[Current Plant Science and Biotechnology in Agriculture] Cellular and Molecular Aspects of the Plant Hormone Ethylene Volume 16 ||

CELLULAR AND MOLECULAR ASPECTS OF THE PLANT HORMONE ETHYLENE

Current Plant Science and Biotechnology in Agriculture

VOLUME 16

Scientific Advisory Board: P.S. Baenziger, University of Nebraska-Lincoln, Lincoln, Nebraska, USA K. Barton, Agracetus Corp., Middleton, Wisconsin, USA F. Cannon, Biotechnica Int., Cambridge, Massachusetts, USA A. Galston, Yale University, New Haven, Connecticut, USA J. Lyman Snow, Rutgers University, New Brunswick, New Jersey, USA C.P. Meredith, University of California at Davis, California, USA N.C. Nielsen, Purdue University, West Lafayette, Indiana, USA J. Sprent, University of Dundee, Dundee, UK D.P.S. Verma, The Ohio State University, Columbus, Ohio, USA

Aims and Scope The book series is intended for readers ranging from advanced students to senior research scientists and corporate directors interested in acquiring in-depth, state-of-the-art knowledge about research findings and techniques related to plant science and biotechnology. While the subject matter will relate more particularly to agricultural applications, timely topics in basic science and biotechnology will also be explored. Some volumes will report progress in rapidly advancing disciplines through proceedings of symposia and workshops while others will detail fundamental information of an enduring nature that will be referenced repeatedly.

The titles published in this series are listed at the end of this volume.

Cellular and Molecular Aspects of the Plant Hormone Ethylene Proceedings of the Intemational Symposium on

Cellular and Molecular Aspects of Biosynthesis and Action of the Plant Hormone Ethylene, Agen, France, August 31-September4, 1992

edited by

J. C. PECH, A. LATCHE and C. BALAGUE

Ecole Nationale Superieure Agronomique, Toulouse Cedex, France

SPRINGER-SCIENCE+BUSINESS MEDIA, B.V.

Library of Congress Cataloging-in-Publication Data

International Symposium an Cellular and Molecular Aspects of Biosynthesis and Action of the Plant Hormone Ethylene 1992 : Agen, Franca>

Cellular and molecular aspects of the plant hormone ethylene : proceedings of the International Symposium an Cellular and Molecular Aspects of Biosynthesis and Action of the Plant Hormone Ethylene, Agen, Franca, August 31st - September 4th, 1992 1 edited by J.C. Pech, A. Latche, and c. Balague.

p. cm. -- <Current plant science and biotechnology in agricultura : 16>

Includes indexes. ISBN 978-90-481-4249-1 ISBN 978-94-017-1003-9 (eBook) DOI 10.1007/978-94-017-1003-9

1. Ethylene--Congresses. 2. Ethylene--Synthesis--Congresses. 3. Plants, Effect of ethylene on--Congresses. 4. Plant hormones--Congresses. I. Pech, J. C. <Jean Clauda> II. Latche, A. <Alain> III. Balague, C. <Claudine> IV. Title. V. Series. OKB98.EBI57 1992 582.13'041927--dc20 93-9351

ISBN 978-90-481-4249-1

Printed on acid-free paper

All Rights Reserved © 1993 Springer Science+Business Media Dordrecht Originally published by Kluwer Academic Publishers in 1993 Softcover reprint ofthe hardcover 1st edition 1993

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.

Table of Contents

Preface J. C Pech, A. Latche, and C Balague

Ethylene Biosynthesis and Fruit Ripening

Structural characteristics of ACC synthase isozymes and differential expression of their genes. Mori H., Nakagawa N, Ono T, Yamagishi H. and Imaseki H.

Monomeric and dimeric forms and mechanism-based inactivation of 1-aminocyc1opropane-l-carboxylate synthase.

xiii

Satoh 8., Mon' H. and Imaseki H. 7

Characterization of the l-aminocyclopropane-l-carboxylate (ACC) synthase isoenzymes (genes) in tomato. Yip W.K 13

Modifying fruit ripening by suppressing gene expression. Theologis A., Oeller P. W. and Min- Wong L. 19

Cloning and expression analysis of an Arabidopsis thaliana l-aminocyc1opropaneI-carboxylate synthase gene : pattern of temporal and spatial expression. Rodrigues-Pousada RA., Van Der Straeten D., Dedonder A. and Van Montagu M. 24

Relationship of ACC oxidase RNA, ACC synthase RNA and ethylene, in peach fruit. Callahan A.M, Fishel D., and Dunn L.J. 31

Maximising the activity of the ethylene-forming enzyme. Smith J.J. and John P. 33

Purification, characterization and subcellular localization of ACC oxidase from fruits. Latche A., Dupzlle E, Rombaldi C, Cleyet-Marel J. C, Lelievre J.M and Pech J.C 39

vi

Purification and characterization of ACC oxidase and its expression during ripening in apple fruit. Dilley D.R., Kuai I., Poneleit L., Zhu Y., Pekker Y., Wilson JD., Burmeister DM., Gran e. and Bowers A. 46

Mechanistic aspects of ACC oxidation to ethylene. Acosta M, Amao MB., Sanchez-Bravo 1., Casas I.L., Vioque B., Femandez-Maculet 1. e., Castellano I.M 53

Apple ACC oxidase : Purification and characterization of the enzyme and cloning of its cDNA. Yang S.F., Dong I.G, Femandez-Maculet I.e. and Olson D.e. 59

Biochemical and molecular characterization of ethylene-forming enzyme from avocado. Christoffersen R.E, McGarvey D.J. and Savarese P. 65

Identification of genes encoding EFE in tomato. Hamilton A.I., Bouzayen M and Grierson D. 71

EFE multi-gene family in tomato plants: Expression and characterization. Bouzayen M, Cooper w., Barry e., Zegzouti H, Hamilton A. 1. and Grierson D. 76

Altered gene expression, leaf senescence, and fruit ripening by inhibiting ethylene synthesis with EFE-antisense genes. Gray I.E, Picton s., Fray R., Hamilton A.I., Smith H, Barton S. and Grierson D. 82

Conversion of l-aminocyclopropane-l-carboxylic acid to ethylene and its regulation by calcium in sunflower protoplasts. Bailly e., Corbineau F, Rona I.P. and Come D. 90

Isolation of a ripening and wound-induced cDNA from Cucumis melo L. with homology to the ethylene-forming enzyme. Watson CF., Balague C., Turner AJ., Pech I.C. and Grierson D. 92

Isolation and characterisation of ethylene-forming enzyme genes from melon. BulII.H, Lasserre E., Brame S and Pech 1. e. 94

Immunocytolocalisation of ACC oxidase in tomato fruits. Rombaldi e., Petitproz M, Cleyet-Marel 1. e., Rouge P., Latche A., Pech 1. e. and Lelievre I.M 96

Biochemical and immunocytological characterization of ACC oxidase in transgenic grape cells. Ayub R.A., Rombaldi e., Petitproz M, Latche A., Pech 1. e. and Lelievre I.M 98

vii

Effect of E8 protein on ethylene biosynthesis during tomato fruit ripening. PeiIarrobia L., Aguilar M, Margossian L. and Fischer R.L. 100

Expression of a bacterial ACC deaminase gene in tomato. Sheehy R.E, Ursin v., Vanderpan S. and Hiatt W.R. 106

Stereospecific reaction of l-aminocyclopropane-l-carboxylate deaminase. HonmaM III

Biochemical and molecular aspects of low oxygen action on fruit ripening. Kanellis A.K., Loulakakis KA., Hassan MM and Roubelakis-Angelakis K.A. 117

Functional analysis of CX-Cellulase (endo-~-I,4-g1ucanase) gene expression in transgenic tomato fruit. Lashbrook C. C. and Bennett A.B. 123

Inhibition of ethylene biosynthesis and suppression of cellulase and polygalacturonase in avocado fruit subjected to low oxygen storage. Metzidakis J. and Sfakiotalds E 129

Cold-induced climacteric rise of ethylene metabolism in Granny Smith apples. Larrigaudiere C. and Vendrell M 136

Regulation by temperature of the propylene induced ethylene biosynthesis and ripening in "Hayward" kiwifruit. Stavroulakis G. and Sfakiotalds E 142

Ethylene involvement in raspberry fruit ripening. Perkins-Veazie P., Nonnecke G.R. and GJadon R.J. 144

Effect of ethylene on sesquiterpene nootkatone production during the maturationsenescence stage in grapefruit (Citrus paradisiMacf.). Garcia-Puig D., Ortuiio A., Sabater F., Perez ML., Porras 1, Garcia-Lidon A.and Del Rio J.A. 146

Ethylene biosynthesis during the ripening of cherimoya (Annona cherimola, Mill). Martinez G., Serrano M, Protei M T., Amoros A., RiqueJme F and Romojaro F. 148

Effects of C02 on ethylene production by apples at low and high 02 concentrations. Levin A., Sonego L., Zutkhi Y. and Ben Arie R. 150

High carbon dioxide treatment before storage as inducer or reducer of ethylene in apples. Pesis E, Ampunpong c., Shusiri B. and Hewett E W. 152

ADH activity, via ethanol, affects ethylene production in tomato pericarp discs. BoJandi R., Gobattoni E, Massantini R. and Mencarelli F. 154

viii

Two-dimensional protein patterns of cherimoya fruits during ripening. Montero L.M., Bscribano MI, Zamorano J.P. and Merodio C

Involvement of ethylene levels in delayed ripening of avocado cv. "Hass" at low temperature. Zamorano P. and Merodio C

Relationship between polyamines and ethylene in cherimoya fruit ripening. Escribano MI, Montero L.M, Zamorano J.P. and Merodio C

Modulation of gene expression under ethylene treatment in the latex of Hevea brasiliensis. Pujade-Renaud v., Perrot-Rechenman c., d'Auzac f., Jacob fL. andGuemJ.

Imrnunodetection of ethylene-induced chlorophyllase from citrus fruit peel. Trebitsh-Sitrit T., Riov f. and Goldschmidt EE.

Ascorbate oxidase of Cucumis melo. Moser 0. and Kanellis AX.

Ethylene Action

Ethylene receptors. Hall MA. , Aho H.M, Beny A. W, Cowan D.S, Harpham N. v.J., Holland MG., Moshkov I Ye., Novikova G. and Smith A.R.

Buckminsterfullerene (C60 buckyball) inhibition of ethylene release from senescing legume foliage and cut carnations. Leshem Y. Y, Rapoport D., Frimer A.A., Strul G., AsafU and FeIner I

Effect of diazocyclopentadiene (DACP) on cut carnations. Sisler £ C, Blankenship SM, Feam J. C and Haynes R.

Reduced sensitivity to ethylene and delayed senescence in a group of related carnation cultivars. Van Doom WG., Woltering £J., Reid MS, Wu MJ.

In vitro study of ethylene binding sites in pea seedlings. Moshkov I E., Novikova G. V, Smith A.R. and HaJl MA.

Stress Ethylene

Fungal xylanase elicits ethylene biosynthesis and other defense responses in tobacco. Anderson J.D., Bailey B.A., Taylor R., Sharon A., A vni A., Mattoo A.K andFuchs Y.

156

158

160

162

164

166

168

174

182

188

195

197

Stress ethylene in Hevea brasiliensis: Physiological, cellular and molecular aspects. d'Auzac J., Bouteau F., Chrestin fl, Clement A, Jacob J.L., Lacrotte R.,

ix

Prevot J.C, Pujade-Renaud V. and Rona J.P. 205

Wound ethylene synthesis in the stress-affected cells. Kacperska A, Kubacka-Zebalska M 211

Ethylene in early signaling phenomena at the plant-microorganism interface. Esquerre-Tugaye M T., Bottin A, Rickauer M, Sancan J.P., Fournier J. and Pouenat ML. 217

Tomato ACC synthase: regulation of gene expression and importance of the C-terminal region in enzyme activity. Mattoo AK, Li N and Liu D. 223

Regulation of ethylene synthesis in maize roots responses to stress. Morgan P. w., Sarquis J.I, He CJ., Jordan W.R. and Drew MC 232

Heavy metal induction of ethylene production and stress enzymes I. Kinetics of the responses. Wech J., Vangronsve1d J. and C1ijsters fl 238

Heavy metal induction of ethylene production and stress enzymes. II. Is ethylene involved in the signal transduction from stress perception to stress responses? Vangronsve1d J., Wech J., Kubacka-Zeba1ska M and C1ijsters fl 240

Flooding resistance and ethylene. I. An ecophysiological approach with rumex as a model. Rijnders J. G., Voesenek L.A CJ., Van Der Sman AJ.M and B10m C W.P.M 247

Flooding resistance and ethylene. II. Application of an advanced laser-driven photoacoustic technique in ethylene measurements on flooded romex plants. Visser E.J. w., Voesenek L.A CJ., Harren F.J.M and B10m C W.P.M 249

Flooding resistance and ethylene. III. The role of ethylene in shoot elongation of rumex plants in response to flooding. Banga M, Voesenek L.AC.J. and B10m C W.P.M 251

Effect of saline stress on growth of Iycopersicon escu1entum plants and its relation with endogenous ethylene metabolism. Botella F., Del Rio J.A and Ortuno A 253

Ethylene biosynthesis in "Hayward" kiwifruit infected by Botrytis cinerea. Niklis N, Sfakiotakis E. and Thanassou1opou1os CC 255

Ethylene, stress and enzymatic activities in Hevea latex: the diversity of responses. PrevOt J. C, Clement A, Pujade-Renaud v., Siswanto and Jacob J.L. 257

x

Molecular and physiological characterisation of the role of ethylene during pathogen attack of tomato fruit. Cooper w., Bouzayen M, Barry C, Hamilton A.J., Rossall S. and Grierson D. 259

The use of antisense transgenic tomato plants to study the role of ethylene in responses to waterlogging. English P.J., Lycett G. w., Roberts J.A., Hall K. C and Jakson MB. 261

Research on the diurnal courses of abscissic acid, l-aminocyclopropane carboxylic acid and its malonyl conjugate contents in needles of damaged and undamaged spruces. Yang C, Wessler A. and Wild A. 263

Flower Senescence. Abscission.

Hormonal and tissue-specific regulation of cellulase gene expression in abscission. Tucker M.L., Matters G.L., Koehler SM., Kemmerer E.C., Baird S.L. and Sexton R. 265

Changes in gene expression during leaf abscission. Roberts J.A., Taylor .I.E., Coupe s.A., Harris N. and Webb S. T.J. 272

Rapid ethylene-induced gene expression during petal abscission. Evensen KB .• Clark D.a and Singh A. 278

Abscission studies in a new mutant of navel oranges. Zacarias L, Tadeo F.R., Bono R. and Primo-Millo Ii 284

Ethylene regulation and function of flower senescence-related genes. Woodson W.R., Brandt A.S., Itzha/d H., Maxson J.M, Wang H., Park K Y. and Larsen P.B. 291

Cloning of ethylene biosynthetic genes involved in petal senescence of carnation and petunia, and their antisense expression in transgenic plants. Michael MZ., Savin K. w., Baudinette S. C, Graham M w., Chandler S.F., Lll C. Y., Caesar C, Gautrais 1, Young R., Nugent aD., Stevenson KR., O'Connor EL.J., Cobbett CS. and Cornish Ii C 298

Interorgan regulation of post-pollination events in orchid flowers. Nadeau J.A., Bui A.Q., Zhang X and O'Neill S.D. 304

Roles of ethylene, ACC and short-chain saturated fatty acids in inter-organ communication during senescence of Cymbidium flowers. Woltering E.J. 310

The role of ethylene in the abscission and ripening of raspberry fruit Rubus idaeus cv Glen Clova. Sexton R., Burdon J.N. and Bowmer J.M 317

Expression of two ACC synthase mRNAs in carnation flower parts during aging and following treatment with ethylene.

xi

Ht:J1sirens H., Somhorst D and Woltering E.J. 323

Promoting the activity of arginine decarboxylase and ornithine decarboxylase by ethylene and its significance to the control of abscission in citrus leaf explants. Goren R., Hubt:nnan M, Levin N. and Altman A. 325

Expression ofEFE antisense RNA in tomato causes retardation of leaf senescence and most fruit ripening characteristics. Murray A.J., Hobson G.B ... Schuch W. and Bird CR. 327

Growth, Development

The role of ethylene in regulating growth of deepwater nce. Kende H., Hoflinann-Benning S. and Sauter M

Gravity dependent ethylene action.

329

Burg S.P. and Kang B.G. 335

Ethylene and the growth of etiolated seedlings of lupinus albus L. Casas J.L., Amao MB., Garrido G., Acosta M, Sanchez-Bravo J. 341

Various conditions of illumination and ethylene evolution. KefeJi v.l, Rakitina T. Y. A, Vlasov P. v., JaJilova F. and Kalevich A.E. 347

Ethylene and vitrification of Fraxinus explants in vitro. Leforestier F. , Joseph C and Come D. 353

Stimulation of somatic embryogenesis in carrot by ethylene. Nissen P. 359

Relationship between ethylene and polyamine synthesis in plant regeneration. Roustan J.P., Chraibi KM, Latche A. and FaJlot J. 365

Ethylene inhibits the morphogenesis of Vitis vinifera cuttings cultured in vitro. Souli60., Roustan J.P. and FaJlot J. 367

Enhanced ethylene production by primary roots of Zea mays L. in response to sub -ambient partial pressures of oxygen. Brailsford R., Voesenek L.A.CJ., Blom C w.P.M, Smith A.R., HaJl MA. and Jackson MB. 369

Ethylene and phosphorylation of pea epicotyl proteins. Novikova 0. v., Moshkov IE., Smith A.R. and HaJl MA. 371

xii

Knowledge of xylem sap flow rate is a pre-requisite for accurate estimates of hormone transport from roots to shoots. Else M.A, Davies w.I., Hall K. C. and Jackson M.B.

Ethylene synthesis by fruit plants cultured in vitro. Jona R., Fronda A, Cattro A and Gallo A

Index of Authors

Index of Keywords

373

375

377

381

Preface

The International Symposium on "Cellular and Molecular Aspects of Biosynthesis and Action of the Plant Hormone Ethylenc" ,vas held in Agen, France from August 31 st and September 4th, 1992. The planning and management of the scientific and social programme of the Conference were carried out jointly by the "Ethylene Research Group" of ENSAlIN"P (Toulouse) and Agropole Congres Service (Agen).

Since the last meetings in Israel (1984) and in Belgium (1988), ethylene physiology has gone through a period of exciting progress due to new developments in cellular and molecular bioiogy. New methods and tools have been developed to better understand the role and functions of ethylene in fruit ripening, flower senescence, abscission, piant growth, and cell differentiation. Genes involved in ethylene biosynthesis have been characterized and transgenic plants with altered ethylene production have been generated. The feasibility of delaying fruit ripening or flower senescence by genetic manipulation is now demonstrated, thus opening new perspectives for the postharvest handling of plant products.

Some progress has also been made on the understanding of ethylene action. However, much remains to be done in this area to elucidate the ethylene signal transduction pathway.

Around 140 scientists from 20 countries attended the Symposium. They presented 47 oral reports and 40 poster demonstrations. All of them are published in these proceedings.

It has been a pleasure for us to organize this important Symposium and to edit this book. We hope that it will remember a week of intense and fruitfull discussions to those who attended the meeting and will provide to others a guide to the most recent developments of research on ethylene biosynthesis and action.

We wish to express our thanks to all Discussion leaders and Chairpersons who largely contributed to the success of the mceting. We are spccially grateful to Professor S.F. Yang (USA) and Professor D. Grierson (UK) for their help in the preparation of the scientific programme.

Tue Symposium would not have been possible without the generous financial support provided by the foHowing organizations:

-Commission of the European Communities; -Conseil Regional d'Aquitaine; -Conseil General de Lot-et-Garonne: -Ministere de la Recherche, de la Technologie et de I'Espace; -Ministere de I'Education Nationale et de la Culture: -Compagnie Fran~aise des Produits Industriels (CFPI); -and Louis Vuitton-Moet Hennessy.

xiii

xiv

We also gratefuily acknowledge the help and support from Mairie d'Agt:n, Comite Economique Fruits et Legumes Aquitaine, Les Vignerons de Buzet, and SICA France Prune.

We are particularly indebted to A.M. Perrin-Naffak (Director of IUFM) and R. Caussieu (Administrative Director) who hosted the Conference at the Ecole Normale d'Agen. We also acknowledge the help of the following persons: J.-F. Runel-Belliard, F. Riboulet and the staff of Agropole-Congres, and J. Bull, C. Sigro, M. Bouzayen, J. Raynal and all the members of the "Ethylene research group" at ENSAT.

Toulouse. 1992 Jean Claude. PECH Alain LATCHE

Claudine BALAGuE

STRUCTURAL CHARACTERISTICS OF ACC SYNTHASE ISOZl'MES AND DIFFERENTIAL EXPRESSION OF THEIR GENES.

1 H. MORl, N. NAKAGAWA, T. ONO, N. YAMAGISHI, H. IMASEKI School of Agricultural. Sciences, Nagoya University yhikusa, Nagoya, 464-01 Japan NatiDnal. Institute for Basic Biology

Okazaki, 444 Japan

ABSTRACT. ACC synthase consists of isozymes that are induced by auxin, ripening or wounding. The expression of each gene was specific to stimulus applied to or generated in tissues. Auxin did not induce the expression of the wound-inducible gene, and wounding did not express the auxin-inducible gene. The primary structures deduced from cDNAs for ACC synthases from different sources were compared in their sequence similarity. The overall similarity between two isozymes induced by wounding and auxin in winter squash is only 55%, and that among three isozymes induced by wounding, auxin and ripening in tomato ranged from 54% to 74%. However, eight regions (box 1 to 8) that include the active site of the enzyme are highly conserved among 14 enzymes. Moreover, numbers of amino acid residues between adjacent regions are constant. Sequential deletion of amino acid residues from the C-terminus of the wound-induced enzyme of winter squash increased the relative specific activity 4-fold, when 56 amino acid residues were deleted, but deletion of 65 residues (deletion of the next 9 residues) resulted in a complete loss of activity. Possible function of these regions in ACC synthase is discussed.

1. Introduction

Success of cloning of cDNA for ACC synthase [3-6] has enabled us to assess the specific expression of the genes and structural characteristics of the enzyme. ACC synthases induced by wounding (W-type) and auxin (A-type) were purified from winter squash (SQ, Cucurbita maxima cv. Ebisu) , and cDNAs for the each enzyme have been cloned [1,3]. We also isolated, from tomato, avocado, mungbean and melon, cDNA clones that corresponded to the enzymes induced by wounding, ripening (Rtype) and auxin.

The molecular mass of native ACC synthase is significantly smaller than that of the in vitro translation product or that is expected from the primary structure deduced from cDNA. The W-type enzyme purified from winter squash was 50 kDa in its size, but the product of in vitro translation of mRNA showed 58 kDa on SDS-PAGE [2] and the mass calculated from cDNA was 56 kDa [3]. Based on label/chase experiments

J. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plant Honnone Ethylene, 1-6. © 1993 Kluwer Academic Publishers.

2

and western blot analysis, we proposed that about 60 amino acid residues at the C-terminus were removed both in vivo and in vitro [3]. A possible role of the C-terminal region in the expression of enzymatic activity was examined with a series of mutant proteins in which Cterminal amino acids were variously removed from the primary product of translation. In this report, we describe the characteristic patterns of expression of the genes of winter squash and tomato and also the structural characteristics of ACC synthase from different sources.

2. Materials and Methods

2.1. ISOLATION OF cDNAS

W- (CMW33) and A-type (CMA101) cDNAs from winter squash have been described [1,3]. Oligonucleotide mixtures corresponding to Gln52 to Leu60 (sense) and Glu408 to Met419 (antisense) of SQ-W enzyme were synthesized and used as primers for PCR. Templates for PCR were cDNAs for poly(A)RNAs isolated from wounded, ripe or auxin-treated tissues. The PCR products were sub cloned and used as probes to obtain full-length cDNA from the respective cDNA library.

2.2. RNA BLOT ANALYSIS

Total RNA was extracted from tissues that had been wounded or treated with indicated chemicals. Electrophoresis and northern blotting of RNA (20 /.l. g per lane) were performed by the stand art methods. In some experiments, total RNA was applied to membranes in slots. The membranes were probed with labeled cDNAs that were prepared by either the random primer method or PCR.

2.3. MUTANT ENZYMES DELETED AT THE C-TERMINAL SIDE

A cassette of synthetic DNA that contained XbaI-EcoRI-PstI sites followed by 3 stop co dons placed at every possible reading frame was inserted to the SnaBI site located downstream of the stop codon of CMW33 to yield CMW331. CMW331, that had been subcloned into an expression vector, directed synthesis of active enzyme in E. coli. After digestion with XbaI and PstI, 3'-side of CMW331 was variously deleted by exonuclease III. cDNA clones with different sizes were selected, and the end-point of deletion of each clone was determined by nucleotide sequencing. In this report, D.. #xx will be referred to a mutant protein that lacks xx amino acid residues from the C-terminus. Due to the cassette inserted, the mutant proteins were attached at the C-terminus with additional 1 to 3 amino acids that were not present in the original enzyme.

E. coli DH5a cells transformed with the mutant clones were grown until A600 reached 14 in the absence of IPTG, harvested, resuspended in the same volume of TB/ampicillin medium that contained 1 mM IPTG, and cultured for 30 min. The cells were collected and suspended in the extraction buffer. A small aliquot was treated with the Tris/SDS buffer and directly subjected to SDS-PAGE followed by western blot-

3

ting and immunostaining. Rest of cells were disrupted by sonication. Half of the lysate was used to assay enzymatic activity and another half was used to determine relative amounts of the immunoreactive protein by ELISA.

3. Results

3.1. THE STIMULUS!TISSUE-SPECIFIC EXPRESSION OF THE ISOZYME GENES IN WINTER SQUASH AND TOMATO

In addition to two cDNA clones from winter squash, five different cDNAs were cloned from tomato; one from intact fruits at the pink stage (R-type), one from wounded pink fruits (W-type), and three from auxin-sprayed seedlings (A-type, A3, A5, and A6). 'Since they did not cross-hybridize to each other, expression of the individual gene could be specifically assessed. The W-type and R-type cDNAs of tomato are the same as those isolated by Van der Straeten et al. [6], and Olson et al. [4], respectively.

Qualitative patterns of the expression of those genes are summarized in TABLE 1. In winter squash, W-type gene was expressed in both fruits and stems when the tissues were wounded. The woundinduced expression was much greater in fruit tissues than in stem tissues. However, A-type gene was not expressed by wounding. Auxin induced the expression of A-type gene in stems, but not in fruit slices. Auxin also stimulated the expression of W-type gene in both fruit

TABLE 1. Patterns of expression of ACC synthase genes in winter squash and tomato. Numbers of + mark represent approximate relative amounts of each transcript. For winter squash, fruit represents mesocarp of mature fruits, seedlings are 4-day-old etiolated seedlings and stem sections from subapical portion of the etiolated seedlings.

WINTER SQUASH

Tissue FRUIT SLICES --------------

Gene None IAA ABA

W-type +++ ++++ ++++ A-type

TOMATO

Tissue INTACT FRUIT

Gene MG BR TNG PK

W-type + +++ R-type + +++ A-type

A5 nd nd nd A3 +++ nd nd nd A6 +++ nd nd nd

nd, Not determined

STEM SECTIONS -----------------------None IAA IAA+ABA ABA

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

FRUIT SLICES

MG MG+IAA TNG BR PK

+

+/+

++ ++ ++++ +++++ +/- +

++ nd nd +/- nd nd +/- nd nd

nd nd nd

SEEDLINGS -----------

None IAA

+++

SEEDLINGS

Cont. IAA

++ +++ +/-

4

slices and stem sections, but when intact seedlings were sprayed with an auxin solution, W-type gene was not expressed. These results indicate that the expression of W-type gene is specific to wound stimulus regardless of tissue types, whereas the expression of A-type gene is specific to auxin and to stem, and that auxin stimulates the expression of W-type gene in wounded fruit and stem tissues. Auxin does not trigger the expression of W-type gene in intact stem tissues. Abscisic acid (ABA) did not induce or stimulate the expression of Atype gene, but it stimulated the expression of W-type gene in both frui t slices and stem sections. However, the expression of A-type gene by auxin was suppressed by the presence of ABA. Thus, ABA and auxin stimulate the wound response, but ABA inhibits the action of auxin to express A-type gene.

In tomato, R-type and W-type genes were not expressed in fruits at the mature green and breaker stages and in intact seedlings, but their expression progressively increased as fruits ripened. In pink fruits, the levels of transcripts of the two genes were approximately the same. When fruits were wounded, the expression of W-type gene was greatly stimulated at all stages, although the wound response was greater in more ripened fruits. By contrast, the expression of R-type gene was suppressed after wounding. The genes to 3 different cDNAs obtained from auxin-treated seedlings showed different pattern of expression. A5 gene was expressed in mature green fruits and seedlings only when they were treated with auxin, and its expression seemed to be specific to auxin. A3 and A6 genes were expressed in intact mature green fruits, but their expression appeared to be suppressed after wounding. A3 gene responded to auxin in intact seedlings.

3.2. SEQUENCE SIMILARITY OF ACC SYNTHASE FROM VARIOUS SOURCES

When amino acid sequences of 14 ACC synthases are aligned at the maximum match, the similarity between W- and A-type enzymes from winter squash was only 55%, whereas tomato isozymes showed similarities from 54% to 82% depending on the combination for comparison. There was no indication that isozymes from one species were more similar than any two enzymes from different species, and that the same type of enzymes was more similar than the different types of enzymes. One exception is that W-type enzymes from winter squash and zucchini are nearly identical. However, there are eight regions (Boxes 1 to 8) that are highly conserved among 14 ACC synthases examined. Sizes of the regions ranged from 8 (Box 2) to 18 amino acid residues (Box 3). Moreover, numbers of amino acid residues between adjacent boxes are constant among all enzymes; 31 residues between Boxes 1 and 2, 49 between Boxes 2 and 3, 41 between Boxes 3 and 4, 17 between Boxes 4 and 5, 30-34 between Boxes 5 and 6, 15 between Boxes 6 and 7, and 91 between Boxes 7 and 8. The amino acid residues that are thought to interact with pyridoxal phosphate and SAM (Tyr95, Asp240, Tyr243, Ser276, Lys279, Arg287 and Arg413 in SQ-W enzyme) and that will maintain the proper three-dimensional structure of the enzyme (Pro151, Pr0210, Gly215) were located in these conserved regions. Lys279 in

5

Box 6 is particularly important to express enzymatic activity, because when this lysine was replaced by arginine or glutamic acid by si tedirected mutagenesis, the proteins completely lost activity.

3.3. FUNCTION OF C-TERMINAL REGION IN ENZYMATIC ACTIVITY

Nine 3'-deletion mutant clones derived from CMW331 were selected. They directed, in E. coli cells, the synthesis of mutant proteins that lacked 25, 43, 56, 60, 65, 79, 137 and 180 amino acid residues from the C-terminus of SQ-W enzyme. The relative specific activity of the mutant proteins was significantly increased when 56 amino acid residues were deleted (TABLE 2). f1#56 showed more than 4-fold activity of the wild type enzyme. However, when the next 4 ,residues were removed (f1#60), the activity dropped to 30% of the original activity and removal of further 5 residues (f1 #65) caused a complete loss of activity. Although 9 amino acid residues deleted from f1#56 to f1#65 are not included in the 8 conserved regions, two amino acid residues (Arg430 and Phe434) in the 9-residue segment were conserved, in all ACC synthases.

TABLE 2. Relative specific activity of C-terminal deleted mutant proteins of ACC synthase. Mutant proteins synthesized by E. coli were assayed for their enzymatic activity and relative contents by ELISA. The content of enzyme in an extract from E. coli cells transformed with CMW331 was assigned as 1. Relative specific activities were calculated from the measured activity and the relative content.

Enzyme Wt. #25 #43 #56 #60 #65 #79 #89

Activity 69.4 130.7 545.3 495.9 14.51 0 0 0 (unit/ml) ReI. ,Cont,. 1.00 1.15 2.00 1.63 0.65 0.52 0.45 0.27 ReI.Sp.Act. 69.4 113.7 272.7 304.2 22.5 0 0 0

Rel.Value 1.00 1.64 3.93 4.38 0.32 0 0 0

4. Discussion

In winter squash and tomato, the genes for ACC synthase are consisted of isozyme genes that are expressed in response to different stimuli. Wound-, auxin- and ripening-induced genes were expressed specifically by wounding, auxin and ripening, respectively. Although auxin- and ripening-induced genes also show tissue specific expression, woundinduced genes are expressed specific to wound stimulus regardless of tissue types. Apparently, the function of these isozyme genes is different. The expression of the wound-specific genes in ripening tomato fruits may have resulted from disorganization of the tissue during ripening. The expression of W-type genes is modified by auxin and

6

ABA. Both auxin and ABA stimulate the expression of W-type gene, but ABA inhibits the action of auxin to express A-type gene. The results is consistent with the effect of ABA on other wound responses. It is interesting to know whether ABA inhibits the auxin action to stimulate the expression of W-type gene. Since ethylene is necessary for regular development as well as for wound healing, the presence of the wound-inducible isozyme genes may be essential not to perturb the expression program of other isozyme genes that function for regular development.

Although the overall similarity of the primary structure of ACC synthases from different sources is not high, the enzymes show some common structural characteristics. Eight highly conserved regions found in the primary structure include the amino. acid residues that interact with pyridoxal phosphate and the substrate, and their relative location in the molecule is the same in all enzymes examined. Thus, these conserved regions may be important to form the proper three dimensional structure of the active site. The C-terminal region consisting of about 55 hydrophylic amino acids also seems to have some .function in the expression of enzymatic activity, because removal of the region increased the specific activity of the enzyme. It is presumed that the C-terminal region interact with the surface of the active site domain so that access of the cofactor and the substrate is controlled. However, a few amino acid residues present at the junction to the C-terminal region are essential for activity.

References

[1] Nakagawa, N., Mori, H., Yamazaki, K. and Imaseki, H. (1991) 'Cloning of a complementary DNA for auxin-induced 1-aminocyclopropane-lcarboxylate synthase and differential expression of the genes by auxin and wounding', Plant Cell Physiol., 32,1291-1298.

[2] Nakajima, N. and Imaseki, H. (1988) 'Molecular size of wound-induced 1-aminocyclopropane-l-carboxylate synthase from Cucurbita maxima Duch. and change of translatable mRNA of the enzyme after wound ing', Plant Cell Physiol., 27, 969-980.

[3] Nakajima, N., Mori, H., Yamazaki, K. and Imaseki, H. (1990) 'Molecular cloning and sequence of a complementary DNA encoding 1-aminocyclopropane-l-carboxylate synthase induced by tissue wounding', Pland Cell Physiol., 31, 1021-1029.

[4] Olson, D. C., White, J. A., Edelman, L., Harkins, R. N. and Kende, H. (1991) 'Differential expression of two genes from 1-aminocyclopro-pane-i-carboxylate synthase in tomato fruits' Proc. Nat!. Acad. Sci. USA, 88,5340-5344.

[5] Sato, T. and Theologis, A. (1989) 'Cloning the mRNA encoding 1-aminocyclopropane-l-carboxylate synthase, the key enzyme for ethylene biosynthesis in plants', Proc. Nat!. Acad. Sci. USA, 86,6621-6625.

[6] Van Der Straeten, D., Van Wiemeersch, I., Goodman, H. M. and Van Montagu, M. (1990) 'Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-l-carboxylate synthase in tomato', Proc. Nat!. Acad. Sci. USA, 87, 4859-4863.

MONOMERIC AND DIMERIC FORMS AND MECHANISM-BASED INACTIVATION OF 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE

S. Satoh, H. Mori and H. Imaseki Department of Biological Sciences, Tohoku University, Kawauchi, Aoba-ku, Sendai 980 (S.S.), and National Institute for Basic Biology, Myodaijichyo, Okazaki 444, Japan (H.M. and H.I.)

ABSTRACT. Among ACC synthase preparations of various orIgIns, i.e. those from tomato and winter squash fruits as well a~ those expressed by E.co~i from cDNAs for tomato and winter squash ACC synthase, only the enzyme from tomato fruit tissue existed in a monomeric form, whereas the others in a dimeric form. The monomeric tomato ACC synthase was much less sensitive to the mechanism-based inactivation than the dimeric forms of ACC synthase. We suggest that ACC synthases have a property to form a dimer, but in tomato fruit tissue some modification takes place to the enzyme protein, which makes it remained as a monomer and less sensitive to the mechanism-based inactivation.

1. INTRODUCTION

ACC synthase from plant tissues was reported to present in either monomeric or dimeric form. The two forms can be judged by the difference between the molecular masses determined by sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration column chromatogaphy (GFC). ACC synthases from tomato pericarp and apple flesh show the similar molecular mass of about 50 kDa on SDS-PAGE and GFC analyses (Acaster et al. 1982, Bleecker et al. 1986, Yip et al. 1990), and the results indicate they exist as a monomer. On the other hand, ACC synthases from winter squash and zucchini mesocarp as well as mung bean hypocotyls gave the molecular mass of about 100 kDa when determined by GFC, which was twice that determined by SDS-PAGE (Nakajima et al. 1988, Sato et al. 1991, Tsai et al. 1988), and they may exsit in a dimeric form.

Recent studies on ACC synthase genes and their expression have shown that ACC synthase is synthesized as a peptide of about 55 kDa, then its C-terminal is processed (or simply degraded during purification) to give 45-50 kDa peptide (Nakajima et al. 1988, 1990, Olson et al.1991, Sato et al. 1991, Van Der Straeten et al. 1990). Thus, the dimeric form of ACC synthase is thought to be a homodimer of

7

J. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, 7-12. © 1993 Kluwer Academic Publishers.

8

about 45-50 kDa subunit. ACC synthase is a PLP-requiring enzyme and has an amino-acid

seqence which is conserved among other PLP-utilizing enzymes (Huang et al. 1991, Nakajima et al. 1990, Rottmann et al. 1991, Yip et al. 1990). Aspartate aminotaransferase (AATase) is the most extensively studied PLP-utilizing enzyme. This enzyme is active in a dimeric form, which has two active sites, one site on each subunit, because some amino acids that constitute each of the active sites are distributed to two subunits. For example, Tyr70 of one subunit binds to a-carboxyl group of aspartate that is bound to the active site of the other subunit, and vice versa. From the analogy of AATase, Huang et al. (1991), working with ACC synthase from zucchini mesocarp, proposed that ACC synthase must dimerize to express enzymatic activity. Indeed, Tyr70 of AATase could be referred to Tyr95 of ACC synthase from zucchini mesocarp (Huang et al.1991) and to Tyr92 of that from tomato pericarp (Rottmann et al. 1991). However, their proposition does not seem to be applicable to tomato and apple enzymes, which are present in the monomeric form with enzymatic activity.

In the present study, we investigated the monomeric and dimeric forms and catalytic function of ACC synthase using various preparations from tomato and winter squash fruits as well as those expressed by E.coli from cloned cDNAs. We found that only the enzyme from tomato peri carp was present in a monomeric form, whereas the others in a dimeric form, and the monomeric tomato enzyme was less sensitive to the mechanism-based inactivation of the enzyme than the other dimeric ones. We discussed these findings in relation to the possible modification of ACC synthase in tomato peri carp tissue.

2. MATERIALS AND METHODS

Tomato (Lycopersicon escuLentum Mill.) ACC synthase was prepared from wounded peri carp of ripe fruits as described previously (Satoh and Yang, 1988) with some modification. In brief, the partially purified preparation was subjected to HPLC on a semi-preparative anion-exchange column (J.T. Baker, WP-PEI, 10 mm dia. x 25 cm) with a potassium acetate gradient. The preparation was further purified with the line 5b immunoaffinity gel (Bleecker et al. 1986). A crude sample of winter squash ( Cucurbita maxima Duch.) ACC synthase was prepared from wounded mesocarp.

ACC synthase was also prepared from a fusion protein of ACC synthase with maltose binding protein (MBP) by cleaving it with Factor Xa. The chimeric gene for the fusion protein was constructed by placing cDNA for ACC synthase at the downstream of the gene for MBP in a plasmid pMAL cRI (New England Biolabs, Inc.), and expressed by E. coLi XL1-Blue. We used four kinds of cDNAs for ACC synthase; two were cDNAs for tomato enzymes, which corresponded to the cDNAs cloned from ripe and wounded peri carp by Van Der Straeten et al. (1990) and from ripe, but not wounded, peri carp by Olson et al. (1991), and the other two were cDNAs for winter squash enzymes induced by wounding (Nakajima et al. 1990) and by auxin-treatment (Nakagawa et al. 1991).

For convenience, we named ACC synthases expressed from these cDNAs, in that order; L.esc-ACCS-1, L.esc-ACCS-2, C.max-ACCS-1 and C.maxACCS-2, respectively.

GFC/HPLC was carried out on a TSKgel G3000SW (Toso, 7.5 mm dia. x 60 cm).

Mechanism-based inactivation of ACC syntase was investigated at 200 /.l M AdoMet in the presence of BSA at 1 mg/ml.

3. RESULTS AND DISCUSSION

3.1. Separation by HPLC with a WP-PEI column of ACC synthase from ripe and wounded tomato pericarp

9

We found that ACC synthase preparation extracted from wounded pericarp of ripe tomato fruits was separated into two fractions by HPLC with a semi-preparative WP-PEI column. We can expect the presence of more than one kind of ACC synthase protein in the enzyme preparation, since it was recently demonstrared that two ACC synthase ,genes, encoding L.esc-ACCS-1 and L.esc-ACCS-2, were simultaneously transci bed in wounded peri carp of ripe tomato tissue (Mori et al. unpublished, Olson et al. 1991). Although we do not know yet the relationships of these two fractions to the products of two genes, we named them PEI-I and PEl-II according to the order of elution and used both of them in the following experiments.

3.2. Monomeric and dimeric forms of ACC synthase

Figure 1 shows the elution profiles after GFC/HPLC of ACC synthases, and the values for molecular mass for respective enzymes: Tomato PEI-I and PEl-II were eluted with an identical retention time, and their molecular mass was -estimated as 53 kDa. L.esc-ACCS-1 and L.esc-ACCS-2 had a similar retention time, the estimated molecular masses being 93 kDa and 110 kDa, respectively.

On the other hand, ACC synthase from wounded mesocarp of winter squash was eluted at a molecular mass of 110 kDa. Also, C.max-ACCS-1 and C.max-ACCS-2, had similar molecular masses of 105 kDa and 80 kDa, respectively. In addition, an extra small peak of 210 kDa was found with C.max-ACCS-1 and that of 270 kDa with C.max-ACCS-2, respectively. But, We did not investigate further these extra ACC synthase peaks.

Figure 2 shows the SDS-PAGE profile of purified ACC synthase. Molecular masses for both PEI-I and PEl-II were estimated to be 48 kDa. All ACC synthase preparations from the transformed E. coli gave two or more overlapped bands, indicating that each sample was a mixture of two or more proteins with very similar molecular masses. The average molecular ~ass was 45 kDa for L.esc-ACCS-1; 48 kDa for L.esc-ACCS-2; 48 kDa for C.max-ACCS-1; and 45 kDa for C.max-ACCS-2. These values were smaller by 6-8 kDa than those calculated from respective cDNA; that is 54.7 kDa, 53.5 kDa, 55.9 kDa and 54 kDa, respectively, in that order (Nakagawa et al. 1991, Nakajima et al. 1990, Olson et al.1991, Van Der Straeten et al. 1990. It was thought

10

that the peptide of corresponding length was eliminated from ACC synthase by proteolysis at its C-terminal in E.coLi cells and/or during purification, since its N-terminal was covalently bound to MBP until cleavage by Factor Xa at the later step of purification.

These findings confirmed the previous results that ACC synthase was obtained as a monomer of about 50 kDa from tomato peri carp and as a homodimer of about 50 kDa subunit from winter squash mesocarp. Moreover, we found that the ACC synthases which were expressed by E. coLi from cloned cDNAs existed as a dimer for both tomato and winter squash enzymes. The loss of C-terminal peptide of several kDa did not affect the formation of the dimeric form. It is noteworthy that even tomato ACC synthase could form a dimer, when it was expressed by E. coLi from its cDNA.

3.3. Mechanism-based inactivation of monomeric and dimeric forms of ACC synthase

ACC synthase has an unique characteristic that it is inactivated by its substrate AdoMet during catalytic action. We compared the halflives for the mechanism-based inactivation among ACC synthases. Half-lives for PEI-I and PEl-II were identical and estimatd as 180 min. The half-life was longer than that (66 min) obtained previously for ACC synthase extracted from wounded tomato peri carp (Satoh and Yang 1988). L.esc-ACCS-1 and L.esc-ACCS-2 were inactivated with the half-lives of 15 min and 8 min, respectively. These values were one order of magnitude smaller than that obtained for the enzyme from tomato tissue. On the other hand, winter squash enzymes showed similar half-lives irrespective of their origin, that is, 32 min for the enzyme from wounded mesocarp; 30 min for C.max-ACCS-1; and 16 min for C.max-ACCS-2.

Thus it has become evident that the monomeric form of ACC synthase, i.e. PEI-I and PEl-II, has a longer half-life for the mechanism-based inactivation than the dimeric forms of ACC synthase. In other words, it~an be said that the monomeric ACC synthase is less sensitive to the mechanism-based inactivation than the dimeric ACC synthase.

Based on the present observations, we suggest that although both tomato and winter squash ACC synthase protein has a property to form a dimer, some modification might have taken place to ACC synthase in tomato pericarp cells, that makes it remained as a monomer and less sensitive to the mechanism-based inactivation, whereas no such the modification takes place in winter squash mesocarp tissue and in E. coL i.

Previously Satoh and Yang (1988) suggested that the mechanism-based inactivation acts as the initial reaction in the rapid decrease of ACC synthase activity in plant tissues. From this proposition, it would be expected that the half-life of ACC synthase in vitro agrees to that in vivo. The half-life of ACC synthase in wounded peri carp of ripening tomato peri carp was reported to be 114 min (Kende and Boller 1981). This in vivo value roughly corresponds

Hol. _ass (kO.) 440 158 67 43 .. .. .. ..

20 0 . 002 PEl-I

10 0.001

Hol . aass (kO.) 440 158 67 43

0 .. .. .. .. 10 0.008

PEl - II WS 0.10

~ ~ ..... ~ .. -;: 5 0.004

2-

:i 0

0 ~ - 0 5 ... .: c

~ 400 ~ 100 L.esc - ACCS- l

0.02 u C .•• x- ACCS - l u § ...

