]. Further, when the fruits were returned to air from hypoxia the commencement of protein and mRNA synthesis was directly proportional to the O2 pressure, indicating that the effects of hypoxia continues after the fruits are returned to air [8, 13-15]. Similarly, the staining intensity of the anoxic ADH isoforms increased with decreasing O2 pressure, while the rate of their subsequent disappearance in air was inversely related to the O2 pressure: the lower the 0 2 pressure, the longer the time needed for their elimination [8, 14, 15, 20].

In "Gala" apples which have been kept for long periods under low O2 the rate of C2~ evolution when the fruits were transferred back to air is greatly depressed (Fig. 7) and in extreme cases the fruits fail to produce significant amounts of C2~. The results, thus, indicate that the suppressive effects of low O2 on fruit ripening remain even when the fruits are returned to air. This aspect is an important consideration

250

because prolonged storage under low O2 may prevent the proper development of the quality characteristics of the particular fruits.

0,50

0,40

0,30

~ 0,20 ~ u

-= AIR 0,10

I 0,00

0 8 15 21 31 38 49 64 79

Days Post Storage

Figure 7. Ethylene evolution of "Gala" apples. Fruits were stored for 215 days under 1.5% O2, then transferred to air.

8. References

1. Andrews, D.L., Drew, M.C., Johnson, J.R., and Cobb, B.G. (1994) The response of maize seedlings of different age to hypoxic and anoxic stress. Changes in induction of Adhl mRNA, ADH activity and survival of anoxia, Plant Physiol. 105, 53-60.

2. Blackman, F.F. (1954) Analytic Studies in plant respiration, Cambridge University Press, London, New York.

3. Chen, X. and Solomos, T. (1996) Effects of hypoxia on cut carnation flowers (Dianthus carophyllus L.): longevity, ability to survive under anoxia, and activities of alcohol dehydrogenase and pyruvate kinase, Postharv. BiOI. and Technol. 7, 317-329.

4. Chevillote, P. 1973. Relation between the reaction of cytochrome oxidase - oxygen and oxygen uptake of cells in vivo, Theor. BioI. 39, 277-295.

5. Crank, 1. (1970) The mathematics of diffUsion, Claredon, Oxford. 6. Drew, M.C., Saglio, P.H., and Pradet, A (1985) Larger adenylate energy charge and ATP/ADP

ratios in aerenchymous roots of zea mays in anaerobic media as a consequence of improved oxygen transport,Planta 165, 51-58.

7. E1-Mir, M.H. (1996) Enhancement oftolerance of avocado fiuits to extreme low oxygen atmospheres following hypoxic acclimation, M.S. Thesis, Mediterranean Agronomic Institute of Chania, Chania, Greece.

251

8. Fidler, J.C., Wilkinson, B.G., Edney, KL., and Sharples, R.O. (1973) The biology of apple and pear storage. Research Rev. No.3. Commonwealth Bureau of Agricultural and Plant Crops, East Mailing, Maidstone, Kent.

9. Hassan, M.M. (1993) Some biochemical and molecular aspects oflow oxygen action on fiuit ripening, M.S. Thesis, Mediterranean Agronomic Institute ofChania, Chania, Greece.

10. Isherwood, FA (1973) Starch-sugar interconversion in Solanum tuberosum, Phytochem. 12,2579-2591.

11. Kader, AA (1986) Biochemical and physiological basis for effects of controlled and modified atmospheres on fiuits and vegetables, Food Technol. 40,99-104.

12. Kanellis, AK, Solomos, T., and Mattoo, AK (1989) Changes in sugars, enzymatic activities and phosphatase isoenzyme profiles of bananas ripened in air or stored in 2.5% O2 with and without ethylene, Plant Physiol. 90,251-258.

13. Kanellis, AK, Solomos, T., and Mattoo, AK (1989) Hydrolytic enzyme activities and protein pattern of avocado fiuit ripened in air and in low oxygen with and without ethylene, Plant Physiol. 90, 259-266.

14. Kanellis, AK, Solomos, T., Mehta, AM., and Mattoo, AK (1989) Decreased cellulase activity in avocado fiuit subjected to 2.5% O2 correlates with lower cellulase protein and gene transcript levels, Plant Cell Physiol. 30, 829-834

15. Kanellis, AK, Solomos, T., and Roubelakis-Angelakis, KA (1991) Suppression of cellulase and polygalactorunase and induction of alcohol dehydrogenase isoenzymes in avocado fiuit mesocarp subjected to low oxygen stress, Plant Physiol. 96, 269-274.

16. Kanellis, AK, Loulakakis, KA, Hassan, M., and Roubelakis-Angelakis, KA (1993) Biochemical and molecular aspects of the low oxygen action on fiuit ripening, in C. J. Pech, A Latche and C. Balague (eds.), Cellular and Molecular Aspects of Biosynthesis and Action of the Plant Hormone Ethylene, Kluwer Academic Publishers pp. 117-122.

17. Kanellis, AK (1994) Oxygen regulation of protein synthesis and gene expression in ripening fiuits: Future outlook, Proc. Int. Symp. on Postharvest Treatment of Fruits and Vegetables, Oosterbeek, Netherlands, Oct. 19-22, 1994.

18. Kennedy, M.GA and Isherwood, FA (1975) Activity of phosphorylase in Solanum tuberosum during low temperature storage, Biochem. 14, 667-670.

19. Liu, F.W. and Long-Jum, C. (1986) Responses of daminozide-sprayed McIntosh apples to various concentrations of oxygen and ethylene simulated CA storage, J. Amer. Soc. Hort.. Sci. 111, 400-403.

20. McNabb, D.S., Xing, Y., and Guarente, L. (1995) Cloning of yeast HAP5: a novel subunit of a heterotrirneric complex required for CCAAT binding, Genes & Dev. 9, 47-58.

21. Mapson, L.W. and Burton, W.G. (1962) The terminal oxidases of potato tuber, Biochem. J. 82, 19-25.

22. Mapson, L. W. and Robinson, J.E. (1966) Relations between oxygen tention, biosynthesis of ethylene, respiration and ripening changes in banana fiuit, J. Food Technol. 1,215-225.

23. Roberts, D. V. (1977) Enzyme kinetics, Cambridge University Press, Cambridge, London, New York. 24. Sachs M.M. (1991) Molcular responses to anoxic stress in maize, In M.B. Jackson, D.D. Davies, and

H.Lammbers (eds.), Plant Life Under Oxygen Deprivation, Academic Pub!., The Hague. ppI29-139. 25. Sachs, M.M., Freeling, M.M., and Okimoto, R. (1980) The anaerobic proteins of maize, Cell 20, 761-

767. 26. Saglio, P.H., Drew, M.C., and Pradet, A (1988) Metabolic acclimation to anoxia induced by low (2-4

kPa partial pressure) oxygen pretreatments (hypoxia) in root tips of Zea mays, Plant Physiol. 86, 61-66.

27. Solomos, T. (1982) Effects of low O2 concentration on fiuit respiration: Nature of respiratory diminution, in D.G. Richardson and M. Meheriuk (eds.) Controlled Atmospheres for Storage and Transport of Perishable Agricultural Commodities, pp. 161-170.

28. Solomos, T. (1993) Effects of hypoxia on "Gala" apple ripening, in Proc. of Sixth International Atmosphere Research Conforence, Cornell University, Ithaca, New York. Vo!. 1 :20-30.

29. Solomos, T. (1994) Some biological and physical principles underlying modified atmosphere packaging, in R.C. Wiley (ed.), Minimally Processed Refrigerated Fruit and Vegetables, Chapman and Hall, New York, London, pp. 183-225.

30. Solomos, T. and Gross, KC. (1996) The effects of hypoxia on respiration and the onset of senescence in cut carnation flowers {Dianthus caryophyllus), Postharvest BioI. Techn. (in press).

252

31. Sowokinos, J. (1990) Stress-induced alterations in carbohydrate metabolism, in M.E. Vayda, and W.D. Park (eds.), Molecular and Cellular Biology o/the Potato, CAB. International, Wallington, u.K. pp. 137-158.

32. Storey, K.B., and Storey, J.M. (1990) Metabolic rate of depression and biochemical adaptation in anaerobiosis, hibernation and estivation, Quat. Rev. Bioi. 65, 145-175.

33. Tucker, M.L. and Laties, 0.0. (1985) The dual role of oxygen in avocado fiuit respiration: Kinetic analysis and computer modeling of diffusion-affected respiratory kinetics, Plant Cell Environ. 8, 117-127.

34. Zhou, D. (1989) Oxygen and ethylene interaction on respiration of sweet potato roots. M.S. Thesis, University of Maryland, College Park.

35. Zhou, D. (1994) Biochemical aspects oflow oxygen storage of potato tubers at low temperatures and molecular cloning of potato acid invertase, Ph.D. Thesis, University of Maryland, College Park.

36. Zittomer R.S. and Lowry C.V. (1992) Regulation of gene expression by oxygen in Saccharomyces serevisiae, Microbiolog. Reviews 56, 1-11.

ETHYLENE REGULATION BY THE NITRIC OXIDE (NO' ) FREE RADICAL:

A POSSmLE MODE OF ACTION OF ENDOGENOUS NO'

E. HARAMATY AND Y.Y. LESHEM Department of Lifo Sciences, Bar-Jlan University Ramat Gan 52900, Israel

1. Introduction

Nitric oxide (NO), a potentially toxic relatively unstable free radical gas, has emerged to be a major factor in several pivotal regulatory pathways [3]. Recent research mainly in mammalian systems has indicated that NO and its formative enzyme nitric oxide synthase (NOS) are endogenous bioregulators, both stimulatory and inhibitory [cf. review, 6].

This research endeavored to determine whether as in mammals, higher plants also produce NO and if so, to ascertain its mode of action with emphasis on possible interaction with ethylene evolution. We surmised that this goal could be achieved by employment of a recently developed NO specific probe, and also by application to plants of NO-producing compounds - S-nitroso-N-acetylpenicilamine (SNAP) and N­tert-butyl-a-phenylnitrone (PBN) [1,4].

2. Experimental Approach

2.1. PLANT MATERIAL

2.1.1. Pea Foliage Experiments were performed on 21-day-old pea plants (Pisum sativum Linn. cv. P.F. 70A, USA) grown on a vermiculite medium. Kinetics of ethylene response in senescing pea foliage and details of growth media and conditions have been detailed elsewhere [7]. When included, 10.3 M SNAP was applied, as detailed later: preliminary trials having indicated that this is the optimal effective concentration.

2.1.2. Carnation Flowers Since the carnation is a flower species highly sensitive to C2Rt and whose senescence is temporally accompanied by increments of both ACC synthase and ACC oxidase resulting in petal C2Rt evolution which encodes genes for p-galactosidase, p­glucosidase and gluthatine-S-transferase [12], it was considered ideal for the present research: Mode of experimentation and C2Rt assay were as outlined elsewhere [8].

253

A. K. Kanellis et al. (eds.), Biology and Biotechrwlogy of the Plant Hormone Ethylene, 253-258. © 1997 Kluwer Academic Publishers.

254

Treatment with the NO releasing compound PBN [1] was at 10-3, 10-5, and 10-7 M applied to the buffer medium which also contained 2mM ACC. Two control treatments were also included both without PBN - one containing, the other lacking, ACC. Freshly cut flowers were placed in 25 ml Erlenmeyer flasks containing the treatment media as detailed above for the pea foliage trials. All treatments were quadruplicated. Ethylene, as in the pea experiments, was assayed for the initial 24 h during which the major upsurge of this gas occurs [8]. Results are presented in Figure 4.

2.2. NO DETERMINATION

NO was directly measured by employing an ISO-NO Nitric Oxide Sensor (WPI -Florida, USA) equipped with a 2 mm NO specific probe. Experiments usually employed 4 replicates, each replicate consisting of three plants severed from their roots and placed in 25 ml vials containing the buffer medium outlined above.

The vials containing the plants were then placed under 0.5 I plastic bell jars and sealed with rubber serum caps. Bell jars were also equipped with an adaptor inlet through which the NO probe was inserted and, aided by an O-ring held in place directly over the pea foliage, the purpose being to monitor NO emission, if any, immediately after its release from the plants, and thus to obviate a sensor misreading the conversion of NO to N02, the half life of NO being ca. 5s [11]. Preliminary trials carried out in bell jars purged with N2 provided essentially similar results thus indicating registering only of NO and not of N02 to which the probe also possesses a limited degree of sensitivity.

2.3. ETHYLENE DETERMINATION

At given periods, employing a hypodermic syringe, 1 ml gas samples were withdrawn through the rubber serum caps of the bell jars (the same ones with an NO probe inlet which were simultaneously employed to monitor NO emission) containing the pea foliage to assess ethylene production. To prevent moisture accumulation on the bell jars' interior from carnation transpiration, an open vial containing 5 g anhydrous CaCh was also placed in each. Ethylene measurement was performed on a Varian Model 3400 FID gas chromatograph equipped with an alumina column and a Varian 4290 integrator. Injection temperature was 150°C and column temperature llO°C. Readings presented are four replicate means.

2.3.1. NO and Ethylene Production Under Stress Emission of C2~ and NO were measured in three groups of plants which were severed from their roots and thereupon: a) immediately placed in the Epps buffer solution under the bell jar as outlined above; b) allowed to wilt at 24°C for 1 h in dry air before placing in the buffer medium; c) allowed to wilt for 2 h.

255

3. Results and Discussion

3.1. INfER-RELATIONSHIP BETWEEN ENDOGENOUSLY PRODUCED NO ANDC2~

Figure 1 clearly indicates endogenous evolution of NO from pea foliage where emission proceeds simultaneously with that of ethylene: not only is C2~ emission decreased as expected by lack of precursor ACC, but also that of NO. Since both co­ordinate axes are on equimolar scale, it is also noteworthy that NO emission exceeds that of ethylene.

When pea plants underwent moisture stress prior to placing in the buffer medium, depending on duration, plants responded by increase of NO emission [Fig. 2). This effect could be interpreted either as a stress-inducing response since C2~ too induces stress [9] or alternatively, as a possible stress-coping strategy.

~

~ .c C2H4

Z~ 300 0::I: NO oo(!) 00--w :e~

200 W O:I: Z~ ~a::: OLL 100 ~-::I: 0 N' U::E

c

Figure 1. Endogenous evolution of NO and ~lLt in senescing pea foliage. 2 mM ACC was present in or absent from the buffer medium. This experiment was repeated 3 times, each producing similar results; the above figure represents those of one typical trial. Standard deviations did not exceed 11.5 percent of given data points.

3.2. EFFECTS OF THE NO RELEASING COMPOUNDS SNAP AND PBN ON ErnYLENE EVOLUTION

If indeed NO and C2~ are metabolically interlinked, then addition of NO releasing compounds to the buffered growth solution should effect C2~ emission by the plants. Figure 3 indicates that C2~ production was markedly decreased by NO. These results

256

also imply that NO release by the plant is a stress-coping response rather than a stress­inducing one.

'-.r:. ~ J: 160 (!)

Z -0 W

(J) 3= !:Q::c

140 <.!) <.!) ~(J) Z Z WW

~ j:: c::: -J -J

OlL. 0 ~ ~ a::: Z ."j I- ..: ..: '- Z .c .c ~ 0 N

:E u

c:

Figure 2. Effect of water stress on NO emission from stressed pea foliage. Results presented are of one of three typical experiments. Standard deviations did not exceed 8% of given data points.

~ 400

Cii!i: !a(!) 2-11.111.1 IIJ~ z:J: IIJ(/) -l1lJ )-0: :J:IL. I--r 11.1 .. co 11.12 2: c ~ -l 11.1 0:

0 30 60 90 120 TIME-MIN

Figure 3. Effect of the NO releasing compound SNAP on ~H4 enuSSlon in senescing pea foliage. See Materials and Methods for experimental details. Results presented are 4 replicate means.

257

In the carnation experiment (Fig. 4), it can be seen that while as expected, addition of ACC to the control medium induced both flower senescence and a marked increase of ethylene, PBN markedly reversed both processes. This is clearly apparent where 10.3 M PBN produced flowers as fresh as those of the non-ACC treated controls and significantly reduced ~Rt. Figure 4 moreover indicates that this PBN effect is furthermore concentration-related, its effects gradually wearing off with concentration decrease of from 10-3 to 10-7 M. This observation taken together with those seen in Figures 1 and 2 suggests decisive role of NO in control both of ethylene production and physiological effects in plants.

200

A

278 220 247 282

B c o

* Numbers above flowers indicate cumulative C2K. ppm/gr fresh wt)'in bell jars during the initial 22 h period

E

Figure 4. Effect of the NO releasing compound PBN on senescence and ethylene evolution of carnation flowers. The depicted flowers are one replicate typical of four and reflect degree of flower senescence 7 days after cutting.

Key: A - Control; B - Control + ACC; C - 10-3 M PBN; D - 10" M PBN; E - 10.7 M PBN. (C, D and E also included ACC).

4. Conclusions

Taken together, the above in vivo and in vitro data indicate that NO is endogenously produced in plants and that in the present experimental system this free radical gas reacts stoichiometrically with C2Rt. It functions, in the present experimental system, to down-regulate stress ethylene. These findings are in keeping with an earlier report of ours [7] indicating a like trend and which also presented preliminary data suggesting presence of a plant NOS. One interlinking observation [Fig. 1] still awaiting elucidation is the mode of enhancement of both C2Rt and NO by ACC.

Concerning mode of stoichiometIy between NO and C2Rt, one possible interpretation may be oxidation caused by NO (or by one of its peroxynitrite derivatives) on either ACC oxidase or one of its cofactors such as ascorbate or Fe2+ [2].

258

This key enzyme in ethylene fonnation may well be the target for NO attack, since as a gas, NO transportation in plants may be apoplastic, the plant apoplast being directly proximal to the plant cell wall where, by immuno-cytological fluorescence staining techniques ACC oxidase has been found to be mainly located [5]. NO induced oxidation of auxin required for ACC synthase action may be another possibility as is that of the finding that the protonated NO adduct - hydroperoxynitrite (HOONO) causes formation of methionine sulphoxide [10]. This may deplete stores of S­adenosine methionine for conversion into ACC and result in reduced ethylene production.

5. Acknowledgments

The authors wish to thank Levana Cohen, Zahit Eitan, Ya'ara Oppenheimer and Inessa Solomonicki for their aid in the carnation experiments.

6. References

1. Chamulitrat, W., Jordan, S.J., Mason, R.P., Saito, K., and Culter, R.G. (1993) Nitric oxide fonnation during light-induced decomposition ofphenyl-N-tert-butylnitrone, J. Bioi. Chem. 268, 11520-11527.

2. Christoffersen, R.E., McGarvey, P.J., and Savarese, P. (1993) Biochemical and molecular characterization of ethylene forming enzyme from avocado, in J.C. Pech, A Latche, and C. Balague (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, Kluwer Academic Publishers, Dordrecht, pp. 65-70.

3. Feldman, P.L., Griffith, O.W., and Stuehr, D.J. (1993) The surprising life of nitric oxide, Chem. Eng. News 71, 26-38.

4. Hery, P.J., Horowitz, J.D., and Louis, W.J. (1989) Nitroglycerin-induced tolerance affects multiple sites in the organic nitrate bioconversion cascade, 1. Pharmacal. Exp. Ther. 248, 762-768.

5. Latche, A, Dupille, E., Rombaldi, C., Cleyet-Marel, J.C., Lelievere, J.M., and Pech, J.C. (1993) in J.C. Pech, A Latche, and C. Balague (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, Kluwer Academic Publishers, Dordrecht, pp. 39-45.

6. Leshem, Y.Y. (1996) Nitric oxide in biological systems. Plant Growth Regulation 18, 155-159. 7. Leshern, Y.Y. and Haramaty, E. (1996) The characterization and contrasting effects of the nitric oxide

free radical in vegetative stress and senescence of Pisum sativum Linn. foliage, J. Plant Physiol. 148, 258-263.

8. Leshern, Y.Y., Rapaport, D., Frimer, AA, Strul, G., Asaf, u., and Feiner, I. (1993) Buckministerfullerene (C-60 carbon allotrope) inhibits ethylene evolution from l-aminocyclopropane­I-carboxylic acid (ACC)-treated shoots of pea (Pisum sativum), broad bean (Viciafaba) and flowers of carnation (Dianthus caryophyllus), Ann. Bot. 72, 457-461.

9. Leshem, Y.Y., Sridhara, S., and Thompson, J.E. (1984) Involvement of calcium and calmodulin in membrane deterioration during senescence of pea foliage, Plant Physiol. 75, 329-335.

10. Pryor, WA, Jin, x., and Squadrito, G.L. (1994) One- and two-electron oxidations of methionine by peroxynitrite, Proc. Natl. Acad. Sci. USA 91, 11173-11177.

11. Snyder, S.H. (1992) Nitric oxide: frrst in a new class of neurotransmitters, Science 257,494-496. 12. Maxson, J.M and Woodson, W.R.. (1996) Transcriptional regulation of senescence-related genes in

carnation flowers, in Biology and Biotechnology of the Plant Hormone Ethylene, AK. Kanellis, C. Chang, H. Kende, D. Grierson (eds), Kluwer Academic Publishers, Dordrecht, (in press)

ETHYLENE SYNTHESIS IN TOMATO PLANTS EXPOSED TO OZONE

The Role of Ethylene in Ozone Damage

.. I I 2 2 J. KANGASJARVI , J. TUOMAINEN, C. BETZ, D. ERNST, C. LANGEBARTELS2, H. SANDERMANN, JR.2,

IDepartment of Ecology and Environmental ScienceUniversity of Kuopio, Box 1627, FIN-70211 Kuopio, Finland, 2Institut fur Biochemische Pjlanzenpathologie, GSF-Forschungszentrum fur Umwelt und Gesundheit, D-85764 Oberschleiiheim, Germany

1. Introduction

Ozone (03) concentrations in the lower atmosphere have increased in both Europe and North America during last decades. 0 3 affects both agriculturally important plants and natural vegetation in two ways; lower elevated concentrations affect mostly physiological processes such as photosynthesis, while higher, short term peaks, so called 0 3 episodes, cause direct damage to the plants. It has been estimated that in the United States 0 3 causes more crop losses than all other air pollutants combined [6]. Once ozone enters plant leaves, it is rapidly decomposed in the apoplast and a plenitude of activated oxygen species is formed, such as the superoxide anion, hydroxyl ion and hydrogen peroxide, thought to be responsible for the harmful effects of 0J. Plants contain several antioxidative enzymes that can deal with activated oxygen species, but they are mostly located intracellularly and, at least in some species and circumstances, the increases in their activities are correlated with damage formation, not with protection from 0 3 [20].

Plants exposed to elevated atmospheric 0 3 undergo several biochemical changes even when no damage can be detected [8]. Ozone induces similar defense reactions as pathogens or elicitor treatments, for example accumulation of basic and acidic isoforms ofPR proteins [15]. Induction of the ethylene-responsive basic isoforms is early, when the acidic isoforms respond only later at the time of lesion formation [19]. Ethylene emission is one of the fastest plant responses to 0 3 [12,13] and it seems to play an important role in determining plant's ozone sensitivity [reviewed in 8], but the ultimate basis of the sensitivity still remains unknown. Wellborn and Wellborn [21] have studied ethylene emissions and antioxidative mechanisms in six pairs of ozone­sensitive and -tolerant plant strains/cultivars from different species. The only consistent similarity between all these pairs was increased ethylene emission from the sensitive plants after 0 3 treatment. Thus, studies of ethylene synthesis and its regulation in ozone-exposed plants may elucidate the basis of plant ozone sensitivity.

259

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 259-265. © 1997 Kluwer Academic Publishers.

260

We have studied ozone-induced ethylene synthesis in tobacco [11,22] and tomato plants (fuomainen et al., manuscript, Betz et al., unpublished) first, to characterize the early events and regulation of ethylene synthesis by 0 3• Our ultimate aim is to reveal the events leading from ozone exposure to stress ethylene formation, and to study the role of ethylene in 0 3 -induced tissue damage. For that we are using tomato cultivars altered in ethylene biosynthesis or perception by an anti sense transgene or mutation.

2. Experimental Procedures and Plants

In our experiments tomato (Lycopersicon esculentum Mill. cultivars 'Roma', 'Ailsa Craig' and 'Pearson') plants are first grown in ozone-free air. Seven week old plants are exposed to a single ozone pulse (0 - 250 ± 10 nl rl) for 0.5 to 5 h in growth chambers as described [11]. Samples collected from middle-aged leaves (length 15 to 30 cm) have been used for measurements of ethylene evolution, ACC concentration and ethylene biosynthesis enzyme activities. Gene expression for S-adenosyl­methionine (SAM), ACC and ethylene synthesis has been analyzed with gene-specific probes for SAM synthetase and ACC synthase, and pTOM13 for ACC oxidase.

3. Ethylene Biosynthesis Induced by Ozone

Under our conditions, ozone concentration has to exceed a threshold value before it causes visible damage in the plants. Exposure of tomato plants to 85 nl r 1 0 3 does not cause any visible symptoms, but 165 and 250 nl r 1 0 3 for 5 h induce leaf injwy and epinasty on middle-aged leaves no. 3 through 6. The injwy develops between 15 and 48 h after the onset of treatment as white necrotic lesions, mainly around the major veins. In the cultivar 'Roma' damage comprises about 30% of the leaf area on the affected leaves at 250 nl r 1 0 3 when at 165 nl r 1 0 3 the damaged area is significantly smaller. Young leaves as well as old leaves do not develop any 0 3 injwy, which is in accordance with several previous studies [see 8]. Visible injwy has been observed only in plants where ethylene biosynthesis has significantly been induced within one hour after the beginning of exposure.

Stress ethylene synthesis induction by 0 3 has been studied in these plants at metabolite, enzyme activity and mRNA levels. ACC synthase (ACS) activity increases within one hour at 165 and 250 nl rl ozone, while 85 nl rl 0 3 does not have any effect (Fig. 1). ACC concentrations and ethylene emission have a time course and dose response similar to ACS activity with maximum levels at 2 h and no response at the low 0 3 concentration. By 24 hours all the parameters have decreased to the control plant levels. Concentrations of conjugated ACC are also affected by 0 3: During the first hours of exposure they decrease significantly below the concentrations of control plants and increase back by the end of the treatment (Table 1).

40.-------~------------------_, -a-control

o 2 3 4 5

tirre [h]

Figure 1. Effect of ozone exposure on ACC synthase activity in tomato. Plants were exposed to clean air or to three ozone concentrations (85, 165 and 250 nl r1) for 5 h. The response of ACS activity to ozone exposure was analyzed from samples from middle-aged leaves collected during the exposure. Standard errors (n = 3) are given when greater than symbol width.

4. Regulation of Ethylene Synthesis in 0 3 - exposed Plants

4.1. GENE EXPRESSION

261

We have analyzed the expression of genes for three ethylene biosynthesis enzymes, SAM synthetase, ACC synthase (ACS) and ACC oxidase (ACO) from plants exposed to 200 nl rl 0 3. The results of these experiments are combined in Table 1. ACC oxidase was the fastest ethylene biosynthesis gene to respond. Its mRNA levels increased dramatically and were 95% of the maximum already at 30 minutes and maximal at one hour, decreasing thereafter. The temporal response of ACO transcripts in 0 3 exposed leaves was quite similar to the A CO 1 induction in wounded tomato leaves [3]. Increases in the mRNA levels for SAM synthetase were detected for only one of the three genes, SAM3. From the four tomato ACS genes analyzed, transcript levels for only LEA CS2 were specifically increased by 0 3 exposure, the other ACC synthase genes studied, LEA CS3, LEA CS4 and LEA CS5 did not show any response. Both SAM3 and LEACS2 had their maximal mRNA levels at two hours. Induction of SAM3 by ozone is most probably not only for increased ethylene synthesis but also mirrors the detected increase in polyamine synthesis, and methylation reactions for secondary compounds and lignin formation under stress conditions [13].

262

Table 1. Relative changes in biochemical parameters and mRNA levels for ethylene biosynthetic genes in ozone-exposed tomato leaves. The increases in mRNA levels for SAM3, LEACS2 and LEACO, ACC synthase activity, ACC and conjugated ACC concentrations and ethylene evolution are indicated relative to control plants at the same time points.

Time after the beginning of 0 3 exposure [h)

Response

SAM3 LEACS2 ACC Conjugated ACC ACS activity

0.5

LEACO ++++ Ethylene evolution (+)

(+) +

++ ++++ +++

2 5

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

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

Ozone-induced reactions in plants show similarity to both wound- and pathogen­induced reactions, ethylene synthesis is also increased under both stress types. This prompts the question, whether 03-induced ethylene synthesis is similar to the latter or the former type of stress. LEACS2 responds to both pathogen infection [17] and wounding of fruits [9], but it is not the only wound-responsive tomato ACC synthase gene; both LEA CS4 and LEA CS5 are also induced by wounding in fruits. However, under our conditions the corresponding transcripts were not detected in ozone-exposed tomato leaves arguing that 0 3 does not, at least during the 5 h exposure period, induce ethylene gene expression typical to wound response. Scblagnhaufer et af. [14] have isolated an 03-induced potato ACC synthase cDNA that encodes a protein 96% identical to the LEACS2. Both the tomato and potato ozone-inducible ACS genes are also induced by pathogen infection [5,17]. Furthermore, in tobacco both xylanase [1] and TMV [10] induce ACC synthase gene whose deduced amino acid sequence is 89% identical to the LEACS2 protein. These similarities, and other pathogen defense responses in 0 3 exposed plants [13] suggest that 03-induced ACS induction in tomato is similar to the pathogen response and that 03-induced ethylene synthesis is regulated in a specific way that is conserved across genera at least in the Solanaceae family.

4.2. EN2YME ACTIVITY CHANGES

The order of events in 03-induced ethylene production in tomato plants (Table 1) is intriguing. Under our conditions the mRNA levels for ACO in the three tomato cultivars used have increased distinctly prior to that of ACS. This suggest that ACO is, at least partly, responsible for the early induction of ethylene synthesis in 0 3-exposed plants. Furthermore, in our experiments the proportional ACS activity increase at one hour has been above the relative induction-level of LEACS2, thus the gene activation might be preceded by ACS activation at the protein level.

263

It is known that protein phosphorylation can regulate ACS activity in tomato suspension cells [4,18]; phosphatase inhibitors increased the ACS activity even in the absence of the elicitor and phosphorylase inhibitors prevented the elicitor-induced increase. When we infiltrated tomato leaves with the phosphatase 1 and 2A inhibitor calyculin A, ACS activity increased rapidly with similar rate as in ozone-exposed plants (Fig. 2). Control plants showed only a weak induction after 30 minutes of treatment caused by the abscission of the leaves, but showed no changes in activity thereafter. Furthermore, infiltration of the leaves after one hour of 0 3 exposure with the protein kinase inhibitor K-252a prevented the further ACS activity increase. These results suggests that the 03-induced rise in ethylene emission could initially be controlled by ACS protein activation and increased LEACO mRNA level (at 0.5-1 h) and only later (at 2 h) LEACS2 additionally regulates ethylene emission.