50 0. 01

.1 200 c :;-

0 ~ 0

200 l . esc - ACCS-2

0 . 02 400

100 200

~~~~~~~--~o 50 60 80 90

fr. No . (0.2 .1/fr.)

Figure 1. Gel filtration chromatography of ACC synthases on a high pressure TSKgel G3000SW column.

Figure 2. SDS-PAGE profile of purified ACC synthases. PEI-I and PEIII were used after elution from the line 5b immunoaffinity gel. Bands with an arrowhead are ACC synthases, and other two bands are the heavy and light chains of IgG. ACC syn- --thases expressed in E. coLi were used after the purification by GFC/HPLC shown in Figure 1.

kDa

e92.5

e66.2

11

12

to that of monomeric ACC synthase, but not of dimeric one. Therefore, we further suggest that the possible modification of ACC synthase in tomato tissue plays an important role in the regulation of ethylene production through changing the sensitivity of the enzyme to the mechanism-based inactivation. We do not know yet the nature of the possible modification to ACC synthase, and the problem is currently under investigation.

4. REFERENCES

Acaster, M. A. and Kende, H. (1982) Plant Physiol. 72, 139-145. Bleecker, A. B., Kenyon, W. H., Sommerville, S. C. and Kende, H.

(1986) Proc. Natl. Acad. Sci. USA 83, 7755-7759~ Huang, P-L., Parks, J. E., Rottmann, W. H. and Theologis, A. (1991)

Proc. Natl. Acad. Sci. USA 88, 7021-7025. Kende, H. and Boller, T. (1981) Planta 151,476-481. Nakagawa, N., Mori, H., Yamazaki, K. and Imaseki, H. (1991) Plant

Cell Physiol. 32, 1153-1163. Nakajima, N., Nakagawa, N. and Imaseki, H. (1988) Plant Cell

Physiol. 29, 989-998. Nakajima, N., Mori, H., Yamazaki, K. and Imaseki, H. (1990) Plant

Cell Physiol. 31,1021-1029. Olson, D. C., White, J. A., Edelman, L., Harkins, R. N. and Kende,

H. (1991) Proc. Natl. Acad. Sci. USA 88, 5340-5344. Rottmann, W. H., Peter, G. F., Oeller, P. W., Keller, J. A., Shen,

N. F., Nagy, B. P., Tayler, L. P., Campbell, A. D. and Theologis, A. (1991) J. Mol. Biol. 222, 937-961.

Sato, T., Oeller, P. W. and Theologis, A. (1991) J. Biol. Chern. 266, 3752-3759.

Satoh, S. and Yang, S. F. (1988) Plant Physiol. 88, 109-114. Tsai, D. S., Arteca, R. N., Bachman, J. M. and Philips, A. T. (1988)

Arch. Biochem. Biophys. 264, 632-640. Van Der Straeten, D., Van Wiemeersch, L., Goodman, H. M. and Van

Montagu, M. (1990) Proc. Natl. Acad. Sci. USA 87, 4859-4863. Yip, W-k., Dong, J-G., Kenny, J-W., Thompson, G. A. and Yang, S. F.

(1990) Proc. Natl. Acad. Sci. USA 87, 7930-7934. Yip, W-K., Dong, J-G. and Yang, S. F. (1991) Plant Physiol. 95, 251-

257.

CHARACTERIZATION OF THE l-AMINOCYCLOPROPANE-I-CARBOXYLATE (ACC) SYNTHASE ISOZYMES (GENES) IN TOMATO

w. K. YIP Department of Botany University of Hong Kong Pokfulam Road Hong Kong

ABSTRACT. Following the radiolabeling of the active site with Ado[carboxyl- 14C]Met or with NaB3H4 , and the purification of the radiolabeled ACC synthase by immunoaffinity column, we.have isolated and sequenced from tomato fruit, two highly conserved active-site tryptic peptides, which differred in only one amino acid residue. This result provide evidence for the existent of two isoforms of ACC synthase in tomato. Based on two conserved peptide sequences (SNPLGTT and MSSFGLV) deduced from the known ACC synthase gene sequences, polymerase chain reactions (PCR) were performed using cell culture mRNA as template for the first strand cDNA. Four ACC synthase gene fragments encoding the active site domain were isolated. By employing the ribonuclease protection assay, we have studied the expression of each of the four ACC synthase transcripts in tomato fruits as influenced by ripening and wounding, and in vegetative hypocotyls as influenced by auxin treatment. Our results indicate that ACC synthase is encoded by a multigene family and each gene is differentially induced by developmental, environmental and physiological factors.

1. Introduction

The plant hormone ethylene regulates many aspects of plant growth and development. Adams and Yang [1] have established that ethylene is biosynthesized via the following sequence: methionine --> AdoMet --> 1-aminocyclopropane-1-carboxylic acid (ACC) --> C2 H4 • Ethylene production in plant tissue is normally very low but is greatly promoted at certain developmental stages, such as fruit ripening, by auxin treatment or under certain environmental stress [2]. In all cases the increasing ethylene production resulted from increased synthesis of ACC from AdoMet catalyzed by ACC synthase (S-adenosyl-Lmethionine methylthioadenosine-lyase. EC 4.4.1.14). ACC synthase is believed to be a pyridoxal 5'-phosphate (PLP)-utilizing enzyme [3], the proposed reaction mechanism involves the formation of a Schiff base between the PLP and AdoMet, followed by an a,y-elimination, yielding ACC and methylthioadenosine [1-4]. The substrate of ACC

13

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14

synthase AdoMet also serves as a suicide inactivator. Satoh and Yang [5] demonstrated that when a partially purified ACC synthase preparation isolated from tomato fruit was incubated with Ado[3,4_14C] and the resulting protein was analyzed by SDS/PAGE, only one radioactive protein was observed. This protein was judged to be ACC synthase based on the observations that its molecular mass was 50 kDa and that it was specifially bound to a monoclonal antibody against ACC synthase prepared by Bleecker et al. [6]. Later work showed that ACC synthase was also radiolabeled with Ado[carboxyl-14C ]Met but not with Ado[methyl-14C]Met [7].

ACC synthase cDNA clones have been isolated from various tissues including wound fruit tissues of tomato, winter squash and zucchini and from ripening apple and tomato fruit, and many others (for a review, see [8]). The expression of two ACC synthase genes in wounded, ripe tomato pericarp was shown by the isolation of two different ACC synthase cDNA clones by Van Der Straeten et al. [9] and by Olson et al. [10]. In addition, five genomic genes encoding ACC synthase in tomato were reported by Rottmann et a1. [11] ..

In this study, we have probed the active site of ACC synthase isozymes in tomato with Ado[carboxyl-14C ]Met and NaB3H4. Using appropriate primers, we have obtained four ACC synthase-related gene fragments by PCR amplification of cDNA derived from mRNA of tomato fruit and tomato suspension cell culture. Employing the ribonuclease protection assay, we investigated the level of each transcript in response to ripening and wounding of tomato fruit and to auxin treatment of tomato seedlings.

2. Experimental Procedures

2.1. Immunogurification of ACC synthase. ACC synthase was isolated from ripe and wound (Lycopersicon esculentum Mill.) fruits as described [SJ. One unit of enzyme activity is defined as 1 nmol of ACC produced per hr. Immunoaffinity agarose gel was prepared by passing 2 ml of the ascitic fluid (monoclone line 5b, a gift from Dr. Hans Kende, Michigan state University) to protein G-agarose (Pharmacia); the bound antibodies were then covalently linked to the protein G matrix with dimethylpimelimidate [12J.

2.2. Active-site labeling of ACC synthase. For NaB3 H4 reduction, a sample of ACC synthase preparation (SO,OOO units containing 100 ug of ACC synthase protein) was passed through an immunoaffinity gel column (O.S-ml bed volume in 1-ml pipette tip). The affinity column was loaded and then washed with 10 roM sodium phosphate buffer (pH 7.0) containing 10 uM PLP. The immunoaffinity gel was then transferred to a 1.S-ml Eppendorf tube to which 2S mci of NaB3H4 (14 Ci/mmol; 1 ci = 37 GBq) was added. After 30 min at OOC, 1 mg of unlabeled NaBH4 was added, and incubation was continued for another 30 min. After the gel was washed with 20 roM NH4HC03 , the adsorbed radiolabeled ACC synthase was eluted with 4 bed volumes of 2% SDS in H20. For Ado[carboxyl-14C]Met labeling, a sample of ACC synthase (30,000 units containing 60 ug of ACC synthase) was similarly passed through a

15

column containing 0.3 ml of irnrnunogel. After the gel was washed extensively with 100 rnM Hepes buffer (pH 8.5) containing 10 uM PLP, 10 uCi of Ado[14C]Met (55 mCi/rnrnol), which had been preheated at 1000 C for 7 min in 0.05 M H2S04 [7], was added to the irnrnunogel. After 6 hr with gentle shaking at 30 °c, 600 nrnol of preheated AdoMet was added, and the incubation was allowed to proceed for another 6 hr.

2.3. Trypsin digestion And peptide separation. Radiolabeled ACC synthase was eluted from the irnrnunoaffinity column with 2% SOS [12]. The dried ACC synthase (ca. 60-100 ug) was suspended in 2 M urea containing 50 rnM NH4HC03 (pH 8.0), and digested with TPCK-treated trypsin (a final 5% trypsin-to-protein ratio). The tryptic peptides were separated on a C4 reverse-phase HPLC column with a linear acetonitrile gradient with a flow rate of 1 ml/min [13].

2.4. Peptide sequencing. Edman degradation of the purified radiolabeled peptide was performed by using an Applied Biosystems model 477A protein sequencer, with on-line separation of phenylthiohydantoin-amino acids on an Applied Biosysterns model 120 phenylthiohydantoin-amino acid analyzer. To detect the radioactivity release, fractions (30%) from each sequencer cycle were determined in a liquid scintillation counter.

2.6. Polymerase ~ reaction. PCR primers containing restrictionsite sequences: 1. 5'-CT(GGATCC)G-TCAAAYCCNYTRGGCAC-NAC-3' and 2. 5'CTC(AAGCTT)-ACNARNCCRAARCTYGACAT-3', that derived from the conserved peptide sequences of SNPLGTT and MSSFGLVS were used. poly(A)+ RNA isolated from cell culture, fruit tissues and auxin-treated hypocotyls, as described previously [14], were used as templates for the first strand cDNA synthesis. DNA fragments were synthesized by PCR by the method of Wang et al. [15].

2.7. Cloning of the ~ products. Amplified PCR products were cloned into th~ BamHI and HindIII sites in pBluescript II SK(+) (Stratagene). The sequences of the cloned fragments were determined by DNA sequencing using sequenase 2.0 kit from United States Biochemical.

2.8. Ribonuclease protection assay. The relative levels of ACC synthase mRNAs in poly(A)+ RNA preparations were examined using the ribonuclease protection kit from Ambion (Austin, TX) according to the manufacturer's instructions. [a-32p]UTP-labeled antisense RNA probes were transcribed from BamHI-digested plasmid DNA (1 ug) and were subsequently purified by 8 M urea/5% polyacrylamide gel electrophoresis. The probe was eluted from the gel and 1.2 x 105 cpm was then hybridized with the poly(A)+ RNAs. The hybridization products were treated with a mixture of and ribonuclease A and HI and were then analyzed by polyacrylamide gel electrophoresis and autoradiography.

3. Results and Discussions

16

After radiolabeing and immunopurification, only one radiolabeled band was observed in the SOS/PAGE. This protein was judged to be ACC synthase based on its molecular mass of 48 kOa and on the observations that it was specifically radiolabeled with AdoMet and specifically bound to a monoclonal antibody (5b) against tomato ACC synthase. After trypsin digestion of this radiolabeled protein, only a single radiolabeled peptide peak was isolated. However, Edman degradation of this peptide peak revealed two 12-amino acid sequences which differred in only one amino acid residue (at position 6) as shown in Table 1.

Table 1. Active site peptide sequences of tomato ACC synthase

H -ser-Leu-ser-Lys'-Asp-LeU-Gly-Leu-pro-Gly-Phe-Arg-coOH 2 ,

H2-ser-Leu-ser-Lys -Asp-Met-Gly-Leu-Pro-Gly-Phe-Arg-COOH

'Location of bound radioactivity when the enzyme was radiolabeled with NaB3H4 or Ado[carboxyl-14C]Met.

It is logical to conclude that position 4 is a lysine-PLP adduct after reduction with NaBH4. ACC synthase radiolabeled with AdoMet yielded the same tryptic peptide, indicating that it is the same lysine at the active site binds the PLP in native enzyme and covalently links to the 2-aminobutyrate portion of AdoMet durng the inactivation process [16].

The discovery of two active site tryptic peptides provides the initial evidence for the existent of two ACC synthase isozymes express in tomato fruit tissues.

Using the appropiate primers, we have obtained four DNA fragments representing tomato ACC synthase homologs and their deduced amino acid sequences are shown in Figure 1. pBTASl and pBTAS4 were identical to those full-length sequences previously reported by Van Oer Straeten et al. [9] and Olson et al. [10] from tomato fruit, whereas pBTAS2 and pBTAS3 represent new cONA sequences.

pBTAS1 pBTAS2 pBTAS3 pBTAS4

pBTAS1 pBTAS2 pBTAS3 pBTAS4

snplgttLDKDTLKSVLSFTNQHNIHLVCDEIYAATVFDTPQFVSIAEILDE 52 snplgttLNRNELELLLTFVDEKGIHLISDEIYSGTVFNSPGFVSVMEVLIE snplgttMSRNELNILNTFAMTKNIHIVSDEIYAGTYSDSPKFVSIIDALID

KISTFTNEHNIHLVCDEIYAATVFNPPKFVSIAEIINE . . * ... ** ... **** .. * * ***

QEMTYC-NKDLVHIVYSLSKDMGLPGFRVGIIYSFNDDVVNCARKmssfglv 103 KNYMKTRVWERVHIVYSLSKDLGLPGFRIGAIYSNDEMVVSAATKmssfglv RKLEKSKMWNQVHIVSSLSKDLGLPGFRVGMIYSNNETLIHAQTKmssfglv DN---CINKDLVHIVSSLSKDLGFPGFRVGIVYSFNDDVVNCARKmssfglv

. **** *****.*.****.* .** .... *

Fig. 1. The aligned amino acid sequences of tomato ACC synthase gene fragments (pBTASl, 2, 3, and 4). For details, see [14].

Ribonuclease protection assays were used to examine the expression of these transcripts under three different conditions of enhanced ethylene production - namely, during fruit ripening, in response to

17

mechanical wounding in fruit tissue, and auxin stimulation in vegetative tissue. The results are shown in Figure 2 and Figure 3. Transcripts of pBTASl accumulated massively during ripening and wounding but only slightly in response to auxin treatment. Although pBTAS4 was assoicated with fruit ripening, it was unresponsive to auxin treatment in vegetative tissue. In constrast, the expression of pBTAS2 and pBTAS3 was greatly promoted in auxin-treated vegetative tissue but was absent from fruit tissue. While the expression of pBTAS2 was moderately dependent on wounding, pBTAS3 was unresponsive to wounding. These data support the view that ACC synthase is encoded by a mUltigene family and that the members are differentially expressed in response to developmental, environmental, and hormonal factors.

baaes 340-320 -

B

12 ·3456123456

c o

t 2 3 ,. oS 6 1 ~ 3 4 5 "6

Fig. 2. Expression of four genes of ACC synthase family in tomato fruit and tomato cell culture as assessed by the ribonuclease protection assay. Two ug of polY(A)+ RNA was used in each assay. Probes were pBTASl (A), pBTAS2 (B), pBTAS3 (C), and pBTAS4 (0). Lane I, undigested probe without RNA; lane 2, RNA from green tomato fruit; lane 3, RNA from ripe tomato; lane 4, RNA from wounded ripe tomato; lane S,RNA from S-day-old suspension cell culture; lane 6, digested probe without RNA.

Fig. 3. Expression of four members of ACC synthase gene family in auxintreated tomato seedling hypocotyls as assessed by the ribonuclease protection assay. Probes were pBTASl (A), pBTAS2 (B), pBTAS3 (C), and pBTAS4 (0). Lane I, O.S ug of poly(A)+ RNA from untreated hypocotyls; lane 2, O.S ug poly(A)+ RNA from hypocotyls treated with O.S rnM

indoleacetic acid .

bases 340 -320-

A B c o 2121212

18

4. References

1. Adams, D.O. and Yang S.F. (1979) Ethylene biosynthesis: identification of 1-

aminocyclpropane-l-carboxylic acid as an intermediate in the ccnversion of methionine

to ethylene. Proc. Nat1. Acad. Sci. USA 76, 170-174.

2. Yang, S.F. and Hoffman, N.E. (1984) Ethylene biosynthesis and its regulation in higher

plants. Ann. Rev. Plant Physiol. 35, 155-189.

3. Yu, Y.B., Adams, D.O. and Yang, S.F. (1979) 1-Aminocyclopropane-1-carboxy1ate synthase, a

key enzyme in ethylene biosynthesis. Arch. Biochem. Biophys. 198, 281-286.

4. Ramalingham, K., Lee, K., Woodard, R.W., Bleecker, A.B. and Kende, H. (1985)

Stereochemical course of the reaction catalyzed by the pyridoxal phosphate-dependent

enzyme 1-aminocyclopropane-1-carboxylate synthase. Proc. Natl. Acad. Sci. USA 82, 7820-

7824.

5. Satoh, S. and Yang, S.F. (1988) S-Adenosylmethionine-dependent inactivation and

radiolabeling of l-aminocyclopropane-l-carboxylate synthase isolated from tomato

fruits. Plant Physiol. 88, 109-114.

6. Bleecker, A.B., Kenyon, W.H., Somerville, s.c. and Kende, H. (1986) Use of monoclonal

antibodies in the purification and characterization of l-aminocyclop~opane-l

carboxylate synthase, an enzyme in ethylene biosynthesis. Proc. Natl. Acad. Sci. USA

83, 7755-7759.

7. Satoh, S. and Yang, S.F. (1989) Specificity of S-adenosyl-L-methionine in the inactivation

and the labeling of l-aminocyclo-propane-l-carboxylate synthase isolated from tomato

fruits. Plant Physiol. Arch. Biochem. Biophys. 271, 107-112.

8. Theo10gis, A. (1992) One rotten apple spoils the whole bushel: the role of ethylene in

fruit ripening. Cell 70, 181-184.

9. Van Der Straeten, D., Van Wiemeersch, L., Goodman, H.M., and Van Montagu, M. (1989)

Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-1-carboxylate

synthase. Proc. Nat1. Acad. Sci. USA 87, 4859-4863.

10. Olson, D.C., White, J.A., Edelman, L., Harkins, R.N. and Kende, H. (1991) Differential

expression of two genes from l-aminocyclo-propane-l-carboxylate synthase in tomato

fruits. Proc. Natl. Acad. Sci. USA 88, 5340-5344

11. Rottmann, W.H., Peter, G.F., Oeller, P.W., Keller, J.A., Shen, N.F., Nagy, B.P., Taylor,

L.P., Campbell, A.D. and Theologis, A. (1991) 1-Aminocyclopropane-1-carboxylate

sy"nthase in tomato is encoded by a multigene family whose

transcription is induced during fruit and floral senescence. J. Mol. BioI. 222, 937-961

12. Yip, W.K., Dong, J.G. and Yang, S.F. (1991) Purification and characterization of 1-

aminocyclopropane-1-carboxy1ate

synthase from apple fruits. Plant Physiol. 95, 251-257.

13. Yip, W.K., Dong, J.G., Kenny, J.W., Thompson, G.A. and Yan9, S.F. (1990) Characterization

and sequencing of the active site of 1-aminocyclopropane-l-carboxylate synthase. Prec.

Natl. Acad. Sci. USA 87, 7930-7934.

14. Yip, W.K., Meore, T. and Yang, S.F. (1992) Differential accumulation of transcripts for

four tomato 1-aminocyclopropane-lcarboxylate synthase homologs under various

conditions. Proc. Natl. Acad. Sci. USA 89, 2475-2479.

15. Wang, A.M., Doyle, M. and Mark, D.F. (1989) Quantitation of mRNA by the polymerase chain

reaction. Proc. Natl. Acad. Sci. USA. 86, 9717-9721.

16. Satoh, S. and Yang, S.F. (1989) Inactivation of 1-aminocyclopropane-1-carboxylate synthase

by L-vinylglycine as related to the mechanism-based inactivation of the enzyme by S

adenosyl-L-methionine. Plant Physiol. 91, 1036-1039

MODIFYING FRUIT R1PENlNG BY SUPPRESSING GENE EXPRESSION

Athanasios Theologis, Paul W. Oeller and Lu Min-Wong USDA/U.C. Berkeley Plant Gene Expression Center 800 Buchanan Street Albany, CA 94706

1. Introduction

Ethylene is one of the simplest organic molecules with biological activi!)" Its effects on plant tissue are spectacular and commercially important (1,2). This hydrocarbon gas is generally considered to be the fruit ripening hormone (2,3). Because of its effects on plant senescence, large losses of fruits and vegetables are incurred annually in the U.S. The losses are much greater in third world countries because of the lack of sufficient refrigeration and transportation. Consequently, it has always been a goal of plant biologists and of postharvest physiologists, in particular, to be able to prevent or delay fruit ripening in a reversible manner by controlling ethylene action or production. Thus, an understanding of ethylene action and biosynthesis is of fundamental as well as of applied significance. This update summarizes the recent advances in manipUlating key genes in the ethylene biosynthetic pathway to prevent ethylene production and fruit ripening.

Methionine is the biological precursor of ethylene in all higher plants (22), which is converted to ethylene according to the following sequence:

1 2 3 Methionine--> AdoMet---> ACC --> Ethylene.

The rate limiting step in the pathway is the formation of the amino acid ACC from AdoMet, catalyzed by ACC synthase (reaction 2). The final step is the conversion of ACC to CzH4 catalyzed by ACC oxidase (reaction 3). Molecular cloning approaches and expression in heterologous systems allowed the isolation of the genes encoding AdoMet synthase (step 1, [14]), ACC synthase (step 2, [11,16,21]), and ACC oxidase (step 3, [6,7,18,19]).

2 Iohtbition of Fruit Ripening Using Reverse Genetics

Ethylene is thought to regulate fruit ripening by coordinating the expression of genes that are responsible for a variety of processes, including enhancing a rise in the rate of respiration,

Abbreviations: AdoMet, S-adenosylmethionine; ACe, l-aminocyclopropane-l-carboxylic acid; PG, polygalacturonase.

19


20

autocatalytic ethylene production, chlorophyll degradation, carotenoid synthesis, conversion of starch to sugars, and increased activity of cell wall degrading enzymes (5). Throughout the years, various methods for prolonging fruit senescence have been employed, such as ventilation with air under hypobaric pressures (4). The procedure accelerates the escape of ethylene and, by reducing the oxygen tension, also lowers the fruits' sensitivi7 to hormone. Also, inhibitors of ethylene action have been used such as silver ions (Ag ) and carbon dioxide (C02) (22). These approaches are expensive and fail to prevent fruit senescence satisfactorily. In a few cases, however, such as apple, controlled atmosphere storage has been a commercial success. A more desirable solution to the problem will be the construction of a mutant plant whose fruits do not ripen unless they are treated with ethylene. Tomato ripening mutants exist, but their phenotype is not reversible by ethylene (10).

The cloning of genes induced during fruit ripening and of genes involved in ethylene biosynthesis opened the road to the construction of ripening mutants in tomato using reverse genetics. In the absence of gene replacement technology in plants, antisense RNA technology and overexpression of an ACC metabolizing enzyme became the tools of choice (5).

2.1 Antisense RNA

Initially, attempts to inhibit tomato fruit softening by antisense polygalacturonase (PG) RNA, a gene thought to be responsible for cell wall hydrolysis during ripening, failed to give a strong effect (17,18). Expression of PG antisense RNA dramatically inhibited PG mRNA accumulation and enzyme activity, suggesting that PG is not the sole determinant of cell wall hydrolysis (18). Another approach to prevent fruit ripening is to inhibit ethylene production. Hamilton et al. (6) inhibited ACC oxidase activity with antisense RNA In plants that were homozygous for the antisense gene, ethylene production was inhibited by 97% in ripening fruit. In these antisense fruits, the color change was initiated at about the normal time; however, the extent of reddening was reduced. Antisense fruits stored for several weeks at room temperature were more resistant to overripening and shrivelling than control fruits (6). More recently, Oeller et al. (12) used antisense RNA to ACC synthase to inhibit tomato fruit ripening. This approach led to severe inhibition of ethylene production (below 0.1 nVg-h; 99% inhibition) resulting in a tomato fruit mutant with a striking phenotype (Figure 1). This dramatic inhibition of ethylene production can be attributed to the short half life of ACC synthase (8). Antisense experiments are intrinsically "leaky", thus allowing some mRNA to be translated. Consequently, the stability of the encoded polypeptide is an important factor for successful gene inactivation by antisense RNA (12).

During tomato fruit ripening, two ACC synthase genes are expressed, LE-ACC2 and LEACC4 (13,15). Expression of antisense RNA derived from the cDNA of the LE-ACC2 gene resulted in an almost complete inhibition of mRNA accumulation of both ripening-induced ACC synthase genes (12). Control fruits kept in air begin to produce ethylene 50 days after pollination and fully ripen after 10 more days. The red coloration resulting from chlorophyll degradation and Iycopene biosynthesis is inhibited in antisense fruits. Antisense fruits kept in air or on the plants for 90 to 150 days eventually develop an orange color but never turn red and soft or develop an aroma.

The antisense phenotype can be reversed by treatment with ethylene or propylene, an ethylene analog. The treated fruits are indistinguishable from naturally ripened fruits with respect to texture, color, aroma, and compressibility. The duration of C2H4 treatment required to reverse the antisense phenotype is 6 days. Antisense fruits treated for 1 or 2 days

21

- 8 ~

..c •

-e> c 6 -..-c 0 +-' :::J

4 0 > w Q) c Q)

>-..c +-' W

50 60 70 Days after Pollination

Figure 1. Inhibition of ethylene production in detached tomato fruits by antisense ACC synthase RNA (12).

with C2H4 do not develop a fully ripe phenotype compared with control fruits treated similarly.

2.2 ACC Deaminase

Klee et al. (9) used a different approach to inhibit ethylene production. They overexpressed the ACC deaminase gene from Pseudomonas sp. in transgenic tomato plants which metabolizes ACC to a-ketobutyrate (9). This approach led to 90-97% inhibition in ethylene production during ripening. Reduction in ethylene synthesis in transgenic plants did not cause any apparent vegetative phenotypic abnormaliities. However, fruits from these plants showed significant delays in ripening, and they remained firm for at least 6 weeks longer than the non transgenic control fruits (9).

Three important conclusions can be inferred from the tomato fruit mutants: (i) The C2H4-mediated ripening process requires continuous transcription of the necessary genes, which may reflect a short half-life of the induced mRNAs or polypeptides. (ii) Ethylene is indeed autocatalytically regulated. (iii) The hormone acts as a rheostat rather than as a switch for controlling the ripening process. The accumulated evidence from the antisense and the deaminase experiments indisputably demonstrated that the Yang cycle is solely responsible for ethylene synthesis during ripening and that ethylene is the key regulatory molecule for fruit ripening and senescence, not the by-product of ripening (2).

22

The mutant fruits producing low levels of ethylene have proven to be excellent experimental material for assessing which of the ripening induced genes so far cloned are indeed ethylene inducible. The expression of PG and pTOM13 (encodes ACC oxidase) genes, which were previously thought to be ethylene regulated, have been found to be ethylene independent (12). Surprisingly, while antisense fruits express large amounts of PG. mRNA, they fail to accumulate the PG polypeptide, suggesting that ethylene may control the· translatability of PG mRNA or the stability of the PG polypeptide (20).

The mutants also told us that at least two signal transduction pathways are operating during tomato fruit ripening. The ethylene independent (developmental) pathway is responsible for the transcriptional activation of genes such as PG, ACC oxidase, and chlorophyllase. The ethylene dependent pathway, on the other hand, is responsible for the transcriptional and posttranscriptional regulation of genes involved in lycopene and aroma biosynthesis, respiratory metabolism, ACC synthase gene expression, and translatability of developmentally regulated genes such as PG (20).

3. The Future

The use of antisense technology and overexpression of metabolizing enzymes such as ACC deaminase in controlling fruit ripening is only the first step towards controlling fruit senescence. Expression of antisense RNA using regulated promoters may eliminate the use of exogenous ethylene for reverting the mutant phenotype. However, the development of gene transplacement technology by homologous recombination should allow the creation of non-leaky ripening mutants with long term storage potential. The prospect arises that inhibition of ethylene production using reverse genetics may be a general method for preventing senescence in a variety of fruits and vegetables.

4. Acknowledgements

This work was supported by grants to AT. from the NSF (DCB-8645952, -8819129, -8916286), the NIH (GM-35447) and the USDA (5835-2841O-DOO2).

5. References

1. Abeles, F. B. (1973) "Ethylene in Plant Biology", Academic Press, New York. 2. Biale, J. B. and Young, R. E. (1981) Respiration and Ripening in Fruits-- Retrospect and

prospect. In: J. Friend, M. J. C. Rhodes, eds., "Recent Advances in the Biochemistry of Fruits and Vegetables", Academic Press, London, pp. 1-39.

3. Burg, S. P. (1962) The physiology of ethylene formation. Ann. Rev. Plant Physioi 13:265-302. 4. Burg, S. P. and Burg, E. A (1966) Fruit storage at subatmospheric pressures. Science 153:314-

315. 5. Gray, J., 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. 6. Hamilton, A J., Lycett, G. W. and Grierson, D. (1990) Antisense gene that inhibits synthesis

of ethylene in transgenic plants. Nature 346:284-287.

23

7. Hamilton, A J., 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. Kende, H. and Boller, T. (1981) Wound ethylene and l-aminocyclopropane-l-carboxylate synthase in ripening tomato fruit. Planta 151:476-481.

9. Klee, H. J., Hayford, M. B., Kretzmer, K. A, Barry, G. F., Kishmore, G. M. (1991) Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants. Plant Cell 3:1187-1193.

10. McGlasson, W. B. (1985) Ethylene and fruit ripening. Hort. Sci. 20-.51-54. 11. Nakajima, N., Mori, H., Yamazaki, K. and Imaseki, H. (1990) Molecular cloning and sequence

of a complementary DNA encoding l-aminocyclopropane-l-carboxylate synthase induced by tissue wounding. Plant Cell PhysioL 31:1021-1029.

12. 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.

13. Olson, D. c., White, J. A, Edelman, L., Harkins, R. N. and Kende, H. (1991) Differential expression of two genes for l-aminocyclopropane-l-carboxylate synthase in tomato fruits. Proc. Natl. Acad. Sci. USA 88:5340-5344.

14. Peleman, J., Boerjan, W., Engler, G., Seurinck, J., Botterman, J., Alliote, T., Van Montagu, M. and Inze, D. (1989) Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase. Plant Cell 1:81-93.

15. Rottmann, W. E., Peter, G. F., Oeller, P. W., Keller, J. A, Shen, N. F., Nagy, B., Taylor, L. P., Campbell, A D. and Theologis, A (1991) l-aminocyclopropane-l-carboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence.l Mol. Bioi. 222:937-961.

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

17. Sheehy, R. E., Kramer, M. and Hiatt, W. R. (1988) Reduction of polygalacturonase activity in tomato fruit by antisense RNA Proc. Natl. Acad. Sci. USA 85:8805-8809.

18. Smith, C. J. S., Watson, C. F., Ray, J., Bird, C. R., Morriss, P. C., Schuch, W. and Grierson, D. (1988) Antisense RNA inhibition of polygalacturonase gene expresSion in transgenic tomatoes. Nature 334:724-726

19. Spanu, P., Reinhardt, D. and Boller, T. (1991) Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes. EMBO J: ·10:2007-2013.

20. Theologis, A (1992) One rotten apple spOils the whole bushel: the role of ethylene in fruit ripening. Cell 70:1-4.

21. Van Der Straeten, D., Van Wiemeersch, L., Goodman, H. M. and Van Montagu, M. (1990) Cloning and sequence of two different cDNAs encoding l-aminocyclo-propane-lcarboxylate synthase in tomato. Proc. NatL Acad. Sci. USA 87:4859-4863.

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

CLONING AND EXPRESSION ANALYSIS OF AN ARABIDOPSIS THALIANA 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE GENE: PATTERN OF TEMPORAL AND SPATIAL EXPRESSION

R.A. RODRIGUES-POUSADA, D. VAN DER STRAETEN, A. DEDONDER *, and MARC VAN MONTAGU Laboratorium voor Genetica and *Laboratorium voor Plantenfysiologie Universiteit Gent K.L. Ledeganckstraat 35 B-90OO Gent Belgium

ABSTRACT. A genomic clone of one member of the Arabidopsis thaliana (L.) Heynh. l-aminocyclopropane-l-carboxylate synthase gene family (aec1) was isolated and sequenced. Genomic DNA gel blotting suggested the existence of an ACC synthase multigene family in Arabidopsis. The other members are distantly related to aec1. The existence of at least another gene was confirmed by the isolation of a cDNA (aee2) from a flower-specific cDNA library. Expression of the aec1 gene was studied by reverse-transcription polymerase chain reaction (RT-PCR) on total RNA. The mRNA accumulated strongly in young leaves and flowers. Wounding of young leaves did not induce the aec1 gene. Ethylene exposure of flowering plants led to an induction of aec1 expression. An aec1 promoter-6-g1ucuronidase fusion was introduced in A. thaliana by the root transformation method. Temporal and spatial regulation of expression of aec1 were analyzed, revealing a developmental control both in shoot and root and confirming results obtained by RT-PCR analysis. A role for aec1 in the developmental control of ethylene synthesis is suggested.

1. Introduction

Ethylene is a plant growth regulator known to be involved in several stages of plant development (Abeles, 1973; Yang and Hoffman, 1984). The direct precursor of ethylene in higher plants is 1-aminocyclopropane-1-carboxylic acid (ACC) of which synthesis is catalyzed by ACC synthase, a pyridoxal-5' -phosphate-dependent enzyme. This is the rate-limiting step of the pathway (yang & Hoffman, 1984). With the recent cloning of the enzymes involved in the biosynthesis of ethylene (for a review, see Van Der Straeten and Van Montagu, 1991; Theologis, 1992), more information about the molecular regulation of the formation of this hormone has been made available. From a molecular point of view, it is intriguing how different conditions during development, or exposure to external stress factors, lead to the induction of the same key regulatory enzyme, ACC synthase. The genes encoding this enzyme have been cloned from a number of different plant species: tomato (Van Der Straeten et al., 1990; Olson et aI., 1991; Rottman et aI., 1991; Yip et aI., 1992), zucchini (Huang et aI., 1991), winter squash (Nakajima et aI., 1990; Nakagawa et aI., 1991), apple (Dong et aI.,

24


25

1991; Kim et aI., 1992), carnation (par\<: et aI., 1992), mung bean (Botella et aI., 1992; Kim et aI., 1992), and Arabidopsis (Van Der Straeten et aI., 1992). In most cases, the existence of ACC synthase mUltigene families has been demonstrated. The genes appear to be differentially regulated although some coordination has been observed. These studies have provided us information about general aspects of ACC synthase inducibility. Nevertheless, our knowledge of the tissue and cell specificity of the expression of ACC synthase(s) during plant development remains poor. In this report, we present data based on reverse transcriptase polymerase chain reaction CRT-PCR) and promoter-fi-glucuronidase (GUS) fusion techniques, that provide us with a first insight in those and other questions related to the role of ethylene in development of higher plants.

2. Material and Methods

2.1. LIBRARIES, GENE CLONING, AND SEQUENCE ANALYSIS

A cos mid library of Arabidopsis thaliana (ecotype Columbia) was a gift from N. Olszewski (University of Minnesota). Screening was performed with the same oligonucleotides and under the same conditions as published previously (Van Der Straeten et aI., 1990). A flower-specific cDNA library of ecotype C24 (provided by M.H. Goldman and D. De Oliveira, Plant Genetic Systems, Gent, Belgium) was screened as described elsewhere (Van Der Straeten el aI., 1992). DNA sequencing analysis was performed as reported previously (Van Der Straeten et aI., 1992).

2.2. PLANT MATERIAL

Arabidopsis thaliana (ecotype C24) plants were grown in vitro at 22°C and 60% relative humidity under white-fluorescent light (photoperiod 16 h light/8 h dark, at a fluence rate of 75 J.tmollm2/s1). Plant material used for the preparation of total RNA for the RT-PCR experiments is described elsewhere (Van Der Straeten et aI., 1992).

2.3. RNA ISOLATION AND REVERSE TRANSCRIPTASE POLYMERASE CHAIN REACTION CRT-PCR)

Total RNA was isolated as reported (Rodrigues-Pousada et a!., 1990). RT-PCR was performed as in Van Der Straeten et al. (1992).

2.4. NUCLEIC ACID HYBRIDIZATION ANALYSIS

Nuclear DNA preparation, DNA gel blotting, probe preparation, and hybridizations were essentially as reported (Van Der Straeten et aI., 1990; Van Der Straeten et aI., 1992).

2.5. HISTOCHEMICAL II-GLUCURONIDASE ASSAYS

Histochemical assays were according to Jefferson et a!. (1987) except that all material analyzed was pre-fixed in cool 90% acetone for 10-20 minutes at room temperature, and then incubated 24-48 hours, at 37°C in 1 mM 5-bromo-4-chloro-3-indolyl-B-D-glucuronide (in 50 mM sodium phosphate buffer, pH 7.0).

26

3. Results

3.1. ISOLATION AND CHARACTERIZATION OF ARABlDOPSIS ACC SYNTHASE GENES

Screening of a genomic cosmid library of Arabidopsis with oligonucleotides derived from tomato ACC synthase peptide sequences (Van Der Straeten et aI., 1990) led to the isolation of the aec1 gene and its identification by similarity to other ACC synthases (Van Der Straeten el aI., 1992); 5613 bp of the aec1 gene were sequenced, 1432 bp upstream from the initiation codon, and 1993 bp downstream from the stop codon. The gene contains 3 introns. The position of the exon-intron junctions are identical to those in the tomato ACC synthase genes (Rottmann et aI., 1991). A partial cDNA of another Arabidopsis ACC synthase gene was isolated from a flower-specific cDNA library using a 2.2-kb BamHI fragment of aec1 as a probe, and was designated AT-ACC2 (Van Der Straeten et aI., 1992). Its 159 nucleotides are 79% identical to the corresponding region of aec1. The deduced amino acids in this region are 83 % identical.

The aec1 gene is a member of the ACC synthase gene family, but without high similarity to any other family member. Hybridization of the aec1 2.2-kb BamHI fragment to Arabidopsis nuclear DNA under high-stringency conditions revealed one BglII fragment and two EeoRI fragments, each with half the intensity of the BglII band (Fig. 1, lanes 1 and 2). Confirmation of a single gene pattern was obtained by hybridizing Arabidopsis genomic DNA with a PCR -generated fragment, of which the sequence resides mostly in the 3' -untranslated region (data not shown). In the latter cases a single band was observed upon EeoRI and XbaI restriction. However, when repeated under low-stringency conditions (53°C) with the 2.2-kb BamHI fragment several extra bands became apparent (data not shown), indicating the existence of several related members of the ACC synthase gene family.

'BE'rBE"BE'~ -.-.. ------.-kb

~ -6.0

-1..0

-3.0

-2.0

Fig. 1. Genomic DNA gel blot analyses of A. thaliana (ecotype C24), rice (cv. Taipei), tomato (cv. Orlando), and tobacco. From left to right: A. thaliana, 1 fLg nuclear DNA; rice, 3 fLg nuclear DNA; tomato, 5 fLg total DNA; tobacco, 20 fLg nuclear DNA. B. BglII; E, EeoRI. The filter was hybridized with a 2.2-kb BamHI fragment of the aecJ gene.

3.2. STUDY OF THE EXPRESSION OF THE ACC] GENE AS DETERMINED BY RT-PCR

Due to the very low abundance of ACC synthase messengers, RT-PCR was used for analysis of mRNA levels (Delidow et aI., 1989). Different sets of oligonucleotides were used chosen either in conserved or in divergent regions of the gene (Van Der Straeten et aI., 1992). Fig. 2A presents a DNA gel blot of a RT-PCR reaction on different total RNA samples using a set of primers (Van Der Straeten et aI., 1992), which most likely amplify specifically aecJ mRNA. The signal was very high in young leaves and in flowers, but very weak in roots of flowering plants, and absent in siliques and etiolated seedlings (Fig. 2A, lanes 1-7). The same pattern was

27

found when a set of primers corresponc\ing to conserved regions was used. (Fig . 2B, 1-7). When mature flowering plants were exposed to a continuous flow of 10 ppm

ethylene an early induction could be observed (2 hours; Fig. 2A, lane 9), but was almost back to basal level after 8 hours (lane 11). Control plants did not show any signal (Fig. 2A, lane 8). When the non-specific set of oligonucleotides was used, a biphasic induction occurred, with a first peak at two hours, and a second, strong induction at 12 hours.

In Figure 3, the effect of wounding on Arabidopsis ACC synthase mRNA levels is shown. Panel A presents a DNA gel blot of samples treated with the pair of primers, which specifically amplifies aeel mRNA. The signals obtained between 30 minutes and 8 hours wounding, did not show significant variation except a slight repression at 30 minutes. However, a clear induction was observed when the non-specific set of oligonucleotides was used, with a peak at 4 hours after wounding (Fig. 3B).

3 4 5 6 7 8 9 10 11 12 bp

-608

bp

............. ~-220

Fig. 2. RT-PCR analysis of expression of the aeel gene performed on total RNA from different organs of Arabidopsis and from mature plants after ethylene exposure with (A) and without (B) specific primers. Lane 1, 500-bp marker; lane 2, young leaves; lane 3, roots; lane 4, flowers; lane 5, mature-green siliques; lane 6, ripe siliques; lane 7, etiolated seedlings; lane 8, mature plants; lane 9, mature plants after 2 hours ethylene exposure; lane 10, 4 hours; lane 11, 8 hours; lane 12, 12 hours of ethylene exposure.

Fig. 3. RT-PCR analysis of expression of the aeel gene performed on total RNA from wounded, young Arabidopsis leaves with (A) and without specific primers. Lane 1, young leaves; lanes 2 to 8, 0.5, 1, 1.5,2,3,4, and 8 hours wounding; lane 9, control on 5 ng cosmid DNA, carrying aeel.

3.3. TISSUE-SPECIFIC EXPRESSION OF THE Accl GENE DETERMINED BY PROMOTER/GUS

FUSIONS

We analyzed the expression of the aeel promoter by qualitative measurements of the Il-Glucuronidase activity at several stages of Arabidopsis thaliana development (Rodrigues-Pousada et aI., in preparation). GUS activity was detected at the earliest 4-5 days after seed germination in the hypocotyl-root junction, in the root tip, and in the young cotyledons. The activity in roots increased steadily until full development of the root system was reached at about 30-35 days. Expression at the level of the root tip disappeared after 9-12 days. The expression was strongly correlated with the initiation and formation of lateral roots . Lateral root tips did not show detectable expression after having emerged through the main

28

root. In the aerial part of the plant, the expression increased steadily to a maximum peak at about 30-35 days and was consistently stronger in the youngest tissues. This peak of expression coincided significantly with the peak observed at the level of the roots. Thereafter, a significant reduction of expression was observed reaching almost undetectable levels in all the rosette leaves. This coincided with the transition to the reproductive cycle. At this stage, GUS activity was detected only in the youngest tissues: young stem leaves, floral buds and immature siliques. The expression was also high near the floral meristems and especially in young flower buds. In mature green or ripe siliques expression was very low to undetectable. In open flowers, expression was low in the sepals and even less in petals and anthers. High expression was observed in the upper half of the style, near the stigma, and in the receptacle. Wounding of the rosette leaves in either vegetative or reproductive stages of the plant showed no influence on the expression of GUS.

4. Conclusion

We have analyzed the pattern of temporal and spatial expression of one member of the A. thaliana ACC synthase gene family. Both RT-PCR and promoter/GUS fusion techniques led us to the same major conclusions. First, there is a clear association between expression of this gene and developing tissues like young floral buds, shoot or inflorescence meristem, young developing leaves and early stages of lateral root formation. Second, wounding of either young or old leaves does not seem to have any effect on the expression of accl. However, the significance of the expression of acel in young developing tissues in relation to the role of ethylene in plant development remains to be proved. In view of recent evidence suggesting that ACC oxidase is not necessarily a constitutive enzyme in all tissues (Smith et al., 1986), one still has to consider the possibility that regulation of ACC oxidases exists in developing tissues, and might or might not lead to the conversion of ACC into ethylene. In vivo determinations of ACC oxidase activity will provide us with an answer to this question. It is, however, tempting to speculate that with both ACC synthase and oxidase being active in young tissues, the role of ethylene may be in the determination of cell size and/or shape, as suggested earlier (Ortufio et al.,199i~·Sanchez-Bravo et al., 1991). A more detailed analysis of the factors affecting aeel expression as well as a study of the kinetics of appearance of the activity may provide some clues on this issue.

Acknowledgements

We thank Raimundo Villarroel sequencing and Wim Van Caeneghem for excellent technical assistance. We are grateful to Martine De Cock for lay-out of the manuscript and to Karel Spruyt and Vera Vermaercke for preparing the figures. RARP is indebted to JNICT (portugal) for a PhD scholarship. DVDS is a Research Associate of the National Fund for Scientific Research (Belgium).

29

References

Abeles, F.B. (1973) Ethylene in Plant Biology, Academic Press, New York. Botella, J.R., Schlagnhaufer, C.D., Arteca, R.N. & Phillips, A.T. (1992) 'Identification abd

characterization of three putative genes for 1-aminocyclopropane-l-carboxylate synthase from etiolated mung bean hypocotyl segments', Plant Mol. BioI. 18,793-797.

Delidow, B.C., Peluso, J.J. & White, B.A. (1989) 'Quantitative measurement of mRNAs by polymerase chain reaction', Gene Anal. Techn. 6, 120-124.

Dong, J.G.,Kim, W.T., Yip, W.K., Thompson, G.A., Li, L., Bennett, A.B., & Yang, S.F. (1991) 'Cloning of a cDNA encoding 1-aminocyclopropane-l-carboxylate synthase and expression of its mRNA in ripening apple fruit', Planta 185, 38-45.

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

Huang, P.-L., Parks, J.E., Rottmann, W.H. & Theologis, A. (1991) 'Two genes encoding 1-aminocyclopropane-l-carbox y late synthase in zucchini (Cucurbita pepo) are clustered and similar but differentially regulated', Proc. Natl. Acad. Sci. USA 88, 7021-7025.

Jefferson, R.A., Kavanagh, T.A. & Bevan, M.W. (1987) 'GUS fusions: B-glucuronidase as a sensitive and versatile gene fusion marker in higher plants', EMBO J. 6, 3901-3907.

Kim, W.T., Silverstone, A., Yip, W.K., Dong, J.G. & 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.

Nakajima, N., Mori, H., Yamazaki, K. & Imaseki, H. (1990) 'Molecular cloning and sequence of a complementary DNA encoding 1-aminocyclopropane-l-carboxylate synthase induced by tissue wounding', Plant Cell Physiol. 31, 1021-1029.

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

Olson, D.C., White, J.A., Edelman, L., Harkins, R.N. & Kende, H. (1991) 'Differential expression of two genes for 1-aminocyclopropane-l-carboxylate synthase in tomato fruits', Proc. Natl. Acad. Sci. USA 88, 5340-5344.

Ortuiio, A., Del Rio, J.A., Casas, J.L., Serrano, M., Acosta, M. & Sanchez-Bravo, J. (1991) 'Influence of ACC and Ethephon on cell growth in etiolated lupin hypocotyls. Dependence on cell growth state', BioI. Plant. (Praha) 33, 81-90.

Park, K.Y., Drory, A. & Woodson, W.R. (1992) 'Molecular cloning of an 1-aminocyclopropane-lcarboxylate synthase from senescing carnation flower plants', Plant Mol. BioI. 18, 377-386.

Rodrigues-Pousada, R., Van Montagu, M. & Van Der Straeten, D. (1990) 'A protocol for preparation of total RNA from fruit', Technique 2, 292-294.

Rottmann, W.H., Peter, G.F., Oeller, P.W., Keller, J.A., Shen, N.F., Nagy, B.P., Taylor, L.P., Campbell, A.D. & Theologis, A. (1991) , 1-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.

Sanchez-Bravo, J., Ortuiio, A.M., Perez-Gilabert, M., Acosta, M. & Sabater, F. (1992) 'Modification by ethylene of the cell growth pattern in different tissues of etiolated lupine hypocotyls', Plant Physiol. 98, 1121-1127.

Smith, C.J.S., Slater, A. & Grierson, D. (1986) 'Rapid appearance of an mRNA correlated with ethylene synthesis encoding a protein of molecular weight 35 000', Planta 168, 94-100.

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

Van Der Straeten, D., Van Wiemeersch, L., Goodman, H.M. & Van Montagu, M. (1990) 'Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-l-carboxylate synthase in tomato', Proc. Natl. Acad. Sci. USA 87, 4859-4863.

30

Van Der Straeten, D. & Van Montagu, M.(l99l) 'The molecular basis of ethylene biosynthesis, mode of action and effects in higher plants', in B.B. Biswas & J.R. Harris (eds.), Plant Genetic Engineering, Subcellular Biochemistry, Voll7, Plenum Publishing Corporation, New York, pp. 279-326.

Van Der Straeten, D., Rodrigues-Pousada, R.A., Villaroel, R., Hanley, S. Goodman, H. & Van Montagu, M.(1992) 'Cloning, genetic mapping and expression analysis of an Arabidopsis thaliana ACC synthase', Proc. Natl. Acad. Sci. USA 89, in press.

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

Yip, W.-K., Moore, T. & Yang, S.F. (1992) 'Differential accumulation of transcripts for four tomato 1-aminocyclopropane-1-carboxylate synthase homologs under various conditions', Proc. Natl. Acad. Sci. USA 89, 2475-2479.

Wang, H. & Woodson, R.W.(1991) 'A flower senescence-related mRNA from carnation shares sequence similarity with fruit ripening-related mRNAs involved in ethylene biosynthesis', Plant Physiol., 96, 1000-1001.

RELATIONSHIP OF ACC OXIDASE RNA, ACC SYNTHASE RNA, AND ETHYLENE IN PEACH FRUIT.

A.M. CALLAHAN*, D. FISHEL and L.J. DUNN, USDA-ARS, Appalachian Fruit Research Station, Kearneysville, WV, 52430 USA

We would like to be able to regulate the softening process in peach fruit in order to obtain higher quality fruit with adequet shelf-life. The longer the fruit is left on the tree to ripen, the more flavor is developed but, the longer the fruit is left. on the tree less shelf-life of the fruit remains. A series of experiments with peach fruit were undertaken in order to understand the relationship of ethylene to ACC synthase RNA and EFE RNA and their relationships to softening. In experiments that decrease the amount of these RNAs in tomato fruit, ripening and softening are delayed (Hamilton et al. (1990), Oeller et al. (1991)).

Northern analyses of peach RNA from developing and ripening fruit indicate that peach ACC oxidase (Callahan et al. (1992) and ACC synthase (Dong, Callahan,and Yang, unpublished) RNAs accumulate as the fruit ripen at 134 DAB (fully colored and sized) and more specifically at the time the fruit have softened to less than 40 newtons (N). The first experiment measured ethylene evolvolution, fruit firmness, ACC synthase RNA, and EFE RNA in individual fruits taken at different stages of softening. The results demonstrate no good correlation with any of the parameters except the amount of ethylene and fruit fmnness. Ethylene evolution increases when the fruit is <50 N. Duplicate RNA preparations were made from each fruit to look at within the fruit variation. The results indicate vary little variation.

The second experiment measured firmness, ACC synthase RNA, and EFE RNA on mature, preclimacteric fruit that had been treated with ethylene, 2,5-norbornadiene, or both to see if any treatments had an effect. ACC oxidase could be detected by seven hours after treating with ethylene. Norbornadiene also appears to induce ACC oxidase RNA but at a lower level. The combination of ethylene and norbornadiene results in an additve effect. The presense of ethylene also caused the fruit to soften by 48 hours. None of these combinations induced accumulation of detectable ACC synthase RNA.

The regulation of ACC synthase and oxidase RNA accumulation is not simple. The amounts of either are not directly linked to the amount of ethylene evolved, the stage of fruit softness, or to each other. There are most likely many other factors that influence the level of ethylene and fruit softening. In addition it appears from the preliminary data presented here that ACC oxidase RNA accumulates within seven hours of treatment by ethylene. This accumulation is not inhibited by norbornadiene and in fact is stimulated by that compound. The fruit ripening specific ACC synthase RNA is not stimulated by the presense of ethylene. Our conclusion from these experiments is that regulation of RNA accumulation differs for each enzyme in the

31

J. C. Pech et at. (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, 31-32. © 1993 Kluwer Academic Publishers.

32

ethylene biosynthesis pathway. We would like to pursue this idea in the future by both looking at the regulatory regions of the genes and by ffurther examining the rates of accumulation under different phyiological situations.

The authors would like to acknowledge and thank Drs. J. Dong and S. F. Yang for the isolation and use of the peach fruit ACC synthase clone in this study.

Callahan, A.M., Morgens, P.H., Wright, P., and Nichols, K.E. (1992) 'Comparison ofpch313 (pTOM13 homolog) RNA accumulation during fruit softening and wounding of two phenotypically different peach cultivars' Plant Physiol. 100: in press.

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.

Oeller, P.W., Wong, L.M., Taylor, L.P., Pike, D.A., and Theologis, A. (1991) 'Reversible inhibition of tomato fruit senescence by antisense 1-aminocyclopropane-1-carboxylic synthase', Science 254:437-439.

MAXIMISING THE ACTIVITY OF THE ETHYLENE-FORMING ENZYME

J.J. SMITH AND P. JOHN Department of Agricultural Botany Plant Science Laboratories University of Reading Reading RG6 2AS UK

ABSTRACT. Activity of the ethylene-forming enzyme extracted from melon can be stabilised by the inclusion of 30% glycerol in media, so that there is virtually no los~ of activity when the enzyme is stored at O·C for 24 hr. When enzyme preparations from melon are desalted by passage through a column of Sephadex, activity is reduced. The lost activity can be restored by the inclusion of dilute N aOH to the reaction mixture, which gives a 6-fold stimulation of the EFE activity from melon. The EFE activity is also stimulated by the addition of HC03'/C02 to the reaction mixture, with a 25-fold increase in activity for melon and an 8-fold increase in activity for apple.

1. Introduction

The ethylene-forming enzyme (EFE) is responsible for the generation of ethylene from its immediate precursor, l-aminocyclopropane-l-carboxylic acid (ACC). Activity of the EFE in vitro was first demonstrated in extracts of melon fruits [1] when it was revealed that activity required the presence of ascorbate and Fe2+. Subsequently we described the properties of the enzyme partially purified from melon [2], and recent studies have been made of the EFE from fruits of apple [3,4 and 5] and avocado [6]. During the purification of the melon EFE two problems presented themselves. First there was a loss of enzyme activity when the enzyme remained for a few hours near to O·C. Secondly, when the enzyme was desalted by passage through a column of Sephadex, there was a loss of activity. In the present paper we describe how the enzyme can be stabilised by the inclusion of glycerol, and how the loss of activity observed on desalting led us to identify a requirement by the EFE for HC03'/C02 for maximum activity in vitro.


2.1. ENZYME PREPARATION

Ripe fruits of melon (Cucumis melo L. var. Galia) and apple (Malus domestica Borlch. var. Golden delicious) were prepared and frozen as in [2]. Except where indicated in legends, enzyme extraction was as follows. When melon was used the ground pericarp (200 g) was homogenised with sand in 200 ml of extraction buffer, which contained 0.1 M Tricine pH 8.0 and 30% (v/v) glycerol, until the temp. reached 0-2·C. The homogenate was filtered through a double layer of

33

J. C. Pech et aL (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, 33-38. © 1993 Kluwer Academic Publishers.

34

muslin before centrifugation at 21,000 g for 20 min. Aliquots of the extract were then frozen at -70°C until used as the enzyme extract. When apple was used, the enzyme was prepared as for melon, except that 100 g of ground pulp was homogenised with sand in 200 ml of extraction buffer, which was supplemented with 5% (w/v) PVPP [3] and 30 mM sodium ascorbate. Except where indicated, thawed aliquots (2.5 ml) of extract were desalted by passage through Sephadex G-25 PD-lO columns (pharmacia) previously equilibrated with the appropriate incubation buffer specified in the legends to the figures and table. Protein was determined by the method of Bradford [7] using BSA as a standard.

2.2. ASSAY OF EFE ACTIVITY

The EFE activity was assayed by measuring with a GC the ethylene produced [1], after incubation for 1 hr at 300C in a reaction mixture (1 ml) containing 0.1 M Tricine (PH 7.5), 30 % (v/v) glycerol, 0.1 mM FeS04, 30 mM sodium ascorbate and ImM ACC. All determinations were made in triplicate. The results are expressed as means, with tabulated data including ± s.e.

3. Results and Discussion

3.1. STABILISATION BY GLYCEROL

When an extract from melon fruits remained near to OOC in a buffer containing no glycerol, activity declined so that after 24 hr about 90% of the original activity had been lost (Fig. 1). Increasing the concentration of glycerol present resulted in a progressive enhancement of enzyme stability so that with 30% (v/v) glycerol, over 90% of enzyme activity was retained after 24 hr. Presumably this stabilisation is similar to that of other globular proteins which preferentially hydrate to minimise their contact with glycerol, and in this way stabilise their active structure [8].

o O%glycerol ~ • 100/oglycerol " I::, 20%glycerol .~ 240 '" 30% glycerol

~2~::;:::>1~"--",

" c 12

U 40 o W O~~--~--~~F~~ It 0 2 4 6 24

Time after initial incubation(hr)

Figure 1. Effect of glycerol on the EFE stability. Melon extracts were prepared (20 g tissue: 25 ml degassed buffer) using 0.1 M Tricine (PH 8.0) containing 30 mM sodium ascorbate, O.lmM FeS04 and glycerol as indicated. Extraction was conducted under a flow of N2 to exclude O2, After centrifugation the supernatants were stored at ODC until 973 Itl containing about 500 Itgprotein was taken for assay.

3.2. A LOW MOLECULAR WEIGHT FACTOR REQUIRED BY THE EFE

When an extract of melon fruit was passed through a column of Sephadex, the EFE activity typically fell by 25-50%. The low molecular weight material eluted from the column enhanced activity when it was added to the desalted enzyme, so that the original activity of the crude extract returned (Fig. 2). The low molecular weight material also stimulated activity of the crude extract (Fig. 2). When the low molecular weight fra~tion obtained from the melon tissue was added to th~ apple extract, either before or after passage through a column of Sephadex, we

35

observed a stimulation of activity comparable to that recorded with the desalted melon extract (Fig. 2).

o -factor Melon ~ .factor Apple

Figure 2. Effect of passage through Sephadex and the addition of melon factor, on the activity of the EFE from melon and apple. Crude enzyme was prepared in buffer containing 10% (v/v) glycerol and 30 mM sodium ascorbate, supplemented with 5% PVPP [3] for apple. Melon factor was prepared from 16 g melon tissue in 20 mI 0.1 M Tricine pH 8.0. Aliquots (2.5 mI) of the melon factor supernatant (21,000 g for 20 min), were passed through Sephadex and eluted with 8 ml equilibration buffer (PH 7.5). Melon factor was collected in the eluate fractions 6-8 mI. All enzyme extracts (500 JLI containing 114 JLg protein for apple and 364 JLg protein for melon) were assayed ± 473 JLI melon factor.

After a preliminary search through a range of possible low molecular weight cofactors, we discovered that simply adding 10 mM NaOH to the reaction vessel prior to the addition of the desalted enzyme extract, stimulated the EFE activity to its pre-Sephadex level (Fig. 3). The amount of NaOH added is insufficient to cause a permanent alkalisation of the reaction medium greater than 0.2 pH units. The interaction between NaOH and the EFE is a complex one. One possible factor contributing to the stimulatory effect observed is that NaOH provides a transient alkali shock to the EFE which has a renaturing effect, but more detailed studies on the enzyme structure are required to substantiate this possibility.

g UJI u. UJ

5 10 15 20 [NaOHllmMI

~

1200......----------..

>-0 after passage

through Sephadex

UJ 00~-~,~0-~20~~~t~~60~ [NaHCn;llmMI

Figure 3. Effect of NaOH addition on the EFE activity. An enzyme extract (900 JLI containing 473 JLI protein) was assayed either before or after passage through Sephadex.

Figure 4. Effect of N aH C03 on the EFE activity. An ~nzyme extract (873 JLl) was assayed either directly (528 JLg protein), or after passage through Sephadex (349 JLg protein).

36

3.3 ACTIVATION BY BICARBONATE/CARBON DIOXIDE

Activity of the desalted extract could also be restored by adding HCO;. Increasing concentrations of HC03- added to the reaction mixture progressively stimulated the EFE activity to levels which were even greater than those observable with the crude extract in the presence of HC03- (Fig. 4). A stimulatory effect of HC03- addition was also observed with the EFE extracted from apple (Table 1), and in a variety of other fruit (M.A. Moya-Leon and T. Itturriagagoitia, unpublished data), but in subsequent experiments the effect was examined in detail only with the EFE extracted from melon.

Table 1. Effect of NaHC03 on the EFE extracted from apple fruits, before and after desalting the enzyme preparation.

Enzyme preparation

EFE activity (nl/mg protein/hr)

+ 30 mM NaHC03

Crude extract Desalted

139 ± 2 65.2 ± 1.4

581 ± 8 520 ± 52

Enzyme extract (873 ttl) was assayed with 0.1 M Tricine (PH 7.5) directly (194 ttg protein) or after it had been desalted by passage through a column of Sephadex (169 ttg protein).

Carbon dioxide gas injected into the gas phase of the reaction vessels also stimulated the EFE activity (Fig. 5), but was slightly less effective than HC03-. Moreover HC03- provided a more convenient mode of addition than CO2, and therefore was employed in the remainder of the study.

~700r--~==~..., c: iii e 0.500 OJ E "-C -;300

:~ g UJ 100 u. UJ 0 0'--~5 ~10~15:--:2"7:0......J

[C02odded I (% I

Figure 5. Effect of the CO2 concentration on the activity of the EFE. An enzyme extract (973 ttl containing 409 ttg protein) was assayed in vials where the gas phase was modified by injection of CO2,

atmospheric pressure being maintained by removal of the equivalent volume of air.

37

The present finding that addition of CO2 or HC03- is essential for maximum activity in vitro of the EFE is consistent with the previous observation that the in vivo conversion of ACC to ethylene is modulated by CO2 [9,10], with the non-enzymic, free-radical driven generation of ethylene from ACC [10], and with the doubling of avocado EFE activity in vitro on addition of NaHC03-, when the enzyme was assayed in a modified atmosphere [6].

3.3.1. Enzyme kinetics. As well as stimulating the V max of the EFE, HC03- addition also increased the apparent K". of the enzyme with respect to ACC from about 24 IlM to about 250 IlM, and with respect to oxygen from about 1.4% to about 3.3%. However despite the apparent lowering of the affinity of the enzyme towards its substrates, the rate of the reaction was greater in the presence of HC03- than in its absence over the whole range of substrate concentrations tested. The influence of HC03-/C02 on the apparent Km values for ACC may explain (a) the wide variation in these values that have been reported previously [11] with the enzyme assayed in vivo, when the HC03-/C02 Ievels were not controlled; and (b) the low values reported recently for the EFE extracted from apple [3,4] and avocado [6] when the enzyme was assayed in the absence of added HC03-/C02•

It was shown previously [2] that the reaction time-course of the melon EFE is non-linear. Rubisco is a CO2-activated enzyme that can have a non-linear time-course, and in this case addition of HC03- stabilises enzyme activity [12]. The melon EFE was also severely inactivated if preincubated in the presence of ACC, Fe2+ and ascorbate (Fig. 6), but this catalytic inactivation was not greatly affected by the inclusion of HC03-, despite the striking effect of HC03- on the catalytic turnover of the EFE.

~

£800 "c

'" ~600 0> c E 2

w g LL W

Adcllt,ons dUring 30 min preincubation

Figure 6. Inability of NaHC03 to prevent the inactivation of the EFE observed during an incubation under catalytic conditions. An enzyme extract (923 III containing 378 Ilg protein) was preincubated for 30 min in the presence of ACC and the cofactors indicated. The reaction mixture was completed by addition before assay of ACC, FeS04,

sodium ascorbate and 30 mM NaHC03

as required.

3.3.2. Carbon dioxide or bicarbonate as the activating species. The enzyme Rubisco is activated by CO2, and not HC03- [13]; while Photo system II electron transport is stimulated by HC03- and not CO2 [14]. We have used specific structural analogues to both CO2 and HC03- to help identify the species active with the EFE. The gas carbonyl sulphide (COS) has been used to define the role of CO2, and formate has been used to define the role of HC03- (Smith and John, submitted for publication). CO2 rather than HC03- could be the species that activates EFE since COS inhibits EFE strongly, while formate inhibits EFE weakly. However because of the lack of any CO2-dependent reversibility of the COS inhibition, this conclusion must remain tentative until other, more direct evidence becomes available.

In conclusion our data show that high concentrations of glycerol are a valuable aid to stabilization of the EFE from melon, and in order to maximize the EFE activity in vitro HCOJIC02 must be present.

38

4. Acknowledgements

We are grateful to Drs Brockway, Prescott, Schofield and Ververidis for valuable discussions, NATO for a grant enabling collaboration with ENSA, Toulouse, Drs Dilley and Pech for preprints of papers, and the EC ECLAIR Programme for financial support.

5. References

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

2. Smith, J.J., Ververidis, P. and John, P. (1992) 'Characterization of the ethylene-forming enzyme partially purified from melon', Phytochemistry, 31, 1485-1494.

3. Kuai, J. and Dilley, D.R. (1992) 'Extraction, partial purification and characterization of 1-aminocyclopropane-l-carboxylic acid oxidase from apple (Malus domestica Borkh.) fruit' Postharvest BioI.TechnoI., (in press).

4. Fernandez-Maculet, J.C. and Yang, S.F. (1992) 'Extraction and partial characterization of the ethylene-forming enzyme from apple fruit', Plant Physiol 99, 751-754.

5. Dupille, E., LatcM, A., Roques, C. and J.-C. Pech (1992) 'In vitro stabilisation and purification of the ethylene-forming enzyme from apple fruits' Compte Rendus Acad. Sci., 315,77-84

6. McGarvey, D.G. and Christoffersen, R.E. (1992) 'Characterization and kinetic parameters of ethylene-forming enzyme from avocado', J. BioI. Chern., 267, 5964-5967.

7. Bradford, M.M. (1976) , A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding', Analyt. biochem., 72, 248-254.

8. Gekko, K. and Timasheff, S.N. (1981) 'Mechanism of protein stabilisation by glycerol: Preferential hydration in glycerol-water mixtures' Biochem., 20, 4667-4676.

9. Kao, C.H. and Yang, S.F. (1982) 'Light inhibition of the conversion of 1-aminocyclopropane-l-carboxylic acid to ethylene in leaves is mediated through carbon dioxide', Planta, 155,261-266.

10. McRae, D.G., Coker, J.A., Legge, R.L. and Thompson, J.E. (1983) 'Bicarbonate/COz-facilitated conversion of l-aminocyclopropane-l-carboxylic acid to ethylene in model systems and intact tissues' Plant Physioi. 73, 784-790.

11. Yip, W.-K., Jiao, X.-Z. and Yang, S.F. (1988) 'Dependence of in vivo ethylene production rate on l-aminocyclopropane-l-carboxylic acid and oxygen concentrations', Plant Physiol. 88,553.

12. Laing, W.A., Ogren, W.L. and Hageman, R.H. (1975) 'Bicarbonate stabilization of Ribulose I,S-diphosphate carboxylase', Biochem. 14, 2269-2275.

13. Lorimer, G.H. and Pierce, J. (1989) 'Carbonyl Sulphide: An alternative substrate for but not an activator of Ribulose-l ,S-bisphosphate carboxylase', J. BioI. Chern, 264, 2764-2772.

14. Blubaugh, DJ. and Govindjee. (1986) 'Bicarbonate, not COz, is the species required for the stimulation of Photosystem II electron transport', Biochim. Biophys. Acta, 848, 147-151.

PURIFICATION, CHARACTERIZATION AND SUBCELLULAR LOCALIZATION OF ACC OXIDASE FROM FRUITS.

A. LATCHEt, E. DUPILLEI, C. ROMBALDII,

lC. CLEYET-MAREL2, J.M. LELIEVREI* and lC. PECH' IENSAT. 145 Av. de Muret, 31076 Toulouse France; 2INRAIEN!J'AM, 9, Place Viala, 34060 Montpellier Fran~e; *Present Address: INRA Technologie, 84140 Montfavet France.

ABSTRACT. ACC oxidase from Golden delicious apple fruits has been purified l70-fold to homogeneity in a 5-step procedure and some of its kinetic parameters have been determined. Antibodies raised against a recombinant ACC oxidase from pTOM13 cDNA recognise enzyme of apple fruit with high specificity. The enzyme appears to be non glycosylated. Evidence is given by various methods, including immunocytology, that most of ACC oxidase of ripening fruits was located in the aploplasm.

1. Introduction

The last step in the biosynthesis of ethylene is catalyzed by the ethylene-fonning enzyme (EFE), also called l-aminocyclopropane-l-carboxylic acid oxydase (ACC oxidase). It has long be considered that this enzyme was membrane-bound and required structural integrity [1]. However, it was recently recovered as a soluble enzyme in melons [14], thus opening new perspectives for its characterization and purification. We have previously described a method for stabilizing ACC oxidase in vitro and conditions for its purification [5]. In this paper, We report on its purification to homogeneity, its partial amino acid sequencing and some of its biochemical characteristics. We also raised antibodies which allowed specific recognition on western blotting and on immunocytology.


2.1. EXTRACTION, PURIFICATION AND ASSAY OF ACC OXIDASE

The methods used for the extraction, purification and assay of apple fruit ACC oxidase in vitro were essentially the same as described previously [5] except that they were adapted to larger amount of plant material (1 kg instead of200g). The activity of ACC oxidase in vivo was estimated by incubating disks (1 cm x 2 mm) in 100 mM Tris-HCI buffer (pH 7.0) supplemented with either 0.6 M or 2.5 M sorbitol, 50 f.lM cycloheximide (CH) and 1-mM ACC. After I h, flasks were aerated with air and sealed 30 min before measuring ethylene. Treatment with 2,4,6-trinitrobenzenesulfonic acid (TNBS) was perfonned by

39


40

first incubating disks for 20 min in the presence of 0.6 M sorbitol and 2.5 mM TNBS. Ethylene produced over 20 min was measured after aeration and addition of 1 mM ACC.

2.2. RELEASE OF ACC OXIDASE FROM FRUIT DISKS

~isks of melon fruits (1 cm x 2 mm) were dipped for a short time into a medium consisting in 100 mM Tris-HCl (pH 7.4), 10% glycerol (v/v) and 50 ~M CH and dryblotted. Five disks were incubated in 3 mL of the same medium under gentle shaking for 90 min at 25°C. ACC oxidase activity was measured in supernatant (50 OOOg, 15 min) as described in [2]. PEPC was extracted from whole tissue by grinding disks (1g) in liquid nitrogen and homogenizing in 3 mL of 100 mM Tris-HCI (pH 8.0) containing 10% glycerol (v/v), 5 rnM MgCI2 and 5 mM OTT. PEPC activity was measured on the 50

OOOg supernatants of total extract and bathing medium according to [15].

2.3. PREPARATION OF ANTISERUM

A recombinant ACC oxidase protein was prepared using the pTOM13 clone, in pT7-7 plasmid [8]. The plasmid was cut with Hind IIlIPst I, and the purified insert was treated with Ora I to remove most of the 3' non-coding region. The resulting 1 kb fragment was directly ligated into Sma I-linearized pT7-7, generating the pT7-TOM13 construct which was used for transformation of strain K3 8/pGP 1-2 of E. coli [13]. Protein inclusions were obtained as described by Fritsch et al. [6] and purified by preparative SOS-PAGE. The gel powder containing the polypeptide was injected into rabbits to generate polyclonal antiserum. Antibodies were purified by affinity chromatography according to Harlow and Lane [7].

2.4. ELECTROPHORESIS, IMMUNOBLOTTING , DETECTION OF GLYCOPROTEINS AND SEQUENCING

Proteins were separated by SOS-PAGE on 12% gels and electrophoretically transferred to nitrocellulose membranes. Immunoblotting was performed using alkaline phosphatase (Biorad). Glycoproteins were detected on transfer blots of the purified protein using the biotinylated lectins/immunoperoxidase reagents (Vectatstain ABC). The purified protein was digested with trypsin for 15 h at 37°C in 0.1 M ammonium bicarbonate buffer (pH 8.0) and the peptides released were separated by reverse-phase HPLC. Amino acid sequencing was performed on an Applied Biosystems model 470A peptide sequencer.


3.1. PURIFICATION AND CHARACTERIZATION OF APPLE FRUIT ACC OXIDASE.

One of the major problems encountered during purification of ACC oxidase was the rapid loss of activity. We found that the sequestration of iron by means of 1, 10-phenanthroline resulted in complete stabilization [5]. It became then possible to undertake purification of the enzyme. Figure 1 shows the progress of complete purification in a 5-steps procedure.

41

These include ammonium sulphate precipitation, hydrophobic interaction chromatography, DEAE ion exchange, molecular sieving and anion exchange on Mono Q. At the last step, the protein appeared as a single band on silver staining. It had a specific activity of around 500 pKatals per mg of protein, with almost a 200-fold purification. The protein was recognized as a single band on immunoblotting (Fig. I) of the purified protein (lane 8), but also of the ammonium sulphate precipitate (lane 3) by antibodies raised against the recombinant ACC oxidase.

KDa 1

31.0

21.5

14.4

2 3 4 5 678 Figure 1: Silver-stained SDSPAGE showing the progress of purification and the imrnuno blot analysis of apple fruit ACC oxidase. lane 1, MW markers; lane 2, (NH4hS04 precipitate;

lane 3, imrnunoblot oflane 2; lane 4, Phenyl Sepharose; lane 5, DEAE MemSep; lane 6, Superose 12; lane 7, Mono-Q; lane 8, imrnunoblot of lane 7.

Some of the kinetic and biochemical properties of the enzyme have been determined: optimum activity around pH 7.4 (in Tris-HCI buffer) and 26°C; apparent Kms of 20 11M

for ACC, 0.5 mM for ascorbate and 0.311M for Fe2+ These values are in agreement with those obtained from apple [9] or avocado [10]. The molecular weight of the protein was estimated at around 39kD while predictions made from cDNA sequences gave 35 kD [4], indicating possible post-translational modification of the protein. A potential glycosylation site is present in all sequences derived from various ACC oxidase-related cDNAs and was predicted to be sufficiently exposed by hydrophobic cluster analysis (not shown). However, the protein did not react with a mixture of 7 biotinylated lectins. Furthermore, it was found at sequencing that the asparagine residue of this potential glycosylation site exhibited no N-linked substitution. This peptide was the following: HisLeu-Pro-Ser-Ser-Asn-I1e-Ser-Glu. It corresponded exactly to amino acids 94 to 102 predicted from apple cDNA [4].

3.2. SUBCELLULAR LOCALIZA nON

We have previously suggested that plant cells contain two sites at which ACC can be converted into ethylene: an extracellular site directly accessible to ACC from the apoplasm, and an intracellular site preferably accessible to intracellular ACC. It was also demonstrated that plasmolysis and digestion of cell wall led to the suppression of the extracellular site [2]. Plasmolysis therefore represents a means to discriminate between the two sites. Another method discrimination consists in the use of membrane-impermeant

42

protein-binding reagents which cannot ,cross the plasma membrane at least in short time, like TNBS. Table 1 shows that TNBS greatly inhibits ethylene production of climacteric apple fruit disks kept in isotonic solutions (0.6 M sorbitol) by 68%, while it doesn't cause any additional inhibition when applied to plasmolyzed tissues (2.5 M sorbitol) in which ethylene production is exclusively internal. It appeared that both plasmolysis and TNBS had very little or no inhibitory effect on ethylene production of melon fruit disks at the preclimacteric stage while ethylene synthesis was still low (Fig. 2). At later stages, when fruits entered the climacteric period, TNBS and high osmoticum greatly affected ethylene evolution. The part of ethylene which remained unaffected by these treatments roughly corresponded to the initial basal ethylene production of preclimacteric fruits. We therefore conclude that the sharp increase in ethylene synthesis during ripening of climacteric fruits is probably due to a large extent to the apoplastic ACC oxidase. Similar data were obtained in the case of apple fruits (not shown). We were also able to show that ACC oxidase could be released by bathing of melon disks in the medium used for extraction of the enzyme (Fig. 3). In our experimental conditions, there was no contamination by the cytosolic marker enzyme PEPC. The amount of activity released was increasing during the climacteric and reached more than 20% of the initial activity of tissues at the maximum climacteric. However, these experiments were performed in the absence of CO2, recently discovered as a co-factor of ACC oxidase [12 ], so that the activity of the enzyme being released was probably largely underestimated (Fig. 3).

50 r------------------------,~---------------------, 18

>-15 -';

~'i' 12 <l;, --CII.t:

VI--9 10....1 'as

'x 6

0

U U

3 c:(

0 2 3 4 5 6 7 8

oL-~~~~~~L-~~~~~~~~~~~ o 23456780

Ripening stages Ripening stages

Figure 2: Effect of plasmolysis and TNBS on ethylene production during ripening of melon. A: disks incubated in the presence of 0.6 M sorbitol (e) or 3.0 M sorbitol (0); B: disks pretreated (0) or not (e) with 2.5 mM TNBS.

43

Table I: Inhibition of ethylene fonnation in apple fruit disks by TNBS. Tissues were preincubated or not with 2.5 mM TNBS prior to ethylene measurements.

>--.; ~-c(!

Dl GI-VIS: Ia--c...l ._ c: )(-

0 (.) (.) c(

Conditions

0.6 M sorbitol -TNBS +TNBS

2.5 M sorbitol -TNBS +TNBS

175

150

125

100

75

50

25

0 0 2 3 4 5 6 7

Ripening stages

8 9

ethylene fonnation

nmoles . h- 1 . g-I

15.9 5.1 4.7 4.6

inhibition

%

68 70 71

Figure 3: Changes in activity of ACC oxidase offmit disks measured in vivo (e) and of ACC oxidase released in the bathing medium (0) PEPC activity released in the bathing medium was 0 and 4% of whole tissue at stages 1 and 7 respectively. Each value represents the mean ( ± SD) of thrce independant replicates .

Figure 4. Immunocytolocalization of ACe oxidase in ripe tomato fmits tissues by fluorescence labelling (FITC). A: tissues treated with antibodies raised against the recombinant ACC oxidase; B: tissues stained by calcofluor white.

44

The subcellular localization of the epzyme was further studied by immunocytology in tomato fiuits using antibodies raised against the recombinant ACC oxidase and labelling with secondary tluorescent antibodies. The labelling is extremely important in the cell wall space (Fig. 4A) and co'incides exactely with the staining of cell wall polysaccharides of the same cells by calcotluorwhite (Fig. 4B). A secondary site is present in the cytoplasm but it is hardly visible due to the small volume of this compartment. Further data on immunocytolocalization of ACC oxidase are given by Rombaldi et al. [11].

The presence of large amounts of ACC oxidase protein in the apoplasm IS m contradiction with all cDNA predictions so far published. None of the predicted sequences have a consensus signal for excretion through the reticulum. Alternative excretion pathways may operate in this case. Structure predictions of the protein indicate the presence of a helix hydrophobic domains which may be involved in exportation of the protein or interaction with membrane [3]

4. Acknowledgements

Authors are grateful to Drs D. Grierson and A Hamilton (University of Nottingham, UK) for providing us with pTom13 clone, to G. Borderie (UPS, Toulouse, France) for protein sequencing, to Dr G. Truchet and F. de Billy (CNRSIINRA, Castanet Tolosan, France) for their help in immunocytology, and to Dr P.John (University of Reading, UK) for fruitfull discussions. This work was supported by the Ministere de l'Education Nationale, the EEC (ECLAIR grant AGRE 015) and the NATO (grant 383/88).

5- References

J- Apelbaum, A, J.A., Anderson, J.D., Solomos, T., Lieberman, M. (1981) Some characteristics of the system converting 1- aminocyclopropane-l-carboxylic acid to ethylene. Plant Physio!. 61, 80-84 2- Bouzayen, M, Latche, A, Pech, J.e. (J990) Subcellular localization of the sites of conversion of l-aminocyclopropane-l-carboxylic acid into ethylene in plant cells. Planta 180, 115-180 3- Bouzayen, M., Cooper, W., Barry, C., Zegzouti, H., Hamilton, A, Grierson, D. (1993). EFE multigene family in tomato plants: expression and characterization. In: Cellular and Molecular Aspects of Plant Hormone Ethylene (J.C. Pech, A Latche & C. Balague, eds) Kluwer Acad. Pub., Dordrecht, The Netherlands, pp. 16-81 4- Dong, J.G., Olson, D., Silverstone, A, Yang, S.F. (1992) Sequence ofa cDNA coding for a l-aminocyclopropane-l-carboxylate oxidase homolog from apple fruit. Plant PhysioL 98, 1530-1531 5- Dupille, E., Latche, A., Roques, e., Pech, J.C. (1992) Stabilisation in vitro et purification de I'enzyme formant l'ethylene chez la pomme. C. R. Acad. Sci. Paris 315, 11-84 6- Fritsch, E.F., Maniatis, T., Sambrook, J. (1989) Molecular Cioning : A laboratory manual, 2 nd edn., Cold Spring Harbor Lab., Cold Spring Harbor, NY

45

7- Harlow, E., Lane, D. (1988) Antibodies - A laboratory manual, Cold Spring Harbor Lab, Cold Spring Harbor, NY 8- K6ck, M., Hamilton, AJ., Grierson, D. (1991) cthl, a gene involved in ethylene synthesis in tomato. Plant Mol. BioI. 17, 141-1425 9- Kuai, J., Dilley, D.R. (1992) Extraction, partial purification and characterization of 1-aminocyclopropane-l-carboxylic acid oxidase from apple fruit. Postharvest Biology and Technology 1, 203-211 10- McGarvey, 1., Christoffersen, R.E (1992) Characterization and kinetic parameters of ethylene-forming enzyme from avocado fruit. 1. BioI. Chem. 267, 5964 -5967 11- Rombaldi, C., Petitprez, M., Cleyet-Marel, J.C., Rouge, P. Latche, A., Pech, J.C., Lelievre, 1.M. (1993). Immunocytolocalisation of ACC oxidase in tomato fruits. In: Cellular and Molecular Aspects of Plant Hormone Ethylene (J.C. Pech, A. Latche & C. Balague, eds) Kluwer Acad. Pub., Dordrecht, The Netherlands, pp. 96-97 12- Smith, 1.1., John, P. (1992). Activation of l-aminocyclopropane-l-carboxylate oxidase by bicarbonate/carbon dioxide. Phytochemistry (in press) 13- Tabor, S. (1990) Current Protocols in Molecular Biology (F.A. Ausubel et al.) Green Publishing, Wiley Intersciences NY, ppI6.2.1-16.2.11 14- Ververidis, P., John, P. (1991) Complete recovery in vitro of ethylene-forming enzyme activity. Phytochem. 30, 725-727 15- Vidal, 1., Cavalie, G. (1974) Mise en evidence de formes isofonctionnelles de la PEP carboxylase chez Phasoo/us vulgaris L. Physioi. Veg. 12, 175-188

PURIFICATION AND CHARACTERIZATION OFACC OXIDASE AND ITS EXPRESSION DURING RIPENING IN APPLE FRUIT

David R. Dilley, Jianping Kuai, Loelle Poneleit, Yali Zhu, Yevgenia Pekker, Ian D. Wilson, Douglas M. Burmeister, Christopher Gran, and Alex Bowers Postharvest Physiology Laboratory Horticulture Department Michigan State University E. Lansing, MI 48824

1. ABSTRACT

1-aminocyc1opropane-1-carboxylic acid (ACC) oxidase was purified to apparent homogeneity from ripened apple fruits (Malus domestica Borkh. cv. Golden Delicious). Ascorbate is required as a cosubstrate (I(." = 0.4 mM); L-ascorbic acid-6-palmitate inhibited ACC oxidase competitively (KI = 20JiM) with respect to L-ascorbate. The enzyme is markedly activated by CO2 (KA = 0.65%). Fe2+ is required (KA = 3JiM); C02+ is inhibitory (K; = 3JiM) competitively with respect to Fe2+. ACC oxidase activity was positively correlated with the development of the ethylene climacteric in apples; in vivo or in vitro activity was not detectable in preclimacteric apples. Immunoblots of proteins and of in vitro translation products of poly(A)+ RNA from apples at progressive stages of the ethylene climacteric indicated that ACC oxidase and the mRNA encoding ACC oxidase was nil in unripe fruits but accumulated markedly during the ethylene climacteric.

2. INTRODUCTION

The biosynthetic pathway for ethylene in higher plants has been established (Adams and Yang, 1979; Kende, 1989). The rate limiting step is normally the conversion of Sadenosylmethionine to ACC by ACC synthase (Kende, 1989). ACC is converted to ethylene by ACC oxidase and in some fruits this is the rate-limiting step (Ye and Dilley, 1992). The purification and characterization of ACC oxidase has been problematic until recently. Ververidis and John (1991) extracted ACC oxidase activity from melon fruits and found that Fe2+ and ascorbate were needed to determine enzyme activity in vitro. We have purified ACC oxidase from apples (Kuai and Dilley, 1992) and have characterized many of its properties (Kuai et aI., 1992). McGarvey and Christoffersen (1992) have characterized some of the properties of ACC oxidase from avocado fruits. Smith et al. (1992) reported further purification and characterization of melon ACC oxidase. Dupille et al. (1992) and Fermlndez-Maculet and Yang (1992) and Dong et al. (1992) have also purified apple ACC oxidase to homogeneity. Dong et al. (1992) discovered that CO2 is required for enzyme activity.

46

J. C. Pech et at. (eds.), Cellular and Molecular Aspects of the Plant Honnone Ethylene, 46-52. © 1993 Kluwer Academic Publishers.

47

~,------------------------------. 3. MATERIALS AND METHODS

20 The enzyme purification procedures and other experimental methods are described ~ elsewhere (Kuai and Dilley, 1992; Kuai et al., 1992).

T 15

'" E

jo 10

4. RESULTS AND DISCUSSION "0 E "

4.1 Substrate and Cofactor Dependence

, '" E

30

Zi- 10

"0 E

5

10 o ull M-6-P

2 3 4 Aocorbic acid cone. mM

1 b

o 75 u~ M-6-P

The K", values for ACC (6J,lM) and 02 (0.3%), pH optimum (7.2) and evidence for stereoselectivity for 1R,2S analogs of ACC were reported earlier (Kuai and Dilley, 1992). Enzyme activity was dependent upon ascorbate concentration (Fig. 1a and b). The apparent K", for ascorbate was O.4mM. The ACC oxidase requirement for ascorbic acid is structurally specific. Some ascorbic acid analogs inhibitory to ethylene production with L-ascorbate included in the assay medium at 5 mM were: L-ascorbate-6-palmitate (KI = 20J,lM), dehydroascorbate (KI = 0.68 mM) and L-ascorbate-2-S04

(KI = 1.21 mM). Analogs that substituted for L-ascorbate at 5mM were: Disoascorbate, D-ascorbate, L-isoascorbate, D-erythroascorbate and Mg-ascorbate-6-P04• The most inhibitory analog, ascorbate 6-palmitate, competitively inhibited activity (KI = 20J,lM) with respect to ascorbate (Fig. la, b, c).

~ ~ U ~ ti 'Ml~ '¥ 1~ ~ ~ (Acsoreie acid cone, mMr

Figure la, band c. Effect of ascorbic acid concentration on ACC oxidase activity and inhibition by ascorbic acid-6-palmitate [ascorbate alone (0); plus 0.08mM (D) or O.lmM (l» ascorbate-6-palmitate].

1 c ] 2.0

1.S

t E '"

1.0 a

O.S 20 uM

-0.04 -0:02 0.00 0.02 0.04 0.06 0.08 Aoeoreie aeid-6-palmitate cone, mM

o

0.10 0.12

48 16'-.-------------------,

Dong et a1. (1992b) have found that ascorbate is stoichiometrically converted to dehydroascorbate as ACC is converted to ethylene.

Enzyme activity was dependent 'e upon Fe2+ concentration (KA = 3J.lM) and was competitively inhibited by C02+ (~ = 3#M) as shown in Figure Id, e, and f.

Figure Id, e and f. Effect of Fe2+ concentration on the activity of ACC oxidase and inhibition of C02+ [Fe2+ alone (.); plus 0.08J.lM (.) or 24J.lM (.) C02+].

4.2 Carbon Dioxide Activation of ACC Oxidase

Low levels of CO2 activate ACC oxidase up to 10- to 12-fold (Fig. 2a). Dong et al. (1992) have found similar results and have determined that CO2 is essential for the catalysis of ACC to ethylene. The mechanism of CO2 activation may be similar to the carbamylation mechanism of CO2 activation of Rubisco (Lorimer and Miziorko, 1980). CO2 activation occurs at near ambient CO2 levels in air; the KA = 0.65% v Iv. Preincubating the enzyme with gaseous CO2 prior to assaying activity with or without supplemental CO2 was found to remove a lag-period of about 15 min. until CO2

activation was apparent (Fig. 2b). This lag-period is similar to that observed for the CO2 activation of Rubisco (Lorimer et al., 1976). Delaying the time of addition of CO2 to the reaction mixture resulted in a time-related decrease in the extent of activation (data not shown). We have also observed that an apparently 'dead' enzyme may be partially reactivated by CO2 alone and markedly reactivated with

80

50

E~ 30 :.:

1d

0.1

1e

-200

1f

o 4

0.2 O.J 0.4 fez+ cone, mM

0.5 0.6

12

x 10

, .. ~ 8

i

E 6

o uM eo2+

-lOp' 0 100 (Fe· canc. mM)-'

200 300

D

D

K, = ;3 uM

Ii 12 18 20 Co" cane. uM

24 28

CO2 plus dithiothreitol (data not shown). Order of addition of substrates and cofactors indicated that CO2 is responsible for some

~~--------------------------------,

50

yet proven structural modification ' ... of the enzyme which is essential ~ 40

for the effective oxidative catalysis 'E' of ACC to ethylene (data not z 30

shown). CO2 increases the cS apparent KIn for ACC, ascorbic acid and 02 (data not shown). Our preliminary studies of the mechanism of CO2 activation of ACC oxidase suggest that a carbamylation mechanism is involved.

10

20

o

COz HEADSPACE CONC. "

o o

Figure 2. CO2 activation of ACC 15o.-2- b-------------------------------, + 551 CO2 headspace

oxidase. a) Effect of CO2

concentration on enzyme i at

activity. Inset: double E

reciprocal plot of the data ~ 100 u

yielding a KA = 0.65% ::I e CO2, b) Effect of Co

preincubating ACC z oxidase with 5% CO2, (0) ~ 50

no preincubation; (.) E c:

preincubation of enzyme only with 5% CO2 for 1 hr at O°C.

SIr co. preincubation

49

4.3 Gene Expression of Apple ACC Oxidase

0~~~~~~~3~0-T~~~~~~~60~r-~~· TIME. min

In vivo and in vitro ACC oxidase activity increased markedly (Fig. 3) as the climacteric developed and accumulated extensively as the apples ripened.

50

Figure 3. In vivo and in vitro activity of ACC oxidase of Golden Delicious apples as a function of the internal ethylene concentration in the fruits.

Western blots showed that ACC oxidase was not detected in preclimacteric apples but accumulated as the climacteric developed (Fig. 4). Poly(A)+ RNA from Mutsu apples at six stages of ripening was translated in. vitro using a rabbit reticulocyte system with 3SS-methionine. One major radiolabeled polypeptide with a molecular mass similar to that of ACC oxidase was immunoprecipitated with ACC oxidase PAbs (Fig. 5).

50,.-----------------;-----,-200 3

:~ :2 ~ JO dj ,

~i"/ i 10 ------.- -----.---:o----..!-... ~ ....

o 0_1 10 100

Int. mol E1hyle.,. (ul/I)

o 1000

Figure 4. Western blot analysis of ACC oxidase from Golden Delicious apple fruit during the development of the ethylene climacteric. a) Lane 1, 0.2J-d1-1; lane 2, 6J-d1-1; lane 3, 100J-d1-1; lane 4, 300JAI-1; lane 5, 500J-d 1-1• 5J.1.g of protein was loaded on each lane. Lanes 6, 7 and 8 as lane 5 plus 10, 25 or 50 ng of ACC oxidase, respectively.

K.O

94 67

43

30

20.1 -

14.4 -

1 2 3 4 5 6 7 8

Figure 5. Immunoprecipitation of in vitro translated proteins of poly(A) + RNA extracted from cortical tissue of Mutsu apple fruits at progressive stages of the ethylene climacteric. Lane 1, 0.1JJ1 1.1; lane 2, 1.3JJ1 1.1; lane 3, 10JJ1 1.1;

lane 4, 122J.ll 1.1; lane 5, 308JJ1 1-1; lane 6, 607 JJ11-1•

Lane 7, 8 and 9 were the same as lane 6 except that 1 ng, 10 ng, 100 ng of pure ACC oxidase was included in the immunoprecipitation solution, respectively.

1 2 :3 4

51

5 678 9

The intensity of the radiolabeled polypeptide decreased when pure ACC oxidase purified from two-D gel was included in the immunoprecipitation reaction (lanes 7, 8, 9), indicating that pure ACC oxidase and the radiolabeled polypeptide competed for ACC oxidase antibody. We conclude that the precipitated radiolabeled polypeptide is ACC oxidase. The in vitro translated ACC oxidase increased as the ethylene climacteric developed (Fig. 5), indicating that the level of the translatable mRNA for ACC oxidase increased during ripening.

52

S. LITERATURE CITED Adams, D.O. and Yang, S.F. 1979. Ethylene biosynthesis: Identification of 1-

aminocyc1opropane-1-carboxylic acid as an intermediate in the conversion of methionine to ethylene. Proc. NatI. Acad. Sci., USA 76:170-174.

Dong, J.G., Fernandez-Maculet, J.e. and Yang, S.F. 1992. Purification and characterization of 1-aminocyc1opropane-1-carboxylate oxidase from ripe apple fruit. Proc. NatI. Acad. Sci. USA (in press).

Dupille, E., Latche, A., Rogues, C. and Pech, J.C. 1992. In vitro stabilization and purification of the ethylene-forming enzyme from apple fruit. C. R. Acad Sci., Paris. 315:77-84

Fernandez-Maculet, J.e. and Yang, S.F. 1992. Extraction and partial characterization of the ethylene-forming enzyme from apple fruit. Plant PhysioI99:751-754.

Kende, H. 1989. Enzymes of ethylene biosynthesis. Plant PhysioI. 91:1-4. Kuai, J. and Dilley, D.R. 1992. Extraction, partial purification and characterization of 1-

aminocyc1opropane-1-carboxylic acid oxidase from apple fruit. Postharvest BioI. and Tech. 1:203-211.

Kuai, J., Zhu, Y., Pekker, Y. and Dilley, D.R 1992. Purification and characterization of 1-aminocyc1opropane-1-carboxylate oxidase from apple fruits. Plant PhysioI. (In press).

Lorimer, G.H., Badger, M.R and Andrews, T.J. 1976. The activation of ribulose-1,5-bisphosphate carboxylase by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested mechanism, and physiological implications. Biochemistry 15:529-536.

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 Mi+. Biochemistry 19:5321-5328.

McGarvey, D.J. and Christoffersen, RE. 1992. Characterization and kinetic parameters of ethylene-forming enzyme from avocado fruit. J. BioI. Chern. 267:5964-5967.

Smith, J.R., Ververidis, P. and John, P. 1992. Characterization of the ethylene-forming enzyme partially purified from melon. Phytochem 31:1485-1494.

Ververidis, P. and John, P. 1991. Complete recovery in vitro of ethylene-forming enzyme activity. Phytochem.30:725-727.

Ye, W. and Dilley, D.R 1992. Development of ACC oxidase activity during maturation and ripening of 'Paulared', 'Empire', and 'Law Rome' apples. Postharvest BioI. and Tech. 1:195-202.

MECHANISTIC ASPECTS OF ACC OXIDATION TO ETHYLENE

M. ACOSTA1, M.B. ARNA01, J. SANCHEZ-BRAVO 1 , J.L. CASAS2, B. VIOQUE3, J.C. FERNANDEZ-MACULET3, J.M. CASTELLAN03. IDepartment of Plant Biology. University of Murcia. Santo Cristo, 1. E-30001 MURCIA (SPAIN). 2Department of Environmental Sciences and Natural Resources. University of A1icante. P.O.Box 99. E-03080 ALICANTE (SPAIN). 3Instituto de 1a Grasa y sus Derivados (C.S.I.C.). P.O.Box 1078. SEVILLA (SPAIN).

ABSTRACT. The two-electron oxidation of l-aminocyclopropane-l-carboxylic acid (ACC) to ethylene may proceed through a concerted or stepwise mechanism, this latter being that probably occurring in vivo. An essential feature of this reaction is the production of ACC-free radicals as intermediate. If these ACC-free radicals are liberated into the reaction medium, they will readily react with dissolved oxygen resulting in the formation of an ACC-derived hydroperoxide. Subsequently to the hydroperoxide formation, a decrease in the yield of ethylene and the appearance of other compounds, such as 3-hydroxypropylamide (HPA) , might occur. HPA has been found not only in stepwise-type reactions, such as those mediated by peroxidase, but also in concerted reactions, such as the oxidation by hypochlorite or hydrogen peroxide. In this work we investigate the mechanism of ACC oxidation by the two kinds of reactions and discuss some physiological implications.

1. Introduction

The ethylene (C2H4) formation from ACC involves a twoelectron (e-) oxidation. This process might be:

a) Concerted:

b) Stepwise: ACC -----> ACC· -----> ACC+ e

It seems that in vivo the reaction proceeds in a stepwise manner. When dideuterated ACC is supplied to apple slices, an equal mixture of cis- and trans-dideuteroethy1ene is obtained, indicating that the reaction proceeds with a loss of estereochemistry. However, the oxidation with sodium

53

1. C. Pech et al. (eds.), Cellular and Molecular Aspects ojthe Plant Hormone Ethylene, 53-58. © 1993 Kluwer Academic Publishers.

54

hypochlorite follows a concerted mechanism, giving cisdideuteroethylene with retention of configuration (Adlington, et al., 1983, Pirrung, 1983, McKeon and Yang, 1987).

The aim of this work was to investigate the mechanism of ACC oxidation to ethylene. This was carried out by using an in vitro system to produce ACC-free radicals and analyzing their final fate and the factors affecting the yield of ethylene. Because it is known that peroxidase produces the uni-electronic oxidation of its substrate, we use this enzymatic system to assess the production of ACC free radicals.


Purified horseradish peroxidase (HRP) type VI, and ACC were obtained from Sigma. Hydrogen peroxide (30%, v/v) was from Merck. Compound I of peroxidase was prepared immediately before each experiment by mixing equimolecular amounts of HRP and hydrogen peroxide (Acosta et al., 1991a, 1991b). Oxygen consumption was measured in an oxygen monitor equipped with a Clark electrode. The typical reaction media were made in 50 mM glycine-HCl buffer (pH 4.5). Before the start of the reaction, media were saturated with air by bubbling. Temperature was maintained at 30°C. Ethylene was determined by gas chromatography. The identification of the final reaction products using labelled ACC was performed by mass spectrometry and nuclear magnetic resonance (NMR) as previously described (Vioque and Fernandez-Maculet, 1990).


The conditions under which ACC functions as a substrate of peroxidase (HRP) were investigated by following the oxygen consumption in the reaction medium (Acosta et al., 1991a). The oxygen consumption occurred only when both Mn2+ and H202 were present, though H202 can be substi tuted by the active form of the enzyme, namely, Compound I of peroxidase. The oxygen consumption was dependent on enzyme concentration, the optimum ACC/Mn2+ ratio being 1/1. The addition of free radical scavengers (n-propyl gallate, salicylic acid) to the reaction medium led to inhibition of the oxygen consumption, suggesting that the reaction proceeds with a radical intermediate. In this system, ethylene production from ACC can be measured but as a very minor product of the reaction (less than 1% of the consumed ACC).

Mass spectrometry and NMR analysis of the final products of the reaction using [2,3- l4C] as a tracer, revealed the presence of 3-hydroxypropylamide as the major product of the ACC oxidation (Vioque and Fernandez-Maculet, 1990).

55

These results showed that ACC is a substrate of peroxidase, in a reaction that can be depicted as in Scheme I. In the Scheme it can be observed the two al ternati ves routes: one towards ethylene formation and the other towards the ACC hydroperoxide (ACCOOH). This hydroperoxide can react with peroxidase yielding the corresponding alcohol (HIBA). Both compounds (the hydroperoxide and the -imino-carboxylic acid)

ACC ACC Elh7 1• n •

H,N COOH • - NH, - NH

A -----<'--> '~COOH ~> '~COOH£> H.C=CH •• Ne-COOH L...->. H

ACC' Ace

E < __ '\.-,,"-~/<L- Compound I I

ACCOO NH. ~Ace' '00... JI.. ~ 'eOOH

AeeOOH~

AeeOH~ / (ACC

Compound I z·

Mn

ACC' + O. ----, ACeOO' ~: ACCOOH Un

. NH. HRP NH.).O z NH.

HOn.. JI.. -'\-' Ho... JI.. ~, He... ! ~ 'COOH Co-I ~ 'eOOH ~

ACCOOH HIBA HPA

Scheme I: Proposed mechanism for ethylene and HPA formation. Inset: Peroxidative cycle using ACC as substrate showing the formation of ACC-hydroperoxides. (Additional abbreviations used: HIBA: 4-hydroxy-2-imine-n-butyric acid).

are unstable and through oxidative produce 3-hydroxypropyl-amide (HPA) as product.

Therefore, a global reaction could follows:

°2

decarboxylation can the final reaction

be formulated as

ACC' ------> ACCOOH ------> HPA + C02 H+

The detection of HPA can be useful to check the stability of ACC solutions (i. e. the ACC autooxidation). A 2 mM ACC solution stored 3 days at room temperature may yield up to 10% HPA, depending on the pH. In addition, the concerted mechanism can also show variable percentages of HPA (Table 1), suggesting the possibility of side reactions affecting the yield of ethylene. In this sense, mechanisms favouring the attack of H202 at very basic pH, or including pyridoxal phosphate (PLP) might be taken into account (Mazelis and Ingraham, 1962, Smith and Marshall, 1988).

56

Some remarks should be noted about the physiological interest of these reactions. The low yield of ethylene given by the oxidation of ACC catalyzed by peroxidase allows us to conclude that a weak relationship exists between this

Table 1. Percentage of different compounds in the reaction medium after oxidation of ACC in the contidions of (A) Lizada & Yang (1979) and (B) Boller et al. (1979).

[ACCh C2 H4 HPA Others

(A) NaOCljHg 2+ 4.2% 82.4% 10.2% 3.2%

(B) PLPjMn2+ 26.0% 62.0% 12.0% H20 2

Initial ACC= 5 nmoles; [ACCh= remaining ACC

enzymatic system and the ethylene-forming enzyme recently found in vivo (Smith et al., 1998). However, the effective breakdown of ACC and the formation of HPA suggest a possible role of this reaction in ACC catabolism. At least an analogy with indolyl-3-acetic acid oxidation catalyzed by peroxidase can be established if we compare the structures of the final reaction products:

H I

H-C-CH OH I 2

C~ H-N/ '-':0

I H

3-Hydroxy-propylamide (HPA) 3-Hydroxymethyl-oxindole

Where might this reactions take place? Probably in those cellular spaces where an interaction between ACC and peroxidase in vivo could exist, e.g., apoplast, vacuole or in the xylem sap (ACC can move from roots to shoots in some conditions). Nonetheless, the answer will only be obtained when HPA is found as an endogenous product in plants.

On the other hand, some observations can be made about the implications of the in vivo mechanism of ACC conversion to ethylene. Thus, if the process must be stepwise, our results indicate that ACC' can not leave the active center of the enzyme. In such a case, the model proposed by Baldwin et al. (1985) involving a transition metal has to be considered. Models can be proposed (Scheme II) with oxygen as a final electron acceptor allowing the conservation of the metal

57

valence in the catalytic center (A). In addition, other alternatives implying the participation of ascorbic acid (AscH) can be proposed (B)(C). The production of H202 coupled to the release of ethylene, in some of these models, could have a new physiological interest (McGarvey and Christoffersen, 1992).

(B)

0; - 0=. 0.-I· I I

E '\ AlcH -> E As" --> E

'-ACC' '-ACC·./'. - '-ACC· AscH

i 0. 0. 0;

j (A)

I .,.E ,"

I 1-__ > E \e I

-->E --> C,H •• H,O,

(C)

Scheme II: oxidation.

'-Acc '-ACC' '-ACC·

- + 2e • 2H

E + ACCOOH

Possible involvement of ascorbate in ACC

Vegegative or young tissues with a higher level of ascorbate peroxidase can detoxify the cytoplasmic increase in H202 with a high efficiency (Kevers et aI., 1992). Thus, these tissues would behave in a different way with respect to those senescent tissues with lower levels of ascorbate peroxidase. The confimation of one or other mechanism in a future will open interesting physiological perspectives.

4. Acknowledgements

This work has been supported by Comision Interministerial de Ciencia y Tecnologia (CICYT). Project ALI89-0293.

5. References

Acosta, M., Casas, J.L., Arnao, M.B. and Sabater, F. (1991a) 'l-Aminocyclopropane-l-carboxylic acid as a substrate of peroxidase: condi tions for oxygen consumption, hydroperoxide generation and ethylene production' , Biochim. Biophys. Acta 1077, 273-280.

Acosta, M., Arnao, M.B., Casas, J.L., Del Rio, J.A., Vioque, B. and Fernandez-Maculet, J.C. (1991b) 'Mechanism of ACC

58

oxidation by peroxidase', in J. Lobarzewski, H. Greppin, C. Penel and Th. Gaspar (eds), Biochemical, Molecular and Physiological Aspects of Plant Peroxidases, University of Geneva (Switzerland), 88, 121-124.

Adlington, R.M., Baldwin, J.E. and Rawlings, B.J. (1983) 'On the stereochemistry of ethylene biosynthesis', J. Chem. Soc., Chem. Commun. 290-292.

Baldwin, J.E., Jackson, D.A., Adlington, R.A. and Rawlings, B.J. (1985) 'The stereochemistry of oxidation of 1-aminocyclopropane-l-carboxylic acid' J. Chem. Soc., Chem. Commun. 206-207.

Boller, T., Herner, R.C. and Kende, enzymatic formation of an aminocyclopropane-l-carboxylic 303.

H. (1979) 'Assay for and ethylene precursor 1-

acid' ,. Planta 145, 293-

Kevers, C., Goldberg, R., Van den Driessche, T. and Gaspar, Th. (1992) 'A relationship between ascorbate peroxidase activi ty and the conversion of l-aminocyclopropane-lcarboxylic acid into ethylene' J. Plant Physiol. 139, 379-381.

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

Mazelis, M. and Ingraham, L.L. (1962) 'The pyridoxal phosphate-dependent oxidative decarboxylation of methionine by peroxidase', J. BioI. Chem. 237, 109-112.

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

McKeon, T.A. and Yang, S.F. (1987) 'Biosynthesis and metabolism of ethylene', in P.J. Davies (ed.), Plant Hormones and Their Role in Plant Growth and Development, Kluwer Academic Publishers, Dordrecht, pp. 94-112.

Pirrung, M.C. (1983) 'Ethylene biosynthesis. 2. Stereochemistry of ripening, stress, and model reactions', J. Am. Chem. Soc. 105, 7207-7209.

Smith, T.A. and Marshall, J.H.A. (1988) 'Oxidation of aminoacids by manganous ions and pyridoxal phosphate', Phytochyemistry 27, 1611-1613.

Smith, J.J., Ververidis, P. and John, P. (1992) 'Characterization of the ethylene-forming enzyme partially purified from melon', Phytochemistry 31-1485-1494.

Vioque, B. and Fernandez-Maculet, J.C. (1990) '3-Hydroxypropylamide formation from 1-aminocyc1opropane-lcarboxylic acid by a cell-free ethylene forming system from olive leaves', Physiol. Plant. 79, 487-490.

APPLE ACC OXIDASE: PURIFICATION AND CHARACTERIZATION OF THE ENZYME AND CLONING OF ITS eDNA

S.F. YANG, J.G. DONG, J.C. FERNANDFZ-MACULET and D.C. OLSON Dept. Vegetable Crops, University of California Davis, CA 95616, U.SA.

ABSTRACT. ACC oxidase was isolated and purified from ripe apple fruit to an apparent homogeneity with a specific activity of 20 nmoVmg-min. This purified enzyme migrated as a simple band of 35 kDa on SDS-PAGE and of 39 kDa on gel filtration. The enzyme activity, as in vivo, required CO:z/HC03-; CO2 being the active species. The purified enzyme displayed absolute requirement for Fe2+ and ascorbate. The stoichiometry of the reaction was detennined to be: ACC + ascorbate + 02 ~ C2I4 + HCN + dehydroascorbate + C02. We have recently isolated an apple cDNA, pAE12, which is homologous to tomato ACC oxidase cDNA. The purified enzyme was recognized on Western blot by a polyclonal antibody raised against a synthetic peptide whose sequence was derived from pAE12. When the purified enzyme was cleaved with CNBr and one of the fragments was sequenced, its sequence matched that predicted from pAE12. When preclimacteric apple fruit was treated with ethylene, parallel increases in in vitro ACC oxidase activity, in vivo ACC oxidase activity and the level of pAE12 transcript were observed. These observations support the conclusion that the isolated ACC oxidase protein is encoded by pAE12.

1. Introduction

The final step in the biosynthesis of ethylene is catalyzed by ACC oxidase (also known as ethylene-forming enzyme). Although ACC oxidase activity is readily demonstrated in vivo by supplying tissues with ACC, isolation of authentic ACC oxidase in cell-free preparation encountered many difficulties. Only very recently important advances have been achieved in the molecular biology and isolation of ACC oxidase. Hamilton et al. (6) suggested that a tomato gene, pTOM13, encodes ACC oxidase. Later work conf'mned that pTOM13 confers ACC oxidase activity when expressed in yeast (5) or Xenopus oocytes (15). Ververidis and John (17) were the first to report that authentic ACC oxidase activity can be demonstrated from melon fruit when the enzyme was assayed in the presence of Fe2+ and ascorbate. Since then ACC oxidase has been isolated from apple (4, 8) and avocado fruit (12) but not purified.

In this work we describe the purification of ACC oxidase to near homogeneity, demonstrate its requirement for CO2, detennine the stoichiometry of the enzymic reaction, and present data showing that ACC oxidase is encoded by pAE12, an apple cDNA homologous to tomato ACC oxidase gene (3).

59


60


2.1. ACC OXIDASE ASSAY

The standard reaction mixture contained in a volume of 0.5 mL, 100 mM MOPS (PH 7.2), 20 J1M FeS04, 1 mM sodium ascorbate, 4% CO2 (in the gas phase), 10% glycerol and 10-50 ~ enzyme preparation. After incubation for 20 min at 30·C, a gas sample was taken for ethylene determination.

2.2. ANALYSIS OF REACTANTS AND PRODUCTS

Ethylene CO2 and 02 were determined with a gas chromatograph, an infrared analyzer and an oxygen electrode, respectively. ACC and dehydroascorbate w~ measured as previously described (9, 16). The HCN produced in the reaction was adsorbed onto a filter paper wetted with 1 N NaOH and colorimetrically determined (18).

2.3. ENZYME PURIFICATION

Apple fruit was homogeneized in 400 mM phosphate buffer (pH 7.2) containing 10 mM sodium bisulfite, 4 mM 2-mercaptoethanol and 3 mM ascorbate. After centrifugation at 28,000 g for 30 min, the pellets were resuspended in buffer A (25 mM MOPS, pH 7.2, 1 mM OTI, 3 mM ascorbate, 30% glycerol). Triton X-l00 was added to a final concentration of 0.1 % and the resulting supernatant was loaded onto a OEAE-Sepharose column and the enzyme was eluted with 100 mM ammonium sulfate in buffer A. The ACC oxidase-containing fractions were subsequently loaded onto a phenyl-Sepharose column. ACC oxidase was then eluted with 20 mM ammonium sulfate in buffer A and furtherly chromatographed on a Sephadex G-150 column.

2.4. PEPTIDE SYNTHESIS AND PRODUCTION OF POLYCWNAL ANTISERUM

A 14-residue oligopeptide corresponding to residues 104 to 116 of the deduced amino acid sequence ofpAE12 (3) plus a cystein residue at the C-terminus was conjugated to BSA via the SH group of the cystein residue. A rabbit was immunized subcutaneously with 100 J1g of the conjugated peptide in the presence of complete Freund's adjuvant. After two boosters, the rabbit blood was collected and the antiserum prepared by routine procedures.

2.5. IMMUNO-BLOTTING

After SOS-PAGE, protein preparations were transferred onto a nitrocellulose filter. Following incubation with fetal bovine serum, a solution of the antiserum was added, and the blots were detected by the peroxidase reaction.

2.6. PEPTIDE SEQUENCING

A SOS-PAGE purified ACC oxidase was degraded with CNBr in 70% formic acid. Total digests were resolved by SOS-PAGE and transferred onto a Problot nylon filter. One of the peptide fragments was excised and microsequenced by Edman degradation.

2.7. RNA BLOTTING AND HYBRIDIZATION

61

Total RNA was isolated from apple fruit as previously described (2) and spotted onto ZetaProbe membranes. The membranes were heated in a vacuum oven and hybridized to radiolabelled pAE12 cDNA by routine procedures.


3.l. PURIFICATION OF ACC OXIDASE AND ITS RELATIONSHIP WITH pAE12

ACC oxidase was purified 180-fold with 37% recovery from apple fruit following a 3-step chromatography (Table 1). The enzyme activity was associated with the pellet fraction when the fruit was homogeneized. After the resuspension of the pellet in 25 mM MOPS buffer (pH 7.2) with 0.1% Triton X-100, the enzyme becomes soluble but unstable. Hence, it is important to include glycerol (30%, v/v) in the preparation and to remove the detergent as soon as feasible. This was achieved by passing the solubilized enzyme through a DEAE-Sepharose column, which also afforded a 39-fold purification. The resulting enzyme preparation became stable and was furtherly purified by hydrophobic interaction chromatography and by molecular sizing. The final preparation had a specific activity of 20 nmoVmg-min. When the enzyme preparation at different purification steps was subjected to SDS-PAGE analysis, a 35-kDa polypeptide was enriched and at the final stage of purification only a single band was detected. The molecular size of the native protein was determined to be 39 kDa by Superose-FPLC gel flltration, indicating that apple ACC oxidase exists in a monomeric form.

Table 1. Purification of ACC oxidase from 10 kg mesocarp tissue of ripe apple fruit.

Purification Total Total Specific Recovery Purification step activity protein activity

(units*) (mg) (units/mg) (%) (fold)

Pellet fraction 2,122 18,200 0.11 100

DEAE-Sepharose 1,307 303 4.31 61 39

Phenyl-Sepharose 956 166 5.76 45 52

Sephadex G-150 793 40 19.8 37 180

*One unit is defmed as that which converts 1 nmol of ACC to ethylene per min at 30·C.

We have recently cloned an apple cDNA, pAE12, which is highly homologous to tomato ACC oxidase cDNAs (3). In order to determine whether the ACC oxidase purified in this investigation is encoded by pAE12, we prepared a polyclonal antibody against a synthetic oligopeptide whose sequence (PDLEEEYRKTMKE) was deduced from pAE12. On Western blots, the 35-kDa polypeptide was detected by this antibody, indicating that the isolated enzyme protein has antigenic determinants in common with the antigen encoded by pAE12. Furthermore, the amino acid composition of the purified enzyme agreed reasona-bly well to that predicted from the pAE12 sequence. To obtain direct evidence of the identity of ACC oxidase to the pAE12 product, the purified enzyme was subjected to CNBr cleavage and the peptide fragments were separated by SDS-PAGE. A predominant peptide fragment with a molecular weight of 12 kDa was isolated and

62

sequenced for 20 cycles; the amino acid sequence (KEFAVELEKLAEKLLDLLCE) precisely matched that predicted from pAE12 (residues 115 to 134).

3.2. ACTN ATION OF ACC OXIDASE BY CARBON DIOXIDE

In addition to substrates ACC and 02, the enzyme displayed absolute requirement for Fe2+, ascorbate and C02 for its activity. As in vivo (7), ACC oxidase activity increased with increasing C02 concentration to a maximum at 4% CO2 in the gas phase (Fig. 1); at this C02 concentration the activity was more than 10 times higher than that in the ambient air (0.03% C02). When NaHC03 was substituted in the reaction mixture for C02, the enzyme activity was similarly stimulated; bicarbonate above 10 mM gave maximal activity.

50

40

:a 30 !§ '-'

g 20 0

;>..

~ 10

0 0 2 3 4

CO2 (%)

Figure. 1. Dependence of ACC oxidase activity on C~ concentration. The reaction tubes were preincubated for 15 min under various concentrations of atmospheric C02 as indicated, and the reaction was initiated by the injection of the enzyme preparation. The reaction was carried out according to the standard assay described in "Materials and Methods" except that the concentration of MOPS was increased to 200 mM so that variation in the pH caused by C02 can be minimized.

In order to determine which species, CO2 or bicarbonate, serves as the activator, the enzyme reaction was carried out at low temperature (5°C) in the presence of either CO2 or HC03- plus/minus carbonic anhydrase. Our results indicate that CO2 is the active species.

In Rubisco C02 has been shown to activate the enzyme by forming a carbamate with the E

amino group of a lysyl residue of the enzyme, while decarbamation causes inactivation of the enzyme (10, 11). We are currently investigating whether the activation of ACC oxidase by C02 involves a similar reaction.

3.3. STOCHIOMETRY

Peiser et al. (13) have established that ACC is metabolized in vivo to equivalent amounts of ethylene (from C-2, 3), HCN (from C-l) and C02 (from the carboxyl carbon). In this study using the purified enzyme preparation, we found that for each mol of ACC oxidized,

63

one mol of 02 was consumed and equimolar amounts of dehydroascorbate, ethylene, HCN and C02 were formed. The stoichiometry of the reaction can be formulated as follows (AH2 = ascorbate; A = dehydroascorbate):

Fe2+, CO2

ACC+02 +AH2 ) C214+C~ +HCN +A+2H20

These results show that, as in vivo, ACC oxidase in vitro catalyzes the oxidation of ACC to ethylene, C~ and HCN and are in agreement with the notion that the enzymic reaction catalyzed by ACC oxidase may proceed via N-hydroxylation. Since it does not require 2-oxoglutarate (1, 14), ACC oxidase can be refered to as ACC N-monooxygenase with ascorbate as a donor. It is to be noted that the purified ACC oxidase has a low specific activity (20 nmol/mg-min). It is possible that it may' require other as yet unidentified cofactors for maximal activity.

3.4. INDUCTION OF ACC OXIDASE AND pAE12 TRANSCRIPTS BY ETHYLENE IN PRECLIMACTERIC APPLE FRUIT

When preclimacteric apple fruit was treated with exogenous ethylene (70 J.LLIL) for 0, 12, 24 and 48 hours, its in vivo ACC oxidase activity increased with increasing duration of the treatment, and this increase was accompanied by a parallel increase in extractable ACC oxidase activity and in the level of the pAE12 transcript (Fig. 2).

20 ~ in vivo enzyme activity • in vitro enzyme activity 0 RNA 100

,.... ,.... 16 ~ ..c:

~ 'r;]

g '-' .8 ~ 12 75 G) .> > .= ·i ~ ~ j '-'

8 50 5 >< :a 0 u

~ u < 4 25

0 0 12 24 48

Time (h)

Figure 2. Development of in vivo and in vitro ACC oxidase activities and accumulation of RNA hybridizing to pAE12 in preclimacteric ,,~le fruit treated with 70 ~ ethylene for various periods. Total RNA (10 ~ per slot) was hybridized to P-labelJed pAE12 cDNA. Hybridizable RNA was determined by densitometric scanning after exposing the blots to Kodak X-my film.

64

4. References

1. Dong, J.G., Fernandez-Maculet, J.C. and Yang, S.F. (1992) 'Purification and characterization of ACC oxidase from apple fruit', Proc. Nad. Acad. Sci. USA (in press). 2. Dong, J.G., Kim, W.T., Yip, W.K., Thompson, G.A., Li, L., Bennett, A.B.and Yang, S.F. (1991), 'Cloning of a cDNA encoding ACC synthase and expression of its mRNA in ripening apple fruit', Planta 185, 38-45. 3. Dong, J.G., Olson, D, Silverstone, A. and Yang, S.F. (1992) 'Sequence of a cDNA coding of an ACC oxidase homolog from apple fruit', Plant Physiol. 98, 1530-1531. 4. Fernandez-Maculet, J.C. and Yang, S.F. (1992) 'Extraction and partial characterization of the ethylene-forming enzyme from apple fruit', Plant Physiol. 99, 751-754. 5. Hamilton, A.J., 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. 6. 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. 7. Kao, C.H. and Yang, S.F. (1982) 'Light inhibition of the conversion of ACC to ethylene in leaves is mediated through carbon dioxide', Planta 155,261-266. 8. Kuai, J. and Dilley, D.R. (1992) 'Extraction, partial purification and characterization of ACC oxidase from apple fruit', Post. BioI. Technol. 1,203-211. 9. Lizada, M.C. and Yang, S.F. (1979) 'A simple and sensitive assay for 1-aminocyclopropane-l-carboxylic acid', Anal. Biochem. 100, 140-145. 10. Lorimer, G.H., Badger, M.R. and Andrews, T.J. (1976) 'The activation of Rubisco by carbon dioxide and magnesium ions. Equilibria, kinetics, a suggested mechanism, and physiological implications', Biochemistry 15, 529-536. 11. 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 C02 and Mg2+', Biochemistry 19,5328-5334. 12. McGarvey, D.J. and Christoffersen, R.E. (1992) 'Characterization and kinetic parameters of ethylene-forming enzyme from avocado fruit' , J. BioI. Chem. 267, 5964-5967. 13. Peiser, G.D., Wang, T.T., Hoffman, N.E., Yang, S.F., Liu, H.W. and Walsh, C.T. (1984) 'Formation of cyanide from carbon 1 of ACC during its conversion to ethylene', Proc. Nad. Acad. Sci. USA 81, 3059-3063. 14. Smith, J.J., Ververidis, P. and John, P. (1992) 'Characterization of the ethyleneforming enzyme partially purified from melon', Phytochemistry 31,1485-1494. 15. Spanu, P., Reinhardt, D. and Boller, T. (1991) 'Analysis and cloning of the ethyleneforming enzyme from tomato by functional expression of its mRNA in Xenopus laevis oocytes', EMBO J. 10, 2007-2013. 16. Terada, M., Watanabe, Y., Kunimoto, M., Hayashi, E. (1976) 'Differential rapid analysis of ascorbic-2-sulfate by dinitrophenylhydrazine method', Anal. Biochem. 84, 604-608. 17. Ververidis, P. and John, P. (1991) 'Complete recovery in vitro of ethylene-forming enzyme activity', Phytochemistry 30, 725-727. 18. Yip, W.K. and Yang, S.P. (1988) 'Cyanide metabolism in relation to ethylene production in plant tissues', Plant PhysioI. 88,473-476.

BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ETHYLENE FORMING ENZYME FROM AVOCADO.

R.E. CHRISTOFFERSEN, DJ. MCGARVEY1, P. SAVARESE2 Department of Biological Sciences University of California Santa Barbara, CA 93106 U8.A.

ABSTRACT. Ethy1ene-fonning enzyme (EFE) catalyzes the final step in biosynthesis of the phytohonnone ethylene in higher plants, the oxidation of 1-aminocyclopropane-1-carboxy1ic acid (ACC). EFE extracted from avocado fruit was partially purified and shown to be a soluble, Fe(Il) ascorbate-dependent enzyme. A ripening-related cDNA from avocado, pA VOe3, shows sequence similarity to several Fe(ll) and ascorbate-dependent oxidases including EFE from tomato. The A VOe3 antigen was shown to be a soluble protein suggesting its identity with the EFE enzyme. The A VOe3 protein was overexpressed in E. coli with the recovery of EFE activity from the cell extracts. Using residue-specific chemical modification, a histidine critical for catalytic activity was detected. This is likely to be one of three histidine residues highly conserved among sequence-related Fe(ll) ascorbate-dependent oxidases.

1. Introduction

Ethylene plays a critical role in regulating the growth and development of plants. This simple gaseous plant growth regulator has been implicated in senescence, abscission, growth, rooting, fruit ripening, and many other aspects of plant development. As would be expected for a potent signal molecule, biosynthesis of ethylene is highly regulated during the plant life cycle. Manipulation of the ethylene biosynthetic pathway can have dramatic effects on plant development as shown by antisense experiments in tomato using cDNAs for the ethylene biosynthetic enzymes, ACC synthase (Oeller et al. 1991) and ethylene fonning enzyme (Hamilton et al. 1990).

The pathway of ethylene biosynthesis is from S-adenosylmethionine ~ 1-aminocyclopropane-1-carboxylic acid (ACC) ~ ethylene (Yang and Hoffinan 1984; McKeon and Yang 1987; Kende 1989). The key enzymes involved are ACC synthase and ethylene fonning enzyme (EFE or ACC oxidase), respectively. While ACC synthase has been successfully studied in vitro for some time, the EFE enzyme has been very difficult to characterize due to its instability or

Ipresent Address: Institute of Biological Chemistry, Washington State University, Pullman, WA 99164 U.S.A.

2Permanent Address: Universita Tuscia, Istituto di TEcnologie Agroalimentari, Via Lellis, 1-01100, Viterbo, Italy

65

J. C. Pech et al. (eds.), Cellular and Molecular Aspects o/the Plant Hormone Ethylene, 65-70. © 1993 Kluwer Academic Publishers.

66

inactivity on homogenization. Only recently has EFE become amenable to in vitro biochemical study. The observation that pTOM13 antisense tomato fruit have suppressed levels ofEFE was an key element in the demonstration of the identity between EFE and pTOM13-related polypeptides. Derivatives of the pTOM13 cDNA were subsequently expressed in both Xenopus eggs (Spanu et al. 1991) and yeast (Hamilton et al. 1991) with both expression systems proving that pTOM13 and authentic EFE are identical. A crucial observation was made by Hamilton et al. (1990) when they reported that the pTOM13 polypeptide has homology to flavanone-3-hydroxylase from snapdragon. This implied that EFE might be a member of a class of hydroxylases that require Fe(ll) and ascorbate as cofactors, similar to the flavanone-3-hydroxylase. Ververidis and John (1991) were the first to demonstrate authentic EFE activity in soluble extracts from melon fruit by adding Fe(ll) and ascorbate as cofactors to the enzyme assays.

A ripening-related cDNA, pA VOe3, from avocado was previously identified by differential hybridization (McGarvey et al., 1990). This cDNA is highly similar to the pTOM13 sequence and thus is likely to represent the orthologous gene family member from avocado. In order to characterize the in vivo polypeptide derived from the pAVOe3 cDNA, a B-galactosidase/AVOe3 fusion was constructed in an E. coli expression vector, the polypeptide from E. coli was isolated, and antiserum specific to the A VOe3 coding sequence was generated. With this antiserum it was shown that the A VOe3 antigen accumulates during avocado fruit ripening in a manner consistent with it being an ethylene biosynthetic enzyme (McGarvey et al. 1992).

Authentic EFE from supernatants of avocado fruit was detected using Fe(ll) and ascorbate as cofactors. Further characterization of the EFE from avocado fruit showed it consisted of two separable fractions, EFEI and EFE2 (McGarvey and Christoffersen 1992). The low Km for ACC and the ability to discriminate between stereoisomers of ABC (ACC analog) strongly suggests that this Fe(ll)- and ascorbate-dependent activity is the authentic EFE that operates in vivo during avocado fruit ripening.

2. Subcellular Localization of the A VOe3 Antigen and ACC Oxidase (EFE)

Antiserum generated to the E. coli expressed A VOe3/f3-galactosidase fusion polypeptide has been used to characterize the A VOe3 protein extracted from avocado fruit. Fractionation of avocado fruit extracts by differential centrifugation under a variety of conditions has failed to indicate an association of the A VOe3 antigen with any membrane fraction with the A VOe3 antigen always retained in the lOOK g supernatant. This behavior of the A VOe3 antigen parallels the fractionation of the avocado ACC oxidase enzyme activity.

It is possible to produce protoplasts from avocado fruit in high yield with recovery of intact cells approaching 90 % (percival et al., 1991). These cells have been extensively washed by floatation centrifugation during protoplast preparation and thus loosely-bound cell wall proteins would be lost during purification of the protoplasts. These purified cells retain the A VOe3 antigen as judged by immunoblot analysis when equivalent amounts of protein derived from either protoplasts or total fruit tissue are compared. This strongly suggests that the A VOe3 antigen is not likely to be localized in the cell wall of avocado fruit.

Inspection of the predicted A VOe3 polypeptide sequence reveals a very hydrophilic protein with few hydrophobic domains (Fig 1). The N-terminus does not have a typical signal peptide region that would normally be found on a secreted polypeptide such as the avocado cellulase (Bennett and Christoffersen, 1986). While vacuolar targeting signals in plants are ill-defined, the

67

A VOe3 polypeptide does not have any significant similarity to any of the short motifs that have been identified as potential signals (Chrispeels and Raikhel, 1992). The A VOe3 translation product migrates in SDS-P AGE with the same mobility as the A VOe3 antigen extracted from avocado fruit (McGarvey et aI., 1992). This suggests that no significant proteolysis during posttranslational processing of the AVOe3 polypeptide occurs as has been observed for a number of proteins that are either secreted or are targeted to the vacuole.

1 13 HPhobic · · V'lV M. {I,. & • .'\J ...... 0 V q / 9 'rr"P.{N '(Gp i\ly v h \Jt

-3 HPhilic I ' • • • I • • I I I I • • I I

o 100 200 300

Fig 1 Kyte-Doolittle analysis o/the AVOe3 polypeptide

The avocado ACC oxidase (EFE) has an in vitro pH optimum around pH 7.2-7.5 and is completely inactive at any pH below 6 (McGarvey and Christoffersen, 1992). While the pH of the apoplast may vary depending on the physiological state of the plant, maintenance of a plasma membrane potential requires it to generally stay below 6 (Taiz and Zeiger, 1991). Thus the ACC oxidase would be unlikely to operate in the apoplastic environment. This would be particularly true under situations were auxin-induced acidification of the cell waIl was occurring. A similar argument can be made to exclude the interior of the vacuole as a potential site of ACC oxidase due the low pH environment. Taken together, the available evidence points to the cytoplasm as the most likely subcellular site of physiologically active ACC oxidase.

The avocado ACC oxidase extracted from ripening fruit can be separated into two distinct fractions, designated EFEl and EFE2, by ammonium sulfate precipitation (McGarvey and Christoffersen, 1992). EFEI is in the 40-50% ammonium sulfate cut while EFE2 is completely soluble in 100% saturated ammonium sulfate with the two fractions being present in equivalent proportions. Immunoblot analysis of a fractionation using a series of increasing steps of ammonium sulfate showed that the A VOe3 antigen is restricted to only the fractions containing EFEI and EFE2. This suggests that these two ACC oxidase enzymes share very similar structural features. The reason why two seemingly very similar polypeptides behave so differently on ammonium sulfate fractionation is unknown. One possible hypothesis is that a posttranslational modification of the same primary polypeptide generates the two forms of mature ACC oxidase observed in fruit.

3. ACC Oxidase is a Member of a Fe(II) Ascorbate-requiring Oxidase Superfamily

The A VOe3 cDNA was expressed in E. coli using a T7 RNA polymerase promoter. The cells carrying this transcriptional fusion have authentic ACC oxidase activity that is identical to the ACC oxidase activity extracted from avocado fruit (McGarvey, Savarese, and Christoffersen, manuscript submitted). This result demonstrates the identity between the A VOe3 polypeptide and the avocado ACC oxidase and thus confirms the implied relationship described in the previous section. Searching the GenBank or the PIR databases reveals a number of polypeptide sequences with significant similarity to the A VOe3 sequence. These sequences cluster into distinct protein families with > 60 % identity among the members of each family. This collection

68

~"/JII niduJans

Pen/cl1/Ium ~

~lICf8IfIOtIIum

SInIptDm)'l*! clavu/lglHus

~~

SInIptDm)'l*! /Ipman//

1..--__ FIllIIObacIstIum OIl. SC 12.154

Apple

] Tomato

carnation

Avocado

~Iin N synthase

ACCOllldaae (Ethylene forming enzyme)

Bartey

] F1avaOOlle ~ydm~ Petunia

Snapdragon

Hyoscyamus n/gIIr -- Hyoscyamine 6jI-Ilydmxyiase

Tomato -- ES(?)

Maize -- A2 (Flavan-3.4-ds-d1ol C2-1lydroxylaae?)

Fig 2 Sequence relationships among superfamily of Fe (II) and ascorbate requiring oridases from plants, fUngi and bacteria.

of similar polypeptide sequences represent a superfamily of Fe(II) ascorbate-requiring oxidases although it should be noted that not all Fe(II) ascorbate-requiring oxidases are included in this superfamily.

Among the known members of the ACC oxidase family, sequence identity is > 70%. These sequences come from widely divergent dicots and thus it may not represent the maximal possible difference among ACC oxidase sequences from monocots and lower plants. A multiple alignment of the entire superfamily was generated using the GCG program PILEUP. A number of conserved domains were identified using a sliding window that measures the sequence similarity among the aligned residues at each position (McGarvey, Savarese and Christoffersen, manuscript submitted). An absolutely conserved histidine residue is detected in three separate domains. The Fe(II) ligands of the isopenicillin N synthase (IPNS) were investigated in detail using EPR and NMR spectroscopy (Ming et al., 1990, 1991; Jiang et al., 1991). Three histidine residues have been postulated to be the Fe(Il) protein ligands of IPNS. To determine whether histidine residues are critical for catalytic activity of avocado ACC oxidase we have utilized histidine-specific chemical modification to inactivate the enzyme (McGarvey, Savarese and Christoffersen, manuscript submitted). These results strongly suggest that ACC oxidase contains a critical histidine residue at the active site. The enzyme is protected from chemical inactivation only the presence of Fe(II) and ACC together. This implies that the modified histidine may participate in forming the active site of ACC oxidase and could be a Fe(II) ligand. A hypothetical active site Fe center of ACC oxidase is presented in figure 3. This model is based on that previously proposed by Ming et al. (1991) for IPNS. Further studies are necessary to determine the validity of this model using spectroscopic techniques as well as site-directed mutagenesis of the three conserved histidine residues.

69

Fig 3. Hypothetical Fe center of ACC oxidase

Acknowledgments

The authors would like to thank Drs. N.O. Reich and D.D. Kaska for helpful discussions. This work was supported by the USDA-CRG program grant #90-37261-5658.

References

Bennett, A. B., and Christoffersen, R E. (1986). Synthesis and processing of cellulase from ripening avocado fruit. Plant Physiol. 81, 830-835.

Chrispeels, M. J., and Raikhel, N. V. (1992). Short polypeptide domains target protein to plant vacuoles. Cell 68, 613-616.

Hamilton, A 1., Lycett, G. W., and Grierson, D. (1990). Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346, 284-287.

Hamilton, A. J., Bouzayen, M., and Grierson, D. (1991). Identification of a tomato gene for the etheylene-forming enzyme by expression in yeast. Proc. Natl. Acad. Sci. USA 88, 7434-7437.

Jiang, F., Peisach, J., Ming, L.-1., Que, L., Jr., and Chen, V. J. (1991). Electron spin echo envelope modulation studies of the Cu(Il)-substituted derivative of isopenicillin N synthase: A structural and spectroscopic model. Biochemistry 30, 11437-11445.

Kende, H. (1989). Enzymes of ethylene biosynthesis. Plant Physiol. 91,1-4. McGarvey, D. 1., and Christoffersen, R E. (1992). Characterization and kinetic parameters of

ethylene-forming enzyme Hom avocado fruit. 1. BioI. Chern. 267, 5964-5967. McGarvey, D. J., Yu, H., and Christoffersen, R E. (1990). Nucleotide sequence of a ripening

related cDNA from avocado fruit. Plant Molec. BioI. 15, 165-167. McGarvey, D. J., Sirevag, R, and Christoffersen, R E. (1992). Ripening-related gene from

avocado fruit - ethylene-inducible expression of the messenger RNA and polypeptide. Plant Physiol. 98, 554-559.

McKeon, T. A., and Yang, S. F. (1987). Biosynthesis and metabolism of ethylene. In Plant Hormones and Their Role in Plant Growth and Development, P. J. Davies, ed. (Dordrecht: Martinus Nijhofi), pp. 94-112.

Ming, L.-J., Que, L., Jr., Kriauciunas, A, Frolik, C. A, and Chen, V. J. (1990). Coordination chemistry of the metal binding site of isopenicillin N synthase. Inorg. Chern. 29, 1111-1112.

Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., and Chen, V. J. (1991). NMR studies of the active site of isopenicillin N synthase, a non-heme iron(Il) enzyme. Biochemistry 30, 11653-11659.

70

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.

Percival, F. W., Cass, L. G., Bozak, K. R., and Christoffersen, R. E. (1991). Avocado fruit protoplasts: A cellular model system for ripening studies. Plant Cell Reports 10, 512-516.

Spanu, P., Reinhardt, D., and Boller, T. (1991). Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its messenger RNA in Xenopus-Iaevis oocytes. EMBO J 10,2007-2013.

Taiz, L., and Zeiger, E. (1991). Plant Physiology. (Redwood City: Benjamin/Cummings). Ververidis, P., and John, P. (1991). Complete recovery in vitro of ethylene-forming enzyme

activity. Phytochem 30, 725-727. Yang, S. F., and Hoffman, N. E. (1984). Ethylene biosynthesis and its regulation in higher

plants. Ann. Rev. Plant Physiol. 35, 155-189.

IDENTIFICATION OF GENES ENCODING EFE IN TOMATO

AJ.HAMILTON I, M. BOUZAYEN',& D. GRIERSON!. !AFRC Research Group in Plant Gene Regulation, Department of Physiology and Environmental Science, University of Nottingham School of Agriculture, Sutton Bonington, Loughborough, LE12 5RD, U.K. tENSAT, 145 Av. Muret, 31076 Toulouse, France.

ABSTRACT. In this article we relate how tomato genes encoding the ethylene forming enzyme were identified by first inhibiting their expression with antisense genes and then by demonstrating their functional expression in a heterologous host. These results have shed much light on the nature of this enigmatic enzyme including its cellular location and mechanism of action.

1. Introduction

A major obstacle to a better understanding of ethylene biosynthesis has been the recalcitrance of the ethylene-forming enzyme (EFE) to biochemical purification. Consequently, isolation of the gene(s) encoding EFE has been impossible by the conventional routes of screening either a cDNA library with an oligonucleotide probe (based on the amino acid sequence of the protein) or a cDNA expression library with an antibody raised to the purified protein. However, molecular biology has offered an alternative strategy to identify genes related to ethylene synthesis and action. The clone pTOM13 had been isolated from a ripe tomato fruit cDNA library (Slater et aI., 1985) and shown to be homologous to an mRNA which accumulated in leaves after mechanical wounding. It was suggested at this point that the protein encoded by sllch mRNAs may be ethylene related (Smith et al., 1986). The full sequence of the cDNA was determined (Holdsworth et al., 1987) and shown to contain an open reading frame encoding a protein of 33.5 kDal., which was similar to the tigure of 35 kDal., obtained by hybrid release translation experiments (Slater et al., 1985). Computer analysis of this polypeptide sequence revealed no obvious membrane spanning regions or signal peptide consensus sequence, indicating that it would probably be located in the cytosol. Three classes of genomic clones were isolated (Holdsworth et al., 1988) but comparisons of these with sequences in several databases revealed no homology to any genes of known function. Expression studies showed that levels ofpTOM13-homologous RNA correlated positively with ethylene evolution in both wounded leaves, ripening fruit (Holdsworth et al., 1987) and senescing leaves (Davies and Grierson, 1989), further supporting the hypothesis that the pTOM13 protein is involved in ethylene physiology.

71


72

2. Identification Of Gene Function With Antisense Genes

In order to obtain more information about the function of proteins encoded by this gene family, an antisense gene was created from the pTOM13 cDNA and the Cauliflower mosaic virus 35S promoter and polyadenylation signals. When introduced into tomato plants by Agrobacterium tumefaciens mediated transformation, this gene caused a reduction ethylene synthesis and in particular reduced EFE activity (Figure 1a and Hamilton et al., 1990) .

100 .---- 2h 8h 14h 19h 26h 100% - - - -

~ 8

'C

,....-34%

~ 1:> ... M .s 6 S

~ .. !.

= J ! 4 .. J =il ,., u ~

2

o ~ o 1 2

Number of Antisense Genes Time (hours)

a. b.

Figure 1. a: EFE activity in wounded leaf discs excised from tomato plants containing 0, 1 or 2 pTOM13 antisense genes; b: Conversion of ACC to ethylene during batch culture by untransformed S. cerevisiae (0) compared to S. cerevisiae expressing the pRC13 cDNA () .... represents the increase in cell density.

The most likely explanation of these results was that pTOM13 homologous RNA encodes at least part of EFE. Further evidence for this was the observation that the gene encoding the enzyme flavanone 3-hydroxylase shared significant homology with pTOM13 (A. Prescott, personal communication cited in Hamilton et at., 1990). This was significant because the conversion of ACC to ethylene had previously been suggested to occur via a hydroxylation reaction (Yang, 1985).

73

3. Functional Heterologous Expression Of EFE

The pTOM13 cDNA contains several cloning artefacts at its 5' end (Hamilton et aI., 1991). These probably are at least partly responsible for the failure to obtain functional expression of this clone in E. coli (G. Stewart, personal communication.) A corrected cDNA (PRC13) was created by fusing the 5' end of a genomic clone (Kock et aZ., 1991) to the 3' end of the cDNA. This was then shown to encode a functional EFE when expressed in Saccharomyces cerevisaie (figure Ib and Hamilton et aZ., 1991): Furthermore, this EFE activity exhibited strong stereodiscrimination between the cis and trans isomers of the ACC analogue l-amino-2-ethylcyclopropane-l-carboxylic acid (AEC) (Table 1).

Table 1. Production of I-butene from AEC stereoisomers by S. cerevisiae expressing the pRC13 cDNA

Substrate I-butene produced (pI. h· l )

- ascorbate ± ascorbate

Trans-AEC o ± 190 4907 ± 270

Cis-AEC 14 ± 5 59 ± II

Table 1. I ml cultures of transformed S. cerevisiae (7 x 107 cells ml- l ) were incubated (± 50mM ascorbate) with ImM trans or cis racemic mixtures of AEC at 30°C. I-butene produced after 2 h was determined by gas chromatography. Data are means (± SE) of three replicates.

Table 2. Factors influencing the conversion of ACC to ethylene by S. cerevisiae expressing pRC13 DNA

Factor

control

CoCl2

PA

PeSO.

ascorbate

ascorbate + PeSO.

Ethylene produced (nl. h- l )

5.79 ± 0.4

0.92 ± 0.09

0.42 ± 0.07

7.15 ± 0.62

24.47 ± 1.39

26.49 ± 2.85

Table 2. I ml cultures of transformed S. cerevisiae (8 x 107 cells ml- l ) were incubated with ImM ACC and 25 }lM CoCl2 or 50 mM ascorbate or 200}lM I,IO-phenanthroline (PA). Transformed cells used as control were incubated in the presence of I mM ACC only. After 1 hour at 30°C the ethylene evolved was measured by gas chromatography .. Each value represents the mean (±SE) of three replicates.

74

Yang's hypothesis that the conversion of ACC to ethylene involved a hydroxylated intermediate was further supported by the observation that both iron and ascorbate enhanced ethylene production (Table 1) and this enhancement demonstrated AEC stereo selectivity (Table 2): both iron and ascorbate are necessary for the function of flavanone-3-hydroxylase. The inhibition of EFE activity by phenanthroline is probably a result ofthis compounds ability to chelate iron. The results of the EFE gene expression in yeast are in agreement with those of Spanu et al. (1991) who demonstrated EFE activity in Xenopus laevis oocytes encoded by transiently expressed pTOM13 homologous RNA.

4. Conclusions

The results presented clearly show that genes homologous to the pTOM13 cDNA encode the complete EFE in tomato. Previous experiments on EFE have led to the conclusion that it required intact membranes for activity (Yang, 1985) however the predicted structure of any of the three polypeptides encoded by the three pTOMl3-homologous genes from tomato strongly suggest that EFE.is located in the cytosol. The above evidence which suggests that EFE is a hydroxylase has prompted other investigators to try to purify EFE using techniques which previously had been used to successfully extract flavanone-3-hydroxylase. Both Ververidis and John (1991) and Fermindez-Maculet and Yang (1992) have now successfully extracted EFE in a soluble form. The latter group have suggested that the previously observed requirement for membrane integrity was a result of their sequestration of iron and ascorbate (required by EFE for activity) and the loose binding of EFE to these membranes during cell disruption. The antisense results demonstrate the usefulness of this simple technique both for the identification of gene function and for the creation of novel phenotypes. The ability to express pTOM13 homologous genes in Saccharomyces cerevisiae will allow the detailed analysis of the EFE protein by site directed mutagenesis. It should also allow the biochemical discrimination between the three different EFEs present in tomato. Finally, the analysis of the DNA or RNA sequences which control the expression of EFE will lead to the identification of the factors and events which trigger the onset of ethylene biosynthesis.

5. References

Davies, K.M., Grierson, D. (1989) Identification of cDNA clones for tomato (Lycopersicon esculentum) mRNAs that accumulate during fruit ripening and leaf senescence in response to ethylene. Planta 179, 73-80. Fermindez-Maculet, J.e. and Yang, S.F. (1992) Extraction and partial characterisation of the ethylene-forming enzyme from apple. Plant Physio!. 99, 751-754 Hamilton, AJ., Lycett, G.W., Grierson, D. (1990) Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346, 284-287. Hamilton, AJ. Bouzayen, M., Grierson, D. (1991) Identification of a tomato gene for the ethylene forming enzyme by expression in yeast. Proc. Nat!. Acad. Sci., USA, In press. Holdsworth, MJ., Bird, C.R., Ray, 1., Schuch, W., Grierson, D. (1987) Structure and expression of an ethylene-related mRNA from tomato. Nucleic Acids Res. 15, 731-739.

75

Holdsworth, M.1., Schuch, W., Grierson, D. (1988) Organisation and expression of a wound /ripening-related small multigene family from tomato. Plant Mol. BioI. 11, 81-88. Kock, M., HamiJWn, A.1., Grierson, D. (1991) eth 1, a gene involved in ethylene synthesis in tomato. Plant Mol. BioI. 17, 141-142. Slater, A., Maunders, M.1., Edwards, K., Schuch, W., Grierson, D. (1985) Isolation and characterisation of cDNA clones for tomato polygalacturonase and other ripening related proteins. Plant Mol. BioI. 5, 137-147. Spanu, P., Reinhardt, D. and Boller, T. (1991) Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its messenger-RNA in Xenopus Laevis oocytes. EMBO J. 10, 2007-2013 Ververidis, P. and John. P (1991) Complete recovery in vitro of ethylene-forming enzyme activity. Phytochemistry. 30, 725-727 Yang, S.F. (1985) Biosynthesis of ethylene, in Current Topics in Plant Biochemistry and Physiology, Vol. 4, pp.126-138. eds D.D. Randall, D.G. Blevins & R.L. Lasson, University of Missouri, Columbia, U.S.A.

EFE MUL TIGENE FAMILY IN TOMATO PLANTS: EXPRESSION AND CHARACTERIZATION

M. BOUZAYEN1, W. COOPER2, C. BARRy2, H. ZEGZOUTP, A.J. HAMIL TON2 & D. GRIERSOW 1 ENSA Toulouse 145 A venue de Muret 31076, Toulouse, France. 2UniversityofNottingham, Sutton-Bonington, LE125RD, U. K.

ABSTRACT. Decisive progress in the understanding of ethylene metabolism was made when a cDNA clone (pTOM13) was identified by Hamilton et a/. (1990) as encoding EFE. Previous screening of a tomato genomic library revealed three genomic clones, refered now to as BTH1, ETH2 and ETIl3. The nucleotidic sequence of ETH3 is unknown while those of ETH1 and E71I2 have already been published. Moreover, BTEl1 gene has been shown to encode pTOM13 mRNA whereas no data were available so far concerning the expression of the two other genes. Following sequencing of ETIl3 we report here the comparative analysis of the derived amino acid sequences encoded by this gene family and show that the three predicted proteins share the same hydrophilicity pattern. However, ETH2 protein displays significantly higher isoelectric point than the two others.We also describe the isolation and cloning of ETH2 cDNA and thus provides experimental evidence for its expression at the RNA level. Gene specific oligonucleotides were designed and used as primers in PCR reactions to amplify about 200 bp of the 3' untranslated regions. The amplified fragments were cloned in a transcription vector generating appropriate probes for subsequent study of the expression of EFE genes in tomato plants .

1. Introduction

Fruit ripening is under control of the plant hormone ethylene which also plays a major role in leaf senescence and responses to wounding and in invasion by pathogens. The biosynthetic pathway of ethylene in plants involves the conversion of S-adenosylmethionine to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC-synthase and the oxidation of ACC into ethylene by the ethyleneforming enzyme (EFE) (1).

Whereas it is now established that during plant development various ACC-synthase are specifically expressed and their respective genes identified (2-4), no gene encoding ACe-oxidase was available until recently. Significant progress was achieved when transgenic tomato plants transformed with the pTOM13 antisense gene, were shown to produce significantly lower level of ethylene during fruit ripening and in wounded leaves (5) than controls. More recently, direct identifiction of an EFE encoding gene was given by functional expression ofpTOM13 cDNA in S. cerevisiae (6) and in xenopus oocytes (7). Screening the tomato genomic library with pTOM13 probe revealed three genomic clones refered to as E71I1, ETH2 and E71I3. The clones E1Tll and E71I2 have already been fully sequenced but not E71I3 (8,9).

76 J. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, 7(H! 1. © 1993 Kluwer Academic Publishers.

77

After sequencing of ETH3 genomic clone , we report here the comparison of the predicted proteins encoded by the three genes. We also provide direct evidence for expression of E71l2 gene in tomato by cloning its cDNA following PCR amplification. In the last part of this work we describe the construction of specific probes for EFE genes.


2.1 PLANT MATERIAL Tomato plants (Lycopersicon esculentum Mill, variety Alisa Craig) were grown as described previously (10) from seeds of homozygous line maintained at Sutton-Bonington since 1978.

2.2 RNA EXTRACTION AND Poly(A)+ ISOLATION

Total RNA was extracted either from whole plantlets or from tomato fruit and leaves according to (11) with the following modification. Four grammes of plant tissue were grounded in liquid nitrogen and supplemented with one volume of extraction buffer (100 mM Tris-Cl, 20 mM Aurintricarboxylate, 200 mM LiCI2, 100 mM EDTA and 100 mM 2-mercaptoethanol. The

mixture was extracted twice with an equal volume ofphenol/chioroformlisoamylalcohol (25,24,1). After centrifugation, RNA was recovered from the aqueous phase by precipitation with LiCl2 3 M.

The pellet was washed with 3 M LiCI2, dissolved in 2% potssium acetate and precipitated in

ethanol. This total RNA fraction was subsequently used for Poly(A)+ selection using oligo(dT)cellulose as described in (12).