-0- control (ozone) 60 ----e- 200 nlll ozone

C -0- control (calyculin A)

~ 40 -.- calyculin A

l:i.. ~

.2> 20

~ ~ t: .s

0 Q

0 2 3 4 5

tirre [h]

Figure 2. ACC synthase activity induced by ozone and protein phosphatase inhibitor Calyculin A in tomato plants. Ozone-treatment: Plants were exposed to a single 5h pulse (200 nl rl) of ozone or to pollutant-free air. Calyculin A treatment: Leaflets from middle-aged leaf no. 4 were infiltrated in vacuo with 0.2 11M calyculin A or buffer for the indicated periods of time. Leaf samples were collected during the treatments and ACS activity was measured from them. Means ± S. E. (n = 3).

5. Ethylene and Plant Ozone Damage

Depending on the plant species and the 0 3 concentrations used, two genetically regulated mechanisms, premature senescence and programmed cell death (pcd), have been proposed as basis for ozone injury [8]. In studies with potato [5,14] ozone­induced ethylene synthesis was suggested to activate premature leaf senescence by affecting Rubisco SSU mRNA and protein. Ozone-induced tissue damage in tomato under our conditions and in tobacco [11], however, is more similar to the pcd occurring in the HR during incompatible plant-pathogen interactions where ethylene can have an important role in symptom development by controlling or promoting the spread of cell death [1,2,17].

264

Considering possible modes of action for ethylene in ozone damage, leaf senescence and fruit ripening can be used as good models for comparison. It has been shown that, for example, when controlling leaf senescence, ethylene operates together with other factors. They all are necessary for the induction of senescence related genes, and, none of them alone is sufficient to bring about the process [7]. We believe that in plant- 0 3 interactions ethylene together with other, damage-related factors is required in the tissue damage formation and spread as visible lesions. It has indeed been shown that at least two distinct processes are involved in the 03-induction of plant defense responses [16]. Accordingly in tissue damage, if one of the components, like ethylene in 03-tolerant tobacco [11], or other 03-tolerant plant selections [21] is absent, cell death will not occur, or spread from the few individual cells initially damaged in both 0 3 sensitive and insensitive plants [20].

In the dose-response experiment ACS activity (Fig. 1) and ACC levels were similar in plants exposed to both 165 nl r1 and 250 nl r1 0 3 concentrations, even though tissue damage was more pronounced at the higher concentration. The model proposed above explains this difference. If ethylene's role is in controlling and promoting the spread of cell death, the magnitude of the damage will not correlate to the rate of ethylene evolution but instead to the other factors. Our results with tomato and other species suggest that 0 3 provokes similar damage-related processes as elicitors in plant­pathogen interactions and doesn't seem to act primarily as directly damaging agent.

6. References

1. Avni, A, Bailey, BA, Mattoo, AK. and Anderson, J.D. (1994) Induction of ethylene biosynthesis in Nicotiana tabacum by a Trichoderma viride xylanase is correlated to the accumulation of 1-aminocyclopropane-l-carboxylic acid (ACC) synthase and ACC oxidase transcripts, Plant Physiol. 106, 1049-1055.

2. Bailey, B.A, Avni, A and Anderson, J.D. (1995) The influence of ethylene and tissue age on the sensitivity ofXanthi tobacco leaves to a Trichoderma viride xylanase, Plant Cell Physiol. 36, 1669-1676.

3. Barry, C.S., Blume, B., Bouzayen, M., Cooper, W., Hamilton, AJ. and Grierson, D. (1996) Differential expression of the l-aminocyclopropane-l-carboxylate oxidase gene family of tomato, Plant J. 9, 525-535.

4. Felix, G., Regenass, M., Spanu, P. and Boller, T. (1994) The protein phosphatase inhibitor calyculin A mimics elicitor action in plant cells and induces rapid hyperphosphorylation of specific proteins as revealed by pulse labeling with e3p]phosphate, Proc. Natl. Acad. Sci. USA 91, 952-956.

5. Glick, RE., Schlagnhaufer, C.D., Arteca, RN. and Pell, E.J. (1995) Ozone-induced ethylene emission accelerates the loss ofribulose-l,5-bisphosphate carboxylase/oxygenase and nuclear-encoded mRNAs in senescing potato leaves, Plant Physiol. 109,891-898.

6. Heagle, AS. (1989) Ozone and crop yield, Annu. Rev. Phytopathol. 27, 397-423. 7. John, 1., Drake, R, Farrel, A, Cooper, W., Lee, P., Horton, P. and Grierson, D. (1995) Delayed leaf

senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis, Plant J. 7, 483-490.

8. Kangasjarvi, J., Talvinen, J., Utriainen, M. and Katjalainen, R (1994) Plant defense systems induced by ozone, Plant, Cell Environ. 17,783-794.

9. Kende, H. (1993) Ethylene biosynthesis, Annu. Rev. Plant Physiol. Plant Mol. Bioi. 44,283-307. 10. Knoester, M., Bol, J.F., van Loon, L.C. and Linthorst, H.J.M. (1995) Virus-induced gene expression

for enzymes of ethylene biosynthesis in hypersensitively reacting tobacco, Mol. Plant-Microbe Interact. 8, 177-180.

265

11. Langebartels, C., Kerner, K, Leonardi, S., Scbraudner, M., Trost, M., Heller, W. and Sandermann, H., Jr. (1991) Biochemical plant responses to ozone I. Differential induction of polyamine and ethylene biosynthesis in tobacco, Plant Physiol. 95, 882-889.

12. Rodecap, KD. and Tingey, D.T. (1983) The influence of light on ozone-induced 1-aminocyclopropane-l-carboxylic acid and ethylene production from intact plants, Z. Pjlanzenphysiol. 110,419-427.

13. Sandermann, H., Jr. (1996) Ozone and plant health, Annu. Rev. Phytopathol. 34, 347-366. 14. Schlagnhaufer, C.D., Glick, R.E., Arteca, R.N. and Pell, E.J. (1995) Molecular cloning of an ozone­

induced l-aminocyclopropane-l-carboxylate synthase cDNA and its relationship with a loss of rbcS in potato (Solanum tuberosum L.) plants, Plant Mol. BioI. 28, 93-103.

15. Scbraudner, M., Ernst, D., Langebartels, C. and Sandermann, H., Jr. (1992) Biochemical plant responses to ozone III. Activation of the defense-related proteins i-l,3,g1ucanase and chitinase in tobacco leaves,PlantPhysiol. 99, 1321-1328.

16. Sharma, Y.K, Leon, J., Raskin, I. and Davis, KR. (1996) Ozone-induced responses in Arabidopsis thaliana: the role of salicylic acid in the accumulation of defense-related transcripts and induced resistance, Proc. Natl. Acad. Sci. USA 93, 5099-5104.

17. Spanu, P., Boller, T. and Kende, H. (1993) Differential accumulation of transcripts of 1-aminocyclopropane-l-carboxylate synthase genes in tomato plants infected with Phytophthora infestans and in elicitor-treated tomato cell suspensions, J. Plant Physiol. 141,557-562.

18. Spanu, P., Grosskopf, D.G., Felix, G. and Boller, T. (1994) The apparent turnover of 1-aminocyclopropane-l-carboxylate synthase in tomato cells is regulated by protein phosphorylation and dephosphorylation, Plant Physiol. 106,529-535.

19. Thahnair, M., Bauw, G., Thiel, S., Doehring, T., Langebartels, C. and Sandermann, H. (1996) Ozone and ultraviolet B effects on the defense-related proteins [3-1,3-glucanase and chitinase in tobacco, J. PlantPhysiol. 148,222-228.

20. Tuomainen, J., Pellinen, R., Roy, S., Kiiskinen, M., E1oranta, T., Krujalainen, R. and Kangasjarvi, J. (1996) Ozone affects birch (Betula pendula Roth) phenylpropanoid, polyamine and active oxygen detoxifYing pathways at biochemical and gene expression level, J. Plant Physiol. 148, 179-188.

21. Wellburn, FAM. and Wellburn, AR. (1996) Variable patterns of antioxidant protection but similar ethene emission differences in several ozone-sensitive and ozone-tolerant plant selections, Plant, Cell Environ. 19,754-760.

22. Yin, Z.-H., Langebartels, C. and Sandermann Jr., H. (1994) Specific induction of ethylene biosynthesis in tobacco plants by the air pollutant, ozone, Proc. Royal Soc. Edinburgh 102D, 127-130.

INVOLVEMENT OF ETHYLENE IN PROTEIN ELICITOR-INDUCED PLANT RESPONSES

lD. ANDERSON', F.C. CARDINALE2, lC. JENNINGS', H.A. NORMAN', A. A VNI3, U. HANANIA3, AND B.A. BAILEY' Plant Sciences Institute, Beltsville Agricultural Research Center, Beltsville, MD, USA l ; Di. Va.P.R.A., University of Turin, Turin, Italy; Botany Department, Tel Aviv University, Tel Aviv, Israel3

1. Introduction

Plant vegetative tissues respond to a number of chemicals, e.g., plant hormones, proteins, carbohydrates, heavy metals, etc. by producing ethylene. In some cases, e.g., tobacco, an ethylene pretreatment is known to enhance the response to some of these chemicals [12]. The enhancement of the response by ethylene pretreatment is generally lost within 24 to 48 h after the plant tissues are placed in an air atmosphere, but the tissue is capable of responding to another treatment of ethylene [7, 12].

Various chemicals have been found to be potent ethylene action inhibitors. A very effective one, silver [11], is currently used to extend shelf life of flowers. However, as a heavy metal, silver has substantial limitations to its use. A volatile ethylene action inhibitor, norborodiene, was introduced by Sisler et al. [25]. This is very effective in inhibiting ethylene action, but is limited because it needs to be constantly present. Recently, Sisler et al. [24, 26] introduced a new group of very active, volatile ethylene action inhibitors that seem to tightly bind to ethylene recognition sites and thereby eliminate the need to have the tissue in constant contact with the inhibitor. Plants treated with nanomolar levels or less of I-methylcyclopropene (I-MCP) appear to remain insensitive to ethylene for several days. Thus, these ethylene antagonists have great promise for practical use as well as for studying basic regulation of ethylene action.

We have tried to utilize I-MCP to determine if ethylene plays a role in elicitor induced responses (ethylene biosynthesis, cellular necrosis, and transcript levels) in different plants. I-MCP (100 nLlI...) treatment of tomato plants for 16 h, blocked epinasty by a subsequent treatment of ethylene. The inhibition of ethylene-induced epinasty lasted at least 4 days in plants treated with I-MCP (Cardinale, et al., unpublished data). In this paper, we discuss the influence of I-MCP treatment on the activity of the Trichoderma xylanase elicitor in tomato and tobacco leaf tissue.

267

A. K. Kanellis et al. (eds.). Biology and Biotechnology o/the Plant Hormone Ethylene. 267-274. © 1997 Kluwer Academic Publishers.

268

2. Results and Discussion

It is well known that ethylene plays a role in the response of the Trichoderma viride xylanase-induced responses of sensitive tobacco plants [7, 12]. However, it is not understood if ethylene action is needed for the xylanase-induced responses. In some cases ethylene and the elicitor induce the same responses, e.g. PR-protein synthesis. But in specific cases the PR-protein induced by xylanase is independent of the ethylene-induced PR-protein [21], indicating a very complex interaction. Xylanase treatment reduces PR-I basic transcript level in ethylene pretreated tissues. The complexity of the interaction is further complicated by the age of the tissue being studied [7]. The induction of ethylene biosynthesis and tissue necrosis by xylanase are both enhanced by ethylene pretreatment in young or mature leaves, but not in older, senescent (yellowed) ones (Table I). Ethylene pretreatment of young tobacco leaf tissue seems to mimic the aging process in these tissues as far as sensitivity to xylanase is concerned.

Table I. Relative increase in xylanase-induced responses in ethylene pretreated leaves compared to air pretreated leaves of different ages. Data were summarized from Bailey et aL [7].

LeafAge Ethylene Production Necrosis (Days) (Relative Units) (Relative Units)

4 + (7) + (7) 8 +++++ ++

11 +++++ +++ 14 +++++ +++ 17* +++ + 21 ++ + (7) 25** +(?) + (?)

"'Leaves are beginning to yellow; "''''Leaves are yellow.

Ethylene pretreatment enhances the production of ACC synthase transcripts in response to xylanase in mature tobacco leaves, but ethylene pretreatment does not enhance significantly the levels of this transcript in older, senescent leaves in response to the elicitor (Fig. I).

Ethylene treatment promotes the accumulation of PR-I basic transcript in both mature and senescent leaves without elicitor treatment (Fig. I). While elicitor treatment also stimulates PR-I basic transcript accumulation, elicitor treatment of ethylene pretreated tissue actually decreases the amount of transcript. At this time we do not understand the cause of the reduction in PR-I basic transcript level. Possibly in the ethylene treated tissue there is no new synthesis of this transcript in response to elicitor treatment, turnover rates are changed, or these actually represent different gene products that are regulated separately. Thus, there could be several possible mechanisms working. Such results indicate the complexity in interpreting such interactions.

269

I ~ <I "

.......-m-~ ~ ---:m-.......... Mitanl

... .......-m-~ ~---:m-

........ MIIhue

~,'"

f uSO

J ~

.................................. -;m- -:m-~ ---:mx-

Mata ..

Figure 1. Effect oftissue age on induction of ethylene biosynthesis, ACC synthase, and PR-l basic transcripts by ethylene and/or xylanase in Xanthi tobacco. Plants were incubated in air or ethylene (12 "IlL) for 16 h prior to xylanase treatment. Leaves from nodes 9, 10, and 11 were detached and treated with xylanase (2.5 "gig tissue) for 3 h 15 min approximately 8 (Mature) or 25 (Senescent) days after their emergence and expansion from the meristem. Ethylene production from each leaf was measured for 15 min after 3 h 15 min of xylanase treatment. Total RNA was isolated from each leaf and 10 "g total RNA was separated on fonnaldehyde gels and hybridized with probes for ACC synthase (clonepTACC-13) [5], PR-l basic [141 and the 168 ribosomal RNA from Xenopus .[27J. We isolated 13.5 "g(S.D.= 6.2 "gig) total RNA per gram ofsene8cent tobacco leaf and 31.2 "g (S.D.=5.1 "gig) total RNA per gram of mature tobacco leaf. Blots were scanned on a Molecular Dynamic's Densitometer and equalized as a percent of maximum for each blot for each probe. The data (% maximum) was then corrected by the mean total RNA isolated from 8 or 25 day old leaves and presented as % maximum on a per gram fresh weight basis. Three eaves were sampled for each treatment combination. The bars represent plus or minus one standard deviation.

The enhanced ability of tobacco leaves to produce ethylene upon subsequent treatments of elicitor begins to be lost as soon as the tissues are removed from ethylene and is lost by 24 to 48 hours [12]. The reduction in ethylene biosynthesis is probably do to loss in accumulation of ACC synthase transcripts in response to an elicitor (Fig. 2).

270

Time (h) o 8 24 48

----C214 + + - + + + + + + -EIX +-+ +-+-+-+-

ACC Synthase

PRot Basic

rRNA

Figure 2. Time course for decay of ethylene pretreatment effect on the xylanase interaction with Xanthi tobacco. [Figure from Bailey et aL, 7]. Plants were incubated in air or ethylene (12 IlVL) for 16 h. Plants were removed from the air or ethylene atmosphere for 0, 8, 24 or 48 h prior to treatment of detached leaves with xylanase. The ninth, tenth, and eleventh leaves from the base of each plant were detached and treated with xylanase (2.5 Ilgig tissue) for 3 h 15 min approximately 8 days after their emergence and expansion from the meristem. Ethylene production from each leafwas measured for 15 min after 3 h 15 min of xylanase treatment. Total RNA was isolated from each leaf and 10 Ilg total RNA was run on formaldehyde gels and successively hybridized with probes for ACC synthase, PR-l basic and the 16s ribosomal RNA from Xenopus.

Tomato leaves respond to xylanase elicitor by producing ethylene and tissue necrosis, but do not respond much to a pretreatment of ethylene [4]. We have used the ethylene action inhibitor, I-MCP, to study the role ethylene action plays in the elicitor response in tomato. This inhibitor presumably binds to the ethylene binding site [24, 26], a homologue of ETRI and EINI [23]. Elicitor-treated tomato tissue treated with I-MCP produces more ethylene than non-l-MCP treated tissue (Fig. 3). This stimulation of ethylene production occurs in tissues given I-MCP either as a pretreatment or concomitant with the elicitor treatment. This stimulation of ethylene production by inhibitors of ethylene action (silver ion) is known for senescing tobacco leaves [2] , as well as hormone-induced ethylene production [3]. This seelDS to be a broad based effect across species and different ethylene action inhibitors [e.g., 19]. Ethylene overproducing Arabidopsis mutants (etol) also respond [18].

We compared the effect of EIX, ethylene and I-MCP treatments on tobacco (Xanthi), tomato (Bonny Best) and pepper (Bell-type) leaf tissue. We measured ethylene production, levels of ACe oxidase and ACC synthase transcript levels, and tissue necrosis (Table 2). All three plant types responded to xylanase. Leaf tissues from all plant-types responded to EIX by an increase in ACC oxidase transcript level. As far as ethylene production is concerned, pepper responded a little to an ethylene

271

pretreatment, tobacco responded very well to an ethylene pretreatment, and tomato did not respond much, if at all. Tomato leaf tissue responded the best to EIX after I-MCP treatment. Pepper also responded well, but tobacco showed little response to I-MCP. Xylanase induced tissue necrosis in all plant-types. Of all the treatments, xylanase induced the most necrosis in tobacco plants pretreated with ethylene. The rate and degree of necrosis did not seem to be affected by I-MCP treatment. Thus, it does not appear that ethylene plays much of a role in elicitor-induced tissue necrosis, except in the case of tobacco.

80.-----------------------------------~

c: 60 0-'p ...; u ~ -6 ~ 0 ..... ... C) 40

0.. .......

~o GJ E - c: ~-.ti 20

Pretreatment, Post-treatment

!ill Air, Air

• Air, l-MCP

~ l-MCP, Air

~ l-MCP, l-MCP

3 6 9

Time After EIX Treatment (h)

24

Figure 3. Effect of I-methylcyclopropene on Xylanase-induced ethylene production by tomato leaves. Leaves were used from greenhouse grown tomato plants that were either treated for 16h in a chamber of air with ethylene removed with or with 100 nUL I-MCP. Leaves were treated with Xylanase by the hanging drop method and then placed in glass vessels, stopper and in some 100 nUL I-MCP was added Ethylene production was monitored at intervals.

How does this fit together? EIX, as well as other elicitors, interacts with sensitive plants to induce signal transduction pathways. Different elicitors (e.g., fungal proteins [1,6], yeast extract-derived elicitor [15,16,17], Cu++ [5], and plant growth regulators [20)) turn on signal transduction pathways that in some respects are similar, but are different even in the same tissues [17]. The recognition of chemical stimuli by plant cells and how the plant cell reacts to them is very complicated. Our working model suggests that the elicitor must be specifically recognized by the cell. In this case there appears to be a 44 kD plasma membrane associated protein that recognizes EIX in sensitive cells (Hanania and Avni, unpublished data). This plasma membrane protein appears to occur only in sensitive cells and might represent the gene product of the

272

dominant gene that gives sensitivity to EIX [4, 9]. In tomato, the sensitivity gene maps to the lower portion of chromosome 7 [4].

The binding of the elicitor sets in motion several complex signal transduction processes. Rapid responses (within minutes) that have been measured in tobacco tissue culture cells include: increases in extra cellular pH of the media, increase in K+ efflux and an increase in ea++ uptake, [10]. Shortly after, there is an efflux of ACC into the medium and a decrease in ethylene production. Tomato cells respond a little differently in that they are induced to produce ethylene [17]. In tobacco there is an accumulation of acylated sterol glycosides [22] and N-acylphosphatidylethanolamine [13], indicating changes in plasma membrane lipids. These relatively rapid responses are followed by ethylene biosynthesis, PR-protein biosynthesis, phytoalexin synthesis and cell necrosis and death [8]. At this time, we do not know if these are just unrelated responses or integral parts of the signaling pathway.

Table 2. Effect of air, ethylene and I-methylcyc1opropene on xylanase responses in fully expanded leaves of tobacco, pepper and tomato. Intact plants or isolated leaves were used fresh or pretreated with air, ethylene (10 /LUL), or I-methylcyc1opropene (100 nUL) for 16 h prior to treating with elicitor. Elicitor treatment was administered by placing a freshly cut petiole into a solution of elicitor for various periods of time and then placing the treated leaf into a sealed vessel for ethylene measurement. In other experiments, leaves were harvested after elicitor treatment for RNA extraction and Northern blotting.

SPECIES PRETREATMENT

Tobacco Air Tomato Pepper

Tobacco Ethylene Tomato Pepper

Tobacco I-MCP Tomato Pepper

* Transcript level. ? Not detected with the tobacco probe.

3. Summary

Ethylene Production

XYLANASE RESPONSE

ACC Synthase*

ACC Oxidase*

Necrosis

(-----------------------Relative Units-----------------------)

++ + ++ ++ ++ ? ++ + + ? ++ +

+++++ +++ ++ ++++ ++ ? ++ +

+++ ? + +

++ ? +++ ++ ++++++ ? +++ + +++++ ? ++ +

Does ethylene playa role in elicitor-induced responses? In some species, e.g., tobacco leaves it appears to, but in others (tomato) it appears that it doesn't. In tobacco,

273

ethylene enhances the ability of the tissue to respond to the elicitor. Ethylene treatment must extend for 6 to 8 hours. This enhancement is lost when plant tissues are removed from ethylene and exposed to air. But the ethylene action inhibitor, 1-MCP, did not have much effect on xylanase induced responses, i.e., it did not prevent or slow the development of responses. If ethylene does playa role in elicitor-induced responses, one would expect that I-MCP, would have made the tissue less responsive to xylanase. But it had little if any effect. In tomato, ethylene treatment only slightly enhanced the xylanase responses. I-MCP enhanced xylanase-induced ethylene biosynthesis, though it did not prevent or slow tissue necrosis. If one interprets this enhancement of elicitor-stimulated ethylene biosynthesis by I-MCP to be an interference in ethylene feedback regulation, then one would have to conclude that ethylene does play a role in elicitor induced responses, but only in a pathway induced by the elicitor.

4. References

1. Anderson, J.D., Bailey, BA, Dean, J.F.D., and Taylor, R. (1990) A fungal endoxylanase elicits ethylene biosynthesis in tobacco (Nicotiana tabacum L. cv. Xanthi) leaves, in H.E. Flores, RN Arteca, JC Shannon (eds.), Polyamines and Ethylene: Biochemistry, Physiology, and Interactions. American Society of Plant Physiologists. pp. 146-156.

2. Aharoni, N. and Liebennan, M. (1979) Ethylene as a regulator of senescence in tobacco leaf discs, PlantPhysiol. 64, 801-804.

3. Aharoni, N., Anderson, J.D., and Liebennan, M. (1979) Production and action of ethylene in senescing leaf discs, Plant Physiol. 64, 805-809.

4. Avni, A, Avidan, N., Eshed, Y., Zamir, D., Bailey, BA, Stommel, J.R., and Anderson, J.D. (1994) The response of Lycopersicon esculantum to a fungal xylanase is controlled by a single dominant gene,PlantPhysiol. (Supp.) 105, 872.

5. Avni, A, Bailey, BA, Mattoo, AK., and Anderson, J.D., (1994) Induction of ethylene biosynthesis in Nicotiana tabacum by a Trichoderma viride xylanase is correlated to the accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase transcripts, Plant Physiol. 106, 1049-1055.

6. Bailey, BA (1995) Purification of a protein from culture filtrates of Fusarium oxysporum that induces ethylene and necrosis in leaves ofErythroxylum coca, Phytopathology 85, 1250-1255.

7. Bailey, B.A, Avni, A, and Anderson, J.D. (1995) The influence of ethylene and tissue age on the sensitivity ofXanthi tobacco leaves to a Trichoderma viride xylanase, Plant Cell Physiol. 36, 1669-1676.

8. Bailey, BA, Dean, J.F.D., and Anderson, J.D. (1990) An ethylene biosynthesis-inducing endoxylanase elicits electrolyte leakage and necrosis in Nicotiana tabacum cv. Xanthi leaves, Plant Physiol. 94, 1849-1854.

9. Bailey, BA, Korcak, R.F., and Anderson fD. (1993) Sensitivity to an ethylene biosynthesis­inducing endoxylanase in Nicotiana tabacum L. cv. Xanthi is controlled by a single dominant gene, PlantPhysiol.l0l,1081-1088.

10. Bailey, BA, Korca.k, R.F., and Anderson, J.D. (1992) Alterations in Nicotiana tabacum L. cv. Xanthi cell membrane function following treatment with an ethylene biosynthesis-inducing endoxylanase, Plant Physiol. 100, 749-755.

11. Beyer, E.M. Jr. (1979) A potent inhibitor of ethylene action in plants, Plant Physiol. 58: 268-271. 12. Chalutz, E., Mattoo, AK., Solomos, T., and Anderson, J.D. (1984) Enhancement by ethylene of

cellulysin-induced ethylene production by tobacco leaf discs, Plant Physiol. 74, 99-103. 13. Chapman, K.D., Conyers-Jackson, A, Moreau, R.A, and Tripathy, S. (1995) Increased N­

acylphosphatidylethanolamine biosynthesis in elicitor-treated tobacco cells. Physiologia Plantarum 95, 120-126.

274

14. Eya!, Y., Oded, S., and Fluhr, R. (1992) Dark-induced accumulation of a basic pathogenesis-related (PR-l) transcript and light requirement for its induction by ethylene, Plant Molecular Biology 19, 589-599.

15. Felix, G., Grosskopf; D.G., Regenass, M., Basse, C.W., and Boller, T. (1991) Elicitor-induced ethylene biosynthesis in tomato cells. Characterization and use as a bioassay for elicitor action, Plant Physiol. 97, 19-25.

16. Felix, G., Grosskopf; D. G., Regenass, M., and Boller, T. (1991) Rapid changes of protein phosphorylation are involved in transduction of the elicitor signa! in plant cells, Proc. NatL Acad. Sci. USA. 88,: 8831-8834.

17. Felix, G., Regenass, M., and Boller, T. (1993) Specific perception of subnanomolar concentration of chitin fragments by tomato cells: Induction of extracellua!ar alkalinization, changes in protein phosphorylation, and establishment of a refractory state, Plant J. 4,307-316.

18. Guzman, P. and Ecker, J.R. (1990) Exploiting the triple response of Arabidopsis to identifY ethylene­related mutants, Plant Cell 2, 513-523.

19. Kende, H. (1993) Ethylene biosynthesis, Annu. Rev. Plant Physiol. Plant MoL BioI. 44, 283-307. 20. Klee, H. and Estelle, M. (1991) Molecular genetic approaches to plant honnone biology, Annu. Rev.

PlantPhysiol.42, 529-551. 21. Lotan, T. and Fluhr, R. (1990) Xylanase, a novel elicitor of pathogenesis-related proteins in tobacco,

uses a non ethylene pathway for induction, Plant Physioz. 93, 811-817. 22. Moreau, RA, Powell, M.J., Whitaker, B.D., Bailey, BA, and Anderson, J.D. (1994) Xylanase

treatment of plant cells induces g1ycosylation and fatty acylation of phytosterols, Physiologia Plantarum 91, 575-580.

23. Roman, G., Lubarsky, B., Kieber, J.J., Rothenberg, M., and Ecker, J.R. (1995) Genetic analysis of ethylene signal transduction in Arabidopsis thaliana: five novel mutant loci integrated into a stress response pathway, Genetics 139,1393-1409.

24. Sisler, E. C., Dupille, E., and Serek, M. (1996) Effect of I-methylcycloppropene and methylenecyclopropane on ethylene binding and ethylene action on cut carnations, Plant Growth Regulation 18, 79-86.

25. Sisler, E.C., Raphael G., and Huberman, M. (1985) Effect of 2,5-norbornadiene on abscission and ethylene production in citrus leaf explants, Plant Physioz. 63, 114-120.

26. Sisler, E. C., Serek, M., and Dupille, E. (1996) Comparison of cyclopropene, I-methylcyclopropene, and 3,3-dimethylcyclopropene as ethylene antagonists in plants, Plant Growth regulation 18, 169-174.

27. Sollner-Webb, B. and Reeder, R.H. (1979) The nucleotide sequence of the initiation and tennination sites for ribosomal RNA transcription in Xenopus laevis, Cell 10, 485-499.

CHANGES IN IN VIVO AND IN VITRO ACC OXIDASE ACTIVITIES DURING CHILLING AND SUBSEQUENT WARMING AS EXEMPLIFIED BY VIGNA RADIATA SEEDLINGS

F. CORBINEAUI, R. BOGATEK2, S. RADICEI, M.A. PICARDI and D. COMEI 1 Universite Pierre et Marie Curie, Physiologie Vegetale Appliquee, Tour 53, 1 er etage, 4 place Jussieu, 75252 Paris cedex 05, France, 2 University of Warsaw, Institute of Experimental Plant Biology, ul. Pawinskiego 5a, 02-106 Warsaw, Poland

1. Introductiou

Chilling injury is commonly observed in many species of tropical and subtropical origin [1, 2]. The critical temperature for chilling injury is usually between 0 and lOoC, but it depends on species, developmental stages, organs and tissues [1, 2]. Damage increases with exposure time, however a feature of this phenomenon is its reversibility following short exposure to low temperature [3].

Oxidative processes and free radical accumulation are usually invoked in molecular and cellular damage induced by a wide range of stresses including chilling [4-7]. Free radicals generate changes in insaturated fatty acids which affect the structure of cell membranes and therefore their functional properties. These membrane alterations can play an important role in the regulation of the in vivo ACC oxidase activity [8-10].

Production of ethylene is widely used as an indicator of various stresses including chilling [11, 12]. Chilling treatment can enhance ethylene production by plant tissues after transfer to warmer temperatures [12-17]. This phenomenon is generally associated with an accumulation of l-aminocyclopropane I-carboxylic acid (ACC) resulting from a stimulation of ACC synthase activity which occurs during the chilling treatment or after subsequent warming [12]. Recent data [18, 19] have demonstrated that ACC oxidase can also accumulate in Granny Smith apples during chilling and be activated during warming up. However, prolonged chilling results in a marked reduction in ethylene production and ACC oxidase activity in various chilling-sensitive tissues, and this reduction becomes progressively irreversible [20, 21].

The aims of the present paper are (i) to precise some cellular damage induced by chilling, (ii) to investigate the effects of chilling on in vivo and in vitro ACC oxidase activities as exemplified by Vigna radiata seedlings which are known to be chilling sensitive [21, 22], and (iii) to precise whether the changes in ACC oxidase activity induced by chilling remain reversible after subsequent warming.

275 A. K. Kanellis et al. (eds.), Biology and Biotechrwlogy o/the Plant Hormone Ethylene, 275-281. © 1997 Kluwer Academic Publishers.

276

2. Membrane Damage and Lipid Peroxidation

Injury to the plasmalemma and intracellular membranes seems to be the primary event that is common to cellular damage induced by various environmental stresses.

Electrolyte leakage from tissues can be used to indicate the effectiveness of membranes as barriers to solute diffusion and to evaluate membrane injury [23,24]. In Vigna radiata, treatment of seedlings at 1°C induces a gradual increase in electrolyte leakage (expressed as percentage of total leachable electrolytes) from hypocotyl segments (Table 1). Leakage reaches 26%, 39% and 61 %, respectively, after 4, 8 and 11 days of chilling at 1°C. Increase in electrolyte leakage is also associated with chilling injury in papaya [15], cucumber [5, 25] and melon [25]. These results suggest that membranes are damaged by chilling in chilling-intolerant tissues.