2.3 FIRST STRAND cDNA SYNTHESIS AND AMPLIFICATION BY PCR METHOD.

First strand cDNA was generated by reverse transcription of poly(A)+ RNA and used as template for polymerase chain reaction according to the protocol of (12). The reaction was carried out using gene specific oligonucleotides as primers.

2.4 MOLECULAR CLONING

The cloning vector pT7/T3 a.-18 or pBluescriptII SK was digested with the XbaJ restriction enzyme and ligated to the amplified cDNA treated with the same enzyme using the protocol of (12). The ligation product was used to transform E.coli strain DH5a.™ competent cells (GibcoBRL) and the recombinant bacteria were selected according to the manufacturers' procedure.


3.1 COMPARATIVE SEQUENCE ANALYSIS OF EFEI, EFE2 AND EFE3 PROTEIN

The complete sequence of ETH] and ETH2 genes have been already published (8,9) . Sequencing of the full genomic clone of ETH3 revealed complete homology to the coding region of the elicitorinduced cDNA clone isolated from tomato cell culture (7).

78

50 EFEl MENFPIINLE KLNGDERANT MEMIKDACEN WGFFELVNHG IPHEVMDTVE EFE2 MENFPIINLE KLNGaERvaT MEklnDACEN WGFFELVNHG IPHEVMDTVE EFE3 MENFPIINLE nLNGDERAkT MEMIKDACEN WGFFELVNHG IPHEVMDTVE

51 100 EFEl KMTKGHYKKC MEQRFKELVA SKGLEAVQAE VTDLDWESTF FLRHLPTSNI EFE2 KITKGHYKKC MEQRFKELVA kKGLEgVeVE VTDmDWESTF FLRHLPsSNI EFE3 KITKGHYKKC MEQRFKELVA SKGLEAVQAE VTDLDWESTF FLRHLPTSNI

101 150 EFEl SQVPDLDEEY REVMRDFAKR LEKLAEELLD LLCENLGLEK GYLKNAFYGS EFE2 SQIPDLDdvY R-VMRDFrKR LEKLAEELLD LLCENLGLEK sYLKNTFYGS EFE3 SQVPDLDEEY REVMRDFAKR LEKLAEELLD LLCENLGLEK sYLKNAFYGS

151 200 EFEl KGPNFGTKVS NYPPCPKPDL IKGLRAHTDA GGIILLFQDD KVSGLQLLKD EFE2 KGPNFGTKVS NYPPCPKPDL IKGLRAHTDA GGIILLFQDD KVSGLQLLKD EFE3 KGPNFGTKVS NYPPCPKPDL IKGLRAHTDA GGIILLFQDD KVSGLQLLKD

201 250 EFEl EQWIDVPPMR HSIVVNLGDQ LEVITNGKYK SVLHRVIAQT DGTRMSLASF EFE2 grWIDVPPMR HSIVVNLGDQ LEVITNGKYK SVmHRVIAQK DGTRMSLASF EFE3 EQWIDVPPMR HSIVVNLGDQ LEVITNGKYK SVmHRVIAQT gGTRMSLASF

251 300 EFEl YNPGSDAVIY PAKTLVEKEA EE-STQVYPK FVFDDYMKLY AGLKFQAKEP EFE2 YNPGnDALIY PApaLVEKDA EEHnkQVYPK FmFDDYMKLY AnLKFQAKEP EFE3 YNPGnDAVIY PApsLi---- EE-SkQVYPK FVFDDYMKLY AGLKFQpKEP

301 321 EFEl RFEAMKAMES -----DPIAS A EFE2 RFEAMKAMES -----DPIAI A EFE3 RFEAMKAMEa NVELVDqIAS A

Figure 1: Derived amino acid sequences of EFE proteins. The numbers represent the position of the amino acids in the EFE 1 protein. Changes in amino acids of EFE2 and EFE3 as compared to EFEI are indicated by lower-case letters. Amino acid deletions are indicated by gaps (-). The putative glycosylation site is underlined.

Alignement of the amino acid sequences derived from the three genes shows that they share the same position for the ATG giving the longest reading frame, with EFE3 being longer by one amino acid (Fig. 1). EFEI and EFE3 proteins are very similar with regards to their content in charged group but diverge from EFE2 which contains higher proportion of basic amino acids positively charged at neutral pH. Therefore EFEI and EFE3 share the same isoelectric point of 4.99 and 5.09 respectively, while EFE2 has a significantly higher pI of 6.14. Sequence analysis indicates that none of the three proteins have an obvious transit peptide or hydrophobic membrane-spanning domain. However several common measures of protein secondary structure, ploted in figure 2 according to (13) representation, outline the domains where an a-helix could be predicted. Thus, a short domain at the C-terminal region shows a hydrophobic pattern and a probability for a short ahelix to nucleate. The region laying between amino acid 120 and 145 shows a possibile formation of an amphipatic a-helix which would be long enough to span the membrane lipid bilayer. In addition this region contains multiple leucine residues raising the probability for a leucine zipper

79

formation. Taking these informations into account, the possibility cannot ruled out that the protein might be excreeted or associated with membrane structure (14).

o I

100 I

200 I

300 t _, ...... __ .... ..--_'_111_ ........ ____ ._, .. _ ............. _=-__ ......... ....,.....,· .... · ............... ,,.._·· .... " ....... ·_ ..... __ .............. _ ......... " .. "' ............................... __ ..

"1IIIIIIIIII.I.lI.lIIII.I.lIIIIIIII,lr lll . .III.11111.1IJIIII.l11II11III..I.lI..I1Llnl.II,I.III.1I.,III.lIIIIII.IIJIIIIII""I"IfIIIIM'lIInll,IIIII.I .. .I111UlII"I'I"III.III.I"I,,,IIIII.lIIII,,,Ii-II..ml'Tl"JiUr.lnlll.llllllllJ~IIII'l11I1111I1II1I1.l1II1II1"IIIJ

Amino acid

Alpha Forming

Beta Forming

Figure 2 Repesentation of the protein secondary structure. according to the criteria defined in (13) The upper panel gives the predicted EFE 3 protein with amino acid numbering. The middle panel of the plot shows the propensity measures for alpha-helix and beta-sheet where the dashed lines are for alpha structures and solid lines for beta, Thc lower panel represents the residues that are alphahelix forming and breaking, as defined in (13).

3.2 ISOLATION AND CLONING OF ETH2cDNA

While the ETHI gene has been clearly identified as encoding pTOM13 mRNA, no data is available showing the expression of ETH2 and BTH3 genes. The perfect homology of the coding sequence of BTH3 with the elicitor-induced clone brings indirect evidence for the expression of ETH3 in tomato cells and led us to focuse our study on the expression of BTH2. Since sequence analysis of the three genes showed substantial divergence in the 3' untranslated regions, two oligonucleotides specific for E1H2, were designed and used as primer for PCR amplification. Amplification was successfully achieved using template cDNA isolated from a whole tomato plantlet. The PCR fragment was digested by XbaJ and ligated to the cloning vector pBluescriptII SK prelinearized by XbaJ before transformation into E. coli strain DH5a.. The new generated plasmid, named pEFE2, was extracted from recombinant colonies and the nature of the cloned insert was determined by partial sequencing of the 5' and 3' ends using T3 and T7 primers. The cloned fragment was recovered by BglIl digestion of pEFE2 plasmid and ligated into the Bglll site between the phosphoglycerate kinase promoter and terminator of the yeast expression vector.

3. 3 CONSTRUCTION OF SPECIFIC PROBES FOR EFE GENES

Study of differential expression of EFE genes required the construction of specific probes. Two oligonucleotides, encompassing the 3' untranslated region and specific for each gene, were designed for use as primers in PCR reactions to amplify about 200 bp fragment of the 3' untranslated regions. The amplified fragments were cloned in transcription vector pT7/T3 a.-IS and their orientation checked by sequencing both strands using T3 and T7 primers. For probe synthesis,

80

plasmids were linearized before use as template for T7 RNA polymerase. The RNA probe was

labelled by incorporation of 32p_UTP in the reaction mixture. Specificity of the probe was checked by hybridizing the RNA transcript either to the DNA insert cut from the vector by the appropriate restriction enzyme or to the DNA insert obtained by PCR amplification using T3 and T7 oligonucleotides. The DNA slot blot presented in Figure 3, was probed as described above and washed at low stringency allowing detection of possible cross-hybridization between the three probes. The results show strict specificity of the probe when the template DNA corresponds to the precise 3' untranslated region of each gene. Howevcr, when PCR amplified fragments were used as template, slight cross-hybridization between Eml and Em3 probes was observed. The cloning pattern of these fragments implies that the T3 and T7 oligonucleotides used as primers in PCR reaction will also amplify about 60 bp of the vector generating some sequence homology between ETH 1 and Em3 probes which in low stringency condition could give rise to cross hybridization .

3 1 3 2 1 3 2

Figure 3 Specificity of the probes tested by cross-hybridization in slot blot analysis. The insert is either amplified by PCR (upper loading) or cut from the cloning vector by appropriate restriction enzyme digestion (lower loading). The probes generated from ETHI, ETH2 and ETH3 genes are numbered 1, 2 and 3 respectively.

3. 4 CONCLUSIONS AND PROSPECTS

This study showed that EFE proteins encoded by the ETH multigene family in tomato plants share strong homology at the amino acid sequence. All three genes are shown to be functionnal since they are expressed at the RNA level. Cloning of their respectives cDNA and subsequent expression in Scerevisiae will allow the comparative study of the biochemical properties of the three proteins. The use of ETH1,2 and 3 specific probes in northern analysis will lead to the identification of EFE genes being expressed during normal and abnormal plant developpment. Tissues specific expression will be studied by in situ hybridization. Finally, plant transformation with E11£1,2 and 3 specific fragments in antisense construction,currentiy undertaken, will soon reveal the respective role of each gene in controlling different physiological processes known to be under ethylene regulation.

4 Acknowledgements

Authors are grateful to A Evans (Sutton-Bonington, U K.) for providing substantial help in computer-based analysis of the protein secondary structure. This work was supported by a grant

81

from Agricultural and Food Research Council (U K) to D.G. and a European Economic Community Training Fellowship to M.B.

5 References

1- Yang, S.F., Hof'finan N.E.(1984) Ethylene biosynthesis and its regulation in higher plants. Ann. Rev. Plant Physiol. 35, 155-189. 2- Nakagawa, N., Mori, H., Yamazaki, K., Imaseki, H. (1991).Cloning ofa complementary cDNA for auxin-induced 1-aminocyclopropane-l-carboxylate synthase and differential expression of the gene by auxin and wounding. Plant Cell Physiol. 32, 1153-1163 3- Olson, D.e., White, lA, Edelman, L., Henskens, RN and Kende, H. (l991).Differential expression of two genes for l-aminocyclopropane-1-carboxylate s)'TIthase in tomato fruits. Proc. Natl. Acad. Sci. USA, 88, 5340-5344 4- Huang, P.L., Parks, J.E., Rottman, W.H., Theologis, A (1991). Two. genes encoding 1-aminocyclopropane-l-carboxylate synthase in zucchini are clustered and similar but differentially regulated. Proc. Natl. Acad.Sci. USA 88, 7021-7025. 5- Hamilton, AJ., Lycett, G.W., Grierson, D. (1990) Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346, 284-287 6- Hamilton, A, Bouzayen, M., 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 7- Spanu, P., Reinhart, D., Boller, T. (1991). Analysis and cloning of the ethylene-forming enzymc from tomato by functional expression of its mRNA in Xenopus laevis oocytes. EMBO J., 10, 2007-2013 ' 8- Holdsworth, M.J., Schuch, W., Grierson, D. (1988). Organisation and expression ofa wound! ripening-related small multigene family from tomato. Plant Mol. BioI. 11, 81-88 9- Kock, M., Hamilton, AJ., Grierson, D. (1991). ethl, a gene involved in ethylene synthesis in tomato. Plant Mol. BioI. 17, 141-142 10- Grierson, D., Slater, A, Speir, J.R., Tucker, GA (1985) The appearance of polygalacturonase mRNA in tomatoes : one of a series of changes in gene expression during development and ripening. Planta 163,263-271 11- Wadsworth, GJ., Redunbaugh, H.G., Scandalios, lG. (1988) A procedure for the small-scale isolation of plant RNA suitable for RNA blot analysis. Anal. Biochem. 172, 279-283 12- Fritsch, E.F., Maniatis, T.R., Sambrook, 1. (19,89) Molecular cloning: a laboratory manual. Cold Spring Harbour Lab., New York. 13- Chou, and Fasman (1978) Adv. Enz. 47, 45-147 14- Latche, A., Dupille, E., Rombaldi, e., Cleyet-Marel, J.e., Lelievre, J.M., Pech, J.e. (1993) Purification, characterization and subcellular localization of ACC oxidase from fruits. In: Cellular and Molecular Aspects of Plant Hormone Ethylene (J. e. Pech, A Latche & e. Balagut\' eds) Kluwer Acad. Pub., Dordrecht, The Netherlands, pp. 39-45

ALTERED GENE EXPRESSION, LEAF SENESCENCE, AND FRUIT RIPENING BY INHIBITING ETHYLENE SYNTHESIS WITH EFE-ANTISENSE GENES

lE. GRAY, S. PICTON, R. FRAY, A.J. HAMILTON, H. SMITH, S. BARTON, AND D. GRIERSON University of Nottingham, AFRC Group in Plant Gene Regulation, Faculty of Agricultural and Food Sciences, Dept. Physiology and Environmental Sciences, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK.

ABSTRACT. Genes involved in changes in colour, flavour, texture, and aroma are expressed during ripening of fleshy fruits. In tomato, and other climacteric fruits, ethylene synthesised at the outset of ripening is involved in regulating expression of some of these ripening genes: "Many ripening-related mRNAs have been cloned and several, including polygalacturonase, pectinesterase, ACC synthase, ethyleneforming enzyme (EFE), and phytoene synthase have been identified and sequenced (for review see Gray et al., 1992). We have used an antisense-RNA strategy, developed originally for the polygalacturonase gene (Smith et al., 1988, 1990) to identify cDNA and genomic clones for EFE (Hamilton et al., 1990, 1991). Reducing EFE expression in transgenic tomatoes inhibits ethylene synthesis and slows ripening and over-ripening offruits. Ripening can be accelerated by adding ethylene to detached fruit. In the EFEantisense plants, leaf senescence is delayed and wound-ethylene synthesis is also reduced. Inhibiting ethylene synthesis caused a reduction in carotenoid production in fruit, which was associated with reduced expression ofphytoene synthase required for the production of ~-carotene and Iycopene (Bird et al., 1991).

A naturally occurring tomato mutant deficient in carotenoid production has been characterised and shown to contain a mutation in the phytoene synthesis gene expressed in fruit. Constitutive over-expression of a wild-type sequence in mutant transgenic plants restores carotenoid production in ripening tomatoes and leads to unscheduled carotenoid synthesis in other cell types. The potential for using sense and antisense genes to .identify and assign functions to ripening genes and to alter the physiology and biochemistry of fruits is discussed.

1. Introduction

Transgenic plants, and in particular those expressing antisense genes, have been important in analysing the function of encoded ripening-related enzymes (Smith et 01., 1988, 1990a ,1990b; Sheehy et 01., 1988; Hamilton et 01., 1990; Bird et 01., 1991; Oeller et 01., 1991; Tieman et 01., 1992; Hall et 01., 1993). An antisense gene causes specific inhibition of target genes which share complementary sequences. However, this technique does not require knowledge of the functions of the polypeptides encoded by the targeted genes. Hence, it is possible to produce a specific mutation, by inhibiting gene expression using mRNAs which have been cloned but whose function is unknown. In this chapter we shall discuss the phenotype of plants transformed with an antisense ethylene- forming enzyme gene and the use of a phytoene synthase gene to complement a mutation inhibiting carotenoid biosynthesis.

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1. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plallt Hormone Ethylene, 82-89. © 1993 Kluwer Academic Publishers.

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2.1. ALTERED LEAF SENESCENCE AND FRUIT RIPENING IN PLANTS EXPRESSING AN EFE-ANTISENSE

TRANSGENE

The technique of antisense inhibition has been used to help determine that a ripening-related cDNA clone, pTOM13, encodes the ethylene-forming enzyme (Hamilton et al. 1990, also see chapter in this volume by Hamilton et at). The resulting pTOM13 antisense plants had a low ethylene phenotype, and EFE activity was inhibited in a gene dosage dependant manner in plants heterozygous and homozygous for the antisense gene. These plants produced very low levels of ethylene during fruit ripening and on wounding of leaves or fruits (Hamilton et al., 1990).

In addition to changes observed in the physiology of ripening fruit, which are described below, homozygous EFE-antisense plants were also used to investigate the involvement of ethylene in other developmental processes. The rate of germination of EFE-antisense seed was found to be unaffected (S .Picton, unpublished data), but alterations were observed in the onset of foliar senescence. In young EFE-antisense plants the onset of leaf senescence was temporally delayed (Figure 1) by at least one week but once initiated continued at a rate equivalent to that observed for the wild-type controls (Picton et al., 1993).

Figure 1. Phenotypic changes in leaf senescence of EFE-antisense plants. Tomato seed was pre-germinated and grown in compost without further nutrient addition. Plants were removed from a random block for photography at 8 weeks post germination. The wild-type plant (left) already displays advanced senescence of the lower leaves (arrow) whilst the EFE-antisense plant (right) shows much less advanced senescence of similar leaves.

84

The fruit borne by homozygous EFE-antisense plants showed substantially reduced ethylene evolution during ripening (Hamilton et al., 1990). They appeared physiologically normal at all developmental stages prior to ripening, and the onset of ripening occurred at a similar age to wildtype fruits (Hamilton et al., 1991; Picton et al., 1993). Transgenic fruit allowed to ripen on the plant changed from green to red over a period of several days but, after ripening on the plant for several weeks, lycopene accumulation was reduced when compared to wild-type controls (Figure 2). This contrasts with the senescence results where chronological onset was delayed but subsequent progression of senescence was unaffected. EFE-antisense fruit, allowed to ripen fully on the plant, demonstrated an increased resistance to over-ripening characteristics such as shrivelling, splitting and subsequent spoilage (Picton et al., 1993). Obviously, these qualities could prove of use in the development of fruit with increased shelf-life.

When EFE-antisense fruits were removed from the plant prior to ripening, the delayed ripening phenotype was enhanced being dependant upon the developmental stage at which the fruit were removed (Picton, 1993). EFE-antisense fruit, detached at a mature-green stage and incubated in air, showed a substantial reduction in the accumulation of total carotenoids, particularly lycopene (Figure 2) and remained orange in colour. All of these detached fruit failed to achieve normal pigmentation, even after prolonged periods of ripening (Picton et al., 1993). Like the fruit ripened on the plant, the detached EFE-antisense fruit showed increased resistance to post-harvest shrivelling and subsequent spoilage.

Application of exogenous ethylene to the detached EFE-antisense fruit increased the accumulation of \ycopene such that the fruit appeared indistinguishable from wild-type controls. However, extraction and spectrophotometric measurement of carotenoids, particularly lycopene (Figure 2), indicated that application of ethylene alone was insufficient to completely reverse the EFEantisense phenotype. These ethylene-treated antisense fruit also showed resistance to over-ripening when compared to wild-type controls, again suggesting that application of ethylene alone can not fully reverse the EFE-antisense phenotype (Picton et al., 1993) This raises the possibility of the existence of another factor which affects fruit ripening and is associated with attachment to the plant. Such a ripening factor may be either an importable ripening enhancer or an exportable inhibitOl;". Although it has been suggested that ethylene is the complete trigger for ripening in tomato fruit (Oeller et al., 1991), our observations with vine-ripened and detached air- and ethylene-treated EFE-antisense fruit suggest ethylene acts as a switch in association with another factor and also as a rheostat, modulating the level of specific gene expression. It seems probable that in these experiments the low levels of ethylene produced by attached EFE-antisense fruit was sufficient, in conjunction with this plant associated factor, to trigger most aspects of ripening. Higher levels of ethylene would presumably be required for complete ripening to occur. When antisense fruit are detached from the plant and treated with high levels of ethylene, the ripening process is triggered but remains incomplete due to the absence of the plant associated factQr.

Another ripening-related cDNA clone, pTOM5, has recently been identified as encoding the enzyme phytoene synthase (Bird et al., 1991) which catalyses the formation of phytoene, the primary C40 precursor for carotenoid biosynthesis in plants. This was shown both by the yellow fruit phenotype of pTOM5 antisense plants and by its sequence homology to identified prokaryotic genes (Bird et al., 1991; Armstrong et al., 1990). Both the pattern and peak accumulation of homologous phytoene synthase mRNA appear largely unaffected in vine-ripened EFE-antisense fruit (Figure 3). However, the accumulation of this mRNA is substantially reduced in detached

85

EFE-antisense fruit that show reduced l,Ycopene accumulation. Application of ethylene to detached EFE-antisense fruit led to increased accumulation of the pTOM5 mRNA (Figure 3) and may provide an explanation for the observed increase in lycopene accumulation.

: A : B

: :

. , I. • ,+1 8'·4 8,·1 8r+2I ., a • 8r+2 .,+4 It •• B,d.

N C N D

i .. ~ .. .. = c

i - -,. E ,. iii 1: .. E .. Ii:

., B. 8,+2 .,+. .,., .,.21 ", a. aut 8,+. If.' .,+ ..

N E

N F

'" ., .,+2 .,+4 .,.1 .,.21 N, " 8'.2 8,+4 .,.. 1,+21

Ripening stage

Figure 2. Effect of EFE-antisense genes and ethylene addition on levels of pigments in detached and attached ripening fruit. Total chlorophylls (panels A and B), total carotenoids (panels C and D) and lycopene (panels E and F) were extracted and quantified from fruit ripened on the plant (left panels, wild-type, .. ; EFE-antisense A) or fruit detached from the plant at a mature-green stage (right panels, wild-type, .; EFE-antisense incubated in air, 0; or EFE-antisense incubated in air with 20~ 1.1 ethylene, 0 ).

86

Fruit ripened on the plant

mg 1 3 5 7 29

Fruit ripened off the plant o ..

o -o ..

o

<> -

mg 1

-3 5 7

Days from onset of colour change

29

Figure 3. Changes in the accumulation ofphytoene synthase (pTOMS) mRNA in EFE-antisense fruit. Accumulation of pTOM5 homologous mRNA was quantified at various stages of ripening of fruit on the plant (left panel, wild-type fruit, .. ; homozygous EFE-antisense fruit, t.) or fruit detached at mature-green and ripened in air or ethylene (right panel, wild-type fruit, .; EFE-antisense fruit incubated in air, 0; EFEantisense fruit incubated. in air plus ethylene, 0). Graphs represent data obtained from densitometric analysis of slot-blots shown below each paneL

2.2. COMPLEMENTATION OF THE YELLOW FLESH MUTANT PHENOTYPE

There are a number of known tomato fruit colour mutants which result from blockages in the carotenoid synthesis pathway. For example, the yellow flesh mutant is unable to synthesize the red pigment Iycopene due to a block early in the pathway. The fruit of these plants ripen to a yellow colour and their flowers are pale yellow (Jenkins, 1955, Darby, 1978).

Two pTOM5 genes have been cloned from tomato, GTOM5, from which pTOM5 mRNA is probably transcribed, and another gene, clone F, which has only limited homology (Ray et ai., 1992). Genomic DNA blots probed with pTOM5 indicated that there is a rearrangement of the GTOM5 sequence in the yellow flesh mutant genome, and gel-blot analysis of the pTOM5 mRNA

87

showed a difference between the size oft~e mutant and wild-type messages, such that an mRNA of 850 nucleotides in yellow flesh and 1600 in the wild-type hybridized to pTOM5 (Fray, unpublished data).

To characterise the molecular basis of the yellow flesh mutation, the mutant pTOM5 mRNA was cloned and sequenced. First strand eDNA was made from mutant fruit mRNA and the pTOM5 messages were amplified by peR. The mutant pTOM5 mRNA was found to contain a region of sequence at its 3' end that was not derived from the wild-type message. When this novel sequence from the yellow flesh message was used to probe a gel-blot of digested genomic DNA an intensely hybridizing ladder of bands was seen (Fray, unpublished data). This hybridizing DNA sequence could represent a highly abundant motif such as a transposable element whose transposition may have originally been the cause of the yellow flesh mutation.

The results of the DNA and RNA blots, and the sequence data, demonstrated that the yellow flesh GTOM5 gene and its mRNA are different from those of the wild-type tomato. This, together with the knowledge that pTOM5 encodes an enzyme involved in carotenoid biosynthesis, suggested that the yellow flesh mutant phenotype may have arisen from a lesion in a pTOM5 gene. To confirm that a defective phytoene synthase gene is responsible for the yellow flesh phenotype, the mutation was complemented by constitutively expressing a pTOM5 transgene in the yellow flesh background. yellow flesh explants were transformed with a sense pTOM5 construct and the resulting transformed mutant plants showed a range of ripe fruit colouration from yellow fruit, indistinguishable from the parent plant, to full red pigmentation (Figure 4). As over-expression of a pTOM5 eDNA clone in transgenic plants restored synthesis of the carotenoid lycopene in ripening fruit, it therefore follows that the yellow flesh mutant phenotype is due to the production of aberrant mRNAs for phytoene synthase. These studies on the manipulation of the expression of the pTOM5 genes, or other genes in the carotenoid pathway, may make it possible to enhance the colour of existing tomatoes.

Figure 4. Complementation of the yellow flesh mutant phenotype mRNA. Three yellow flesh lines transformed with a pTOM5 construct showing no complementation (left), partial (centre) or full complementation (right) of the mutant phenotype. Fruit were photographed when fully ripe.

88

3. Summary

In this chapter we have highlighted the use of transgenic tomato plants to study cloned genes both of known and unknown function. Such experiments with the pTOM13 and pTOM5 ripeningrelated cDNAs have led to their identification as encoding the ethylene-forming enzyme and phytoene synthase. The pTOM13 experiments have led to the creation of novel plants with a low ethylene phenotype which have altered fruit ripening and leaf senescence properties. These plants will be of considerable use in studying the role of ethylene in many plant developmental processes. Finally, the identification of phytoene synthase genes has allowed the complementation of a previously characterised but molecularly undefined ripening mutation yellow flesh.

4. Acknowledgement

This work was supported by the Agricultural and Food Research Council.

5. References

Armstrong GA, Alberti M, Hearst JE (1990). 'Conserved enzymes mediate the early reactions of carotenoid biosynthesis in nonphotosynthetic and photosynthetic prokaryotes.' Proc Natl Acad Sci USA, 87: 9975-9979.

Bird CR, Ray JA, Fletcher JD, Boniwell JM, Bird AS, Teulieres C, Blain I, Bramley PM, Schuch W (1991). 'Using antisense RNA to study gene function: Inhibition of carotenoid biosynthesis in transgenic tomatoes.' Bio-technology 9: 635-639.

Darby LA, Ritchie DB, Taylor IB (1978). 'Isogenic lines of the tomato 'Ailsa Craig'.' Glasshouse Crops Res Ann Rep 1977: 168-184.

Gray JE, Picton S,Shabbeer J, Schuch W, Grierson D (1992). 'Molecular biology offruit ripening and its manipulation with antisense genes'. Plant Molecular Biology 19: 69-87.

Hall LN, Tucker GA, Smith CJS, Watson CF, Seymour GB, Bundick Y, Boniwell JM, Fletcher JD, Ray JA, Sc1W~h W, Bird CR, Grierson D (1993). 'Antisense inhibition of pectin esterase gene expression in transgenic tomatoes.' The Plant Journal. In Press.

Hamilton AJ, Lycett GW, Grierson D (1990). 'Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants.' Nature 346: 284-287.

Hamilton AJ, Bouzayen M, 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.

Jenkins JA, MacKinney G (1955). 'Carotenoids of the apricot tomato and its hybrids with yellow and tangerine.' Genetics 40: 715-720.

Oeller PW, Wong LM, Taylor LP, Pike DA, Theologis A (1991). 'Reversible inhibition of tomato fruit senescence by antisense l-aminocyc1opropane-l-carboxylate synthase.' Science 254: 437439.

Picton S, Barton SL, Bouzayen M, Hamilton A Grierson D (1993). 'Altered fruit ripening and leaf senescence in tomatoes expressing an antisense ethylene-forming enzyme transgene.' The Plant Journal. In press

Sheehy RE, Kramer M, Hiatt WR (1988). 'Reduction of polygalacturonase activity in tomato fruit by antisense RNA.' Proc Natl Acad Sci USA 85: 8805-8809.

89

Smith CJS, Watson CF, Ray J, Bird CR,lvlorris PC, Schuch W and Grierson D (1988). 'Antisense RNA inhibition of polygalacturonase gene expression in transgenic tomatoes.' Nature 334: 724-726.

Smith CJS, Watson CF, Bird CR, Ray J, Schuch W, Grierson D (l990a). 'Expression of a truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants.' Mol Gen Genet 224: 477-48l.

Smith CJS, Watson CF, Morris PC, Bird CR, Seymour GB, Gray JE, Arnold C, Tucker GA, Schuch W, Harding S, Grierson D (1990b). 'Inheritance and effect on ripening of antisense polygalacturonase genes in transgenic tomatoes.' Plant Mol BioI 14: 369-379.

Tieman DM, Harriman RW, Ramamohan G, Handa AK (1992). 'An antisense pectin methylesterase gene alters pectin chemistry and soluble solids in tomato fruit.' The Plant Cell 4: 667-679.

CONVERSION OF ]-AMINOCYCLOPROPANE-]-CARBOXYLIC ACID TO ETHYLENE AND ITS REGULATION BY CALCIUM IN SUNFLOWER PROTOPLASTS

e. BAILLYl, F. CORBTNEAUl, .T.P. RONA2. D. COMEl 1 Physiologie Vegetale Appliquee 2ElectTophysiologie des membranes UniveTsite Pierre et Marie Cnrie URA CNRS 1180 TOUT 53, leT etage Universite Paris VII 4, place Jussieu 2, place Jussieu 75252 Paris cedex 05 75251 Paris cedex 05 . France France

The conversion of l-aminocyclopropane-l-carboxylic acid (ACC) to ethylene is known to correspond to a reaction of oxidation catalysed by an enzymatic complex referred to as ethyleneforming-enzyme (EFE) (Yang and Hoffman,1984). The aims of the present work were to investigate the main characteristics and the regulation by calcium of EFE activity in protoplasts isolated from hypocotyls of sunflower (Helianthus annuus L.) seedlings.

[n the air and in the presence of 1 mM ACC, ethylene production by protoplasts depended on their origin along the hypocotyl. It increased from the base (0.59 ± 0.11 nl h-l 1(1i protoplasts-1) to the top (1.52 ± 0.47 nl h-1 1(1i protoplasts-1). Therefore, in all experiments the complete hypocotyls, the length of which was 9-10 cm, were used.

In the air, ethylene production was very low without supply of exogenous ACC, but it increased with increasing concentration of ACC and was maximal (ca 1.1 ± 0.2 nl h-1 1(1i protoplasts-1) in the presence of 1 to 10 mM ACC. Calculated Km for ACC, using Hill equation, was 14 11M. The conversion of ACC to ethylene was impossible in complete anoxia, but it increased with increasing oxygen tension, reaching a maximum (ca 0.8 ± 0.1 nl h-1 1(1i protoplasts-1)in atmospheres containing 21 to 50% .oxygen. The Km value for oxygen, estimated from Hill plot, was 8.9%.

Exogenous CaCl2 strongly enhanced ACC conversion to ethylene at the concentrations of 0.5-5 mM (Fig. 1). Higher concentrations had no effect. Increased exogenous CaCIz resulted in a rise in intracellular Ca++ (Fig. 1) and in respiration of protoplasts, but did not significantly affect the affinity of EFE for ACe. BaCl2 also enhanced the conversion of ACC to ethylene, whereas KCl and MgCl2 had no effect.

Increased exogenous CaCl2 induced depolarization of protoplast membrane, however results obtained with 2,4 DNP and vanadate suggested that the membrane potential value was not directly involved in ACC conversion to ethylene (fable I).

ACC dependent ethylene production was clearly inhibited by the calmodulin antagonists TFPand W7. Bepridil, an inhibitor of Ca++ channels, also reduced ethylene production.

The results obtained clearly show the large dependence of ethylene production rate on ACC and oxygen concentrations. Km values are in agreement with those determined in other plant materials (Yang and Hoffman, 1984; Yip et al., 1988). As in sunflower seedling hypocotyls (Bailly et al., 1992), Ca++ ions seem to be involved in the activity of EFE in protoplasts. However, the

90

J. C. Pech et al. (eds.J, Cellular and Molecular Aspects of the Plant Honnone Ethylene, 90-91. © 1993 Kluwer Academic Publishers.

91

200 2.0 ~ -; •

180 is. S

.::- 1.5 e g Go c 160 . 0 CI .. .. '0 140 1.0 III

t .a + . 120 + • :z: u .. 0.5 u

100 'i .. I!

60 0.0 i

CaCIZ (M)

Figure 1. Effects of exogenous CaCl2 concentration on ethylene production by protoplasts incubated in the air in the presence of 1 mM ACC (I), and on the intracellular concentration of Ca++ (2). Means of 5 measurements ± SD. In the control medium containing 0.1 mM CaCl2 the ethylene production was 0.70 ± 0.05 nl h-l Ia; protoplasts-1.

TABLE 1. Effects of exogenous CaCI2, 2,4 DNP and vanadate on ethylene production by protoplasts and on the membrane potential Incubation medium Ethylene (nl h-l

106 proto.-1)

CaCl2 2,4 DNP Vanadate (ruM) (ruM) (mM) 0.1 (control) 0 0 0.70 ± 0.05 1.0 0 0 1.21 ± 0.09 0.1 0.1 0 0.08 ± 0.02 0.1 1.0 0 0.03 ± 0.01 0.1 0 0.05 1.14 ± 0.15 0.1 0 0.5 0.98 ± 0.08

Membrane potential (m V)

-17.21 ± 1.80 -4.66 ± 1.14

-11.50 ± 3.00 -4.31 ± 2.00

-15.97 ± 4.00 -6.74 ± 2.80

depolarization of membrane cannot be the explanation of the stimulatory action of Ca++. The inhibitory effects ofTFP and W7 suggest that calmodulin, then protein phosphorylation, is engaged in the regulation ofEFE activity.

Bailly, C., Corbineau, F. and Come, D. (1992) 'The effects of abscisic acid and methyl jasmonate on l-aminocyclopropane-l-carboxylic acid conversion to ethylene in hypocotyl segments of suntlower seedlings, and their control by calcium and calmodulin', Plant Growth Reg. 60, in press. Yang, S.F. and Hoffman, N.E. (1984) 'Ethylene biosynthesis and its regulation in higher plants', Annu. Rev. Plant. Physiol. 35, 155-189. Yip, W.K., Siao, X.Z. and Yang, S.F. (1988) 'Dependence of in vivo ethylene production rate on l-aminocyclopropane-l-carboxylic acid content and oxygen concentrations', Plant Physiol. 88, 553-558.

ISOLA nON OF A RIPENLl\lG AND WOUND~INDUCED eDNA FROM Cucumis melo L.WITH HOMOLOGY TO THE ETHYLENE FORMING ENZYME

e.F. WATSONl, e. BALAGUE2, A.J. TURNERl, J.e. PECH2 and D. GRIERSONl.

1 Departemellt of PES, University of Nottingham, Sutton Bonington,

LOllghborollgh, UK. 2 ENSA T, Laboratoire Ethylene et Maturation des Fruits, Toulouse, France

Ethylene is an important plant growth regulator which is synthesized naturally during climacteric fruit ripening [Yang and Hoffmann, 1984]. The ability to extend the storage life and prevent spoilage of climacteric fruit by inhibiting ethylene biosynthesis has already been demonstrated in transgenic tomato plants expressing antisense EFE [Hamilton et a1., 1990] and ACC synthase [Oeller et a1., 1991] genes. In order to try and extend the storage life of melon fruit by inhibiting ethylene synthesis our initial aim was to isolate an EFE clone from a climacteric melon fruit cDNA library, using the heterologous pTOM13 (EFE) probe from tomato.

One melon clone, pMEL1, hybridized strongly to pTOM13 and contained a 1230 bp insert with a predicted open reading frame of 318 amino acids. The deduced amino acid sequence of pMEL1 exhibited a high degree of conservation with the EFE and relalied genes from tomato (eth1, eth2, and pHTOM5: 78%, 80%, and 82% respectively), and avocado (pAVOE3: 71%). pMEL1 was used as a hybridisataion probe in northern analysis of total RNA from unwounded leaf, preclimacteric and ripe fruit and from wound-induced ripe melon fruit and leaf tissue. A single RNA transcript approximately 1.3kbp in size was detected in ripe fruit and wound-induced .fruit and leaf tissue. Maximal expression of pMEL1 RNA occurred in wound-induced ripe fruit (Fig.l, lane 6). Using an antibody raised against tomato EFE (pTOM13) overexpressed in E. coli, a single 35.5 kDa protein was recognized by the EFE antibody on western analysis of total protein extracts from ripe tomato and melon fruit (Fig. 2, lanes 1 and 2). A 35 kDa polypeptide translated from in vitro transcribed pMEL1 was immunoadsorbed by anti-EFE serum and analyzed by SDSPAGE (Fig. 2, lane 4). This immunoadsorbed polypeptide product was very similar in size to the predicted 35.3 kDa pMEL1 cDNA protein product. Northern analysis of RNA extracted from melon leaf and fruit tissue indicates that pMEL1 hybridizes to a mRNA expressed in response to tissue wounding and the fruit climacteric. It is unclear whether these transcripts are transcribed from the same or two different but related genes. The derived protein sequence of pMEL1 is highly conserved with that of the tomato EFE gene ethl [Kock et a1., 1991]. Additionally we have shown that the 35 kDa in vitro translation polypeptide product from pMEL1 and a 35.5 kDa protein from a ripe melon crude protein extract

92


93

cross react with a polyclonal antibody raised against tomato EFE. Collectively these results suggest that there is a high degree of structural conservation between EFE in tomato and melon, and that pMELI encodes a melon EFE that undergoes very little posttranslational modification.

By transforming melon plants with an antisense pMEL 1 transgene it will be possible to demonstrate whether reduced levels of pMELI RNA lead to an inhibition of ethylene biosynthesis and a delay in the onset of additional ripening parameters.

2 3 4 5

Figure 1.

6

Figure 2.

KDa

69

30

FIGURE 1. Northern blot of total RNA from unwoundeu melon leaves (lane 1) and wounded leaves (lane 2), unripe (lanes 3, 4) and ripe melon fruit (lane 5), and wound-induced ripe melon fruit (lane 6). The northern blot was hybridized to the pMELI cDNA insert.

FIGURE 2. Western blot detection of ammonium sulphate precipitated total proteins from ripe tomato (control, lane 1), and ripe melon (lane 2) fruit using a tomato EFE antibody. Polypeptide products translated from in vitro transcribed pMELI (lane 3) were immunoadsorbed by anti-EFE serum using a Protein A-sepharose affinity colums (lane 4). and analyzed by SDS-PAGE. Markers (lane 5).

REFERENCES.

Hamilton et aI., 1990. Nature 346, 284-287. Kock et aI., 1991. Plant Mol. BioI. 17, ]41-142. Oeller et aI., 1991. Science 254, 437-439. Yang and Hoffmann, 1984. Annu. Rev. Plant. Physiol. 35, 155-189.

ISOLA TION AND CHARACTERISATION OF ETHYLENE-FORMING ENZYME GENES FROM MELON

J.H.BVLL, E.LASSERRE, S.BRAME, J.C.PECH. ENSAT 145 avenue de 11luret. 31076 Toulouse Cedex. France.

We aim to study the regulation of the melon (Cucumis melo L) Ethylene-Fonning Enzyme (EFE) gene men a, which encodes a cDNA (pMELl) known to be expressed in fruit (Watson et al. poster 16, this meeting). This cDNA clone exhibits strong homology, both at the nucleotide and amino-acid levels, to the tomato clone pTOMI3, shown to express EFE in yeast (Hamilton et al. 1991).

Melon is a climacteric fruit, though with comparatively poor methods for controlling post-harvest maturation. We plan to assess melon ripening control by antisense RNA : antisense pMELl may be more efficient at inhibiting ethylene production than antisense pTOMI3. In addition, controlling this expression by use of pMELl's own, putative fruit-specific, promotor would minimise effects of ethylene reduction on the development of the plant. To this end we have obtained clones of genomic DNA, by library screening and inverse PCR (IPCR), containing promotor fragments.

Positively hybridising clones from a genomic library have been obtained, and are now undergoing characterisation by sequencing. As judged by Southern blotting, 2 different genes have been obtained. We have also isolated and fully sequenced a PCR-generated fragment using primers homologous to the ends of pMEL 1 cDNA. This generated a fragment of about 1.5 kb compared to the cDNA of 1.2 kb. It contains 3 introns, which have Igt .... ag/ borders and which are in identical positions to those contained in the tomato genes GTOMA (Holdsworth et al. 1987) and ethl (Koch et a1.l991) indicating a 3-intron ancestral gene. The genomic and cDNA sequences contain two small differences in the 3' untranslated region. These are insertions in the genomic sequence of one and six base pairs, probably due to cultivar divergence.

For promotor isolation we have found inverse IPCR to be a useful technique. This amplifies DNA adjacent to a known sequence. Using IPCR we have obtained putative promotors fragments of about 270 and 800 base pairs. So far only the shorter of these has been fully sequenced. It contains putative CAAT and TATA boxes at about -140 and -120 relative to the first ATG translation initiation codon. Upstream of these are a region of CTT repeats, and a GAAnnTTC sequence, found in heat shock promotors in tomato and yeast (Sharf et al. 1990). The structureofmella is given in figure 1.

Transient transfonnation assay following microprojectile bombardment is a tested technique for dissecting specific promoter activity in plant organs and tissues. Ideally, using a promoter -GUS fusion, the number and intensity of blue spots, following incubation with the chromogenic substrate X-gluc, approximate to the strength of a given promoter in different tissues (Hamilton

94

1. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, 94-95. © 1993 Kluwer Academic Publishers.

95

Nsi! CT l'epeats

ATG

CAAT TATA ~ ~ I ...... ~,/ I I I I

sil 38 Bell !>lsi!

~G(E ~ ~ ~ ., ( ~~~~~I-4~ ____ ~~~_[====J-----

-217 o " 210 437 543 I

871 1003 1293 1541

Figure 1. Stmcture ofmella showing exons (open bars), introns, features of the promoter sequence, and restriction sites and primers (numbered arrows) used for PCR.

et al. 1992). Accordingly, mella 5' upstream sequence was amplified by PCR and subcloned into a derivative of plasmid pBIlOI (Clontech), upstream of the GUS reporter gene and nos terminator. This plasmid was used in transient expression assays following shooting of DNA coated tungsten particles into melon tissues. The results showed that this fragment directs GUS expression in all tissues tested (young leaves, roots and stems, and ripe fruit), despite its supposed role in controlling expression in fruit. Northern blots of Watson et al. (posterl6) indicated that pMELl, or a close relative of it, is induced by wounding as well as in fruit ripening, raising the possibility of induction by bombardment itself. Further dissection of the promotor together with transient assays may allow us to demark sequences involved in directing fruit or wound expression. In addition we are attempting to introduce reporter-gene and antisense constmcts stably into melon plants following Agrobacterium- mediated transformation.

Hamilton AJ, Bouzayen M, Grierson D (1991) Identification of a tomato gene for the ethyleneforming enzyme by expression in yeast; Proc. NatL Acad. Sci. 88, 7434-7437. Hamilton DA, Roy M, Rueda J, Sindhu RK, Sanford J, Mascarenhas JP (1992) Dissection ofa pollen-specific promoter from maize by transient transformation assays. Plant Mol. BioI 18, 211-218. Holdsworth, MJ, Schuch W, Grierson D. (1987) Nucleotide sequence ofan ethylene-related gene from tomato. Nucl. Acids Res li 10600 Kock M, Hamilton A, Grierson D. (1991) ethl, a gene involved in ethylene synthesis in tomato. Plant Mol BioI. 17141-142 ScharfKD, Rose S, Zott W, SchOff F, Nover L (1990) Three tomato genes code for heat stress transcription factors with a region of remarkable homology to the DNA binding domain of the yeast HSF. EMBO J 9,4495-4501.

IMMUNOCYTOLOCALISATION OF ACC OXIDASE IN TOMATO FRUITS.

e. ROMBALDII, M. PETITPREZI, le. CLEYET-MAREU,

P. ROUGE3, A. LATCHEI, J.C. PECHI and lM. LELIEVREI* iENSAT, 145, Av.de MUTet F-31076 TOULOUSh~' 2INRAIENSAM, 9, Place ViaJa, F-34060 MONTPhLLlhR; 3paculte de Pharmacie, UPS, G.des maraichcrs, F-31066 1VULOUSh:' "Present address: INRA, F-84140 MONFFA VET.

ACC oxidase has long been considered as an insoluble membrane-bound enzyme. It is now clear that it is in fact soluble [1], but its subcellular localization has not been elucidated yet. However, we had demonstrated earlier that plant cells can generate ethylene from either apoplastic or intracellular ACC, indicating that two potential sites exist for ethylene formation [2].The discovery that the pTOM13 cDNA was encoding EFE in tomato fruits [3] opened new possibilities for the study of ACC oxidase. Two types of polyclonal antibodies were raised against: (i) a synthetic peptide (VEKEAZEESTQUY) considered as a suitable epitope by secondary structure predictions and (ii) a recombinant ACC oxidase polypeptide overproduced in E coli from pTOM13. Antibodies were purifed by affinity column chromatography and eventually saturated with excess of antigenes. Periearp tissue of both normal and transgenic tomatoes transformed with antisense pTOM13 cDNA (kindly provided by A Hamilton and D. Grierson, Nottingham, UK) were processed for immunoc}10localization as described in [41.

Immunofluorescence labelling of pink tomato cells shows that ACC oxidase is largely located at the cell wall (Fig. IA) as demonstrated by superimposition with calcofluor white staining (Fig. IB). Immunogold labelling used as an alternative method (Fig. 10) and toluidine blue staining used for visualisation of cell structure (Fig. IE) lead to the same conclusion. Cells treated with saturated antibodies (Fig. 1 C) or with the pre-immune serum (not shown) gave very weak autofluorescence background. Interestingly, transgenic tomatoes with reduced expression of ACC oxidase and allowed to develop a pink color after harvest exhibited considerable reduction of labelling (Fig. IG) as compared to normal pink tomatoes (Fig. IF). Electron microscopy performed on tissues fixed in paraformaldehyde 2% and glutaraldehyde 0.2% also demonstrates that the immunolabelling is mostly located at the cell wall (Fig. I). Cells treated with saturated immumserum exhibit no labelling (Fig 11). A view of cell ultrastucture after osmium tetraoxide fixation is given in figure IH. References: 1- Ververidis P., John P. (1991) Phytochemistry, 30:725-727. 2- Bouzayen M., Latche A., Pech I.C. (1990) Planta, 180: 175-180. 3- Hamilton A., Bouzayen M., Grierson D. (1991) Proc. Natl. Acad. Sci. USA, 76:7434-7437. 4- Vandenbosch K.A. (1991) In: Electron microscopy of plant cells (lL. Hall and C. Hawes ed.). Acad. Press, New York, p 181-218.

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97

Figure 1: ImmunocytoJocalization of ACC oxidase in tomato fruit. Labelling with: A: antisynthetic peptide antibodies (PAb) + fluoresceine isothiocyanatc (FITC); B: calcofluor white (same cells as in A); C: saturated PAb + PITC; D: anti-recombinant EFE antibodies (RAb) + immunogold (IG); E: toluidine blue (same cells as in D); F: RAb + IG of normal and G: antisense tomatoes (epipolarized light). Electron microscopy ofH: ultrastructure, 1: RAb + IG and J: saturated RAb + IG (same cells as in I). v: vacuole; cw: cell wall; cyt: cytoplasm; __ : gold particles.

BIOCHEMICAL AND IMMUNOCYTOLOOICAL CHARACTERIZATION OF ACC OXIDASE IN TRANSGENIC GRAPE CELLS.

RA AYUB, C. ROMBALDl, M. PETIPREZ, A. LATCHE, lC. PECH and lM. LELIEVRE* hNSA T, 145, A v. de MUTe!, 31076 Toulouse, France; "Prescnt addrcss:INRA Technologic, 84140 Mantmvel, France.

We have previously shown that grape cells (Vilis vinilera L. cv Gamay) grown in vitro generate ethylene mostly from intracellular ACC [ll In contrast ripening tomato fruits synthesize ethylene mainly from the apoplasm [2 J. In the present work, we have transformed Gamay grape cells with the tomato pTOM13 cDNA encoding tomato ACC oxidase and corresponding to a mRNA highly expressed during ripening 13]. The aim of this study is to determine where the pTOMl3-encoded protein is targeted in transgenic grape cells.

Cells were co-eultured in the presence of A. tumcmcicns containing the binary vector pGA643 and the pTOM 13 insert under the control of the 35S promotor. After 24h the bacterial cells were washed off and the plant cells were plated on solid medium containing 150J..lg/ ml of kanamycin and 750J..lg/ ml of carbenicillin. Preparation of antiserum and immunoblotting were performed as in [4) and immunocytolocalization as in [2]. Southern hybridization performed under high strigcncy conditions revealed that the pTOM13 probe hybridized to DNA extracted from kanamycinresistant cells but not from untransformed cells (data not shown), thus demonstrating that cells were transgenic.

Ethylene production of transformed cells is about 2-fold higher during the exponential growth phase (days 3 to 6) than that of untransformcd cells (Fig. I). Immunoblot analysis of the 100 OOOg supernatant of protein extracts show that ACC oxidase protein is much more abundant in transformed than in 1U1transformed cells (data not shown). Ethylene formation of untransformcd cells was inhibited by about 30% by plasmolysis (Table I) while transformed cells were by far less sensitive (14%). These data indicate that the rise in activity due to transformation was probably due to an increase in the internal plasmolysis-insensitive ACC oxidase; apoplastic ethylene production being plasmolysis-sensitive [I]. Light microscopy imm1U1ocytolocalisation revealed a -major site in the cytoplasm and a minor one at the cell wall in both types of cells. However ACC oxidase protein appeared to be more ab1U1dant in the cytoplasm of transformed cells, indicating that accumulation of the pTOM13 protein has occured in the cytoplasm.

These data are in agreement with the absence of a consensus signal sequence for excretion of the pTOM13-encoded protein. However, in ripening tomatoes, ACC oxidase is mainly located at the apoplasm [2]. How ACC oxidase is targeted to this compartment in ripening fruits and not in transgenic cells in culture remains an open question.

98 J. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, 98-99. © 1993 Kluwer Academic Publishers.

99

16r---------------__ ~ 1 4

Table I: Effect of plasmolysis (0.8M sorbitol) on ethylene production by transformed and untransformed grape cells

Ethylene production inhibition

?

O ~O --~?C---~~----~6--~a I)A VS

Figure I: Ethylene production of un- Untransformed

transformed ( ) and transformed () Transformed

grape cells during the log phase of

the growth cycle.

nl. h-1. gFW-l % unplasmolyzed plasmolyzed

4.8 ± 0.1

9.2 ± 0.3

3.3 ± 0.1

7.9 ± 0.6

31

14

Fig 2: Immunocytolocalization of ACC Oxidase by fluorescence labelling (FITC) in untransformed (A) and transformed (8) cells treated with antibodies raised against the recombinant ACC Oxidase (x 100).

References

1- Bouzayen, M., Latche, A., Peeh, le. 1990, Planta, 180, 175-180. 2 - Rombaldi, e., Petitprez, M., Cleyet-Marel, le., Rouge, P. Latehe, A., Pech, le., Lelievre, lM. (1993). Immunocytolocalisation of ACC oxidase in tomato fruits. In: Cellular and Molecular Aspects of Plant Hormone Ethylene (le. Pech, A. Latche & e. Balague, eds) KIuwer Acad. Pub., Dordrecht, The Netherlands, pp. 96-97 3 - K6ek, M., Hamilton, A., Grierson, D. 1991, Plant Mol. BioI. , 17, 141-142 4 - Latche, A., Dupille, E., Rombaldi, e., Cleyet-Marel, le., Lelievre, lM., Pech, le. (1993). Purification, characterization and subcellular localization of ACC oxidase from fruitsJn: Cellular and Molecular Aspects of Plant Hormone Ethylene (le. Pech, A. Latche & e. Balague, eds) Kluwer Aead. Pub., Dordrecht, The Netherlands, pp. 39-45

EFFECT OF E8 PROTEIN ON ETHYLENE BIOSYNTHESIS DURING TOMATO FRUIT RIPENING

Pei'iarrubia, L., Aguilar, M., Margossian, L., and Fischer, R.L. Department of Plant Biology University of California Berkeley, California 94720 USA

ABSTRACT. The ripening of many fruits is controlled by an increase in ethylene honnone concentration. E8 is a fruit ripening protein that is related to a large family of iron (II) dioxygenases. To detennine the function of E8 we have transfonned tomato plants with an E8 antisense gene. We find that reduction of the E8 protein produces an increase in ethylene evolution specifically during the ripening of detached fruit.

1. Introduction

The gas honnone ethylene influences very profoundly the growth and development of plants. It produces effects such as inhibition of growth, loss of geotropic response, acceleration of respiration, onset of rooting, leaf abscission, fruit ripening, and flower senescence. Moreover, ethylene is involved in the response of plants to wounding, pathogen attack, and environmental stress. Ethylene production is influenced by plant hormones, plant metabolites, and a wide variety of environmental stresses (Theologis, 1992).

In fruits such as tomato, the onset of ripening is controlled by an increase in ethylene production (Theologis, 1992). The pathway for ethylene biosynthesis has been elucidated, and the rate-limiting step is the conversion of S-adenosylmethionine to 1-aminocyclopropane-1-carboxylic acid (ACC) by the ACC synthase enzyme. The induction of ethylene production is due to de novo synthesis of ACC synthase. ACC synthase is encoded by a large gene family. In tomato, there are at least six ACC synthase genes, some of which are specifically expressed during fruit ripening. The expression of multiple ACC synthase genes at the onset of tomato fruit ripening results in increased production of ACC. ACC is then oxidized to ethylene by ACC oxidase, also called the ethylene forming enzyme. Inhibition of ethylene biosynthesis by production of antisense RNA to ACC synthase (Oeller et aI., 1991) and ACC oxidase (Hamilton et aI., 1990), or by deamination of ACC (Klee et aI., 1991), represses tomato fruit ripening. Thus, ethylene plays an essential role in tomato fruit ripening.

Ethylene regulates its own biosynthesis (Yang and Hoffman, 1984). In ripening the feedback is positive, whereas in wound-induced ethylene production the feedback is negative. In most cases it is thought that ACC synthase activity determines the rate of ethylene biosynthesis, although there are competing reactions that may serve to reduce ethylene production. In certain situations during plant development ACC is

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101

removed by malonylation (Liu et al.I 1985) and ethylene is oxidized to a variety of products (Hall, 1991).

To begin to understand the mechanism of ethylene action during ripening, we have cloned and analyzed a variety of ethylene-responsive genes. One gene, designated E8, is transcriptionally activated at the onset of ripening, coincident with the increase in ethylene biosynthesis. The E8 predicted polypeptide is a member of a family of dioxygenases found in plants and microorganisms (McGarvey et al., 1992), and is related to tomato ACC oxidase, sharing 34% amino acid sequence identity over 295 residues (Deikman and Fischer, 1988). We have inhibited E8 mRNA and protein accumulation by producing E8 antisense RNA in transgenic tomato plants (Penarrubia et al., 1992). We find that a reduced level of E8 protein results in overproduction of ethylene during the ripening of detached tomato fruit.

2. Results

2.1 ETHYLENE OVERPRODUCTION IN FRUIT FROM TRANSGENIC PLANTS

To determine if E8, like the related ACC oxidase, plays a role in ethylene production, we measured the rate of ethylene evolution in fruit from transgenic plants. As shown in Figure I, at the onset of ripening (day 0), fruit from two independently transformed tomato lines (line number 125 and 191) and from untransformed plants evolved equivalent rates of ethylene. However, 2 days later, the rate of ethylene evolution was approximately 6-fold higher in fruit from transgenic plants compared to untransformed controls. The effect of the antisense gene was transitory, and by the fifth day the rate of ethylene evolution was essentially the same in fruit from transgenic and untransformed plants. These results suggest that inhibition of E8 protein accumulation in detached ripening fruit results in ethylene overproduction. Moreover, we have shown that ethylene overproduction cosegregates with a reduction in E8 protein (Peiiarrubia et al., 1992). Taken together, these results make it very unlikely that ethylene overproduction is due to somaclonal variation.

50

40 ....... ] 30 b.O '-'

........ 20 ~

10

0 0 1 2 3 4 5 6 7

Days

102

Figure 1. Ethylene evolution rate of f\1lit from independently transformed lines. Fruit were harvested at the onset of color change and the rate of ethylene evolution was measured daily. (.), ethylene evolution rate of fruit from line number 125 transgenic plants (N=77); (.), ethylene evolution rate of fruit from line number 191 transgenic plants (N=4); (0), ethylene evolution rate of fruit from untransformed plants (N=15).

2.2 ETHYLENE OVERPRODUCTION OCCURS SPECIFICALLY DURING FRUIT RIPENING

Many plant tissues, including fruit, that are exposed to abiotic or biotic stress rapidly produce high levels of ethylene (Hyodo, 1991). To begin to distinguish between fruitspecific and stress-induced ethylene overproduction, fruit were harvested well before the onset of ripening and ethylene evolution rate was analyzed daily. As shown in Figure 2, the antisense gene had no effect on ethylene production in unripe fruit. That is, the ethylene evolution rate for fruit from both transgenic and un transformed plants was the same, 0.1 nl/(g hr), for 4 days before the onset of ripening. However, after the onset of ripening, ethylene production in fruit from transgenic plants was approximately 6-fold higher than untransformed controls. As with fruit harvested at the onset of ripening (Figure 2), ethylene overproduction in fruit from transgenic plants was transitory, lasting approximately two to three days. These results suggest that the effect of the E8 antisense locus on ethylene biosynthesis is specific to fruit ripening and does not represent a generalized response to stress associated with harvest and/or storage.

30

.......... 20 M

..c:: co ......, ...... -~ 10

0 -4 -3 -2 -1 0 1 2 3

Days

Figure 2. Ethylene evolution rate of fruit from transgenic plants harvested prior to the onset of ripening. Unripe fruit were harvested approximately 4 days before the onset of color development and ethylene evolution rate was measured daily. (.), Ethylene evolution rate of fruit from line number 125 transgenic plants (N=11); (0), ethylene evolution rate of fruit from un transformed plants (N=5).

3. Discussion

3.1 RELATIONSHIP BETWEEN E8 AND DIOXYGENASES

103

Although we have not yet analyzed the E8 protein biochemically, DNA sequence analysis indicates that E8 is a member of a family of dioxygenase enzymes: flavanone 3-hydroxylase, hyoscyamine 6b-hydroxylase, isopenicillin N-synthase and the ethylene forming enzyme (McGarvey et al., 1992). Members of this oxidase family all require Fell and a reductant, usually ascorbate, for catalytic activity. Three histidine and one cysteine residues are conserved throughout the family and may interact with the Fe(II) cofactor. The structural similarities of this extended family suggest that the E8 protein may have a metal cofactor and the ability to react with molecular oxygen and 2-oxoglutarate. The reactions catalized by the enzymes of this large family of dioxygenases are as different as their substrates. The predicted amino acid sequence of E8 is most related to hyoscyamine 6b-hydroxylase (97 matches out of 363 residues) that requires iron (II), ascorbic acid, and 2-oxoglutarate for maximum activity. The ethylene forming enzyme is also a member of this family and is related to E8 (McGarvey et aI., 1992). In experiments presented here we show that an E8 antisense gene suppresses the accumulation of E8 protein in fruit harvested from transgenic plants (Figure 2).

3.2 POSSIBLE MECHANISMS FOR E8 ACTION

The effect of E8 is specific to the burst of ethylene produced during the ripening (Figure 1,2). The onset of ethylene overproduction in fruit from transgenic plants is coincident with the smaller increase of ethylene produced in fruit from untransformed plants. Moreover, the burst of ethylene overproduction in fruit from transgenic plants and the smaller burst observed in fruit from untransformed plants are transitory. One possibility is that reduction in E8 protein results in a metabolic imbalance leading to stress-inducible ethylene production specifically during fruit ripening. Alternatively, E8 protein could be involved in negative feedback regulation of ethylene biosynthesis during ripening.

It is well known that ethylene stimulates its own biosynthesis at the onset of ripening (Yang and Hoffman, 1984). However, in a variety of fruit and vegetative tissues, ethylene also negatively controls its own production, a process termed autoinhibition (Yang and Hoffman, 1984). For example, wound-inducible ethylene production is greatly reduced by exogenous ethylene and/or ethylene analogs (Riov and Yang, 1982), and inhibition of ethylene perception either biochemically (Atta-Aly et aI., 1987; Chi et ai., 1991) or by mutation (Guzman and Ecker, 1990) can result in ethylene overproduction. Although the mechanisms of autoinhibition are not fully understood, ethylene has been shown to inhibit its production by reducing the level of ACC, the precursor of ethylene (Riov and Yang, 1982). This may occur by reducing ACC synthase activity (Y oshi and Imaseki, 1982) and/or by promoting the malonylation of ACC to an inactive form, malonyl-ACC (Liu et ai., 1985). In addition, ethylene is oxidized to a variety of products, although recent experiments suggest that in most plants ethylene oxidation does not have a significant effect on ethylene concentration or the mode of action of ethylene (Hall, 1991).

Normally, the rate limiting step for ethylene production is determined by the level of ACC synthase activity. Preliminary experiments suggest that the level of ACC synthase enzyme activity is elevated in E8 antisense fruit (Aguilar and Fischer, unpublished results). One possibility is that the lack of E8 protein constitutes a stress, and that E8 antisense fruit respond by overproducing ethylene. Alternatively, lack of E8 protein may result in impaired ethylene perception, resulting in ethylene overproduction.

4. Methods

4.1 PLANT MATERIAL

104

Tomato seed (Lycoperssicon esculentum cv. Ailsa Craig) were obtained from the Glasshouse Crops Research Institute and plants were grown under standard greenhouse condition. Fruit were harvested at the indicated stage and stored at 28oe.

4.2 DETERMINATION OF ETHYLENE EVOLUTION RATE

Individual fruits were placed in 500 ml containers that were sealed and incubated 1 hour at room temperature. A 1 ml sample from the closed atmosphere was removed, and the ethylene content was determined by gas chromatography (Hach/Carle). In figures, error bars represent standard errors. Where error bars are not shown, the standard error was no greater than the size of the symbol.

5. Acknowledgments

We express our gratitude to Barbara Rotz, John Franklin and Lou Hancock for providing excellent greenhouse services. This research was supported by an National Science Foundation Grant (DCB04353) and a United States Department of Agriculture Grant (91-37304-6507). L. P. was funded by a Fulbright Fellowship from the Spanish Ministry of Science and Education.

6 . References

Atta-Aly, M.A., Saltveit, M.E., and Hobson, G.E. (1987). 'The effect of silver ions on ethylene biosynthesis by tomato fruit tissue'. Plant Physiol. 83,44-48.

Chi, G.-L, Pua, E.-C., and Goh, C.-J. (1991). 'Role of ethylene on de novo shoot regeneration from cotyledonary explants of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro.'. Plant Physiol. 96, 178-183.

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'. The EMBO J. 7, 3315-3320.

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

Hall, M.H. (1991). Ethylene metabolism. In The Plant Hormone Ethylene, Mattoo, A.K. and S4ttle, J.e. eds (Boca Raton: CRC Press) pp. 65-80.

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

Hyodo, H. (1991). The biochemistry of ethylene biosynthesis. In The Plant Hormone Ethylene, Mattoo, A.K. and Suttle, J.C. eds (Boca Raton: CRC Press) pp. 43-63.

Klee, 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'. The Plant Cell 3, 1187-1193.

Liu, Y., Hoffman, N.E., and Yang, S.F. (1985). 'Ethylene promotes the capability to malonate l-aminocyclopropane-l-carboxylic acid and D-arnino acids in preclimacteric tomato fruits'. Plant Physiol. 77, 891-895. .

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McGarvey, D.J., Sirevag, R, and Christoffersen, R.E. (1992). 'Ripening-related Gene from Avocado fruit: ethylene inducible expression of the mRNA and polypeptide'. Plant Physiol. 98, 554-559.

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.

Penarrubia, L., Aguilar, M., Margossian, L., and Fischer, R.L. 1992. 'An Antisense Gene Stimulates Ethylene Hormone Production during Tomato Fruit Ripening'. The Plant Cell 4, 681-687.

Riov, J., 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.

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

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

Yoshi, H., and Imaseki, H. (1982). 'Regulation of auxin-induced ethylene biosynthesis. Repression of inductive formation of ACC synthase by ethylene'. Plant Cell Physiol. 23, 639-649.

EXPRESSION OF A BACTERIAL ACC DEAMINASE GENE IN TOMATO

R. E. SHEEHY, V. URSIN, S. VANDERPAN and W. R. HIATI Calgene Fresh, Inc. 1920 Fifth St. Davis, CA 95616 USA

ABSTRACT. Pseudomonas sp. ACP contains an inducible l-aminocyclopropane-lcarboxylate (ACC) deaminase which allows this organism to grow on ACC as a nitrogen source. ACC is the immediate precursor in the biosynthetic pathway of the hormone ethylene in higher plants. Ethylene regulates a variety of growth and developmental processes, and in particular senescence and fruit ripening. We have isolated a bacterial gene encoding ACC deaminase and chimeric gene constructs which utilize an enhanced 35S promoter have been introduced into tomato. Expression of the ACC deaminase gene in tomato is described.

1. Introduction

Recently, a number of groups have isolated genes in the ethylene biosynthetic pathway [1-4]. Modulation of these genes and ethylene production utilizing antisense RNA expression has been demonstrated [I, 4]. An alternative strategy involves reducing the availability of ACC by a shunt which removes the precursor from the pathway. This occurs naturally in plants by ACC malonyltransferase which forms an inactive derivative of ACC. We and others [5, 6] have cloned genes from Pseudomonas encoding ACC deaminase to create a novel pathway in plants by which ACC is diverted from the ethylene biosynthetic pathway. Here, we describe expression of a bacterial ACC deaminase gene in transgenic tomato as a means of regulating ethylene production and fruit ripening.


2.1 ACC DEAMINASE GENE CONSTRUcr pCGN1493

The ACC deaminase gene was cloned from Pseudomonas sp. ACP as described by Sheehy et al. [5]. The expression cassette, pCGN2187, contains a double cauliflower mosaic virus (CaMV) 35S promoter and the tml3' region. In order to prepare convenient restriction sites for insertion of the ACC deaminase gene, oligonucleotides complementary to bp 625 to 642 of the deaminase sequence [5] and containing a BamHI site, and complementary to bp 1618 to 1641 and containing a SstI site were synthesized. The resulting ACC deaminase gene fragment was inserted into BamHI-SstI-digested pCGN2187.

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J. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plant Honnone Ethylene, 106-110. © 1993 Ktuwer Academic Publishers.

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The completed expression cassette was digested with Pst! and the fragment containing d35S-5'/ACC deaminase/tml-3' was ligated into PstI-digested pCGNl547 [7]. The structure of the resulting plant expression vector, pCGN1493, is shown in Fig. 1. pCGN1494 is identical to pCGN1493 except that it contains only a single copy of the d35S-ACCD-tml 3' cassette.

2.2 ACC DEAMINASE GENE CONSTRUCT pCGN1497

Binary vector pCGN1497 is similar to pCGN1493 except that the DNA sequence surrounding the A TG initiation codon of the ACC deaminase gene was modified to resemble a plant consensus sequence [8]. The BamHI-NheI fragment of pCGNI472 [5] was replaced with a PCR fragment containing adenine at bp -3 and guanine at bp 4 relative to the start of the ORF.

2.3 TOMATO TRANSFORMATION

Binary vectors were introduced into Agrobacterium tumefaciens strain LB4404 and subsequently used to transform tomato by the method of Fillatti et ale [9].

2.4 ANALYTICAL METHODS

Expression of ACC deaminase protein [5] and mRNA [10] were measured as described.

3. Results

3.1. ACC DEAMINASE mRNA 1N TRANSFORMED PLANTS

pCGN1493 (Fig. 1) contains the Pseudomonas ACC deaminase ORF placed adjacent to a CaMV enhanced (double) 35S promoter for high-level constitutive expression.

Transformed tomato plants resulting from cocultivation with the ACC deaminase binary vectors were screened for expression of deaminase mRNA and representatives are shown in Fig. 2. As shown in lanes 1 and 3, an RNA of the expected size was identified in plants transformed with the pCGN1493 and pCGN1494 constructs.

pCGN1497 is identical to pCGN1493 except that the region surrounding the initiation A TG codon has been modified to resemble a consensus plant ribosome binding site. Specifically bp -3 has been replaced with adenine and bp 4 with guanine. As shown in lane 5 of Fig. 2, this modification did not appear to have an effect on the steady-state ACC dearninase mRNA level.

3.2 ACC DEAMINASE LEVELS 1N TRANSFORMED PLANTS

Leaf preparations from transformed plants were further analyzed for levels of ACC deaminase protein. As shown in Fig. 3, a polypeptide of the expected molecular weight and reactive with anti-ACC deaminase serum was present in transformed plants.

\08

pCGN1 493

LB

Figure 1. Binary vector pCGN1493. RB, right border; LD35S, long untranslated leader enhanced CaMV 35S promoter; ACCD, ACC deaminase ORF; TML 3', Agrobacterium tml 3' termination region; mas 3', Agrobacterium mannopine synthase 3' termination region; npt, neomycin phosphotransferase; mas 5', mannopine synthase promoter region; LB, left border.

1 2 3 4 5

Figure 2. Steady state ACC deaminase mRNA levels in leaf tissue from independent transformed tomato plants. Approximately 20 J..Lg of total RNA from leaves was applied to each lane and analyzed by Northern blot analysis with a radioactive ACC deaminase DNA probe. Lanes 1-3 are plants transformed with pCGN1493/94 and lanes 4 and 5 are plants transformed with pCGN1497.

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1 2 3 4 5 6 7

Figure 3. Levels of ACC deaminase in transfonned tomato plants. Leaf extracts were prepared and subjected to Western blot analysis as described [5]. Lanes 1 and 2 are plants transfonned with pCGN1494 and lanes 3-7 are plants transfonned with pCGN1497. The arrow indicates the expected size of ACC deaminase.

A comparison of ACC deaminase protein levels in approximately 200 1493, 1494 and 1497 transfonnants indicated an approximate 5-fold increase in protein levels in 1497 plants. The presence of guanine at bp 4 results in the replacement of asparagine with aspartic acid. E. coli extracts from strains producing each fonn of the enzyme were analyzed for pH optimum and Km values and showed no differences between the original and modified fonns of the enzyme (M. Honma, personal communication).

4. Discussion

We have successfully expressed a bacterial ACC deaminase gene from Pseudomonas sp. ACP in tomato plants. Expression appeared to be enhanced at the translational level by modification of the Kozak consensus sequence, presumably due to enhanced ribosome binding.

The ACC deaminase gene described here is highly conserved relative to the clone described by Klee et al. [6] with 88% homology at the amino acid level and 76% homology at the nucleic acid level. Klee et al. [6] demonstrated that expression of ACC deaminase in tomato reduced ethylene production and delayed fruit ripening.

We demonstrate here that a Pseudomonas sp. ACP gene is effectively expressed in tomato leaves. Such expression results in the decreased biosynthesis of ethylene in wounded leaf tissue (data not shown). Self-pollinated progeny of initial transfonnants homozygous for the ACC dearninase gene are being identified and analyzed for the effect of reduced ethylene biosynthesis on fruit ripening.

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5. References

1. Oeller, P.W., Min-Wong, L., Taylor, L.T., Pike, D.A. and Theologis, A. (1991) 'Reversible inhibition of tomato fruit senescence by antisense RNA', Science 254,437-439.

2. Rottmann, W.H., Peter, G.F., Oeller, P.W., Keller, J.A., Shen, N.F., Nagy, B.P., Taylor, L.P., Campbell, A.D. and Theologis, A. (1991) 'I-AminocyclopropaneI-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.

3. Van Der Straeten, D.V., Van Wiemeersch, L., Goodman, H.M. and Van Montagu, M. (1990) 'Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-l-carboxylate synthase in tomato', Proc. Natl. Acad. Sci. USA 87, 4859-4863.

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

5. Sheehy, RE., Honma, M., Yamada, M., Sasaki, T., Martineau, B. and Hiatt, W.R (1991) 'Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding l-aminocyclopropane-l-carboxylate deaminase', J. Bacteriol. 173, 5260-5265.

6. Klee, 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', The Plant Cell 3, 1187-1193.

7. McBride, K. and Summerfelt, K. (1990)' Improved binary vectors for Agrobacterium-mediated plant transformation', Plant Mol. BioI. 14, 269-276.

&. Lutcke, H.A., Chow, K.C., Mickel, F.S., Moss, K.A., Kern, H.P. and Scheele, G.A. (1987) 'Selection of AUG initiation codons differs in plants and animals', EMBO J. 6, 43-48.

9. Fillatti, J., Kiser, J., Rose, R and Comai, L. (1987) 'Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumejaciens vector', Biolfechnology 5, 726-730.

10. Sheehy, RE., Kramer, M. and Hiatt, W.R (1988) 'Reduction of polygalacturonase activity in tomato fruit by antisense RNA', Proc. Nat!. Acad. Sci. USA 85, 8805-8809.

STEREOSPECIFIC REACTION OF l-AMINOCYCLOPROPANE-l-CARBOXYLATE DEAMINASE

M.HONMA Laboratory of Biochemistry Faculty of Agriculture Hokkaido University Sapporo, 060 Japan

ABSTRACT. Some microorganisms have an inducible enzyme, l-aminocyclopropane-lcarboxylate (ACC) deaminase which catalyzes the degradation of ACC to a-ketobutyrate and ammonia. Bacterial ACC deaminase also degraded (IS, 2S)-2-methyl-, (IS, 2S)-2-ethyl, (IS )-2,2-dimethyl-, and (I S, 2R)-2-methyl-ACC. These reactions are consistent with the deamination of D-serine and a-proton exchange reaction of o-alanine with solvent. The latter reaction was demonstrated by NMR spectra and appearance of 51 Onm band in absorption spectrum. Furthermore the presence of D-alanine made the ACC deaminase more sensitive to thiol reagent, iodoacetamide. A reactive thiol group was identified to be Cys162 in a sequence of 338 residues by modification with N -(iodoacetamidoethyl)-aminonaphthalene sulfonic acid. The homology of sequences between the ACC deaminase and tryptophan synthase [3-subunit is discussed.