Various results [23, 24] suggest that lipid peroxidation is a cause of membrane deterioration during chilling, and that the damage would result in loss of semipermeability, accumulation of thiobarbituric acid-reactive substances such as malondialdehyde (MDA), and increase in ethane production [5, 11, 24, 26]. In Vigna radiata seedlings, for example, treatment at 1°C increases lipid peroxidation as ethane production by hypocotyl segments progressively increases during the treatment and MDA accumulates after 13 days of chilling (Table 1). Such phenomena are not observed with seedlings exposed to lOoC (Table 1).

3. In vivo and in vitro ACC Oxidase Activities

3.1. EFFECTS OF CHILLING

Decrease in the ability of tissues to oxidize ACC into ethylene can also be a good indication of membrane injury since the in vivo activity of ACC oxidase is known to depend on membrane integrity or properties [8-10].

TABLE 1. Effects of duration of exposure of VIgna radiata seedlings to 1 DC and IODC on MDA content, ethane production and electrolyte leakage. Measurements were performed with hyPocotyl segments. Means on or 4 replicates ± SD.

Treatment MDA Ethane Leakage Temperature Duration (mmol g'! FW) (nJ h'! g'! FW) (%totaI (DC) (days) leakage) No treatment (control) 16.1 ± 0.7 0.35 ±0.01 11.3 ± 1.4 1 2 15.5 ± 1.2 0.50±0.01 13.9±2.8

4 15.9 ± 2.3 0.68±0.06 26.0 ± 4.2 8 10.0 ± 0.5 0.75 ±0.12 39.3 ± 5.2

11 15.4± 0.1 1.01 ± 0.35 61.3 ± 3.5 13 28.6 ± 2.1 3.50 ± 0.30 68.6±8.6

10 2 16.2±0.2 0.30 ± 0.02 13.8± 3.6 4 16.8 ± 2.1 0.35 ±0.09 12.6 ± 1.6 8 10.3 ± 1.2 0.39 ± 0.04 11.8 ± 2.2

11 8.1 ± 1.4 0.43 ± 0.10 12.6 ± 1.2 13 9.3 ± 0.1 0.28 ± 0.02 12.8±2.6

277

Exposure of Vigna radiata seedlings to 1°C results in a sharp decline in in vivo ACC oxidase activity in hypocotyl segments (Fig. lA). A 2-day treatment at 1°C induces a reduction by about 70% of the initial enzyme activity, and hypocotyl segments are completely unable to convert exogenous ACC to ethylene after 8 to 10 days of chilling treatment (Fig. lA). This decrease in the ACC-dependent ethylene production is associated with a similar decrease in in vitro ACC oxidase activity (Fig. IB). Both in vivo and in vitro ACC oxidase activities are reduced by about 40% after 2 days of exposure of seedlings to 10°C, but thereafter they remain constant (Figs. lA and IB). Etani and Yoshida [21] have also shown with the same plant material that ACC-dependent ethylene production declines to less than 50 % of that of the unchilled tissues after 1 day at O°C. The effect of chilling on in vivo ACC oxidase activity depends on the species and the temperature of exposure. The activity of the enzyme declines after 11 weeks at 12°C in oil palm somatic embryos [17], after 5 days at 2.5°C in cucumber fruits [12, 14], and after 3 days and 3 hours at O°C in leaves of Passiflora edulis and Episcia reptans, respectively [20].

In mung bean seedlings, the loss of the ability of hypocotyls to convert ACC to ethylene is associated with a decrease in in vitro ACC oxidase activity (Fig. lB), suggesting that the enzyme is directly affected by chilling.

(J 0 1ft N I-cC

~ ";"

01

";" .c i! ..... or

:r: N

(J

60 10 (J 0 0

50 CO)

~ 8

40 i IL

";" 6

30 01

";" 4 .c

20 i! ..... or 2 10 :r: N

()

0 0 0 2 4 6 8 10 12 14 0 2 4 6 8 10

TIME AT 1°C OR 10"C (DAYS) TIME A 11°C OR 10"C (DA YS)

Figure 1. Effects of duration of exposure of Vigna radiata seedlings to 1°C and lOOC on in vivo (A) and in vitro (8) ACC oxidase activities in hypocotyl segments. Means of 4 replicates ±SD.

12

278

3.2. EFFECTS OF SUBSEQUENT WARMING

Although the reduction of in vivo ACC oxidase activity by chilling is not a characteristic of chilling sensitive materials, since it is observed also in seedlings of Pisum sativum, a cold-resistant species [21], the degree of recovery of this activity upon subsequent warming might be a good indication of the degree of chilling damage. Restoration of in vivo ACC oxidase activity during warming chilled mung bean hypocotyls depends on the duration and the temperature of chilling (Fig. 2 and Table 2). Warming at 25°C for 4 h is enough to completely restore this activity in hypocotyl segments from seedlings previously exposed to lOoC for 2 or 8 days (Fig. 2). In contrast, hypocotyl segments from seedlings exposed to 1°C for 2 days do not completely recover in vivo ACC oxidase activity after 4 and even 24 h of warming, and those from seedlings chilled at 1°C for 8 days remain unable to convert exogenous ACC to ethylene (Fig. 2).

125 r--------------,

CONTROL 100

2 DAYS AT 10·C

8 DAYS AT10·C -

75 f- 2 DAYS AT l·C

-

50 -

25 f- -

C 0 424 0 424 0 424 0 424

DURATION OF SUBSEQUENT WARMING (HOURS)

Figure 2. Effects of duration of wanning at 25°C on in vivo ACC oxidase activity in hypocotyl segments from Vigna radiata seedlings previously exposed for 2 and 8 days to 1 and 10°C. Results are expressed as percentages of ACC oxidase activity measured in h?'!'ocotyl segments from control unchilled seedlings (69.0±7.5 nl ethylene h· g.t FW). Means of3 replicates

279

The partial or complete recovery of in vivo ACC oxidase activity during warming chilled mung bean hypocotyls is associated with an increase in in vitro ACC oxidase activity, whereas such an increase is not observed with seedlings treated at 1°C for 8 days (Table 2). The restoration of in vitro ACC oxidase activity during warming is markedly inhibited in the presence of cycloheximide (Table 2), suggesting that it requires protein synthesis. These results are in agreement with data obtained by Etani and Yoshida [21] with the same plant material. Activation of in vivo ACC oxidase activity in rewarmed Granny Smith apples also requires de novo synthesis of protein [18].

The irreversible loss of in vivo ACC oxidase activity caused in hypocotyls of mung bean seedlings by chilling at 1°C is associated with an increase in electrolyte leakage and thereafter an enhancement of ethane production and an accumulation of MDA (Table 1). These events indicate deterioration of membrane integrity and activation of lipid peroxidation. Such a relation between loss of the ability of tissues to convert ACC to ethylene and alteration of membrane structure and organization has often been interpreted as the consequence of an association of the enzyme responsible for this reaction with cell membranes [8, 10, 27]. Considering the recent characterization of the ACC oxidase as a soluble enzyme [28, 29], the in vivo regulation of its activity might involve trans-membrane gradients [9].

TABLE 2. Changes in in vitro ACC oxidase activity in Vigna radiata hypocotyl segments warmed at 25°C for 4 and 24 h into water or in the presence of 0.5 mM cycloheximide (CH) after exposure of seedlings to 1 and 10°C for 2 and 8 days. Means of3 replicates ± SD.

Treatment of seedlings Duration In vitro ACC oxidase activity Temperature Duration of warming (01 ethylene h·1 g.1 FW) after warming eC) (days) (h)

withoutCH withCH

No treatment (control) 11.03±1.31 1 2 0 S.22 ± 1.10

4 6.03 ± 1.12 5.04±2.10 24 9.0S ±0.25 1.58 ± 0.81

8 0 1.72 ± 0.28 4 1.63 ± O.SI 1.58 ± 0.31

24 1.52 ± 0.31 1.21 ± 0.21

10 2 0 6.20 ± 0.94 4 8.39 ± 1.10 6.08 ± 1.05

24 12.32 ± 1.04 2.S9 ± 0.56

8 0 7.03 ± 0.43 4 8.72±0.SI 6.S7 ± 1.51

24 8.93 ±0.20 3.13 ± 0.40

4. Conclusion

As many organs of tropical and subtropical plants [1-3], Vigna radiata seedlings are very susceptible to chilling injury when temperature is lower than lOoC [21, 22]. A

280

sequence of cellular and metabolic damage is associated with chilling of chilling­intolerant tissues and strong evidence exists regarding the involvement of lipid peroxidation as an early response to chilling stress (Fig. 3). However, impairment of the ability of Vigna radiata hypocotyls to convert ACC to ethylene (i.e. in vivo ACC oxidase activity) during chilling results from both a deterioration of membrane properties and a decrease in the activity of ACC oxidase which seems to be chilling­sensitive.

The recovery of in vivo ACC oxidase activity during rewarming involves de novo protein synthesis and probably endocellular adjustments [18]. Critical features of chilling intolerance are probably the inability to maintain physiological properties of membranes and to limit free radical damage during chilling. In this point the scavenging enzymes and/or the antioxidant compounds might be essential. The possibility of repairing injUI)' upon rewarming, in particular to regain membrane integrity, would be also very important in resistance to chilling.

10C CHILLING 1 °c and 1 DOC

u~ \ ~ REDUCED \ \ PROTEIN

~ROX\AOON ~ liESO

LOSS OF ALTERATION OF MEMBRANE ACC OXIDASE INTEGRITY ACTIVITY

~/,,/ REDUCTION OF IN

VIVOACC OXIDASE ACTIVITY

Figure 3. Damage occurring during chilling and affecting the in vivo ACC oxidase activity in Vigna radiata seedlings placed at 1°C (a temperature which induces chilling injury) and lOOC (a non-chilling temperature). Dark arrows and pale arrows correspond to the effects of 1°C and 10°C, respectively.

5. References

1. Lyons, I.M. (1973) Chilling injury in plants, Annu. Rev. Plant Physiol. 24, 445-466.

281

2. Salveit, M.E. and Morris, L.L. (1990) Overview of chilling injury in horticultural crops, in C. Y. Wang (ed), Chilling injury of horticultural crops, CRC Press, Boca Raton, Florida, pp. 3-15.

3. Levitt, J. (1980)Responses of plants to environmental stresses, Vol. 1. Chilling,freezing and high temperature stresses, Academic Press, New York.

4. Omran, R.J. (1980) Peroxide levels and the activities of catalase, peroxidase, and indoleacetic acid oxidase during and after chilling cucumber seedlings, Plant Physiol. 65, 407-408.

5. Parkin, KL. and Kuo, S.-J. (1989) Chilling-induced lipid degradation in cucumber (Cucumis sativa L. cv Hybrid C) fruit, Plant Physiol. 90, 1049-1056.

6. McKersie, B.D. (1991) The role of oxygen free radicals in mediating freezing and desiccation stress in plants, in E.J. Pell and KL. Steffen (eds.), Active Oxygen/oxidative Stress and Plant Metabolism, ASPP, Rockville, pp. 107-118.

7. Prasad, T.K, Anderson, M.D., Martin, BA, and Stewart, C.R. (1994) Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide, Plant Cell 6, 65-74.

8. Odawara, SA, Watanabe, H., and lmaseki, H. (1977) Involvement of cellular membrane in regulation of ethylene production, Plant Cell Physiol. 18, 569-575.

9. John, P., Porter, AJ.R., and Miller, AJ. (1985) Activity of the ethylene-forming enzyme measured in vivo at different cell potentials, J. Plant Physiol. 121, 397-406.

10. Porter, AJ.R., Boriakogiu, J.T., and John, P. (1986) Activity of the ethylene forming enzyme in relation to plant cell structure and organization, J. Plant Physiol. 125,207-216.

11. Kimmerer, T.W. and Kozlowski, T.T. (1982) Ethylene, ethane, acetaldehyde, and ethanol production by plants under stress, Plant Physiol. 69, 840-847.

12. Wang, C.Y. (1989) Relation of chilling stress to ethylene production, in P.H. Li (ed.), Low Temperature Sstress Physiology in Crops, CRC Press, Boca Raton, Florida, pp. 167-189.

13. Cooper, W.C., Rasmussen, G.K, and Waldon, E.S. (1969) Ethylene stimulated by chilling in Citrus and Persea sp., Plant Physiol. 44, 1194-1196.

14. Wang, C.Y. and Adams, D.O. (1980) Ethylene production by chilling cucumbers (Cucumis sativus L.), Plant Physiol. 66, 841-843.

15. Chan, H.T., Sanxter, S., and Couey, H.M. (1985) Electrolyte leakage and ethylene production induced by chilling injury of papayas, H ortScience 20, 1070-1072.

16. Wang, C.Y. (1987) Changes ofpolyamines and ethylene in cucumber seedling in response to chilling stress, Physiol. Plant. 69, 253-257.

17. Corbineau, F., Engelman, F., and Come, D. (1990) Ethylene production as an indicator of chilling injury in oil palm (Elaeis guineensis Jacq.) somatic embryos, Plant Sci. 71, 29-34.

18. Larrigaudiere, C. and Vendrell, M. (1993) Short-term activation of the conversion of 1-aminocyclopropane-l-carboxylic acid to ethylene in rewarmed Granny Smith apples, Plant Physiol. Biochem. 31, 585-591.

19. Lelievre, J.-M., Tichit, L., Fillion, L., Larrigaudiere, C., Vendrell, M., and Pech, J.-C. (1995) Cold­induced accumulation of l-aminocyclopropane I-carboxylate oxidase proteins in Granny Smith apples, Postharvest BioI. Technol. 5,11-17.

20. Chen, Y.Z. and Patterson, B.D. (1985) Ethylene and l-aminocyclopropane-l-carboxylic acid as indicators of chilling sensitivity in various plant species, Aust. J. Plant. Physiol. 12, 377-385.

21. Etani, S. and Yoshida, S. (1987) Reversible and irreversible reduction of ACC-dependent ethylene formation in mung bean (Vigna radiata [L.] Wilczek) hypocotyls caused by chilling, Plant Cell Physiol. 28, 83-91.

22. Bagnall, D.J. and Wolfe, JA (1978) Chilling sensitivity in plants: Do the activation energies of growth processes show an abrupt change at a critical temperature,J. Exp. Bot. 29,1231-1242.

23. Simon, E. W. (1974) Phospholipids and plant membrane permeability, New Phytol. 3, 377-420. 24. Marangoni, AG., Palma, T., and Stanley, D.W. (1996) Membrane effects in postharvest physiology,

Postharvest BioI. Technol. 7,193-217. 25. Murata, T. (1990) Relation of chilling stress to membrane permeability, in C.Y. Wang (ed.), Chilling

Injury of Horticultural Crops, CRC Press, Boca Raton, Florida, pp. 201-209. 26. Kuo, S. and Parkin, KL. (1989) Chilling injury in cucumbers (Cucumis sativa L.) associated with

lipid peroxidation as measured by ethane evolution, J. Food Sci. 54, 1488-1491. 27. Mattoo, AK and Lieberman, M. (1977) Localization of the ethylene-synthesizing system in apple

tissue, Plant Physiol. 60, 794-799. 28. Ververidis, P. and John, P. (1991) Complete recovery in vitro of ethylene-forming enzyme activity,

Phytochem. 30, 725-727. 29. Fernandez-Maculet, J.C. and Yang, S.F. (1992) Isolation and partial characterization of the ethylene­

forming enzyme from apple fruit, Plant Physiol. 99, 751-754.

IMPACT ASSESSMENT FOR ETHYLENE EMISSIONS AT A PETROCHEMICAL SITE

T. R JACK! R K. McBRIEN! AND B. DOWSLEy2 INOVA Research & Technology Corp., 292816 St NE, Calgary, Alberta, Canada, 2Ecological Services for Planning Ltd., Guelph, Ontario, Canada

1. Introduction

NOVA Chemicals Ltd. is a major producer of ethylene and polyethylene in North America. While significant programs for reducing emissions are in place at all production facilities, ethylene releases do occur at certain sites. At the Moore petrochemical facility in Ontario, Canada, ethylene emissions have been traditionally assessed against a guideline established by lhe Ontario Ministry of Environment and Energy (OMEE). This guideline is intended to protect local vegetation from the effects of ethylene emissions and states a ground level concentration for ethylene below which vegetation impact is not anticipated. The current guideline is 120 micrograms ethylene/m3 (6 hour average).

Application of a fixed concentration guideline has several shortcomings. It creates a year round compliance effort by both industry and regulator even though the potential impact on local vegetation is confined to the growing season. The approach also focuses on concentration alone while risk to vegetation depends on dosage which is a function of both concentration and time of exposure. Further variations in ethylene sensitivity due to synergies between the action of ethylene and other factors such as ground level ozone, sulfur dioxide, herbicides or various environmental conditions are ignored. These shortcomings can be addressed by directly assessing vegetation for ethylene effects around a petrochemical facility.

2. Vegetation Impact

In 1995, ground level concentration monitoring at the Moore site was supplemented by a field study to assess the potential impact of ethylene emissions on vegetation. This provided an opportunity to shift from simple regulatory compliance with a fixed concentration guideline to a risk management approach based on direct assessment of possible damage. Risk based management offers a more realistic and effective way to protect the environment provided emission sources, dispersion and receptor responses are understood.

283 A. K. Kanellis et al. (etis.), Biology and Biotechrwlogy of the Plant Hormone Ethylene, 283-288. @ 1997 Kluwer Academic Publishers.

284

The vegetation impact study was based on the use of indicator plants located in five plots. Three of the plots, referred to here as the "test" plots, were located near or on the petrochemical site, north, south and east of the production facility. Two of the plots, the "control" plots, were located at distances of 3 and 12 kIn away in locations unlikely to receive emissions from the Moore facility. The strategy was to compare observations made in the test plots with those of the control plots to see if proximity to the petrochemical facility resulted in measurable effects which could be related to ethylene emissions. The study was founded on extensive field work carried out by the OMEE in the late 1970's and early 1980's [1].

Indicator plants included two cultivars of petunia (White Joy and White Cascade), cucumbers (Straight Eight) and soybeans (Asgrow 2242). Petunias and cucumbers were started in a greenhouse and tranplanted into raised beds of a common soil trucked to each plot. Soybeans were seeded into the local soil at each plot after existing vegetation had been removed, nutrient added and the soil rototilled. The soybean seeds were treated with a commercial rhizobial inoculant. Petunia and cucumber beds were regularly weeded and watered while the soybeans were grown under ambient conditions. Two of the test plots were adjacent to air monitoring stations which continuously track ground level ethylene concentrations by gas chromatography with a sensitivity of 10 ppb (w/v).

The cultivars of petunia, White Joy and White Cascade have a well characterized response to ethylene exposure [1]. These cultivars have different sensitivities to ethylene, White Joy being more sensitive. Comparing cultivar responses allowed generic factors such as local variations in rainfall to be distinguished from ethylene specific effects. For petunias, flower diameter and bloom lifespan were monitored as indicators of ethylene exposure. Bloom lifespan was calculated for six permanently tagged plants of each cultivar at each plot using the method of Harper and Jones [1]. The lifespan for a given measurement period was taken as being equal to the number of live blooms at the previous measurement period divided by the number of dead blooms in the current measurement period, multiplied by the number of days since the previous measurement period. Dead blooms were removed by pinching the pedicel below the sepals to ensure that they were not recounted in the next observation period. Bloom diameters were measured on six randomly chosen plants of each cultivar in each plot by holding the bloom between two fingers and laying a ruler across the face of the bloom. Fifteen randomly selected blooms were measured on each plant. Early in the study, when a few plants did not have fifteen blooms, all blooms were counted.

Cucumbers were chosen for the study because they are popular in local gardens. The ratio of female to male cucumbers flowers was monitored on up to twenty plants at each plot. As plants became excessively large and tangled they were pruned back to one vine to facilitate data collection.

Measurements on petunias and cucumbers were recorded weekly between mid-June and mid-September except for two weeks in the growing season when more frequent measurements were done.

Soybeans, while not a sensitive indicator plant for ethylene effects [1], were included in the study due to their local commercial importance. Soybeans are routinely cultivated immediately adjacent to the petrochemical site and even on spare land on the

285

site itself Internode stem lengths and the number of bean pods produced per plant on up to twenty marked plants in each plot were monitored weekly. The final yield was measured for plants in an area of 3m x 2m in the interior of one test and one control soybean patch to avoid edge effects. Harvested plants were clipped off at ground level and fed into a small plot combine. The beans were collected and weighed.

Data were analyzed statistically using Costat (CoHort Software, Berkeley, California) and Systat (Intelligent Software, Evanson, Illinois) software. Means were calculated for each recorded variable for each of the five experimental plots. Multiple comparisons of the means were made using the Tukey-Kramer procedure [3]. Plot mean values were considered statistically different from one another when the difference exceeded the Least Significant Difference (LSD) value calculated for each measurement date.

Measurements made in three test plots on or near the petrochemical facility and two control plots at a distance included: - petunias: flower diameter and lifespan (two cultivars) - cucumbers: female/male flower ratio - soybeans: internode stem lengths and pods per plant

Soybean yields were measured at one control plot and one test plot at the end of the study.

2.1. PETUNIAS AND CUCUMBERS

At no time did the plants in the test plots near or on the petrochemical site appear different visually than those in the control plots far from the facility. Indeed in most observation periods, statistically significant differences were not observed between any of the five plots for any of the measurements in the study, Table 1.

TABLE 1. Comparison of results for test plots and control plots for petunias and cucumbers

Observation # of times Test plot < Control

Petunias (White Joy) -bloom diameter 51•2

-lifespan 32

Cucumbers -ratio female/male flowers 0

# of times Control < Test plot

2 1

# times without significant difference

110f14 50f8

70f9

IWhite Cascade showed similar effect in the same plot within 24 h on two of these occasions suggesting that ethylene was not the cause of the effect in these instances. 2 Anticipated response for ethylene.

Normalized bloom diameter measurements for each petunia cultivar recorded during two separate weeks of intensive measurements were subjected to cluster

286

analysis. Systat software was used to create dendrograms to test the relatedness of the results for the five plots using the Euclidean distance single linkage method. Cluster analysis was also applied using the normalized bloom diameter values with two clusters specified. In these analyses, the plots were clustered together to minimize overall variance [4]. In both treatments, control and test plots were intermixed and did not cluster separately. This indicates that for the two one week periods of intensive monitoring, proximity to the petrochemical site was not a key factor in determining petunia bloom size. This suggests that ethylene did not impact local vegetation on a continuous or even routine basis. Closer inspection of the weekly data was needed to assess whether occasional short lived episodes of ethylene exposure caused temporary effects in the plots.

Cases where significant differences were observed in weekly monitoring data between at least one test plot and at least one control plot are presented in Table 1. In eleven instances a significant difference was seen but it was not always a test plot which had smaller, shorter lived petunia flowers or a higher proportion of female cucumber flowers. This suggests that factors other than ethylene exposure affect these measurements at the level of distinction achieved in this study. The distribution of responses between test and control plots suggests that the differences seen are more likely due to background variability than exposure to ethylene emissions coming from the petrochemical facility. This hypothesis was confirmed by comparing vegetation observations with the actual occurrence of ethylene exposures at the plots in question (see Section 3.2. below).

2.2. SOYBEANS

Soybeans proved difficult to monitor on a weekly basis. Due to an unusually wet spring the soybeans were seeded at different times over a two week period in the various plots. Variation in emergence resulted in the plants in the five plots developing at different rates. The plants in various plots began to develop bean pods at different times in the weekly monitoring. Further, three of the plots (one control and two test plots) did not have uniform seed germination. This resulted in gaps in the canopy of the soybean plot which allowed plants to grow laterally rather than being confined to vertical growth by contiguous neighbors on all sides. These differences in growth pattern and stage of development confused weekly data precluding any detailed interpretation in terms of possible ethylene exposures over the course of the study. In general by the end of the study period, average internode lengths and the average number of pods per plant in test plots near the petrochemical site were greater than or indistinguishable from the corresponding measurements for control plots (Tables 2 and 3).

Two plots (one control and one test plot) had uniform plant densities with a closed leaf canopy comparable to commercial fields. These plots were assayed for yield. Comparison of a test plot near the petrochemical site with a control plot 12 km distant showed that yields were similar (2.9 and 2.7 tones/ha respectively at 7% moisture). These yields are slightly in excess of the three year average for the region [2] and indicate that the yield was not affected by proximity to the petrochemical complex.

TABLE 2. Soybean internode lengths (em)

PWT INTERNODE 11 INTERNODE 12 INTERNODE 13 Mean n s. d Mean n s. d Mean n s. d.

Control 6.4" 20 2.4 5.0 .. b 16 1.9 4.1 .. b 11 1.7 (12km)

4.3b Control 15 1.2 4.1" 14 1.5 3.4" 10 1.0 (3km)

6.2b., South Test 6.1" 20 1.2 20 0.95 6.0' 20 1.5 East Test 6.2" 20 0.91 6.3b., 20 1.2 6.0' 19 0.85 North Test 8.4' 19 2.0 6.7' 18 2.4 5.5b., 14 2.4

s. d. = standard deviation. n = sample size. a,b,c: means followed by the same letter are not significantly different Tukey-Kramer: for node 11, LSD (0.05) = 1.04; for node 12, LSD (0.05) = 1.04 and for node 13, LSD (0.05) = 0.99.

TABLE 3. Number of bean pods per plant for soybeans

PWT Number ofPodsIPlant

Control (12 km) Control (3 km) South Test Plot East Test Plot North Test Plot

Mean

458

33" 69b

65b

36" s.d. = standard deviation. n = sample size.

n s. d.

20 14 20 11 20 25 20 24 20 13

a,b,c: means followed by the same letter are not significantly different. Tukey-Kramer: for number of pods, LSD (0.05) = 11.5.

3. Correlation with Ethylene Emissions

287

To confirm the irrelevance of ethylene to the significant differences seen in vegetation plot measurements (Table I), measured ground level concentrations of ethylene were calculated as six hour averages over the entire study period for the two test plots adjacent to air monitoring stations.

No correlation was seen between periods of ethylene exposure as measured by gas chromatography and the observation of vegetation effects. On two occasions at each of these plots, ethylene concentrations exceeded the OMEE guideline for periods of up to four or six hours without any corresponding observation of ethylene effects in the petunia or cucumber plants. Conversely on occasions when reduced petunia size or lifespan was seen in a test plot relative to a control plot or the ratio of female to male cucumber blooms was greater in a test plot than a control plot (Table 1), ethylene concentrations remained below the guideline level or even at background levels. Failure to see any correlation between ethylene exposures exceeding guideline and vegetation impact effects suggests that a guideline of 120 microgramslm3 (6 hour

288

average) is conservative and that the effects seen in this study are indeed due to factors other than ethylene as suggested above.

This discrepancy between guideline expectations and observations may be related to the distinction between concentration and dosage. Ethylene exposures near the petrochemical complex generally occur in short lived spikes lasting only a few hours at any specific location. Such short exposures can give notable six hour average concentrations without perhaps persisting long enough within the six hour period to provoke a response even in sensitive vegetation. Further dose response information is needed to establish a meaningful correlation between ethylene exposure and observations of vegetation effects in the field.

4. Conclusions

Direct assessment of ethylene effects in vegetation test plots on or near the petrochemical site and in control plots remote from the facility showed that proximity to the site was not a factor based on measurement of petunia bloom diameter or lifespan, cucumber flower sex ratio or soybean yields.

No correlation was seen between ethylene exposure based on gas chromatograph measurements at two of the test plots and the vegetation effects observed.

A guideline for ground level concentration of ethylene set at 120 microgramslm3 (6 hour average) was found to be conservative. This may be due to the distinction between concentration and dosage.

5. Acknowledgments

The authors wish to acknowledge the assistance of Randy Jones, phytotoxicologist with the OMEE whose extensive past experience [1] assessing field effects of ethylene on vegetation provided valuable guidance and Professor B. Hale of the University of Guelph who advised on experimental design and data interpretation.

6. References

1. Harper, D.S. and Jones, R.D. (1984). Ethylene-related Vegetation Assessment Studies in the Sarnia Area; 1976-1981, . ARB-55-84-PHYTo. Air Resources Branch, Phyotoxicology Section, Ontario Ministry of the Environment, Toronto.

2. OMAFRA (1994). 1993 Agricultural Statistics for Ontario. Publication 20. Ontario Ministry of the Agriculture, Food and Rural Affairs, Toronto.

3. Sokal, R. and Rohlf, F. J. (1981). Biometry. The Principles and Practice. Statistics in Biological Research. Second Edition. W. H. Freeman and Company. New Yark.

4. Wilkinson, L. (1988). SYSTAT. The System for Statistics. Systat Inc., Evanston.

POTENTIAL APPLICATIONS OF CONTROLLING ETHYLENE SYNTHESIS AND PERCEPTION IN TRANSGENIC PLANTS

H. J. KLEE AND D. TIEMAN Department of Horticultural Sciences, 1143 Fifield Hall, University of Florida, Gainesville, Florida 32611, USA

1. Introduction

The actions of hormones such as ethylene on plant developmental processes can be modulated at several levels. There is ample evidence that regulation of hormone biosynthesis and catabolism are critical to many aspects of growth and differentiation. Researchers have also speculated that hormone action must be mediated at the level of sensitivity [3,25]. Differential sensitivity can be exhibited in two contexts. First, adjacent cells in an organ can respond differentially to a hormone as in abscission layer formation. Second, sensitivity of an organ to a hormone can change over time as in fruit ripening. How a plant regulates the variation in tissue or organ sensitivity to plant hormones spatially and temporally is not clear. Here, we describe our current knowledge of how sensitivity to ethylene changes through development of tomato fruit. Further, having access to genes that mediate ethylene sensitivity, we can ask whether we can take these genes and manipulate ethylene sensitivity in heterologous plant species i.e., do components of the ethylene signal transduction pathway work in a "mixed" system?

2. Ethylene Synthesis

Ethylene plays an important regulatory role in integrating the developmental effects of internal signals and external stimuli in plants. It has been implicated as having a major role in regulating such diverse developmental processes as seed germination, fruit ripening, abscission and senescence [I}. Ethylene is induced in response to most external stresses and usually causes a slowing of the plant's growth rate in what is perceived as less than optimal growth conditions. The ethylene biosynthetic pathway is well characterized [16}. S-adenosylmethionine is converted to l-aminocyclopropane-1- carboxylate (ACC) and then to ethylene by the successive action of ACC synthase and ACC oxidase. The ethylene biosynthetic pathway appears to be regulated at the level of ACC synthase gene transcription [13,17,20} and distinct ACC synthase genes

289

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 289-297. © 1997 Kluwer Academic Publishers.

290

respond to different developmental and environmental stimuli by controlling the availability of ACC [10,20,24].