Metabolism of l-aminocyclopropane-l-carboxylate (ACC) is achieved in three ways by each of following enzymes[l], ethylene forming enzyme and ACC malonyltransferase in plants and ACC deaminase from microorganisms. The third enzyme catalyzing the fragmentation of ACC to a-ketobutyrate and ammonia was induced in several microorganisms and had not been found in higher plants originally, but a transgenic plant having ACC deaminase was reported to show a reduction in ethylene level in the plants[2]. The ACC deaminase purified homogeneously or partially from Pseudomonas sp. Hansenula saturnus, and Penicillium citrinum showed different ~ values (1.5mM, 2,6mM, and 4.6mM at pH8.5) and the same stereospecificity in cyclopropane bond cleavage. Several stereochemical problems in steps of enzymatic ACC deamination were previously analyzed [3,4], but the mechanism of cyclopropane bond cleavage remains unresolved. A nucleophilic addition/elimination mechanism[5] was suggested to work at pro-S methylene of ACC. The ACC deaminase was found to have a sensitive thiol group which can serve as nucleophile and to be completely inhibited by some thiol reagents[6]. Here is described and discussed modification of the thiol group through the stereospecific reaction of the ACC deaminase, identification of the reactive thiol group, and comparison of the ACC deaminase with tryptophan synthase [3-subunit.

Methods

Enzyme, Chemical Modification, Proteolytic Digestion, and Peptide Isolation. ACC dearninase was prepared from Pseudomonas sp. by the method described previously[7,8]. Deaminase activity was assayed by measurement of a-keto acid with 2,4-dinitrophenylhydrazine[7] .

In order to fix pyridoxal phosphate at a binding site and to modify thiol groups of the enzyme. the ACC deaminase was reduced with sodium borohydride. dialyzed against 0.1 M

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potassium phosphate, pH7.5, denatured in 6M guanidine hydrochloride and 2-mercaptoethanol (1 ~l/mg of protein) over night, and mixed with vinylpyridine (1.5 ~l/mg of protein). After a mixture was allowed to stand for a hour, it was acidified with acetic acid by pH3 and diluted with a equal volume of water.

Modified enzyme was purified by reversed-phase chromatography with C4 column and 0.1 % trifluoroacetic acid-acetonitril (0-40%). A purified protein fraction was evaporated to dryness and dissolved in 8M urea in O.OIM Tris, pH9.0. Lysyl endopeptidase (20 ~g/mg of protein) in a equal volume of 0.01 M Tris, pH9.0, was added and incubated for 24 hours at 30°e.

Peptides in the resulting mixture were separated with C8 column and 0.1 % trifluoroacetic acid-acetonitril (0-40%) and used for protein sequence analysis.

Results

Reaction of ACC Deaminase with ACC Analogs. Previous studies[3,4] on enzymatic dearnination of 2-methyl-ACC and 2-ethyl-ACC indicated that of the four possible stereoisomers, only the IS, 2S isomer was a suitable substrate for the ACC deaminase to convert into a-ketocaproate and a-ketovalerate (K = 18.ImM and 12.5mM, V max = 38% and 16.4% of ACC for (1 S, 2S)-2-ethyl-ACC[3] and (J. S, 2S )-2-methyl-ACC[4], respectively). The regiospecificity of cyclopropane bond cleavage was decided by these results (Fig. 1). Besides these substrate, the ACC deaminase reacted to both racemic mixtures of (IS) and (1R )-2,2-dimethyl-ACC, and of (1S , 2R) and (1 R, 2S )-2-methyl-ACC. The above regiospecificity indicates that the actual substrate are (1 S)2,2-dimethyl-ACC and (1 S, 2R )-2-methyl-ACC (Km = 64.1mM and 3.9ffiM, V max = 1.1 % and 0.04% of ACC, respectively). These reactivities indicate the size of a substrate binding site around the pro-S methylene of ACe. The direction from a-carbon to pro-S methylene carbon in ACC corresponds to that from a-carbon to a-hydrogen in D-serine. This was proved by reaction of D-serine with ACC dearninase to form pyruvate (Km = 19.5mM, V max = 4.74% of ACC).

pro-R

~ \.-IeOOH

~ 0. .-IeOOH R~

I \ \NH. pro-S ' I'Ii. H

2

Fig. 1 ACC (I) and (IS, 2S)-2-alkyl-ACC(2)

Reaction with o-Alanine. As described above, ACC deaminase catalyzed deamination of D-serine, 3-chloro-D-alanine, and O-acetyl-D-serine[5]. Deamination of these substrates depends on proton elimination from a-carbon of each amino acids (Fig. 2). This proton elimination was substantiated by NMR spectrum of D-alanine incubated with the ACC dearninase in D20 (Fig. 3). Figure 3 shows that signals of a-proton of o-alanine disappeared after the incubation and a-proton of L-alanine was not exchanged under the same conditions. The incubation of the ACC deaminase with D-alanine showed the appearance of a new absorption band at 510nm. It is known that the 51 Onm band expresses formation of a quinoidal form of pyridoxal phosphate which generates after a-proton elimination of the aldimine. The absorbance at 510nm depended on concentration ofD-alanine (concentration for half value, Ko.s = 450mMand 310mM at 20°C and 30°C, respectively).

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003PO H~' H

N H H

N \ OH 'COOH

~3PO

N ' ,H N~

\

OH COOH

ACC [)- serine

Fig. 2. Aldimine complexes of ACC and D-serine

L-alanine D-alanine

5 4 3 2 o 5 4 3 2 o ppm

Fig. 3. NMR spectrum of alanine incubated with ACC deaminase in Dp. The ACC deaminase, 5.35 units / 200111, and 1M L- or o-alanine were incubated in D20 containing 0.05M potassium pyrophosphate, pD8.S, and ImM NaN3 for 4 days at 30°C.

Sensitive Thiol Group of the ACC Deaminase. It was already reported that the ACC deaminase was almost completely inhibited by 2mM S,S'-dithiobis (2-nitrobenzoic acid) at O°C and restored by lOmM dithiothreitol[6]. In case of iodoacetamide, concentration of SmM caused just 30% inactivation after incubation for 120min at pH8.5 at 20°C. However, the presence ofD-alanine stimulated the inactivation by ImM iodoacetamide remarkably as shown in Fig. 4, while L-alanine served protectively against the inactivation. The rate of inactivation stimulated by D-alanine obeyed first-order kinetics and the concentrations ofD-aianine for a half stimulation of the rate of inactivation by ImM iodoacetamide were calculated to be S40mM at 2Q°C and 320mM at 30°C.

To identify a sensitive thiol group of the ACC deaminase, N -(iodoacetamidoethyl)-Iaminonaphthalene-S-sulfonic acid (IAEDANS) was used as a inactivator instead of iodoacetamide. The ACC deaminase was incubated with O.lmM IAEDANS and SOOmM D-alanine for 120 min at 30°C at pH8.S to obtain 70% inactivation finally, following addition of adequate amount of 2-mercaptoethanol to remove free IAEDANS and dialyzation against O.OS M potassium pyrophosphate, pH8.S. The inactivated enzyme was reduced, denatured, pyridylethylated, and digested as described in Methods. Proteolytic digest of the modified enzyme was analyzed by reversed-phase chromatography as shown in Fig. S. Differences between both chromatograms of the modified enzyme and control enzyme without the modification by IAEDANS are an appearance of KII and a decrease of K9. The absorbance at 3SOnm indicates that the KII peptide involves the thiol group modified by IAEDANS. Sequencing of these two peptides indicated that a modified thiol group was on a Cysl62 residue.

114

100

~

>- 80 +-' 2 > 70 :;::; () ro (]) 60 > +-' ro 3 (]) 50 a:

0 30 60 90 120

Time (min)

Fig. 4. Effect of D-alanine on inactivation of ACC deaminase by iodoacetamide. The ACC deaminase was incubated with 1 mM iodoacetamide in the presence of (1) 0, (2)75, and (3) 300 mM D-alanine at 30°C at pH8.5 before enzyme assay.

Time (min)

Fig. 5. Chromatograms of lysylendopeptidase digest of modified ACC deaminase. Proteolytic digests from the ACC deaminases with (A) and without (B) inactivation by IAEDANS were analyzed by reversed-phase chromatography with C8 column and 0.1 % trifluoroacetic acid-acetonitril (0-40%). Absorbances at 325nm, 350nm and 216nm were monitored for phosphopyridoxyl group, modification site by IAEDANS, and peptide, respectively. Each peptide was numbered Kl to K12 from right in (A). Sequences of K9 and KII were decided as follows

K9 PYAIPAGCSDHPLGG KII PYAIPAGXSDHPLGG

TRPB-5al ACCD-Ps ACCD-Ps2

TRPB-5al ACCD-Ps ACCD-Ps2

TRPB-Sal ACCD-Ps ACCD-Ps2



115

H 1 H 2 51 S 2 HTTLLNP -YFGEFGGKYVPQILKPALNQLEEAFVRAQKDPEFQAQFADLLKNYAGRPTALTKCQNIT AGTRITLYLKRED - - -LLHGGAH IfNLQRFPRYPLTFG- - - - - - - - - - - - - - - - - - - - - - - - -PTPIQPLARLSKHLGG- - - -- - - - - - - - - - -KVHLYAKREDCNSGLAFGGN _ •• N •• E ••••••• - - - - - - -- - - -- - - - - - - - - - -- - - .5 •• T •• K ••• Q •••• -- - - - - - - - - - - - - - •• E ••••••• " ••••••••

H3 JL H4 _5_4_ ..JlL ~ _ KTN - -QVLGQALLAKRKGKSEIIAETGAGQHGVASALASALLGLKCRIYKGAK - - - - -DVERQSPNVFRKRLlfGAEVIPVHSGSATLKDA XTRKLEYLIPEALAQGCDTLVSIGGIQSNQ-TRQVAAVAAHLGKKCVLVQENWVNYSDAVYDRVGNIQKSRILGADVRLVPDGFDIGFRR ... •••••••••••• IE •••••••••••••••• - •••••••••••••••••••••••••••••••••••• E •••• K •••••• DAA ••••• I.P

_H_6_ 2L JL H8 ...ll H9 _ CNEALRDWSGSYETAHYKLGTAAGPHPYPTIVREFQRKIG- - - - - - - -EETKAQlLDKEGRLPDAVlACVGGGSNAlGMFADFlNDT-SV SWEDALESV- - -- - - - - - - -RAAGGKPYAlPAGCSDHPLGGLGPVGFAEEVRAQEAE-LGFKFDYVVVCSVTGSTQAGMVVGFAADGRAD ., .K.IfSD. -----------VEQ •••• FP •••••• E •• Y ••••••••••••• Q •• K. - ••••••• r. ..................... SK

o ~ ... ... ... ----H.1!L ~ H 11 GLlGVEPGGHGIETGEHGAPLKHGRVGIYFGMKAPIIKQTADGQIEESYSISAGLDFPSVGPQHAYLNSIGRADYVSITDDEALEAFKTLC RVIGVDASAKPAQTRE-QITRlARQTAEKVGLEI\.DIKRADVVLDERFAGPEYGL~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ PNEGTLEAlRLCA N ••• r. ..... E •• KA- •• L •••• H ••• L.E.G:1l.TEE ••••• T ••• Y..... • ••••••••••• G

H 12 S10 H 13 RHEGIIPALESSHALAHALKIIIIREQPEKEQLLVVNLSGRGDKDIFTVHDILKARGEl RTEGIILTDPVYEGKSIfHGKlEIIVRNGEFPEGSRVLY AHLGGVPALNGYSFIFRDG- -SL •• V ••••••••••••••••••• R ••••••• K •••••••• A •••• A ••• L •• N.--

Fig. 6. Comparison of amino acid sequences between ACC deaminase and tryptophan synthase [3-subunit. Amino acid sequence of the ACC deaminase was compared with those of Salmonella tryptophan synthase ~-subunit including second-structural data[9] and of ACC deaminase from a different strain of Pseudomonas sp.[2]. Sites of tryptic cleavage were indicated by arrows.

Discussion

The sequence around a pyridoxal phosphate binding site of the ACC deaminase involves similar parts as that of tryptophan synthase ~-su bunit whose three-dimensional structure was published[9].

ACC deaminase (34~52) L Y AKREDCNSGLAFGGN XT Tryptophan synthase [3-subunit (73~88) L YLKRED LLHGGAHXT

The tryptophan synthase ~-subunit catalyzes tryptophan synthesis from indole and L-serine, and deamination of L-serine. The latter reaction is the same type as deamination of D-serine by the ACC deaminase. Sequences of both enzymes are aligned in Fig. 6 to set the pyridoxal phosphate binding sites in the same position, giving 24% homology of the ACC deaminase. Three sites of tryptic cleavage, Lys272, Arg27S, and Lys283 were identified in a hinge region of the tryptophan synthase ~-subunit[9]. A site of tryptic cleavage was found in the region of the ACC deaminase corresponding to the hinge ofthe [3-subunit. These observations indicate a similarity in the three-dimensional structure between both enzymes. It was shown that the ~-subunit is composed of two domains, N-domain and Codomain, which were divided between Phe204 and Gln20S, and that the pyridoxal phosphate site and active site were at the interface between both domains. The reactive thiol group on Cys162 of the ACC dearninase is located near a position corresponding to the dividing point between the N- and Codomains of the [3-subunit.

Mechanistic studies of the ACC deaminase suggested a nucleophilic addition/elimination route for the cyclopropane bond cleavage[S]. In this route, cyclopropane bond cleavage is achieved by attack of an active site nucleophile at a pro-S methylene carbon of ACC. The

116

above discussion implies the possibility for the Cys 162 to react as the nucleophile for cyclopropane bond cleavage. Further studies are re.quired to resolve this question.

References.

[1] Yang, S. F. and Hoffman, N. E. (1984) 'Ethylene Biosynthesis and its regulation in higher plants', in W.R. Briggs, R. L. Jones, and V. Walbot (eds.) Annu. Rev. Plant Physioi. 35, 155-189.

[2] Klee, 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', The Plant Cell, 3,1187-1193.

[3] Honma, M., Shimomura, T., Shiraishi, K., Ichihara, A., and Sakamura, S. (1979) 'Enzymatic deamination of d-coronarnic acid: stereoselectivity of 1-arninocyclopropane-l-carboxylate deaminase', Agric. BioI. Chern. 43, 1677-1679.

[4] Lill, H.-W., AlIChus, R., and Walsh, C.T. (1984) 'Stereochemical studies on the reactions catalyzed by the PLP-dependent enzyme 1-arninocyclopropane-I-carboxylate deaminase', 1. Am. Chern. Soc. 106,5335-5348.

[5] Walsh, c., Pascal, R.A. Jr., Johnston, M., Raines, R., Dikshit, D., Krantz A., and Honma, M., (1981) 'Mechanistic studies on the pyridoxal phosphate enzyme l-aminocyclopropane-l-carboxylate deaminase from Pseudomonas sp.', Biochemistry, 20, 7509-7519.

[6] Honma, M., (1985) 'Chemically reactive sulfhydryl groups of 1-aminocyclopropane-l-carboxylate dearninase', Agric. BioI. Chern., 49, 567-571.

[7] Honma, M., and Shimomura, T., (1978) 'Metabolism of l-arninocyclopropane-lcarboxylic acid', Agric. BioI. Chern., 42,1825-1831.

[8] Honma, M., (1983) 'Enzymatic determination of l-arninocyclopropane-1-carboxylic acid', Agric. BioI. Chern., 47, 617-618.

[9] Hyde, C. c., Ahmed, S. A., Padlan, E. A., Miles, E. W., and Davies, D. R. (1988) 'Three-dimensional structure of the tryptophan synthase (X2 ~ Multienzyme complex from Salmonella typhimurium', J. BioI. Chern., 263, 17857-17871.

BIOCHEMICAL AND MOLECULAR ASPECTS OF LOW OXYGEN ACTION ON FRUIT RIPENING

A.K. KANELLISl, K.A. LOULAKAKISl, M.M. HASSANl,2 AND K.A. ROUBELAKIS-ANGELAKIS3 11 nstitute of Molecular Biology and Biotechnology, FORTH, p.o. Box 1527,711 10 Heraklion, Crete, 2Mediterranean Agronomic Institute of Chania, Crete, 3 Dept. of Biology, University of Crete, Heraklion, Greece

ABSTRACf. The effect of different oxygen levels on preclimacteric and ripening initiated avocado fruits was studied at total protein and gene expression levels. Low oxygen induced the accumulation of new polypeptides in both types of fruits as was evident by two dimensional PAGE (2-D) of total proteins and SDS-PAGE of in vitro translation products, whereas it suppressed the development of new polypeptides only in initiated fruits. The synthesis of cellulase was irrespective of low oxygen concentration in preclimacteric fruits and oxygen dependent in initiated fruits. Alcohol dehydrogenase isoenzymes were induced in both types of fruits correlated with elevated mRNA levels.

1. Introduction

In plants a response to hypoxic and anoxic conditions involves an adaptive mechanism that enables survival in low oxygen stress for several days (Sachs and Ho (1986). Within this context, it is well established that oxygen levels below those in air retard the rate of ripening and softening of climacteric fruits (Mattoo et al. (1988). The mode of action of low oxygen in delaying fruit ripening is not clear. However, since previous evidence indicates that oxygen is required for the synthesis and the action of ethylene (Mattoo et al. (1988) it is a reasonable assumption that the retarding effects of low oxygen on fruit ripening reflect a diminution of ethylene synthesis and action. It should be stressed, however, that the physiological ramifications of low oxygen action are broader than fruit ripening. For instance, hypoxic conditions inhibited the accumulation of RNA, protein and DNA synthesis associated with wounding in potato tubers (Butler et aI. 1990). In a number of plant tissues, anoxia causes marked alterations in profile of proteins, stability of mRNA species and gene expression (Sachs and Ho (1986).

In view of the above evidence and the limited experimental data concerning the mode of action of low oxygen on fruit ripening (Mattoo et al. (1988), we have initiated studies aimed at investigating the biochemical and molecular aspects of low oxygen action on fruit ripening. Our previous results have shown that transferring initiated avocado fruits to 2.5% oxygen for 6 days suppressed the activity (Kanellis et al. (1989b), immunoreactive protein and abundance of mRNA of cellulase (Kanellis et al. 1989c). The above treatment also produced an alternation in the profile of avocado total proteins, which involved suppression, enhancement and induction of new polypeptides (Kanellis et al. (1989b), (1991). In addition, it has been shown that the range of oxygen levels (2.5-5.5%) which suppressed the appearance of ripening enzymes at the protein and mRNA levels was similar to those oxygen levels that induced the synthesis of new isoenzymes of alcohol dehydrogenase (Kanellis et aI. (1991).

In this report, in order to distinguish the interactions between oxygen and ethylene, preclimacteric and ripening initiated with propylene avocado fruits were exposed to different low oxygen levels and the steady state protein pattern was studied by 2-D as well as protein accumulation and gene expression at the level oftrans1atab1e and accumulated mRNA.

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Preclimacteric avocado fruits (Persea americana, cv Hass) were enclosed in glass jars and placed at 2()oC under a steady flow of humidified air. In the first experiment, different oxygen levels of 0, 1,3,5, 10 and 21% were introduced to individual jars for 48 hr. In the second experiment. ripening of fruits was initiated by introducing 200 J.ll/l propylene. When the rate of ethylene production was at the climacteric peak, the exogenous supply of propylene was discontinued. At this point, a set of three fruits was transferred to 0, 1,3,5,10 and 21% oxygen for 48 hr.

Total protein extraction, SOS-PAGE, silver staining and immunological detection methods were as described (Kanellis et al. (1989 b), (1989 c). Two-O gels (native PAGE, first dimension! SOSPAGE, second dimension) were performed according to Sachs et al. (1980) . Alcohol dehydrogenase (AOH) extraction, PAGE and activity staining were done as descibed previously ( Kanellis et al. (1991). RNA extraction and hybridization were performed according to Kanellis et al. 1989c). Northern blots were hybridized with avocado cellulase pAV363, maize ADH pB428 and sucrose synthase pCB16 probes. For in vitro translations, the kits from Amersham and Promega for preclimacteric and initiated fruits, respectively, were used.

If kD -71,0 ___ ----66,2

-43/J

-30,0

I - 17,2

-43/J

-30,0

-17,2

Figure 1. Silver-stained total proteins from preclimacteric avocado fruits separated by 2-0 native! SOS-P AGE. A, 1 % oxygen; B, 5% oxygen; C, 10% oxygen; 0, air. Open-arrowheads indicate polypeptides which accumulated in low oxygen.

119


The deprivation of oxygen induced a change in the polypeptides synthesized under low oxygen regimes. Open-arrowheads (Fig. lA) indicate the appearance of new polypeptides as well as the increase in staining intensity of pre-existing in air fruits. The profIles of preclimacteric fruits treated with 10% oxygen were similar to those in air, which is in agreement with previous results (Kanellis et aI. (1991). Similarly low oxygen altered the polypeptide pattern of initiated fruits (Fig. 2). However, polypeptides which were expressed in air (Fig. 2D, closed-arrowheads), were suppressed in low oxygen held fruits (Fig. 2, A,B). Thus it appears that low oxygen induced the appearance of new polypeptides in both preclimacteric and initiated avocado fruits, whereas it suppressed the synthesis of ripening polypeptides.

Figure 3A illustrates in vitro translation products of poly(A)+ RNA from preclimacteric avocado fruits, which were subjected to 0, 1,3,5, 10 and 21 % oxygen for 48 hrs. The closedarrowheads on the left-hand side show that there are mRNAs which are present mainly in 0 % oxygen although many mRNAs are present in all lanes. It is also evident that low oxygen did not cause detactable suppressive effect on pre-existing mRNAs in preclimacteric avocado fruit. However, initiated avocado fruits kept in different oxygen levels showed that both activation and suppression of mRNAs took place (Fig. 3B). Closed-arrowheads on the left-hand side indicate the appearance of new mRNA whereas open-arrowheads on the right-hand side show the accumulation of new mRNA in ripening fruit which were suppressed in low oxygen held fruits and especially in those fruits kept in 0, 1 and 3% oxygen.

B kD -77,0 -66,2

-43,0

-17,2

o -77P - 66,2

-43,0

-30,0

-1 7,2

Figure 2. Silver-stained total proteins from ripening initiated with propylene avocado fruits separated by 2-D native/ SDS-PAGE. Letters are the same as those described in the legend to Figure 1. Closed-arrowheads indicate polypeptides which were suppressed in low oxygen.

120

o I

-

3 5 10 21 t I I •

A

..

.. .. -

o 3 1021 I I I I

B

kD -119,0

- 84,0 - 64,0

- 48,0

_ 36,4

- 29,7

Figure 3. Auorogram of a SDSPAGE of in vitro translation products of poly (A)+ RNA isolated from preclimacteric (A) and initiated avocado fruits (B) held under different oxygen regimes for 48 hr. Closedarrowheads indicate the appearance of new mRNA and openarrowheads the suppression of ripening polypeptides .

Protein and mRNA accumulation of cellulase, which is synthesized during ripening, was studied under low oxygen (Fig. 4). The synthesis of cellulase protein of preciimacteric and especially that of 56 kD which corresponds to the secretory form (Bennett and Christoffersen (1986), was irrespective of oxygen tensions. Fruits held in air or in low oxygen contained either the 56 kD alone or both the 56 kD and 54 kD, the mature protein. There was no apparent effect of low oxygen on the accumulation of cellulase forms. Thus, 48h treatment with low oxygen did not prevent the synthesis of pre-existing proteins and this is also consistent with the steady state amount of poly(A)+ RNA of cellulase (Fig. 4). In initiated fruits, however, low oxygen and especially 0 and 1 % effectively prevented the accumulation of cellulase protein and its poly(A)+ RNA. Thus, it seems that low oxygen suppressed new protein synthesis and po1y(A)+ RNA accumulation of a de novo synthesized and ethylene regulated protein. It can be also seen from Figure 4 the shift from the 56 kD to 54 kD of cellulase forms. These results in combination with the data in Figure 5, in which the ratio of 54 kD to 56 kD in air, 2.5% oxygen and 2.5%+ 100 IJ.I/l ethylene is plotted over a 6-day period, suggested that ethylene accelerated the processing of cellulase protein. That is, this ratio was more or less the same in air and low oxygen, whereas it increased in ethylene treated fruits indicating faster accumulation of the 54 kD form.

Protein

m RNA blot

A B ~-----'i 1"", ------,

5 10 21 0 j It.

3 5 10 21 , l

Figure 4. Cellulase protein and mRN A changes in preclimacteric (A) and initiated avocado fruits (B).

3

010 2 00

'" '" ""'" "''''

1 ~ 1 Z,5"1. Oz

6 8 10 AT 20·C

121

Figure 5. Pattern of accumulation of the two immunoreactive cellulase proteins present in avocado fruits ripened in air or held in 2.5% oxygen and 2.5% +100 !J.lIl ethylene. An immunoblot was densitometrically scanned and the peak areas were plotted. Arrow on day 7 indicates the return of treated fruits back to air.

In low oxygen stressed fruits, induction of protein synthesis and gene expression took also place. Alcohol dehydrogenase protein was expressed in low oxygen in both preclimacteric and initiated avocado fruits (Fig. 6). This increase in ADH protein corresponded to its elevated mRNA levels (Fig. 7). In addition, sucrose synthase, another anaerobic protein, was expressed at the mRNA level in avocados held in anoxia (Fig. 7).

In conclusion, it appears, from the analysis of the steady state of protein and mRNA patterns in both preclimacteric and ripening initiated avocado fruits, that low oxygen exerts its suppressive effect mainly on de novo synthesized proteins of ripening fruits, whereas it induces the synthesis of new proteins irrespective of the developmental stage of the fruit.

A B .------------,Irl------------~

o I

3 5 1021 0 I I I I I

3 5 10 21 I I II

Figure 6. Induction of ADH isoenzymes by low oxygen. A, preclimacteric and B, initiated avocado fruits.

ZM 0 1 3 II 10 21

_ ADH

, . ~. ~ '.,. - ss

Figure 7. Northern blot analysis of ADH and sucrose synthase in preclimacteric avocado fruits held in low oxygen. ZM, mRNA from Zea mays as controls.

122

4. Acknowledgment

We thank Dr. M. Tucker for cellulase pA V363 clone, Dr. A. Bennett for avocado cellulase antibody, Dr. M. Freeling for maize ADH and sucrose synthase probes and Mr. G. Chronakis for technical assistance. We thank also Mr 1. Metzidakis for supplying the avocados.

s. References

Bennett, A.B. and Christoffersen, R.E. (1986) 'Synthesis and processing of cellulase from ripening avocado fruit', Plant Physiol. 81, 830-835.

Butler, W., Cook, L., and Vayda, M.E. (1990) 'Hypoxic stress inhibits multiple aspects of the potato tuber wound response', Plant Physiol. 93, 264-270.

Kanellis, A.K., Solomos, T., and Mattoo, A.K. (1989a) 'Changes in sugars, enzymic activities and acid phosphatase isoenzyme profiles in bananas riperied in air or stored in 2.5% oxygen with and without ethylene', PlantPhysiol. 90, 251-258.

Kanellis, A.K., Solomos, T., and Mattoo, A.K. (1989b) 'Hydrolytic enzyme activities and protein pattern of avocado fruit ripened in air and low oxygen with and without ethylene', Plant Physiol. 90, 259-266.

Kanellis, A.K., Solomos, T., Mehta, A.M., and Mattoo, A.K. (1989c) 'Decreased cellulase activity in avocado fruit subjected to 2.5% oxygen correlates with lower cellulase protein and gene transcript levels' , Plant Cell Physiol. 30 , 817-823

Kanellis, A.K., Solomos, T., and Roubelakis-Angelakis, K.A. (1991) 'Suppression of cellulase and polygalacturonase and induction of alcohol dehydrogenase isoenzymes in avocado fruit mesocarp subjected to low oxygen stress', Plant Physiol. 96, 269-274.

Mattoo, A.K., Kanellis, A.K., Solomos, T., and Mehta, A.M. (1988) 'Low oxygen atmospheres and gene expression in relation to ethylene action', In R. Jona (ed) Proc. Symp. "Physiology

of Fruit Drop Ripening Storage and Postharvest Processing of Fruits", Turin (Italy) 3-4 Oct. 1988.

Sachs, M.M. and Ho, T.-H.D. (1986) 'Alteration of gene expression during environmental stress in plants', Annual Review Plant Physiol. 37,363-373.

FUNCfIONAL ANALYSIS OF CX-CELLULASE (ENDO-,B-1,4-GLUCANASE) GENE EXPRESSION IN TRANSGENIC TOMATO FRUIT.

CORALIE C. LASHBROOK AND ALAN B. BENNETT Mann Laboratory, Department of Vegetable Crops University of California Davis, California 95616 USA

ABSTRACf. The degradation of cell wall polymers by specific cell wall hydrolases during fruit ripening is thought to be required for tomato fruit softening, with the products of cell wall dissolution possibly influencing additional aspects of fruit ripening behavior. Pectin degradation during ripening is not sufficient for tomato fruit softening, suggesting the involvement of cell wall hydrolases other than polygalacturonase. We are investigating the role of Cxcellulase-mediated cell wall polymer degradation in tomato fruit ripening and softening. Oligonucleotide probes corresponding to two conserved domains of avocado fruit and bean abscission zone Cx-cellulases were used to screen a cDNA library from ripe tomato fruit. cDNAs encoding several related members of the tomato fruit Cx-cellulase gene family were isolated. Cell and Cel2 are distinct gene products with significant sequence differences and exhibit different mRNA expression patterns during ripening. Both Cell and Cel2 cDNAs show significant sequence identities to cellulases of plant, bacterial and fungal origin. We have transformed tomato plants with antisense constructs of both cellulases and have identified plants in which levels of endogenous Cell mRNA have been reduced by 90%. We are currently evaluating the physiological consequences of reduced Cell expression.

Introduction

Tomato fruit ripening is a complex developmental process involving significant changes in cellular architecture and metabolism. Hallmarks of ripening include chlorophyll degradation, lycopene accumulation, cell wall dissolution and fruit softening. Such ripening events are dependent upon the coordinated expression of developmentally regulated genes, a number of which are induced or modulated by ethylene (1,2). Additional control over the ripening process may be exerted by the actual products of ripening-induced gene expression.

We are taking a molecular genetic approach to the study of cell wall degradation in ripening tomato fruit and have focused our studies on the noncellulosic polymers of the plant cell wall. The role of cell wall hydrolases in degrading the cell wall matrix has been recently reviewed (3). Compositional analyses of cell walls isolated over the course of ripening suggest that this dissolution is largely restricted to its pectic and hemicellulosic components( 4).

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J. C. Pech et al. (eds.), Cellular alld Molecular Aspects of the Plant Honllone Ethylene, 123-128. © 1993 Kluwer Academic Publishers.

124

POLYGALACTURONASE

During ripening, pectic polymers within the&rimary cell walls are extensively modified by the enzyme polygalacturonase PG). However, molecular genetic approaches to determining the role of PG in ripening-induced wall hydrolysis have revealed that PG cannot be the sole determinant of fruit softening or the textural changes that accompany softening. Firstly, a PG gene has been introduced into the tomato ripening mutant rin, which normally fails to express polygalacturonase or soften. Expression of the transgene in the mutant background resulted in normal patterns of polygalacturonase expression yet the fruit failed to soften (5). Secondly, while antisense suppression ofPG expression significantly affects the structure of the pectic fraction of transgenic fruit cell walls (6,7), influencing tomato processing characteristics (8), fruit expressing such transgenes soften normally (6). Based on these observations, we have recently begun to address the roles that non-pectolytic cell wall hydrolases may play in the establishment of ripe fruit structure and function.

CX-CELLULASE

During tomato fruit ripening, cell wall hemicelluloses are extensively degraded. Temporally associated with the dissolution of hemicelluloses during ripening is an induction of Cx-cellulase activity in tomato fruit tissue (9,10). The temporal relationships between hemicellulose degradation, Cx-cellulase induction and the softening of tomato fruit suggests a possible role for Cx-cellulases in the establishment of ripe fruit texture.

Cx-cellulases are endo-/3-1,4-glucanases. The use of the term cellulase to describe this class of enzymes is unfortunate because there is at present no indication that these enzymes have the capacity to degrade appreciable levels of crystalline cellulose. Indeed, the term Cx specifically denotes a class of endoglucanase enzymes capable of hydrolyzing the /3-1,4 linkages of solublized cellulose. The term Cx was initially devised by microbiologists to distinguish the activities of this type of microbial endoglucanase from a second class of cellulases capable of directly attacking crystalline cellulose (11). Activity of the latter group of enzymes, the Cl-cellulases, is believed to be a prerequisite for microbial Cx-cellulose-catalyzed hydrolysis of cell wall cellulose. In addition, both microbes and plants secrete a wide variety of non-cellulolytic Cx-cellulases that are thought to act upon the /3-1,4 glycosidic linkages of cell wall hemicelluloses. Cx cellulases of elongating pea internodes and ripening avocado fruit have been shown to be capable of degrading xyloglucans in vitro, suggesting that this may be one Cx-cellulase substrate present in plant systems (12,13). Unfortunately, neither pea nor avocado are suitable systems for molecular genetic manipulation. We are thus investigating the role of Cx-cellulases in tomato, which represents an ideal system for molecular genetic studies of fruit ripening.

Results and Discussion

CLONING STRATEGY

In order to determine the extent to which Cx-cellulases in ripening tomato fruit participate in cell wall structural changes, we have isolated cDNA clones encoding several of these enzymes and have initiated analysis of transgenic plants in which their expression is altered. Our cloning strategy exploited the availability of sequence information from two previously cloned Cx-cellulases from ripening avocado and bean abscission zones (14,15). We examined these two sequences and identified regions over which amino acid homology was completely conserved. Oligonucleotide probes corresponding to two such domains were used to screen a cDNA library derived from red ripe tomato fruit (Figure 1) and the resulting cDNA clones were subclassified into groups based on cross-hybridization. Sequence analysis of all clones revealed that three were derived from a common parent sequence we have called Cell , two clones corresponded to the parent sequence Cel 2, and one corresponded to a sequence we have called Ce13. Full length cDNAs of Cell and Cel 2 have now been sequenced and compared to the cx-cellulases of bean and avocado. Cell and Cel2 are approximately 50% identical to each other, with somewhat higher identities exhibited to cellulases of bean and avocado. Cel 3 has not been completely characterized at this time.

Amino acid sequence: #1 #2

AVOCADO

BEAN

GGYVDAGDN I III II I I I GGYVDAGDN

/ ~

CWERPEDMD I IIIII I II CWERPEDMD

/ ~ Oligonucleotide: GGI GGI TAT TAT GAT GCI GGI GAT AA TGT TGG GAA CGI CCI GAA GAT ATG GA

TOMATO

(pTCEL1)

CCC C C G GC

GGA GGC TAC TAT GAT GCT GGT GAC AA TGT TGG GAA AGA CCA GAA GAC ATG GA

~ / ~ / GGYVDAGDN CWERPEDMD

Figure 1. Conserved amino acid domains in avocado and bean Cx-cellulases and corresponding oligonucleotides used to isolate tomato fruit Cx-cellulase cDNAs. The conserved sequences are labeled as amino acid sequences #1 and #2, with #1 corresponding to the more N-terminal domain. Each sequence was used to design the corresponding degenerate oligonucleotide that in turn was used to isolate the tomato fruit Cx-cellulase cDNAs Cell, Cel2 and Ce13. The sequence and deduced amino acid sequence corresponding to the conserved Cx-cellulase sequences are also shown.

125

126

EXPRESSION ANALYSIS

The mRNAs of both Cell and Cel2 are induced at the mature green stage of ripening, with accumulation of mRNA appearing to precede or coincide with the induction of ethylene biosynthesis. The pattern of Cell mRNA accumulation throughout fruit ripening was also found to parallel the timing of expression of polygalacturonase (Figure 2). However, the maximal level of mRNA accumulation was approximately lOO-fold less for Cell than for PG mRNA. Cel 2 mRNA, present at higher levels than that for Cell, exhibits similar accumulation patterns but its presence is maintained throughout the ripening process. These temporal patterns of mRNA accumulation are similar to the progression of fruit softening, consistent with the view that these enzymes may contribute to the textural changes that accompany ripening.

0> a.

L 0-

Q) 0> 0:: .:::L C '"tJ I 0- 0> 0 C .:::L 0:: 0> L

<.::> Q) "- c a. 0>

Probe: <.::> "- :J .- - 0- > ~ (D ..-. a.. -' 0::: 0

PG

CX-Case

Figure 2. Cell mRNA accumulation in ripening tomato fruit. Two ug of poly(A)+ mRNA was blotted to nitrocellulose and hybridized with 32P-Iabeled Cell (lower panel) or polygalacturonase (upper panel) cDNA insert. Quantitative analysis of hybridization indicated that polygalacturonase mRNA is approximately lOO-fold more abundant than Cell mRNA at the maximal level of induction for both mRNAs.

PRIMARY SEQUENCE ANALYSIS

Sequence relationships to plant cellulases. Sequence comparisons of our tomato cellulases with Cx cellulases of bean and avocado reveal the presence of multiple domains in which both the sequence and its relative position in the cDNA are highly conserved (Fig 3). What is the functional significance of these domains? Do they represent sequence motifs specific to cellulases, general to hydrolases, or common to diverse enzyme classes? Do they afford any information as to the catalytic mechanisms or substrate specificities underlying Cx-cellulase function? With the recent discovery that striking stuctural similarities exist between Cxcellulases of plant, bacterial and fungal origin (16), we believe clues leading to the answers to such questions may reside in the extensive microbial Cx-cellulase literature.

El

E2

5.5. N~xtension Catalytic core

~ KGWHDAGDY 14.4% DDRLWAAA ~£~:~

GGWYDAGDI-I 32.2%

P. fIuomsc8ns

CelA

c.~

CelD

c. sifNrorarium

CelZ

-.~------~I'=M~\=r' 1~:'X[~/~~~~V~/~/Ac.~ GGWYDAGDH 36.5% DELTWAS! fFmJtbf/L'Y// L.J CelF

I GGYYDAGDN 100% DEUWAAA I 501 AA

I GGYYDAGON 50.9% DEUWAAA 494AA

I GGYYDAGDN 68.4% DEUWAAA 495AA

IGGYYDAGDN 49.6% DEUWAAA I 489AA

Lescu!6nttJm

Cell

P. IInIfJrican8

Cell

P. KI~

BACl

L9SCUlonI/Im

Cel2

Figure 3. Homologous catalytic core domains of plant and bacterial cellulases of Family E. Although all protein coding regions shown contain conserved domains in similar positions, overall primary sequence identity (expressed relative to tomato Cell) varies extensively. Structural relationships between the E1 and E2 subfamilies are evident at the predicted secondary structural level (17). S.S.=signal sequence. CBO=cellulose binding domain.

127

128

Sequence relationships to microbial cellulases. Molecular cloning of cellulase genes from diverse prokaryotic sources has revealed that these organisms secrete a wide variety of cell wall digestive enzymes with diverse substrate specificities. Most bacterial genes cloned to date are endo-,8-1,4-glucanases, with fewer genes representing the closely related endo-,8-1,4-xylanases or cellobiohydrolases. Detailed sequence analysis of a large number of cloned bacterial and fungal cellulase genes has recently led to the establishment of a cellulase classification system into which plant Cx-cellulases are beginning to be assigned. Virtually all cellulases sequenced to date can be placed in one of six cellulase families, denoted Families A-F, based on primary and predicted secondary protein structural information within the catalytic cores of these enzymes(17). Sequence analysis of avocado cellulase recently led to its inclusion in Family E (16). A schematic representation of sequence similarities that exist between tomato Cell, Cel2 and other members of Family E is shown in Figure 3. It has recently been proposed that both plant and bacterial members of the E family of cellulases may have evolved from a common ancestor (18).

Functional analysis a/tomato cellulase gene expression. We are taking a two pronged approach towards establishing the substrate specificities present in the tomato cellulase gene family. We are currently overexpressing these cellulases in yeast to generate cellulase protein whose capacity to degrade model substrates can be evaluated. Secondly, we have generated transgenic tomato plants harboring antisense constructs of Cell and Cel2 in whose fruit cell walls lesions of normal cell wall structure may become apparant. We have identified transformants in which Cell mRNA levels have been reduced by 90%, and are initiating studies aimed at determining the physiological consequences of reduced Cell expression in transgenic plants. Analysis of the primary transformants harboring antisense constructs of Cel2 will follow.

References

1. Brady, CJ. 1987. Ann Rev Plant Physio138:155-178 2. Speirs, J and CJ Brady. 1991. Aust J Plant Physiol18: 519-532 3. Fischer, RL and AB Bennett. 1991. Ann Rev Plant Physiol Plant Molec BioI 42: 675-703. 4. Huber, D. 1983. J Am Soc Hort Sci 108:94:4%-498. 5. Giovannoni, J, D DellaPenna, AB Bennett, and RL Fischer. 1989. Plant Cell 1:53-63. 6. Smith, CJS, CF Watson, PC Morris, CR Bird, GB Seymour, JE Gray, C Arnold, GA Tucker,

W Schuch, S Harding and D Grierson. 1990. Plant Molec BioI 14: 369-379 7. Sheehy, RE, M Kramer and WR Hiatt. 1988. Proc Natl Acad Sci USA 85: 8805-8809 8. Kramer, M, RA Sanders, RE Sheehy, M Melis, M Kuehn and WR Hiatt. 1990. In:

"Horticultural Biotechnology" New York: Wiley-Liss, Inc. 9. Hall, CB. 1963. Nature 200:1010-1011

10. Hobson, GE. 1968. J Food Sci 33: 588-592 11. Reese, ET, RGH Siu and HS Levinson. 1950. J BacterioI59:485-12. Hayashi, T, YS Wong and GA Maclachlan. 1984. Plant Physiol 75: 605-610 13. Hatfield, Rand D Nevins. 1986. Plant and Cell Physiol27: 541-552 14. Tucker, ML, ML Durbin, MT Clegg and LN Lewis. 1987. Plant Molec BioI. 9: 197-203. 15. Tucker, ML and S Milligan. 1991. Plant Physiol 95: 928-933 16. Beguin, P . 1990. Ann Rev Microbiol44: 219-248 17. Henrissat, B, M Claeyssens, P Tomme, L Lemesle and J-P Momon. 1989. Gene 81: 83-95 18. Navarro, A. M-C Chebrou, P Beguin and J-P Aubert. 1991. Res MicrobioI142:927-936

INHIBITIo.N o.F ETHYLENE BIo.SYNTHESIS AND SUPRESSION o.F CELLULASE AND Po.LYGALACI1JRo.NASE IN AVo.CADo. FRUIT SUBJECTED TO. Lo.W o.XYGEN STORAGE.

J. Metzidakis* Subtropical Plants and o.live Trees Institute, Chania, 73100-GREECE

E. Sfakiotakis Laboratory of Pomology Aristotle University, Thessaloniki S4006-GREECE

Key Words: Persea americana, low oxygen, ethylene biosynthesis, softening, activity of polygalactouronase, cellulase.

ABSlRACf. This research was undertaken to study the mode of action of low Oz in delaying ripening of avocado fruit. Hass avocado fruits were treated with propylene in various Oz concentrations (1%, 2%, S%, and 21%) at 20°C and ethylene production and ripening of the fruit were followed by measuring accumulation of ACC, EFE activity and the polygalacturonase (pG) and cellulase (Cx) activities respectively. Low Oz delayed the softening of propylene treated fruits and prevented the rise in EFE activity'. Furthennore, low 02 suppressed the rise in PG and Cx activities. Gel electrophoresis and western blot analysis showed that the levels of PG and Cx proteins, were drastically reduced in fruits held in low Oz (1% and 2%).

Introduction

Ethylene is undoubtedly the most multifunctional of plant hormones. It plays a key regulatory role in important physiolOgical events, hence, there is considerable interest in understanting the regulation of ethylene formation system on the molecular level (Yang and Hoffman (1984), Brady (1987».

Kidd and West (I94S) suggested that low oxygen may interfere with the production and/or action of ethylene. Burg and Burg (1967) provided experimental evidence as an indication of a requirement for oxygen in the biological action of ethylene. Furthennore, it was shown that the biological potency of ethylene was reduced at levels of low oxygen that did not affect tissue respiration. Banana and avocado fruit when stored in low oxygen atmosphere, following a brief incubation with ethylene, show diminished rate of sugar accumulation and suppression in the activities of acid phosphatase, polygalacturonase and cellulase as compared to fruit ripening in air, also that low oxygen interferes with the accumulation of cellulase protein and its mRNA in avocado fruit. (Kanellis (1987), Kanellis, et al. (1989a, b».

Softening is one of the most pronounced of the many changes that accompany ripening of avocado fruit. The softening process is associated with an increase in the activities of hydrolytiC enzymes such as cellulase, polygalacturonase and acid phosphatase. On this basis, a role for cellulase in fruit cell wall degradation has been suggested (Awad and Young 1979, Bennett and Christoffersen 1986, Pesis et al. (1978». The increase in cellulase activity during avocado ripening appears to be due to de novo synthesis of the protein. Furthermore, the levels of mRNA of cellulase were shown to closely correlate with the increase in the level of cellulasse protein and activity during avocado ripening, indicating that cellulase synthesis is regulated at the level of transcription. (Christoffersen et al. (1984), Tucker and Laties (1984), Tucker et al. (1987».

Low oxygen atmospheres are widely used in commercial practice to extend the storage life of fruits and vegetables (Kader (1986). Very litle information is, however, available on the

"'This work was conducted to fulfil partly the requirements for M. S. and Ph. D. at Mediterreanean Agronomic Institute-Chania and Aristotle University-Thessaloniki respectively.

129

J. C. Pech et al. (eds.J, Cellular and Molecular Aspects of the Plant Honnone Ethylene, 129-135. © 1993 Kluwer Academic Publishers.

130

biochemical and molecular mechanisms by which low oxygen exerts its action in delaying the rate of ripening/senescence of fruits.

In this paper a study was conducted with avocado fruit, to determine the effect of low oxygen on ethylene biosynthesis and on activities of ex and PG during the ripening.

Materials and Methods

Plant Material. Freshly harvested avocado fruits (Persea americana Mill.. cv Hass) were obtained from the Institute of Subtropical Plants and Olive Trees. Chania-Crete. Twenty-four hours after picking fruit were transfered to Postharvest Pomology Researche Fasility at the University Farm in Thessaloniki. Uniform first grade fruit (weight range 200-220 g) were selected, weighed and treated with benomyl (600 ppm) for decay control.

Experimental Treatments. In order to study the effect of low oxygen on ethylene biosynthesis and on activities of ex and PG during the ripening. 8-10 fruit. were placed in 35 L chambers for continuous aeration (240 ml !min), at 20° C. with the folowing treatements: air (21% 02) without propylene (C3H6). air+ C3H6. 5% Oz+C3H6. 2% Oz+C3H6, 1% Oz+C3H6. In all experiments the concentration of 130 tAlIl of propylene was used. The desired treatments of 02 were attained by mixing gases from cylinders. The storage atmosphere were monitored by an infrared analyser for CO2 consentrations and paramagnetic analyser for 02 consentrations. At periodic intervals 2-3 days. 8-10 fruit were removed from each chamber for internal gases (ethylene and carbon dioxide) analysis, for flesh firmness measurements. for ACC and EFE analysis, for protein extraction and immunodetection and enzymes assays. All treatments were run with 3 replicates.

Measurements of internal ethylene and carbon dioxide concentrations. Fruits (8-10) were removed from each treatment and placed immediatelly in water for internal gas sampling. An air samle of 2 ml withdrawn from each fruit submerged in water and 1 ml sample was used for ethylene analysis and carbon dioxide measurements. Internal ethylene concentration (lEC) was measured using a Varian Aerograph gas chromatograph Model 3300 with hydrogen flame ionization detector and a Porapac Type N (80f1(lO) 1 m x 1/8" column for ethylene separation.

Measurements of ripening parameters. After the withdrawing of gas samples for IEC and ICDC analysis the fruit plased at room temperature and flesh firmness without removing the epidermis by a Chatillon pressure tester using the conical tip.

ACC determination. Five g of tissue was taken from 3 fruit for ACC analysis. The samples homogenized with 20 ml acetone, infiltrated through Whatman No 1 paper in vacuum and after removal of acetone under vacuum at 40°C. the ACC content was determlned according to the method of Uzada and Yang (1979).

Measurements of EFE activity. It was determined by measuring the conversion of added ACC to ethylene (Cameron et al. (1979). Cylinders of 0.5 g (7 mm and 10 mm thick) peel and pulp tissue were cut and were incubated at 20°C for 30 min in vial containing 350 mM manitol, in presense or absence of 1 mM ACC. Gas samples were taken for ethylene analysis by gas chromatography.

Preparation of fruit extract for enzyme assays. One g fme powder was allowed to thaw on ice in 10 ml of 50 mM Na - acetate (pH 5). containing 5 mM Ii - mercaptoethanol, 0.4 M NaCL, 0.5 mM PMSF and 10 fLM leupeptin and 0.25 % Triton X-loo. The mixture after homogenizing for 15 min, was centrifuged at 20000 x g for 20 min and the supernatant used as the cell-free extract.

Assay of Cellulase. Cellulase activity was determined by measuring spectrophotometrically the rise in the liberated reducing sugars , following the method of Nelson (1944). The reaction mixture consisted of 100 fLl enzyme source and 100 fLl of 1 % carboxylmethylcellulose in 0.1 mM Na-acetate, PH 5.5. The reaction was carried out at 25°C for 10 min.

131

Assay of Polygalacturonase. It was detennined following the method of Nelson (1944). The reaction mixture consisted of 100 ILl enzyme source and 100 ILl of 1 % pectic acid In 0.1 M Na- acetate, PH 5. The reaction was carried out at 25°C for 10 min.

Total protein extraction and SDS-PAGE. Frozen tissue powder (lg) was thawed In 4ml of buffer containing 50 mM Tris-HCI, pH 7.4, 0.2M NaC, 20mM NaHC03, 20mM MgS04, lotA.M EDTA, 5mM jJ-mercaptoethanol, O.5mM PMSF, lotLM leupeptin, and 10% (v/V) glycerol. The mixture was allowed to stand on ice for 15min with ocasional stirring and then centrifuged at 20,OOOXg for 20min. The clear supernatant was used for fractionation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAOE) using 10-18% gradiend polyacrylamide gels and Laemmli buffer system (Laemmli (1970). Following electrophoresis, the gels were stained with 0.5% (w/V) Coomassie Brilliant Blue R-250 in 50% (v/V) methanol -10% (v/V) acetic acid and destained In 20% (v/V) methanol and 7% (v/V) acetic acid.

Protein blotting and immunocletection- Transfer of electrophoreticalIy resolved proteins on SDS-polyacrylamide gels to nitrocellulose filters (0.1 14M pore size, Schleicherr and Schuell) was carried out essentially as described by Towbin et al.(1979). After protein transfer, the nitrocellulose filters were washed in phosphate buffered saline (pBS: 0.80% NaCl, 0.02% KCl, 0.115% Na2HP04, 0.02% KH2P04) for 15 min. Unbound sites on the filter were blocked for 2 hours at room temperature with 2% (wlv) BSA in PBS. The filters were washed three times, each for 5 min in PBS supplemented with 0,05% (v/V) Tween-20, and then incubated with cellulase antiserum (Bennett and Christoffersen (1986), at a dilution of 1: 15,000 in PBS containing 1% (wlv) BSA and 0,05% (v/V) Tween-20 for 1 hour at 20° C . After rinsing , with 0.05% (v/V) Tween-20 In PBS, the filters were immersed for 1 hour In PBS solution containing horseradish peroxidase conjugated to anti-rabbit IgO (1: 2,000 dilution), 1 % (w Iv) BSA, and 0,05% (v/V) Tween-20.

Protein Detennination. Protein concentration was measured by the Bradford (1976) method using BSA dissolved In the extraction buffer as a standard.

Results

The effect of low oxygen concentrantion on ethylene production. Fruits ripened In the humidified air stream after the propylene treatment, showed typical

climateric rise in autocatalytic ethylene production, followed by a decline, which concided with the advanced softening of the fruits (Fig. 1 A). Low 02 atmospheres with propylene, decreased the rate of ethylene production and this decrease was Inversely related to the 02 concetrations used. Low oxygen concentration delayed the oncet of climacteric and reduced the peakheight of IEC. A slight reduction of IEC was found at 5% Oz treatments. In the 1% 02 IEC was suppressed drastically. However, ethylene production under such ultra low oxygen concentration supported high IEC (17.6 !LVI In the 7th day) wich was above the threshold concentration to Induced ripening. (Fig. 1 A )

The effect of low oxygen concentrantion on ACC accumulation and EEE activity. Avocado fruits are characterized by its autocatalytic ethylene production during ripening,

and this process is controlled through ethylene biosynthesis. The EFE activity in the air ± propylene, Increased markedly during the autocatalytic ethylene production and then declined. Low Oz prevented the rise in EFE activity, espesially the concentration 1 % 02 (Fig. 1 B). PrecIimacteric fruits contained only trace amounts of ACC ( 0.04 nmoles/g fresh weight) and have low EFE activity. The ACC level In the air ±. propylene ,increased markedly during the climacteric rise and then declined. In the low Oz (2% and 1%), ACC accumulates from the 3 rd day and reached the maximum price 18-20 nmoles /g.

Effect of low oxygen concentrantion, on the Jiuit softening. Low oxygen concentration reduced both ethylene production and ripening of the fruit at

20°C, as was evaluated by IEC and fumness measurements. Fruit kept at 20°C with propylene In air lost the Initial fumness by 50% (about 7 kg) In 3.5 days (Fig. 2A). The fruit reached the same value of firmness, with the 5% Oz In the 5.5th day and with 2% Oz

132

in 10 days. The 1% <>2 treatment showed a small reduction of the Initial finnness (from 13.8 to 9.3 kg) in the 15th day when the experiment was terminared. Effect of low D2 on the activity and accumulation of ex and PO protein during ripening of avocado. The activities of the enzymes PG and ex suppressed by low 02 concentrations (1%, 2% and 5%) (Fig. 2 B, C). Preclimacteric avocado fruits had very low activity of these enzymes, which increased and reached high levels in fruits ripening in air.

During avocado ripening in air ± propylene , there is an increase in ex and PG activity, this increase is due to de novo synthesis of these enzymes (Kanellis (1987). The results (Fig. 3 A, B) show that ex and PG proteins, was very low / or absent in preclimacterlc whereas they accumulated in large amounts three days after the application of propylene. PG antiserwn appears to react with three main PG polypeptidies with a molecular mass of 48 and 46 and 44 KOa, which increase during ripening. The exact relationship of these three polypeptidies is not Known.

The levels of ex and PG immunoreactive proteins were dramatically diminished in fruits held in 2% and 1 % <>2, during the 15 day (Fig. 3A and 3 B).

~ 200

U 8 150

!I! 100 ~

~ g

:<; 15

i ~ 10 .s

25

~2O .. ~ 15 I LIID 0.05 .s 8 10

< 5

A

5 10 15 20

B

5 10 20

c

°0~~--~-~~5~----1~0-----1~5----~20 DAYS

Figurel. Effect of low oxygen concentration (21% , 5%, 2%, 1%) in internal ethylene concentration (A) ,changes in the level of EFE activity (B) and ACC accumulation (C), of " Hass'" avocado fruit treated in a flow-through system with 130 J.I.I/l propylene at 20° C.

~

:g w Z

~ iL

15 A

10

B

20

40

I LIID 0.05 __ --::_::; ......... Ai'

__ - ~1'VIft.

..,.. _ .... _ ,.U""IIMoI&+rt

'-.02%02+Pr .... - .•. _._ .. :Jd .....

tI' .,.... .................... ...~ 1%02.fPr :.: ... -~::g.;.;.:g.: ...................... <1

0~0----~5----~10~----1~5----~20 DAYS

Figure 2.EtIect of low oxygen concentration (1 %, 2%, 5%, and 21 %) at 20°C in Finnness (A) ,and in activities of ex (B) and PG (C) enzymes, of ''Hass'" avocado fruit treated in a flow-through system with 130 J.I.I/l propylene.

ex

PG

1'W2tPr

03 7 10 3 7 10 3 710 3 7 103 710

21%02 21%02+Pr S%02+Pr 2%0z+Pr 1%0z+Pr

o 3 7 10 3 7 10 3 7 10 3 7 10 3 7 10 DAYS AT 20° C

133

A

B

Figure 3. Suppresion by low Oz of cellulase (Cx) and polygalacturonase (pG) proteines in "Hass" avocado fruits. Blots of total proteins (20 f,l.g/lane) were proped with polyclonal antibodies ralsed against Cx ( A) and PG (B).

Discussion

Avocado as climacteric fruit, is characterized by autocatalitic ethylene production during ripening, and this process is controlled through ethylene biosynthesis. It has been shown also that low oxygen concentration reduced ethylene production and softening of the propylene treated fruit. It is now well established that in the pathway for the biosynthesis of ethylene production the conversion of ACC to ethylene is oxygen dependent (Yang and Hoffman, 1984). In our experiment low oxygen concentration (1% Oz), althought reduced ethylene production, supported moderate IEC.

In the 1 % Oz the propylene treared fruit was unable to soften. The strong inhibition of fruit softening by 1 % Oz can not be only attributed to the reduced ethylene production, but rather to the interference with ethylene action. 'This gives support to the action model proposed by Burg and Burg (1967). Ethylene is thought to bind to a receptor, forming an activated complex before it can elicit its physiologycai action and the affinity of ethylene to the receptor depends upon Oz.

The low levels of ACC in preclimacteric fruit suggest that availability of ACC is a limiting factor regulating ethylene production. This suggests that EFE and ACC synthase are constitutive of the tissue, but ACC is not available to EFE, probably because of cellular comparmentation (Boller et al. (1979, Guy and Kende (1984». The decrease on EFE activity during the climacteric peak could be caused by autoinhibition by ethylene or by loss of mebrane integrity, which would cause the increase of ACC after the peak of the ethylene production. The data demonstrate that, in the low Oz concentrations, the suppressed EFE activity, is the limiting factor for the low ethylene production and for ACC accumulation.

A temporal relationship was reported to exist between the amount of polygalacturonase, the rate of softening and pectin degradation. Similarly, a correlation was made between the increase in polygalacturonase activity and loss of electron density in the middle lamella region of cell walls in avocado (platt-Aloia et al. 1980) and apples (Ben Arie et al. (1980). In several fruit tissues, the development of cellulase activity closely parallels the loss of fIrmness, suggesting a role for this enzyme in fruit softening. Supporting evidence for this sugggestion is the demonstration that only a mixture of cellulase and polygalacturonase would transform unripe pear tissue into one with a texture very close to the naturally ripened product (pesis et al. (1978), Awad and Young (1979». The data demonstrate that ripening of avocado fruit is associated with substantial increases in the activities of polygalacturonase,

134

and cellulase, consistent with previously published reports (Kanellis 1987, Kanellis et al. (1989). Furthennor, it is shown that the development of these enzyme activities is suppressed in fruits stored in low Oz atmospheres.

The retarding effect of low Oz on the enzymic activities seen here, may be mediated through a possible decrease in the rate of production of metabolic energy as a result of the decrease in respiration. However, this may not be the case ,because this decrease in respiration may not reflect a restriction of the electron transport chain, but rather a diminution of the biologycal efficacy of ethylene . Thus, it is likely that low oxygen-mediated repression of the enzymic activities may be a result of its interference with the action of ethylene. In the case of cellulase, this low 02 effect is posibly at the pretranslational level since the accumulation of cellulase mRNA was supressed. Propylene might induce the expression of cellulase and PG at low Oz. It seems that a bimolecular reaction, in the catalytic sense, between 02 and propylene lor ethylene ,might influence gene expression during fruit ripening (Kanellis et al 1989).

Low 02 not only suppressed the induction of new polypeptides assocciated with nonnal ripening but also induced the accumulation of some others The suppression of a number of other polypeptides in response to low 02 environment may be a result of either suppression of translation due to the dissociation of polysomes, as was reported for maize and lor repression of the expression of mRNA, or both. Low oxygen atmospheres modify respiration, inhibit ethylene action and elicit distinct changes in protein profiles and gene expression. (Kanellis (1987), Kanellis et al (1989), Sachs et al (1980)).

Accumulation and activity of polygalacturonase and cellulase are inhibited in fruit stored in low oxygen atmospheres, the inhibition being mediated via an effect on ethylen action.

Acknowledgments We would like to thank Dr. A. Kanellis for his help with the immunological work

associated with this project. This work was supported by grants from the Greek Ministry of Agriculture and the Mediterranean Agronomic Institute of Chania.

References

Awad, M., and Young, R. E. (1979) 'Postharvest variation in cellulase, polygalacturonase and pectinmethylesterase in avocado (Persea americana Mill. cv Fuerte) fruits in relation to respiration and ethylene production', PI. Physiol. 64, 306-308.

Ben-Aire, R., Kislev, N. and Frenkel, C. (1979), 'Ultrastructural changes in the cell walls of ripening apple and pear fruit', Plant Physiol. 64, 197-202. Bennet, A. B. and Christoffersen R. E. (1986), 'Synthessis and processing of cellulase from ripening avocado fruit' Plant Physiol.81. 830-835.

Boller, T., Herner R. C., nd Kende H. (1979), 'Assay for and emzymatic fonnation of an ethylene precursor, 1-Aminocyclopropane-1-Carboxyllc Acid' Planta 145, 293-303.

Bradford ,M. M. (1976),'A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the prinCiple of protein-dyebinding'Anal Biochem 72, 248-254.

Brady, C. J, (1987) 'Fruit ripening', Ann. Rev. Plant Physiol. 38, 155 -178. Burg, S. P. and Burg E. A. (1967) 'Molecular requirements for biological activity of ethylene', Plant Physiol. 42, 144-152.

Cameron, A. C. C., Fenton A. G., Y. Yu., Adams D., and Yang S.F. (1979), 'fucreased production of ethylene by plant tissues with ACe HortScience 14, 178-180.

Christoffersen, R.E., Tucker M.L. and Laties G.G. (1984) 'Cellulase gene expression in ripening avocado fruit the accumulation of cellulase mRNA and protein as demonstrated by cDNA hybrldizationand immunodetection', Plant Mol. BioI. 3, 385-392.

Guy, M. and H. Kende (1984) 'Conversion of l-aminocyclopropane-l-carboxyl acid to ethylene by isolated vacuoles of Pisum sativum L.', Planta 160, 281-287.

Kader, A. A. (1986) 'Biochemical and physiological basis for effects of controlled and modified atmospheres on fruits and vegetables', Food Tecnology 40, 99-104.

Kanellis, A. K. (1987) 'Stydies on the suppressive effect of 2.5% 02 on the rate of ripening, activitiess of hydrolytic enzymes and gene expression in ripening fruit Ph.D, Dissertation, University of Marylant.

135

Kanellis, A. K. Solomos T., Mattoo A. K. (1989a) 'Hydrolytic enzyme activities and protein pattern of avocado fruit ripened in air and in low oxygen, with and without ethylene', Plant Physioi. 90, 257-264.

Kanellis A. K., Solomos, T., Mehta, M.T., Mattoo. A.K. (1989b) 'Decreased cellulase activity in avocado fruit subjected to 2.5% <>2 correlates with lower cellulase protein and gene transcript levels', Plant Cell Physiol 30, 817-823.

Kidd, F. and West, C. (1945) 'Respiratory activity and duration of life of apples gathered at different stages of development and subsequently maintained at a constant temperature', Plant Physioi. 20, 467-504.

Uzada M.C.C. and Yang S. F. (1979) 'A simple and Sensitive Assay for lAminocyc1opropane-l-CarboxylicAcid', Anal. Biochem. 100, 140-145.

Laemmll, U.K. (1970) 'Cleavage of structural proteins during the assembly of the head of bacteriophage T, Nature 227, 680-685.

Nelson, N. (1944) 'A Photometric adaptation of the Somogyi method for the detennination of glucose', J. BioI, Chem 153, 375-380.

Pesis, E., Y. Fuchs, and G. Zaubermn (1978) 'Cellulose activity and fru it softening in avocado', Plant. Physioi. 61, 416419.

Pratt Aloia Ka, Thomson W. W, Young R. E (1980) 'Ultrastractural changes in the walls of ripening avocados: transmition seaning and freeze fracture microscopy', Bot Gaz 141, 366-373.

Sachs MM, Freeling M, Okimoto R. (1980) 'The anaerobic proteins of maize',Cell 20, 761-676.

Towbin, H., Staehelin T. , and Gordon J. (1979) 'Electrophoresic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications', Proc. Nat. Acad. Sci. USA 76, 4350-4354.

Tucker, M. L, Laties, G. G. (1984) 'Interrelationship of gene expression, polysome prevalence and respiration during ripening of ethylene and/or cyanide treated avocado fruit, Penea americana cultivar Hass', Plant Physioi. 74, 307-315.

Tucker, M. L., Durbin M. L., Clegg M. T.and Lewis N.L. (1987) 'Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene familly', Plant Mol. BioI. 9, 197-203.

Yang, S.F. and Hoffman N.E. (1984) 'Ethylene biosynthesis and its regulation in hither plants', Ann. Rev. Plant Physioi. 35, 155-189.

COLD-INDUCED CLIMACTERIC RISE OF ETHYLENE METABOLISM IN GRANNY SMITH APPLES.

C.LARRIGAUDIERE and M.VENDRELL Centro de Investigation y Desarrollo,

Dpt de Agrobiologia y Biologia molecular, Jordi Girona, 18-26,

08034 BARCELONA, SPAIN.

ABSTRACT

Changes in ethylene biosynthesis induced by cold treatment (4QC) have been studied on Granny Smi th apples. Cold treatment caused a sharp increase in ACC content both in peel and pulp tissues. It was paralleled by only a slight increase in ethylene production and MACC levels showing the inhibition of EFE and malonyl transferase activity at 4QC. Changes in ethylene biosynthesis were accompanied by significant changes in protein synthesis and particularly by the accumulation at 4QC of two polypeptides of 50 and 55 kD and by the disappearance of polypeptides near 35 kD. Rewarming caused a sharp increase in ethylene production probably related to the activation of EFE. In contrast to non treated fruits, rewarmed fruits presented a typical climacteric evolution. This climacteric behaviour was representative of a rapid maturation of the EFE system at 4QC.

INTRODUCTION

Granny Smith apples show an atypical climacteric behaviour. At room temperature, they ripen slowly, producing only low ethylene and COl levels. Thus, they act as an intermediate between normal climacteric fruits and climacteric fruits which require cold temperature to ripen.

It is now obviously recognized, that cold treatment can induce the climacteric process. This action on the ethylene biosynthetic pathway is evident for pears (Blakenship and Richardson, 1985). The increase in ethylene production was found to be the result of an increased capacity to produce ACC (Knee et al.,1983). However, ACC synthase activity remained generally low in cold conditions and increase only after transfer to a warmer temperature (Knee, 1987). On the other hand, EFE activity was inhibited in cold conditions (Wang and Adams,1980) and seems to playa secondary role.

As regards to apples, this effect of cold treatment is less 136

1. C. Pech et at. (eds.), Cellular and Molecular Aspects o/the Plant Honnone Ethylene, 136-141. © 1993 Kluwer Academic Publishers.

137

described. Knee (1983) studied the effects of low temperature on several apples cuI ti vars and found that cold treatment resulted in earlier rise in ethylene production only on Golden Delicious apples. Similar resul ts were recently described in Granny Smith apples by Jobling et al., (1991). These results suggest that apple fruit could respond similarly to pears.

The aim of this work was to study the effect of cold temperature on the physiology of Granny Smi th apples. It was focussed on the effect on ethylene biosynthetic pathway, climacteric behaviour of the fruit and relation with protein synthesis.

MATERIAL AND METHODS

Plant material and storage conditions: Granny Smith apples (Malus domestica Borkh L. cv Granny Smith)

were obtained from Lleida and harvested at commercial maturity (202 days after full bloom). Fruits were stocked in a controlled chamber whose RH was about 85% and at a temperature of 4QC or 22QC.

Determination of ethylene, C0 production and ACC levels: Ethylene production was estimated by placing fruits individually

in a 350 ml flask continuously ventilated with air. 1 ml gas samples were taken and injected into a gas chromatograph. CO~ concentrations were measured by connecting the effluent air from the v~ntilated flasks to an IRGA. ACC and MACC levels were determined as described by Hoffman et al. (1982).

Assay of EFE: EFE was determined in vivo by measuring the conversion of

administered ACC to ethylene. Disks preparation was done as described by Mansour et al. (1986) except for buffer composition which was: 0.4 M mannitol 10 mM MES-KOH, pH=6.2 and 0.4 M mannitol, 50 mM MES-KOH, pH=7.0 for peel and pulp respectively. The wound ethylene production induced by preparation of the disks was observed only after 8 h (results not shown) and did not affect our measurements.

Preparation of protein extract and electrophoresis: Protein preparation and electrophoresis were done as described

by Dominguez-Puigjaner et al. (1992) but without the phenol and EDTA washing procedures.

RESULTS

Cold-induced changes in Granny Smith apples:

- effect on the ethylene hiosynthetic pathway: Ethylene production in fruits held continuously at 4QC was low

138

but higher than at 22QC (table 1). This enhancement was paralleled by an increase in ACC levels both in pulp and peel tissues. However, and in spite of these high ACC levels, MACC levels were low at 4QC and EFE activity remained also low.

TABLE 1: Effect of cold temperature on the ethylene biosynthetic pathway.

CONTROL SKIN FLESH

C2H4 (nl/g.h) 0.4

ACC (nmoles/g) 0.5 1.3

MACC (nmoles/g) 6.9 2.0

EFE (nmoles/g.h) 0.1 0.1

- effect on protein synthesis:

--.- -.--

---SKIN

4'C I -. I 22'C

."'- .~

-'- -FLESH

COOLED SKIN FLESH

1.4

13.1 11.6

2.1 0.5

0.2 0.1

kDa

- 66

-45

-35

- 24

-14

Fig.l: SDS-PAGE of total polypeptides of Granny Smith apples held 6 weeks at 4QC, 22QC or 4QC and 1 day at 22QC (-+).

Cold treatment induced the accumulation of two polypeptides of about 50 and 55 kD in both peel and pulp tissues (fig.1). It also induced the disappearance of some polypetides of about 35 kD, particularly in peel tissue. Short-term transfer to 22QC (1 day at 22QC) did not induce notable changes in protein synthesis.

Stress response after rewarming:

After transfer to 22QC, Granny Smith apples showed a sharp increase in ethylene production (fig. 2A). This increase was not a consequence of an enhancement in ACC levels but rather a result of an activation of EFE activity (fig.2B). EFE activation was rapid but different between pulp and peel tissues.

A

TIME (HOURS)

::;: flO

.....

2

... 1.5 ~ ~

= ~

] 1 ..s t ~ 0.5 ~ :.l :z. :.l

139

B

PULP

PEEL

22QC

1 2 4 6

TIME (HOURS)

Fig.2: Short term activation of ethylene production (A) and EFE activity (B) after transfer to 22QC. -A: fruits kept at 4QC (~), 22QC (0), or 1 day after rewarming (-to; -B: non chilled ( •• ), and rewarmed fruits(~O) •

Cold induced climacteric rise in ethylene and CO2 production:

Subsequent ripening behaviour of rewarmed fruits at 22QC was greatly modified. In contrast to non chilled fruits, rewarmed fruits produced a large amount of ethylene and CO2 (fig.3). Climacteric evolution was similar for the 5 and 10 days chilled fruits which reached their climacteric peak at the same time (14 days after harvest) •

30

:;: 20 "Q ......

% ~ 10

A

5 10 20 30 TIME (DAYS)

30

::;: 20 ~ ~

! o· ~ 10

~

" ,I"~ ~ I f 1',

B

,~ l \, ....,~......... -"'.""~" ....... " ,~' ...: /' ,,-.. 5d '/. . "

), " ,-'* I 0 • 10d

f I 0/ "" I 0-_-0 0 / 0 22QC '0/ I ,,/ '0

'-01 I 0

5 ' I • . _~_.-- --._e_._e_. 42C

t t 5 10 20

TIME (DAYS)

30

Fig.3: Cold induced climacteric behaviour in Granny Smith apples. Effects on ethylene (A) and CO2 production (B). (.): cool control, (O):non chilled fruits, (*): 5 days chilled, (.): 10 days chilled.

140

DISCUSSION

The promotion of ethylene biosynthesis in Granny Smith apples by exposure to low temperatures corroborates earlier observations made on apple cultivars (Kneeet al.,1983 ; Jobling et al.,1991). Similarly to Golden Delicious, Granny smith apples accumulates ACC when held at 4QC. This enhancement is probably the result of an activation of Ace synthase related to a stress response. It can also be a consequence of two enzymatic inhibitions which occurs at 4QC. First, an inhibition of EFE probably due to the cold-induced alteration of the membrane properties. Second, an inhibition of malonyl transferase enzyme at 4QC.

Chi lIed Granny Smith apples exhibited an important change in protein synthesis. At 4QC, two entirely new proteins of 50 and 55 kD accumulated in the fruit. In contrast, a protein of 35 kD abundantly synthetized by unchilled fruits, declined significantly in chilled fruits. The physiological significance of such protein changes are still unknown. Complementary works are in progress to determine its relation with the ethylene biosynthetic pathway. They should probably allow to show the likely relationship between the 50 kD protein increase at 4QC and ACC synthase (Yip et al., 1991) and between the 35 kD protein increase at 22QC and EFE activity (Watkins et al., 1990 ).

After transfer to 22QC, Granny Smith apples showed a sharp increase in ethylene production and in EFE activity. This EFE activation could represent a "maturation" process in accordance with the model described by Me Murchie et al.(1972), and which probably combines the destruction of ripening inhibitors (Reid et al., 1973) and others processes as de novo synthesis and endocellular adjustments (unpublished data).

All these changes in the ethylene biosynthetic pathway seriously affected the ripening behaviour of the fruit. In contrast to non chilled fruits which produced only low C,H4 and CO2 levels, chilled fruits acted like typical climacteric 'fruits. This cold-induced climacteric behaviour in Granny Smith apples is of interest, because a regulatory influence of temperature on the onset of ethylene production could provide a good model to study the induction of the climacteric process.

REFERENCES

Blakenship, S.M. and Richardson, D.G. (1985), "Development of ethylene biosynthesis and ethylene-induced ripening in d'Anjou pears during cold requirement for ripening", J. Amer. Soc. Hort. Sci., 110, 520-523.

Dominguez-Puigjaner, E., Vendrell, M. and Ludevid, M.D.(1992), "Differential protein accumulation in banana fruit during ripening", Plant Physiol., 98, 157-162.

Hoffman, N.E. and Yang, S.F. (1982), "Identification of 1-

141

(malonylamino)cyclopropane-l-carboxylic acid as a major conjugate of l-aminocyclopropane-l-carboxylic acid, an ethylene precursor", Biochem.Biophys.Res.Commun., 104, 765-770.

Jobling, J., McGlasson, W.B. and Dilley, D.R. (1991), "Induction of ethylene synthesizing competency in Granny Smith apples by exposure to low temperature in air", Postharvest Biol.Techn., 1, 111-118.

Knee, M., Looney, N.E., Hatfield, S.G.S. and Smith, S.M. (1983), "Initiation of rapid ethylene synthesis by apple and pear fruits in relation to storage temperature", J. of Exp.Bot., 34(146), 1207-1212.

Knee, M. (1987), "Development of ethylene biosynthesis in pear fruit at -IQC", J. of Exp.Bot., 38(195), 1724-1733.

Li eberman, M. (1979), "Biosynthesis and action of ethylene", Ann.Rev.Plant Physiol., 30, 533-591.

Mansour, R., Latche, A., Pech, J.C. and Reid, M.S. (1986), "Metabolism of 1-aminocyclopropane-l-carboxylic acid in ripening apple fruits", Physiol. Plant., 66, 495-502.

Mc Murchie, E.J., Mc Glasson, W.B. and Eaks, I.L. (1972), "Treatment of fruit with propylene gives information about the biogenesis of ethylene", Nature, 237, 235-236

Reid, M.S., Rhodes, M.J.C. and Hulme, A.C. (1973), "Changes in ethylene and CO2 during the ripening of apples", J. Sci. Food Agric., 24, 971-979.