In climacteric fruits, ethylene promotes ripening by coordinately inducing the expression of many genes whose products are involved in the ripening process [14,15,23]. Ethylene synthesis can be effectively reduced by the use of antisense genes for either ACC synthase [18] or ACC oxidase [7] or by expression of a bacterial ACC­degrading enzyme, ACC deaminase [11]. In ethylene inhibited fruit the time that it takes to proceed from breaker stage (the stage at which the fruit first exhibits external color change) to full red is inversely proportional to the rate of ethylene synthesis (H. Klee et al., unpublished). We have produced many hundreds of transgenic ethylene­reduced tomato plants by the introduction of antisense ACC synthase, antisense ACC oxidase and the ACC deaminase gene. The most extreme example is illustrated by the antisense ACC synthase fruit described in Oeller at al. [18]. These fruits are 99% inhibited for ethylene synthesis and never fully ripen without exogenous ethylene application. Progressively higher rates of ethylene synthesis result in faster and more complete ripening. Although the introduction of any of these genes yield fruit that are delayed in ripening, the antisense ACC synthase and ACC oxidase genes are more likely to produce extreme fruits that never fully ripen. Introduction of the ACC deaminase gene is less effective in the complete elimination of ethylene synthesis, presumably because the enzyme is competing with the fairly abundant ACC oxidase for substrate. However, lines with extremely reduced ethylene levels are not necessarily commercially desirable since they require continuous exposure to high levels of ethylene over an extended period to achieve full ripening. Thus, the actual commercial target is a fruit that is only partially reduced in ethylene synthesis and will exhibit delayed ripening without the necessity for prolonged applications of ethylene for ripening. With this conclusion in mind, it appears that any of these three transgenes should deliver a commercially useful extended shelf life fruit.

J. Ethylene Perception

Although ethylene acts to coordinate and hasten the ripening process of climacteric fruits, several lines of evidence suggest that other levels of control precede the increase in ethylene synthesis. Experiments conducted on avocado [5], apple [8], and tomato [28] indicate that immature fruits do not respond to ethylene by ripening. These fruits do perceive ethylene since some ethylene-inducible genes such as ACC oxidase are activated when fruits are exposed to ethylene but they do not initiate the developmental sequence that leads to ripening. Even in mature tomato fruits which ripen more quickly when exposed to exogenous ethylene, ripening does not initiate uniformly. Rather, it proceeds from the locules to the pericarp. Then, pericarp ripening proceeds from the blossom end to the stem end. Since ethylene is readily diffusible within the fruit, a reasonable explanation for asynchronous ripening is differential regulation of ethylene sensitivity. Thus, it appears that fruit and fruit tissues can respond to exogenous ethylene in different ways depending on their developmental state.

291

Differential developmental control of ethylene sensitivity is not limited to fruits. In many flowers, tissue responsiveness to ethylene increases with maturation and the mechanisms controlling the differential responsiveness are likely to be similar, if not identical, with those controlling fruit ripening. In the tomato Nr mutant, flower senescence and abscission are affected along with fruit ripening, indicating that a single gene can control these processes. Typically, flowers begin to respond to ethylene at the time that the stigma becomes receptive to pollen. For example, young carnation flowers are essentially unresponsive to exogenous ethylene while mature flowers exhibit petal inrolling and older flowers respond by wilting and initiating autocatalytic ethylene synthesis [2]. Similar development of ethylene responsiveness can be observed in geranium flowers [6].

Most of our knowledge of the ethylene signal transduction pathway is based on genetic analyses performed inArabidopsis. The combination of excellent genetics and a simple and reliable assay has resulted in identification of a large number of ethylene insensitive mutants. The primat)' screen involves germinating seeds in the dark in the presence of ethylene or ACC. Wild-type seeds germinated in these conditions exhibit the so called triple response: shortened and thickened hypocotyls, inhibition of root elongation, and a pronounced apical hook. Ethylene insensitive mutants, in contrast, behave like seedlings germinated in the absence of ethylene or ACC with narrow elongated hypocotyls and roots, with no apical hook. Several of the genes involved in the ethylene signal transduction pathway, including etr 1, have been isolated using positional cloning.

3.1. ETRI

Recent developments have focused on what appears to be the first component of the ethylene signal transduction cascade, ETRI. All of the mutant alleles of ETRI are dominant and genetic analyses indicate that it is at or near the earliest step in ethylene perception. The gene encoding ETRI was isolated using map based cloning [4]. Sequence comparisons indicated that ETRI is homologous to a class of prokaryotic proteins involved in signal transduction referred to as two component histidine kinases. In bacteria, this class of proteins is responsible for sensing many environmental cues such as the presence ofa variety of nutrients or changes in osmolarity [19].

By analogy to the bacterial two component proteins, the ETRI protein can be divided into three distinct domains, represented schematically in Figure 1. The first domain, consisting of approximately amino acids 1- 313, encodes a sensor domain that is responsible for ligand binding. This domain contains three hydrophobic, potential membrane spanning regions. All of the known mutations in ETRI causing ethylene insensitivity lie within this region of the protein. The second ETRI domain encodes a putative autophosphorylating histidine kinase. Finally, the third domain, called either the receiver or response regulator, contains an aspartic acid residue that can act as a receiver for the phosphate from the histidine kinase domain. Although the third domain can act as a phosphate receiver in bacteria, it is not responsible for transducing the signal from the histidine kinase to the next step in the phosphorylation cascade.

292

Indeed, the response regulator domain is not even present in some two component systems [27]. Rather, it may act as a modulator of phosphate transfer to the actual receiver, which is a separate entity. In this way, the response regulator domain might act as a buffer for signal transduction by acting as a dead end for phosphate transfer.

In bacteria, the histidine kinases act as dimers with one subunit phosphorylating the other. Schaller et al. [22] have shown that ETRJ also forms dimers. This dimerization could account for the observation that all of the known mutations in ETRJ are dominant. Presumably, a mutant subunit of ETRJ can inactivate a wild type subunit by forming an unproductive dimer. While such a model can explain the data, it has yet to be demonstrated conclusively. Isolation of only dominant mutations could also be due to functional redundancy in the receptor gene family (see below). It is also possible that the ETRJ protein acts to negatively regulate the pathway in the absence of ethylene. In this model, ethylene would bind to ETRJ, leading to shutoff of the protein and a derepression of the pathway. A mutant unable to bind ethylene would be locked in the "on" position, effectively preventing derepression. Definitive biochemical proof that the ETR1 gene encodes an ethylene receptor has been obtained. Schaller and Bleecker [21] expressed the ETRJ protein in yeast cells and demonstrated that the protein binds ethylene. The dosage-dependent binding in yeast parallels the curve for growth inhibition responses for ethylene in Arabidopsis. Ethylene binding was localized to the amino terminal hydrophobic domain and was eliminated in the ethylene unresponsive Etr J -J mutant protein.

3.2. THE TOMATOETRJ GENE FAMILY

While a great deal of effort has gone into understanding ethylene synthesis during fruit ripening, very little attention has been paid to ethylene perception. We initiated a search for tomato mutants that were altered in their ability to perceive ethylene. Never ripe (Nr) is a semi-dominant mutation originally identified by the inability of its fruit to undergo ripening. Our analysis of Nr indicated a number of pleiotropic effects indicative of ethylene insensitivity throughout the plant [12]. For example, hypocotyl elongation is not inhibited by exogenous ethylene. The mutant is greatly impaired in pedicel abscission. There are also significant delays in leaf and flower petal senescence. In the Pearson cultivar, Nr fruit are phenotypically indistinguishable from the best ACC synthase antisense line, A1l.1, described by Oeller et al. [IS].

Efforts were next focused on identification of the ETRJ homologous sequences in tomato with an emphasis on the Nr mutant. The Arabidopsis ETRJ genomic clone was used to probe southern blots of DNA from an F2 segregating population derived from a cross between Lycopersicon esculentum and L. pennellii. Five independent hybridizing sequences mapping to chromosomes 7,9, 10, 11 and 12 were identified [29]. It has yet to be demonstrated that all five loci contain functional ETRJ homologous sequences. However, the first three genes that have been cloned all contain very significant ETRJ sequence identity. What was particularly significant about the mapping is that one of the loci is tightly linked to the Nr mutation on chromosome 9, within O.S cM. The

293

phenotypic similarity of etr 1-1 and Nr strongly suggested that the molecular basis of Nr is a mutation in an ETRI homologue.

Figure 1. Schematic diagram of the domains of the Arabidopsis Etrl and ERS proteins compared to the three

available tomato ETR-homologous genes. The sensor domain contains three regions capable of spanning a

membrane. The signalling domain contains the histidine kinase region that becomes phosphorylated and then

transfers the phosphate to the aspartate of an unknown substrate. The receiver domain contains an aspartate (D)

that is capable of receiving the phosphate from the histidine kinase. Numbers in each block represent the percent

similarity and identity, respectively, of the amino acids in each region of a protein to that ofETRl.

Screening of a ripening tomato fruit cDNA library was accomplished by hybridization with the full length Arabidopsis ETRI genomic clone. A full length cDNA encoding a protein with similarity to ETR1 was identified. A gene-specific hybridization probe enabled determination of the map location as being chromosome 9, near the Nr locus. The predicted protein encoded by this cDNA is 68% identical and 81% similar to the ETRI sequence. However, the protein lacks the response regulator domain found in ETR1 (Fig. 1). Significantly, all four of the amino acids that, when changed, result in dominant mutations in the Arabidopsis gene are conserved in the tomato protein. DNA sequence comparisons of the tomato Nr mutant and isogenic wild type gene revealed a single nucleotide change that causes a proline to leucine

294

switch at amino acid 36 in the Nr mutant protein. This proline is conserved in the Arabidopsis ETRI protein. When the mutant tomato cDNA under the control of the constitutive CaMV 35S promoter was introduced into wild type tomato plants, the resulting transgenic plants exhibited ethylene insensitivity, conclusively demonstrating that the Nr phenotype is caused by a mutation in this ETRI-related member of the ethylene receptor family [26].

The NR gene of tomato lacks the response regulator domain found in the Arabidopsis ETRI protein. A similar gene, designated ERS (ethylene response sensor) [9], has been identified in Arabidopsis. This protein also lacks a response regulator domain and mutations in ERS can confer dominant ethylene insensitivity. Conversely, we have characterized two additional tomato gene family members, LeETRI and LeETR2, both of which contain response regulator domains (Figure 1). Thus, the family of ethylene receptors appears to be complex both in structure and number.

3.3. NORTHERN ANALYSIS OF FRUIT NREXPRESSION

We used the NR cDNA as a probe to examine the expression pattern of NR mRNAs during fruit ripening. RNA from green and ripening fruit of wild-type and mutant tomato lines was prepared. Ethylene inducibility in mature green fruit was also examined. We found that the NR mRNA is both developmentally regulated and ethylene inducible. NR gene expression increases substantially from green to ripening stages and is blocked in the Nr mutant. NR mRNA expression is induced by ethylene treatment in mature green fruits but not immature green fruits. The data are consistent with a model in which NR gene expression is part of the feedback induction that occurs during ripening. Thus, not only would ethylene synthesis be positively feedback regulated but ethylene perception would also be upregulated in response to ethylene. At this time it cannot be determined whether higher levels of receptor would have a positive or a negative effect on ethylene response since the NR protein could either activate or suppress downstream signal transduction components but ripening occurs as levels of the receptor increase.

The existence of a class of genes, represented by NR and E8, that are ethylene regulated in a developmentally dependent manner illustrates the fundamental change in ethylene sensitivity that must occur upon achieving competence to ripen. That the ethylene receptor itself is regulated in this manner is particularly intriguing. Developmental regulation of the ethylene signal transduction pathway would provide a molecular basis to explain the early observations of differential ethylene responsiveness of immature versus mature tomato fruits. It is important to note that expression of NR is not precisely like E8. While ethylene inducibility is developmentally controlled, there is a detectable basal level of expression of NR in other tissues. But when the fruit must rapidly and coordinately express many genes, the NR gene product is greatly increased. One possible explanation for the differential ethylene responsiveness of immature and mature fruit tissue may be quantitative. The components of ethylene signal transduction may be rate limiting to some but not all ethylene regulated genes in immature fruits. Also, different ETR1-homologous proteins may affect different

295

pathways. While this concept must still be tested, it does appear that we must expand the concept of climacteric autocatalytic ethylene synthesis to include the ethylene receptor.

4. Transgenic Plants Altered in Ethylene Perception

In parallel, we have introduced both the mutant Nr gene and the Arabidopsis etr 1-1 mutant cDNA under the control of the CaMV 35S promoter into tomato plants and assessed the effects of the trans genes on the ability of these plants to respond to ethylene. Our logic in undertaking these parallel experiments was that the heterologous Arabidopsis protein might not effectively interact with the next downstream component of ethylene signal transduction and that the homologous tomato gene might be necessary for effective control. This is not the case. Both the Arabidopsis etrl-l (Wilkinson et al., manuscript submitted) and the tomato Nr mutant [26] genes work effectively in transgenic tomatoes to confer ethylene insensitivity. Transgenic plants expressing the Arabidopsis elr 1-1 mutant cDNA are phenotypically indistinguishable from the Nr mutant plants in terms of seedling triple response, flower abscission and petal wilting, and fruit ripening.

As further proof that the Arabidopsis mutant gene could work in a heterologous plant species, the gene has been introduced into petunia plants. In petunia, flower corolla wilting typically occurs within 48 h after pollination and is known to be an ethylene-mediated response to a pollination-associated signal. Transgenic plants expressing the etr 1-1 protein exhibited no ethylene-induced corolla wilting either following pollination or in response to exogenously applied ethylene. Thus, it appears that the transgene will be effective in increasing shelf life substantially for many floricultural crops that senesce in response to ethylene.

5. Conclusions

There is clearly hormonal regulation of fruit development at the level of perception and strong evidence that regulation of ethylene perception is a major control point in the commitment to fruit ripening. The tomato fruit is an excellent model for differential regulation of hormone perception during development. It has long been postulated that phytohormone effects could be modulated at the level of tissue sensitivity. We can state that the concept of autocatalytic ethylene regulation must be modified to include not only synthesis of the hormone but modulation of the receptor. As our understanding of the regulation of the tomato ethylene receptor family advances, it should be possible to unambiguously define the role of ethylene and ethylene perception in fruit ripening and senescence. Understanding the regulation of this signal transduction pathway will lead to an ability to selectively block processes in plant development to create plants with greater economic value. Finally, the manipulation of ethylene perception by transgenes has progressed from theory to reality.

296

6. References

1. Abeles, F.B., Morgan, P.W., and Saltveit, M.E. (1992) Ethylene in Plant Biology, Academic Press, San Diego, pp. 264-296.

2. Barden, L. E. and Hanan, J. J. (1972) Effect of ethylene on carnation keeping life, J. Am. Soc. Hortic. Sci. 97,785-788.

3. Bradford, K. and Trewavas, A (1994) Sensitivity thresholds and variable time scales in plant honnone action, Plant Physiol. 105, 1029-1036.

4. Chang, C., Kwok, S. F., Bleecker, A B., and Meyerowitz, E. M. (1993) Arabidopsis ethylene­response gene ETRJ: similarity of products to two-component regulators, Science 262, 539-544.

5. Eaks, I. L. (1980) Respiratory rate, ethylene production and ripening response of avocado fruit to ethylene or propylene following harvest at different maturities, J. Am. Soc. Hortic. Sci. 105, 744-747.

6. Evensen, K, B. (1991) Ethylene responsiveness changes in Pelargonium X domesticum florets, Physiol. Plant. 82,409-412.

7. Hamilton, A, J., Lycett, G. W., and Grierson, D. (1990) Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants, Nature 346, 284-287.

8. Harkett, P., Hulme, A, Rhodes, M., and Woohorton, L. (1971) The threshold value for physiological action of ethylene on apple fruits, J. Food Technol. 6,39-45.

9. Hua, J., Chang, C., Sun, Q., and Meyerowitz, E. M. (1995) Ethylene insensitivity conferred by Arabidopsis ERS gene, Science 269,1712-1714.

10. Kieber, J. J. and Ecker, J. R. (1993) Ethylene gas, it's not just for ripening anymore, Trends in Genetics 9, 356-362.

11. K1ee, H. J., Hayford, M. B., Kretzmer, K. A, Barry, G. F., and Kishore, G. M. (1991) Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants, Plant Cell 3, 1187-1193.

12. Lanahan, M. B., Yen, H. -C., Giovannoni, J. J., and K1ee, H. J. (1994) The Never ripe mutation blocks ethylene perception in tomato, Plant Cell 6, 521-530.

13. Liang, X, Abel, S., Keller, J, A, Shen, N. F., and Theologis, A (1992) The l-aminocyclopropane-l­carboxylate synthase gene family of Arabidopsis thaliana, Proc. Natl. Acad. Sci USA 89, 11046-11050.

14. Lincoln, J. E. and Fischer, R. L. (1988a) Regulation of gene expression by ethylene in wild-type and rin tomato (Lycopersicon esculentum) fruit, Plant Physiol. 88,370-374.

15. Lincoln, J. E. and Fischer, R. L. (1988b) Diverse mechanisms for the regulation of ethylene-inducible gene expression, Mol. Gen. Genet. 212, 71-75.

16. McKeon, T. and Yang, S. F. 1987 Biosynthesis and Metabolism of Ethylene, in P. Davies (ed.), Plant Hormones and Their Role in Plant Growth and Development, Martinus Nijhoff, Boston, p. 94-112.

17. O'Neill, S., Nadeau, J. A, Zhang, X S., Bui, A Q., and Halevy, A H. (1993) 1nterorgan regulation of ethylene biosynthetic genes by pollination, Plant CellS, 419-432.

18. Oeller, P. W., Min-Wong, L., Taylor, L. P., Pike. D. A, and Theologis, A (1991) Reversible inhibition of tomato fruit senescence by antisense RNA, Science 254,437-439.

19. Parkinson, J. (1993) Signal transduction schemes of bacteria, Cell 73, 857-871. 20. Rottmann, W. H., Peter, G. F., Oeller, P. W., et al. (1991) l-aminocyclopropane-l-carboxylate

synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence, J. Mol. Bioi. 222, 937-961.

21. Schaller, G. E. and Bleecker, A B. (1995) Ethylene binding sites generated in yeast expressing the Arabidopsis ETRJ gene, Science 270, 1809-1811.

22. Schaller, G. E., Ladd, A N., Lanahan, M. B., Spanbauer, J. M., and Bleecker A B. (1995) The ethylene response mediator ETRI from Arabidopsis forms a disulfide-linked dimer, J. Bioi. Chem. 270,12526.

23. Slater, A, Maunders, M. J., Edwards, K., Schuch, W., and Grierson, D. (1985) Isolation and char.wterization of cDNA clones for tomato polygalacturonase and other ripening-related proteins, Plant Mol. BioI. 5,137-147.

24. Theologis, A (1993) One rotten apple spoils the whole bushel: the role of ethylene in fruit ripening, Cell 70, 181-184.

25. Trewavas, A (1983) Is plant development regulated by changes in the concentration of growth substances or by changes in the sensitivity to growth substances? TIBS 8,354-357.

26. Wilkinson, J. Q., Lanahan, M. B., Yen, H. -C., Giovannoni, J. J., and K1ee, H. J. (1995) An ethylene­inducible component of signal transduction encoded by Never-ripe, Science 270, 1807-1809.

297

27. Winans, S., Mantis, J., Chen, C., Chang, C., and Han, D. (1994) Host recognition by the virA, virG two-component regulatory proteins of Agrobacterium tumefaciens. Research in Microbiology 145:461-473.

28. Yang, S. F. (1987) The role of ethylene and ethylene synthesis in fiuit ripening, in W. Thompson, E. Nothnagel, and R. Huffaker (eds.), Plant Senescence: Its Biochemistry and Physiology, The American Society of Plant Physiologists, Rockville, MD, p. 156-165.

29. Yen, H. -C., Lee, S., Tanksley, S., Lanahan, M., Klee, H., and Giovannoni, J. (1995) The tomato Never-ripe locus regulates ethylene-inducible gene expression and is linked to a homologue of the Arabidopsis ETRJ gene, Plant Physiol. 107, 1343-1353.

REGULATION OF ETHYLENE SYNTHESIS AND PERCEPTION IN TOMATO AND ITS CONTROL USING GENE TECHNOLOGY

C.S. BARRY, B. BLUME, A. HAMILTON, R FRAY, S. PAYTON, A. ALPUCHE-SOLIS AND D. GRIERSON BBSRC Research Group in Plant Gene Regulation, The University of Nottingham, Sutton Bonington Campus, Loughborough, LEI2 5RD, UK

1. Ethylene Biosynthesis

Ethylene synthesis in plants is regulated by two enzymes, ACC synthase and ACC oxidase [l3]. Both enzymes appear to be encoded by multigene families in several plant species and individual members have been shown to exhibit differential regulation [2, 9, 14, 16, 17, IS, 20, 23, 24, 27, 2S]. Such diversity allows the plant the potential to regulate ethylene synthesis in a flexible manner in different organs throughout development and in response to various environmental stimuli and stresses. As a means to begin to understand the regulation of ethylene synthesis we have focused on the isolation, identification and characterisation of ACC oxidase genes from tomato.

ACC oxidase was first identified by reverse genetics. The ripening-related eDNA clone, TOMl3, inserted in the antisense orientation and expressed in transgenic tomatoes under the control of a constitutive promoter, was shown to down regulate endogenous ACC oxidase activity in a gene dosage dependent manner [6]. Functional expression of TOMl3 and a related clone, pHI'OMS, in transgenic yeast and Xenopus oocytes respectively, revealed that both clones were able to convert ACC to ethylene with the stereospecificity of the endogenous enzyme activity [7, 26]. Three ACC oxidase genes have been isolated from tomato using the TOMl3 eDNA probe [9]. These are now called LEA COI , LEAC02 and LEAC03. They are very similar in structure having four exons separated by three introns. The protein coding regions of these genes show a high degree of sequence identity to one another [2, 3], however, the 5'-flanking sequences share very little homology, indicating that the three genes may be differentially regulated. The 3' -untranslated regions of each of the genes also show very little homology to one another. These have been cloned and used as gene-specific probes in conjunction with the ribonuclease protection assay to analyse tomato ACC oxidase gene expression during fruit ripening and in leaves during senescence and in response to mechanical wounding [2]. The quantitative analysis of these results is summarised in Table 1.

299

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 299-306. © 1991 Kluwer Academic Publishers.

300

TABLE 1. Relative abundance of ACC oxidase transcripts in leaves and fiuit

Gene Leaves' Fruitb

U W 0 M B + + W S G R 3 8

K LEAC 2 3 3 1 6

01 7 8 0 2 8

LEAC N N N N N N N 02 D D D D D D D

LEAC N N 1 N 3 N 03 D D 3 D D

Following ribonuclease protection analysis ACC oxidase transcript levels were quantified using the Ambis radioanalytical imaging system All scans were counted for 16.65 h and the results are shown as net counts per minute. The figures were corrected for the number of uracil residues in each probe and are rounded up to the nearest integer. Measurements for a given organ were all perfonned with the same probe on the same day. 'OW, unwounded leaves; W, leaves after wounding for two hour.;; OS, leaves at the onset of senescence as defined by John etaL [12]. bpruit harvested at mature green (MG), the fir.;t visible sign of colour change, that is, breaker stage (BRK) and three (+3) and eight (+8) days after the onset of colour change. 'ND, transcript not detectable.

LEA COl transcripts are at a low concentration in young green leaves but accmnulate 11-fold two hours after wounding. LEAC02 and LEAC03 transcripts are absent in young leaves and expression is not induced in response to wounding. LEA COl also appears to be the principal gene expressed during leaf senescence with transcript levels increasing 27-fold above those seen in young leaves. However, LEAC03 is also expressed but to approximately half the level to which the LEACOI transcript accmnulates. Kinetic analysis of ACC oxidase gene expression in response to wounding and during leaf senescence is shown in Figures 1 and 2. The expression of LEA COlin response to wounding is only transient. Transcripts begin to accmnulate within 30 minutes, peak after about two hours and then begin to decline by four hours post wounding. This pattern of expression coincides with previous data obtained for ethylene synthesis in wounded tomato leaves [8]. In contrast to expression following wounding, the accumulation of LEA COl transcripts during leaf senescence is prolonged over a period of about two weeks. Expression is low in green leaves, increases at the onset of senescence and remains elevated at the mid senescence stage before declining slightly at the advanced stage when the leaves are fully yellow. Hence there appear to be differences in ACC oxidase gene expression in leaves in response to wounding and during senescence. LEACOI expression is both elevated and prolonged in senescing leaves when compared to young wounded leaves. Additionally, LEAC03 is also transiently expressed at the onset of leaf senescence whereas transcripts are undetectable in unwounded and wounded young green leaves.

Figure 1. Ace oxidase gene expression in wounded leaves. Twenty-five micrograms of tota1 RNA extracted from unwounded leaves (0) or from leaves wounded for 30 min (30), 1 h (1), 2 h (2) and 4 h (4) were hybridized to radiolabelled Ace oxidase gene-specific RNA ~~and~A~ffipm=ed$~~ in [2). U, undigested ~be hybridized to yeast RNA; D, digested ~be hybridized to yeast RNA

301

AGO 1 AG02 AGOJ UOGOMA \lOGOMA \lOGO ... "

I

Figure 2. Ace oxidase gene expression during leaf senescence. Twenty-five micrograms oftota1 RNA extracted from leaves offour developmental stages were hybridized to radiolabelled Ace oxidase gene-specific probes and ~ A analysis W$ perl'onned $ descri~ in [2). U, undigested ~be hybridized to yeast RNA; D, digested probe hybridized to yeast RNA; G, 0, M and A, RNA extracted from green leaves (G) and from leaves at the onset (0), mid(M) and advanced (A) stages of senescence $ defined by Jolm et al [12].

The pattern of ACC oxidase gene expression is remarkably similar between fruit ripening and leaf senescence (Table 1 and [2]). LEA CO J transcripts are rare in green fruit but begin to accumulate at the breaker stage, as the fruit begin to ripen, peak at the breaker +3 stage and remain elevated until the fruit are fully ripened. LEA C02 does not appear to be expressed in fruits of any developmental stage whilst the expression of LEAC03 again seems to be transient being restricted to the breaker stage of ripening, and occurs at a low level. Fruit ripening and senescence are two examples of plant development where ethylene has been shown to stimulate its own production in a process termed autocatalysis. The transient expression of LEA CO 3 at the onset of ripening and leaf senescence suggests that this gene may have a role in regulating autocatalytic ethylene production in plants.

2. Ethylene Perception

The use of multigene families to control ethylene synthesis in plants appears to be matched by a multiplicity of ethylene receptors. The identification of the ETR gene in Arabidopsis and its characterisation as an ethylene receptor [4, 25] has led to the cloning of homologous genes from Arabidopsis and tomato. To date four genes have been cloned from Arabidopsis [Rna et al., this volume]. Five genetic loci have been

302

identified in tomato and three cDNAs have been reported to have been cloned [29, 30]. The ETR gene encodes a membrane-spanning protein with a similar structure to bacterial two-component regulators [4]. The protein consists of an amino terminal domain, a putative histidine kinase domain and a receiver domain [5]. Subsequently, a second receptor gene (ERS) was identified from Arabidopsis, which has homology to ETR but lacks the receiver domain [11]. A cDNA, designated TXTR-14, showing homology to ERS has been cloned from tomato and a mutation in this gene, which maps to chromosome nine, was shown to be responsible for conferring the ethylene insensitive phenotype displayed by the Never-ripe mutant of tomato [29]. A cDNA (tETR) identical to the Never-ripe sequence was independently cloned from a tomato fruit library [21]. The levels of mRNA homologous to tETR were measured by ribonuclease protection assay. The mRNA was undetectable in unripe tomato fruit but increased greatly in abundance at the onset of ripening and declined thereafter as the fruit fully ripened (Fig. 3). There was a similar increase in mRNA levels during flower senescence and, to a lesser extent, in floral abscission zones [21]. These expression patterns indicate that at least one component of the ethylene perception system shows differential expression. No tETR mRNA could be detected prior to the initiation of ripening, leading to the prediction that a second ethylene-perception gene must be expressed in green fruit, in order to explain their sensitivity to ethylene. This has since been confirmed by the isolation of a second ETR gene from tomato that appears to be constitutively expressed in green and ripening fruit [H. Klee, this volume].

M B 3 5 7

Figure 3. Expression oftETR during fruit ripening. The abundance of the tETR mRNA was measured in fruit pericarp by RP A analysis. Expression was analysed in fruit at mature green (M), breaker (B) and three (3), five (5) and seven (7) days post breaker.

3. Reduction in Ethylene Synthesis or Response by Gene Modification

Two strategies have been employed to generate transgenic tomatoes with reduced ethylene production, involving four separate enzymes. In the first strategy, the production of the biosynthetic enzymes ACC synthase [19] or ACC oxidase [6, 22] was inhibited by antisense genes. In the ACC synthase experiments by the Theologis

303

group, a transformant believed to contain ten antisense genes was identified. Fruit from this plant produced extremely low levels of ethylene. They failed to ripen and only showed a slight change in colour after many weeks [19]. Addition of ethylene, however, restored colour change and other ripening attributes. ACC oxidase antisense plants that produced 5% of normal ethylene were generated and characterised [6, 22]. In these experiments the reduction in A CO activity was shown to be due to a reduction in mRNA abundance and an antisense-gene-dosage effect was noted. If fruit were picked at the breaker stage, they changed colour only partially and did not ripen fully. The extent of ripening could be partially, but not completely, restored by adding ethylene [22]. If fruit were left to ripen on the vine, they ripened normally, but failed to over-ripen (Fig. 4). These results indicate that low levels of ethylene are beneficial for ripening, but higher amounts lead to rapid over-ripening and spoilage. The different behaviour of detached and attached ACC oxidase antisense fruit could be explained either by the escape of ethylene through the calyx scar of detached fruit, or might indicate the need for an additional ripening factor supplied by the parent plant. Interestingly, in the ACC oxidase antisense plants, leaf senescence was shown to be delayed by 7-10 days, compared to controls [12, 22].

Recently, ethylene production has also been inhibited by sense-gene suppression of ACC synthase [10] and ACC oxidase (Alpuche-Solis et al. unpublished results). The data presently available indicate, therefore, that ethylene biosynthesis can be effectively down regulated in transgenic plants by gene silencing, using either antisense- or sense­gene suppression. Furthermore, targeting either the ACC synthase or the ACC oxidase steps have been shown to be successful.

In the second strategy, also tested in tomatoes, bacterial genes are expressed in transgenic plants to inhibit ethylene production. Endogenous ACC is metabolised by the introduction of genes encoding either ACC deaminase or S-adenosyl-L-methionine hydrolase. These enzymes deplete the pool of ACC available, thus reducing ethylene production and slowing down ripening [15, Kramer et al. this volume].

Based on knowledge of ethylene physiology, it can be predicted that ethylene control by gene silencing offers a significant benefit for prolonging storage life and preventing deterioration of a range of climacteric fruits, vegetables and flowers. In addition to tomatoes, it is being developed commercially for banana [Kipp and May, this volume], melon [1] and other crops. The general conclusion that emerges from these experiments is that a small amount of ethylene is beneficial, whereas large quantities are generally deleterious because they lead rapidly to spoilage. Now that the genes for ethylene receptors have been cloned, this opens up the possibility of modifying ethylene responses by manipulating receptor levels or activities. This may be achieved by gene silencing, or by introducing mutant receptor genes that lead to impaired function. (II Klee, this volume).

Several important questions remain to be answered concerning the precise levels of ethylene to be aimed for in particular crops. If ethylene is completely inhibited, fruit will not ripen. This may be due to an incomplete development of the ethylene perception or response mechanism. Although the inhibition of ripening can be partly alleviated by adding external ethylene after picking it is not yet clear whether full production of flavour and other quality attributes still occurs. Low ethylene fruits are likely to remain on the plant for longer,

304

however, thus allowing them to accmnulate more sugar, and this would be expected to have a beneficial effect on the taste. A significant amount of fine-tuning is likely to be necessmy in order to establish the appropriate levels of ethylene or ethylene receptors that will permit the development of beneficial ripening changes but prevent the deterioration and spoilage that normally rapidly follows the ripening process. Fortunately, genetic modification of ethylene biosynthesis and perception allows the possibility to generate transgenic plants with different levels of ethylene production or response. The best transgenic lines can then be selected for commercial use, once the optimum level of genetic modification has been assessed.

Figure 4. Effect of ACC oxidase antisense genes on tomato ripening and spoilage. The tomatoes shown are the same variety and same age, grown under identical conditions. Those on the right are the regular variety allowed to ripen on the plant for several weeks. Those on the left contain two copies of an ACC oxidase antisense gene that reduces ethylene production to about five per cent of the control level. These fiuit ripen fully, but do not undergo over-ripening and deterioration.

305

4. References

1. Ayub, R, Ollis, M., Ben Amor, M., Gillot, L., Roustan, J-P., Laiche A, Bouzayen, M., and Pech, J-C. (1996) Expression of ACC oxidase antisense gene in cantaloupe melons and its consequences for fiuit ripening on and off the vine, Nature Biotechnology 14, 862-866.

2. Bally, C.S., Blume, B., Bouzayen, M., Cooper, W., Hamihon, AJ., and Grierson, D. (1996) Differential expression of the l-aminocyclopropane-l-carboxylate oxidase gene family of tomato, Plant J. 9, 525-535.

3. Bouzayen, M., Cooper, W., Bally, C.S., Zegzouti, H., Hamihon, AJ., and Grierson, D. (1993) EFE multigene family in tomato plants: expression and characterisation, in J-C. Pech, A Laiche and C Balague (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, K1uwer Academic Publishers, Dordrecht, pp.76-81.

4. Chang, C., Kwok, S.F., Bleecker, AB., and Meyerowitz, E.M. (1993) Arabidopsis Ethylene-response gene ETRl: Similari1y ofproduct to two-componentregulators, Science 262,539-544.

5. Chang, C. and Meyerowitz, E.M. (1995) The ethylene hormone response in Arabidopsis: A eukaryotic two­component signaling system, Proc. NatL Acad Sci. USA 92, 4129-4133.

6. Hamihon, AJ., Lycett, G.W., and Grierson, D. (1990) Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants, Nature 346, 284-287.

7. Hamilton, AJ., Bouzayen, M. and Grierson, D. (1991) Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast,Proc. NatL Acad. Sci. USA 88, 7434-7437.

8. Holdsworth, M.J., Bird, CR, Ray, J., Schuch, w., and Grierson, D. (1987) Structure and expression of an ethylene-related mRNA from tomato, NucL Acids. Res. 15, 731-739.

9. Holdsworth, MJ., Schuch, w., and Grierson, D. (1988) Organisation and expression of a wound / ripening­related small multigene fiunily from tomato, Plant. MoL BioL 11, 81-88.

10. Howie, w., Lee, K, Joe, L., Baden, C., McGugin, C., Bedbrook, J., and Dunsmuir, P. (1996) Suppression of ACC S}TIthase. Commercialization of ripening-impaired fresh marl:et tomatoes, in D. Grierson, G. W. Lycett and GA Tucker (eds.), Mechanisms and applications of gene silencing, Nottingham University Press, Nottingham, pp. 119-125.

11. Hua, J., Chang, C., Sun, Q., and Meyerowitz, E.M. (1995) Ethylene insensitivity conferred by Arabidopsis ESR gene, Science 269, 1712-1714.

12. John, 1., Drake, R, Farrell, A, Cooper, w., Lee, P., Horton, P., and Grierson, D. (1995) Delayed leaf senescence in ethylene-deficient ACC-oxidase antisense tomato plants: molecular and physiological analysis, PlantJ. 7,483-490.

13. Kende, H. (1993) Ethylene biosynthesis, Ann. Rev. Plant PhysioL 44, 283-307. 14. Kim, W.T. and Yang, S.F. (1994) Structure and expression of cDNAs encoding l-aminocyclopropane-l­

cruboxylate oxidase homologs isolated from excised mung bean hypocotyls, Planta 194, 223-229. 15. K1ee, HJ., Hayford, M.B., Kre1zmer, KA, Bally, G.F., and Kishore, G.M. (1991) Control of ethylene

synthesis by expression of a bacterial enzyme in transgenic tomato plants, Plant Cell 3, 1187-1193. 16. Lasserre, E., Bouquin, T., Hernandez, J.A, Bull, J., Pech, J.C., and Balague, C. (1996) Structure and

expression of three genes encoding ACC oxidase homologs from melon (Cucumis melo L.),Moi Gen Genet 251,81-90.

17. Liang, X Abel, S. Keller, J.A, Shen, N.F., and Theologis, A (1992) The l-aminocyclopropane-l-carboxylate S}TIthase gene family of Arabi do psis thaliana, Proc. NatL Acad. Sci. USA,89, 11046-11050.

18. Lincoln, J.E., Campbell, AD., Oetiker, J., Rottmann, W.H., Oeller, P.w., Shen, N.F., and Theologis, A (1993) LE-ACS4, a fiuit ripening and wound-induced l-aminocyclopropane-l-carboxylate S}TIthase gene of tomato (Lycopersicon esculentum),J. BioL Chem. 268, 19422-19430.

19. Oeller, P.W., Min-Wong, L., Taylor, L.P., Pike, DA, and Theologis, A (1991) Reversible inhibition of tomato fiuit senescence by antisense RNA, Science, 254, 437-439.

20. Olson, D.C., Oetiker, J.H., and Yang, S.F. (1995) Analysis ofLE-ACS3, a l-aminocyclopropane-l-carboxylic acid S}TIthase gene expressed during flooding in the roots of tomato plants, J. BioL Chem. 270, 14056-14061.

21. Payton, S., Fray, RG., Brown, S., and Grierson, D. (1996) Ethylene receptor expression is regulated during fiuitripening, flower senescence and abscission,PlantMoL BioL 31, 1227-1231.

22. Picton, S., Barton, S.L., Bouzayen, M., Hamilton, AJ., and Grierson, D. (1993) Altered fiuit ripening and leaf senescence in tomatoes expressing an antisense ethylene-forming enzyme transgene, Plant J. 3, 469-481.

23. Pogson, B.J., Dovvns, C.G., and Davies, KM. (1995) Differential expression of two l-aminocyclopropane-l­cruboxylic acid oxidase genes in broccoli after harvest,PlantPhysioL 108,651-657.

306

24. RoUmann, W.H., Peter, G.F., Oeller, P.W., Keller, I.A, Shen, N.F., Nagy, B.P., Taylor, LP., Campbell, AD., and Theologis, A (1991) l-aminocyclopropane-l-<:arboxylate synthase in tomato is encoded by a multigene family who6e transcription is induced during fiuit and floral SIiIDIIlSCelJI. J. MoL BioL 222, 937-961.

25. Schaller, O.E. and Bleecker, AB. (1995) Ethy1ene-binding sites generated in yeast expressing the Arabidopsis ErR1 gene, Science 270, 1809-1811.

26. Spanu, P., Reinhardt, D., and Boller, T. (1991) Analysis and cloning of the ethylene-fonning enzyme from tomato byfunctiona1 expression ofits mRNA inXenopus laevis oocyIes,Embo. J. 10,2007-2013.

27. Tang, X, Wang, H., Brandt, AS., and Woodson, W.R (1993) Organisation and sbudure of the 1-aminocyclopropane-l-<:arboxy1ate oxidase gene fiunily from Petunia hybrida, Plant. MoL BioL 23, 1151-1164.

28. Tang, X, Gomes, AM.T.R, Bhatia, A, and Woodson, W.R (1994) Pistil-specific and ethylene-regulated expression of l-aminocyclopropane-l-carboxy1ate oxidase genes in peflInia flowers. Plant Cel~ 6, 1227-1239.

29. Wilkinson, I.Q., Lanahan, MB., Yen, H-C., Giovamoni, 1.I., and K1ee, H.l. (1995) An ethylene-inducible component of signal transduction encoded by Never-ripe, Science 270, 1807-1809.

30. Yen, H-C., Lee, S., Tanksley, S.D., Lanahan, M.B., K1ee, H.I., and Giovamoni, J.J. (1995) The tomato Never-ripe locus regulates ethylene-inducible gene expression and is 1inked to a homolog of the Arabidopsis ErR1 gene,PlantPhysioL 107, 1343-1353.

REDUCED ETHYLENE SYNTHESIS AND RIPENING CONTROL IN TOMATOES EXPRESSING S-ADENOSYLMETHIONINE HYDROLASE

M. G. KRAMER, J. KELLOGG, W. WAGONER, W. MATSUMURA, X. GOOD, S. PETERS, G. CLOUGH* AND R. K. BESTWICK Agritope, Inc., 8505 SW Creekside Place, Beaverton, Oregon, USA 97008-7108, *Hermiston Agricultural Research Extension Center, Hinkle Rd. Hermiston, Oregon

1. Introduction

Using standard Agrobacterium binary vectors, we have introduced a SAMase-encoding gene derived from E. coli bacteriophage T3 into the tomato genome. This results in transformed tomato plants that exhibit significantly reduced levels of S­adenosylmethionine (SAM), the substrate for conversion (through ACC synthase) to 1-aminocyclopropane-1-carboxylic acid (ACC) which is the first committed step in ethylene biosynthesis. Lack of a sufficient pool of SAM for conversion to ACC in fruit results in tomatoes with significantly reduced ethylene biosynthetic capabilities and a modified ripening phenotype. Typically, this phenotype is characterized by fruit in which ripening on the vine is delayed while ripening off the vine may be essentially suspended.

The results presented here are based upon transgenic cheny tomato lines transformed with gene construct pAG5420. This construct contains a version of the SAMase gene (sam-k) modified in the 5' region of the gene with a Kozak consensus sequence [4]. This construct encodes a functional SAMase protein. Since SAM plays a central role in numerous biosynthetic pathways in plants, expression of SAMase is under the control of an organ specific (fruit) and temporally regulated (post­climacteric) promoter (a modified E8 promoter) in pAG5420 [4]. The efficacy of this strategy is demonstrated by the fact that the organ specific and temporal expression pattern of ethylene biosynthesis precisely matches the SAMase expression kinetics (ethylene synthesis is inversely correlated to SAMase expression) and provides an explanation of the observed modified ripening phenotype (Fig. 4).

1.1. PLANT MATERIALS

Tomato cultivar Large Red Cheny was used as the genetic background for all experiments and analysis reported here. Tomato cultivar Large Red Cheny is an open pollinated cheny tomato line available in the Public Domain through various

307 A. K. KQ1/J!llis et al. (etis.), Biology and Biotechnology of the Plant Hormone Ethylene, 307-319. © 1997 Kluwer Academic Publishers.

308

commercial outlets. It is characterized as an indetenninate small fruited cherty type variety with an average fruit size of 1.5 inches in diameter. Fruit ripen to a deep red approximately 70 days post transplanting and are borne in clusters on highly productive plants [1].

1.2. THE GENE: sam-k

The sam-k gene is derived from an E. coli bacteriophage T3 gene [5] modified in the 5' region with a Kozak consensus sequence and encodes a functional SAMase protein. The SAMase protein catalyzes the conversion of S-adenosylmethionine (SAM) to methylthioadenosine (MTA). The tissue specific and developmentally regulated expression system employed to express the sam-k gene only in ripening fruit insures that the only impact on the plant of SAM hydrolysis is the down regulation of ethylene production in ripening fruit through the reduction of the pool of SAM available for conversion by ACC synthase (Figs. 3a, 3b and 4).

1.3. THE ENzyME: S-ADENOSYLMETHIONlNE HYDROLASE

S-adenosylmethionine (SAM) is an ubiquitous nucleotide used in many activities in all cells [6, 9]. SAM acts as a co-factor in a variety of reactions and as a methyl group donor in specific transmethylation reactions. Among these reactions are the biosynthesis of biotin, rare nucleotides, 5'-methylthioadenosine (MTA), polyamines and the production of the plant hormone ethylene. SAM also acts as a methyl donor during modifications of proteins, lipids, polysaccharides and nucleic acids. SAMase hydrolyzes SAM to homoserine and 5'-methylthioadenosine [3]. In the course of ethylene biosynthesis, 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor to ethylene, is produced from SAM by the enzyme ACC synthase. As the pool of SAM is depleted by the action of SAMase, neither ACC nor ethylene are produced. A diagram of the ethylene biosynthetic pathway is shown in Figure 1. The effect on the methionine recycling pathway of sam-k gene expression is illustrated in Figure 2.

Methionine ----+) SAM----+) ACC ---+) Ethylene

Figure 1. Ethylene biosynthetic pathway in plants. Ethylene synthesis is an offshoot of the methionine recycling pathway where S-adenosylmethionine (SAM) is converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by the enzyme ACC synthase. ACC is subsequently oxidized to ethylene by the enzyme ACC oxidase.

ACC r SAM - ~ Synthase 3Pi \..... ... _ ACC+C2 H4

ATP SAMase \

MET~ ~ M\A ~ ~ Homoserine.../' ~

a-KMB

HCOOH~ P~

MTR-I-P

MTA Nucleosidase

MTR

MJa~e/ATP

~~n ADP

Ade

Figure 2. The methionine recycling pathway in plants. The bold line shows the effect of sam-k expression on the pathway where the reaction products re-enter the cycle Good et al. [4]. Abbreviations: MET, methionine; SAM, S-adenosylmethionine; ACC, 1-aminocyclopropane-l-carboxylic acid; MT A, 5'-methylthioadenosine; MTR, 5-methylthioribose; Ade, adenine; KMB, a-ketomethylthiobutyric acid. The E8: sam-k chimera expresses a functional S-adenosylmethionine hydrolase (SAMase) protein in a fruit specific, developmentally regulated marmer (Fig. 3a, b).

2. Results

2.1. EXPRESSION OF sam-k IN TRANSGENIC TOMATO

309

Stable insertion of the sam-k transgene into the tomato genome results in the production of functional SAMase protein in a fruit specific and temporally regulated manner (Fig. 3a, b). Using this expression system, SAMase is induced at the onset of ripening. The expression pattern of SAMase in this system closely lnirrors the pattern of ethylene expression normally found in ripening fruit (Fig. 4). Transgenic fruit express the SAMase protein in breaker to light red fruit and expression is attenuated in fruit that are fully ripe. The fact that E8: sam-k gene expression follows the normal pattern of ethylene expression means that the SAMase protein is a transient species and final concentrations in the ripe fruit are lninimal (Fig. 5).

310

Kd 144 87

7.1

1 2 3 4 5

Figure 3a. Protein blot analysis demonstrating tissue specific expression of the sam-k gene in tomato. A 20 ng sample of purified SAMase (lane 1). and 9 f.Lg samples of protein extracted from leaf (lane 3). stem (lane 4). and flower (lane 5) were resolved on an SDS-PAGE gel and blotted to a nylon membrane. The resulting blot was probed with a monoclonal antibody specific to the SAMase protein and visualized using chemiluminescence according to the manufacturer's instructions.

pAG-5420

14·2

Figure 3b. RNAse protection assays were used to demonstrate the pattern of sam-k gene expression in ripening tomato fruit Tomato fruit were picked at the breaker stage (Br) and allowed to ripen to the orange (Or) or red (Ri) stage. Between 0.7 and 1.0 f.Lg of poly A+ mRNA was used in each reaction. The 510 bp protected probe was resolved on a denaturing polyacrylamide gel and visualized by autoradiography.

10 35-1

-.::-..c:: ~ 75 :s "0 Q) (.)

5 :J "0

JIt·f·f·· e a.. Q)

25 c Q)

>. ..c:: U::i

0

0 2 4 6 8 10

Days Post-breaker

Figure 4. Ethylene production from transgenic tomatoes transformed with pAG 5420 (transgenic fruit from of line 35-1). The graph represents ethylene produced by transgenic fruit (diamonds) and Large Red Cherry controls (squares). The values for transgenic fruit represent the average of three fruit from the individual Ro plant. The values for the controls represent the average of six fruit from two different plants. Error bars represent one standard deviation of the data.

2.2. FIELD TESTS OF SAMase TOMATOES

311

Field trials of transgenic cherry tomatoes have been conducted to produce seed and plant material for genetic and molecular analysis, demonstrate and characterize the modified ripening phenotype produced, generate horticultural performance data relative to non-transgenic controls and to produce seed of selected lines for advancement in variety development programs. Analytical results of cherry tomato trials carried out in 1992 and 1993 are reported here.

2.3. FRUIT DEVELOPMENT AND QUALITY EVALUATIONS

An example of fruit development and quality evaluations that have been carried out are shown in Tables 1 and 2. These data are derived from field-grown transgenic cherry tomatoes. The parameters evaluated included pH, soluble solids, titratable acidity, average fruit weight, heat unit accumulation to maturity, and yield.

312

Table 1. Summary offruit development evaluations of transgenic cherry tomato lines

Heat units required from transplant to: Genotype Fruit Set Mat. Green Breaker Pink Red

Large Red 707c 1398bcd 1531d 1629c 1675d Cherry Ctr\. 35-1-N 756abc 1419bc 1709a 1824ab 1901ab IOA-l 814a 1464a 1582cd 1838ab 1860abc 22A-I-D 764abc 1384cd 1692ab 1792ab 1852abc 22A-I-1 742bc 1362d 1598bcd 1700bc 1765bcd 22A-I-J 717bc 1391bcd 1611abcd 1708bc 1782bcd 22A-I-K 748bc 1362d 1639abc 1728abc 1799bcd 22A-I-L 777ab 1391bcd 1669abc 1772ab 1844abc 35-1-J 764abc 1425b 1704ab 1857a 1951a 40A-I-B 732bc 1376d 1598bcd 1713bc 1751cd 40A-I-D 758abc 1398bcd 1638abc 1729abc 1805bcd

Means followed by different letters are significantly different at p=O.05 using Duncan's Multiple Range Test. Heat units based upon a 45°F base, 90°F maximum

Table 2. Summary offruit quality evaluations of transgenic tomato cherry lines

Soluble Solids Titratable Average Fruit Genotype pH (brix) Acidity (meg) Weight (g) Large Red 4.29d 6.93 b 157.5 abc 19.3abc CherryCtrI. 35-1-N 4.35bcd 6.75b 134.5cde 17.7cd IOA-l 4.36bc 8.30a 172.0a 14.7e 22A-I-D 4.34bcd 7.18b 160.0ab 18.2bcd 22A-I-1 4.32cd 6.58b 132.5de 19.8abc 22A-I-J 4.33cd 6.88b 142.0bcde 18.8abcd 22A-I-K 4.33cd 6.83b 151.0abcd 18.0bcd 22A-I-L 4.37bc 6.80b 150.0abcd 17.1d 35-1-J 4.48a 5.93c 123.0e 19.9ab 40A-I-B 4.38bc 7.15b 156.5abc 19.5abc 40A-I-D 4.39b 6.63b 143.0bcde 20.7a

Values reported represent the means of five replications. Means followed by different letters are significantly different at p=O.05 using Duncan's Multiple Range Test.

2.4. YIELD CHARACTERISTICS

As part of the horticultural evaluation and phenotype characterization carried out at field test locations, yield characteristics have been evaluated by quantitative harvest at specific developmental stages. In this example, transgenic cherry tomatoes were evaluated over three consecutive harvests carried out on specific dates. Fruit which had reached at least the breaker stage by the harvest date were harvested, sorted by color, counted and weighed. Yield results were effected by a genotype by harvest date interaction. To resolve this interaction, data was sorted by both variables for analysis. Results indicate that total yield of several transgenic lines was negatively affected due

313

to the extended time necessary to reach maturity and that the distribution of fruit maturity at harvest was significantly impacted. Relative to control Large Red Cherry lines, transgenic lines yielded the majority of their fruit at the breaker to pink stage at each harvest. The modified ripening phenotype significantly slowed the rate at which the fruit of transgenic lines ripened from breaker to full red. Results of yield measurements are found in Table 3.

Table 3. Summary of yield measurements in transgenic cherry tomato lines

Harvest 1

Color Stage Breaker Pink Red Total

Genotype klielot klielot kWelot kWelot Large Red .25 .48 2.70 a 3.45 a CherryCtrI. 35-1-N .11 .15 .34e .62e lOA-l .14 .19 .37e .72e 22A-I-D .26 .44 .33e 1.04de 22A-I-1 .19 .47 1.33c 2.01bc 22A-I-J .28 .43 1.90b 2.64ab 22A-I-K .28 .54 1.1400 1.99bc 22A-I-L .28 .48 1.00cd 1.78bcd 35-1-J .06 .16 .66e .90de 40A-I-B .39 .59 .98cd 2.02bc 40A-I-D .32 .44 .67de 1.46cde

Means followed by different letters are significantly different at p=O.05 using Duncan's Multiple Range Test.

Harvest 2

Color Stage Breaker Pink Red Total

Geno~e kWelot kWelot klielot kWelot Large Red .58bed .92abcd 3.01 a 4.52 a CherryCtrI. 35-1-N .40cd .58ed .34 d 1.32f 10A-l .29d .53d .83ed 1.69def 22A-I-D .66bc 1.06abc 1.14bed 2.89bcde 22A-I-1 . 56bcd 1.35a 2.I7ab 4.09ab 22A-I-J .68bc .99abcd 1.25bcd 2.97bcd 22A-I-K 1.07a 1.3Ia 1.44bcd 3. 84abc 22A-I-L .73b 1.20ab 1.77bc 3.79abc 35-1-J .40ed .75bcd .4Id 1.5gef 4OA-I-B .65bc 1.19ab .86cd 2.75cde 40A-I-D .62bc 1.0labed 1.00cd 2.69ede

Means followed by different letters are significantly different at p=O.OS using Duncan's Multiple Range Test.

314

Harvest 3

Color Stage Breaker Pink Red Total

Genotype kgIplot kg/plot kg/plot kgIplot Large Red .32c 1.92cd 5.26 a 7.63 ab Cherry Ctrl. 35-1-N .85ab 2.80abc .83d 4.56 ef lOA-l .44bc 1.42d .95d 2.91f 22A-1-D .84ab 2.37bcd 1.63cd 4.90e 22A-I-I LOla 1.35a 3.71b 8.44a 22A-1-J .17ab 3.57a 2.75bc 6.73abcd 22A-I-K .86ab 3.08ab 2.92b 7. 15abc 22A-1-L .78ab 3.28ab 2.81b 6.98abc 35-1-J .89a 3.25ab 1.29d 5.58cde 40A-1-B 1.03a 3.26ab 1.24d 5.69cde 40A-I-D .98a 3.61a 1.61cd 6.42bcd

Means followed by different letters are significantly different at p=0.05 using Duncan's Multiple Range Test.

2.5. NUTRITIONAL ANALYSIS

We have also measured a set of nutritional components in the SAMase tomatoes and controls. Table 4 summarizes a selected set of the components measured and the values obtained. Values obtained for the vitamin and nutrient constituent analysis fall well within the range normally reported for fresh tomatoes [2, 7, 8]. Total protein concentration and ,the relative proportions of the individual soluble amino, acids are unchanged in SAMase tomatoes relative to controls. Amino acid levels in both the transgenic and control lines in all cases fall well above the normal range reported for fresh tomatoes. This is most likely due to the normal ranges reported having been measured in large fruited genotypes and the values determined are a result of genotypic differences between large fruited genotypes and cherry tomato genotypes.

2.6. CONCENTRATION OF SAMase IN TRANSGENIC TOMATO FRUIT

Protein blot analysis of SAMase tomato fruit was used to determine the concentration of S-adenosylmethionine hydrolase in the final fruit product. Using a monoclonal antibody, SAMase was detectable only in orange fruit and was undetectable in breaker and ripe stage fruits. In order to determine the concentration of SAMase in transgenic fruit, digital image analysis of a ptotein blot (Fig. 5) was used to quantitate the signal observed in transgenic orange fruit relative to signals observed for a standard curve of known concentrations ranging from 200 pg-600 pg of a SAMase fusion protein. The results of the digital image analysis of individual Protein blots give an average concentration of 25.6 pglJ.lg (n=4) of total fruit protein in orange fruit. This results in a SAMase protein concentration of 0.0026% of the total protein found in ripening tomato fruit. No SAMase protein was detected in green fruit. Given the expression system, this is an expected result. While no SAMase protein was detected in breaker or ripe fruit, we know from the RNAase protection assays (Fig. 3b) that the sam-k gene

315

is indeed expressed during these ripening stages. The results of numerous protein blots used in detecting SAMase have demonstrated a SAMase detection limit of approximately 100 pg. As a result, we conclude that while RNA is being transcribed at both the breaker and ripe stages, the actual protein concentration at these ripening stages is less than 100 pgl15 ~g of total fruit protein. We therefore, conclude that each ripe tomato of approximately 20 grams contains approximately 0.098 grams of total protein of which less than 0.65 j.Lg is SAMase.

Table 4. Nutritional components in representative SAMase tomatoes and Large Red Cherry control lines.

Normal Range Measured Range Measured Range

Constituent [2,7,8,] SAMase Large Red Tomatoes Ch~ Controls

Vitamin A (IV) 192 - 3833 330 - 2131 1100 - 2727 VitaminC 8.4 - 59.0 21.6 - 27.05 17.53 - 23.37 (mgll00g)

Calcium 4.0-21.0 4.90-7.10 4.90-7.30 (mgl100g)

Iron 0.35-0.95 0.79-0.93 0.79-0.95 (mgl100g) Sodium 1.0-33.0 19.0-24.0 10.0-20.0 (mgll00g)

Dietary Fiber % 0.8-1.85 1.20-1.60 1.50-1.70 Simple Sugars 1.60-10.00* 2.24 2.66 Total Protein (%) 1.2-1.6 1.5 Soluble Amino Acids

(mgl100g)

Alanine 2.80-3.40 24.3-27.7 21.6-23.3 Arginine 2.30-6.60 18.3-24.8 15.3-24.9 Aspartic Acid 29.0-53.0 95.5-104 92.0-96.9 Glutamic 69.0-252.0 393-419 395-407

Acid Glycine 1.10-2.30 15.6-18.5 13.4-15.3 Histidine 3.30-5.70 12.0-13.7 12.0-12.9 Isoleucine 3.00-4.60 17.7-20.4 15.2-16.6 Leucine 2.30-9.70 25.4-29.8 21.8-24.4 Lysine 4.00-10.90 23.2-26.3 19.1-21.6 Methionine 0.50-1.70 4.86-5.91 4.21-4.85 Phenylalanine 7.20-11.40 17.8-20.9 16.0-17.6 Proline 0.60-1.60 17.4-20.0 14.1-16.0 Serine 5.70-7.40 22.3-26.0 19.0-21.5 Threonine 6.6 ** 28.4-31.5 24.6-26.3 Tyrosine 1.50-3.90 12.6-14.9 10.6-12.4 Valine 0.80-7.40 17.1-20.1 15.7-17.2

*Redenbaugh; et aI, 1992 **Threonine ranges are not reported in the literature cited, rather a single value is

provided for fresh tomatoes.

316

1 2 3 4 5 6 7 8 9

Figure 5. Protein blot analysis of S-adenosylmetbionine levels in various stages of transgenic tomato fiuit. A standard curve containing known concentrations of a SAMase fusion protein spiked into 15flg of total fiuit protein extracted from red stage non-transgenic control fiuit. Samples were prepared and loaded to give the SAMase equivalent of the fusion protein as follows: Lane (1) 200 pg, (2) 300 pg, (3) 400 pg, (4) 500 pg, (5) 600 pg. Samples containing 15 flg of protein extracted from transgenic fiuit tissue at various ripening stages were prepared and loaded as follows: Lane (6) breaker, (7) orange, (8) ripe, (9) green.

2.7. PROTEIN DIGESTIBILITY

In order to demonstrate that the SAMase protein is readily digestible in the human intestinal tract it is necessary to carry out digestibiJity studies using simulated gastric fluid. Digestibility studies of the SAMase protein were carried out using a SAMase fusion protein produced as described in the manufacturers instructions (New England Bio Labs, Inc. Product Catalog, 1992) and purified by affinity column chromatography. Figure 6 demonstrates inactivation of SAMase protein by simulated gastric fluid.

2.8. HEAT INACTIVATION OF THE SAMase PROTEIN

In order to demonstrate that the SAMase protein is heat labile and therefore not likely to be allergenic, it is necessary to measure SAMase enzymatic activity after heating. 14C labeled S-adenosylmethionine was reacted with a SAMase fusion protein under conditions favorable to SAMase enzymatic activity [5]. The reaction was spotted onto thin layer chromatography plates and chromatographed as described in Hughes et al. [5]. Autoradiography of the TLC plate was followed by quantitative measurement of radioactivity in each spot using liquid scintillation. Results shown in Table 5

317

demonstrate that after heating for 0.5 minutes SAMase enzymatic activity drops to background levels.

1 2 3 4 5 6

A

B

c

Figure 6. Results of S-adenosylmethionine hydrolase digestibility studies. Simulated gastric fluid (0.32% pepsin, 0.2% NaCL pH 1.2) either without (A) or with (8) pepsin was added to 25 nglmJ ofSAMase fusion protein and incubated for 0 min. (lane 1).5 min. (lane 2). 10 min. (lane 3). 20 min. (lane 4). 30 min. (lane 5) or 60 min. (lane 6) at 37°C. (C). Non-transgenic tomato fruit control prepared to 25 IJ.g/mJ of total protein in simulated gastric fluid without pepsin. A1iquots of these reactions were blotted to nitrocellulose using a dot blot apparatus. 'The bound proteins were reacted with an anti-SAMase monoclonal antibody under standard immunoblot conditions. Bound antibody was visualized using chemiluminescence and exposure to film.

Table 5. Heat inactivation of S-adenosylmethionine hydrolase activity

SAMPLE (Rxn. time in SAM MTA SAM + MTA %

minutes) (cpm) (cpm) (cpm) Conversion Neg. Control 203349 4756 207884 2.29 (buffer Only) SAMase o min. 27104 175735 202618 86.73 0.5 min. 203846 5541 209166 2.65 1 min. 207838 6340 213956 2.96 5 min. 203344 6439 209563 3.07 15 min. 209064 6180 215024 2.87 30 min. 202937 6020 208736 2.88

3. Discussion

We report here a genetic engineering strategy to control plant ethylene synthesis using S-adenosylmethionine hydrolase which is applicable to all higher plants. The use of S­adenosylmethionine hydrolase in conjunction with ripening specific promoters. as demonstrated in tomato, is especially useful in climacteric fruit where extended

318

ripening is economically desirable. Because the expression of SAMase is limited to ripening fruit the strategy avoids possible adverse biochemical effects of constitutive SAMase expression and/or constitutive ethylene inhibition. In addition, since the effect of SAMase is to "short circuit" the branch of the methionine recycling pathway that produces ethylene, there should be no accumulation of ACC or ACC metabolites. The use of an ethylene responsive promoter to drive sam-k gene expression means that SAMase induced ripening inhibition is initiated at the onset of the respiratory climacteric allowing the fruit to initiate ripening prior to the expression of the ethylene control response. This is an important consideration in the production of commercially acceptable fruit and vegetable products.