Wang, C. Y. and Adams, D.O. (1980), "Ethylene production by chilled cucumbers (Cucumis sativus L. )", Plant Physiol., 66, 841-843.

Watkins, C., Picton, S. and Grierson, D. (1990), "Stimulation and inhibi tion of expression of ripening-related mRNAs in tomatoes as influenced by chi lling temperatures". J. Plant Physiol., 136, 318-323.

Yip, W.K., Dong, J.G. and Yang, S.F. (1991), "Purification and characteri zat ion of l-aminocyclopropane-l-carboxylate synthase from apple fruits", Plant Physiol., 95, 251-257.

REGULATION BY TEMPERATURE OF THE PROPYLENE INDUCED ETHYLENE BIOSYNTHESIS AND RIPENING IN "HA YW ARD"KIWIFRUIT

G. STAVROULAKIS and E. SFAKIOTAKIS Laboratory of Pomology, Aristotle Univpr.itv Thessaloniki, 54006 GREECE

ABSTRACT. The present study was undertaken to detennine the critical temperature and the step in the ethylene biosynthetic pathway at which low temperature exerts its inhibitory effect in ethylene production. Hayward kiwifruit treated with propylene (150 ppm) at temperatures from 0° to 35°C showed (with Arrhenoius plot) a critical range (11°-14.8°C) above of which autocatalytic ethylene production was proceeded with an accumulation of AeC and an increase in EFE activity. Below the critical temperature range propylene was unable to trigger autocatalytic ethylene production and the inhibition was rather attributed to limited ACC production than to the low EFE activity. By reducing the storage temperature in the propylene treated fruit the rate of ripening (softening, changes in SSC) was retarded and the number of days to ripen was prolonged.

Introduction

It is known from a previous study that low temperature (10° and O°C) blocks initiation of autocatalitic ethylene production (induced by propylen) but not the ripening of kiwifruit. The present study was undertaken to deternine the critical temperature and the step in the ethylene biosynthesis pathway at which low temperature exerts its inhibitory effect in ethylene production.


Kiwifruit (cv Hayawrd) were harvested from a commercial orchard in Pieria, North Greece. The fruits were placed in 5 liter jars with 3 replications for control and propylene treated fruits. Each set of six jars was kept in separate water bath at 0°, 5°, 7°, 9°, 11°, 13°, 15°, 17°, 19°, 26° and 35°C connected in a continues flown-through system (100 ml/min) air or air+130 ppm propylene. Ten fruit samples were removed from each chamber for Internal ethylene concentration (1EC), ftnnness (Fm), Soluble solids content (SSC) ACC and EFE activity, at different intervals for each temperature.

Resultl and Discussion

The results showed that: (1) Propylene (130 ppm) in kiwifruit induced: (a)ripening by accelerating softening and sse changes and (b) autocatalysis of ethylene biosynthesis by stimulating ACe production and increased EFE activity. (2) By reducing the storage temperature in the propylene treated fruit the number of days to ripen was increased (Fig 1A) by redarding the rate of ripening as was evaluated with measurements of flesh softening (Fig lB) and the sse (Fig 1C) changes. The rate of softening gave a Q10 = 4.8 in the temperature range 0°-10°C and a Q10 = 1.3 in the range 1O-200 e, whereas there was no softening change in the range 20°-35°C. (3) Autocatalysis of ethylene production was induced by propylene at temperatures above 15°C as was evaluated by measuring IEC (Fig. 10). However below 15°C there was a strong inhibition of the propylene induced ethylene

142

J. C. Pech etal. (eds.), Cellular and Molecular Aspects of the Plant Honnone Ethylene, 142-143. © 1993 Kluwer Academic Publishers.

143

production. (4) The rate limiting factor at low temperatures for the ethylene biosynthesis of the propylene treated kiwifruit was rather ACC production (Fig. tE) than decreased activity of EFE (Fig. IF). (5) Arrhenius plots showed that a critical temperature for ethylene biosynthesis was between IF and 14.8°C (G). (6) Following the propylene treatment initiation of flesh softening and the concominant changes of SSC were noticed with a small lag period (20-25 hrs) whereas in all temperatures above 15°C the increase in IEC and ACC were noticed with a lag period 60-90 hrs following the propylene treatment. (1) Our data suggested that ripening and ethylene biosynrhesis in kiwifruit are regulated by two independend mechanisms.

60 A

00

8 B

l II> II> W .. Z ::=;; a: ii:

00

y - 50.2911 - 4.001Ox + 0.11716J<"2 - 1.1392.-3x"3 A"2 _ 0.988

10 20 30

y~ 7.205-0.67 4x+O .025x2-0.000296x3 R~.995

10 20 30

200 D ::J ::. a ()

!!! 100 w II>

~ a: ()

~ 00 30 10

E ~5000 ()

~3000 w II>

~1000 a: ()

~

0 10 20 30

€ '" ~ c

!z 12 • ~ 20 F y _ 1.01115 - 022514. + 5.81990-2<"2 - 1.1741.-3."3

() A"2 - 0.901 w !z 0 10 ()

II> a ::::i

8 g y~ 6.782+O.353x-O.00499x2

R~0.978

w -' m 60 ~ -' 10 20 30 g TEMPERATURE ·C

Figure 1. Observed response of kiwifruit to propylene (130 !JIlL) at different temperatures (from 0° to 35°C) versus the control fruit. (A) Time (days) taken to ripen (reach firmness of 0.8-0.4 kg). (8) Flesh softening as it was measured with firmness (7.9 mm plunger) changes. (C) SSC changes. (0) Increase in IEC (ppm). (E) Increase in ACe (% of control fruit). (G) Increase in EFE activity. (G) Arrhenious plots of autocatalytic ethylene production with the max values of ethylene production at aU temperatures.

.. 15 E .s W IL w

~ W II>

~ a: ()

~

10

00

• . .

10 20 30 40

TEMPERATURE ·C

Ea - 111.06 KJlmoI.

; ... ~ i Ea - 4.135 KJlmoie

33 ~11T)·W435 36 37

ETHYLENE INVOLVEMENT IN RASPBERRY FRUIT RIPENING

P. PERKINS-VEAZIEl, G.R. NONNECKE2, and R.J. GLADON2.

1 USDA-ARS, South Central Agricultural Laboratory, Lane, OK 74555

2 Department of Horticulture, Iowa State Univ.ersity, Ames, IA 50011, USA

Ethylene is involved in softening and abscission of raspberry drupes from receptacle tissue (Robbins et al., 1989; Burdon and Sexton, 1990a,b). The objective of this research was to determine the relationships between respiration rate, ethylene production rate, and ethylene action during raspberry ripening.

Fruit of six color stages were harvested with intact pedicels and stored at 2C until used. Color stages were green, yellow (yellowish drupes), mottled (yellow and red drupes), pink, red and dark red, as described previously (Perkins-Veazie and Nonnecke, 1992). Four replicates of four fruit, each with intact pedicels were used for each color stage. To prevent water stress, pedicels with attached fruits were placed in vials containing distilled water. COz and CZH4 production rate determinations were made daily for five days (Perkins-Veazie and Nonnecke, 1992). Ethylene-forming enzyme (EFE) activity was determined from separate freshly harvested drupe and receptacle tissues that corresponded to each color stage. A 0.5 mM aminooxyacetic acid (ADA) solution was applied to raspberry fruit via pedicels for 12 h and fruit simultaneously were exposed to 0 or 10 ~l.l-l CZH4 for 6 h.

Respiration at the green stage was high (155 ml.kg-l.h- l ) and declined gradually to 90 ml.kg-1.h-1 as the fruit ripened. The rate of respiration did not increase two- or more-fold concomitant with color development and this indicates that raspberry fruit are nonclimacteric. Ethylene production was low in green and yellow fruit «0.1 ~l.kg-l.h-l), higher during the transitions to mottled and pink fruit colors (1 to 16 ~l·kg-1.h-1), and comparable to pink fruit during the transitions to red and dark-red stages (13 to 16 ~l.kg-l.h-l).

When fruit parts of mottled to dark red fruits were detached, CZH4

production rates were low (3 to 8 ~l.kg-l.h-l) for drupes, but they were many- fold greater for detached receptacle tissues (Table 1). EFE activity increased during ripening and the effect was more pronounced in receptacle than in drupe tissue (Table 1).

144


145

Yellow or mottled fruit treated with AOA exhibited delayed color development, even in the presence of exogenous ethylene (Table 2). AOA treatment did not affect ethylene production, but it reduced EFE activity. Initial and final respiration rates of yellow fruit did not differ. In mottled fruit, treatment with AOA and/or CZH4 reduced the final respiration rate. Exposure of immature fruit to ethylene did not increase ethylene production or EFE activity (Table 2).

These results indicate that endogenous EFE activity is sufficient for sUbsequent ethylene production and that both drupes and receptacles are involved in raspberry fruit ripening.

Burdon, J.N. and Sexton, R. (1990a) 'Fruit abscission and ethylene production of red raspberry cultivars', Scientia Hort. 43, 95-102.

Burdon, J.N. and Sexton R. (1990b) 'The role of ethylene in the shedding of red raspberry fruit', Ann. Bot. 66, 111-120.

Perkins-Veazie, P.M. and Nonnecke, G. (1992) 'Physiological changes during ripening of raspberry fruit', HortScience 27, 331-333.

Robbins, J., Moore, P.P., and Patterson, M. (1989) 'Fruit respiration rates and firmness of red raspberry and related Rubus genotypes', Acta Horticulturae 262, 311-317.

Table 1. Ethylene production and EFE activity of detached 'Heritage' raspberry drupes and receptacles.

Color Ethylene Production EFE Activity

Stage Drupes Receptacle Drupes Receptacle

(~l CZH4okg-loh-l)

Mottled 8.2±0.6 45.5±2.7 2.6±0.5 3.0±9.9

Pink 6.5±1.1 72.3±2.8 6.6±0.5 l4.0±8.3

Red 3.l±0.9 48.6±7.5 8.7±0.9 30.3±5.6

Dark Red 6.8±2.8 49.2±10.2 l3.9±3.l 26.6±6.2

Table 2. Effect of simultaneous 0.5 mM AOA pulse treatment (12 h) and exposure to ethylene on raspberry fruit ripening after 36 h.

Initial Ethylene EFE Final

Fruit AOA CZH4 Res12iration Production Activ. Fruit

Color TMT Exposure Initial Final Initial Final Color

(~lol-l) (ml COz 0 kg -1 0 h -1) (~l CzH40kg-loh-l)

Yellow 0 87 89 0 6.0 5.1 Pink

+ 0 93 85 0 4.8 0.2 Mottled

10 94 8~ 0 6.3 3.2 Red

+ 10 95 93 0 4.8 0.1 Pink

Mottled 0 85 82 2.9 7.0 3.4 Red

+ 0 88 74 1.3 8.6 0.3 Pink

10 93 76 0.6 11. 0 5.4 Dk Red

+ 10 95 75 1.4 6.7 0.5 Pink

EFFECT OF ETHYLENE ON SESQUITERPENE NOOTKATONE PRODUCTION DURING THE MATURATION-SENESCENCE STAGE IN GRAPEFRUIT (Citrus paradisi Macf.).

D. GARCIA PUIG (1), A. ORTU&O (1), F. SABATER (1), M.L. PEREZ (1), I. PORRAS (2), A. GARCIA LIDON (2), J.A. DEL RIO* (1). (1) Dept. Biologia Vegetal, Univ. de Murcia, Campus de Espinardo, Murcia, Spain., (2) Dept. Citricultura, C.R.I.A., La Alberca, Murcia, Spain.

In a previous work, we described how the synthesis of nootkatone, a bicyclic sesquiterpene ketone, which is an important constituent of commercial flavourings and fragances, is correlated with the development of morphological differentiation in the cells of Citr~s paradisi calli (1) and the maturation-senescence process in the fruit (2),(3).

In this paper, we analyse the effect on the synthesis and/or accumulation of this secondary metabolite caused by changes in the maturation-senescence process when the picked or unpicked grapefruit are treated with different concentrations of ethylene and ethylene-releasing compounds, such as ethephon (2-chloroethyl-phosphonic acid) .

Trees of Citr~s paradisi (var. Star Ruby) were sprayed with an aqueous solution of ethephon (200 ppm) using 5 l/tree. The wetting agent used was polyethylene glycol at 0.1%.

In other assays, the fruits of Citr~s paradisi were picked and then treated with ethylene atmosphere (100 ppm) for 48 h, or treated with an aqueous solution of ethephon (480 ppm) for 30 min. In this last case, the wetting agent was triton X-lOO at 0.1%.

In each assay, the nootkatone present in the rind of the fruit was isolated using n-pentane as extractant. The general conditions for this and for GC quantification, identification by MS and microscopic study were similar to Those described in previous reports (1),(2),(3).

Treatments with ethylene or ethephon before nootkatone synthesis begins in grapefruit (28 weeks after anthesis), both in picked (Fig.l) and unpicked fruits (data not shown) increases the levels of nootkatone found in the rind.

146


O~-+--+--+--+--+--+--+~ o 10 20 30 40 50 50 70 'I"IME <daya)

FLgure 1. -noolkOolone Ln lreOoled ",Llh

Levels pLcked

elhylene

147

ppm) or ethephon ( ... BO

of

fruLl (100

ppm).

The lLme refers lo number of dOoys sLnce lrea.l menlo DOola. represenl lhe mea.n va.lues ± SE(n=9>'

The possible role of ethylene in the accumulation of this sequiterpene can be correlated with its accelerating effect on the processes involved in maturation-senescence, such as increases in the oil cavity area, morphological changes in the cells near this cavity (Table 1) and possible ultrastructural changes in the plastids (data not shown).

Ta.ble 1. - Evoluti.on of i.n lhe exoca.rp of unpi.cked of ",eek. a.fler a.nlhe.i.s. I

equa.lori.a.l cellula.r di.a.melers, i.ndi.cOoled.

severOol morphologi.cOol

frui.ls. Ti.me refers a.nd II i.ndi.ca.le lhe

resp.li.vely. ±

pa.rOomelers

lo number polOor a.nd

SE i.s

Ti.me Trea.lmenl aU ca.2vLly (/-1m)

2nd cellula.r la.yer nea.r lo ca.vi.ly (/-1m)

90

... 2

Conlrol Elhephon

Conlrol Elhephon

2CS,159±19,P1CS

9 ... ,12cS±7,257

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I

....... 7±12. cS

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72. B±25. 5

B5 .... ±19. 1

II 15. 9±9. 7

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20.9± .... 5

19. 1±2. B

In accordance with these results, Nootkatone is proposed as an indicator of maturation-senescence in grapefruit, especially in the rind. ACKNOWLEDGEMENTS: CICYT (BI089-0297), Spain. D.G.P. has a grant of University of Murcia, Spain. REFERENCES (1) Del Rio, J.A.; Ortufio, A.; Garcia Puig, D.; Iborra,

J.L. and Sabater, F. (1991). Plant Cell Reports 10: 410-413 .

(2) Del Rio, J.A.; Ortufio, A.; Garcia Puig, D.; Porras, I.; Garcia Lid6n, A. and Sabater, F. (1992a). J. Agric. Food Chern. (in press).

(3) Del Rio, J.A.; Garcia Puig, D.; Ortufio, A.; Sabater, F.; Garcia Lid6n, A. and Porras, I. (1992b). VII Proc. Int. Soc. Citriculture (in press).

ETHYLENE BIOSYNTHESIS DURING THE RIPENING OF CHERlMOYA (Annona cherimola, Mill).

MARTINEZ, G., SERR.t\NO, M., PRETEL, M.T., AMOROS, A., R1QUELME, F. and ROMOJARO, F. Centro de Edafologia y Biologia Ap/icada del Segura. Avda. de fa Fama 1. Murcia. Spain.

Climacteric fruits are characterized by a rapid increase in the rate of ethylene biosynthesis at the beginning of the ripening process. It has been considered that this hormone is of capital importance at the start of this process, as it controls some modifications that will induce the fruit to acquire very good organoleptic characteristics of consumption (Yang and Hoffinan, 1984; Amoro s et a/., 1989a).

The most important problem in the marketing of cherimoya is the rapid ripening and browning occuring a few days after harvesting (Brown et aI., 1988), which depends on both, the concentration of phenolic components and the activity of polyp he no I oxidase (PPO) (Coseteng and Lee, 1987; Martinez- Cayuela et al., 1988). We have studed the biosynthetic pathway of ethylene and the possible relationship between ethylene bios)-nthesis and brovming.

Ethylene and C02 production were detemlined daily. Ethylene was quantified on a HewlettPackard cromatograph and C02 was quantified in a non dispersive infrared analyzer Beckman model 865. Three cherimoyas with an individual ethylene production rate close to the average of the whole set were taken daily. The luminiscence, browning (according to Baloch et al., 1986) and total chlorophylls (Moran, 1982) were measured in the skin of each fruit. In the flesh, the following were measured: fimmess, sugars (Dubois et al. 1956),starch (enzynmtic kit), 0 Brix, pH, acidity and, also, ACC levels, ACC-synthase (Mansour et aI., 1986) and EFE. Each measurement was carried out twice on each fruit.

Cherimoya behaves like a climacteric fruit after harvesting since shows respiration and ethylene crises connected with its ripening (Figure I). On the first and second days free ACC levels were low (0.40-0.81 nmols g-I FW) but they reached maximal values during the third and forth days (6.40 and 8.96, respectively), coinciding with the maximal rate of ethylene production. Furthermore the percentage of ACC in conjugated form (MACC) was high (85% of totaI ACC) during the preclirnacteric and postclirnacteric periods and less than 40% in the climacteric.

In preclirnacteric fruits (first day) the enzynmtic activities ACC synthase and EFE were very low. Later both increased dramatically, rcaching a maximal rate on the third day, which coincided with the maximal rate in ethylene production. In the postclimacteric stage, both ACC s)-nthase and EFE decreased (Figure 2). Browning of the skin increased after harvesting, but this was not due to

148

J. C. Pech eta/. (eds.), Cellular and Molecular Aspects of the Plant Honnone Ethylene, 148-149. © 1993 Kluwer Academic Publishers.

149

a decrease of chlorophyll, since the chlorophyll level remained stable through the entired period. However, the browning rate developed two days after harvest, increasing from 0.1 to 0.3 at the end of the experiment. The most important increases were produced between the second and third day and later also between the fourth and fifth day. These coincided respectively with the increase and the important dt"-crease of ethylene emission (Figure 1). Decreases in luminiscence were coincident with the rises in browning rate and there was a close correlation between both L parameter of color by reflection and browning (r=0.947). Thus it is possible to use the measured of luminiscence by reflection as a degree of browning. There was also a temporal coincidence between the onset of ethylene production, the degradation of starch, the loss of firmness and the build up of total sugars.

,~;. ~ ~ _OIl " -, -- ;cr .... .10;: 5 .. " .ao! S:: ..

" ,

>- III .J:! 0

u .... -- ltIlytao 10 - CO2

• • • Oays after harnat

Figure I . Rate of ethylene production (00) and respiratory acti"ity (--) dunng the post-harvest ripening of cherirnoya. Tho figures corrospond to an average measiurc :!: SE of about 20 fruits

~ClU • '8 .. SlIP! ":cr

0 I

,," 10_ . ~ ' .. .,;.,;

i 0'" • • .. CI ... " i>. Ul s ... • :! I 'u • u • ~ou ~ a •

CI ; u'" u ~

• s • Oays after banwt

Figure 2 : Evolution of ACC synthase (nmols ACC mg_1 prot 30 min-I) (black bars) and EFE (n! ethyleneg-I h-I) (grated bars) activites during postharvest ripening of cherimoya at 20°C. The figures of ACC synthase are the mean ± ES of the determinations made up twice on three fruits. For EFE activity the figures are the average ± ES ofthc estimations made on three fruits.with five repetitions on eachonc.

A.,\40ROS ct al. (1989). 1. Hortc. Sci., 64(6), 673-677. BALOCH et aI. (1983). 1. Sci. Food Agric. 24, 389-398. COSETENG and LEE (1987). 1. Food Sci., 52(4), 985-989. DUBOIS et aI. (1956). Anal. Chem., 28, 350-356. MANSOUR et aI. (1986). Physiol. Plant. 66(3), 495-502. MARTINEZ-CAYUELA ct aI. (1988). 1. Food Sci., 53(4),1191- 1194. MORAN, R. (1982). Plant Physiol., 69, 1376-1381. YANG AND HOFFMAN (1984). Ann. Rev. Plant Physiol., 35, 155- 189.

EFFECTS OF CO 2 ON ETHYLENE PRODUCTION BY APPLES AT LOW AND HIGH O2 CONCENTRATIONS

A. Levin, L. Sonego, Y. Zutkhi & R. Ben Arie Dept. of Fruit & Vegetable Storage Bet Dagan 50250 Israel

Introduction

Ethylene, an endogenous plant growth regulator, plays a central role in the ripening of fruit (Biale and Young, 1981). The control of ethylene production is an important aspect of the commercial storage of fruit. The retardation of fruit ripening by raised levels of CO2

is generally attributed to its inhibitory effect on ethylene production and/or action. The process by which CO2 may inhibit production of ethylene is still unknown. The effect of CO2 has generally been studied at reduced O2 levels (Kader, 1986). The present study reports on the effect of CO2 on the biosynthesis of ethylene in "Jonathan" apples during controlled atmosphere (C.A.) storage at low and high O2 levels.

Experimental

Ethylene biosynthesis in "Jonathan" apples was examined under conditions of raised CO2 concentrations. A flow-through system was used with 9 ethylene-scrubbed air mixtures introduced into 4 x 30 L plastic containers at O°C. Fruit was removed from storage after 3, 5, and 7 months. Internal ethylene concentration (IEC), 1-aminocyclopropane-1-carboxylic acid (ACC) and ethylene-forming enzyme (EFE) activity in the fruit were determined (Lizada and Yang, 1979; Yang and Hoffman, 1984) immediately after removal from storage, and after 1 week at 20°C.

Results

The average levels of ethylene measured in the containers during the first 8 weeks of storage in C.A show the inhibitory effect of CO2 •

This is also indicated by the IEC data throughout storage. Ethylene production was totally inhibited by 20% CO2 at 15% O2 and by 10% CO 2 at 3% O2 • The significant inhibition incurred by 3% O2 was enhanced by added CO 2 •

Increasing levels of CO 2 proportionately reduced the ACC 150

J. C. Pech et at. (eds.), Cellularalld Molecular Aspects of tile Plalll HOn1l0lle Ethylelle, 150-151. © 1993 Kluwer Academic Publishers.

content of fruit in storage and after shelf life. In air, CO2 , ACC levels increased during storage. Reduced O2

accumulation only in fruits stored without CO 2 • This reduced O2 was lost during shelf life.

151

or without enhanced ACC

effect of

At both O2 levels, EFE activity was stimulated by increasing CO2 from 0 to 10%, but was inhibited by 20% CO2 • Without CO2 ,

EFE activity was inhibited by 3% O2 , but not by 15%. The inhibition of EFE activity incurred by reduced O2 , was overcome by 10% CO2

after 3 months and by 5% CO2 after 7 months in storage. Whereas inhibition of EFE activity by 20% CO2 increased with time, its stimulation by lower CO 2 levels was unaffected by duration of storage.

Conclusions

1. Ethylene production by apples was inhibited by increasing CO 2 from 0% to 20% at both low and high O2 tensions.

2. The Ace content was similarly reduced by increasing CO2

levels, even at reduced O2 , which enhanced ACC accumulation but only in the absence of CO 2 •

3. EFE activity was stimulated by CO 2 up inhibited by 20% CO 2 at both O2 tensions.

4. The inhibition of be attributed to activity.

References

ethylene production by its inhibitory effect

to 10%,

may ACC

but was

therefore synthase

Biale, J.B. and Young, R.E. (1981) "Respiration and ripening of fruits - retrospect and prospect," in L. Friend and M.J.C. Rhodes (eds.), Recent Advances in the Biochemistry of Fruit and Vegetables, Academic Press, London, pp. 1-39.

Kader, A.A. (1986) "Biochemical and physiological basis for effects of controlled and modified atmospheres on fruits and vegetables." Food Technol. 40(5), 99-104.

Lizada, M.C.C. and S.F. Yang. (1979) "A simple and sensitive assay for 1-aminocyclopropane-l-carboxylic acid." Anal. Biochem. 100, 140-145.

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

HIGH CARBON DIOXIDE TREATMENT BEFORE STORAGE AS INDUCER OR REDUCER OF ETHYLENE IN APPLES

E. PESIS 1 , C. AMPUNPONG2 , B. SHUSIRI 2 and E.W. HEWETT2

1Dept . of Fruit & Vegetable storage, The Volcani Center 2P,O Box 6, Bet Dagan 50250, Israel. Dept. of Horticultural Science, Massey University Palmerston North, New Zealand.

It was shown in several varieties of apples that application of high CO2 levels (12-30%) at cold temperatures (0-4°C) for around 2-4 weeks prior to storage at O°C delayed softening and ripening as well as ethylene and CO2 production (Couey and Olsen, 1975; Bramlage et al., 1977). Application of high CO2 or low 02 concentrations at 20°C for a short period as prestorage treatment to various fruits, was shown to be an effective tool for maintaining fruit quality by reducing ethylene production, maintaining firmness and increasing aroma volatiles during storage (Wills et al., 1982; Pesis et al., 1991; Lurie and Pesis, 1992).

In this research we study the effect of application of extreme high CO2 concentration (95%) for very short periods (1-4 days) at 20°C on apples ripening and quality in shelf life (Golden Delicious) and in cold storage followed by shelf life (Braeburn).

Golden Delicious apples were enclosed one day after harvest in high CO2 atmosphere (95% CO2 , 4% N2 and 1% 02) at 20°C for 24, 36 and 48 h. After removal from the CO2 pretreatments, the fruits were left in air at 20°C for further examination. Braeburn apples were treated the same but for periods of 24, 48, 72, and 96h. After treatment the fruits were transferred to cold storage for 6 weeks at 5°C followed by shelf life of 10 days at 20°C. The examination includes measurements of: firmness; colour; total soluble solids; acidity; taste; CO2 and ethylene production by whole fruits; acetaldehyde, ethanol and ethyl acetate measured by GC in extracted juice (Lurie and Pesis, 1992). All data presented are means of 5 measurements.

High CO2 pretreatment induced CO2, ethylene, ethanol and ethyl acetate production in Golden Delic~ous apples during storage at 20°C (Table 1). Treated apples became softer and yellower but more tasty than the untreated fruits after 2 weeks at 20°C.

152

J. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plallt Hormone Ethylene, 152-153. © 1993 Kluwer Academic Publishers.

153

TABLE 1. CO2 , ethylene, ethanol and ethyl acetate levels in Golden Delic10us apples 6 days after treatment at 20°C.

Treatment CO2 Ethylene Ethanol Ethyl acetate (mg/kg.h) (ul/kg.h) (ppm) (ppm)

0 h CO2 4.92 0.00 0.0 0.88 24 h CO2 10.35 36.16 165.9 9.18 36 h CO2 8.18 23.50 48 h CO2 7.03 8.41 564.8 11 .59

Carbon dioxide and ethylene production was reduced and volatiles induced by high CO2 pretreatment of Braeburn apples during 2 weeks of shelf life at 20°C, after 6 weeks cold storage (Table 2). The treated fruits remained firmer but yellower than the control ones.

TABLE 2. ethylene, and ethyl acetate levels and firmness of Braeburn

CO2 , apples 2 days after removal from cold storage of 6 weeks.

Treatment CO (mgdg.h)

o h CO2 8.43 24 h CO2 7.65 48 h CO2 8.01 72 h CO2 6.66 96 h CO2 5.61

In conclusion, high ethylene production and temperature. However, CO2 in both temperatures.

Ethylene Ethyl acetate Firmness (ul/kg.h) (ppm) (N)

42.34 3.75 61.44 27.29 4.03 65.95 23.70 30.17 69.77 24.07 54.57 69.97 27.81 43.44 76.00

C~2 pretreatment can reduce or induce r1pening of apples depending on the storage pretreatment induces volatiles production

Bramlage, W.J., Bareford, G.D., Dewey, D.H., Taylour, S., Porritt, S.W., Longheed, E.C., Smith, W.H. and McNichols, F.S. 1977: Carbon dioxide treatment for 'McIntosh' apples before CA storage. J. Amer. Soc. Hort. Sci. 102, 685-662

Couey, H.M. and Olsen, K.L. 1975. Storage response of 'Golden Delicious' apples after high carbon dioxide treatment. J. Amer. Soc. Hort. Sci. 100, 148-150

Lurie, S. and Pesis, E. 1992. Effect of acetaldehyde and anaerobiosis as postharvest treatments on the quality of peaches and nectarines. Postharvest BioI. Technol. 1, 317-326.

Pesis, E., Zauberman, G., Avissar, I. 1991. Induction of feijoa certain aroma volatiles by postharvest application of acetaldehyde or anaerobic conditions. J. Sci. Fd. Agric. 54, 329-337.

ADH ACTIVITY! VIA ETHANOL, AFFECTS ETHYLENE PRODUCTION IN TOMATO PERI CARP DISCS

R.Botondi, E. Gobattoni, R. Massantini, F. Mencarel li - Istituto Tecnologie Agroalimentari, Universita di Viterbo, Via DeLellis, 01100 Viterbo (Italy)

Exposure of tomato fruits to ethanol vapors delays the ri pening by inhibiting the ACC oxidase activity via a sugge sted modification of membrane microstructure. Alcohol dehy drogenase (ADH) activity increases several folds in green -tissue exposed to hypoxia; in contrast, the increase is small under ethanol treatment. ADH activity has been postu lated being affected by ethylene in yeast but Morrell and -Greenway (1989) have shown no relation exists between ethy lene and ADH in rice coleoptiles. Because in ripening fruit~ ethanol, acetaldehyde and ethylene increase and ADH activi ty rises in ripening tomato fruits (Longhurst et al. 1990)~ we have studied if a relation exists,in tomato fruit peri carp discs treated with ethanol vapor, between ethylene and ADH. Pink tomato fruits (~YQ22~~§~Q2~_~§Q~1~~t~~ Mill.,Fl hybrid Montecarlo) were picked from plants grown at a field close to the laboratory. They were washed in a solution of commercial bleach (1:20 to water), rinsed with water and air dri-ed in a sterile transfer hood. One-cm epidermal peri carp discs were excised and ethanol treatment conducted as de-scribed by Saltveit (1989) but 0.5mL of absolute ethanol in a 3,5 L glass jar kept sealed for 7 hours was used.20 uL of water, 5 mM 2-aminoethoxyvinylglycine (AVG) or 10 mM 4-methylpyrazole (4-MP), inhibitors of ACC-synthase and ADH re-spectively, were applied to each disc after 1.5 hours from the jars opening. After the absorption, discs were placed into glass vials (3 discs in 25 mL free volume) capped (1/2-1 h) with a rubber stopper and ethylene production was fol-lowed for 8 or 24 hours (1 mL head-space gas sample injected in Carlo Erba Fractovap 4200 adapted with FID and alumina column kept at 100°C). ADH (EC 1.1.1.1) activity was evaluated at different times from the end of ethanol treatment by following the procedure of Longhurst et al. (1990) but using liquid nitrogen and increasing 6-folds the extract concentration. ADH activity is expressed as the amount of en-

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J. C. Pech et af. (eds.), Cellular and Molecular Aspects a/the Plant HomlOne Ethylene, 154-155. © 1993 Kluwer Academic Publishers.

zyme required to produce 1 umole of NADH/min. Experiments were run twice obtaining similar results.

155

Ethanol treatments enhanced ADH activity (2.2-fold)compared the water-treated discs soon after the end of treatment and inhibited greatly the ethylene production. 8 hours later, ADH activity increased significantly and apply of 4-MP par tially inhibited the increase; in water-treated discs, ADH-activity was completely inhibited by 4-MP. In both cases, 4-MP reduced the ethylene production. Apply of AVG decreased significantly the ethylene production in water-treated discs but not in ethanol-treated ones which already showed lower levels of ethylen~. Apply of ACC stimulated ethylene 4-5-fold in water-treated discs and only 2-fold in ethanol ones. Ethanol inhibits ethylene production but stimulates ADH acti vity when the concentration into the tissue is very high. -By the time 4-MP is applied, ADH activity is reduced but not completely inhibited and ethylene is ~i~atly decreased. This behaviour seems to indicate ethanol itself inhibits ethylene production,and not via acetaldehyde, depending on concentra tion. Our supposition is confirmed by water-treated discs -data where the apply of 4-MP inhibits completely ADH and re duces ethylene production. (50% less than control).

Research sup'ported by National Research Council of Italy, Special Project RA1SA, Sub-project N°4

References

Longhurst, T.J., Tung H.F. and Brady,C.J. (1990) Developmen tal regulation of the expression of alcohol dehydrogenase -in ripening tomato fruits. J. Food Biochem. 14, 421-433. Morrell,S. and Greenway, H.(1989) Evidence does not support ethylene as a cue for synthesis of ADH and pyruvate decar boxylase during exposure to hypoxia. Aus. J. Plant PhysioT. 16,469-475. Saltveit, M.E. Jr (1989) Effect of alcohols and their inter action with ethylene on the ripening of epidermal pericarp -discs of tomato fruits. Plant Physiol. 90, 167-174.

TWO-DIMENSIONAL PROTEIN PATTERNS OF CHERIMOYA FRUI'I'S DURING RIPENING

L. M. MONTERO, ~I. I. ESCRIBANO, J. P. ZAMORANO & C. MERODIO Instituto del Frio (CSIC). Ciudad Universitaria, sin. 28040. Madrid Spain

Fruit ripening is characterized b, a series of highly-coordinated physiological and biochemical changes. Some of these changes appear to be regulated by the expression ot ripening-related genes which control the synthesis of different mRNAs and proteins (DellaPenna et a1., 1986). Differential accumulation of proteins at specific ripening stages has been reported in tomatoes (Grierson et a1., 1986), avocados (Christoffersen et a1., 1984), apples (Lay-Yee et a1., 1990), and bananas (Domillf]UeZPuigjaner et a1., 1992). To date no reports about modifications in the protein pattern of an;- Jnnond fruit during ripening have been published. In the present study we analyzed the evolution of several physiological parameters (respiration rate. ethylene production and flesh firmness) in cherimoya fruit in relation to changes in protein pattern observed at different ripening stages.

Respiration rate and ethylene production in cherimoya fruits (llnnona cherimo1a Mill., cv. Fino de Jete) were determined by gas cromatography. Flesh firmness was measured in an Instron Food Testing instrument. An efficient protein extraction method was developed to obtain total protein from pulp tir;sues. Four protein precipitation methods, using acetone. trichloroacetic acid, phenol-ammonium acetate-methanol, and ethanol, were evaluated to select a quantitative and highly-resolutive precipitation procedure for electrophoretic analyses. Proteins (40 pg) were separated on a 129" SD8-polyacrylamide gel accordinq to the method of Laemmli (1970). Two-dimensional electrophoresis was performed as described by O'Farrell (1975), with minor modifications. Ampholytes ranged in both a pH from 5-8 and 3.5-10. Gels were stained with Coomassie brilliant blue and/or silver following commonly used protocols.

Based on yield results, the acetone method appeared to be the most efficient for total protein recovery. However. electrophoretic analyses revealed better resolution of proteins separated following the phenolammoni 11m acetate-methanol method, espec i all y for proteins ranq i ng in molecular weight (~IW) from 30 to 67 kIl. This method showed the best yield/resolution rate and wa~; selected for subsequent electrophoretic analyses. Changes in protein pattern were studied on fruits at: a) early ripening stage (first respiration peak), b) maximum ethylene production stage, and c) last ripening stage (second rer;piration peak).

156


157

SDS-PAGE protein patterns obtained after Coomassie brilliant blue (CBB) staining revealed a preferential accumulation of polypeptides at the maximum ethylene production stage, ranging in MW from 20 to 36 kD. Major differences were observed in polypeptides of 21, 23, 31, 32 and 35 kD. A more sensitive double CBB-sil ver staining, however, showed relevant accumulation of polypeptides in unripe fruit (24 and 33 kD), and fruits at the early (26 and 27 kD) and at the last ripening stage (34 kD).

Two-dimensional electrophoresis improved the resolution of polypeptides separated by SDS-PAGE. Some polypeptides ranging from 40 to 67 kD showed different quantitative trends during ripening. Thus, in a pI range from 5.20 to 5.85, a set of polypeptides of 50 kD were significantly present in unripe fruit, decreased at the early ripening stage, then accumulated at the maximum ethylene production stage, and finally declined at the last ripening stage. Three other groups of polypeptides (41, 43, and 58 kD) accumulated specifically at the maximum ethylene production stage. Finally, a group of 40 kD polypeptides (pI range 4.20-4.55) decreased during ripening, whereas another group of 63 kD (pI range 7.35-8.15) accumulated at the maximum ethylene production stage, but maintained a relatively high level at the last ripening stage.

Improved resolution and more significant changes were found in polypeptides ranging from 22 to 36 kD, which appeared, accumulated, or dissapeared at specific ripening stages. Major differences were found in polypeptides with MW close to those detected by SDS-PAGE analysis. The changes observed in the level of some of these polypeptides at specific ripening stages were highly associated with the rise of ethylene production, as well as with trends in respiration rate and decline of flesh firmness. These results suggest that the modification of cherimoya fruit protein pattern could be coordinated with physiological and biochemical changes occurring during ripening.

Dominguez-Puigjaner, E., Vendrell, M., and Ludevid, M.D. (1992). 'Differential protein accumulation in banana fruit during ripening' . Plant Physiology 98, 157-162.

Christoffersen, R.E., Tucker, M.L., and Laties, G.G. (1984). 'Cellulase gene expression in ripening avocado fruit: the accumulation of cellulase mRNA and protein as demonstrated by cDNA hybridization and immunodetection'. Plant Molecular Biology 3, 385-391.

DellaPenna, D., Alexander, D.C., and Bennet, A.B. (l986). 'Molecular cloning of tomato fruit polygalacturonase: analysis of polygalacturonase mRNA levels during ripening'. Proc. Natl. Acad. Sci., USA 83, 6420-6424.

Grierson, D., Maunders, M.J., Slater, A., Ray, J., Bird, C.R., Schuch, W., Holdsworth, M.J., Tucker, G.A., and Knapp, J.E. (1986). 'Gene expression during tomato ripening'. Phil.Trans.R.Soc.Lond. B 314, 399-410.

Laemmli, U.K. (1970). 'Cleavage of structural proteins duri~ the assembly of the head of bacteriophage T4'. Nature 227, 680-685.

Lay-Yee, M., DellaPenna, D., and Ross, G.S. (1990).'Changes in mRNA and protein during ripening in apple fruit (Malus domestica Borkh cv. Golden delicious)'. Plant Physiology 94, 850-853.

O'Farrell, P.H. (1975). 'High resolution two-dimensional electrophoresis of proteins'. J. Biological Chemistry 250, 4007-4021.

INVOLVEMENT OF ETHYLENE LEVELS IN DELAYED RIPENING OF AVOCADO CV. 'HASS' AT LOW TEMPERATURE.

P.Zamorano and C.Herodio Instituto del Frio (CSIC) Dept.Refrigeration of Vegetable Products Ciudad Universitaria sin. 28040-MADRID SPAIN

Low temperature storage of avocados (Persea americana ~i11.) is someway limited by the ocurrence of chilling injury (CI). The CI symptons of avocado include atypical respiration and ethylene production patterns and failure to soften properly upon warming after storage (Wang,1990).

Induction of cellulase (enzyme with a cardinal role in avocado softening) by C,H l has been reported by Tucker et al. (1985) . Changes in the avocado gene expression have been shown by Christoffersen et al. (1982). Starret & Laties (1991) reported that propylene (analogue ot ethylene) strongly affected three species of mRNA that showed changes during avocado ripening .However, the polypeptide profile ot avocados ripened in low oxygen was not modified by C;H, supply (Kanellis et al. (1989).

Since ethylene hastens the softening of avocado fruits, is involved in ripening-related changes in the polypeptide profile (specially those affecting cell wall degrading enzymes) and increases CI ocurrence during cold storage of avocados (Zauberman et al. (1973) we have studied the relationship between flesh firmness, CO; and C;H, production and the protein pattern of avocados cv. 'Hass' stored at 7°C.

Late season,mature avocado fruits were stored in cold-storage rooms at 7° and 20°C, 90go R.H. Twenty-five fruits at each temperature were enclosed in respiration chambers, and external ~H, and C~ production were determined by gas chromatography. Flesh firmness was measured using an Instron Testing Machine.Three representative fruits were halved,cut into pieces and frozen in liquid N1 • Samples were ground to a fine powder and stored at -70°C.

Total protein extraction was made according to Kanellis et al. (1989). Proteins were analyzed by SDS-PAGE using 10 to 15 96 gradient polyacrilamide gels. Silver-stained gels were scanned using the Biolmage System (Millipore Corporation).

The rates of CO, and C;H, production were lowered ,and softening was delayed at 7°C. The drop in flesh firmness coincided with the beginning of a rise in CO; and was preceded and tollowed by peaks ot C;H,. Since ethylene was below 1.2 pl/Kg/h during cold storage it is shown that high levels of C;~ production are not required tor avocado softening.

158


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The protein profiles showed that most of the polypeptides are present at both temperatures.Some of the main changes at 20° are resembled at 7°C, but delayed. Polypeptides with MW of 15.8.21,23.5 and 25.8 kD ,increasing during ripening • also accumulated after 32 days at 7°C. Polypeptides of 18.2 , 23 and 24 kD, whose levels d.ecreased at 20° showed similar profile at 7°C, but were still detectable after 39 days. The 25.8 kD polypeptide could

correspond to that of 25.7 kD reported by Kanellis et al (1989) to increase during ripening in air but decreasing in low oxygen atmosphere.

Based on the data presented and on evaluation of fruit ripeness,it appears that the climacteric ethylene is not essential to trigger the ripening of avocado fruits.It has been suggested that low levels of wound ethylene could sensitize avocado discs, so that a low level of CIH. would trigger or mediate ripening (Starret and Laties, 1991).It would also be possible that the low increases in the ethylene production at 7°C could sensitize the tissue, allowing the avocado fruits to ripen at this temperature. The lack of climacteric ethylene at low temperature would lead to a lack of clear correlation between ripening parameters, but could not prevent ripening. The suggestion that both synthesis and degradation of proteins are involved in the ripening process is supported by the similar polypeptide profiles, but delayed at low temperature.

Christoffersen,R.E., Warm,E. & Laties,G.G.(1982).'Gene expression during fruit ripening in avocado'. Planta 155, 52-57.

Kanellis,A.K., Solomos,T. & Mattoo,A.K.(1989).'Hydrolitic enzyme activities and protein pattern of avocado fruit ripened in air and in low oxygen. with and without ehylene'. Plant Physiol. 90, 257-266.

Starret,D.A. & Laties,G.G.(1991). 'The effect of ethylene and propylene pulses on respiration, ripening advancement, ethylene-forming enzyme , and l-aminocyclopropane-I-carboxylic acid synthase activity in avocado fruit'. Plant Physiol.95,921-927.

Starret, D.A. & Laties,G.G.(1991). 'Involvement of wound and climacteric ethylene in ripening avocado discs' . Plant Physiol. 97 , 720-729.

Tucker,M.L., Christoffersen,R.E., Woll,L. & Laties, G.G. (1985) 'Induction of cellulase by ethylene in avocado fruit' ,in J.A.Roberts & G.A.Tucker (eds.),Ethylene and Plant Development,Butterworths,London.pp 163-171.

Wang,Chien Y. (1990). Chilling Injury of Horticultural Crops. eRe Press, Boca Raton, Florida.

Zauberman, G. & Fuchs. Y . (1973) . 'Ripening processes in avocados stored in ethylene atmosphere in cold storage' .J.Amer.Soc.Hort.Sci. 98, 477-480.

RELATIONSHIP BETWEEN POLYAMINES AND ETHYLENE IN CHERIHOYA FRUIT RIPENING

H.I. Escribano, L.M. Montero, J.P. Zamorano and C. Merodio U.E.I. Refrigeracion de Productos Vegetales, Instituto del Frio Ciudad Universitaria s/n, 28040 Madrid Spain

Ethylene and polyamines are known to have opposite effects on fruit ripening: ethylene production begins only after the levels of certain endogenous polyamines decline (Winer and Apelbaum, 1968; Saftner and Baldi, 1990). The precursor S-adenosyl methionine is found in the biosynthesis of both compounds (Evans and Malmberg, 1989).

While polyamine action is not exactly known, some authors suggest that the inhibitory effect on ethylene production involves modification of either activity or synthesis of ACC synthase (Apelbaum et al., 1982). Another contention is that polyamines act on EFE activity, specifically on the enzyme complex or indirectly through membrane interaction (Saftner,1989).

This paper presents an analysis of trends in certain physiological parameters (ethylene production and flesh firmness), and how they relate to changes in polyamine levels during cherimoya (Annona cherimola, Hill. cv. Fino de Jete) fruit ripening. The free polyamines (unhydrolized supernatant), soluble conjugates (hydrolized supernatant) and bound polyamines (hydrolized pellet) were analyzed in mesocarp cherimoya tissue as described in Escribano and Legaz (1988), with some modifications. After tissue homogenization in cold 5 % perchloric acid (400 mg tissue/ 1 ml acid), dansylated polyamines were separated by HPLC on a 4.6 mm x 15 cm reverse phase Cli column. The polyamines were eluted with a 21-min, 70 % to 90 % methanol in water gradient and detected by fluorescence spectrophotometry (excitation 350 nm, emission 495 nm). The identity of the dansyl polyamines was determined by retention times related to an internal standard (1,6-hexanediamine).

Three relevant ripening phases have been defined for cherimoya fruits (Montero et al., unpublished results). We have shown that putrescine, spermidine and spermine are present during the early ripening stage (first respiration peak). The levels of conjugated and bound putrescine were very low or nondetectable, whereas free and conjugated spermine levels were observed to be quite similar. Spermine was the predominant polyamine, accounting for approximately 53 % of the total polyamine content. Free, conjugated and bound spermidine were likewise present in these unripe fruits.

Attempts to identify a specific polyamine pattern related to 160

J. C. Pech et al. (eds.), Cellular and Molecular Aspects o/the Plant Hormone Ethylene, 160-161. © 1993 Kluwer Academic Publishers.

161

ripening have - to date - addressed only free polyamines. During ripening at 20 DC, cherimoya fruit softens rapidly, with

maximum ethylene levels being reached on the 4th day after harvest. Two days after harvest, prior to peak ethylene production, high spermidine levels decreased sharply, while the putrescine titer increased considerably. No changes were observed in spermine levels until the 4th day of the ripening period, during the second respiratory peak, when they rose by 44 %.

In order to determine whether the increase in the putrescine titer, the main free polyamine, is related to ripening or occurs with time regardless of the ripening process, the polyamine rate was analyzed. The polyamine rate is highly correlated (r=0.99) to the ripening process (as defined by flesh firmness) aad is a dependent process.

This study seems to indicate that, as in avocado and tomato fruits (Winer and Apelbaum, 1986; Saftner and Baldi, 1990), in cherimoya fruit a decrease in polyamines, specifically in spermidine, is required for ethylene production. Furthermore, we report here that the rapid softening of cherimoya fruit may be related to the relatively high level of putrescine during ripening.

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Apelbaum, A., Icekson, I., Burgoon, A.C. and Lieberman, M. (1982) "Inhibition by polyamines of macromolecular synthesis and its implication for ethylene production and senescence processes", Plant Physiol. 70, 1221-1223.

Escribano, M.I. and Legaz, M.E. (1988) "High performance liquid chromatography of the dansyl derivatives of putrescine, spermidine and spermine", Plant Physio1. 87, 519-522.

Evans, P.T. and Malmberg, R.L. (1989) "Do polyamines have roles in plant development?", Annu. Rev. Plant Physiol. Plant Mol. BioI. 40, 235-269.

Saftner, R. (1989) "Effects of organic amines on a-aminoisobutyric acid uptake into the vacuole and on ethylene production by tomato pericarp slices", Physio1. Plant. 75, 485-491.

Saftner, R. and Baldi, B.G. (1990) "Polyamine levels and tomato fruit development: possible interaction with ethylene", Plant Physiol. 92, 547-550.

Winer, L. and Apelbaum, A. (1986) "Involvement of polyamines in the development and ripening of avocado fruits", J. Plant Physio1. 126, 223-233.

MODULATION OF GENE EXPRESSION UNDER ETHYLENE TREATMENT IN THE LATEX OF Hevea brasiliensis

v. PUJADE-RENAUD, C. PERROT-RECHENMAN, J. D'AUZACo, J.L. JACOBI, J. GUERN. Institut des Sciences Vegetales. C.N.R.S .. Bat 22, Avenue de la terrasse. 91198 Gif sur Yvette, France 1 instilu! de Recherche sur Ie Caoutchouc, C.l.RA.D., av du Val de Montjerrand B.P. 5035. 34032 Montpellier Cedex France ° Laboratoire de Physiologie Vegetale Appliquee, U.S. T.L. place E. Bataillon 34095 Montpellier Cedex 5, France

The ethylene releaser ethephon is commonly used in rubber tree farming to stimulate latex production (5). Two main effects of ethylene treatment have been observed:

- extended latex flow (due to increased water uptake and delayed coagulation phenomena), - activation of general metabolism, allowing more efficient regeneration of latex after tapping.

Many different metabolic pathways are affected, directly or not, by ethylene; in particular, glycolysis is activated (8), as well as protein synthesis, as suggested by an increase in ribosome polymerisation index (4) and in the incorporation oflabelled aminoacids in proteins (6). Moreover, some enzymatic activities in latex have been shown to be specifically modified by ethylene. In the present work, Our goal was to investigate ethylene-induced modifications of gene expression in the latex cells. In vitro translation oflatex mRNA indicated that the level of specific RNA species is modified after ethylene treatment, in both genotypes studied, namely OTt (a middle producer which responds quite well to ethylene treatment) and PB217 (an initially low producer but highly responsive to ethylene treatment). Monodimensional gel electrophoresis of the in vitro translation products (which reflects the mRNA pattern) was sufficient to visualize significant modifications in the intensity of a number of bands, either increasing or decreasing, suggesting that some specific genes are over-expressed whereas others are repressed, as a consequence of ethylene treatment.

Homologous and heterologous probes, choosen accordingly to the knowledge already achieved in biochemistry and physiology of the latex cells, allowed identification of a few of these ethyleneresponsive genes: a glutamine synthetase (OS) probe from soybean (7) showed, in both genotypes studied, ethylene-induced accumulation of the corresponding OS mRNA in latex. In the same way, ethylene-induced accumulation of chitinase and superoxide dismutase (Mn-SOD) could be evidenced in both genotypes using a bean chitinase probe (2) and an homologous Mn-SOD probe (Miao and Oaynor, to be published). Only a slight accumulation (2 to 5 folds) of he vein transcripts had been described for OTt, this genotype having already a very high initial level of hevein mRNA (1). In genotype PB217, the increase obtained (using the same homologous probe) was about 5 or 6 folds the initial level. Accumulation was also observed for the 3.3 kb transcript of the

162

J. C. Pech etal. (eds.), Cellular and Molecular Aspects of the Plant Hormone Ethylene, 162-163. © 1993 Kluwer Academic Publishers.

163

corresponding mRNA in latex. The fact that neither the AKin 5-3 protein kinase nor the other HRGPs transcripts were modified guaranties that the stimulations observed for the 3.3 kb HRGP transcript and the other probes were significant.

"hydroxyproline rich glycoproteins" (HRGPs) family, whereas the other transcripts were not affected by the treatment, as described for elicitor-treated bean cells (3). A probe coding for the protein kinase AKin 5-3 (Thomas and Kreis. to be published) showed no modification of the

The accumulation of glutamme synthetase mRNA that we observed could account lOr the increase of potential and specific activities of this enzyme measured in the latex after ethylene treatment (article in preparation). As the glutamine synthetase is involved in amino-acids supply, its stimulation is probably related to the ethylene-induced intensification of proteosynthesis required for latex regeneration. Chitinase, hevem and Mn-SOD might be involved m mecharusms 01 latex coagulation. In latex, both coagulating factors (like hevein) and substances with anti-coagulating properties (like chitinase) are present. The latex stability depends on the relative proportion of these elements but first of all, it depends on their compartmentation in the laticifers. The coagulating factors are stocked in the vacuolysosomes called lutordes and become active only when released in the rubbercontaining cytoplasm, upon lutoIde bursting (which happens mainly during tapping). Ethylene treatment efficiently reduces lutorde bursting. Considering our results, the stimulation of the coagulating factor hevein by ethylene is surprising as ethylene treatment globally promotes higher latex stability. It seems that chitinase stimulation could partly counteract hevein stimulation. Moreover ethylene contributes to increase lutoIde stability by its stimulatory effect on superoxide dismutase, which is known to confer protection against peroxidative degradations of membranes.

Our results show that ethylene regulates several speCific genes at the RNA level (probably through transcriptional regulation). We have already identified a few of them using a Northern blot approach. These genes are involved in various metabolic phenomena such as mechanisms of latex coagulation, protein synthesis (through ammonium fixation), or in other less understood reactions to stress. Several other probes, corresponding to potentially ethylene-regulated genes have still are to be tested. Some of the most interesting heterologous probes will be used to fetch the homologous corresponding clones from a latex cDNA library, in order to provide markers for Hevea genetic improvement and early selection of perform ant hybrids, in terms of yield, responsiveness to ethylene stimulation, resistance towards bark dryness induced by over-exploitation ... etc. These probes will also give tools to study the mechanisms of ethylene control at the gene level.

1. Broekaert, W., Lee, H.-I., Kush, A., Chua, N.-H. and Raikhel, N. (1990).Proc. Natl. Acad. Sci. USA; Vol 87, pp 7633-7637.

2. Broglie, K. E., Gaynor, J. J. and Broglie, R. M. (1986). Proc. Natl. Acad. Sci. USA; Vol.83, pp 6820-6824.

3. Corbin, D. R., Sauer, N. and Lamb, C. J. (1987). Molec. Cell BioI.; Vol.7, pp 4337. 4. Coupe, M., Lambert, c., Primot, L. and d'A.uzac, J. (1977). Phytochem.; Vol 16, pp 1133-

1136 5. d'Auzac, J. and Ribailler, D. (1969). C. R. Acad. Sci. Paris, Vol 268, pp 3046-3049. 6. Gidrol, X.(l984). PhD Thesis. 3eme cycle, Universite Aix-Marseille II 7. Miao, G.-H., Hirel, B., Marsolier, M.-C., Ridge, R. W. and Verma, D. P. S. (1991).

The Plant Cell; Vol.3, pp.1l-22. 8. Tupy, J. (1973). c. R. Acad. Sci. Paris, Vol 268, pp 3046-3049.

IMMUNODETECTION OF ETHYLENE- INDUCED CHLOROPHYLLASE FROM CITRUS FRUIT PEEL

T. TREBITSH-SITRIT, J. RIOV, E.E. GOLDSCHMIDT Department of Horticulture, Faculty of Agriculture The Hebrew University of Jerusalem Rehovot 76100 ISRAEL

Chlorophyllase (chlorophyll-chlorophyllido-hydrolase, EC 3.1.1.14) (Chlase) catalyzes the conversion of chlorophyll to chlorophyllide by removing the phytol group. In vivo and in vitro studies indicate that Chlase plays a key role in the breakdown of chlorophyll. Accumulation of chlorophyll ide , which is the immediate product of the Chlase reaction, and additional catabolic products that could be derived only from chlorophyll ide have been detected in ethylene-treated Citrus fruit (1,5) and leaves of several plant species (2,4). Additionally, Chlase activity was found to increase during ethylene-enhanced degreening (1,3). Nevertheless, little is known about the regulation of Chlase activity.

Our research focused on Citrus Chlase since the enzyme is fairly abundant in ethylene-treated Citrus fruit peel. Chlase was purified 400-fold from acetone powder of chromoplast fraction of ethylenepretreated orange (Citrus sinensis L. Osbeck, cv. 'Valencia') by ammonium sulfate fractionation, gel filtration on Sephadex G-200, hydrophobic column chromatography on Phenyl Sepharose CL-6B and a preparative nondenaturing SDS-PAGE. The purified enzyme had a specific activity of 174 ~mol chlorophyll a/min/mg protein, and showed one band on SDS-PAGE. The molecular mass of the purified Chlase varied according to the treatment conditions prior to the SDS-PAGE; 25 kD for nondenatured enzyme and 35 kD for denatured enzyme. Rabbits were immunized with the purified enzyme. The antiserum was highly specific and recognized both the denatured and nondenatured Chlase on western blots (Figure 1). Also, the antibodies recognized the native soluble enzyme as evidenced by immonoprecipitation of more than 70% of Chlase activity from the solute, using serum deluted 1:50.

The chlorop~yll content in the flavedo decreased from 19.2 in control to 6.5 ~g/cm in ethylene-treated fruit. Concomitantly, Chlase activity increased 100-fold from 0.3 to 3.6 units/g FW, respectively. While the Chlase protein could not be detected on immunoblots of denatured and nondenatured preparations from control fruit, a significant band of Chlase protein was detected in ethylene-treated fruit (Figure 1), indicating that ethylene induces de novo synthesis of Chlase.

164


165

. , ,

80 60 Oil -ASE ACTIVITY

Immunoblot of Chlase in total proteins extract from Citrus peel. Total proteins were solubilized from acetone powder by 1% sodium cholate. Lanes land 5, total proteins isolated from air control fruit (10 ~g,

specific activity of .0364); Lanes 2 and 6, total proteins isolated from ethylene treated fruit (10 ~g, specific activity of 1.545); Lanes 3 and 7, purified chlorophyllase (1 ~g) isolated from ethylene-treated citrus peel; Lane 4, prestained molecular weight standards (Bio-Rad), 106, 80, 49.5, 32.5, 27.5, 18.5 kD. Nitrocellulose was visualized by a standard peroxidase staining procedure. Enzyme activity was localized by assaying Chlase activity in slices of the resolved gel. 1. Amir-Shapira D, Goldschmidt EE Altman A (1987) Chlorophyll

catabolism in senescing plant tissues: In vivo breakdown intermediates, suggest different degradative pathways for Citrus fruit and parsley leaves. Proc Natl Acad Sci USA 84:1901-1905

2. Matile P, Duggelin T, Schellenberg M, Rentsch D, Bort1ik K, Peisker C, Thomas H (1989) How and why is chlorophyll broken down in senescent leaves. Plant Physiol Biochem 27:595-604

3. Purvis AC, Barmore CR (1981) Involvement of ethylene in chlorophyll degradation in peel of citrus fruit. Plant Physiol 68:854-856

4. Rise M, Goldschmidt EE (1990) Occurrence of ch1orophyllides in developing, light grown leaves of several plant species. Plant Sci 71:147-151

5. Shimokawa K, Hashizume A, Shioi Y (1990) Pyropheophorbide A as a catabolite of ethylene-induced chlorophyll a degradation. Plant Physiol 93:S-150

ASCORBATE OXIDASE OF CUCUMIS MELO

O. MOSER' and A. K. KANELLIS Institute of Molecular Biology and Biotechnology P.O. Box 1527 711 10 Heraklion, Crete Greece

ABSTRACf. Ascorbate oxidase (AO), an enzyme involved in the development of melon fruit (Cucumis melo) was purified by following different chromatographical steps. Some of the enzyme properties are given.

AO, an enzyme widespread in plants (Stark and Dawson, 1962), catalyses the oxidation of ascorbic acid in dehydroascorbic acid. However, its physiological role has not been fully defined yet. It was described as participating in the reorganization of the cell wall during the characteristic rapid growth of young cucurbit fruits (Lin and Varner, 1991).

We found that in flowers and in very young melon fruits AO showed high enzymatic activity. Moreover, we could observe an increase of AO activity during maturation of fruit concomitant with an increase of ethylene production. It seems, therefore, that the enzyme may also be involved in fruit ripening processes.

To understand the function of AO, we purified the enzyme. For this purpose, melon female flowers were collected, ground and homogenized in extraction buffer (50 mM K Phosphate buffer pH 6.8, 1.5% Polyvinylpolypyrolidone, 0.5 mM PMSF, 4 IlM leupeptin) and the enzyme solubilized. The soluble fraction was concentrated and dialysed against 10 mM K Phosphate buffer pH 6.8 containing 0.5 mM PMSF (buffer A).

This sample was applied onto a DEAE-Sephacel column which was equilibrated with buffer A. Figure 1 (panel A) shows that AO did not bind to DEAE-Sephacel column but this step allowed a significant elimination of contaminant proteins. The pooled active fractions were directly loaded onto a CM-Sepharose column (panel B) equilibrated with buffer A. AO was eluted by a linear salt gradient. AO fraction was then applied to a ConASepharose column (panel C). AO was retained on this glycoprotein- affinity resin and eluted with 10 mM methyl manno-pyranoside. Fractions containing AO activity were dialysed and concentrated. This sample was passed through a Sephacryl S-300 column equilibrated with buffer A containing 150 mM NaCl. AO containing fractions (panel D) were dialysed and concentrated. This final gel filtration step purified AO to homogeneity as was evidenced by silver stained SDS-PAGE (data not shown). The native Mr of the protein was estimated to be 137 kD and its subunit 68 kD. Based upon these data, AO from Cucumis melo appeared to be a homodimer protein.

Some properties of the purified protein were analysed. Figure 2 demonstrates that the

, This work was funded by the ECLAIR programme AGRE - 0015 from the European Community

2 O.M. was supported by a mobility grant from the European Community, DO XII - F3

166

J. C. Pech et al. (eds.). Cellular and Molecular Aspects of the Plant Honllolle Ethylene. 166-167. © 1993 Kluwer Academic Publishers.

167

100 B 100

75 75 E 50 50 >-

E ~ = 25 25 ~ co

0 0 ~ ~ 10 20 0

~ 100 100 ~

75 75

-50 50

25 25

0 0

0 10 20 30 20 40 60 80 Fraction number

Figure 1: Purification of AO from melon flowers. A) DEAE-Sephacel- B) Carboxy-methyl Sepharose-C) Concanavalin-A Sepharose- D) Sephacryl S-200 chromatographies. The elution profIle of: total proteins (fine lane) and AOactivity (bold lane) is indicated. Solid horizontal bars represent pooled active fractions.

optimal temperature for AO activity was located in a range between 37 - 42'C. The enzyme remained partially active at elevated temperatures (40% activity at 6O'C).

When we analysed the effects of pH on AO activity, we could conclude (panel B) that the enzyme activity was optimal in pH range 5.5 to 6.5.

In conclusion, AO has been purified to homogeneity by applying anion exchange-, cation exchange-, affmity- and gel ftltration chromatographies. This protein is a homodimer of 137 kD (68 kD per subunit). Since AO activity and protein increased during maturation, AO may playa role in the ripening process of melon fruit. Further studies are under way to understand the putative role of AO in fruit growth and development.

References

>-100 ~ 80 ~ 60 ~ 40 ~ 20

00 20 40 60 'C

>-100 ~ 80 ~ 60

~ 40 20

~ 0 2 4 6 8 10 pH

Figure 2: Effects of temperature (A) and pH (B) on AO activity.

Lin L.S. and Varner J.E. (1991) 'Expression of ascorbic acid oxidase in zucchini squash (Cucurbitapepo L)' Plant physiology 96, 159-165

Stark G.R. and Dawson C.R. (1962) 'On the accesibility of sulfhydryl groups in ascorbic acid oxidase' J. BioI. Chern. 237, 712-716.

ETHYLENE RECEPTORS

M.A. HALL, H.M. Aho, A.W. Berry, D.S. Cowan, N.V.I. Harpham, M.G. Holland, LYe. Moshkovl , G. Novikoval and A.R. Smith Department of Biological Sciences, The University College of Wales, Aberystwyth, Dyfed SY23 3DA, Wales, u.K. 1 Timiriazev Institute of Plant Sciences, Academy of Sciences, Botanicheskaya 35, 127276 Moscow, Russia.

ABSTRACT. Current progress in research on ethylene receptors in our laboratory is described, including the sequencing of the receptor protein from Phaseolus, the use of antibodies to identify homologous proteins in other species, the use of ethylene-insensitive mutants of Arabidopsis to probe receptor functionality and investigations on the role of phosphorylation in the modulation of receptor activity.

Introduction

Binding proteins for ethylene having appropriate affinities and specificities for the gas and its physiologically active analogues were discovered independently and simultaneously just over a decade ago (Jerie, 1979; Bengochea et al., 1980a&b; Sisler, 1979). Since that time a consistent pattern has emerged in that all species investigated so far appear to possess at least two classes of ethylene binding site having essentially identical affinities for ethylene (KD ::: 10-11 M) and its analogues and differing only in their rate constants of association and dissociation (U12 association approximately 8 min and 13 h respectively). Much information is now available on these sites but so far the only rigorous structural information available is for the slow associating site from Phaseolus vulgaris.

The Ethylene Binding Protein from Phaseolus vulgaris

The ethylene binding protein (EBP) from developing cotyledons of Phaseolus vulgaris is mainly situate in membranes of the endoplasmic reticulum and protein body membranes with possibly a small fraction associated with plasmamembrane (Evans 1982a,b). Unlike the auxin-binding protein from maize which is also associated predominantly with endoplasmic reticulum, the EBP from Phaseolus is an integral membrane protein and, since it is highly hydrophobic, can only be extracted using detergents. This feature has also rendered difficult both purification and assessment of molecular weight. On the

168

J. C. Pech eta!' (eds.!, Celiularand Molecular Aspects of the Plant Hormone Ethylene, 168-173. © 1993 Kluwer Academic Publishers.

other hand the fact that this particular site has low rate constants of association and dissociation makes it possible to prelabel the protein and monitor its purification.

169

Using a new electrophoretic technique to separate partially purified protein, we are able to distinguish two bands on gels, both of which bind ethylene, having molecular weights of26 and 28 kDa respectively. These appear to associate to yield proteins ofMW 52 and 56 kDA (see below). Fully denaturing electrophoresis yields bands at about 13 and 14 kD. Previously, we had calculated from data obtained by ultracentrifugation on isokinetic sucrose sucrose gradients and by gel permeation chromatography (Thomas et al., 1985) that the molecular weight was in the range 50-60 kDa and it seems highly probable therefore that the proteins concerned are tetramers.

We now have sequence information on the 28 kDa component which indicates that its two subunits are similar but not identical, it is moreover clear that the both the 28 and 26 kDa components are glycosylated and probably phosphorylated to some extent (see below). Searches of databases demonstrate no significant homology of the 28 kDa component with any known protein. Efforts are now in hand to sequence the 26 kDa band and to clone the appropriate gene(s) using expression libraries with the antibodies described below; we are about to commence work using oligonucleotide probes based on the sequence data.

To what extent the fast associating sites are homologous with the slow associating form is unclear, although the difference in properties may reflect a difference in subcellular location rather than a fundamental difference in protein composition. Thus, in peas whereas the slow associating form is invariably membrane-bound, a significant proportion of the fast associating form is either loosely membrane-bound or soluble.

Antibodies to the Ethylene Binding Proteins from Phaseolus

We have raised polyclonal antibodies to both the 28 and 26 kDa components. The antibodies are highly specific, both types recognise the 28 and 26 kDa bands as well as the basic subunits and the 52 and 56 kDa bands. However, while this indicates a degree of homology between the two proteins it should be noted that each antibody shows a much stronger affinity for the component to which it was raised.

It also appears that homologous proteins are present in other species, including peas, Arabidopsis, rice and tomatoes. It is not yet clear however whether both components are present in each species. Thus in peas we have so far only been able to detect the 28 kDa component whereas in tomato both components appear to be present.

It is unclear as to whether either or both of these components are functional receptors (but see below) nor, if they are, whether they have different functions or whether one represents a precursor of the other; certainly the 26 kDa component is less abundant than that at 28 kDa.

170

Ethylene Insensitive Mutants of Arabidopsis

We have available a range of Arabidopsis mutants (the eli series) showing a range of sensitivities to ethylene, one of which, eli 5, is completely insensitive to ethylene up to a concentration of 10,000 ~11-1 (Harpham el al., 1991). The mutations are pleiotropic and all developmental responses are affected more or less equally.

In common with the other species studied, Arabidopsis possesses both fast and slow associating sites and their distribution in the mutants has been investigated (Sanders el aI., 1991). This endeavour has however, proved difficult to achieve experimentally. Thus the in vivo binding assay we have developed (Sanders el al., 1989) is disproportionately affected by endogenous ethylene production (Sanders el al., 1991) although the use of inhibitors of ethylene biosynthesis can resolve most of the problems. However, several of the mutants produce ethylene at rates higher than the wild type and, in eli 5 in particular, ethylene biosynthesis is freed from autoinhibition (incidentally indicating receptor involvement in this process) such that rates between four and fifteen times higher than wild type are observed (Table 1).

TABLE 1. Ethylene production by wild type and eli 5 Arabidopsis plants

Rate of ethylene production (nmol g-1 fw h-1

Wild type eli 5

Incubation time (h) 0-1 1 - 4 4 - 20

0.17 ± 0.02 0.71 ± 0.02

0.10 ± 0.01 0.92 ± 0.05

0.06 ± 0.01 0.98 ± 0.07

Adapted from Sanders el al., Ann. Bot. 68: 97-103 (1991).

This leads to artifactually low figures for binding and even the use of inhibitors of ethylene biosynthesis do not reduce emanation rates to comparable levels with wild-type so treated (Table 2). Thus, the figures in Table 2 must be treated with some caution. On the other hand certain broad conclusions may be drawn. Firstly, it appears that differences in ethylene binding between the mutants may be qualitative as well as quantitative. Thus, even allowing for the error introduced by ethylene production the amount offast associating sites is reduced in eli 5 relative to wild type; the concentration of slow associating sites on the other hand is relatively unaltered. Conversely eli 13, a relatively insensitive mutant, has the highest recorded concentration of fast associating sites but no detectable slow associating sites. Moreover, in this case interference by endogenous ethylene can be discounted since the rates are very low and barely different from those in the wild type.

TA

BL

E 2

. E

thyl

ene

bind

ing

in w

ild ty

pe a

nd m

utan

t pl

ants

of

Ara

bido

psis

.

Gen

eral

ord

er o

f E

thyl

ene

bind

ing

Eth

ylen

e pr

oduc

tion

se

nsit

ivit

y to

(p

mol

g-1

fw

h-l

) in

trea

tmen

t et

hyle

ne

Con

trol

T

reat

men

t (A

VG

+ C

oCl2

+ 1

% 0

2)

(nl g

-1 f

w h

-l)

(lh

) (l

h)

(20

h)

(20h

-lh)

Wild

typ

e 0.

12

0.66

0.

81

0.15

0.

05

eli

3 2

0.10

0.

76

0.71

0.

10

eli

5 6

0.03

0.

28

0.41

0.

12

1.55

eli

8 3

=

0.08

0.

39

0.33

0.

26

eli

10

5 0.

18

0.73

0.

85

0.12

0.

12

eli

13

3=

0.

15

0.89

0.

86

0.09

::::i

172

The most which can be claimed is that insensitivity can be associated with reduced binding (of either the fast or slow associating variety). It must be remembered however that what is being measured here is total binding of two kinds and there is, as yet, no way of determining what proportion of each site represents functional receptor. Equally, in a mutant where the lesion is at a point distant from the binding domain the receptor might be disabled without any effect on total binding.

Conversely, for reasons outlined in the next section, modifications to the receptor may affect its ability to bind ethylene and hence a mutation affecting such modifications could in turn affect binding without actually affecting receptor abundance.

Thus, while mutants such as these do offer the opportunity to explore receptor functionality and factors influencing it, they do not offer an easy route to identifYing the gene(s) for the receptor itself

Ethylene & Protein Phosphorylation

We have shown that ethylene at physiological concentrations stimulates soluble and membrane-bound protein phosphorylation both in vivo and in vitro. The effect is concentration dependent and specific and is reversed by the inhibitor of ethylene action 2,5 norbornadiene. Moreover, both in vivo and in vitro the proteins phosphorylated are electrophoretically homologous to the subunits of the ethylene binding proteins. More significant still is our observation that in endomembrane preparations, stimulation of phosphorylation in vitro reduces ethylene binding while dephosphorylation has the reverse effect. While it is too early to assess the significance of these findings they bear a close resemblance to the situation obtaining with some animal hormone receptors such as (3-adrenergic receptor (Hausdorff et al., 1989). Equally, they may be particularly significant in assessing the binding data from Arabidopsis described above.

Conclusions

Significant progress in the study of ethylene receptors has been made in the recent past and the isolation of the gene(s) involved seems likely to open the way for further exciting advances.

Acknowledgements

The authors are grateful to the Science & Engineering Research Council, u.K. for the award of postgraduate studentships to A WB and MGH, to The Royal Society for the award of Visiting Fellowships to IYeM and GN and the the CEC for the award of a BRIDGE grant enabling the employment ofDSC as a postdoctoral fellow.

173

References

Bengochea, T., Dodds, J.H., Evans, D.E., Jerie, P.H., Niepel, B., Shaari, AR and Hall, M.A (1980a) 'Studies on ethylene binding by cell-free preparations from cotyledons of Phaseolus vulgaris L. I. Separation and characterization', Planta 148, 397-406.

Bengochea, T., Acaster, M.A., Dodds, J.R., Evans, D.E., Jerie, P.H. and Hall, M.A. (1980b) 'Studies on ethylene binding by cell-free preparations from cotyledons of Phaseolus vulgaris L. II. Effects of structural analogues of ethylene and of inhibitors, Planta 148, 407-41l.

Evans, D.E., Bengochea, T., Cairns, AJ., Dodds, J.H. and Hall, M.A. (1982) 'Studies of ethylene binding by cell free preparations from cotyledons of Phaseolus vuglaris L.: subcellular localisation, Plant Cell Environ. 5, 101-107.

Evans, D.E., Bengochea, T., Cairns, AJ., Dodds, J.H. and Hall, M.A. (1982) 'A study of the subcellular localisation of Phaseolus vulgaris L. by high resolution autoradiography', Planta 154,48-52.

Harpham, N.V.J., Berry, AW., Knee, E.M., Roveda-Hoyos, G., Raskin, I., Sanders, 1.0., Smith, AR, Wood, CK and Hall, M.A. (1991) 'The effect of ethylene on the growth and development of wild-type and mutant Arabidopsis thaliana (L.) Heynh., Annals of Botany 67, 104-109.

Hausdorff, W.P., Bouvier, M., O'Dowd, B.F., Irons, G.P., Caron, M.G. and Lefkowitz, RJ. (1989) '/32 Adrenergic receptors are involved in distinct pathways of receptor desensitization', Journal BioI. Chern. 264, 12657-12663.

Jerie, P.R., Shaari, AR and Hall, M.A. (1979) 'The compartmentation of ethylene in developing cotyledons of Phaseolus vulgaris L.', Planta 144, 503-507.