Regulated expression of SAMase in tomato fruit results in a modified ripening phenotype which will provide production flexibility and help prevent losses due to immature and overripe fruit in the field, packing, shipping, handling and distribution systems. In addition, this technology will enable the production of a more physiologically mature fruit able to withstand the rigors of the current production and distribution system resulting in a product of higher overall quality. We have used cherry tomatoes as a model system to demonstrate this technology and begin to quantify the economic benefits that can be achieved through reduced spoilage and losses in the field and in the packing, shipping, handling and distribution systems. We believe that the ripening phenotype has numerous commercial applications in the current fresh tomato production and distribution system. These include but are not limited to the following: - Reduction in producer losses through reduced harvest of immature and/or over

mature fruit. - Improved production dynamics and reduced harvest frequency. - Reduced spoilage and loss through out the distribution system. - Enhanced fruit quality due to harvest of more physiologically mature fruit.

In terms of food safety of genetically engineered products expressing SAMase the following points have been demonstrated: - There are no significant changes in levels of important nutrients or naturally

occurring toxicants. - The S-adenosylmethionine hydrolase protein is present at extremely low levels in

ripe fruit and are equal to or less than levels of natural exposure expected from T3 coliphage infection of intestinal microflora.

- The S-adenosylmethionine hydrolase protein is not known to be toxic, does not have the characteristics of an allergen and is rapidly degraded by heat and gastric conditions. Based upon these results, we conclude that fruits and/or vegetables transformed to

express S-adenosylmethionine hydrolase in a tissue specific, developmentally regulated manner may offer substantial economic benefits to producers and consumers alike and are safe as food.

319

4. References

1. American Horticultural Society. (1982). Tomatoes. Illustrated Encyclopedia of Gardening. Ortho Books, Mt. Vernon, VA p144.

2. Davies, IN. and Hobson, G.E. (1981) The constituents of tomato fruit - the influence of environment, nutrition and genotype. CRC Critical Reviews in Food Sciences and Nutrition, 15, 205-280.

3. Gold, M., et at. (1964) The enzymatic methylation of RNA and DNA Vill. Effects of bacteriophage infection on the activity of methylating enzymes, Proc. Nat. Acad. Sci. USA 52, 292-297.

4. Good, x., Kellogg, JA, Wagoner, W., Langhoff, D., Matsumura, W., and Bestwick, R.K. (1994) Reduced ethylene synthesis by transgenic tomatoes expressing S-adenosylmetbionine hydrolase, Plant Mol. Bioi. 26, 781-790.

5. Hughes, JA et at. (1987) Expression of the cloned coliphage T3 S-adenosylmethionine hydrolase gene inhibits DNA methylation and polyamine biosynthesis inE. coli, J. Bact. 169,3625.

6. Salvatore, F. et at. (1977) The Biochemistry of Adenosylmethionine. New York, Columbia University Press.

7. Souci, S.W., Fachman, W., and Kraut, H. (1986) Food composition and nutrition tables, 1986-1987. Stuttgart, Wissenschaftliche Verlagsgesellschaft mbH.

8. Souci, S.W., Fachman, W., and Kraut, H. (1989) Food composition and nutrition tables, 1989-1990. Stuttgart, Wissenschaftliche Verlagsgesellschaft mbH.

9. Usdin, E. et at. (1979) Transmethylation. ElsevierlNorth Holland Publishing Co., New York.

INTERACTIONS OF EmYLENE AND POLYAMINES IN REGULATING FRUIT RIPENING

R. MEHTAl, A. HANDA2, A. MAITOO1,3

1 Plant Molecular Biology Laboratory, USDAIARS-REE, Beltsville Agric Res Ctr, Bldg. 006, Beltsville, MD 20705, USA and 2Department of Horticulture, Purdue University, W. Lafayette, IN 47907, USA. 3Address correspondence to this author

1. Introduction

Ethylene is one of the simplest organic molecules which affects many aspects of growth, development and senescence of higher plants - biological effects that make it a very versatile plant hormone [21]. For instance, ethylene promotes seed germination [31], leaf senescence [19], flower abscission [27], fruit ripening [6] and development of plant defense systems [5]. In addition, it inhibits cell division and cell differentiation [2, 18, 32]. The biochemical pathway of ethylene synthesis has been elucidated and the enzymes involved have been characterized. The key enzymes regulating the production of ethylene are l-arninocyclopropane-l-carboxylic acid [ACC) synthase, which catalyzes the formation of ACC from S-adenosylmethionine (AdoMet), and ACC oxidase, which catalyzes the synthesis of ethylene from ACC, in the following metabolic sequence: methionine ~ AdoMet ~ ACC ~ ethylene [35]. The genes encoding ACC synthase and ACC oxidase have been cloned and sequenced from a wide variety of horticultural, agronomic and model plants [14, 33]. Also, molecular analysis of Arabidopsis and tomato mutants has identified the genes that encode proteins that either bind ethylene or regulate ethylene signal transduction pathway [14, 34].

Our laboratory is investigating molecular mechanisms involved in the hormonal control of fruit ripening and senescence. Specifically, we are concentrating on interactions that occur prior to the initiation of ripening/senescence. We are particularly interested in understanding the molecular events involved in the interactions between ethylene and other plant growth regulators that control ethylene biosynthesis or perception such as auxin [7], polyarnines [1, 19, 25, 30] and salicylic acid [16], since these impact growth, development and senescence of plants thereby altering physiology of plants. Gibberellic acid, indole acetic acid, cytokinins and polyarnines are generally considered to be growth stimulators whereas methyl jasmonate, abscisic acid and ethylene promote senescence and cell death. Plant cells have evolved mechanisms to balance the levels of these hormones whose synthesis and action are developmentally regulated. The shift from growth to senescence is a commitment related to the relative levels of these two sets of hormones, the balance determined by the tilt in their ratio as depicted in Figure 1.

321

A. K. Kanellis et al. (eds.). Biology and Biotechnology olthe Plant Hormone Ethylene, 321-326. @ 1997 Kluwer Academic Publishers.

322

GA,IAA,Cytokinins, polyamines me-JA, ABA, CaH4

GROWTH SENESCENCE

Figure J. Honnonal seesaw balance concept of plant senescence.

2. Controlling Fruit Quality by Overexpressing Polyamine Genes

Molecular biotechnology has demonstrated direct involvement of ethylene in tomato fruit ripening [24, 33] and the identification of the genes encoding components of ethylene perception [4, 36] and ethylene signal transduction pathway [10, 34]. Furthering these studies will enable delineation of the role(s) ethylene and other plant growth substances have in controlling not only fruit ripening but also wilting of flowers, leaf senescence, and organ abscission.

A characteristic feature differentiating growth phase of a fruit from the ripening one is the inverse developmental relationship in a fruit's ability to accumulate polyamines and to produce ethylene (Fig. 2). Polyamines are ubiquitous and abundant in actively growing tissues [11, l3] and generally decrease during tissue senescence [l3].

Tomato cultivars that accumulate and maintain relatively high levels of polyamines produce relatively low levels of ethylene and have longer shelf-life [9, 26]. Applied polyamines inhibit ethylene production [1, 3, 12, 30], by suppressing the induction of ACC synthase activity [20], which has been correlated to repression of the cognate transcript [17]. On the other hand, ethylene is known to inhibit the key branching enzyme in polyamine biosynthesis, AdoMet decarboxylase [2, l3]. The inhibition of ethylene biosynthesis by polyamines is concentration dependent, the higher polyamine spermine being the most effective inhibitor of ACC synthase. However, the polyamine effect in vitro is transient [17], which could be due to a number of reasons: sequestration of polyamines in the cell at sites away from the target, oxidation of polyamines resulting in lowering their effective concentration [29], or switching on of a cellular mechanism that over-rides the effect of the polyamines. The transduction pathway through which polyamines mediate regulation of ACC synthase expression is unknown at the present. Since polyamines have been shown to interact with RNA polymerase II [11] and components of protein synthesizing machinery [22, 23], involvement of these processes are attractive possibilities. It is possible that high endogenous levels of polyamines in concert with other growth-promoting substances (Fig. 1) are required for repression of ACC-synthase gene(s) in growing tissues, to inhibit the untimely production of ethylene during early development. What commits

323

the plant cell into the ripening! senescence phase is a very important question central to plant biology.

Figure 2. Jntennediary metabolic connection between ethylene biosynthesis and that of polyamines.

Polyamine biosynthesis involves the synthesis of diamines putrescine (put), and cadavarine from basic amino acids [28]. Put can be formed directly from ornithine by ornithine decarboxylase (ODC), or indirectly, through a series of intermediates, including agmatine, from arginine by arginine decarboxylase (ADC), aminopropyl group from decarboxylated AdoMet is donated to form spermidine (spd) and, in turn, spermidine reacts with another aminopropyl group from decarboxylated AdoMet to form the higher polyamine spermine (spm) (Fig. 2). ADC and AdoMet decarboxylase are the key enzymes in that their activities are modulated by light, environmental stresses, and plant hormones. It is noted here that both ethylene and polyamines share AdoMet as a common precursor and methylthioadenosine as a common byproduct (Fig. 2). Reciprocal inhibition of enzymes in the two pathways can provide an effective means of controlling the contribution of each pathway to a particular developmental phase.

One approach that we have used to control ethylene and fruit characteristics is based on the fact that AdoMet is an intermediate in both ethylene and polyamine biosynthesis. During growth and development, when ACC synthase activity is

324

minimal or absent, AdoMet decarboxylase is highly active and channels AdoMet into the polyamine biosynthesis pathway by producing decarboxylated AdoMet. Upon cessation of growth and onset of the senescence phase, ACC synthase expression is induced concomitant with repression of AdoMet decarboxylase, and AdoMet is channeled to ethylene biosynthesis pathway by producing ACC (Fig. 2). This leads to the possibility that overexpression of AdoMet decarboxylase during ripening may result in the production of polyamines and thus effect biosynthetic and some other processes, among other things (Table 1), those relevant to fruit ripening characteristics. The plant would continue to produce polyamines at a developmental stage when their levels would be normally decreasing and those of ACC synthase increasing (and thus of ethylene). These experiments would also reveal if continued polyamine production (via the expression of any particular gene construct) interferes with the expression of ACC synthase, thereby delaying abscission and senescence of different organs.

Table 1. Polyamines in plant growth and development

1. Cell division

High levels before mitosis, suggesting involvement in protein and

mRNA synthesis and DNA methylation

- Increased levels ofODC, ADC and AdoMet decarboxylase

2. Fruit set and development

- Highest levels at anthesis

- Low levels during ripening

- Decreased levels ofODC, ADC and AdoMet decarboxylase

Inhibition by ethylene

3. Seed gennination:

- Increased levels of spd, spm, RNA and protein in embryo during

gennination; decreased levels in cotyledon

- Low polyamines---Iow germination potential

4. Morphogenesis:

- Conjugated polyamines involved in morphogenesis at shoot apex

- Somatic embryogenesis increases with 1-5 mM spdlspm/put

- Tuber fonnation requires putrescine

Root fonnation

Towards this goal, we constructed a chimeric gene containing the coding region of a yeast AdoMet decarboxylase [15] coupled to the tomato ripening-specific promoter E8 [8] and transformed tomato plants using Agrobacterium-mediated gene transfer procedure. Preliminary analysis of transgenic plants have indicated increased levels of specific polyamines in the ripening fruit tissue with a preferential accumulation of spd and spm in the ripe fruit. The ripening and post-ripening period of the transgenic fruit

325

on the vine also appeared to have been prolonged in some of the transformed lines. Also, after harvest, the transgenic fruit showed longer shelf-life and delayed softening. In addition to these, some transformed lines had delayed leaf senescence. One of the unexpected finding was higher levels of lycopene in the transgenic fruit of some lines. A few of the transgenic lines also showed decreased fruit set, smaller fruit size, and very low seed number. Further biochemical and molecular characterizations are in progress. Thus, introduction of an important single gene can influence several different traits in a plant. We are using this approach to obtain novel transgenic fruit, which could also be used as a model to study the interactive roles of polyamines and ethylene in regulating senescence of plants.

3. References

1. Apelbaum, A, Burgoon, AC., Anderson, J.D., Liebennan, M., Ben-Arie, R, and Mattoo, AK. (1981) Polyarnines inhibit biosynthesis of ethylene in higher plant tissue and protoplasts, Plant Physiol. 68, 453-456.

2. Apelbaum, A, Goldlust, A, and Icekson, I. (1985) Control by ethylene of arginine decarboxylase activity in pea seedlings and its implication for honnonal regulation of plant growth, Plant Physioz. 79, 635-640.

3. Ben-Arie, R, Lurie, S., and Mattoo, AK. (1982) Temperature dependent inhibitory effects of calcium and spermine on ethylene biosynthesis in apple discs correlate with changes in microsomal membrane viscosity, Plant Sci. Lett. 24, 239-247.

4. Bleecker, AB., Estelle, MA, Somerville, C., and Kende, H. (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana Science 241, 1086-1088.

5. Boller, T. (1982) Ethylene-induced biochemical defense against pathogens, in P.F. Wareing, (ed.) Plant Growth Substances, Academic Press, London, pp. 303-312.

6. Brady, C.J. (1987) Fruit ripening, Annu. Rev. Plant Physioz. 38, 155-178. 7. Burg, S.P., and Burg, EA (1966) The interaction between auxin and ethylene and its role in plant

growth. Proc. Natl. Acad. Sci. USA 55,262-269. 8. Deikman, J., Kline, R, and Fischer, RL. (1992) Organization of ripening and ethylene regulatory

regions in a fruit-specific promoter from tomato (Lycopersicon esculentum), Plant Physiol. 100,2013-2017.

9. Dibble, ARG., Davies, P.J., and Mutschller, MA (1988) Polyamine content of long-keeping Alcobaca tomato fruit, Plant Physioz. 86, 338-340.

10. Ecker, J.R (1995) The ethylene signal transduction pathway in plants, Science 268,667-675. 11. Evans, P.T., and Malmberg, RL. (1989) Do polyarnines have roles in plant development?, Annu. Rev.

Plant Physioi. Plant Mol. BioI. 40, 235-269. 12. Even-Chen, Z., Mattoo, AK., and Goren, R. (1982) Inhibition of ethylene biosynthesis by

arninoethoxyvinylglycine and by polyarnines shunts label from [3,4_14C] methionine into spermidine in aged orange peel discs, Plant Physioz. 69,385-388.

13. Flores, H.E., Protacio, C.M., and Signs, M.W. (1989) Primary and secondary metabolism of polyarnines in plants, in plant nitrogen metabolism, in Conn, E.E., (ed.) Recent Advances in Phytochemistry, 23, Plenum Press, New York. pp 329-354.

14. Fluhr, Rand Mattoo, AK. (1996) Ethylene-Biosynthesis and Perception. In Critical Reviews in Plant Sciences, CRC Press Inc., Boca Raton (In Press).

15. Kashiwagi, K., Taneja, S.K., liu, T.-Y., Tabor, C.W. and Tabor, H. (1990) Spermidine biosynthesis in Saccharomyces cerevisiae. J. BioI. Chem. 265, 22321-22328.

16. Leslie, CA and Romani, RJ. (1986) Salicylic acid: A new inhibitor of ethylene biosynthesis, Plant Cell Reports 5,144-146.

17. Li, N., Parsons, B.L., Liu, D., and Mattoo, AK. (1992) Accumulation of wound-inducible ACC synthase transcript in tomato fruit is inhibited by salicylic acid and polyarnines, Plant Mol. Bioi. 18, 477-487.

18. Liebennan, M. (1979) Biosynthesis and action of ethylene, Annu. Rev. PlantPhysiol. 30, 533-591. 19. Mattoo, AK. and Aharoni, N. (1988) Ethylene and plant senescence, in L. Nooden, and AC. Leopold,

(eds.) Senescence and Aging in Plants, Academic Press, London, pp. 241-280.

326

20. Mattoo, AI<. and Anderson, J.D. (1984) Wound-induced increase in l-aminocylcopropane-l­carboxylate synthase acitivity: Regulatory aspects and membrane association of the enzyme, in Y. Fuchs and E. Chalutz (eds.) Ethylene: Biochemistry, Physiological and Applied aspects,: Martinus NijhofflDr. W.Junk Publishers, Amsterdam, pp. 139-147.

21. Mattoo, AI<. and Suttle, J.C. (1991) The Plant Hormone Ethylene, CRC Inc., Boca Raton. 22. Mehta, AM., Safuter, R.A, Schaeffer, G.W., and Mattoo, AI<. (1991) Translational modification of

an 18 kilodalton polypeptide by spennidine in rice cell suspension cultures, Plant Physiol. 95, 1294-1297.

23. Mehta, AM., Safuter, R.A, Mehta, R.A, and Davies, P.J. (1994) Identification ofposttranslationa1ly modified 18-kDa protein from rice as eukaryotic translation initiation factor 5A, Plant Physiol. 106, 1413-1419.

24. Deller, P.W., Min-Wong, L., Taylor, L.P., Pike, D.A, and Theologis, A (1991) Reversible inhibition oftomato fruit senescence by antisense RNA, Science, 254, 437-439.

25. Roberts, D.R., Walker, M.A, Thompson, J.E., and Dumbroff, E.B. (1984) The effects of inhibitors of polyamine and ethylene biosynthesis on senescence, ethylene production and polyamine levels in cut carnation flowers, Plant Cell Physiol. 25, 315-322.

26. Safuter, R.A and Baldi, B.G. (1990) Polyamine levels and tomato fruit development: possible interaction with ethylene, Plant Physiol. 92, 547-550.

27. Sexton, R. and Roberts, J.A (1982) Cell biology of abscission, Annu. Rev. Plant Physiol., 33, 133-162.

28. Slocum, R.D. (1991) Polyamine biosynthesis in plants, in R.D. Slocum and H.E. Flores, (eds.) Biochemistry and Physiology ofPolyamines, CRC Press, USA, pp 23-40.

29. Smith, T.A and Marshall, J.H.A (1989) Oxidative decarboxylation of amino acids by plant extracts, Biochem. Soc. Trans. 16,972-975.

30. Suttle, J.C. (1981) Effect ofpolyamines on ethylene production, Phytochem. 20, 1477-1480. 31. Taylorson, R.B. and Hendricks, S.B. (1977) Dormancy in seeds, Annu. Rev. Plant Physiol. 28, 331-

354. 32. Vanden Driessche, T.H., Kevers, C., Collet, M., and Gaspar, T.H. (1988) Acetabularia mediterranea

and ethylene: production in relation with development, circadian rhythms in emission, and response to external application, J. Plant Physiol. 133, 635-639.

33. Van Der Straeten, D., Van Wiemeerscb, L., Goodman, H.M., and Van Montagu, M. (1990) Cloning and sequence of two different cDNAs encoding l-aminocyclopropane-l-carboxylate synthase in tomato,Proc. Natl. Acad. Sci. U&4. 87, 4859-4863.

34. Van Der Straeten, D., Djudzman, A, Van Caenegechern, W., Smalle, J., and Van Montagu, M. (1993) Genetic and physiological analysis of a new locus in Arabidopsis that confers resistance to 1-aminocyc\opropane-l-carboxylic acid and ethylene and specifically affects the ethylene signal transduction pathway, Plant Physiol. 102,401-408.

35. Yang, S.F. and Hoffinan, N.E. (1984) Ethylene biosynthesis and its regulation in higher plants, Annu. Rev. Plant Physiol. 35, 155-189.

36. Zhou, D., Kalaitzis, P., Mattoo, AI<., and Tucker, M.L. (1996) The mRNA for an ETRI homoloue in tomato is constitutively expressed in vegetative and reproductive tissues, Plant Mol. Bioi. 30, 1331-1338.

DIFFERENTIAL EXPRESSION OF ACC OXIDASE GENES IN MELON AND PHYSIOLOGICAL CHARACTERIZATION OF FRUIT EXPRESSING AN ANTISENSE ACC OXIDASE GENE

M. GUIS, T. BOUQUIN, H. ZEGZOUTI, R. A YUB, M. BEN AMOR, E. LASSERRE, R. BOTONDI, J. RAYNAL, A. LATCHE, M. BOUZA YEN, C. BALAGUE and J.C. PECH ENSAT (UA INRA Ethyene et Maturation des fruits) 145, Avenue de Muret, F-31076 Toulouse Cedex, France

1. Introduction

Studies on the molecular factors of fruit ripening have been, so far, almost exclusively restricted to tomato as a model fruit. However, a number of other climacteric fruit, that exhibit a sharp climacteric phase with a very fast ripening rate might also represent valuable models. Cantaloupe Charentais melon is one of these fruit. It is widely cultivated in Southern Europe and has very good organoleptic traits such as the accumulation of large amounts of sugars and the production of abundant aroma volatiles. However it has poor keeping quality and cannot withstand long transportation.

Since ethylene controls the ripening of climacteric fruit, extensive studies have been carried out on the isolation and characterization of genes involved in ethylene biosynthesis. It is now well documented that the two key enzymes of this pathway, ACC synthase and ACC oxidase, are encoded by a large and a small multigene family [1], respectively. Three ACC oxidase genes have been isolated from tomato and shown to be differentially expressed [2, 3]. In the melon, we have also isolated and characterized three ACC oxidase genes and demonstrated their differential expression [4]. We bring here a more complete picture of the expression pattern of the three ACC oxidase genes in melon during the development of a number of plant organs and under the effect of various stimuli.

The isolation of ethylene biosynthetic genes has enabled the control of fruit ripening through biotechnological methods. Reversible inhibition of tomato fruit ripening has been first achieved by down-regulating ACC oxidase [5] and ACC synthase[6] gene. The same result has been also obtained by overexpressing genes of ACC deaminase [7] and S-adenosylmethionine hydrolase [8], both enzymes reduce the

327

A. K. Kanellis et al. (eds.), Biology and Biotechnology of the Plant Hormone Ethylene, 327-337. © 1997 Kluwer Academic Publishers.

328

availability of ACC by metabolizing this ethylene precursor. However, up to now, these methods have only been applied to tomato. In the present paper, we describe the biochemical and physiological characteristics of Cantaloupe Charentais melons harboring an antisense ACC oxidase gene that exhibit strong reduction of ethylene production and inhibition of ripening both on and off the vine.

2. Results

2.1. PLANT MATERIAL

Experiments were carried out using a cultivated pure line (cv Vtdrantais) of Charentais type Cantaloupe melons (Cucumis me/o, Naud). Control and transformed plants were cultivated in a greenhouse and trained in trellis under standard cultural practices for fertilization and pesticide treatments. Agrobacterium-mediated transformation was performed using cotyledons as described previously in [9].

Studies of ACC oxidase (ACO) gene expression were carried out using (i) fruit picked 30 days (unripe) and 40 days (ripe) after pollination, (ii) flowers collected the day of anthesis or at the senescence stage, (iii) etiolated hypocotyls grown in the dark for 14 days, and (iv) 2 week-old plantlets grown on vermiculite substrate and submitted to various stress conditions. Wound effect was allowed to develop for 2 hours after laceration of leaf blades. Salinity stress was performed by adding 250 mM NaCI to the substrate for 4 hours and drought stress by leaving the plantlets for 4 hours on the bench after removal from vermiculite.

2.2. DIFFERENTIAL EXPRESSION OF ACC OXIDASE GENES IN THE MELON

We have previously shown that melon ACO is encoded by a three member multigene family [4]. We present here (Fig. 1) a picture of their differential expression in a number of tissues and under various stimuli. Expression of CMe-AC02 was found only in etiolated hypocotyls. Both CMe-AC01 and CMe-AC03 were expressed in male and hermaphrodite flowers, but CMe-A CO 1 was more strongly expressed in senescing flowers and CMe-AC03 in fully open flowers at anthesis. CMe-AC01 and to a lesser extent CMe-AC03 were expressed in leaves. Their basal level (control lanes) was much lower in leaves than in roots. Salinity, drought, wounding and ethylene all greatly stimulated the expression of CMe-A CO 1 in leaves, while only salinity and drought had an effect on CMe-AC03 expression. In roots, salinity and drought did not stimulate expression of any of ACO genes. Fruit exhibited strong expression of CMe­AC01 only at the ripening stage, while all three genes were expressed in etiolated hypocotyls, with higher expression for CMe-A CO 1.

329

AC031 .... ......

& 0 ~ ~ ~~

B cP v'1. ~o

~ ~ ~o c} vOJ

C CP~ ~1j <;:},o

AC01 r CD AC01 1-.. 1 m­

AC031 .• , .•• 1 m AI <

AC03 CD III

AC01 ~ o

AC03 H,#,~,i.-I ur

o AC01

E AC01

AC02 AC02

AC03 AC03 t~;,1 , .. ~

Figure 1. Differential expression of melon ACC oxidase genes monitored by RT-PCR during development of the plant and under various stimuli. Total RNA from leaves, roots and flowers was isolated according to [10] and total RNA from fruit and etiolated hypocotyls according to [11]. RT-PCR was performed as previously described [4]. (A): male and hermaphrodite flowers at anthesis or at the senescence stage; (B): wounded and ethylene-treated leaves; (C): leaves and roots after NaCI and drought treatments; (D): Fruit; (E) etiolated hypocotyls.

330

2.3. MOLECULAR CHARACTERISTICS OF THE SELECTED TRANSFORMED LINE AND INHERITANCE OF THE TRANS GENE

Several sets of transformation allowed us to generate around 30 primary transformants that were screened for several characters: integration of the transgene, by PCR; resistance of young seedlings to kanamycine; and ploidy level. One particular line was selected for its strong inhibition of wound-ethylene production and self pollinated. Statistical analysis of kanamycin resistance was performed on the Rl population by germinating seeds on kanamycin-enriched medium. The segregaton ratio was close to the theorical 3/1 Mendelian ratio ('1: test significant at P>O.2), indicating that the transgene was inserted at one locus only. Molecular characterization of this line showed that two copies of the transgene had been incorporated in this locus. In paired t-test, the mean values of wound ethylene production of leaves from the two segregation classes (Table 1) were significantly different from each other (P<O.OOI). In addition, there was a very high correlation (r=O.86; P<O.OOl) between the ripening behavior and the level of wound ethylene production.

Table 1. Co-segregation of the non-ripening phenotype and suppression of leaf wound ethylene in Cantaloupe Charentais melons (R! progeny). Wound-induced ethylene production was measured using 5 day-old disk leaves ofR! plants. Leaves were taken from the same rank of the plants (the fiflh rank) and cut into lern' disks using a cork borer. For each assay, 3 disks were placed in 6 ml vials and vacuum infiltrated (Imin, 400 mbars) with a 2 ml solution of 50 mM Tris-HCI (PH 7.0), 100 mM sucrose and 250 11M ACC. After 2 hours pre-incubation, the vials were sealed, and I hour later, I ml gas samples were taken from the head-space and analyzed by gas chromatography.

Fruit Number of plants Leaf ethylene production (nLg-'.h-')

Ripening inhibited 33"< 6.2 +1-3.3b.<

Normal ripening 16"< 29.7 +1- 1O.2b.<

·Chi-square test: ·l=1.53, 0.2<P< 0.3 bMean comparison between two classes of ethylene production (Student t = 8.702; significant at P = 0.00 I) <Coefficient of correlation between the ability to ripen and the level of leaf ethylene production. (r = 0.86; significant at P = 0.001)

331

2.4. ETHYLENE PRODUCTION AND ACC METABOLISM

Figure 2 shows that control untransformed fruit kept on the vine exhibited a climacteric increase in internal ethylene concentration starting from 33 days after pollination and reaching a peak of 87 ppm at 35 days. In contrast, the internal concentration of transgenic fruit remained below 0.5 ppm even at late stages of fruit development (50 days after pollination). The level of inhibition of ethylene production at the climacteric peak was therefore above 99%. Northern blot analysis of total RNA (Fig. 2, insert) showed that transcripts hybridizing to ACC oxidase probe were absent in untransformed fruit at the preclimacteric stage (31 day-old) but strongly accumulated in climacteric fruit (35 day-old). By contrast, transgenic fruit showed no accumulation of mRNAs at any stage of development.

-:2 n. n. - 95 w Z W ......J 75 >-J: ~ 55 w ......J « z 35 a:: w ~

15 z

o

Transformed

Untransformed

31 35

31 33 35 37 39 41 43 45

DAYS AFTER POLLINATION

Figure 2. Ethylene production and ACCoxidase expression at the transcriptionailevel (insert) in control (-) and transgenic melon (- - - -). Internal ethylene concentration of ftuit attached to the vine was monitored using an external gas collector [12]. Gas samples were taken daily from the collector through a serum stopper and ethylene was assessed by gas chromatography. Northern blot assays were performed using total RNA extracted from ftuit according to [9).

332

~~----------------~ A

,I, .... 1'/ ,. .. , .. . ,' I"

, , ,1

32 36 40 44 48 DAYS AFTER POLLINA TION

25r--------------------. B 1-.... 1 ' , I ' . , , .... I' ' ..

32 36 40 44 48 DAYS AFTER POLLINATION

Figure 3. ACC content (A) and ACC synthase activity (8) of control (--) and antisense ACC oxidase melons (- - - -) during development on the vine. ACC was extracted and assayed as described in [13]. ACC synthase was extracted and assayed as already reported [13].

As already found in other climacteric fruit [13, 14] the burst in ethylene production of control fruit was associated with a peak of ACC content, and ACC synthase activity (Fig. 3). Antisense ACO fruit also exhibited a rise in ACC content, starting at the same time as control fruits, and steadily increasing until 48 days after pollination to reach values that were about 10 times higher than control fruits at the climacteric peak (Fig. 3A). Similarly, ACC synthase activity of transgenic fruit started to increase at 31 days after pollination and reached high levels even at late stages of development when its activity on control fruit had sharply declined (Fig. 3B). The level of MACC, the conjugated derivative of ACC, started to increase steadily at 31 days in both wild type and antisense fruits (Fig. 4A). However, in wild type fruits, accumulation of MACC is quicker than in antisense fruits: wild type reached 25 nmoles. g-! FW, 36 days after pollination while in antisense ACO fruits, this amount was reached only after 48 days after pollination. The rapid accumulation ofMACC in wild type melons was correlated with a sharp and transient stimulation of ACC N-malonyltransferase activity (Fig. 4B) while in transgenic fruits, activity remained at a constant basal level throughout. A stimulation of ACC N-malonyltransferase activity by ethylene has already been described [15].

40

~ ~ bD

:; 30

~ '""" U 20 ~ . ~ 0

10

~

>< 200 A ~ B ....

E-<

~ 160

w~

... ·I ~ ~ 120 w---~-= . ~~ .

.! .. ··r >ol! . "t···· ~

~ :::g 0

32 36 40 44 48 32 36 40 44 48 DAYS AFTER POLLINATION DAYS AFTER POLLINATION

Figure 4. MACC content (A) and ACC N·malonyltransferase activity (B) of control (-) and antisense ACC oxidase melons (- - - -) during development on the vine. MACC was extracted and assayed as described in [13]. ACC N-Malonyltransferase wes extracted and assayed as already reported [13].

333

2.5. BIOCHEMICAL EVALUATION OF TRANSGENIC FRUITS ON THE VINE

Sugar accumulation is an important feature of melon fruit sensory quality. Figure 5 shows that total soluble solids (TSS) accumulate at approximately the same rate in wild type and antisense fruit until about 36 days after pollination. Thereafter, TSS increased only slightly in transgenic fruits. Under our conditions, the commercial picking date would be around 33 days. At that date fruit had not reached their maximum sugar content. Interestingly, as ACO antisense fruit can be kept on the vine for a longer period of time without risk of over-ripening, they can be allowed to accumulate higher amounts of sugars. At present, the major stumbling block encountered by the growers of Cantaloupe Charentais melons is that harvesting of the fruits must take place at a precise date. The main implication of this feature is that the harvest date is able to be delayed and the period in which the fruit can be picked from the vine is significantly extended.