Sanders, 1.0., Smith, AR and Hall, M.A (1989) 'The measurement of ethylene binding and metabolism in plant tissue', Planta 179, 97-103.

Sanders,1.0., Harpham, N.Y.J., Raskin, I., Smith, AR and Hall, M.A. (1991) 'Ethylene binding in wild type and mutant Arabidopsis thaliana (L.) Heynh.', Annals of Botany 68,97-103.

Sanders, 1.0., Smith, AR. and Hall, M.A. (1991) 'Ethylene binding in epicotyls of Pisum sativllw L. cv. Alaska', Planta 183,209-217.

Sisler, E.C. (1979) 'Measurement of ethylene binding in plant tissue', Plant Physiol. 64, 538-542.

Thomas, C.J.R, Smith, AR. and Hall, M.A. (1985) 'Partial purification of an ethylene binding site from Phaseolus vulgaris L. cotyledons', Planta 164, 272-277.

BUCKMINSTERFULLERENE (C60 BUCKYBALL) INHIBITION OF ETHYLENE

RELEASE FROM SENESCING LEGUME FOLIAGE AND CUT CARNATIONS·

YA'ACOV Y. LESHEM,l DOV RAPOPORT,1 ARYEH A FRIMER,2 GILA STRUL,2 URI ASAF3 AND ISRAEL FELNER3

Departments of 1 Life Sciences and 2Chemistry, ** Bar-Ilan University, Ramat Gan 52900, Israel and The Racah Institute of Physics, 3 The Hebrew University of Jerusalem 91904, Israel

ABSTRACT. The newly discovered solid form of carbon - COO (buckminsterfullerene) when applied either as colloidal solution or in liposomes significantly reduces ethylene evolution from senescing legume [Pisum sativum and Vida Jabal foliage and from cut carnation (Dianthus caryophyllus) flowers, the liposomes being twice as effective as colloidal solutions. C 60 mode of action is attributed to ethylene adsorption stemming from the interaction between the vast C 60 surface area - 1,317 m2/gr - and the affinity of its carbon atoms for the 7!

component in the ethylene double bond.

INTRODUCTION.

The recently discovered third allotropic form - after graphite and diamond - of carbon, C 60

or buckminsterfullerene, is at present generating much interest amongst physicists and

organic chemists. Its existence as a component of interstellar dust and the theoretical

calculation of its shape (Fig. 1) was suggested by two Soviet astrophysicists, Bochvar and

Galperin (1972), however its actual isolation and synthesis in laboratory conditions by laser

aided combustion of graphite electrodes in a helium atmosphere was achieved more than a

decade later (cfreview by Huffmann 1991). As pointed out by Culotta and Koshland (1991),

this unique compound possesses a broad spectrum of physico-chemical properties including

the ability to generate or extinguish singlet oxygen (depending on state of excitation), the

ability to serve as a free radical scavenger and also probably what is now the most intensely

investigated aspect - the ability when "doped" with metals such as K, Ca or Rb and 11, to serve

as an electrical superconductor at relatively high temperatures -45°K for Rb-l1 co-doped C

60. The latter experiments established solid C 60 as the first three-dimensional organic

superconductor. Subsequent research has indicated that fullerenes are also present in more

than negligible amounts in automobile exhaust and industrial soot from which by

chromatographic procedures based on solubility in benzene or toluene they may be purified.

'This paper was presented jointly at the Neljubov Ethylene Conference in July 1992 in Pushchino, Russia and in August 1992 at

the Ethylene Symposium in Agen, France.

"The Ethel and David Resnick Chair in Active Oxygen Chemistry.

174

1. C. Pech et at. (eds.), Cellularalld Molecular Aspects of the Plam Honnone Ethylene, 174-181. © 1993 Kluwer Academic Publishers.

175

Figure 1. The Buckminsterfullerene and lreterofulJerene family of carbon compounds. C 60 is a soccer -ball shaped caged structure composed of 12 pentagons and 20 hexagons, the generic name assigned in view of the structural similarity to geodesic domes constructed by the U.S. architect, Robert Buckminster Fuller - and hence the appellation "Buckyballs". Higher fullerenes (C> 60) are ellipsoid: to date C>280 bucky-"needle" caged structures are known.

While fullerene research in the above and related fields is being carried out intensely,

biologists as yet have not yet turned their attention to the potential utilization of C 60's

properties and following, in context of ethylene action in plant systems, to the best of our

knowledge is the first report of buckyball action in biological systems. The specific aim of the

present experimentation was an endeavor to shed light on the mode of interaction of C 60,

applied either as colloidal solutions or as a liposome component, with ethylene produced in

senescing plants, these being cut carnations and legume foliage.

METHODS AND RESULTS

These were of 2 categories - 1) Biological. 2) Physical.

1. BIOLOGICAL

Plant Material: Carnation flowers (D. caryophyllus cv. Simona) were freshly cut from 4-month

old carnation beds. Stem length was ca 8 cm, flowers being fully open. Pea foliage (P. sativum

cv. Kelvedon) was taken from 1 month old mother plants grown on a loam vermiculite (5:1)

mixture in a growth chamber at 22°C under an 18 hr photoperiod, light being GRO-LUX

fluorescent light tubes providing a 125 Il Einsteins!cm2 intensity. In the broad bean (Vida

faba) experimental leaf cuttings from identical phyllotaxial sites were taken from plants

growing outdoors. In all experiments 5 flowers or foliage cuttings were employed per

replicate and each treatment consisted of 4 replicates.

Colloidal solution preparation: 5 mg of C 60 (Texas Fullerene Corporation) or of a crude

C 6O:C 70 mixture prepared by the graphite electrode method (Kriitschmer et all990) were

dissolved in 0.5 ml toluene to which 0.1 ml Tween 20 was added. These fullerene mother

solutions were subjected to slow N2 bubbling for 2 hr during which the toluene evaporated.

176

The residue was then dissolved in the buffer systems at concentrations detailed in the

captions of the pertinent Figures.

Liposome preparation: Uni-lamellar liposomes comprised of a mixture of soybean

phosphatidylcholine (PC) and sn-1 palmitoyl, sn-2linoleoyl PC (15:1 WIW) containing C 60

were prepared as follows: 1.5 mg C 60 dissolved in 0.4 ml toluene was added to a chloroform

solution of the above PC species at 1:15 C 6O:PC molar ratio. After chloroform evaporation

in a Rotary Evaporator the buffer as detailed in the captions to the Figures was added at a

concentration of 1 ml buffer125 mg lipid. The mixture was subsequently heated to 30°C and

thereafter subjected to 10 min Vortex agitation to obtain multi-lamellar liposomes. These

were then sonicated for 9 x 30 sec with a MSE Titanium Probe Ultrasonic Disintegrator

Model MK2 at 20 KHz output frequency to obtain uni-Iamellar liposomes which were then

oxygenated by gentle 02 bubbling for 20 min. In both application methods - colloidal

solutions and liposomes - controls were prepared employing identical procedures only lacking

fullerenes.

Fullerene application: This was achieved by a 1.5 hr pulsing of each replicate placed in a test

tube at 22°C after which plants were removed, 5 mm distal ends excised with a sharp

razorblade, the sections gently washed with distilled H20 and cuttings placed in fresh buffer

solutions in 10 ml test tubes. These were then placed under sealed 0.5 I plastic belljars, the

mouths of which were equipped with rubber bacteriological caps through which at given

1500 ___

~--

E ---- ---- ----- •• a. ~---- .------2 1000 --.---------.:.::. •• :..:..:..:..-::.::-.::f;.~ •• :.::..--_7'

c:: _____________ _

.2 U =>

"0 e 0.

or I

U

Figure 2: Carnation ethylene inhibition bv C 60 buckvballs applied as single bi/ayered liposomes.

177

periods 1 ml gas samples were withdrawn with a syringe to assess ethylene production. To

prevent moisture accumulation, an open vial containing 5 gr anhydrous CaCh was placed in

each belljar.

Ethylene measurement: This was performed on a Varian Model 3400 FID gas chromatograph

equipped with a Varian 4290 Integrator. Standard ethylene measuring procedure was

employed.

Biological Experimental Results

Carnations: Fig. 2 indicates a marked concentration dependent inhibition of ethylene release

from the treated flowers.

Legume foliage experimentation: Table 1 indicates that in Vicia as in carnations, C 60

markedly inhibits ethylene evolution.

Table 1: Effect of C 60 on Ethylene Production in Vida faba Foliage: C 60 (25 pg/leaf was fed to cuttings as a colloidal suspension in oxygenated 2 mM buffer EPPS pH 8.5 containing 2 mM ACC and 10-5 M CaCh. 4 replicate means.

Treatment

Buffer Control Buffer + C60

Ethylene Production Cppmlgr fresh wt) after 2 hrs after 4 hrs

0.38 0.11

0.50 0.13

The next experiment carried out on pea foliage compared the relative effectivity of

C 60 as compared to graphite soot fullerene extract. Table 2 indicates that inclusion of C 70

significantly accentuates the ethylene inhibiting effect.

Table 2: Effect of Various Fullerenes (C 60 and C70) on Accumulative Ethylene Production in Pisum sativum Foliage: Concentration and application was as in caption to Table 1.

Ethylene Production Treatment % Fullerene Content* ppmlgr fresh wtLmin

C60 C70 1.5 hr 2.5 hi-

Buffer Control 0.218 0.338

Purified Buckyballs 99.62 0.38 0.03b O.13b

Graphite Soot Fullerene Extract 87.20 12.80 O.ooc O.ooc

*as determined by mass spectrographic analysis. Different letters in vertical columns indicate statistically significant differences at p > 0.01.

178

Fig. 3 presents results of trials assessing the ethylene inhibitory effect of C 60 applied

as colloidal solution to that of C 60 when incorporated into liposomes. The rationale was that

the latter could possibly interact more effectively with membranes and if incorporated into

the membrane could enter the cytosol. Fig. 3 bears out the assumption: C 60 liposome

induced inhibition was clearly more pronounced than colloidal systems.

c: .2 -g c:

~~ 0.-

.~ .,. I.e:

N IJI

U ~ ........ o ...

~~ ~c

0.4

0.3

0.2

0.1

hr

Figure 3. Buckyball inhibition of ethylene in pea foliage: Comparison of C 60 colloidal solutions to C 60 loaded liposomes. Mode of application and C 60 concentrations were as described in Tables 1 and 2.

2. PHYSICAL

C 60 sUrface area calculation: See enclosed box.

SURFACE AREA CALCULATION OF Cao

ICao M.W. a 7201

Mass of 1 proton = 1.67.10.24 gram

1 cage has mass = 720.1.67.10.24 = 1202.4.10-24 gm

= 1.2024.10-21 gm

1 1 gm has 1 .2024. 1 0-21 cages

= 0.832.1021 cages ~

Cage diam = 7.1A. radius = 3.55A

Surface Area of 1 cage = 4" 13.55A)2

= 158.3A2

SURFACE AR~ OF 1 GRAM Cao

= 0.832.1021 .158.3A2

=1.37.1023A2

= 1.317 meter2

179

Ethylene adsorption by C 60: Aliquots of 1 ml colloidal C 60 solutions at concentrations listed

in Fig. 4 and prepared as detailed in Methods in the same buffer system as before but without

ACC, were injected into rubber capped 10 ml infiltration flasks previously filled with

calibrated 10 ppm ethylene gas. Flasks were then subjected to gentle shaking in the dark at 5,

25 and 40°C. After 60 min, 1 ml gas samples were withdrawn with a hypodermic syringe and

ethylene content of the enclosed flask atmosphere determined.

Physical Experimental Results.

Surface area calculation indicates that 1 gr of C 60 has an immense surface area - 1317 m2:

the significance of this parameter will be dealt with in the Discussion. Ethylene adsorption

(Fig. 4) indicates a virtually geometric relationship between C 60 concentration and

temperature - higher temperatures manifested enhanced adsorption. It is of note that within

such a short period as 1 hr, a 10 ppm ethylene atmosphere underwent a 5-10% decrease in

the 25-400C biological temperature range at a 1 mg/l C 60 concentration.

180

10.0

----75 • -c, ......

~~ ~!': -~ ~ 5.0 • ~~

':t~ ~~

2.5 .

0.0 "'0

DISCUSSION

-------

----

-- , ,

,

"

,

, ,

Figure 4. Ethylene adsorption by colloidal buckyball solutions. See text for experimental details. Each column represents a 4 replicate mean.

Experimental evidence indicates that fullerenes significantly reduce plant ethylene evolution

- liposome application being twice as effective as colloidal solutions. Apparent mode of entry

is through the xylem vessels possessing a ± 50,000 A diameter - that of the individual

buckyballs being only 7.1 A (Huffman 1991) and of the employed liposomes ± 250 A, thus

enabling unhindered passage in the transpirational stream. While the C 60 colloids may

subsequently accumulate on the extrafacial leaflet of the plasmalemmal bilayer and not

penetrate the ca 4.0 A space between individual membrane phospholipids (Leshem 1992),

the liposomes albeit larger, may be incorporated into the lipid membrane matrix, their

contents spilling out into the cytosol and hence their greater effectivity.

Concerning mode of action, the adsorption experiment (Fig 4) and the vast, 1317

mZ/g, C 60 surface area (compared to 200 mZ/gr of activated charcoal - Moore 1956), suggest

that physical adsorption of CzH4 by C 60 here plays the major role. In the structural model of

ethylene (as described by Pauling, 1967) the double bond is composed of one sigma and one

pi bond. The latter undergoes strong interaction with the spz C atom array abundant in

graphite and the analogous spherical analogue, the buckyball. Fig. 5 suggests mode of

adsorption bonding. Aggregates of C 60 possibly keep the effective surface at lower levels

than indicated.

181

Figure 5. Proposed mode of ethylene adsorption on C 60. The large C2H4 molecule in the center represents the structural model in which the double bond consists of a peripheral 7! (or banana) bond which has particular affinity for CzH4 molecules, and a central a bond.

Conclusion: C 60 promises to be a powerful tool in control of plant ethylene, acting as an

internal adsorbent or scrubber of the gas as it is being formed. Problems of use in edible

produce, but less so in floriculture still to be addressed are health-related and pertain either

to fullerenes themselves or to their specific solvents - benzene which is carcinogenous or

toluene which is inflammatory. However purified C 60 per se in this respect probably

resembles pharmaceutical use of activated charcoal for gastric ailments, its use as an

industrial decolorizer or in wineries - as an agent to rid certain wines of cloudiness.

Acknowledgement We would like to thank Prof. Y. Yeshurun, Department of Physics, Bar-Han University for his helpful discussions and advice, Debby Barkhan for her technical assistance and Philip Caplan for his aid with the surface area computation.

BIBLIOGRAPHY Bochvar DA and Galperin EG 1973. Carbon 60. Dok. Acad. Niuk SSR 209 610-612 Culotta E and Koshland DE 1991. Buckyballs: wide open playing field for chemists. Science 254 1706-1707 Huffman DR 1991. Solid C 60. Physics Today 4411 22-31 Kratschmer W Lamb DL. Fostiorpoulous and Huffman DR 1990. Solid C 60: a new form of carbon. Nature 347 354-358 Leshem YY 1992. Plant Membranes: A Biophysical Approach to Structure, Development and Senescence. Kluwer Acad. Pub. Dordrecht p. 105 Moore WJ 1956. Physical Chemistry. Longmans London p. 517 Pauling L 1967. The Chemical Bond. Cornell University Press Ithaca p. 88

EFFECT OF DIAZOCYCLOPENTADIENE (DACP) ON CUT CARNATIONS

EDWARD C. SISLER, SYLVIA M. BLANKENSHIP, JEFFREY C. FEARN, ROBIN HAYNES Departments of Biochemistry and Horticul tural Science, North Carolina State University, NCSU Box 7622, Raleigh, NC, 27695, USA

ABSTRACT.

Diazocyclopentadiene (DACP) undergoes photolysis under 5000 lux fluorescent lights with a t=I/20f 28 minutes. Cut carnations are preserved with as little as 0.14 ul/l of pre irradiated DACP, which seems to be as effective as silver thiosulfate. DACP must be exposed to light to be effective, but once irradiated, is effective in the dark. DACP treatment delays and reduces endogenous ethylene production.

1. Introduction

cut carnations have a variable vase life depending on the variety: 7 days for White Sim, and 14 days for Sandra and Chinera (8). All are sensitive to ethylene. Protection against ethylene has been achieved by treatment with silver compounds and, particularly, silver thiosulfate treatment which presumably block ethylene action (5), although the specific mechanism is unknown. Vase life has also been extended with compounds such as aminooxyacetic acid which inhibit ethylene synthesis (2). There are some environmental objections to the use of silver, and ethylene synthetic inhibitors are also sometimes toxic.

Recently, diazocyclopentadiene (DACP) has been shown to be an effective ethylene action inhibitor after illumination with fluorescent light (7). DACP is a volatile compound and would offer some advantages for the preservation of carnations.


2.1. MATERIALS

Cut carnations (Diantus caryophyllus L., cv. White Sim) were obtained from a commercial grower. Diazocyclopentadiene was prepared by the method of Regitz and Liedhegener (4) and the concentration determined using triphenylphosphine. The explosive and light-sensitive compound was stored at -l5°C in the dark as a 4-8% solution in hexane or similar solvent. After putting carnations in 3-liter glass jars, DACP in hexane was placed on paper towels to increase surface area, the jars sealed, and placed in the light. Treatment with silver was as previously described using 4 roM silver thiosulfate (6). All DACP values are gas phase values.

182


183

2.3. ETHYLENE MEASUREMENTS

Ethylene binding measurements were made by the method of Sisler et al. (6) using 14c-ethylene (Amersham 110 mCi romol -1) in a 2.5 1 desiccator. After exposure to 0.5 uCi of 14C-ethylene in the presence and absence of unlabeled ethylene, samples were vented for 4 minutes before placing them in jars containing a scintillation vial. The vial contained mercury perchlorate on a piece of glass fiber to increase the surface area. After 12 hours, scintillation fluid was added and the samples counted. Ethylene production was measured by gas chromatography on alumina.

3. Results

3.1. COMPARISON OF DACP AND SILVER THIOSULFATE IN PRESERVING CARNATION FLOWERS.

Figure 1 compares the effect of 4.0 roM silver thiosulfate and 9 ul/l of preirradiated DACP on the preservation of cut White Sim carnations. There was no apparent difference in the appearance of the two treatments at the end of 12 days. The vase life of the two treatments did not appear to differ. It is suggested that DACP is just as effective as silver thiosulfate for preserving carnations.

3.2. PHOTOLYSIS OF DACP

DACP undergoes a photolysis under fluorescent light to form other products. In order to determine the rate of decomposition of DACP, a small amount was placed in a silica cuvette which could be closed at the top. The rate of decline in absorbance was measured at the maximum absorbance for DACP at 292 nm. The cuvette was placed inside a pyrex container under 375 }.Imol m-2 s-\ (5000 lux) of fluorescent light. Under these conditions, the half-life of DACP was 28 minutes (Fig. 2). Most experiments were carried out with carnations inside glass jars that were not borosilicate, and the decay rate was also measured inside these jars as the absorbance of light by glass could make a considerable difference in the amount of light reaching the compound. The rate was very close to that in a pyrex container. When stored in an inverted flask with a saturated (NH4)2S04 seal, little or no decline in activity was noted after 96 hours at 25°C.

TlJl1C (millules)

Figure 1. Effect of STS (4 roM) and Figure 2. Photolysis of DACP by DACP (9 ul/l) on carnations. 5000 lux of fluorescent light.

184

3.3. EFFECT OF DACP CONCENTRATION ON ETHYLENE BINDING

14C-ethy1ene binding is used to determine the amount of free receptor present in plant tissue. When some other compound is competing for the binding site(s) a lower value is obtained for 14C-ethylene binding.

As the pre incubated DACP concentration is increased, the amount of ethylene binding decreases. When DACP is allowed to compete with 14Cethylene and the results are analyzed by a Scatchard plot (3), an ISO value of 263 u111 is obtained. This gives a Kd value of 195 ulll for DACP. The Scatchard plot reveals only one binding site in carnations (Fig. 3).

After DACP is subjected to photolysis for 3 hours and then allowed to compete with 14C-ethylene, a Scatchard plot gives an ISO value of 0.16 u111 for DACP (and its photolytic products). The Kd value obtained was 0.12 (Fig. 4). This means the amount of DACP (or its derived products) is 1625 times lower than non-irradiated DACP to occupy 1/2 of the sites. Although the active component in irradiated DACP is not known, it is much more effective than non-irradiated DACP.

b4 500 ~O.4r CACP IN THlO DAR.K

] .,.- ., Co 400 I~ 0.3 ~

L'~ ....,

S50.2 ~

= 300 i! :e '" = ~O.1

is c;;J< "-:,'" 0 200 0_00 20 4(J 60 80 100 = U Cuplacccl ('iii)

>. -= 100 Iii

r C,) -or 00 400 800 1200 1600 2000

DACP Concentration (uJ/L) Figure 3. Effect of DACP on 14Cethylene binding and (inset) Scatchard plot of ethylene-DACP competition.

...... 500 [ PRI!-IIUlADIA TI!D CAe? ~ ....,

e • \ Co

400 f ~ .\. --~ \ = 300 :e ~ C K.i.0_12ul1L

is 0 200 '.

= U °0 20 40 60 80 loa >. Diaplaced ('iii) -= 100 Iii r

C,) -or

~.O 0.5 1.0 1.5 2.0 2.5 DACP Concentration uJ/L

Figure 4. Effect of pre irradiated DACP on 14C-ethylene binding and (inset) Scatchard plot of ethylene-DACP competition.

3.4 EFFECTIVE CONCENTRATION OF DACP

When carnations are pre incubated with DACP in the dark, then exposed to fluorescent light (5000 lux) in the air, the lowest concentration of DACP that will protect them against 10 ulll of ethylene is above 700 ul/l. When carnations are exposed to pre irradiated DACP, they are protected by 0.14 ulll of DACP (Fig. 5). Pre irradiated DACP then is about 5000 times more effective than non-irradiated DACP.

3.5. TIME DACP REMAINS BOUND TO THE ETHYLENE BINDING SITE

Figure 6 shows that over a wide range of concentrations, the measured amount of receptor does not decline much after exposure to DACP in the dark for 45 minutes and then assayed with 14C-ethylene. This suggests a rapid turnover as most of the DACP came off the receptor during the 45 minutes. Figure 6 (inset graph) also indicates a short binding time with a half-life of perhaps 15-20 minutes since that amount of delay before exposure to light lowers the amount of binding site inactivated by DACP and light about 50%.

Figure 5. Effect of DACP (top) and pre irradiated DACP (bottom) on cut carnations.

185

600 r ~'-~---.r---___~

110

100

ISO ~ ••

200 ••• ~0~--~l~0----~~---9=0~--'~1.

500 1000 1500 2000 DACP CONCENTRATION ulIL

Figure 6. Effect of DACP on 14Cethylene binding during a 45-min. assay and (inset) effect of vented time on 14C-ethylene binding.

3.6 EFFECT OF HIGH CONCENTRATIONS OF ETHYLENE OF CARNATIONS

When carnations exposed to 9 ul/l of pre irradiated DACP and then exposed to 0, 10, 100, 1000, and 10,000 ul/l of ethylene, no apparent difference is noted. When carnations are exposed to 2800 ul/l of DACP in the dark, then vented and exposed to 5000 lux of fluorescent light, for 4 hours, followed by 0, 10, 100, 1000, and 10,000 ul/l of ethylene, the 1000 and 10,000 ul/l of ethylene, samples show pronounced senescence while the lower concentrations do not (Fig. 7). This suggests the presence of a high-level or modified receptor in the case of preincubation, but not in the case of preirradiation.

Figure 7. Protection of carnations against 0, 10, 100, 1000, and 10,000 ul/l of ethylene by 9 ul/l of preirradiated DACP (upper row) and preincubation (low row) with 2000 ul/l DACP followed by 5000 lux of flurorescent light. Note that pre irradiated DACP completely protects against 10,000 ul/l ethylene but pre incubated with 2000 ul/l DACP does not.

3.7. ABSORPTION SPECTRA OF DACP AND A CARNATION PETAL EXTRACT

Doering and DePuy (1) give an absorption spectra for DACPi however, they present the logarithm of the molar absorptivity. Figure 8 gives the absorption spectra of DACP in the vapor state and also in aIM hexane solution to show the absorption in the region where absorption is small but would be in the range of fluorescent light illumination. An absorption spectra is also given of a carnation petal extract in 95% ethanol. The extract is at the same approximate concentration as in petals and also diluted 1/450th. A comparison can be made of the DACP

186

absorption of light and the transmission of light by carnation petals. It appears that very little light would be transmitted by carnation petals below about 415 nm and lower wavelengths would likely have little effect on DACP unless the light reached the DACP before it did the petal pigments. The absorption spectra of DACP shows the strongest absorption is below 415 nm (292 max in the vapor state), but there is a tail on the spectra that extends nearly to 500 nm. Although the pigments reduce the amount of light getting inside the petals, the pigments do not eliminate it and DACP could undergo a photolysis in carnation petals due to light above about 415 nm.

3.8 ETHYLENE PRODUCTION

Ethylene production does not appear to be altered during the first 5 days by DACP (Fig. 9). The normal rise in ethylene production which occurs after 5 days was delayed several days Ln DACP-treated petals, and the magnitude was considerably less than in control or ethylenetreated petals.

2.0...-------.,---r----,

-DACP - CAR..."fATION

1.6

, , 110 diluuoa

1.2 V J\iL~. ,I r., \.¥ '

08 ./ 1/ \ 1M ACPiIlh . \,/ ,

0.4

\

9~ps

/ " \ \ \ \ \ \

370 410

Wave1e11gtlL (nm)

Figure 8. Absorption spectra of carnation petals; also, 9.2x10-5M DACP in gas phase and 1M hexane.

4. Discussion

50 ...

\ _ control ---&-- DACP

40

30 -20

10

4 6 8 10 12 Days Mter Treatment

Figure 9. Ethylene production by ethylene-treated, DACP-treated, and control carnations.

14

Irradiated DACP is an effective inhibitor of ethylene responses and senescence in cut carnations. Preirradiation of DACP probably increases its effectiveness because the petals absorb a large portion of the light, although some of the reaction likely does take place on the receptor. After venting and exposure to light, the binding sites in carnations pre incubated with DACP are inactivated, and the ethylene response is inactivated. Exposure to pre irradiated DACP protects against 10,000 ulll of ethylene, but carnations preincubated with DACP are not protected at 1000 and 10,000 ulll of ethylene. This may indicate a different mode of inhibition, but likely it means there is a second receptor which is not completely inactivated by preincubation with DACP followed by venting and irradiation. This high-level receptor then might allow senescence to be induced.

5. Acknowledgements

The authors acknowledge support from the Fred C. Foundation and the USDA Competitive Grants Program. Fukutome, Watsonville, CA, supplied the carnations.

6. References

187

Glockner Jr. Mr. Harry K.

1. Doering, V.E. and DePuy, C.H. (1953) 'Diazocyclopentadiene', J. Amer. Chern. Soc. 75, 5955-5957.

2. Fujino, D.W., Reid, M.S. and Yang, S.F. (1980) 'Effects of aminooxyacetic acid on postharvest characteristics of carnation', Acta Hort. 113, 59-68.

3. Presecan, E., Porumb, H. and Lascu, 1. (1989) 'Potential misinterpretation of the competitive binding assays' , Trends Biochem. Sciences 14, 443-444.

4. Ramirez, F. and Levy, S. (1958) 'Reaction of diazocyclopentadiene with triphenylphospine', J. argo Chern. 23, 2036-2037.

5. Regitz, M. and Liedhegener, A. (1967) 'Reaktionen aktiver methylenverbindungen mit aziden-XV. Syntheses von diazocyclopentadiene durch diazogruppenubertagung und einigen reaktionen', Tetrahedron 23, 2701-2708.

6. Reid, M.S., Paul, J.L., Farhoomand, M.B., Kofranek, A.M. and Staby, G.L. (1980) 'Pulse treatments with the silver thiosulfate complex extend the vase life of cut carnations' , J. Amer. Soc. Hort. Sci. 105, 25-27.

7. Sisler, E.C. and Blankenship, S.M. (1992) 'Diazocyc1opentadiene, a light sensitive reagent for the ethylene receptor', Plant Growth Reg. (in press).

8. Sisler, E.C., Reid, M.S. and Yang, S.F. (1986) 'Effects of antagonists of ethylene on binding of ethylene in cut carnations', Plant Growth Reg. 4, 213-218.

9. Wu, M.J., Doorn, W.G. and Reid, M.S. (1991) 'Variation in the senescence (Dianthus caryophyllus L.) cultivars. 1. Comparison of flower life, respiration and ethylene biosynthesis', Scientia Hort. 48, 99-107.

REDUCED SENSITIVITY TO ETHYLENE AND DELAYED SENESCENCE IN A GROUP OF RELATED CARNATION CULTIVARS

WOUTER G. VAN DOORN, ERNST J. WOLTERING, MICHAEL S. REID, MENG-JEN WU

Agrotechnological Research Institute (ATO-DLO). P.O. Box 17,6700 AA Wageningen. Holland. and Department of Environmental Horticulture. University of California. Davis. CA 95616.

ABSTRACT. Three related carnation cultivars (Ginevra, Epomeo and Chinera) were less sensitive to exogenous ethylene than cv. White Sim. The concentration to give 50% of the maximum response (HsJ after a 24 h treatment was 0.21 pI J-I in cv. White Sim and 0.32, 0.48, and 0.65 pll'\ in the cvs. Ginevra, Epomeo, and Chinera, respectively.

When no exogenous ethylene was given the time to petal inroUillg was positively correlated with Hso. It was about 7 days in cv. White Sim, 12 days in cv. Ginevra, 14 days in cv. Epomeo and 15 days in cv. Chinera. A similar delay was found in the climacteric rise in ethylene production. In cv. Epomeo, moreover, the maximum ethylene production during natural senescence was about one third that in cv. White Sim, and in cv. Chinera about one third that in cv. Epomeo.

When treated with a high dose of ethylene for a short period, the cvs. Chinera and Epomeo responded by producing high amounts of ethylene, indicating that all necessary steps for autocalytic ethylene production are present or can be rapidly induced.

The number of binding sites for ethylene, assessed with an isotope competition technique, was similar in the cvs. Chinera and White Sim. Binding affinity per site was less in cv. Chinera and this may be the basis for its lower sensitivity. It is not known, however, whether assesment of the number of binding sites and their affinity reflects the physiologically active receptor(s). The difference between the cultivars may also be in the signal transduction pathway.

1. Introduction

Sensitivity to ethylene may be one of the main determinants of the time to senescence in climacteric fruits and also in cut flowers in which petal senescence is regulated by ethylene. The basis of ethylene sensitivity has not been studied in detail because of a lack of suitable experimental material. An exception is the tomato fruit. Tomato ripening mutants include ripening inhibitor (rin; Robinson and Tomes 1968), non-ripening (nor; Tigche1aar et al. 1973) and never ripe (Nr; Rick 1956). Both rin and nor fruits respond to exogenous ethylene by an increase in respiration, without a detectable increase in ethylene production (Herner and Sink 1973, McGlasson et al. 1975). When no ethylene is given these two mutants do not show a climacteric rise in respiration and ethylene production (Herner and Sink 1973, Ng and Tigchelaar 1977). In the Nr fruit not treated with ethylene, the rise in respiration rate and ethylene production is delayed and the peak of ethylene production is attenuated (Tigchelaar et al. 1978).

The recent finding of ethylene-insensitive mutants in Arabidopsis thaliana and the analysis of the genes involved (Bleecker et al. 1988) may be an important step towards our understanding of sensitivity. As yet it is not clear, however, whether the differences in sensitivity between mutant and wild type are based on changes in the receptor, in the signal transduction pathway (from receptor to the genome), or in the capacity to respond.

188

J. C. Pech etaL (eds.). Cellular and Molecular Aspects of the Plant Honnone Ethylene. 188-194. © 1993 Kluwer Academic Publishers.

189

We found that the vase-life of some carnation cultivars was exceptionally long and hypothesized that this difference might be due to either a reduced production of ethylene and/or a reduced sensitivity. The relatively long life of the cultivar Sandra was due to the absence of the autocatalytic rise in ethylene production, similar to rin or nor tomato fruits. When exposed to exogenous ethylene cv. Sandra was, however, even more sensitive than White Sim. a cultivar with a normal short vase life (Wu et al. 1989, 1991a). The long vaselife of the cultivar Chinera was due to the opposite set of characteristics. This cultivar showed an autocatalytic rise in ethylene production, but was less sensitive to exogenous ethylene (Wu et al. 1991a, b).

In the present paper we report on the pedigree of cv. Chinera, its base level ethylene production, and on the induction of autocatalytic ethylene production by a relatively high level of exogenous ethylene. We also investigated the sensitivity to exogenous ethylene, and the time to petal inrolling in some cultivars closely related to cv. Chinera, and discuss the relevance of measuring the ethylene binding characteristics.


2.1. Plant material and determination of vase-life.

Cut flowering stems of carnation (Dianthus caryophyllus L.) cvs. White Sim, Scania, Cantalupo, Ginevra, Epomeo, and Chinera were obtained from commercial growers in California or Holland, or were grown in a greenhouse at the Department of Environmental Horticulture in Davis, California. After harvest at commercial maturity the stems were transported dry to the laboratory and used the same day. Stems were recut to a length of about 30 cm and placed in water. Experiments started after several hours of rehydration in water. The length of vase life was assessed in a controlled environment of 12 h fluorescent light (15 ).Imol m,2 sec,l) and 12 h darkness, 60% RH and 20°C. The room was ventilated and ethylene concentrations were below levels detectable by gas chromatography and the concentrations of carbon dioxide and oxygen were as in ambient air. The length of vase life was defined as the time to clear petal inrolling.

Except for the cultivars White Sim and SCllInia, which were used as a reference, the cultivars investigated were bred by the Dr. Nobbio Company, at San Remo, Italy. One of the goals of the breeding progranl from which these cultivars emerged was to obtain a longer vase life.

2.2. Ethylene production and sensitivity to exogenous ethylene.

Ethylene production was measured at intervals by removing flowers from the vase-life room, recutting to a stem length of 3-4 cm and enclosing them in glass jars, which were flushed with ethylene-free air before closure. After 0.5 to 2 h of incubation, depending on the stage of senescence, the ethylene concentration in the headspace was measured by gas chromatography.

For determination of sensitivity freshly harvested flowers were placed dry in 70 I stainless steel chambers at 20 ± 1°C in darkness. The carbon dioxide concentration was kept below 0.05% (v/v) and the oxygen concentration at about 21 %. The desired concentration of ethylene was obtained by injection of a known amount of gas (Matheson, Belgium) into the

190

chambers. Effects were measured over a range of elhylene concentrations for 24 h or over a range of exposure times to a saturating concentration (850 pI.l-!). The reduction in time to petal inrolling was determined in lhe above controlled environment and was expressed as lhe number of flowers which showed inrolling. Experiments using a range of concentrations were conducted once for cvs. Cantalupo and Ginevra, twice for cv. Scania, and repeated four times for lhe cvs. White Sim, Epomeo, and Chinera. The results were compared by analysis of variance using Genstat V and T-test at P>O.01.

Elhylene production induced by exogenous elhylene was measured in freshly harvested flowers after 20 h treatment wilh 20 pI r! ethylene at 20°C in darkness, followed by 4 h of equilibration in air, and 0.5 h enclosure in vials.

3. Results

3.1. The pedigree of cvs. Cantalupo, Ginevra, Chinera, and Epomeo.

A non-commercial breeding line wilh a long vase life, 8367, was lhe product of lhree generations of olher non-commercial selections. Crossed wilh lhe red cultivar Faust it produced Chinera (Fig.l), a salmon cultivar wilh dentate petal margins. Epomeo, a red cultivar wilh dentate petal margins, is lhe offspring of Chinera and a non-commercial breeding line 9775.

Cantalupo, a red cultivar wilh relatively smoolh petal margins (comparable to White Sim and Scania) is lhe offspring of non-commercial breeding lines. Crossed wilh '8367' it produced Ginevra. which has dark pink petals wilh smoolh rims.

~~7"~ -~,. i~ )~I ~ ! ...... !

9m"" / I Epomeo I Fig. 1. The pedigree of the carnation cultivars

Epomeo, Chinera, Ginevra, and Cantalupo.

3.2. The time to petal inrolling.

When lhe flowers were not treated wilh exogenous elhylene lhe lenglh of vase life (based on petal inrolling) depended on lhe season. Generally, no differences were found in lhe vaselife of lhe cvs. Cantalupo, White Sim, and Scania, whereas lhe vase-lives of lhe cvs. Ginevra, Epomeo and Chinera were progressively longer. In a typical experiment in which lhe life of lhe various cultivars were compared concurrently, time to inrolling (± SD) of cv. Cantalupo was 9.1 ± 0.8 days, of cv. Ginevra 12.0 ± 2.2 days, cv. Epomeo 13.6 ± 0.7 days and cv. Chinera 14.7 ± 1.4 days, whereas lhe life of cv. White Sim was 7.6 ± 2.8 days and cv. Scania 8.4 ± 1.3 days.

191

3.3. Ethylene production prior to and during senescence.

The basal production of ethylene prior to the autocatalytic phase was always higher in the cvs. Chinera and Epomeo than in cv. White Sim (Table 1).

In cv. Epomeo the onset of increased ethylene production occurred later than in cv. White Sim, and in cv. Chinera it occurred later than in cv. Epomeo (Fig. 2). The maximum rate of ethylene production in cv. Epomeo was lower than in cv. White Sim, and in cv. Chinera lower than in cv. Epomeo (Fig. 2).

Table 1. Ethylene production in cut flowering stems of carnation, before the autocatalytic phase (basal production), and after 20 h treatment with 20 pI 1.1 exogenous ethylene, determined 4 h following treatment.

Cultivar

WhiteSim

Epomeo

Chinera

Ethylene production (nl.g FW'.h·')

Basal Four halter produc- 20 h exoge-tion nous ethylene

0.035 89

0.060 68

0.055 45

3.4. Sensitivity to exogenous ethylene.

j" z: '6

, ~ .. u.. C> >2

:§ <: .~ U ::J

"t:! 0

0.. CD <: CD

m

•

>2 z: W

• White Sim

o Chinera

b. Epomeo

'0

Time (d)

Fig. 2. Ethylene production of cut flowering stems of the carnation cuItivars White Sim, Epomeo, and Chinera, during senescence.

>2 ..

When exposed to a range of concentrations the dose-response curves had the same shape. From the dose-response curves the concentration giving 50% of the maximum response (Hso) was calculated. Cv. Epomeo was less sensitive than the reference cultivars White Sim and Scania, and cv. Chinera less sensitive than cv. Epomeo (Table 2). Although the experiment with the cvs. Cantalupo and Ginevra has thus far only been conducted once, the former is apparently not different from cv. Scania and the latter is apparently intermediate between the reference cultivars and cv. Epomeo.

As shown in Fig. 3 the Hso values were positively correlated with the time to petal inrolling.

192

Table 2. Hso, the concentration of exogenous ethylene giving 50% of the maximal response. Values were calculated from dose-response curves.

Cultivar

Scania 0.15 ± 0.05 a

WhiteSim 0.21 ± 0.02 a

Cantalupo 0.15

Ginevra 0.32

Epomeo 0.48 ± 0.10 b

Chinera 0.65 ± 0.09 c

L.. '5. ,;

i !: :~ 'in r:: .. .. . !: II) r:: II)

>. ..r:: W

0.8

0.6

0.4

0.2

0

Chinera

Ginevra

Whit.~ Cantalupo

ScaOia

10 12 14 16

Vase life (d)

Fig. 3. Relationship between the length of vase-life (based on time to petal inrolling), and insensitivity to exogenous ethylene.

3.5. Exposure to a high exogenous concentration of ethylene.

When treated with 850 ).II tl ethylene the minimum time of exposure necessary for 100% response (= all flowers showing inrolling within 0.5 d after treatment) was about 8 h in cv. White Sim and 13 h in cv. Chinera.

Treatment of cvs. Chinera and Epomeo with a high concentration of exogenous ethylene for 20 h resulted in a considerable increase in ethylene production, about 10-20 times higher than during natural senescence. The production rate was highest in cv. White Sim, lower in cv. Epomeo and still lower in cv. Chinera (Table 1).

4. Discussion

The sensitivity to ethylene in the investigated group of carnation cultivars was inversely related to the time to petal inrolling and to the onset of increased ethylene production. Sensitivity to ethylene, therefore, is a main regulator of the autocatalytic ethylene production. When compared with normally sensitive cultivars, the reduced sensitivity in cv. Chinera resulted in a doubling of the time to inrolling.

Autocatalytic production of ethylene is regulated by sensitivity but may also be regulated by the amount of ethylene produced prior to the autocatalytic phase, and the duration of ethylene production. The longer vase-life of the cvs. Chinera, Ginevra and Epomeo was not due to a lower basal production of ethylene prior to the autocatalytic phase. Basal production was even higher than in the cultivars with a normal short vase-life. A higher than normal basal production is also found when the receptors are blocked by silver. Prior to the autocatalytic phase, therefore, the receptor/signal transduction pathway may be involved in a negative feedback which down-regulates the rate of ethylene production.

193

In the cultivars investigated, insensitivity to ethylene was apparently inherited to a different degree. Insensitivity was apparently conferred through the selection 8367, since the other parental lines of Chinera, Ginevra and Epomeo (Fig. 1) do not have a long vase-life. This was now found for Cantalupo, and has previously been found for cv. Faust and the selection 9775 (Sapia, pers. comm. 1992). The genetical background of the difference in sensitivity between the cvs. Chinera, Ginevra and Epomeo is as yet unknown.

These low sensitivity carnation cultivars resemble the Nr tomato fruit, which shows autocatalytic production of ethylene, although delayed and attenuated (Tigchelaar et al. 1978). We found that the delay in the rise in ethylene production in the cvs. Chinera, Ginevra, and Epomeo was correlated with the degree of attenuation of the ethylene peak. These cultivars also resemble the heterozygous +/nor tomato fruit. Although an autocatalytic increase in ethylene production was not found in nor/nor fruit, in +/nor fruit a peak in ethylene production was observed, which was delayed and attenuated (Ng and Tichelaar 1977).

In the cvs. Chinera and Epomeo autocatalytic ethylene production was rapidly induced by a high exogenous ethylene concentration. The same occurs in normally sensitive cultivars. In carnation petals both enzymes are transcriptionally regulated; mRNAs for both enzymes were present within 6 h of ethylene treatment (Woodson et al. 1992). From the high amounts of ethylene produced in cvs. Chinera and Epomeo we infer that a large amount of de novo ACC synthase and EFE was induced. The results indicate that all necessary genes for autocatalytic production are present in cv. Chinera, and that these can also rapidly be expressed following ethylene treatment. Both transcription and translation, therefore, do not seem to be limiting. Under these conditions the low sensitivity of ethylene perception is overridden. One anomaly, however, remained: during exposure to saturating levels of exogenous ethylene the exposure time needed for expression of the symptoms (petal inrolling) in 100% of the flowers was 13 h in cv. Chinera and 8 h in cv. White Sim. Further analysis of the expression of the ACC synthase and EFE genes may show whether this difference is located before or after transcription.

The results obtained thus far indicate that the main difference between cv. Chinera and cv. White Sim is located before transcription. Analysis of the number of ethylene binding sites, using the isotope competition technique, indicated no difference between cv. White Sim and cv. Chinera (Wu et al. 1991b). In morning glory flowers (Ipomoea nil), and in apples, which become more sensitive to exogenous ethylene during development, the number of binding sites remained constant or even decreased, respectively (Blankenship and Sisler 1989). This method, therefore, may not determine the number of physiologically active receptor sites. The only positive correlation found between sensitivity and the number of binding sites was in an ethylene-insensitive mutant of Arabidopsis thaliana, in which the number of sites was lower than in the wildtype (Bleecker et al. 1988)

The binding affinity per site was lower in cv. Chinera than in cv. White Sim (Wu et al. 1991b). This was apparently the first report of such a difference. Sisler (pers. comm. 1992) also determined binding affinity, in a number of systems in which ethylene sensitivity increased with aging but did not find differences. The above criticism concerning the method for determining the number of binding sites may also apply to calculation of binding affmity.

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The difference in affinity does not necessarily reflect the physiologically active receptor(s) for ethylene.

It is concluded that sensitivity to ethylene is a major determinant in the control of the onset of autocatalytic ethylene production, and of the maximum of the autocatalytic rise. The basis of sensitivity is as yet unknown. It may be related to receptor binding but also to factors in the signal transduction pathway.

Acknowledgements

We thank Dianne Somhorst, Colinda de Beer, Anneke Polderdijk, Jacinto Zimmerman, and Lorenzo Zacarias for their valuable contribution to this project.

5. References

Blankenship, S.M. and Sisler, E.C. (1989) Ethylene binding changes in apple and morning glory during ripening and senescence. J. Plant Growth Regul. 8: 37-44.

Bleecker, AB., Estelle, M.A, Somerville, M.A and Kende, H. (1988) Insensitivity to ethylene conferred by a dominant mutation in Arabidopsis thaliana. Science 211: 1086-1088.

Herner, R. and Sink, K. (1973) Ethylene production and respiratory behavior of the rin tomato mutant. Plant Physiol. 52: 38-42.

McGlasson, W., Dostal, H. and Tigchelaar, E. (1975) Comparison of propylene-induced responses of immature fruit of normal and rin mutant tomatoes. Plant Physiol. 55: 218-222.

Ng, T. and Tigchelaar, E. (1977) Action of the non-ripening (nor) mutant on fruit ripening of tomato. J. Amer. Soc. Hort. Sci. 102: 504-509.

Rick, C. (1956) New mutants. Rep. Tomato Genet. Coop. 6: 22-23. Robinson, R. and Tomes, R. (1968) Ripening inhibitor: a gene with multiple effects on

ripening. Rep. Tomato Genet. Coop. 30: 2-17. Sisler, E.C. (1979) Measurement of ethylene binding in plant tissue. Plant Physiol. 64: 538-

542. Tigchelaar, E., Tomes, M., Kerr, E., and Barman, R. (1973) A new fruit ripening mutant,

non-ripening (nor). Rep. Tomato Genet. Coop. 23: 33. Tigchelaar, E., McGlasson, W. and Buescher, R. (1978) Genetic regulation of tomato fruit

ripening. HortScience 13: 508-513. 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.

Wu, M-J., van Doom, W.G. and Reid, M.S. (1989) Senescence of 'Sandra' carnation. Acta Hortic. 261: 221-225.

Wu, M-J., van Doom, W.G. and Reid, M.S. (1991a) Variation in the senescence of carnation (Dianthus caryophyllus L.) cultivars. I. Comparison of flower life, respiration and ethylene biosynthesis. Scientia Hortie. 48: 99-107

Wu, M-J., Zacarias, L. and Reid, M.S. (1991b) Variation in the senescence of carnation (Dianthus caryophyllus L.) cultivars. II. Comparison of sensitivity to exogenous ethylene and of ethylene binding. Scientia Hortic. 48: 109-116.

IN vrmo Sl'UDY OF ElHYLENE BINDING SI1N> IN PEA. SEEDLINGS

I.E.MOSHKOV, G.V.NOVIKOVA, A.R.SMITH* and M.A.HALL* Institute of Plant Physiology Acad.Sci.Russia, 35 Botanicheskaya str., Moscow 127276, Russia * - Dept. of Biological Sciences, University College of Wales, Aberystwyth, Dyfed SY23 3DA, U.K.

Presently the existence of ethylene binding protein(s) is absolutely clear. Ethylene binding was detected and characterized in some plant species, and ethylene binding protein was isolated from Phaseolus cotyledons [1]. All these studies were based on binding of ethylene in vivo. Therefore, the aim of present work was to study ethylene binding in vitro.

Epicotyl tips of 5-day-old pea seedlings (Pisum sativum L. cv. Alaska) were used for this work because the high concentration of ethylene binding sites in pea seedlings tips had been shown earlier [1] and pea seedlings are extremely sensitive to exogenous ethylene. Seedling tips were homogenized in buffer contained 50 mM Tris-HCI (pH 7.3), 0.1 mM PMSF, and 10% glycerol. Homogenate was filtered through tight nylon and centrifuged at 9,000g for 30 min. Obtained supernatant was separ~ted on membrane enriched fraction (96 kP) and soluble protein fraction (96 kS) by centrifugation at 96,000g for 4 hs. For binding assay we used the technique that was developed for in vivo experiments [2] with some modifications. Preparations (from 5 g of epicotyl tips in a volume of 3-3.5 ml per each point) were placed in 115 ml flasks sealed with size-45 Subaseal. To prevent the incorporation of [14-C]ethylene to metabolism, CS2 (100 ul/l) - inhibitor of ethylene metabolism - was included to incubation buffer. Incubation was conducted at SoC with shaking. The association plots were produced by incubation of samples with [14-C]ethylene for various time intervals. The dissociation plots were produced by incubation of samples for 2 or 20 hs and following ventilation for various time intervals. Then samples were transfered to scintillation vials contained scintillation liquid. In order to estimate Kd values Scat chard analysis was conducted.

Analysis of association and dissociation plots for ethylene binding in vitro with 96 kP fraction showed that there are at least two classes of binding sites in membrane enriched fraction. One class showed high rate constants of association and dissociation (fast sites) and another one had low rate constants (slow sites). These results are similar to that reported for ethylene binding with pea seedling tips in vivo [2]. It was very important to distinguish fast and slow binding sites by their Kd values. The data on association showed that fast sites were sa-

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J. C. Pech et al. (eds.J, Cellular and Molecular Aspects of the Plant Hormone Ethylene, 195-196. © 1993 KliMer Academic Publishers.

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turated with ethylene for 2 hs of incubation, whereas slow binding sites needed for saturation at least 20-h incubation. Preliminary experiments demonstrated that unbound ethylene was removed from sample solution for 4-5-min ventilation. Dissociation of bound ethylene from fast binding sites occured for 1.5-2 hs. So, to determin Kd for these two classes of binding sites we used the following time intervals: for fast sites - 2 hs for association and 5 min for ventilation (only unbound ethylene was removed), for slow sites, respectively - 20 hs and 2 hs (unbound and bound with fast binding sites ethylene was removed). Both classes of binding site= ~ad ~i~n demonstrated to possess approxymately the same ~ values (10 1 -101M) but differ in their concentrations. These data corresponde well to that obtained with pea tips in vivo [2].

Ethylene binding activity was also detected in 96 kS fraction and this is the first reference about soluble ethylene binding sites. 96 kS fraction contained only one class of binding sites - fast sites. But its characteristics - rate constants of association and dissociation, ~ -were similar to characteristics of fast binding sites of 96 kP fraction. As far as experiments with slow binding sites require long-time intervals (about 40 hs), a protein proteolysis is very probable. Therefore, one should not exclude that 96 kS fraction maintains slow binding sites, and we could not detect them under used conditions.

Ethylene binding with both fractions was reversible, and bound [14-C]ethylene could be displaced by [12-C]ethylene or propylene. This displacement of bound ethylene can be evidence of physiological significance of ethylene binding with these fractions.

Thus, present work demonstrates the possibility of ethylene binding in vitro that is very important for isolation of ethylene binding proteins. If in the case of isolation of slow binding sites prelabelling in vivo with [14-C]ethylene is useful [1] only in vitro binding assay can by applaied for identification of fast binding sites in isolation procedure. Furthermore, in vitro assay allows to study an interaction between hormone and receptor directly excluding all possible intermediate steps in their interaction. But this technique also has some disadvantages. An interaction of a ligand with its binding protein can be conducted under nonoptimal conditions. A biJlding protein can partially degradate during isolation or (as in our case) long-time incubation. Some difficulties in experimental technique and inaccurateness in results are related with gaseous state of ethylene and its presence in the atmosphere. Moreover, as it has been shown in another our work [3], binding of ethylene can be affected by phosphorylation level of protein fraction. All these-moments could be reasons of differences in obtained results.

References 1. Hall, M.A., Connern, C.P.K., Harpham, N.V.J. et al. (1990) 'Ethylene:

receptors and action', in J.Roberts, C.Kirk and M.Venis (eds.), Hormone perception and signal transduction in animals and plants, Cambridge, pp. 87-110.

2. Sanders, 1.0., Smith, A.R., and Hall, M.A. (1991) 'Ethylene binding in epicotyls of Pisum sativum L. cv. Alaska', Planta 183, pp. 209-217.

3. Novikova, G.V., Moshkov, I.E., Smith, A.R., and Hall, M.A. 'Ethylene and phosphorylation of pea epicotyl proteins', in this issue.

FUNGAL XYLANASE ELICITS ETHYLENE BIOSYNTHESIS AND OTHER DEFENSE RESPONSES IN TOBACCO.

J.D. Anderson1 , B.A. Baileyl, R. Taylorl, A. Sharon 1 ,

A. Avni2, A.K. Mattoo2 and Y. Fuchs3 Weed Science Laboratoryl, Plant Molecular Biology Laboratory2, and Horticultural Crops Quality Laboratory3. Beltsville Agricultural Research Center, Beltsville, MD. 20705 USA

ABSTRACT. A protein isolated from filtrates of the fungus Trichoderma viride induces ethylene biosynthesis when applied to tobacco (Nicotiana tabacum, cv, Xanthi) leaf tissue. The protein has a molecular weight of 22,000 dalton, a pI of 9.4, and is active as an p-1,4-endoxylanase. The protein is referred to as an ethylene biosynthesis-inducing xylanase (EIX). When applied to cut petioles, EIX is translocated through the xylem and unloaded from the xylem into the leaf mesophyll. In addition to inducing ethylene biosynthesis, it induces several other responses, e.g., ACC synthase gene activation, PR protein accumulation, ion leakage, secondary product formation and tissue necrosis. Not all tobacco varieties respond to EIX. Sensitivity is regulated by a single dominant nuclear gene carried by the cultivar Xanthi.

Introduction

During the past few years there has been an increased interest in understanding how higher plants interact with other organisms, particularly fungi and bacteria [13,15.18]. In some cases, pathogenic microorganisms induce hypersensitive responses, while a number of biotic as well as abiotic molecules (elicitors) are capable of mimicking such responses [4,11,15,16,20]. Often ethylene biosynthesis occurs as an early response in plant defense and in plants treated with elicitors.

We summarize results on the induction of ethylene biosynthesis and numerous other responses by the fungal endoxylanase (EIX). The experimental plant (Nicotiana tabacum, cv, Xanthi) material included whole plants, isolated

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leaves, leaf discs, leaf protoplasts and tissue cultures [4,5,7,14].

Characteristics of EIX

EIX was first isolated and purified from the commercial enzyme, Cellulysin [8,14]. It was subsequently shown to be a xylan inducible enzyme in Trichoderma viride and in several other fungi including plant pathogens [9]. It is a 22 kDa ~-1,4-endoxylanase with a pI of 9.4, lacking sulfur amino acids, and contains less than 1% carbohydrate [9]. The enzyme releases no xylose or xylobiose and does not produce heat stable products from purified xylans capable of inducing ethylene biosynthesis in tobacco leaf discs [10].

EIX Transport Within the Plant

Not only do leaf discs respond to EIX, excised whole leaves when fed EIX through the cut end of the petiole also produced ethylene and showed symptoms of hypersensitive response (HR) [4]. SDS-PAGE and western blot analysis of extracts prepared from these leaves demonstrated that EIX was present throughout the leaf.

Whole plants were treated with EIX through a petiole midway between the top and bottom of the plant and found to undergo a HR in leaves both above and below the treated petiole [4]. Further confirmation that EIX is actually translocated was obtained by tissue printing of stems and petioles and immunological detection of EIX in the xylem [7]. Only those leaves that had EIX translocated to it as shown by tissue prints of petioles had elevated levels of ethylene production and showed tissue necrosis [7]. In addition, autoradiograms of plants treated as above with 125 1 labeled EIX showed presence of the EIX in the vascular tissue of the stem both above and below the petiole of application [19]. However, not all leaves contained radioactivity. Apparently, EIX is translocated to only those leaves that have vasculature connections to the petiole to which EIX was applied. The roots did not accumulate detectable amounts of EIX. EIX administered through cut roots showed a uniform distribution (Sharon et al., unpublished) similar to what was reported for cryptogein [11].

Tissue Responses to EIX

The responses induced by EIX in tobacco tissues are summarized in Table 1. These include induction of ethylene biosynthesis in leaf discs, whole leaves, and leaf

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protoplasts. The increase in ethylene biosynthesis involves an increase in transcript level and ACC synthase activity. A cDNA clone for EIX-induced ACC synthase has been isolated and sequenced [2,3]. Besides ethylene biosynthesis, other processes induced by EIX in tobacco leaf tissue are synthesis of PR proteins [15], capsidiol [13], and salicylic acid [16], as well as undefined pigments [4]; and leakage of K+ and other cellular components [4,5]. Cell cultures give similar responses to EIX as leaf tissues except for induction of ethylene biosynthesis [5,6]. Cell cultures are very suitable for short time-course studies. Responses, e.g., media pH changes, electrolyte and K+ leakage, were detected within a few minutes in susceptible cells. There are relatively rapid effects on membrane lipid components, e.g., peroxidation of fatty acids and acylation of membrane sterols. To date, one major difference between leaves and cell cultures, is the effect of EIX on ethylene production. In Xanthi tissue cultures, ethylene biosynthesis is inhibited. The decrease in ethylene production in suspension cell cultures is explained, at least in part, by the leakage of ACe from the cells into the media and a lowering of the internal pools of ACC.

Ethylene Enhancement of Responses to EIX

Pretreatment of the tissues with exogenous ethylene potentiates or enhances the plants responses to EIX. Ethylene biosynthesis seems to be induced at about the same time in ethylene-pretreated and control plant tissues, but the ethylene-pretreated tissue produces up to 10 times more ethylene. Ethylene-pretreated tobacco tissue is known to produce more ACC and convert it more efficiently to ethylene and malonyl-ACC than non-pretreated tissue [1]. These data are consistent with the presence of more ACC synthase transcript in ethylene-pretreated tissue [3]. The mechanism of ethylene-enhancement of plant's responses to EIX and other elicitors, e.g., Cu++, is being investigated.

Model

How does EIX induce such a multitude of responses in sensitive tissues? We previously proposed a model [1] that we've simplified and modified to include more recent data (Figure 1). This model assumes that EIX has direct access to cell membranes whether in leaves, cell cultures or protoplasts. In whole leaves where EIX is applied to cut petioles EIX moves through-out the leaf with water movement via the xylem. Once EIX reaches sensitive cells it perhaps interacts with specific binding sites. Evidence to date suggests that EIX itself and not a product-of-its-action is

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responsible for its activity. Data supporting this concept include differential sensitivity to boiling in the presence of specific detergents where xylanase activity is lost, but biological activity is retained (Dean et al., unpublished), isolated protoplasts respond to addition of EIX (Table 1), and other purified xylanases, e.g., from Magnaporthe grisea, do not induce ethylene biosynthesis in tobacco tissues.

TABLE 1. Summary of known responses of Nicotiana tabacum L tissues to treatment with EIX.

Response Cultivar Leaf Cell Culture Protoplast

Ethylene biosynthesis Xanthi Incr [14] Decr [5] Incr [a]

ACC Synthase Activity Xanthi Incr [3] Transcript Xanthi Incr [2] ACC level Xanthi Incr [3] Decr [5]

PR Proteins Samson Incr [15]

Phytoalexins Capsidiol Samson Incr [13] Salicylate Xanthi Incr [ 16]

Cell Responses pH Xanthi Decr [4] Incr [5] Ca++ uptake Xanthi Incr [5] ACC leakage Xanthi Incr [5] K+ leakage Xanthi Incr [ 6] Incr [ 6] Electrolyte

leakage Xanthi Incr [ 4] Incr [5]

Membrane Components Lipid Perox Xanthi Incr [b] Sterol Acyl KT-17 Incr [c]

Cell Death Xanthi Incr [ 4] Incr [5] Incr [a]

References a,b, and c are unpublished data of Fuchs et al.; Norman et al.,; and Moreau et al., respectively. Incr increase; Decr = decrease.

The question of where EIX interacts with cells to induce these responses is not understood. The response is specific in that tissues respond to extremely low (nmolar) concentrations of EIX. We have shown that a single dominant gene in Xanthi is responsible for sensitivity to EIX [6]. The simple interpretation is that there are EIX binding sites

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in sensitive tissues not found in insensitive tissues. The nature of signal transduction of EIX-action is not

known. Ca++ is implicated with EIX-induced responses because it's uptake is stimulated while La+++ is a good inhibitor of the response. La+++ is considered to be a Ca++ channel blocker. The results with La+++ should not be interpreted to

----------~~~ C H ••••••• - 2 4 ACC Oxidase

Peroxldatlon ,~___ ~ ~?--~ ACC

(( INUC! ~ ,...-t-...:,.., X tranSi=crlPtlon -:t- translation

~:-- --_:' -- -SAM

Awl translation

j ---------- ~ -- -------- - ...... ,~~--- ---~" ,.. .",. ...............

;~,.. ,~,

/' ~" chltlnase MACC'~ /~" B -1,3 glucanase ! V AC !

//

Figure 1. Proposed model by which a fungal xylanase elicits ethylene biosynthesis and other responses in Xanthi tobacco.

mean that EIX acts at the Ca++ channel, but that Ca++ uptake is a critical factor. Other inhibitors (e.g.,verapamil) of Ca++ metabolism were not effective in influencing EIX-induced responses. The changes in pH, K+, electrolyte leakage and Ca++ uptake may implicate the involvement of various pumps or ATPases in EIX-action. Besides these apparent membrane effects, new transcripts and proteins are also induced.

Attempts are being made to determine the nature of the signal(s) developed by EIX in sensitive plants. It is of interest to determine if all responses are controlled by a single signal or whether the multitude of responses are regulated by a cascade-type reaction where secondary signals, e.g., salicylic acid and jasmonic acid, are produced that

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turn on different processes. Salicylic acid, which is known to accumulate in EIX treated tissues [16], is considered a secondary signal related to systemic acquired resistance and as a possible new plant hormone [17]. Because lipids are known to be affected by EIX (TABLE I), jasmonic acid, a metabolite of lipoxygenase activity on linolenic acid [12] may also be produced. In the area of gene activation, it will be interesting to compare promoter sequences of genes that are induced by EIX to see if the synthesis of new proteins are controlled at this or some other level. Our discovery of a single gene difference in determining sensitivity to EIX [6] should be of great help in establishing the legitimacy of proposed signals developed by EIX-action.

Acknowledgments

We thank Drs. C.J. Baker and M. Edelman for reviewing this manuscript.

REFERENCES.

1. Anderson JD, Bailey BA, Dean JFD, Taylor R (1990) 'A fungal endoxylanase elicits ethylene biosynthesis in tobacco (Nicotiana tabacum L. cv. Xanthi) leaves.' In HE Flores, RN Arteca, JC Shannon, eds, Polyamines and Ethylene: Biochemistry, Physiology and Interactions. A.S.P.P., pp 146-156.

2. Bailey, B.A.,Avni, A., Li,N., Mattoo, A.K., and Anderson, J.D. (1993). 'Nucleotide sequence of the Nicotiana tabacum cv Xanthi gene encoding 1-aminocyclo propane-1-carboxylate synthase. Plant Phyiol.: in press.

3. Bailey, B.A.,Avni, A., Li,N., Mattoo, A.K., and Anderson, J.D. (1992). 'Induction of l-aminocyclo propane -l-carboxylate synthase in tobacco treated with an ethylene biosynthesis-inducing xylanase.' Plant Phyiol.99:472a.

4. Bailey BA, Dean JFD, Anderson JD (1990) 'An ethylene biosynthesis-inducing endoxylanase elicits electrolyte leakage and necrosis in Nicotiana tabacum cv. Xanthi leaves.' Plant Physiol 94: 1849-1854.

5. Bailey BA, Korcak RF, Anderson JD (1992) 'Alterations in Nicotiana tabacum cv. Xanthi cell membrane integrity following treatment with an ethylene biosynthesisinducing endoxylanase.' Plant Physiol: in press.

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6. Bailey BA, Korcak RF, Anderson JD (1993) 'Sensitivity to an ethylene biosynthesis-inducing endoxylanase in Nicotiana tabacum L. cv. Xanthi is controlled by a single dominant gene.' Submitted.

7. Bailey BA, Taylor R, Dean JFD, Anderson JD (1991) 'Ethylene biosynthesis-inducing endoxylanase is translocated through the xylem of Nicotiana tabacum cv. Xanthi plants.' Plant Physiol 97: 1181-1186.

8. Dean JFD, Anderson JD (1991) 'Ethylene Biosynthesisinducing xylanase. II. Purification and physical characterization of the enzyme produced by Trichoderma viride.' Plant Physiol 95: 316-323.

9. Dean JFD, Gamble HR, Anderson JD (1989) 'The ethylene biosynthesis-inducing xylanase: its induction in Trichoderma viride and certain plant pathogens.' Phytopathology 79: 1071-1078.

10. Dean JFD, Gross KC, Anderson JD (1991) 'Ethylene Biosynthesis-inducing xylanase III. Product Characterization.' Plant Physiol 96:571-576.

11. Devergne J-C, Bonnet P, Panabieres F, Blein J-P, Ricci P (1992) 'Migration of the fungal protein cryptogein within tobacco plants.' Plant Physiol 99:843-847.

12. Farmer EE, Ryan CA (1990) 'Inter-plant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves.' Pro Nat Sci Acad (USA) 87: 7713-7716.

13. Fluhr R, Sessa G, Sharon A, Ori N, Lotan T (1991) 'pathogenesis-related proteins exhibit both pathogen-induced and developmental regulation.' In H Hennecke, DPS Verma, eds. Advanced in Molecular Genetics of Plant-Microbe Interactions. Kluwer, Dordrecht, The Netherlands, pp 387-394.

14. Fuchs Y, Saxena YA, Gamble HR, Anderson JD (1989) 'Ethylene biosynthesis-inducing protein from Cellulysin is an endoxylanase.' Plant Physiol 89: 138-143.

15. Lotan T, 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.

2M

16. Malamy, J., Hennig, J, and Klessig, D.F. (1991). 'Salicylic acid and disease defense response in tobacco' Third International Congress of Plant Molecular Biology:1089.

17. Raskin I (1992) 'Salicylate, a new plant hormone' Plant Physiol 99: 799-803.

18. Schottens-Toma IMJ, De wit PJGM (1988) 'Purification and primary structure of a necrosis-inducing peptide from the apoplastic fluids of tomato infected with Cladosporium fulvum (syn. Fulvia fulva).' Physiol Mol Plant Pathol 33: 59-67.

19. Sharon, A., Bailey, B.A., McMurtry, J.P., Taylor, R., and Anderson, J.D. (1993). 'Characteristics of EIX

(ethylene biosynthesis-inducing xylanase) movement in tobacco leaves.' Plant Physiol. In Press.

20. Toppan A, Esquerr~-Tugay~ MT (1984) 'Cell surface in plant-microbe interactions. IV. Fungal glycopeptides which elicit the synthesis of ethylene in plants.' Plant Physiol 75: 1133-1138.

STRESS ETHYLENE IN lfEVEA BRASILIENSIS: PHYSIOLOGICAL, CELLULAR AND MOLECULAR ASPECTS

J. d'AUZAC*, F. BOUTEAU1, H. CHRESTIN2, A. CLEMENT3, J.L. JACOB3, R. LACROTTE3, , 3 2 1 J.C. PREVOT , V. PUIADE-RENAUD ,J.P. RONA.

* Lab. Physiol. Veget., App!. Univ. Montpellier 2,34095 Montpellier Cedex 5, France l. Lab. Electrophysio!. des Membranes,Univ. Paris 7, 75251 Paris Cedex OS, France 2. Inst. Sciences Vegetaies, Bat. 22, Av. de la Terrasse, 91198 Gif sur Yvette, France 3. Inst. Rech. Caoutchouc IRCNCIRAD, BP. 5035, 34032 Montpellier Cedex 1, France

ABSTRACT. Under the effect of ethylene, there is a close parallel between increased latex production and the alkalinisation of laticiferous cytosol, which is parallel with activation of tonoplast ATPase and vacuole (lutoid) acidification. The alkalinisation of the cytosol by several tenths of a pH unit considerably enhances the activity of certain enzymes, including invertase, the key enzyme in glucidic catabolism. Ethylene increases the ATP content, the pool of adenine nucleotides and the myokinase balance shifts towards A TP synthesis. Increase in the proton motive force, related to that of the cytosol/vacuole pH gradient, enhances membrane transport including an H+ /sucrose symport on laticiferous cell plasmalemma. The sink effect of the latificers for sucrose increases in proportion to over-production of latex. The polyribosome level in latex is increased and several enzymes are activated (post-translation or de novo synthesis?) Hevein transcripts analogous with certain agglutinins and heveamines with chitinase/lysozyme activity are more abundant after mechanical stress, addition of ABA or ethylene. Ethylene causes changes in calmodulin distribution between cytosol and organelles and an increase in protein phosphorylation. Repeated treatment with large amounts of Ethrel results in imbalance in latex between toxic oxygen production (02'- H202) and protective systems (superoxide dismutase, catalase, thiols, etc.); this is followed by in situ ~oagulation of latex and degeneration of the laticiferous system. The laticiferous system in Hevea is totally dependent on ethylene stress and forms an ad hoc model for understanding the mechanisms of the action of ethylene.

Introduction

The wounding of l/evea brasiliensis bark by tapping causes endogenous production of ethylene which activates the isoprenic metabolism of the laticiferous cells. A virgin rubber tree produces only a few drops of latex. The repeated traumatism caused by tapping several times a week little by little induces the flow of a few hundred ml of latex at each tapping. The tree responds to tapping. In fact, traumatic ethylene stimulates the enzymatic machinery of the laticiferous vessels which formerly functioned at a slow rate.

The first effective, reproducible treatments for stimulating latex production in adult rubber tree plantations consisted of injections of trace elements (CuS04 and H3B03) and auxinomimetic coating (2,4-D, ANA, MCPA and 2,4,5-T). Gas treatments (ethylene oxide, acetylene and ethylene) were subsequently tested (d' Auzac, 1989). Today, Ethrel is used industrially in rubber tree plantation to increase the quantity of latex produced at each tapping.

It is now known that the mechanical, chemical and phytopathological traumatisms cause ethylene synthesis in most living tissue and in l/evea brasiliensis (paranjothy et ai., 1979). Tapping wounds the tree by cutting the bark and also results in a traumatism. Endogenous and exogenous ethylene is the common feature of all treatments known to date to increase latex production by Hevea.

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1. C. Pech et ai. (eds.), Cellular and Molecular Aspects of the Plant Honnone Ethylene, 205-210. © 1993 Kluwer Academic Publishers.

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Results

1 - ETHYLENE AND THE STIMULATION OF LATEX PRODUCTION

Stimulating treatment increases production, in particular by prolonging latex flow after tapping. Colloid stability of latex appears to increase. Lutoids (lysosomal micro-vacuoles), which form 10 to 15% of the volume of latex, are involved in the coagulation of the colloid suspension of rubber particles, either by releasing positive charges (H+, divalent cations, proteins) (d' Auzac and Jacob, 1989) into the cytoplasm or by inducing clumping phenomena involving hevein (Kush et al., 1991), appear to be stabilised.

2 - IS THE PH THE SECOND MESSENGER IN THE ACTION OF ETHYLENE?

2-1 Parallel between alkalinisation of the cytosol and increased latex production. In addition to increasing latex production, ethylene causes alkalinisation of the cytosol pH and acidification of the vacuole contents and an increase in the pH gradient between the two compartments. Latex pH is kept at between 6.5 and 7.3 (its physiological limits) by a biochemical pH-stat centred on PEPcase and a chemostatic pH-stat using the various proton pumps of the laticiferous cells (Chrestin et al., 1989): a type V ATPase inhibited by nitrate, a Mg2_K+ -dependent pyrophosphatase (Siswanto et al., 1992), an NADH-cyt-c reductase functioning as an outflow pump for intra-vacuolar H+ (d'Auzac et al., 1989) and an ATPase plasmalemma proton pump (Bouteau et al., 1991).

2-2 Activation of the tonoplast ATPase. The kinetics of the effect of Ethrel reveal considerable increase in tonoplast ATPase activity 12 to 14 h after treatment (Gidrol et al., 1988). However, the ATP level falls for the first 24 hours and it is necessary to wait until it increases markedly for vacuole hyperacidification. Increased specific ATPase activity in purified lutoid tonoplast was noted. The Km of the enzyme for ATPMg was unchanged; only V max increased, possibly because of the enhancing of ATPase synthesis induced by ethylene.

2-3 - Availability of ATP and the adenyl pool. The adenyl pool decreased significantly six hours after Ethrel treatment (Amalou et al., 1992). It subsequently tripled in three days and remained at the resulting level for at least a week while the energy charge remained constant throughout the treatment. This phenomenon may be explained by the immobilisation of nucleotide adenines in RNA in correlation with enhancement of the incorporation of marked amino acids by membrane and cytosol proteins. This suggests both the de novo synthesis of nucleotide adenines and the rebalancing of the energy charge through the functioning of adenylate kinase, whose apparent reaction equilibrium constant (Kapp. = [A TP] x [AMP]/[ADP]2) of about 3 before treatment falls sharply and reaches 0.5 six days afterwards.The ATP/ADP ratio logically has a parallel trend and is more or less halved. The great sensitivity of pH to adenylate kinase (Noda, 1973) may account for the modification of its activity by ethylene treatment.