It is well known that wild type Cantaloupe Charentais melons exhibit a very fast softening of the flesh during ripening (Fig. 6). The suppression of ethylene production in transgenic fruits resulted in an almost complete inhibition of softening during development on the vine (Fig. 6). Some cell wall degrading enzymes displayed similar activity in control and transgenic fruits, e.g. pectin methyl esterase (Fig. 7 A) while others, e.g. a-L-arabinosidase (Fig. 7B) or ~-D-galactosidase (not shown) showed a much higher activity in wild type than in transgenic fruits. These data demonstrate that some cell wall degradation patterns are ethylene-independent, while others are ethylene-dependent.

334

16

>:

~ f ····1 .,..-- .-00 ~ >-< ....:I 0 00

~ ....:I ~

B 4 0 00

0

Figure 5. Accumulation of total soluble solids in control (--) and antisense ACO melons (- - --) on the vine. Total soluble solids ~Brix) of mesocarp tissue were assessed by refractometric reading of juice samples made from each individual fruit Values are expressed as the mean standard error of measurements per fruit carried out on three different fruits.

10.0_-----------.

2.0

0.0 L-.L...&......&... ....... ...L....&.. ..... ....&....& ...... 32 36 40 44 48 DAYS AFTER POLLINATION

Figure 6. Finnness of the flesh of control (--) and antisense ACO melons (- - - -) on the vine measured destructively using an Effegi penetrometer. Finnness was quantified either destructively by taking four equid instant measurements in the flesh of fruit cut from the stylar end, using an 8mm cylinder pressure tester penetrometer (Effegi) or non destructively by measuring the compression force required to cause a 2mm defonnation of the equatorial diameter of intact fruit using a Panelaup computer based penetrometer

2.6. POSTHARVEST RESTORATION OF RIPENING BY EXOGENOUS ETHYLENE

Treatment with 50 ppm ethylene was performed on transgenic fruit that were harvested at 40 (Fig. 8A) or 43 days after pollination (Fig. 8B) corresponding to the climacteric peak of wild type fruit. Figure 8A shows that exogenous ethylene treatment was able to restore yellowing of the rind at a rate that was identical to that of control fruit. On the other hand, the non destructive monitoring of the firmness of intact fruit (Fig. 8B) indicated the following: (i) control wild type frui.ts had already displayed significant softening at harvest time and continued to soften during storage in air ; (ii) antisense ACO fruits exhibited some softening during storage in air but always remained approximately twice as firm as wild type fruit; (iii) softening was stimulated in antisense ACO fruit by ethylene treatment (50 ppm) with antisense fruits reaching the same firmness as control fruits at 48 days, i.e. after 5 days treatment.

>< t: >-~[ ~tlI) CI:l~

~.S ~~ E-<-00 0 ~ ;j ....:1.;3

~ S E-< (!)

~-:::E 0 Z! f:: U ~ ~

335

50 50 A B

40

~-----f-----i ~ ~'""' 40

6~ 30 <t:~

~~

~rS Cl~

1-----f -----i 20 - - (!) 00-0 0

~! .-' 10

(:Q

~ <t:

0 I I I I • I 0 32 36 40 44 48 32 36 40 44 48 DAYS AFTER POLLINATION DAYS AFTER POLLINATION

Figure 7. Activity of pectin methyl esterase (A) and ll-L-arabinosidase (B) in control (--) and antisense ACO melons (- - - -) during development on the vine. Glycosidases were extracted according to the method of Watkins et al. [17] as modified by Fils-Lycaon and Buret [18]. ~-D-galactosidase and a-L-arabinopyranosidase activities were assessed using the appropriate p-nitrophenyl glycoside substrate. Pectin methyl esterase was extracted and assayed as in [19].

40r----------------~--------------. 10000 A Q B:

::!J ~~~ ~ BOOO , .

I \ ~\ ~ , \ • tIl ~ • ,

6000 • " E-< , • •

~ • • • • • "-e, >< • +, 'e- .. E-< , U " f::

, , 00 "'", <t: " ....:I '.,. ~

32 36 40 44 48 0

42 44 46 48 50 DAYS AFTER POLLINATION DAYS AFTER POLLINATION

Figure 8. Effect of exogenous ethylene on the reversibility of antisense phenotype for color (A) and firmness (8). Fruit were harvested at 40 (A) or 43 days (8) after pollination and stored at 25°C in air or in 50 ppm ethylene. Color of the rind was monitored with a reflectance meter (Minolta CR-300 chromameter). Measurements were made using the L. *a*b color system, a and b being respectively chromaticity coordinates on a green-red and yellow-blue axis. Firmness was monitored non destructively using a PENELAUP firmness tester. Symbols represent: (e --e) control fiuit in air; (e - - - e) antisense ACO fiuit in air; (11- - - _) antisense ACO fiuit in the presence of 50 ppm ethylene. Vertical dashed line indicates the harvest date. Four independent measurements were made on each fiuit.

336

3. Conclusions

Like other plant species [3, 20], the melon ACC oxidase gene family comprises three members that are differentially expressed. Expression of CMe-A CO 1 was extremely high in fruit and was strongly stimulated by exogenous ethylene. We therefore conclude that, this gene probably plays an important role in autocatalytic ethylene production during fruit ripening. Antisense constructs of CMe-A CO J were able to reduce ethylene production in the fruit by more than 99% and to inhibit ripening. The transgenic fruit generated therefore provide an interesting model to discriminate between patterns of fruit ripening that are ethylene-regulated and ethylene­independent. One of the most intriguing findings was the very high activity of ACC synthase that developed as a function of age in antisense ACO fruit. It can be hypothesized that the corresponding gene could be developmentally regulated and ethylene inhibited. Strong expression of this gene in transgenic fruit would be made possible by the absence of feed-back inhibition by ethylene. In wild type fruit, on the contrary, its expression would be transient and would correspond to the first step of a cascade in which a second ACS gene, now ethylene-responsive, would be put in motion by the low levels of ethylene generated during the expression of the first ACS gene. Work is in progress to test this hypothesis. On the applied side, the newly generated transgenic melons, with extended shelf-life and capacity to ripen on command, have a promising potential for commercial development.

4. Acknowledgments

The authors greatly acknowledge the support of the EU (ECLAIR AGRE-015) and the Midi Pyrenees Regional Council for the generation of antisense ACO melons, of INRA (AlP Matural) for the study of the expression of ACO gene family, and the French Ministry of Agriculture (Aliment Demain) for the evaluation of quality traits. We wish to thank J. Pradier and H. Durtaut for their efficient participation in the molecular and biochemical analysis. We are grateful to Prof. S.F. Yang (UC Davis, USA) for useful discussions made possible by a NATO Collaborative grant (No 930996).

5. References

1. Kende, H. (1993) Ethylene biosynthesis, Annu. Rev. Plant Physiol. Plant Mol. BioI. 44, 283-307. 2. Bouzayen, M., Cooper, W., Barry, C., Zegzouti, H., Hamilton, AJ., and Grierson, D. (1993) EFE

multi gene family in tomato plants : expression and characterization. In Cellular and molecular aspects of the plant hormone ethylene,. J.C Pech, A Latch!., C. Balagm (eds,), K1uwer Academic Publishers, Dordrecht, the Netherlands, pp76-81.

3. Barry, C.S., Blume, B., Bouzayen, M., Cooper, W., Hamilton, AJ., and Grierson, D. (1996) Differential expression of the l-arninocyclopropane-l-carboxylate oxidase gene family of tomato, Plant J. 9, 525-535.

4. Lasserre E., Bouquin T., Hernandez J., Bull J., Pech J.C., and Balague C. (1996) Structure and expression of three genes encoding ACC oxidase homologs from melon (Cucumis melD L.), Mol. Gen. Genet. 251, 81-90.

337

5. Hamilton, AJ., Lycett, G.W., and Grierson D. (1990) Antisense gene that inhibits synthesis of the honnone ethylene in transgenic plants, Nature 346, 284-287.

6. Oeller, P.W., Min-Wong, L., Taylor, L.P., Pike, D.A, and Theologis A (1991) Reversible inhibition of tomato fruit senescence by antisense RNA, Science 254, 437-439.

7. K1ee, H.J., Hayford, M.B., Kretzmer, KA, Barry, G.F., and Kishore G.M. (1991) Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants, Plant Cell3, 1187-1193.

8. Good, x., Kellogg, J.A, Wagoner, W., Langhoff, D., Matsumura, W., and Bestwick, R.K (1994) Reduced ethylene synthesis by transgenic tomatoes expressing S-adenosylmethionine hydrolase, Plant Mol. BioI. 26,781-790.

9. Ayub, R., Guis, M., Ben Amor, M., Gillot, L., Roustan, J.P., Latcht, A, Bouzayen, M., and Pech, J.C. (1996) Expression of ACC oxidase antisense gene inhibits ripening of cantaloupe melon fruits, Nature Biotechnology 14, 862-866.

10. Roby, D., Broglie, K, Gaynor, J., and Broglie, R. (1991) Regulation of a chitinase gene promoter by ethylene and elicitors in bean protoplasts, Plant Physiol. 97,433-439.

11. Balagut, C., Watson, C.F., Turner, AJ., Rougt, P., Picton, S., Pech, J.C., and Grierson, D. (1993) Isolation of a ripening and wound-induced cDNA from Cucumis melo L., with homology to the ethylene-forming enzyme, Eur. J. Biochem. 212,27-34.

12. Salveit Jr, M.E. (1993) Internal carbon dioxide and ethylene levels in ripening tomato fruit attached to or detached from the plant, Physiol. Plant. 89,204-210.

13. Mansour, R., Latcht, A, Vaillant, v., Pech J.C., and Reid M.S. (1986) Metabolism of 1-aminocyclo­propane-I-carboxylic acid in ripening apple fruit, Physioz. Plant 66,495-502.

14. Bufler, G. (1984) Ethylene-enhanced 1-aminocyclopropane-1-carboxylic acid synthase activity in ripening apples, Plant Physiol. 75, 192-195.

15. Liu, Y., Hoffinan, N.E., and Yang, S.F. (1985) Ethylene-promoted malonylation of 1-aminocyclo­propane-I-carboxylic acid participates in autoinhibition of ethylene synthesis in grapefruit flavedo discs,Planta 164, 565-568.

16. Abbal' Ph. and Planton, G. (1990) La mesure objective de la fermete des fruits et legumes In: 9° Colloque sur les recherchesfruitieres. CTIFL-INRA, Publishers, 147, Rue de I'Universite, 75338, Paris, 69-81.

17. Watkins, C.G., Haki, J.M., and Frenkel C. (1988) Activities of polygalacturonase, a-D-mannosidase, and a-D and boD galactosidases in ripening tomato, HortScience 23,192-194.

18. Fils-Lycaon, B. and Buret, M. (1991) Changes in glycosidase activities during development and ripening of melon, Postharvest Biology and Technology 1, 143-151.

19. Taillan, E., Ambid, C., Pech, J.C., and Raynal, J. (1992) Demethylation of pectic substances: relationship to methylesterase activity during brine storage of cherries, J. Fd Sci. 57,682-685.

20. Tang, x., Wang, H., Brandt, AS., and Woodson, W.R. (1993) Organization and structure of the 1-aminocyclopropane-1-carboxylate oxidase gene family from Petunia hybrida. Plant Mol Bioi. 23, 1151-1164.

GENETIC MODIFICATION OF EmYLENE BIOSYNTHESIS AND ETHYLENE SENSITIVITY IN CARNATION

A.C. VAN ALTVORST, A.G. BOVY, G.C. ANGENENT AND J.J.M. DONS Department of Developmental Biology, Centre for Plant Breeding and Reproduction Research (CPRO-DLOj, P.o. Box 16, 6700 AA Wageningen, The Netherlands

1. Shoot Regeneration and Agrobacterium Transformation of Carnation

The work presented in the oral paper comprises the development of a regeneration and transformation system for carnation (Dianthus caryophyl/us L.) [I). One of the more critical factors in the genetic transformation is the ability to regenerate a mature plant from a single cell. Therefore much attention was paid to shoot regeneration of carnation. Explants from leaves were used and adventitious shoot formation was developed at the bases of the explants. It was possible to generate adventitious shoots at the transition region from stem to leaf: the leaf base. The position of leaves on the plant appeared to be important for subsequent regeneration. The youngest leaves (leaf position 1) showed the highest regeneration percentage. The shoot regeneration procedure was efficient, 65 % of the explants showed regeneration with an average of 10 shoots per explant. Regenerants from leaf explants did not show aberrant plant growth. The shoot regeneration procedures were applicable for a wide range of cultivars.

Transformation of carnation was carried out using Agrobacterium tumefaciens. The Agrobacterium strains used in the research project were able to transfer the kanamycin resistance gene. Transformed cells could grow on kanamycin containing medium, while non-transfromed cells died. Shortly after infection, the number of transformation events in a leaf explant was estimated using a reporter gene, the gus gene. Using a histochemical assay, the presence of the introduced gus gene in a cell was visualized as a blue spot. The transformation effiency was dependent on the age of the leaf explant, with the youngest leaves showing the highest number of explants with GUS-positive spots. Four weeks after A. tumefaciens infection, the first GUS-positive shoot primordia were visible. Despite selection on kanamycin-containing medium, only a small fraction of the adventitious shoots was transgenic. Apparently, regeneration was not fully inhibited by kanamycin (Tabel 1). The trangenic plants were GUS-positive and able to root on kanamycin. Control plants however failed to root under these conditions. The transformation percentage (the number of transgenic shoots per 100 leaves) varied from 3 to 4 % after infection with the A. tumefaciens strain

339

A. K. Kanellis et al. (eds.), Biology and Biotechnology olthe Plant Hormone Ethylene, 339-345. © 1997 Kluwer Academic Publishers.

340

AGLO(pCGN7001). The number of introduced gene copies varied between the transgenic carnation plants from 1 to 7. By sexual crossing, it was shown that the introduced genes were inherited in a Mendelian fashion.

The A. tumefaciens-mediated transformation procedure enabled us to apply genetic transformation strategies for improving carnation cultivars. The aim of the research was to develop transgenic carnation plants with an improved vase-life.

Table 1. Tranfonnation of 4400 carnation leaves with pAB250, pAB260, pAB280 and pAB2100 constructs.

Total number of Number of harvested shoots transgenic shoots

6 weeks 325 105

9 weeks 142 56

12 weeks 58 14

Total 525 175

2. The role of Ethylene in the Senescence of Carnation

Control of flower senescence is of great interest to horticulturists in search for methods to improve the postharvest longevity of cut flowers. The senescence process is mediated by a series of highly co-ordinated physiological and biochemical changes, such as increased activity of hydrolytic enzymes, degradation of starch and chlorophyll, loss of cellular compartmentation, and a climacteric surge in respiration. These changes are associated with changes in gene expression and de novo synthesis of proteins [2]. In many plant species the senescence process is regulated by the phytohormone ethylene. In addition, ethylene is an important regulator of plant defense responses, fruit ripening, leaf abscission, seed germination and many other processes in plant growth and development [3]. During carnation flower senescence, the hormone ethylene functions as the central regulator (Fig. 1). The senescence process of carnation flowers is associated with a climacteric increase in the production of ethylene. This ethylene production is autocatalytic, which means that exposure to ethylene stimulates ethylene biosynthesis [4]. Ethylene serves to initiate and to regulate the processes that finally lead to programmed cell death. An overview of the (molecular) mechanisms underlying the regulation of ethylene biosynthesis and ethylene sensitivity is given by Van Altvorst and Bovy [5].

The vase-life of carnation flowers can effectively be increased by pre-treatment of flowers with chemicals that inhibit either ethylene synthesis or the ethylene response. In practice, silver is supplied as an ionic complex with sodium thiosulphate (STS) to inhibit the binding of ethylene to its receptor. Carnation flowers treated with STS do

341

not exhibit a climacteric rise in ethylene production and have an extended vase life. However, because of environmental pollution, the use of STS will be restricted in the near future. Therefore, genetic modification of ethylene biosynthesis or ethylene sensitivity will be good alternatives for the use of chemicals. The Agrobacterium­mediated transformation method was used to introduce genes that inhibit either ethylene synthesis or the ethylene response. In this article, the potential to control the flower senescence process by genetic engineering techniques will be discussed.

(1) SAM synthetase

(2) ACe synthase

(3) ACC oxidase

(4) ACC malonyl-transferase

Ag+

ACC

~. Ethylene

Signal transduction , Gene expression , Protein synthesis ,

Response: flower senescence

Figure 1. Schematic overview of ethylene biosynthesis, signal transduction and effects. ACC: l-aminocyclopropane-l-carboxylic acid; MACC: malonyl 1-cyclopropane-l-carboxylic acid; MTA: 5'-methylthioadenosine; SAM: s-adenosyl­methionine; STS: silver thiosulphate.

+

342

3. Inhibition of Ethylene Synthesis

The ethylene biosynthesis route has been fully elucidated by Yang and Hoffman [6]. Genes encoding SAM synthase, ACC synthase and ACC oxidase have been isolated from several plant species, including carnation. The climacteric increase in ethylene biosynthesis during carnation flower senescence was associated with a dramatic increase in the accumulation of the mRNAs encoding ACC synthase and ACC oxidase, while these mRNAs were undetectable in presenescent petals [7]. This suggests that the autocatalytic ethylene production is regulated, at least in part, at the level of transcription. To inhibit ethylene synthesis we have introduced sense and antisense ACC oxidase constructs in carnation. This should lead to antisense-inhibition or co­suppression of the endogenous ACC oxidase gene. By using different promoters we aim to inhibit ethylene synthesis either in the whole plant, or specifically in the petals.

To inhibit ethylene synthesis a set of DNA constructs was made (Fig. 3). To suppress ACC oxidase gene expression in all flower parts a co-suppression construct was made, in which a sense ACC oxidase cDNA was transcribed from the constitutive CaMV 35S promoter. Since this promoter is active in the whole plant it is possible that also other ethylene-dependent processes, such as defense responses, may be influenced. Therefore, we also made a construct in which the ACC oxidase cDNA was transcribed in an antisense orientation from a flower-specific promoter ifsp).

The various components of the constructs were amplified by means of PCR (P 35S,

P jsp, Tnos, and the gus gene) or by reverse transcriptase PCR (ACC oxidase cDNA). These components were subsequently cloned in binary vector pAB2, a derivative of pCGN7001 [8]. A compilation of the constructs is shown in Figure 3. Construct pAB250 is a control construct in which the flower specific promoter P jsp is fused to the gus gene. This construct is used to monitor the expression of the flower specific promoter. In the antisense-suppression construct pAB260 the ACC oxidase cDNA is cloned in antisense orientation behind the flower specific promoter. Construct pAB280 is a P35s-gus-Tnos fusion to monitor the expression of the 35S promoter. In the co­suppression construct pAB2100, the ACC oxidase cDNA is cloned in sense orientation behind the the 35S promoter.

The four constructs were transformed into cultivar CPRO 89100 by Agrobacterium­mediated transformation of leaf explants [I]. About one third of the regenerated shoots were able to root on kanamycin (Table 1). The transformation efficiency was 4% (4 transgenic plants per 100 leaves). From 4400 infected leaves, we obtained 64 fsp­ACO, 47 35S-ACO and 21 GUS transgenic plants. Southern analysis of 30 transgenic plants revealed that the constructs were integrated correctly and that the number of integrated gene copies varied between 1 and 5 per genome (results not shown).

The transgenic plants were transferred to the greenhouse. Preliminary experiments revealed that the vase-life of fsp-ACO plants was almost twice as long. Transgenic plants showed a vase-life of 14 days while control plants had a vase-life of 8 days. The effect of the introduced genes on the vase-life of these plants will be analyzed in detail. We will investigate the expression of the ACC oxidase gene, study the physiology of

343

the plants and compare the constitutive versus flower-specific inhibition of ethylene synthesis.

pCGN7001

ILB , Gm

pAB2

,LB Gm

Nptli

BamHI

, I BamHI

I MAS-GUS

SaAICledl BamHI

Nptll ,I RB ,

BamHI

h~B ,

Figure 2. Schematic drawing of the T-DNA regions ofplasmids pCGN7001 and its derivative pAB2. LB: left border; RB: right border; Om: gentamycin resistance gene; Nptll: kanamycin resistance gene; MAS-GUS: mannopine synthetase promoter-gus gene fusion.

Sail

pAB250 I Fsp

pAB260 Fsp

pAB280 35S

pAB2100 35S

8amHI

I GUS

eseplxoooV

GUS

EcoRIClal

ITnOS I

ITnos I

ITnos I

ACC oxidase ITnos I

Figure 3. Schematic representation of the SalI/ClaI inserts of plasmids pABSO, pAB60, pAB80 and pABIOO.

344

4. Inhibition of the Ethylene Response

Plants in which the ethylene synthesis is inhibited, either by chemicals or by genetic modification, are still sensitive to exogenous ethylene from the surrounding. This may cause an accelemtion of senescence e.g. during transportation. Plants in which the ethylene response is blocked, e.g. by STS treatment, are insensitive to exogenous ethylene. Therefore, blocking the ethylene response may be more effective to increase flower vase-life than blocking ethylene synthesis.

The ethylene response pathway is much less characterized than the ethylene synthesis pathway. The ethylene response is presumably mediated by binding of ethylene to a specific ethylene receptor whose activation signal is tmnsduced via a protein kinase cascade. Recently, major achievements in the elucidation of the ethylene signal transduction pathway have been made by the analysis of so-called 'triple response' mutants in Arabidopsis thaliana. Etiolated wild-type Arabidopsis seedlings that are exposed to ethylene exhibit three distinct morphological changes: (1) inhibition of stem and root elongation, (2) mdial stem expansion, and (3) loss of geotropism. The screening for triple response mutants resulted in the isolation of A rabi do psis plants that failed to respond to ethylene or that constitutively displayed this response. One of the best characterized ethylene-resistant mutants is etrl [9]. Four dominant mutant alleles of etrl have been identified. Analysis of the etrl-l mutant revealed the absence of all tested ethylene responses in various parts of the plant. This suggests that the ETRl protein acts early in the ethylene signal transduction pathway. In this mutant the ethylene binding was reduced 82%, suggesting that the ETRl protein is the ethylene receptor or a protein that influences the function of the ethylene receptor. Expression of the etrl-l mutant allele in transgenic Arabidopsis plants resulted in dominant ethylene insensitive plants [9].

To inhibit the ethylene response the Arabidopsis thaliana etrl-l gene was expressed in carnation. A similar stmtegy was used as described for the construction of the ACC oxidase constructs: constitutive expression in the whole plant was achieved by using the CaMV 35S promoter, and petal-specific expression was achieved by using the flower-specific fop promoter.

A 4.55 kb etrl-l fragment was amplified from plasmid pETRl-l [9] by means of PCR and cloned behind the CaMV 35S promoter or the flower-specific fop promoter in plasmid pAB2. This resulted in plasmids pBE0210 and pBE0220 (Fig. 4). These two constructs have been transformed to carnation cultivar CPRO 89100. At present 58 fsp-etrl-l, 35 35S-etrl-l and 9 etr-etrl-l transgenic plants have been obtained and tmnsferred to the greenhouse. The effect of the introduced genes on the ethylene sensitivity and the vase-life of these plants plants will be analyzed in the near future.

Sail

pBE0210 I F8P

pBE0220 358

SamHI

I etr1-1

etr1-1

EcoRI Clal

I Tn08 I

I Tn08

Figure 4. Schematic representation of the SalIlClaI inserts of plasmids pAB50, pBE0210 and pBE0220.

5. Acknowledgments

345

The project was supported financially by the Dutch Ministry of Economic Affairs and five Dutch carnation breeding companies, viz Hilverda B.V., M. Lek en zonen B.V., P. Kooij en zonen B.Y., Van Staaveren B.V. and West-Select B.V.

6. References

1. Van A1tvorst, AC., Riksen, T., Koehorst, H., and Dons, J.J.M. (1995) Transgenic carnation plants obtained by Agrobacterium tumefaciens-mediated transfonnation of leaf explants, Transgenic Res. 4, 105-113.

2. Borochov, A and Woodson, W.R. (1989) Physiology and biochemistry of flower petal senescence, Hortic. Reviews 11, 15-43.

3. Reid, M.S. (1987) Ethylene in plant growth, development, and senescence, in Plant Hormones and their Role in Plant Growth and Development, Den Hague, Martinus Nijhoff, pp257-279.

4. Woodson, W.R. and Lawton, K.A (1988) Ethylene-induced gene expression in carnation petals. Relationship to autocatalytic ethylene production and senescence, Plant Physiol. 71, 495-502.

5. Van A1tvorst, AC. and Bovy, AG. (1995) The role of ethylene in the senescence of carnation flowers, a review, Plant Growth Regul. 16, 43-53.

6. Yang, S.F. and Hoffinan, N.E. (1984) Ethylene biosynthesis and its regulation in higher plants, Annu. Rev. Plant Physiol. 35,155-189.

7. Woodson, W.R., Park, K. Y., Drory, A, Larsen, P.B., and Wang, H. (1992) Expression of ethylene biosynthetic pathway transcripts in senescing carnation flowers, Plant Physiol. 99, 526-532.

8. Comai, L., Moran, P., and Maslyar, D. (1990) Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements, Plant Mol. BioI. 15,373-381.

9. Chang, C., Kwok, S.F., Bleecker, AB., and Meyerowitz, E,M. (1993) Arabidopsis ethylene response gene etr 1: similarity of product to two-component regulators, Science 262, 539-544.

MODULATION OF ETHYLENE PRODUCTION IN TRANSGENIC TOBACCO

M. KNOESTER, J.F. BOLi • L.C. VAN LOON AND H.lM. LINTHORSTi

Department of Plant Ecology and Evolutionary Biology. Utrecht University. PO box 80084, 3508 TB Utrecht. The Netherlands; Ilnstitute of Molecular Plant Sciences, Gorlaeus Laboratories, Leiden University, PO Box 9502. 2300 RA Leiden. The Netherlands

1. Introduction

Ethylene is involved in many physiological and developmental processes in plants. as well as in the responses of plants to abiotic and biotic stresses [1, 2]. In hypersensitively reacting tobacco. tobacco mosaic virus (TMV) infection results in increased ethylene synthesis [3]. Associated with TMV infection is the production of acidic and basic pathogenesis-related (pR)-proteins several of which have antifungal activity [4, 5. 6]. Associated with the production of PR-proteins. the plant becomes more resistant to subsequent pathogen attack. a phenomenon known as systemic acquired resistance (SAR) [7, 8]. Under the influence of the primary infection. a signal moves through the plant and induces the resistant state.

The nature of the systemic signal is still unclear. In 1977 Van Loon [9] showed that SAR can be induced artificially in the absence of TMV by local application of the ethylene-releasing chemical ethephon, suggesting that ethylene may playa role in the induction of SAR. This hypothesis is supported by observations that both the appearance of necrotic local lesions and the development of SAR are preceded by a burst of ethylene production [10]. Since the genes encoding the basic PR-proteins are strongly induced by exogenous ethylene [11]. the question was raised whether increased ethylene production in the inoculated leaf is required for the development of SAR. To avoid the use of ethylene inhibitors or action. that may have unwanted side effects, direct interference with ethylene production was sought through genetic modification (for reviews see [12, 13]). The antisense approach has been highly successful in inhibiting ethylene production and ripening in tomato fruits [14]. Ethylene biosynthesis is controlled by two enzymes. SAM is converted to ACC by the enzyme ACS. while in the subsequent step ethylene is released from ACC by the action of ACC-oxidase. also known as EFE. The conversion of SAM to ACC appears to be the rate-limiting step in ethylene synthesis and the early increase in ACS activity precedes the burst in ethylene production [3]. To determine the effect of altered ethylene levels on PR-gene expression and SAR, transgenic tobacco plants were

347

A. K. Kanellis et al. (eds.), Biology and Biotechrwlogy o/the Plant Hormone Ethylene, 347-354. © 1997 Kluwer Academic Publishers.

348

generated containing sense or antisense constructs corresponding to ACS and/or ACO. The relationship between constitutive transgene expression and inducible ethylene production was analysed.

2. Results

2.1. REGULATION OF ACS AND ACO IN TMV-INFECTED TOBACCO

To study ACO and ACS gene expression in tobacco upon TMV infection, cDNA clones encoding ACO were isolated. A lambda ZAP cDNA library from TMV-infected tobacco was screened at low stringency, using a 32P-Iabelled insert from the cDNA clone pRC13 from tomato [15]. Eleven clones were isolated belonging to two different groups according to restriction enzyme analysis. The clone cEFE-26 encoded a 36 kDa protein highly similar to ACOs from other plant species, including tomato (88.5% identity, [16]) and Petunia hybrida (90.9% identity, [17]). Clone cEFE-27, belonging to the other group, was only partially sequenced and appeared more than 90% identical to cEFE-26. Southern blot analysis of genomic tobacco DNA using cDNA inserts for ACO (cEFE-26) and ACS (pACC13, [18]) revealed that both ACO and ACS are encoded by low copy number genes.

To determine whether the increased ethylene production in tobacco upon TMV­infection is the result of induced gene expression , tobacco was inoculated with TMV and analysed for ACO and ACS gene expression in infected and non-infected leaves. Locally the amounts of both ACS mRNA and ACO mRNA increased by 36 hrs after infection, and remained high at later stages of infection. The high expression of ACS and ACO corresponds well with the increased ACC and ethylene production. Systemically ACS mRNA was not detectable at any time from 2-12 days after inoculation. ACO mRNA was transiently increased at 4 d.p.i.. This correlates with enhanced ACO activity in non-inoculated leaves [3, 10].

2.2. EXPRESSION OF SENSE AND ANTISENSE GENES FOR ACS AND ACO IN TRANSGENIC TOBACCO

The tobacco ACS cDNA clone pACC-13 and ACO cDNA clone cEFE-26 were integrated into Samsun NN tobacco in either sense or antisense orientation as single or double constructs. For ACO, the sense and antisense constructs contained the same 997 bp fragment, with most of the coding region between the 35S promoter and the nos terminator. For ACS, the antisense constructs contained the 5' 1002 bp region and the sense construct contained the full-length open reading frame of the cDNA clone. The double constructs contained (anti)sense ACS/ACO cDNAs in tandem with separate 35S promoter and nos terminator.

Tobacco was transformed by Agrobacterium-mediated leaf disk transformation, using kanamycin resistance as a marker. For each construct, 12 to 17 primary transformants were obtained. These were analysed for ACS and/or ACO RNA expression. For northern blots double-stranded probes were used to allow a direct

349

comparison of the transcript levels from the transgene and the endogenous mRNA. ACS and/or ACO transcript levels varied in all types of transformants and of each type five lines differing widely in expression level were selected for further analysis.

Because the amount of ethylene produced upon TMV infection is dependent on the number of lesions, and reproducible infection was difficult to achieve, induction by TMV was mimicked by floating leaf disks on a-aminobutyric acid (aAB) [19]. The ethylene accumulated over a 3-day period was determined relative to the amount produced by non-transformed control plants and results are presented in Table 1. Although most antisense lines had inducible ethylene levels lower than the control plants, in none the ethylene production was dramatically reduced. Line ACS(-)lS had the lowest inducible ethylene production (26% of wild type). Plants containing a double antisense construct did not have lower ethylene levels than plants with single antisense constructs.

All ACS(+) lines had increased ethylene levels. Since the ACO construct in the ACO(+) plants lacks the 3 '-terminal 13 nucleotides of the open reading frame, perhaps leading to the production of an ACO enzyme with decreased activity, it is not surprising that in most plants no overproduction of ethylene was found. However, the majority of the ACO(+) plants had lower levels of induced ethylene production than control plants, suggesting the occurrence of co-suppression. This effect also seems to be responsible for the small decrease in ethylene production in most plants containing the double sense construct.

TABLE 1. Relative ethylene levels of a selection ofT! plants from the 6 types oftransfonnants upon induction by a-aminobutyric acid. The ethylene production of wild type Samsun NN tobacco was set at 100%. (-): antisense, (+): sense 1ransfonnants.

ACS(-) ACO(-) ACS( -)ACO( -)

Line Ethylene Line Ethylene Line Ethylene

15 26 12 42 3 44

13 53 13 70 17 49

6 73 1 86 7 72

7 93 11 97 9 90

9 131 6 173 2 113

ACS(+) ACO(+) ACS( + )ACO( +)

Line Ethylene Line Ethylene Line Ethylene

15 126 10 57 13 42

17 134 6 62 11 66

13 172 5 73 3 83

14 192 16 97 1 100

5 320 9 141 14 175

350

2.3. ACS AND PR-GENE EXPRESSION IN TRANSGENIC PLANTS

To relate ethylene production to ACS and PR gene expression, sense and antisense lines shown in Table 2 were selected for further study. These lines show the largest variation in ethylene levels. Lines ACS(-)I5 and ACS(+)5 were chosen for their low and high ethylene production, respectively (Table 1). Generally the level of transgenic RNA accumulation did not correlate with the ethylene production. Therefore, line ACS(-) 13 , with low antisense RNA expression but a relatively large reduction in ethylene production was also included, as was line ACS(+) 14, which shows low transgene expression but a relatively large ethylene production. Other lines were selected because of their intermediate RNA and ethylene levels.

To analyse the accumulation of ACS sense and/or antisense RNA and PR mRNA after infection, plants from the selected lines were inoculated with 1MV and infected leaves were harvested 3 days later. Total RNA was isolated and expression levels are presented in Table 2. In healthy plants ACS(-) plants 7, 9 and 15 showed decreasing levels of the antisense ACS RNA, whereas in line 13 the antisense RNA was not detectable. After infection, the ACS mRNA levels were only reduced in lines 15 and 13. Surprisingly, no reduction was seen in lines 7 and 9, whereas an almost complete suppression of ACS mRNA expression occurred in line ACS(-)13, which apparently has a relatively low level of antisense RNA expression.

TABLE 2: Expression levels of ACS and basic PR-l in selected ACS(-) and (+) lines. Total RNA was isolated from leaves ofhealthl: Elants or Elants 3 da~ after infection with TMV.

ACS PR-lg mRNA mRNA

Healthy TMV Healthy TMV

Line antisense sense antisense sense

Control ++ ++

ACS(-)7 ++ ++ ++ ++

ACS(-)9 +/++ -/+ ++ ++

ACS(-)13 -/+ ++

ACS(-)15 + + + ++

ACS(+)5 ++++ ++++++ ++

ACS(+)13 +++ +++++ ++

ACS(+)14 +++ +++++ ++

Transgenic plants from ACS(+) lines 5, 13 and 14, contained high levels of ACS sense RNA and these levels further increased after infection. Probably, the ACS mRNA present in 1MV-infected leaves reflects the combination of the constitutive sense RNA from the transgene and the induced mRNA from the endogenous ACS

351

gene. PR-lg expression was analysed as a representative of basic PR proteins induced

(Table 2). Similar results were obtained with a probe corresponding to basic PR-5 (osmotin, data not shown). It is evident that high level expression of ACS sense RNA or increased ethylene production is not associated with constitutive basic PR-gene expression. Moreover similar levels in the control and transgenic ACS(-) or (+) plants of PR-lg mRNA indicate that silencing or overexpression of ACS mRNA does not result in differences in basic PR-gene expression.

To examine if altered ethylene levels affect the development of SAR, three plants from each of the selected lines (Table 2) were inoculated with TMV, while three others were mock-inoculated with water. Ten days after inoculation the plants were challenge-inoculated with TMV on non-infected upper leaves, and lesion sizes on these leaves were measured 4 days later. In most of the transgenic lines lesion size was reduced to the same extent as in non-transformed controls. However, lines ACS(-)7 and ACS(+)14 showed a slightly weaker and stronger SAR, respectively.

TABLE 3: Accumulation ofmRNA for etr1-1, PR-Ia and PR-Ig in transgenic tobacco containing the Arabidopsis mutant etr1-1 gene. RNA was isolated from mock inoculated leaves (healthy) and leaves treated with TMV 2 da~ earlier.

Healthy TMV

Line etrl-I PR-Ia PR-Ig etrl-I PR-Ia PR-Ig

Control + + +++++ ++++++

Tetr3 + +/- + +/++ +++ ++++

Tetr6 + +/- +/++ +++++ ++++++

TetrI8 + + +/++ +++++ +

Tetr20 + + +/++ +++++ +

2.4. MODULATION OF ETHYLENE PERCEPTION

Another way of studying the effect of ethylene is by interfering with ethylene action. The Arabidopsis mutant etrJ-J contains a defective ethylene receptor which results in dominant ethylene insensitivity [20,21]. To test whether the mutant etrJ-J gene can also act in tobacco, we transformed Samsun NN tobacco with an Arabidopsis etrJ-J construct, containing both the promoter and the coding region. After Agrobacterium­mediated leaf disk transformation 21 primary transformants were obtained, of which 12 plants developed spontaneous wilting and stem necrosis. To study etrJ-J and PR­gene expression, leaves were detached from primary transformants and inoculated with either water or TMV. Forty-eight hours after inoculation RNA was isolated and analysed for etrJ-J, PR-la and PR-lg gene expression. The Arabidopsis etrJ-J gene was expressed in 4 selected lines (Table 3). In TMV-infected leaves, expression of the etrJ-J gene was slightly increased. The slight expression of PR-la and PR-lg in healthy plants is a consequence of detachment of the leaves before inoculation. Upon

352

lMV infection expression of the acidic PR-Ia gene was little affected, but basic PR-Ig gene expression was strongly reduced in lines Tetrl8 and Tetr20.

3. Conclusions

We investigated the possibility of down-regulating ethylene biosynthesis by modification of ACS and ACO gene expression levels in order to analyse the effects on PR gene expression and SAR. Transgenic tobacco plants were constructed from cDNA clones to express sense and antisense transcripts corresponding to ACS and/or ACO. This resulted in transgenic plants with varying levels of inducible ethylene production. However, there was no close correlation between the level of transgene expression and the level of ethylene production. E.g. ACS( -)7 showed a relatively high level of antisense ACS RNA (Table 2), but ethylene production was hardly reduced (Tablel). Similarly, the high accumulation of ACS sense RNA in ACS(+)13 plants (Table 2) resulted in only a modest increase in ethylene production (Table I). Neither was a correlation observed between transgene expression and suppression of the endogenous gene. ACS(-)13 did not express measurable antisense RNA, but ACS mRNA accumulation was efficiently reduced (Table 2). In contrast, high level antisense RNA accumulation had hardly any effect on the expression of the endogenous ACS genes of ACS(-) plants 9, 15 and 7 (Table 2). Although both ACS and ACO are transcriptionally regulated [22], the present data support the idea of significant posttranscriptional regulation [23].

The northern blot analysis of Table 2 revealed that variation in ACS mRNA accumulation, resulting in modified ethylene levels, had no effect on PR-gene expression. This was observed for both sense and antisense ACS plants. Although the ethylene levels of the individual plants were not dramatically changed, the difference in ethylene accumulation between ACS(-)I5 and ACS(+)5 plants was about 10 times (Table 1).

The observation that ACS(+) plants, producing higher levels of ethylene do not constitutively express basic PR genes, may be due to the fact a 3-fold increase is insufficient to trigger PR-Ig gene expression. Alternatively, PR-gene expression might be induced only by local, high ethylene concentrations. In the transgenic plants ethylene production is equally increased over the whole leaf, whereas in lMV-infected leaves only the tissue surrounding the developing lesions produces ethylene [10).

The variation in the ethylene production seen in ACS(-) and (+) transgenic plants appear insufficient to draw conclusions about a possible role for the hormone in lMV­induced PR-gene expression or SAR. Interfering with ethylene action by introduction into tobacco of the mutant etrJ-J gene from Arabidopsis appears an attractive alternative. The first results suggest that the mutant Arabidopsis gene also interferes with ethylene signal transduction in tobacco, which opens the possibility to study its effects on PR-gene expression and SAR.

353

4. Acknowledgements

We thank dr. Leo Melchers for providing essential plasmids, Teun Tak:, Aoja Peltenburg and dr. Eric Knegt for help and advice in gas chromatographic analysis.

5. References

1. Abeles, F.B., Morgan P.W., and Saltveit Jr. M.E. (1992) Ethylene in plant biology, Academic Press, Inc. San Diego.

2. Mattoo, AK. and Suttle, J.C. (1991) The plant hormone ethylene, CRC Press, Boca Raton. 3. De Laat, AM.M. and Van Loon, L.C. (1982) Regulation of ethylene biosynthesis in virus-infected

tobacco leaves. II. Time course of levels of intennediates and in vivo conversion rates, Plant Physiol. 69,240-245.

4. Linthorst H.J.M. (1991) Pathogenesis-related proteins of plants, Crit. Rev. Plant Sci. 10, 123-150. 5. Woloshuk, C.P., Meulenhoff, E.J.S., Sela-Buurlage, M., Van den Elzen, P.J.M., and Cornelissen,

B.IC. (1991) Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans, Plant Cell 3, 619-628.

6. Ponstein, AS., Bres-Vloemans, S.A, Sela-Buurlage, M.B., Van den Elzen, P.J.M., Melchers, L.S., and Cornelissen, B.J.C. (1994) A novel pathogen- and wound-inducible tobacco protein with antifungal activity, Plant Physiol. 104, 109-118.

7. Ross, AF. (1961) Localized acquired resistance to plant virus infection in hypersensitive hosts, Virology 14, 329-339.

8. Ross, AF. (1961) Systemic acquired resistance induced by localized virus infections in plants, Virology 14, 340-358.

9. Van Loon, L.C. (1977) Induction by 2-chloroethylphosphonic acid of viral-like lesions, associated proteins, and systemic resistance in tobacco, Virology 80,417-420.

10. De Laat, AM.M., and Van Loon, L.C. (1983) The relationship between stimulated ethylene production and symptom expression in virus-infected tobacco leaves, Physiol. Plant Pathol. 22, 261-273.

11. Brederode, F.T., Linthorst, H.IM., and Bol, IF. (1991) Differential induction of acquired resistance and PR gene expression in tobacco by virus infection, ethephon treatment, IN light and wounding, Plant Mol. BioI. 17, 1117-1125.

12. Mol, J.N.M., Van Blokland, R., De Lange, P., Starn, M., and Kooter, J.M. (1994) Post-transcriptional inhibition of gene expression: sense and antisense genes, in J. Paszkowski ( ed.), Homologous Recombination and Gene Silencing in Plants, Kluwer Academic Publishers, Dordrecht, pp.309-334.

13. Bourque, J.E. (1995) Antisense strategies for genetic manipUlations in plants, Plant Science 105, 125-149.

14. Fray, R.G. and Grierson, D. (1993) Molecular genetics of tomato fruit ripening, Trends Genet. 9, 438-443.

15. Hamilton, AI, Bouzayen, M., and Grierson, D. (1991) Identification of a tomato gene for the ethylene- forming enzyme by expression in yeast, Proc. Natl. Acad. Sci. USA 88,7434-7437.

16. Holdsworth, M.I, Bird, C.R., Schuch, W., and Grierson, D. (1987) Structure and expression of an ethylene-related mRNA from tomato, Nucleic Acids Res. 15, 731-739.

17. Wang, H. and Woodson, W.R. (1992) Nucleotide sequence of a cDNA encoding the ethylene-forming enzyme from petunia corollas, Plant Physiol. 100, 535-536.

18. Bailey, B.A, Avni, A, Li, N., Mattoo, AK., and Anderson, ID. (1992) Nucleotide sequence of the Nicotiana tabacum cv Xanthi gene encoding l-aminocyclopropane-l-carboxylate synthase, Plant Physiol. 100, 1615-1616.

19. Lotan, T. and Fluhr, R. (1990) Xylanase, a novel elicitor of pathogenesis-related proteins in tobacco, uses a non-ethylene pathway for induction, Plant Physiol. 93, 811-817.

20. Bleecker, B., Estelle, M.A, Somerville C., and Kende H. (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana, Science 241, 1086-1089.

21. Chang, C., Kwok, S.F., Bleecker, AB., and Meyerowitz, E.M. (1993) Arabidopsis ethylene repsonse gene ETRl: similarity of product to two-component regulators, Science 262,539-544.

22. Knoester, M., Bol, IF., Van Loon, L.C., and Linthorst, H.IM (1995) Virus-induced gene expression

354

for enzymes of ethylene biosynthesis in hypersensitively reacting tobacco, Mol. Plant-Microbe Interact. 1, 177-180.

23. Spanu, P., Grosskopf, D.G., Felix, G., and BoUer, T. (1994) The apparent turnover of l-amino­cyclopropane-l-carboxylate synthase in tomato cells is regulated by protein phosphorylation and dephosphorylation,Plant Physiol. 106, S29-S3~

Index of Authors

A Alpuche-Solis, A 299 Anderson, J.D. 267 Angenent, G.C. 339 Antunes, M.D. 47 Avni, A 267 Ayub, R. 327

B Bailey, B.A 267 Balague, C. 327 Barry, C.S., 299 Ben Arnor, M. 327 Bennett, AB. 197 Berry, AW. 77 Bestwick, RK. 307 Betz, C. 259 Bihun, M. 113 Blay, R. 93 Bleecker, AB. 63 Blume, B. 299 Bogatek, R. 275 Bol, J.F. 347 Bonghi, C. 149 Botondi, R. 327 Bouquin, T. 327 Bouzayen, M. 327 Bovy, AG. 339 Brummell, D.A 197

C Cardinale, F.C. 267 Casadoro, G. 191 Catala, C. 197 Chang, C. 57 Charng, y.-y. 23 Chauvaux, N. 99 Childs, K.L. 105 Clendennen, S.K. 141

355

Clough, G. 307 Come, D. 275 Corbineau, F. 275 Coupe,S.A. 123,185 Creelman, RA 105

D de Vrije, G.J. 163 Deikman, J. 123 Dewitte, W. 99 Dilley, D.R. 5 Dong, J.G. 23 Dons, J.J.M. 339 Dowsley, B. 283 Drew, M.C. 105

E Ernst, D. 259

F Ferrarese, L. 191 Finlayson, S.A 105 Fray, R 299

G Gerasopoulos, D. 47 Gholland, M. 77 Gibson, E.J. 15 Giovannoni, J.J. 133 Gonzalez-Bosch, C. 197 Good, X. 307 Granell, A 93 Grierson, D. 299 Guis, M., 327

H Haegman, M. 87 Hall, M.A 77

356

Hamilton, A 299 Hanania, U. 267 Handa, A 321 Haramaty, E. 253 Harpham, N.V.l 77 He, C.-l 105 Hedderson, T.Al 15 Hemsley, Rl 77 Hong, S.-B. 175 Hua, l 71

I Ievinsh, G. 217 Iturriagagoitia-Bueno, T. 15

J Jack, T. R 283 Jackson, M.B. 229 Jaworski, S.A 5 Jennings, lC. 267 Jirage, D. 57 John, P. 15

K Kacperska, A 207 Kadyrzhanova, D.K. 5 Kalaitzis, P. 175 Kanellis, AK. 239 Kangasjiirvi, 1. 259 Kellogg, l 307 Kende, H. 31 K~pczynska, E. 113 K~pczynski, 1. 113 Kipp, P. B. 141 Klee, H.l 289 Knoester, M. 347 Koehler, S.M. 175 Kramer, M. G. 307 Kurepa, l 87

L Langebartels, C. 259 Lashbrook, C. C. 197 Lasserre, E. 327

Latche, A. 327 Lay, V. 15 Lee, I.-l 105 Leshem, Y. Y. 253 Linthorst, H.lM. 347 Liu, Y. 23

M Machlickova, I. 99 Matsumura, W 307 Matters, G.L 175 Mattoo, AK. 175,321 Maxson, lM. 155 May, G. D. 141 McBrien, R K. 283 McCully, T.l 5 Mehta, R 321 Meyerowitz, E. M. 71 Morgan, P. W. 105 Moshkov, I. 77 Mullet, lE. 105 Murakami, K.G. 5

N Nath, P. 175 Niklis, N. 47 Norman, H.A 276 Novikova, G. 77

o Orzaez, D. 93 Ozola, and D. 217

p Payton, S. 299 Pech, lC. 327 Peck, S.C. 31 Peters, S. 307 Picard, M.A. 275 Prescott, AG. 15

R Radice, S. 275 Ramina, A 149

Raynal, l 327 Roberts, lA. 185 Rose, lK.C. 197 Ruperti, B. 149

S Sakai, H. 71 Sandennann Jr, H. 259 Saniewski, M. 39 Schofield, C.l 15 Sfakiotakis, E. 47 Smalle, l 87 Smith, A.R., 77 Solomos, T. 239 Stavroulakis, G. 47 Summers, lE. 229

T Tarun, A.S. 1 Taylor, lE. 185 Theologis, A. 1 Thomas, P.G. 15 Tieman, D. 289 Tonutti, P. 149 Trainotti, L. 191 Tucker, M.L., 175 Tuomainen, J. 259

V Van Altvorst, A.C., 339 Van Amerongen, A. 163 Van der Bent, A. 163 Van Der Straeten, D. 87 Van Loon, L.C. 347 Van Montagu, M. 87 Van Onckelen, H. 99 Ververidis, P. 5, 47 Vlachonasios, K.E. 5 Voesenek, L.A.C.J. 229

W Wagoner, W. 307 Whitelaw, C.A. 185 Woltering, E.J. 163

Woodson, W.R. 155

x Xu, R. 123

y Yang, S.F. 23

Z Zegzouti, H. 327 Zhou, D. 175

357

Index of Keywords

A Abiotic or biotic stressors, 207-212 Abscission

cellulase, 176 ETR1,180 specific gene promoters, 175 regulation, 149; 175 related genes, 150; 185; 188 specific genes, 150; 189 peach fruitlet 149; 150 polygalacturonase, 179 zone 149-151; 177; 179; 188-191

ACC concentration, 150; 230; 243; 260; 262 production, 52 translocation, 170

ACC deaminase (S-adenosyl-L­methionine hydrolase), 290; 303; 307;314; 327

ACC oxidase active site, 5; 20 activation by CO2, 6; 19; 23 activity, 8; 10-12; 26; 29; 32; 34; 40-42; 52; 116-118; 119-121; 209; 210; 229; 233; 235; 236; 275; 277; 278-280; 299 antisense plants, 209; 299; 302; 303; 327; 332; 333;342; 348 binding site, 20; 23; 27 gene expression, 149; 209; 299; 300;301; 342 genes, 290; 299; 327;329 kinetics, 28 protein, 245 recombinant, 19; 23-28 stereospecificity, 26 structure-funcrion analysis, 5; 15; 26 mRNA, 106; 108

359

site-directed mutagenesis, 5; 6; 8; 12; 17; transcripts, 208; 264; 273; 300

ACC synthase active site, 2; 3 activity, 2; 3; 33; 35; 42; 43; 52; 118; 121; 261-263; 275; 322; 323; 332 antisense plants, 134; 139; 160; 290;292; 348; 350 expression, 322; 324 genes, 1; 35; 88; 90; 208; 209; 261; 289 transcripts, 229; 268; 269 site-directed mutagenesis, 1; 3; 4 structure-funcrion analysis, 1

Adaptation, 226 Aerenchyma, 109 ahs Arabidopsis mutant, 90 Alcohol dehydrogenase, 246; 249 alel,90 Amaranthus caudatus, 113; 116; 122 Amaranthus retrojlexus, 113; 116; 122 Aminoethoxyvinyl glycine (A VG), 101; 233 l-amino-2-ethylcyclopropane-l­carboxylic acid (AEC), 26 a-Aminoisobutyric acid (AIB), 23; 24 Aminotransferases, 4 Anaerobiosis, 207; 242 Antisense, 134; 139; 160; 290; 296;

305;332;336;337;348;350;353 Apoptosis, 96; 98 Apples, 40; 47 Arabidopsis ctrl, 135; 136; 137 Arabidopsis rosette, 87; 90; 91 Arabidopsis thaliana, 31; 62; 70; 71;

76; 77; 86; 91; 92; 102; 139; 214;

360

227; 265; 274; 296; 305; 325; 344; 353

a-L-arabinosidase, 333; 335 Ascorbate, 6; 8; 17-19; 23; 24; 217-

220; 225-227; 257 Ascorbate peroxidase, 219; 226 Aspartate aminotransferases 1; 3; 4 ATP, 79; 80; 81; 83; 241; 249; 250 AT-ACSJ,90 AT-ACSJ promoter, 91 Autocatalysis, 47 Auxin, 31;202;205 Avocado, 247-249

B BA,81-83 Bananas, 141; 145; 146;246 Bean, 176; 196; 199 Bean abscission cellulase, 176 Botrylis cinerea, 43; 53; 54 Brassica napus, 187; 190

C CA atmospheres, 239 Calyculin A, 263 Cantaloupe melons, 328 Carbon dioxide

activation, 6; 8; 10-13; 17; 19 Carbon dioxide binding site, 23; 27 Carnation, 155; 156;253;339;340 Catalase, 211 Cell, 198; 199; 200 Ce12, 198; 199; 200 Ce13, 200; 201 CEL4,201 Ce17, 202; 203 Cell death, 94 Cell wall, 212

degrading enzymes, 186 Cellulase, 153; 176; 177; 196; 204;

245;249 Charentais, 327; 328; 330;333 Chenopodium rubrum, 41; 43 Chenopodium rubrum, 99; 100; 104 Chilling, 51; 147; 207; 275; 277; 281

Chilling injury, 275; 281 Circadian phenomena, 105 Circadian ethylene production, 106 Climacteric fruit, 141; 243 CMCase, 199 Coronatine, 42; 44 CTRJ, 59; 61; 62; 69; 71; 72; 76; 86;

91; 135; 136; 140; 145; 148;211 Cucumber, 285 Cucumis me/o, 305; 328; 337 Cymbidium flowers, 165-167

D Dendrobium, 40; 41; 44 Desiccation, 207 dgl,202 Differential display, 189; 246 Dioxygenases, 5; 6; 10; 17; 21 DNA binding proteins, 131; 155; 160;

162 DNA fragmentation, 95; 97 Dormancy, 113; 116; 121;326

E E4, 123-131; 160 E4 promoter, 125-127; 160 E8, 123-129; 134; 160; 294; 307; 309;

324 E8 promoter, 124; 307 EGase, 191-203 EIN4, 59; 72-75 Elder, 199 Electric current, 207 Electrolyte leakage, 227; 276; 281 Elicitor, 267; 270; 272; 274 Endoglucanase, 175; 194; 197; 205 Epi, 135-137 EFUE, 156; 159; 160 EFUEBPs, 155; 160 ERS, 58; 59; 62; 70; 72-76; 92; 134;

135;293;294;296;302 Ethephon, 113 Ethrel,99 Ethylene

action inhibitor, 94

auxin-induced, 31 binding, 63; 64; 69; 77; 86; 292; 296 binding site, 58; 63; 64; 86; 296 biosynthesis, 1; 5; 15; 17; 33; 39; 41; 42; 47; 53; 55; 81; 104; 110; 130; 147; 153; 215; 216; 218; 226; 244; 264; 274; 305; 326; 336; 345; 347 diffusion, 166 evolution, 111; 150; 215; 242; 244; 249;250;262 flowering, 99 interorgan signaling, 163; 170 perception, 58; 59; 64; 69; 71; 73-78 production,5; 15;25;26; 34; 39-43; 48- 55; 56; 81; 104; 106; 107; 109; 145; 213; 215; 216; 228; 230; 231; 232;235;269-271;281;311;331 receptors, 58; 65; 71; 70; 74; 177; 181 receptor genes, 57; 58; 61; 74 signal transduction, 57-59; 68-71; 77; 78; 84; 85; 88; 89; 135-138; 145; 211 synthesis, 214; 290; 299 stress, 260

eti, 80 eti5,80-83 Etiolated pea stem, 31; 32 ETRI, 58; 59; 62; 63; 65-77; 86; 88;

92; 134; 135; 138; 139; 148; 168-171; 211; 270; 291-294; 296; 297; 305; 306;326;344;353 modelling, 168; 169 binding site structure, 163

ETR2, 59; 72-75

F Fe2+ ligands, 10; 12 Firmness, 334; 335 Flower, 93; 99 Flower senescence, 93 Flowering, 90; 99

Free radical scavengers, 210 Freezing, 207

361

Fruit, 44; 45; 48; 51; 53; 97; 123; 133; 138; 142; 146; 149; 153; 200; 204; 249; 251; 301; 308; 312; 322; 324; 325;328;330;335 ripening, 123; 133; 146;301;325

G GA3, 106; 111; 116 ~-D-Galactosidase, 156; 253; 333; 335 Gene expression, 97; 134; 161; 246;

247;260 Genetics, 15;62; 76; 87; 92;274;296 Gibberellins, 94; 95 Glucose phosphate isomerase, 246 Glutathione S-transferase (GST), 155-

159 GTP-binding proteins, 79; 80; 83-86 GUS, 156-158; 178; 339; 342; 343

H H20 2, 210; 211; 217-219; 220; 224;

225 hap5,242 Heme, 242 Heavy metals, 207 Histidine kinase, 58 hlsl Arabidopsis mutant, 88 hookless 1 (hlsl) mutation, 88 Hydrogen peroxide, 225 Hydrolase, 149; 151; 175; 191; 245; 303;307 Hypoxia, 242; 245-247; 249

I Invertase, 146;239;246-248;251 Isopenicillin N synthase, 6-8; 21 Interorgan signaling, 163

J Jasmonic acid (JA), 39

362

K Kiwifruit, 47; 48; 50 Kinase, 58;59;61;62;68; 71-74;80; 83-86; 126; 134; 135; 211; 241; 263; 291-293; 302; 344;

L Lactate, 242 Lactate dehydrogenase, 246 Laser-driven photoacoustics, 231 LeETR1,294 LeETR2,294 LeEXT, 202; 203 Lipid peroxidation, 226 Lipoxygenase, 219

M 1-Methylcyclopropene (MCP), 267;

270-273 Methyljasmonate, 39-45

N Never ripe, 95; 139 Never ripe, 292; 296 NO, 229; 253-257 N02,254 nor, 40; 43; 107; 131; 134; 136; 138;

139; 220; 308 2,5 norbornadiene, 114-116; 145; 178;

199;274 NOS, 126; 253; 257 Nutation, 33; 36; 37 Nr mutation, 134; 203; 292

o Oxidative stress, 225; 227 2-oxoacids, 17; 21 2-oxoglutarate, 6; 17; 25 2-oxosuccinate, 17; 18 Oxygen

deficiency, 234 low, 239-240; 243 regulated genes, 247

Ozone, 207; 214; 259;260;262-265

p Particle gun, 176 Pathogenesis related (PR) proteins,

188;259;268;351 Pathogens, 207 PBN (N-tert-butyl-a-phenylnitrone),

253-255; 257 Pea, 31; 199; 253 Peach, 149; 150; 153; 192 Pectin methyl esterase, 335 Pepper, 194 Petunia, 40; 41; 165; 284; 306; 337;

348 Phalaenopsis, 41; 44; 86 Phaseolus vulgaris, 42; 77; 83; 190;

196;204;214;216 Phylogeny, 16;21; 187; 195; 199 Phytochrome b, 105; 106; 108 phyB-l sorghum genotype, 108 Photoperiod, 99-10 1 Pisum sativum, 86; 93; 95; 97; 204;

227;230;232;233;253;258;278 Plant hormones, 64; 76; 91, 191; 198;

289;296;323;345 Pollination, 86 Polyamines, 212; 273, 322; 324-326 Polygalacturonase expression, 179;

190;245 Poplar, 199 Potamogeton pectinatus, 229; 232-

234;237 Primary Dormancy, 113 Promoter, 125; 126; 133; 138; 176;

178 Propylene, 50; 54 Protein phosphorylation, 82 Pyruvate, 242

R Raf, 59; 61; 62; 71; 76; 84-86; 91;

135; 140; 148 Ras,62; 79;84;85;86 Resistance, 207; 210; 212; 225; 280;

330;343;347;353

Respiration, 49; 55; 147; 239 Rhythmicity, 104 rin, 40; 43; 124; 131; 134; 136; 139;

197; 199;200;296 Ripening, 39; 133; 138; 146;204;244;

249;330 RNA differential display, 189; 247 Rosette development, 87; 88; 90

S Saccharomyces cerevisiae, 13; 60;

242;325 Salicylic acid, 194; 325 SAMase, 307-318 Sambucus nigra, 152; 185; 186; 190;

196;205 Secondary Dormancy, 116 Seed germination, 324 Senescence, 40; 41; 72; 82; 87; 89; 93-

97; 129; 137; 142; 150; 155-157; 159; 188; 191; 196; 207; 210; 212; 218; 225; 239; 253; 257; 263; 297; 324;325;328;329;340;342 related genes, 155

Shade Avoidance Syndrome, 107; 108 Signal transduction, 57; 58; 62; 68;

70; 88-92,133,134;296 Silver thiosulphate, 41; 178; 220; 340 Site-directed mutagenesis, 1; 3-8; 12;

17; 19;23;24;26;65;67 SNAP (S- nitroso-N-

acetylpenicilamine), 253; 255; 256 Sorghum bic%r (L) Moench, 105 Soybean, 41; 43; 284; 285-287 Storage, 147;239;244;246;251 Stress

ethylene, 208-213 cold,246 low oxygen, 239-250

Submergence, 207; 214; 229-237 Sucrose phosphate synthase, 246 Sucrose synthase, 246 Superoxide dismutase (SOD), 211

T TeTR, 135; 136 Transcript, 272 Transcription, 123 Transcriptional regulation, 258 Transgenic

363

plants, 91; 123; 133; 134; 146; 178; 289; 290; 294; 295; 299; 302-304; 309;342;350 fruits, 124; 311; 312-314; 331; 332-336

Trichoderma viride, 264; 268; 273 Triple response, 58; 75; 80; 87; 88; 134; 344 Two component regulators, 57; 68 Two component proteins, 58

V Vegetation, 283; 288 Vigna radiata, 275; 276; 277; 278;

279;280;281

W Waterlogging, 207; Wounding, 41; 42; 53;208

X )GETs, 198;202;204 Xylanase, 268; 271; 274; 353 Xyloglucan, 190 Xyloglucan endotransglycosylase, 186; 190; 198

y Yeast, 242 Yeast osmolarity-response pathway, 60

Z Zea mays, 110; HI; 187; 215; 251