2-4 - Alkalinisation of the cytosol by Ethrel and activation of the cytoplasm metabolism. Latex sucrose and its metabolism may be limiting factors in production from rubber trees (reviewed by Tupy, 1989). Some latex enzymes are very finely regulated by variations of several tenths of a pH unit within the physiological pH range. This is the case of the invertase initiating glycolysis, phospho-fructokinase, glyceraldehyde P-dehydrogenase, pyruvate decarboxylase and PEP-case (Chrestin et al., 1989).

Positive correlations between the pH of fresh latex and its invertase activity have also been shown between production and invertase activity and between production and sucrose uptake (Tupy, 1989). In a general manner, exponential negative correlations have been demonstrated between the pH of laticifer cytosol and rubber tree production of latex (Chrestin et al., 1989). The link between alkalinisation of cytosol pH by ethylene and over-production is at least partially accounted for by the activation of invertase without an apparent increase in the synthesis of the latter enzyme. Increase in pH, sucrose content, r-RNA and production were particularly marked after Ethrel treatment whereas there was a fall in latex rubber content, and a fall and then a recovery of the thiols content (Lacrotte, 1991) ~ any disturbance connected with latex flow and the metabolic activation that it causes.

207

2-5 - Increase in the cytosol-vacuole pH gradient and activation of transport phenomena. The effect of ethylene applied at a normal dosage consists mainly of strong intravacuolar acidification and correlated alkalinisation of cytosol (Chrestin et al., 1989). This increases the Proton Motive Force driving secondary transmembrane transport and activates the laticiferous metabolism. Application of ethylene thus increases the sink effect and enhances sucrose supply to the laticifers. Over-production of latex in comparison with a non-treated control was all the more marked when the latex sugar content increased in comparison with the control. When sucrose U_14C was applied to slightly scraped rubber tree bark, the radioactivity of the sucrose or its derivatives was found in the latex less than 3 hours after application. This is not the case with a non-permeable molecule such as mannitol U-14C. This means that sucrose U-14C crosses the laticifer plasmalemma and does not use the apoplast pathway. Absorption kinetics according to the sucrose concentration displayed Michaelis characteristics implying the existence of a transporter. Various inhibitors (NaF, vanadate, DES, DNP) oppose the uptake of sucrose U_14C showing the existence of an active component in plasmalemma sugar transfer, (Lacrotte, 1991). Ethylene increased the sucrose U_14C velocity; its effect is rapid (less than 12 hours). It occurs in situ before tapping.

Membrane potentials of some -113 ± 21 mV were determined by inserting micro-electrodes in very young rubber trees (0 10 mm) (Bouteau et al., 1991). In continuous measurement, the application of sucrose or glucose induced transitory depolarisation of the plasmalemma of +15 to +25 mV and slight alkalinisation (0.1-0.2 pH) of the external surface of the laticifer (waIVplasmalemma interface). Vanadate and DNP caused depolarisation of 10 to 35 mY. Phloridzin, which inhibits H+-sugar transport, is effective in inhibiting glucid transport and hence depolarisation and the accompanying pH slide (Bouteau et al., 1991).

Ethrel treatment of young plants caused hyperpolarisation of -42 ± 12 mV and depolarisation by vanadate of + 50 mY; this was significantly greater than that of the control. Directly or indirectly, ethylene causes vanadate-dependent hyperpolarisation. Plasmalemma A TP-ase is probably involved in the mechanism stimulated by ethylene (Bouteau et al., 1991).

3 - EfHYLENE, CALMODULIN AND PHOSPHORYLATION OF PRafElNS

Calmodulin is present in large concentrations in Hevea brasiliensis latex (more than 5000 units/mllatex). Latex calmodulin has been shown to activate membrane-bound HMG-CoA reductase and NAD-kinase in latex (Wititsuwannakul et al., 1990) (two probable key enzymes in the control of isoprenic synthesis in Hevea latex cells). Calmodulin is essentially cytosolic (+ 87% of total calmodulin) in the latex from normally tapped control trees. Very small but detectable amounts of calmodulin are associated with latex organelles (mainly lutoids). Most of the 32P-labelled phosphorylated proteins (63%) are similarly located in the latex cytosol. After less than 36 hours, stimulation with Ethrel reduced lutoid density; this was accompanied by different distribution of calmodulin and phosphoproteins in cytosol and organelles but without significant change in the calmodulin and phosphoprotein levels in whole latex. Ethrel induced significant calmodulin and phosphoprotein enrichment in organelles at the expense of the cytosol. Over 85% of lutoid calmodulin and phosphoproteins were located in or firmly bound to the membrane fraction. These results indicate that calcium, calmodulin and protein phosphorylation may be involved in signal transduction in Hevea bark tissues in response to ethylene treatment.

4 - EfHYLENE: EFFECT ON THE GENOME

In Hevea brasiliensis, ethylene increases the polymerisation index of latex ribosomes and the incorporation of marked amino acids in latex proteins (Coupe and Chrestin, 1989). In addition, ethylene increases specific tonoplastic ATPase and glutamine synthetase activity (pujade-Renaud, 1992 a and b) but reduces sucrose-synthetase (Tupy and Primot, 1982) and PP-PFK activity (Prevot et al., 1992). Laticifer m-RNAs are 20 to 100 times richer in transcripts encoding rubber biosynthesis enzymes (HMGCoA S, HMG-CoA R) than leaves (Kush et al., 1990). The genes involved in plant defence are also expressed 10 to 50 times more in latex (chitinase, PR-proteins, PAL, chalcone isomerase, cinnamylalcohol dehydrogenase, 5-enoylpyruvylshikimate-3-P-synthetase). According to Kush et al. (1990), the presence of genes encoding plant defence show the response of the tree to the wounding caused by tapping and/or ethylene.

208

Hevein, a 5 KD lutoid protein is structurally quite similar to wheat, barley and Urtica dioica lectins (Broekaert et at, 1990). Tapping wounds, ethephon or ABA cause an increase in hevein transcripts in leaves, stems and latex (Broekaert et at, 1990). Hevein fixes chitin and possesses antifungal properties (Van Parijs et at., 1991). It is also involved in latex coagulation by clumping by binding by Nacetylglucosamine functions to a 23 KD protein fixed to the surface of the rubber particles (Kush et at., 1991).

I-Ieveamine A is a 29 KD basic vacuolar protein which possesses chitinase/lysozyme activities and strong structural similarities to other plant chitinases/lyzozymes (Jekel et at., 1991).

The fact that hevein and heveamines are rare in young, healthy dicotyledons and increase rapidly after ethylene treatment or stress places them in the PR-protein category and suggests that latex plays a defensive role in the plant (Martin, 1991). The high latex content of these proteins may be caused by endogenous (tapping) or exogenous (application of Ethrel) ethylene stress.

5 - ETIlYLENE AND DEGENERATION OF THE LATICIFEROUS SYSTEM

Excessive application of Ethrel in rubber-growing may cause degeneration of the latificerous tissue and the "dry bark" syndrome. Latex is an unstable medium because of its richness in lutoids - lysosomal microvacuole "suicide bags". When their membranes break down, lutoids release into the cytoplasm a number of substances which can cause in situ latex coagulation (d'Auzac and Jacob, 1989) and also lysosomal acid hydrolases which break down certain molecules which are essential to the metabolism. An NAD(P)H-quinone reductase (EC: 1.6.99.5) on lutoid membrane generates superoxide ions (02·-) which are transformed into H202 and OH· (d' Auzac et at., 1987). It causes the peroxidative degradation of unsaturated fatty acids in this tonoplast and finally the bursting of the lutoids. Latex also contains considerable protective enzyme activities (superoxide dismutase (SOD) and catalase) and antioxidants (ascorbic acid and glutathion). The in situ peroxidative degradation of membrane structures is the result of imbalance between toxic peroxidative activities and all the protective activities and molecules in the laticifers (Chrestin, 1989).

The sampling of a Hevea population according to high, medium or low production or the "dry bark" phenomenon has made possible the characterisation (Chrestin et at., 1984) of high producer trees by low cytoplasm NADH oxidase and peroxidase activities, large quantities of antioxygens, strong SOD and catalase activities. Low producer trees and, especially, those moving towards bark dryness display inverse characteristics. Over-use of ethylene to stimulate yield can effectively upset the balance between toxic oxygen and protectors. Under these particularly severe conditions, it were as if ethylene enhanced the synthesis of an enzyme generating toxic oxygen (NADH-02 reductase) and repressed protective enzymes (SOD and catalase) (Chrestin, 1989).

Discussion - Conclusion

In Hevea brasiliensis, traumatic ethylene probably activates in particular the laticiferous metabolism and thus enables continuous production of latex in response to regular tapping. When latex production reaches its normal level, excessive endogenous production of ethylene caused by a chemical (CuS04) or hormonal (2,4-D, ANA, etc.) traumatism or Ethrel results in marked transitory over-production of latex or even a senescence phenomenon in the laticiferous system if too much ethylene is applied.

It is very difficult to distinguish between the primary and secondary effects of ethylene. One's attention is drawn by the effects (direct or indirect) of ethylene on the pH of the two major compartments of latex: cytosol and vacuoles (lutoids). This leads to recalling Felle's hypothesis (1989) according to which the pH may be one of the second hormonal messengers. Irwin et at. (1992) recently showed that changes in the pH of stomatic cells under hormonal influence preceded stomatic movements. Changes in the cytoplasmic pH may be a signal for activation of the metabolism (Chrestin et at., 1989). This is supported by the extreme dependence of the pH on several key enzymes in glucidic catabolism, including an invertase. However, the substantial, contrasting variations in cytosol and tonoplast pH values can hardly be detected until 24 h after treatment. The polysome level increases by 75% and specific activity of tonoplast ATPase by 250% 12 hours after Ethrel application. Total nucleotides increase 2.5-fold 48 h after treatment. The 24-hour period required to detect the major variations in compartmental pH values is

209

ascribed to the need for a combination of greater tonoplast ATPase activity and greater availability of ATP, which requires this time. The variations caused by ethylene in the distribution of calmodulin and phosphorylated proteins between the cytosol and the membrane fractions of latex lead to considering that the transduction of the ethylene signal requires several secondary messengers, as is the case in most extracellular signals. The increase in the specific activity of certain enzymes such as tonoplast ATPase and glutamine synthase and the decrease in others such as PP-PFK and sucrose synthase indicate direct or indirect action of ethylene at genome level. Over-expression of SOD and glutamine synthase and underexpression of sucrose synthase have recently been shown at m-RNA level (pujade-Renaud et al., 1992). High levels of PR-proteins typical of chitinase!lysozyme activity, such as heveamines or chitin-binding protein and hevein probably result from the traumatic ethylene produced after tapping. Excessive ethylene treatment leads to degenerescence/senescence of the laticiferous system in which a modification of expression of the genome also appears to be involved; enzymes producing ions or toxic molecules are enhanced (NADH-quinone reductase and cytosol peroxidase) and protective enzymes such as SOD and catalase reduced. Identical action of ethylene has been observed in the browning/necrosis of Hevea tissue callus in vitro where the addition of ACC enhances polyphenoloxidase, peroxidase and NADH-quinone reductase activities and inhibits SOD, catalase and ascorbate peroxidase activities. Addition of aminooxyacetic acid (AOA) causes the opposite effect to that of ACC (Housti et al., 1992).

Latex, a laticiferous cytoplasm easily obtained in unlimited quantities, is a homogeneous plant material. It is an excellent medium for the use of molecular biology, whose methods can be used to investigate both the isoprenic metabolism and, in particular, hormonal phenomena involving ethylene.

PL\S~I.""LE\L\l-\

JtSLt:~SE -~ -SU~R~E-CSUCtl<os~,\VERTASE (+) tl4 S) \,I!\SI

II.. ~ ( ) GLU + FRU . t !" .... IP F6P):j'1'1

t I Pi'l'fK I ,IIC"OSOL(+) I ,(.) J 16m? J'l

PEI'C<lSC , OAA~I'EI' t (+) t

GLL' + ."1!4 (+)

t ~~~~~~.~~~E Glu" ~lALATE ---... PYRliV.HE

~=====-----'I ,KEr,t'COA . C.-\L\jODL:U:-.r FL\.xno:-.; t-o PROTEI:-': PHO(:{~RYLAT!O~ I!P

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Kapp .\!YOKISASE ( • ) BIOSYNTHESIS

(+)

ETHYLENE EFFECTS ON LATEX METABOLISM POSITIYE( +); NEGATIVE(·)

References

WOUNDING

CllfnNASE I IIEVEIN

IIRGP (+)

SOD

or-SENESCENCE

IN VIVO, IN VHRO

NADII OXYDAS£ (+) PEROXIDASE ( + ) CATALASE (l SOD (:

TIllOLS CONTENT ( - )

Amalou, Z., Bangratz, J. and Chrestin, H. (1992). Ethrel (ethylene releaser) induced correlative increases in the adenyl ate pool and transtonoplastic LlpH within the Hevea latex cells. PlanJ Physiol., 98, in press.

d'Auzac J. (1989). The Hormonal stimulation of latex yield; Historical account. In "Physiology of Rubber Tree Latex", d'Auzac, J., Jacob J.L. and Chrestin H. (eds) CRC Press, pp. 289-293.

d'Auzac, J., Chrestin, H. and Marin, B. (1987). Biochemical and Enzymatic Components of vacuolar membrane: Tonoplast of Lutoids from Hevea Latex. In "Methods in Enzynwlogy" Packer, L. and Douce R. Acad. Press NY, 148, 87-104.

d'Auzac, J. and Jacob, J.L. (1989). The composition of latex from Hevea brasiliensis as a laticiferous cytoplasm. In "Physiology of Rubber Tree Latex", d'Auzac J., Jacob J.L. and Chrestin H. (eds) CRC Press, pp. 60-96.

210

Broekaert, W., Lee, H.I., Kush, A., Chua, N.H. and Raikkhel, N. (1990). Wound-induced accumulation of m-RNA containing a hevein sequence in laticifers of rubber-tree (Hevea brasiliensis ) Proc. Natl. Acad. Sci. USA, 87, 7633-7637.

Bouteau, F., Lacrotte, R., Cornel, D., Monestiez, M., Bousquet, U., Pennarun, A.M. and Rona, I.P. (1991). Electrogenic active proton pump in Hevea brasiliensis laticiferous cell. Its role in activating sucrose/H+ and glucose/H+ symports at the plasma membrane. Bioelectrochemistry and Bioenergetic, 26, 223-236.

Coupe, M. and Chrestin, H. (1989). Physiological and biochemical mechanisms of hormonal (ethylene) stimulation. In "Physiology of Rubber-Tree latex", d'Auzac, I., Jacob, J.L. and Chrestin, H. (eds) CRC Press, pp. 295-319.

Chrestin, H. (1984). Biochemical aspects of Bark dryness induced by over-stimulation of Rubber-Tree with Ethrel. In "Physiology of Rubber-Tree latex" , d'Auzac, 1., Jacob, I.L. and Chrestin, H. (eds) CRC Press, pp. 431-441.

Chrestin, H., Gidrol, X., d'Auzac J., Jacob, J.L. and Marin, B. (1985). Cooperation of a "Davies type" biochemical pH-stat and the tonoplastic bio-osmotic pH-stat in the regulation of the cytosolic pH of Hevea latex. In "Biochemistry and Function of Vacuolar ATPase in Fungi and Plants ". Marin, B. (ed), SpringerVerlag, Berlin 245-249.

Chrest in, H., Marin, B., Jacob, J.L. and d'Auzac, J. (1989). Metabolic regulation and homeostatis in the laticiferous cell, In "Physiology of Rubber Tree Latex", d'Auzac J., Iacob, I.L. and Chrestin, H. (eds) CRC Press. pp. 165-178.

Felle, H. (1989). pH as a second messenger in Plants. In "Second messengers in plant growth and development". W.F. Bross and D.I. Morre (eds). Plant Biology, 6, 145-166.

Gidrol, X., Chrestin, H., Mounoury, G. and d'Auzac, J. (1988). Early activation by ethylene of the tonoplast H+ pumping ATPase in the latex from Hevea brasiliensis. Plant Physiol., 86, 899-903.

Housti, F., Coupe, M. and d'Auzac, J. (1992). The effect of ethylene on several enzymatic activities involved in the browning of Hevea brasiliensis callus: consequences for the embryogenenic potential. Physiologia Plantarum (accepted).

Irving, H.R., Gehring C.A. and Parish, R.W. (1992). Changes in cytosolic pH and calcium of guard cells precede stomatal movements. Proc. Natl. Acad. Sci. USA, 89, 1790-1794.

Jacob, J.L., Prevot, J.C. and Kekwick R.G.O. (1989). General metabolism of Hevea brasiliensis latex. In "Physiology of Rubber Tree Latex", d'Auzac, J., Jacob, J.L. and Chrestin, H. (eds) CRC Press, pp. 101-144.

Kush, A., Goyvaerts, E., Chey, M.L. and Chua N.M., 1990. Laticifer-specific gene expression in Hevea brasiliensis (rubber-tree). Proc. Natl Acad. Sci. USA, 87, 1787-1790.

Kush, A., Tan, H.L., Gidrol, X., Than, C.T. and Chua Nam Hai (1991). Hevein, a lectine like glycoprotein from Hevea brasiliensis (Rubber-Tree) is involved in the coagulation of latex. ISPMB, 1991, TUCSON USA.

Lacrotte, R. (1991). Etude des relations entre la teneur en sucres du latex et la production. Approche des mecanismes du chargement en saccharose des laticiferes d'Hevea brasiliensis. These de Doctorat. Universite Montpellier 2 - France.

Martin, M.N. (1991). The latex of Hevea brasiliensis contains high levels of both chitinases Ilysozymes. Plant Physiol., 95, 469-476.

Noda, L. (1973). Adenylate kinases. Enzymes, 8, 279-305. Paranjothy, K., Sivakumaran, S. and Yong, W.M. (1979). Ethylene formation in excised Hevea discs. J. Rubber

Res. Inst. Malaysia, 23, 159-167. Prevot, J.C., Clement, A., Siswanto, Pujade-Renaud V., Jacob, J.L. (1992). Ethylene stress and enzymatic

activities in Hevea latex: the diversity of responses. Ethylene Symposium. Agen 1992. Pujade-Renaud, V., Perrot-Rechenmann, C., d'Auzac, J., Jacob J.L. and Guern, J. (1992) a. Modulation of genes

expression under ethylene treatment in the latex cells of Hevea brasiliensis. Ethylene Symposium, Agen 1992.

Pujade-Renaud, V., Clement, A., Rechenmann, C., Prevot, I.C., Chrestin, H., Jacob, J.L. (1992) b. Ethyleneinduced increase in Glutamine synthetase activity and its m-RNA level in Hevea brasiliensis latex cells. (to be published).

Siswanto, Prevot, J.C., Clement, A. and Jacob, J.L., 1992. Tonoplast pyrophosphatase in Hevea brasiliensis latex: characteristic and properties. International Natural Rubber Conference, Bangalore, India, in press.

Tupy, I. (1989). Sucrose supply and utilization for latex production. In "Physiology of Rubber Tree Latex", d'Auzac J. and Chrestin H. (eds) CRC Press, pp 179-218.

Tupy, J. and Primot, L. (1982). Sucrose synthetase in the latex of Hevea brasiliensis. J. Exp. Bot., 33, 988-995.

Van Parijs, J., Broekaert, W., Golsdtein, 1.1. and Peumans, J. (1991). Hevein : an antifungal protein from Rubber-Tree (Hevea brasiliensis) latex. Planta, 183, 258-264.

Wititsuwannakul, R., Wititsuwannakul, D. and Dumkong, S. (1990). Hevea calmodulin regulation of the activity of latex 3-Hydroxy-3-methyl Gluraryl Coenzyme A reductase. Phytochemistry, 29, 1755-1758.

WOUND ETHYLENE SYNTHESIS IN THE STRESS-AFFECTED CELLS.

A.KACPERSKA, M.KUBACKA-ZEBALSKA University of Warsaw Plant Stress Physiol. Lab. Krakowskie Przedm. 26/28 PI-OO-927/1 Warsaw Poland

ABSTRACT. In the low temperature- or desiccation- affected tissues, the promotion of ethylene synthesis depends not only on the enhanced synthesis of I-amino cyclopropane-I-carboxylic acid (ACC) but also on a degree of membrane injury. The reversible membrane alterations (potassium or electrolyte leakage not higher than 40%) were associated with a linear increase in ethylene production, whereas irreversible injury (leakage higher than 40%) - with a rapid decrease in ethylene evolution. Simi-lar pattern of the stress-induced changes was observed for lipoxygenase activity. Inhibition of the enzyme activity led to the inhibition of wound ethylene evolution. It is proposed that the stress-promoted synthesis of ethylene in cells, showing reversible membrane alterations, depends both on a high availability of ACC and on a promotion of lipoxygenase-mediated increased production of lipoperoxides. Lipoperoxides or their derivatives, formed in radical-chain reactions, facilitate the chemical conversion of ACC to ethylene.

1. INTRODUCTION

It is now accepted that the stress-induced enhacement of ethylene synthesis is due to the stress-promoted synthesis of 1-aminocyclopropane-l-carboxylic acid (ACC), which is converted to ethylene by ACC oxidase (ethyle-ne forming enzyme, EFE) [22J. Promotion of the ACC oxidase activity by the stress factors was not taken into consideration because it has been stated that the enzyme activity depends on membrane integrity and it is inhibited in tissues subjected to membrane-destabilizing factors, such as dinitrophenol, and low or high temperature [17J.

On the other hand, the enhanced synthesis uf ethylene has been reported for tissues, showing a certain degree of the stress-induced damage [3, 6, 7, 8J. The intent of the present paper is to reconsider previously published results in order: 1) to see to what extend the wound ethylene formation depends on membrane integrity, 2) to find out whether the stress-induced ACC accumulation is the only factor responsible for synthesis of wound ethylene, 3) to discuss the possibility that synthesis of ethylene in the stress-affected tissue may depend on the wound-induced activation of the ACC oxidizing system, possibly other than EFE.

211


212

2. ETHYLENE FORMATION IN TISSUES SHOWING DIFFERENT DEGREE OF MEMBRANE DAMAGE.

Determination of electrolyte efflux from the stressed tissues is a commonly used method for an estimation of membrane injury brought about by different stresses. The stress-induced electrolyte leakage is highly correlated with potassium efflux [11, 12J which points to proton-driving ATPases, being the primary target of a stress action [1].

It was found, that formation of wound ethylene depends on the degree of membrane alterations (Fig. 1). Up to 40% membrane injury, a stimulation of ethylene formation was observed, whereas at higher levels of injury an inhibition of ethylene evolution was noted. The membrane injury not higher than 40% were found to be fully reversible.

Fig. 1. Stimulation of ethylene formation in the prefrozen winter rape (~). or rhododendron (~) leaves. Open or full circles denote nonacclimated and cold-acclimated tissues, respectively. I = index of injury in %, determined with a conductivity method [6J. Data for rhododendron leaves after Harber and Fuchigami [5J, modified.

A si;l;ilar pattern of changes in membrane properties and in ethylene formation was observed in water-stresses plum laves [8J. In sugar beet leaves, subjected to point freezing, an increased ethylene formation was associated with a damage of a leaf surface up to 50% [3J.

3. ACC ACCUMULATION AND ETHYLENE FORMATION.

In the stress-affected cells. overproduction of ACC is observed [17J. This is due to the increased synthesis of ACC.

Increased accumulation of ACC and its conjugated form, MACC, was also observed in desiccated soybean leaves [17J. In these leaves, the stress-induced enhacement of ethylene evolution went along with the increased membrane injury. Feeding of the non-stressed leaves with exogenous ACC resulted in the promotion of ethylene formation, whereas inhibition of ACC synthesis with aminooxy acetate (AOA) prevented the desiccation-induced burst of ethylene evolution [7J. Therefore, it was concluded that stress-promoted synthesis of ACC is indispensable factor in synthesis of ethylene in the stress-injured cells.

On the other hand, there are data indicating that accumulation of ACC in the stress-affected cells not always lead to the enhanced synthesis of wound ethylene (Table 1).

Table 1 Comparison of ethylene, ACC and MACC formation in the cold-grown winter wheat leaves of freezing resistant (R = Mironovska 808) and freezing suscegttible (S = Slavia) cultivars, after transfer to 25 C. (After Machackova et al. [10J, the approximative values, calculated from the figures)

Temperature Ethylene ACC MACC treatment (nl g FW-l) (nmol. g FW-l) (days at 4OC)

R S R S R S

0 11.7 11.5 0.30 0.20 1.6 2.2 5 1.0 1.0 - 0.40 0.45 2.2 4.4

14 0.4 0.3 0.50 0.48 2.7 5.0

213

Winter wheat is classified as a chilling-resistant plant. Contrary to chilling sensitive plants, showing a burst in ethylene evolution upon transfer to warmer temperatures [16J, winter wheat does not suffer from membrane injury when exposed to temperature close to OOC.

It seems, therefore, that the crucial point in the control of ethylene formation from ACC, accumulated in the stressed tissues, is a degree of membrane destabilization by a stress factor: if there is no modification in membrane permeability no stimulation of ethylene production is observed, although ACC synthesis might be promoted.

4. POSSIBLE NATURE OF ACC OXIDIZING SYSTEM IN THE WOUNDED CELLS

The question arises whether in cells, showing reversible membrane injury, conversion of ACC to ethylene depends on activation of ACC oxidase (ethylene forming system, EFE) or on induction of other system.

Previous works showed that EFE is highly structured and requires membrane integrity[17J: the enzyme was found to be easily inactivated by membrane-destabilizing factors. It was assumed that the enzyme is localized in the tonoplast and plasma membrane [17J. In fact, it was found in vacuoles isolated from pea protoplasts [17J. All these facts militate againts the possibility that the stress-induced destabilization of membranes may activate ethylene forming enzyme.

Most recently (see papers of Smith and John, Dupile et al., Rombaldi et. al., in this volume) it was shown that ACC oxidase is a soluble enzyme. The enzyme from ripening apple and tomato fruits is mainly located in the apoplasm and in cell wall space. In a view of these discoveries the dependence of ACC oxidase activity on membrane integrity calls for an explanation. It can not be excluded that the stress-induced changes in membrane permeability may activate apoplastic (or cytosolic)

214

enzyme through increased substrate and/or cofactors avai~abil~ty. The other explanation of the increased ACC conversion to ethylene

in cells, showing a certain degree of membrane injury, is based on data describing the stress-induced modifications in lipoxygenase activity. Lipoxygenase (EC 1.13.11.12) catalyzes the oxygenation of polyunsaturated fatty acidscontainig a cis,cis-1,4- pentadiene system to give unsaturated fatty acid hydroperoxides. The reaction involves the formation of organic radicals [14J and may initiate free radical-chain reactions in biological systems. Its possible involvement in ACC conversion to ethylene in the frost affected and in senescing tissues was postulated previously [2, 6J. From Fig. 2 it may be seen that the pattern of changes in lipoxygenase activity in the frost- and desiccation-affected winter rape leaves was very similar to that observed for ethylene formation

260

220

8180 !! 8,140

1100

60 0~~20~-~~0~~OO~~~

1(%1

Fig. 2. Lipoxygenase activity as affected by the degree of membrane injury (I %) in the prefrozen (circles) or dehydrated (triangles) winter rape leaves. Open and full symbols denote non-acclimated and acclimated leaves [6J.

Similar relationship between ethylene formation and lipoxygenase activity was also noted for soybean leaves exposed to desiccation stress [10J.

Plotting ethylene formation against lipoxygenase activity indicates a positive, highly siginificant, linear correlation between these activities in stress-~t~~£t.ed cells (Fig. 3).

300 y032,68+0,921

~200 -l: 8

lipolyuanase (%)

Fig. 3. Relationshi~between lipoxygenase activity and ethylene formation in plant tissue showing a different degree of the stress-induced membrane damage. Correlation coefficient = 0.78, statistically significant at 0.001 confidence level: (td= 6.49 > ttab = 2.65)

Pretreatment of bean leaves with lipoxygenase inhibitor, phenylhydrazine [4J, prevented leaf cells from the drought-induced wound ethylene formation (Fig. 4).

5 c drought • phenylhydrazine + drought

OL-----~1~5----~~~---,4~5--~

I (;')

Fig. 4. The effects of phenylhydrazine (5mM) on ethylene formation in the drought-affected bean leaves, showing different degree (I, %) of membrane injury.

Observations presented above make us to suggest that in plant

215

cells suffering from chilling, frost, desiccation or other stresses and showing reversible membrane alterations [15], ethylene synthesis depends both on a high ACC availability and on promotion of lipoxygenasemediated increased production of lipoperoxides, which facilitate the chemical (non-enzymatic) conversion ACC to ethylene (Fig. 4).

Stress factor

(chillin~h freezing, desiccation, etc.)

J Enhanced .ynth •• i 8

of ACC I""a,o

Increaaed activity

of ACC 'Itha,o

Accu~,at~

Enhanced non-anz

I 1

l4embran. responses (r .... eraibl. destabilization)

1 Inactivation of EFE

Activation of lipoxYgenase

'''a"o. , J. po,o.i.atio"

tic converaion

of ACC tlothY'O"O

Formation of wound ethylene

Fig. 5. The proposed chains of events leading to formation of wound ethylene

The proposition is in accordance with Lieberman-s [9J observation that lipid peroxidation goes along with ethylene formation, as well as with Shimokawa-s [13] suggestion about two prerequisites for ethylene biosynthesis in vivo: ACC biosynthesis and free radical generation. The proposed link between ACC synthesis, the free radical-generating system and ethylene formation explains many of the features of the reaction, namely its dependence upon oxygen and 02", its sensitivity to radical scavengers and low affinity for ACC. Neither, the substrate stereodiscrimation shown for ethylene forming enzyme (EFE) activity can be expected. The hypothesis presented would also explain many of the similarities in ethylene formation between wounded and senescing tissues. It may also imply that the systems, involved in the control of the last step of synthesis of "basal" and "wound" ethylene, may be different.

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5. REFERENCES

1. Arora, R. and Palta, J.P. (1991) A loss in the plasma membrane ATPase activity and its recovery coincides with incipient freeze-thaw injury and postthaw recovery in onion bulb scale tissue. Plant Physiol. 95, 846-852.

2. Bousquet, J. F. and Thimann K. (1984) Lipid peroxidation forms ethylene from 1-aminocyclopropane-1-carboxylic acid and may operate in leaf senescence. Proc. Natl. Acad. Sci. USA 81, 1724-1727.

3. Elstner, E.F. and Konze. J. R. (1976) Effect of point freezing on ethylene and ethane production by sugar beet leaf discs. Nature 263, 351-352.

4. Gibian, M. J. and Singh, K. (1986) Irreversible inhibition of lipopoxygenase by phenyldiazene, autoxidizing phenylhydrazine and related materials. Biochim. Biophys. Acta 878, 79-92.

5. Harber, R. M. and Fuchigami, L.H. (1986) The relationship of ethylene and ethane production to tissue damage in frozen rhododendron leaf discs. J. Amer. Soc. Hort. Sci. Ill, 434-436.

6. Kacperska, A., and Kubacka-Z~balska, M. (1985). Is lipoxygenase involved in the formation of ethylene from ACC? Physiol Plant. 64, 333-338.

7. Kacperska, A., and Kubacka-Z~balska, M. (1987) Formation of stress ethylene depends both on ACC synthesis and on the activity of free radical-generating system. Physiol. Plant. 77, 231-237.

8. Kobayashi, K., Fuchigami, L. H. and Brainerd, K. E. (1981) Ethylene and ethane production and electrolyte leakage of water stressed "Pixy" plum leaves. HortScL, 16, 57-59.

9. Liberman, M. (1979). Biosynthesis and action of ethylene. Ann. Rev. Plant Physiol. 30, 533-591.

10. Machackova, I., Hanisova, A. and Krekule, J. (1989) Levels of ethylene, ACC, MACC, ABA and proline as indicators of cold hardening and frost resistance in winter wheat. Physiol. Plant. 76,

11. Palta, J. P. and Li, P. H. (1980) Alteration in membrane transport properties by freezing injury in herbaceous plants: evidence against rupture theory. Physiol. Plant. 50, 169-173.

12. Shcherbakova, A. and Kacperska, A. (1983) Water stress In]uries and tolerance as related to potassium efflux from winter rape hypocotyls. Physiol. Plant. 57, 296-300.

13. Shimokawa, K. (1983) An ethylene forming enzyme in Citrus unshiu fruits. Phytochemistry 22, 1903-1908.

14. Siedow, J.N. (1991) Plant lipoxygenase: structure and function. Ann. Rev. Plant Physiol. Plant Mol. BioI. 42, 145-199.

15. Sikorska, E. and Kacperska-Palacz, A. (1980) Frost-induced phospholipid changes in cold-acclimated and non-acclimated rape leaves. Physiol. Plant. 48, 201-206.

16. Wang, C.Y. and Adams, D.O. (1982) Chilling-induced ethylene production in cucumbers (Cucumis sativus L.) Plant Physiol. 69, 424-426.

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

ETHYLENE IN EARLY SIGNALLING PHENOMENA AT THE PLANT-MICROORGANISM INTERFACE

M.T. ESQUERRE-TUGAYE, A. BOTTIN, M. RICKAUER, J.P. SANCAN, J. FOURNIER and M.L. POUENAT Centre de Physiologie Vegetale Universite Paul Sabatier 31062 Toulouse, France

ABSTRACT. An early increase in ethylene production was recorded in melon, tobacco and alfalfa in response to infection by Colletotrichum, Phytophthora and Rhizobium, respectively. This effect was mimicked by elicitor treatment of plant tissues and cells. Measurements of ACC synthase and ACC showed that the methionine pathway of ethylene biosynthesis was activated. The effect of exogenous and of endogenous ethylene on defence induction was investigated.

INTRODUCTION. It has been known for a long time that host plants produce large amounts of ethylene in response to infections by pathogens and wounding. The biological significance of this increased production has been questioned. The purpose of this paper is to illustrate the fact that ethylene is a widespread response of plants to microorganisms and their elicitors. The possible role of the hormone in signal transduction leading to defence will be considered.

1. Ethylene In Plant-Microorganism Interactions

The effect of infection on ethylene production is illustrated through different systems under study in our laboratory, involving two fungal pathogens and one symbiotic bacterium.

The anthracnose disease of Melon, caused by Colletotrichum lagenarium, is characterized by necrotic spots which develop on the aerial part of the host plant, including fruits. A kinetic study performed on melon seedlings shows that the symptoms start to appear 3 to 4 days after spraying a conidia suspension on the host ; spreading of the necrotic lesions over the entire aerial surface leads to seedling death 7 days after inoculation. A time course measurement of ethylene showed that infected whole seedlings start producing large amounts of the hormone 2-3 days after inoculation. Increase in ethylene production was transient, with a maximum 3-4 days post inoculation (Figure lA). ACC and M-ACC were simultaneously increased, thereby indicating that the

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whole methionine pathway of ethylene biosynthesis was activated. The black shank disease of Tobacco, caused by Phytophthora parasitica

nicotianae is characterized by rotting and wilting symptoms which develop from the collar 4 days after inoculation of the root system with a zoospore suspension. Ethylene production is increased prior to symptom appearance (Figure 1B). As in the case of the Melon system, this increase is transient.

A

-----~ 3 ~ 0'

"-(5 E 2 c:: ~

(!) c:: (!)

>, r. ..... w

•

/\ i \

• -=~- -0- -0- -rl __ ~ o o 2 3 4 5 6 7 o

Time (days) post inoculation

B

• !\ / . • 0-0-0-0

2 345 6 7

Time (days) post inoculation

30

20

10

0

0; c::

-0 (!) (!) Ul

"-(5 E

5 (!) c:: (!)

>, r. W

Figure 1. Ethylene production by Colletotrichum lagenarium-infected melon plants (A) or Phytophthora parasitica var. nicotiahae-infected tobacco seedlings (B). Symbols are 0 un infected control plants, • infected plants.

The nitrogen-fixing symbiotic interaction between alfalfa (Medicago sativa) and Rhizobium meliloti was retained as the third system. It differs from the previous ones in that the outcome of the interaction is a symbiotic relationship, and that it involves bacteria. Again, a transient increase in ethylene production, starting 2 days after inoculation of the root system was observed (Table 1). It preceded the appearance of functional nodules by several days.

From the above examples, it follows that ethylene production is an early biochemical marker of compatible interactions, whatever the phenotype of the inte.raction. Reinhardt et al. (1991) have shown that ethylene is increased even earlier during race-cultivar incompatibility than during compatibility in Phytophthora megasperma glycinea-infected soybean seedlings. The biological significance of early ethylene increases is under study.

TABLE 1. Production of ethylene by alfalfa root (Medicago sativa) after inoculation with Rhizobium meliloti : time course study

EthY!ine _1 Time after inoculation (hr) mm.g .h

20 48 72 96

Experiment 1a -13 16.6 11.1 11

Experiment 2b -11.3 15.5 22.2 23.7

a values above the control (-N03=, - Rhizobium) b values above the control (+N03 ' - Rhizobium)

2. Ethylene Induction By Elicitors

120

-8.1

15.6

219

The hypothesis that the transient induction of ethylene production might reflect early events occurring at the cell surface of the partners was first checked in the melon-Colletotrichum lagenarium system (Esquerre-Tugaye et ale 1978). It was found that glycoconjugates released by autoclaving Colletotrichum had the ability to mimick the effect of infection on ethylene production. Since then, elicitors as chemically diverse as oligosaccharides, glycopeptides and peptides have been shown to induce the same phenomenon (Toppan and Esquerre-Tugaye 1984, Mauch et ale 1984, Milat et ale 1990). Elicitation of ethylene is not species specific, i.e. elicitors

isolated from Colletotrichum lagenarium induce ethylene, not only in melons but also in other plants, for example tobacco and soybean ; elicitors prepared from Phytophthora megasperma glycinea, a fungal pathogen of soybean stimulate ethylene production in melon and tobacco as well as in soybean. A detailed study of the methionine pathway of ethylene biosynthesis

was undertaken on a tobacco cell suspension culture treated with elicitors obtained from Phytophthora parasitica nicotianae. It was found that, prior to ethylene emission, there is a burst in ACC synthase activity and ACC synthesis within the very first minutes following addi tion of the elicitor (Rickauer et al. 1990) . Elicitor-induced ethylene was abolished by AVG, while ACC synthase induction was inhibited by cycloheximide, but only slightly affected by actinomycin D. This clearly indicated that the whole methionine pathway is activated by elicitors and that ACC synthase, a key enzyme in the pathway, is controlled at least in part, at a post-transcriptional level. Similar data were reported in the non-host interaction between parsley cells and elicitors of Phytophthora megasperma glycinea (Chappell et al. 1984). Another observation of our study was that elicitation of tobacco cells depends on the physiological stage of the culture only at late growth phase did the cells respond to the elicitor.

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3. Ethylene In Signal Transduction Leading To Defence

Since the initial discovery by Abeles that ethylene treatment triggers chitinase and 13-1,3-glucanase production in bean seedlings (Abeles et al. 1970), several reports have shown that ethylene induces defence responses in plants, notably hydroxyproline-rich glycoproteins (HRGPs), chitinase and 13-1,3-glucanase activity, both at the mRNA and protein levels (Esquerre-Tugaye et al. 1979, Rumeau et al. 1988, Broglie et al. 1986, Ecker and Davis 1987, Takeuchi et al. 1990). The kinetics of ethylene production during infection are consistent with a role for this hormone in defence induction. Indeed, the burst of ethylene recorded in the above pathogenic and symbiotic interactions, precedes the increased deposition of HRGP in the cell wall of melon, and in bean root nodule tissues (Toppan et al 1982, Benhamou et al. 1991). HRGP molecules are supposed to participate in the defenc;- of plants by strengthening the cell wall. Following ethylene production, PR proteins such as chitinases, 13-1,3-glucanases and proteinase inhibitors are also induced upon infection or elicitor treatment of melon and tobacco seedlings. In the melon system, we found that systemic acquired resistance against Colletotrichum was obtained by treatment of the seedlings with ethylene prior to inoculation (Esquerre-Tugaye et al. 1979). - -These data raise the question of the possible involvement of ethylene

in signal transduction leading to defence. Biochemical and molecular approaches aimed at controlling the level of endogenous ethylene may be used for understanding the role of the hormone. Thus, AVG inhibition of elicitor-induced ethylene in melon seedlings resulted in partial inhibition of defence responses involving HRGPs, chitinases and proteinase inhibitors ; HRGP inhibition was rescued by adding ACC to the seedlings (Roby et al. 1985, 1986, 1987). In the tobacco system, however, elicitor-induced proteinase inhibitors were not lowered by AVG (Rickauer et al. 1992). Attempts by other workers to correlate endogenous ethylene to defence induction has also yielded contradictory results (Mauch et al. 1984, Chappell et al. 1984). It seems that the role of ethylen-;- might depend on the host-pathogen interaction under investigation, and on the gene under consideration, as suggested by Lincoln and Fisher (1988) in other studies. Another interpretation of the data obtained through ethylene

inhibition is that several pathways leading to defence might coexist in plants. In this respect, jasmonic acid formed through the lipoxygenase pathway of polyunsaturated fatty acid metabolism, has recently received special attention (Farmer et al. 1992, Gundlach et al. 1992, Rickauer et al. 1992). This lipid derived compound induces defence responses. The possibility that inhibiting the ethylene pathway favors the jasmonic acid pathway and vice versa, should be explored. If this was the case, it would explain why ethylene inhibition does not necessarily result in a lowering of defence induction.

CONCLUSION. Antisense inhibition of ethylene biosynthesis has revealed a powerful tool to assess the critical role played by ethylene in fruit

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ripening and organ senescence. A reappraisal of the relationships between ethylene production and defence induction using such techniques remain to be done. The early and transient synthesis of ethylene in plant-microorganism interactions suggests that the increased production of the hormone is not merely a side reaction reflecting a general stress effect.

REFERENCE

Abeles, F.B., Bosshart, R.P., Forrence, L.E. and Habig, W.H. (1970) Preparation and purification of glucanase and chitinase from bean leaves, Plant Physiol. 47, 129-134.

Benhamou, N., Lafontaine, P.J., Mazau, D. and Esquerre-Tugaye, M.T. (1991) Differential accumulation of hydroxyproline-rich glycoproteins in bean root nodule cells infected with a wild-type strain or a C4-dicarboxylic acid mutant of Rhizobium leguminosarum pv. phaseoli, Planta 184, 457-467.

Broglie, K.E., Gaynor, J.J. and Broglie, R.M. (1986) Ethylene-regulated gene expression molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris, Proc. Natl. Acad. Sci. USA 83, 6820-6824.

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.

Ecker, J.R. and Davis, R.W. (1987) Plant defense genes are regulated by ethylene, Proc. Natl. Acad. Sci. USA 84, 5202-5206.

Esquerre-Tugaye, M.T., Mazau, D. and Toppan, A. (1978) Triggering fungal components of a defense mechanism involving cell glycoproteins in plants. 3rd International Congress of Pathology (Munchen) Paul Parey ed. Berlin and Hambourg p 229.

by wall

Plant

Esquerre-Tugaye, M.T., Lafitte, C., Mazau, D., Toppan, A. and Touze, A. (1979) Cell surfaces in plant-microorganism interactions. II. Evidence for the accumulation of hydroxyproline-rich glycoproteins in the cell wall of diseased plants as a defense mechanism, Plant Physiol. 64, 320-326.

Farmer, E.E., Johnson, R.R. and Ryan, C.A. (1992) Regulation of expression of proteinase inhibitor genes by methyl jasmonate and jasmonic acid, Plant Physiol. 98, 995-1002.

Gundlach, H., MUller, M.J., Kutchan, T.M. and Zenk, M.H. (1992) Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures, Proc. Natl. Acad. Sci. USA 89, 2389-2393.

Lincoln, J.E. and Fisher, R.L. (1988) Diverse mechanisms for the regulation of ethylene-inducible gene expression, Mol. Gen. Genet. 212, 71-75.

Mauch, F., Hadwiger, L.A. and Boller, T. (1984) Ethylene: symptom, not signal for the induction of chitinase and ~-1,3-glucanase in pea pods by pathogens and elicitors, Plant Physiol. 76, 607-611.

Milat, M.L., Ricci, P., Bonnet, P. and Blein, J.P. (1991) Capsidiol and

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ethylene production by tobacco cells in response to cryptogein, an elicitor from Phytophthora cryptogea, Phytochem. 30, 2171-2173.

Reinhardt, D., Wiemken, A. and Boller, T. (1991) Induction of ethylene biosynthesis in compatible and incompatible interactions of soybean roots with Phytophthora megasperma f. sp. glycinea and its relation to phytoalexin accumulation, J. Plant Physiol. 138, 394-399.

Rickauer, M., Fournier, J., Pouenat, M.L., Berthalon, E., Bottin, A. and Esquerre-Tugaye, M.T. (1990) Early changes in ethylene synthesis and lipoxygenase activity during defense induction in tobacco cells, Plant Physiol. Biochem. 28, 647-653.

Rickauer, M., Bottin, A. and Esquerre-Tugaye, M.T. (1992) Regulation of proteinase inhibitor production in tobacco cells by fungal elicitors, hormonal factors and methyl jasmonate, Plant Pl1ysiol. Biochem. 30, 579-584.

Roby, D., Toppan, A. and Esquerre-Tugaye, M.T. (1985) Cell surfaces in plant-microorganism interactions. V. Elicitors of fungal and of plant orIgIn trigger the synthesis of ethylene and of cell wall hydroxyproline-rich glycoprotein in plants, Plant Physiol. 77, 700-704.

Roby, D., Toppan, A. and Esquerre-Tugaye, M.T. (1986) Cell surfaces in plant-microorganism interactions. VI. Elicitors of ethylene from Colletotrichum lagenarium trigger chitinase activity in melon plants, Plant Physiol. 81, 228-233.

Roby, D., Toppan, A. and Esquerre-Tugaye, M.T. (1987) Cell surfaces in plant microorganism interactions. VIII. Increased proteinase inhibitor activity in melon plants in response to infection by Colletotrichum lagenarium or to treatment with an elicitor fraction from this fungus, Physiol. Mol. Plant Pathol. 30, 453-460.

Rumeau, D., Mazau, D., Panabieres F., Delseny, M. and Esquerre-Tugaye, M.T. (1988) Accumulation of hydroxyproline-rich glycoproteins mRNAs in infected or ethylene treated melon plants, Physiol. Mol. Plant Pathol. 33, 419-428.

Takeuchi, Y., Yoshikawa, M., Takeba, G., Tanaka, K., Shibata, D. and Horino, O. (1990) Molecular cloning and ethylene induction of mRNA encoding a phytoalexin elicitor-releasing factor, ~-1,3-endoglucanase, in soybean, Plant Physiol. 93, 673-682.

Toppan, A., Roby, D. and Esquerre-Tugaye, M.T. (1982) Cell surfaces in plant-microorganism interactions. III. In vivo effect of ethylene on hydroxyproline-rich glycoprotein accumulation in the cell wall of diseased plants, Plant Physiol. 70, 82-86.

Toppan, A. and Esquerre-Tugaye, M.T. (1984) Cell surfaces in plant-microorganism interactions. IV. Fungal glycopeptides which elicit the synthesis of ethylene in plants, Plant Physiol. 75, 1133-1138.

TOMATO ACC SYNTHASE: REGULATION OF GENE EXPRESSION AND IMPORTANCE OF THE C-TERMINAL REGION IN ENZYME ACTIVITY

AUTAR K. MATIOO, NING LI and DERONG LID Plant Molecular Biology Laboratory, USDAIARS, Beltsville Agricultural Research Center (W), Building 006, Beltsville, MD 20705, USA

ABSTRACT. We have isolated and sequenced a tomato (Lycopersicon esculentum cv. PikRed) ACC synthase gene and expressed it in Escherichia coli. This gene encodes a wound-inducible, 1.8 kb transcript; the degree of transcript induction in red-ripe fruit was much greater than that observed in early-red fruit. Inhibitors of ethylene perception (2,5-norbornadiene) or biosynthesis (salicylic acid) inhibited accumulation of the transcript, suggesting its positive regulation by ethylene. This may be a part of the molecular basis of autocatalytic ethylene production. Polyamines, which are antisenescence growth regulators, inhibited the induction of the transcript. The ACC synthase expressed in E. coli was not a fusion protein and was enzymatically active. Transformation of E. coli with an ACC synthase gene construct in which 57 amino acids from the C-terminus of the coding region were deleted, produced a truncated protein without enzymatic activity. This suggess that all or some of the C-terminal 57 amino acids are necessary for ACC synthase activity. The E. coli expression system used here seems to be valuable for investigating the structure-function relationships of this important plant protein.

INTRODUCTION

The plant hormone ethylene regulates a number of diverse metabolic processes in plants (see Mattoo and Suttle, 1991). Studies of molecular regulation of its biosynthesis has received a tremendous boost since the molecular cloning of the genes encoding the enzymes l-aminocyclopropane-l-carboxylate (ACC) synthase (Sato and Theologis 1989, Van der Straeten et al. 1990, Nakajima et al. 1990) and ACC oxidase (Hamilton et al. 1990, 1991, McGarvey et al. 1992) that catalyze the last two steps in the following pathway:

methionine ~S-adenosylmethionine (SAM) ---'7"ACC ~ethylene.

Both ACC synthase (Rottmann et al. 1991, Olsen et al. 1991) and ACC oxidase (see these proceedings) genes represent families of multiple genes. In this context, successful expression of functional ACC synthase (Sato and Theologis 1989, Li et al. 1992b) or ACC oxidase (Hamilton et al. 1991) genes in heterologous systems, such as yeast and Escherichia coli., provides a unique opportunity to study each of the gene products comprising these multi-gene families independent of one another.

We have cloned ACC synthase genes from PikRed variety of tomato. One of these genes is wound-inducible and its expression in the tomato fruit is regulated by

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ethylene, salicylic acid and polyamines (Li et al. 1992a). We have expressed it in E. coli as an active, full length, non-fusion protein of 53 kDa, which demonstrates suicidal inactivation by SAM (Li et al. 1992b). We are using the E. coli expression system to study the structure-function relationships of ACC synthase.

Sequence Analysis of PikRed ACC Synthase cDNA Clones

Oligonucleotides representing the upstream 31 bases and the downstream 33 bases (underlined nucleotides at 5' and 3' ends of the sequence in Figure 1) were synthesized and used in conjunction with total cellular RNA isolated from wounded tomato pericarp tissue to perform RNA-PCR's. The PCR products, ranging in size from 1.6, 1.2, 0.7 to 0.6 kb, were cloned in E. coli and the library screened using ACC synthase-specific oligonucleotide probe (Li et al 1992b). Three clones, each containing an 1.6 kb insert, were selected (referred to as pTACC-A2, -Bl and -7C) and sequenced in both directions using double-strand plasmid DNA as template. The complete nucleotide sequence of PikRed tomato ACC synthase is shown in Figure 1. Only two changes were seen in this sequence when compared to the cDNA sequence of ACC synthase from the Mill. cultivar (van der Straeten et al. 1990): T at position 972 versus C and C at position 1203 versus T (Figure 1). These two nucleotide changes in the cDNA sequence result in changes in the deduced amino acid sequence, i. e. pr0322 and leu399 of Mill ACC synthase are leu322 and Pro399 in the Pik-Red ACC synthase (Figure 1). These two changes confirm identical alterations found in Rutgers ACC synthase (Rottmann et al. 1991).

When the deduced amino acid sequence of the Pik-Red ACC synthase was used to search the data bank (Key bank 7.1 of Intelligenetic Suite 5.4), many putative protein modification sites were revealed. These include putative single sites for amidation (amino acids 339-342), asn glycosylation (amino acids 131-134), and cAMP/cGMPdependent protein kinase phosphorylation (amino acids 302-305). However, only the putative casein kinase II phosphorylation site (SfND) seems highly conserved among the ACC synthases sequenced so far (Fig. 1). Phosphorylation of the protein can result in charge heterogeneity (Elich et al. 1992) and may contribute to the existence of different pI forms of ACC synthase.

Wound Induction of ACC Synthase mRNA Accumulation

Wounding of tissues is known to increase the activity of ACC synthase, the induction kinetics of which vary depending upon the conditions used (Kende and Boller 1981, Mattoo and Anderson, 1984). Under our conditions of wounding tomato pericarp tissue, ACC synthase activity reaches a peak by 10 h. To examine whether or not this rapid induction of ACC synthase activity coincides with a corresponding increase in its transcript level, we analyzed total RNA isolated from tomato (early-red stage) pericarp tissue wounded for 1,4, 8 and 16 h. The results are presented in Figure 2A. Hybridization to a single transcript of 1.8 kb was detected in unwounded early-red stage fruit. The level of this transcript increased markedly upon wounding and the highest transcript level was found in tissues wounded for 8 h. In 16-h wounded samples, hybridization to an additional band at 1.25 kb was noted. This hybridization may represent another, late-wounding transcript or a product of degradation of the 1.8 kb transcript. The data were normalized to the hybridization signal obtained using a tomato rRNA probe as an internal control (Parsons and Mattoo 1991) and are presented in Figure 2A (graph). The level of ACC synthase transcript increased approximately 5-fold at 8 h after wounding.

Nco I AAAA~ATG GGA TTT GAG ATT GCA AAG ACC AAC TCA ATC TTA TCA AAA TTG GCT

M G F E A K T N 5 I 15K 1 A 16 56 ACT AAT GAA GAG CAT GGC GAA AAC TCG CCA TAT TTT GAT GGG TGG AAA GCA TAC

T NEE H G ENS P Y F D G W KAY 34 110 GAT AGT GAT CCT TTC CAC CCT CTA AAA AAC CCC AAC GGA GTT ATC CAA ATG GGT

D 5 D P F H P 1 K N P N G V I Q M G 52 164 CTT GCT GAA AAT CAG CTT TGT TTA GAC TTG ATA GAA GAT TGG ATT AAG AGA AAC

1 A E N Q 1 C 1 D 1 lED W I K R N 70 218 CCA AAA GGT TCA ATT TGT TCT GAA GGA ATC AAA TCA TTC AAG GCC ATT GCC AAC

P K G 5 C 5 F G K 5 F K A I A N 88 272 TTT CAA GAT TAT CAT GGC TTG CCT GAA TTC AGA AAA GCG ATT GCG AAA TTT ATG

F Q D Y H G 1 P E F R K A A K F M 106 326 GAG AAA ACA AGA GGA GGA AGA GTT AGA TTT GAT CCA GAA AGA GTT GTT ATG GCT

E K T R G G R V R F D P E R V V M A 124 380 GGT GGT GCC ACT GGA GCT AAT GAG ACA ATT ATA TTT TGT TTG,GCT GAT CCT GGC

G G A T G A N E T F C 1 A D P G 142 434 GAT GCA TTT TTA GTA CCT TCA CCA TAC TAC CCA GCA TTT AAC AGA GAT TTA AGA

D A F 1 V P Spy Y P A F N R D 1 R 160 488 TGG AGA ACT GGA GTA CAA CTT ATT CCA ATT CAC TGT GAG AGC TCC AAT AAT TTC

W R T G V Q 1 I P H C E S 5 N N F 178 542 AAA ATT ACT TCA AAA GCA GTA AAA GAA GCA TAT GAA AAT GCA CAA AAA TCA AAC

KIT S K A V K E AYE N A Q K S N 196 596 ATC AAA GTA AAA GGT TTG ATT TTG ACC AAT CCA TCA AAT CCA TTG GGC ACC ACT

I K V K G 1 1 T N P S N P 1 G T T 214 650 TTG GAC AAA GAC ACA CTG AAA AGT GTC TTG AGT TTC ACC AAC CAA CAC AAC ATC

1 D K D T 1 K S V 1 S F T N Q H N I 232 704 CAC CTT GTT TGT GAC GAA ATC TAC GCA GCC ACT GTC TTT GAC ACG CCT CAA TTC

H 1 V C DEI Y A A T V F D T P Q F 250 758 GTC AGT ATA GCT GAA ATC CTC GAT GAA CAG GAA ATG ACT TAC TGC AAC AAA GAT

V S I A E L D E Q E M T Y C N K D 268 812 TTA GTT CAC ATC GTC TAC AGT CTT TCA AAA GAC ATG GGG TTA CCA GGA TTT AGA

1 V H V Y S L S K D M G 1 P G F R 286 866 GTC GGA ATC ATA TAT TCT TTT AAC GAC GAT GTC GTT AAT TGT GCT AGA AAA ATG

V G I I Y S F N D D V V N CAR K M 304 920 TCG AGT TTC GGT TTA GTA TCT ACA CAA ACG CAA TAT TTT TTA GCG GCA ATG ~

S S F G 1 V S T Q T Q Y F 1 A A M [!J 322 974 TCG GAC GAA AAA TTC GTC GAT AAT TTT CTA AGA GAA AGC GCG ATG AGG TTA GGT

S D E K F V D N F 1 RES A M R 1 G 340 1028 AAA AGG CAC AAA CAT TTT ACT AAT GGA CTT GAA GTA GTG GGA ATT AAA TGC TTG

K R H K H F T N G 1 E V V G K C L 358 1082 AAA AAT AAT GCG GGG CTT TTT TGT TGG ATG GAT TTG CGT CCA CTT TTA AGG GAA

K N NAG L F C W M D 1 R P 1 1 R E 376 1136 TCG ACT TTC GAT AGC GAA ATG TCG TTA TGG AGA GTT ATT ATA AAC GAT GTT AAG

S T F D S EMS 1 W R V N D V K 394 1190 CTT AAC GTC TCG ~ GGA TCT TCG TTT GAA TGT CAA GAG CCA GGG TGG TTC CGA

L N V S [g] G S S F E C Q E P G W F R 412 1244 GTT TGT TTT GCA AAT ATG GAT GAT GGA ACG GTT GAT ATT GCG CTC GCG AGG ATT

V C FAN M D D G T V D A 1 A R 430 1298 CGG AGG TTC GTA GGT GTT GAG AAA AGT GGA GAT AAA TCG AGT TCG ATG GAA AAG

R R F V G V E K S G D K S SSM E K 448 1352 AAG CAA CAA TGG AAG AAG AAT AAT TTG AGA CTT AGT TTT TCG AAA AGA ATG TAT

K Q Q W K K N N L R L S F S K R M Y 466 1406 GAT GAA AGT GTT TTG TCA CCA CTT TCG TCA CCT ATT CCT CCC TCA CCA TTA GTT

DES V 1 S P L SSP P P S P 1 V 484 1460 CGT TAA GACTTAATTAAAAGGGAAGAATTTAATTTATGTTTTTTTATATTTTGAAAAAAATTTGTAAGA

R Bam HI 485 1529 ATAAGATTATAATAGGAAAAGAAAATAAGTATGTAGGATRCtGAGTATTTTCAGA8ATAGTTGTT

225

Figure 1. Composite nucleotide and deduced amino acid sequence of a wound-inducible tomato (cv. PikRed) ACC synthase. The two primers used for RNA-based peR are shown as underlined DNA sequences. The Nco I and Bam HI restriction sites that were created by substitution are also indicated. The conserved putative casein kinase II phosphorylation site (SfND; amino acids 292-295) is underlined in bold.

226

In a similar fashion, the ACC synthase transcript level was analyzed in red-ripe tomato peri carp tissues wounded for longer periods of time (16, 24 , 32 and 40 h). The results showed that the red-ripe fruit maintains the property of inducingACC synthase transcript upon prolonged wounding (Figure 2B). A comparison of the level of induction between time zero and 16 h after wounding revealed a 25-fold induction of ACC synthase transcript in the red-ripe fruit (Figure 2B) in contrast to a 3.6-fold increase in the early-red fruit (Figure 2A). Moreover, by 40 h of wounding, the transcript level increased by 66-fold in red-ripe fruit (Figure 2B, graph). The 1.25 kb RNA is again detectable in the 40-h wounded sample.

ACC Synthl Y-

rRNA

100

OiE >" 80 ~E

l~ 60

"'- 40 co

~t 20

0

A. B. F-----,

0 , 4 8 16 0 16 24 32 40

Hours of Wounding

Figure 2. Accumulation of ACC synthase mRNA upon wounding of tomato fruit. Early-red (A) and red-ripe (B) tomato fruit pericarp tissues were wounded for the indicated times. Total RNA was isolated from these tissues and fractionated on formaldehydecontaining 1 % agarose gels. The RNAs were transferred to nylon membranes, cross-linked by UV irradiation, then hybridized first to the ACC synthase probe (5 X 109 cpm/Jlg), and later to a rRNA probe (Northern blots). The signals on the Xray fIlms were quantified densitometricalJyand the ACC synthase transcript level normalized to the rRNA level in each sample. The ACC synthase transcript levels are given as a % of maximum transcript and are represented by bars under each lane of the RNA blot. The open triangle indicates the position of a 1.25 kb transcript.

Antagonistic Regulation of ACC Synthase Transcript Accumula,tion by Ethylene and Polyamines

Wound-induced ACC synthase gene expression in winter squash was found to be repressed in the presence of ethylene and de-repressed under conditions when ethylene action was inhibited by 2,5-norbornadiene (NBD) (Nakajima et al. 1990). In contrast, the accumulation of the 1.8 kb ACC synthase transcript appears to be ethylene inducible in tomato. Two approaches were used to study the involvement of ethylene in the wound induction of tomato ACC synthase transcript. First, we used NBD to block ethylene action during a given period of wounding, and then compared the ACC synthase transcript level in this sample to an identical sample incubated without NBD. A strong inhibition in the accumulation of ACC synthase transcript was observed (data not shown). Second, we inhibited the production of ethylene by salicylic acid, an inhibitor known to affect ACC oxidase (Leslie and Romani 1986) and the wounding signal in tomato plants (Doherty et al. 1988). The results in Figure 3 clearly show that salicylic acid inhibits the wound-induced

227

increase in the ACC synthase transcript level. These results as well as those obtained using the ethylene action-inhibitor, NBD, demonstrate that ethylene induces the accumulation of ACC synthase transc.ript in wounded tissue.

Salicylic acid -- +

o 9 9 Hours of Wounding

Figure 3. Inhibition of transcript accumulation in fruit tissue incubated in the absence or presence of salicylic acid.

If ACC synthase enzyme activity is regulated at the transcript level, then conditions causing marked inhibition of its transcript accumulation (for instance, the presence of NBD or salicylic acid) would result in lower enzyme activity and in turn lower ethylene production. Indeed, such is the case. In NBD-treated samples, ethylene production was markedly inhibited while in the salicylic acid-treated samples, ACC synthase activity was reduced to 10% of that in the control after 9 h of wounding (data not shown).

The polyamines putrescine, spermidine and spermine, the anti-senescence growth regulators, inhibited the induction of ACC synthase transcript level in wounded pericarp tissue to different degrees (Figure 4). By 3 h of incubation, the transcript level dropped to 65%, 70% and 35% of the control in the presence of putrescine, spermidine and spermine, respectively. But upon further incubation (6 and 10 h), the ACC synthase transcript accumulated to the control levels in putrescine- and spermidine-treated tissues. In spermine-treated tissues, the transcript level was further decreased to 45% of the control by 6 h. However, by 10 h after wounding the transcript level was higher in the sperminetreated tissue than the control (Figure 4, 10-h point, hatched bar). The levels of ACC synthase enzyme activity measured in identical samples are given as open bars in Figure 4 and are plotted next to the quantified (and normalized) data of the transcript levels (hatched bars). These results confirm that polyarnines inhibit the induction of ACC synthase enzyme activity upon wounding (see open bars). However, like their effect on the transcript level, different polyamines had slightly different effects on ACC synthase activity. For instance, the inhibitory effects of putrescine and spermidine lasted only until 3 h after wounding and

228

were reversed completely by IO h after wounding. In contrast, the inhibitory effect of spermine was sustained throughout the wounding period. These results are consistent with previous reports showing that spermine has a stronger inhibitory effect on ethylene production than other polyamines (Apelbaum et al. 1981 , Roberts et al. 1984). In all cases,

A. Putrescine B. Spermidine C. Spermine

140

0 120 !:: 100 c 0 80 0

60 (5

40 ~

20 0

0 II.iIII 0 3 6 10 o 3 6 10 o 3 6 10

Hours of Wounding

Figure 4. Inhibition of wound-induced ACC synthase transcript and enzyme activity by polyamines in red-ripe tomato pericarp tissue. Q, ACC synthase enzyme activity; II, ACC synthase transcript level.

the return of transcript level to control levels or higher, preceded the observed rise in the enzyme activity. This suggests that the two processes are linked. Taken together, these results implicate polyamines as regulators of ACC synthase expression at the transcriptional and posttranscriptionallevels.

The ethylene-inducible nature of ACC-synthase mRNA accumulation might, in fact, explain the autocatalytic regulation of ethylene production. Even though ethylene affects ACC oxidase activity (Hoffman and Yang 1982, Chalutz et al. 1984), we have shown (Li et al. 1992a) that it can regulate ethylene biosynthesis at the ACC synthase transcript level. These data can be outlined in a broader context of ethylene and polyamine regulation as shown in Figure 5. As previously described, ACC synthase induction would lead to the accumulation of ACC in the cell, which gets converted to ethylene by ACC oxidase. Ethylene, thus produced, presumably reacts with its receptor and causes further induction of ACC synthase transcript. Our data indicate that this induction can be nullified if the ethylene receptor is inactivated with NBD. Furthermore, if ethylene production itself is reduced, for instance, by salicylic acid, a diminution in the induction of the ACC synthase transcript results. Also, polyamines interfere with the development of ACC synthase transcript and thus result in the inhibition of ethylene production.

Expression of Functional ACe Synthase in E. coli

A wound-inducible tomato (Mill. cultivar) ACC synthase was previously expressed in E.coli as a 55-kDa polypeptide; however, the identification was based on immunological cross-reactivity and the expressed protein did not show ACC synthase activity (van der Streaten et al 1990). In contrast, ACC synthase-specific clones from winter squash and zuccini were expressed in both E.coli and yeast and the expressed protein was shown to

229

possess enzymatic activity (Sato and Theologis, 1989;Nakagawa et al. 1991). In order to check if we could express a functional PikRed ACC synthase in E. coli, we cloned the PCR-amplified ACC synthase cDNAs into a pCR 1000 cloning vector (Invitrogen) in

ACC Synthase Gene

I 1[ E9 Ethylen .. Receptor

~. Complex

pOlyarnes~mr tHBD I ACC Synthase

dc·SAM e I Ethylene • ~M

S~M --------- - __ ACC --f---EthYlene

Selicylic Acid

Figure 5. A scheme for regulation of ACC synthase transcript and enzyme activity by ethylene and polyamines. Explanation is given in the text. Dashed arrow-lines indicate the forward direction of each metabolic sequence. The thick and thin arrow-lines indicate the step(s) at which ethylene and polyamines cause up (+) or down (-) regulation, respectively. NBD is shown as inhibiting the action of ethylene by binding to ethylene-binding (receptor) protein whereas salicylic acid is shown as an inhibitor ACC oxidase.

which transcription of the introduced gene fragment is driven by a constitutive LacZ promoter in a host cell with an inactivated LacI repressor. The PCR-amplified ACC synthase clones were examined for extracellular content of ACC, the product of an active ACC synthase expressed in the bacterium.

When the open reading frame was in the same direction as the LacZ transcription (sense orientation), ACC was detected in E. coli cultures both intracellularly and extracellularly (PT-ACC-A2 and pT-ACC-Bl). However, when the open reading frame was in the opposite direction to LacZ transcription, ACC was not detected either in the cells or in the medium (Table 1).

Table 1. Expression of tomato ACC synthase in E. coli

Clone

pTACC-A2 pTACC-Bl pTACC-C7 pTACC-B1..1.

Insert orientation

Sense Sense Anti-sense Sense

Insert size (kb) 1.6 1.6 1.6 1.3

ACC content (nmol. ml-1) 79.25±6.25 73.25 ± 1.25

o o

Because the C-terminus of ACC synthase is highly variable (Dong et al. 1991), we speculated that this region may not be important for enzymatic activity. To test this, we constructed a mutant (pTACC-B1..1) from pTACC-Bl clone, deleting 57 amino acids from the c-terminal non-conserved region by double-digestion. The ability of this ACC synthase-deletion mutant to produce ACC was then examined in the same way as for the non-mutated control. This deletion mutant was found not to produce any detectable amount of ACC intracellularly or extracellularly (Table 1). These data suggest

230

that some of the deleted highly-variable region may be essential for enzyme activation, correct assembly of the enzyme, or enzyme activity via its juxtaposition to the active site. The importance of the C-terminus in influencing the ACC synthase activity is being further investigated.

Acknowledgments

We thank Prof. Marvin Edelman and Dr. James D. Anderson for critical comments on the manuscript, Prof. Shyam Dube for discussions on the cloning strategy, and Dr. Barbara Parsons for collaboration with results presented in Fig. 2 .

References

Apelbaum, A, Burgoon, AC., Anderson, J.D., Lieberman, M., Ben-Arie, R., and Mattoo, AK. (1981) 'Polyamines inhibit biosynthesis of ethylene in higher plant tissue and protoplasts', Plant Physiol. 68, 453-456.

Chalutz, E., Mattoo, AK., Solomos, T., and Anderson, J.D. (1984) 'Enhancement by ethylene of cellulysin-induced ethylene production by tobacco leaf discs', Plant Physioi. 74, 99-103.

Doherty, H.M., Selvendram, R.R., and Bowles, D.J. (1988) 'The wound response of tomato plants can be inhibited by aspirin and related hydroxy-benzoic acids', Physioi. Mol. Plant Pathoi. 33, 377-384.

Dong, J.G., Kim, W.T., Yip, W.K., Thompson, G.A, Li, L., Bennett, AB., and Yang, S.F. (1991) 'Cloning of a cDNA encoding l-aminocyclopropane-l-carboxylate synthase and expression of its mRNA in ripening apple fruit', Planta 185, 38-45.

Elich, T.D., Edelman, M., and Mattoo, AK. (1992) 'Identification, characterization, and resolution of the in vivo phosphorylated form of the D 1 photosystem II reaction center protein', J. BioI. Chern 267,3523-3529.

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.

Hamilton, AJ., Bouzayen, M., and Grierson, D. (1991) 'Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast', Proc. Nati. Acad. Sci. USA 88,7434-7437.

Hoffman, N.E., and Yang, S.F. (1982) 'Enhancement of wound-induced ethylene synthesis by ethylene in preclimacteric cantaloupe', Plant Physiol. 69, 317-322.

Kende, H., and Boller, T. (1981) 'Wound ethylene and l-aminocyclopropane-lcarboxylate synthase in ripening tomato fruit', Planta 151,476-481.

Li, N., Parsons, B.L., Liu, D., and Mattoo, AK. (1992a) 'Accumulation of woundinducible ACC synthase transcript in tomato fruit is inhibited by salicylic acid and polyamines', Plant Mol. BioI. 18,477-487.

Li, N., Wiesman, Z., Liu, D., and Mattoo, AK. (1992b) 'A functional tomato ACC synthase expressed in Escherichia coli demonstrates suicidal inactivation by its substrate S-adenosylmethionine', FEBS Lett. 306, 103-107.

Leslie, C.A, and Romani, R.J. (1986) 'Salicylic acid: A new inhibitor of ethylene biosynthesis', Plant Cell Reports 5,144-146.

231

Mattoo, A.K., and Anderson, J.D. (1984) 'Wound-induced increase in 1-aminocylcopropane-l-carboxylate synthase acitivity: Regulatory aspects and membrane association of the enzyme', in Y. Fuchs and E. Chalutz (eds.), Ethylene: Biochemistry, Physiological and Applied aspects, Amsterdam: Martinus Nijhoff/Dr. W.Junk Publishers, pp. 139-147.

Mattoo, A.K., and Suttle, J.C. (1991) The Plant Hormone Ethylene, CRC Publ. Inc., Boca Raton, Florida.

McGarvey, D.J., Sirevag, R, and Christoffersen, RE. (1992) 'Ripening-related gene from avocado fruit', Plant Physiol. 98, 554-559.

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

Nakajima, N., Mori, H., Yamazaki, K., and Imaseki, H. (1990) 'Molecular cloning and sequence of a complementary DNA encoding l-aminocyclopropane-lcarboxylate synthase induced by tissue wounding', Plant Cell Physiol. 31, 1021-1029.

Olsen, D.C., White, J.A., Edelman, L., Harkings, RN., and Kende, H. (1991) 'Differential expression of two genes for l-aminocyclopropane-l-carboxylate synthase in tomato fruits', Proc. Natl. Acad. Sci. USA 88,5340-5344.

Parsons, B.L., and Mattoo, A.K. (1991) Wound regulated accumulation of specific transcripts in tomato fruit: interactions with fruit development, ethylene and light', Plant Mol. BioI. 17, 453-464.

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.

Rottmann, W.H., Peter, G.F., Oeller, P.W., Keller, J.A., Shen, N.F., Nagy, B.P., Taylor, L.P., Campbell, A.D., and Theologis, A. (1991) 'I-Aminocyclopropane-lcarboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence', J. Mol. BioI. 222,937-961.

Sato, T., Oeller, P.W., and Theologis, A. (1991) 'The l-aminocyclopropane-lcarboxylate synthase of Cucurbita. Purification, properties, expression in Escherichia coli, and primary structure determination by DNA sequence analysis', J. BioI. Chern. 266, 3752-3759.

Sato, T., and Theologis, A. (1989) 'Cloning the mRNA encoding 1-aminocyclopropane-l-carboxylate synthase, the key enzyme for ethylene biosynthesis in plant', Proc. Natl. Acad. Sci. USA 86, 6621-6625.

Van Der Straeten, D., Van Wiemeersch, L., Goodman, H.M., and Van Montagu, M. (1990) 'Cloning and sequence of two different cDNAs encoding 1-aminocyclopropane-l-carboxylate synthase in tomato', Proc. Nati. Acad. Sci. USA. 87, 4859-4863.

Regulation Of Ethylene Synthesis In Maize Root Responses To Stress

P. W MORGAN, I.I. SARQUIS, C.-I. HE, W.R JORDAN, AND MC. DREW. Texas A & M University Dept of Soil and Crop Sciences and Dept of Horticultural Sciences * College Station, Texas 778-/3-2-/74 USA

ABSTRAcr. The role and regulation of ethylene synthesis was investigated irr maize roots subjected to stresses that inlubit elongation, promote radial swelling and promote formation of aerenchyma. Physical impedance was imposed by compression of the growing medium around unemerged seedlings at controlled pressures, and ethylene production rates by intact seedlings were assayed with the aid of a continuous flow system. Ethylene production increased before effects on growth were observed, and A VG plus STS restored root extension to 90% of control values. One hour after application of 100 kPa pressure to the medium, ACC and conjugated ACC levels and ACC synthase and ACC oxygenase activities all had increased Sharply. TransientN treatment increased sensitivity to ethylene and initiated formation of aerenchyma. Effects of both -N and hypoxia treatments were blocked with Ag+. Both treatments induced synthesis of cellulase which was prevented by A VG. Perception of impedance stress and effects of -N on ethylene binding capacity and affmity are being studied.

Introduction

Physical impedance inhibits elongation and promotes radial expansion of roots and shoots (1,4,16). These growth effects have been linked to elevated production of ethylene (4,7), and application of small concentrations of the gas have mimicked the effects of impedance (4,7). However, a role of ethylene in responses of plants to physical impedance was inconsistent with some other evidence (10,17), and we initiated a new study of the phenomena using pressure to compress the growing medium around emerging maize seedlings (13.15). Oxygen deficiency and brief deficiency of N or P induced formation of aerenchyma in maize roots (3,6,8). This apparent acclimation to stress was linked to elevated ethylene synthesis for seedlings exposed to hypoxia (3) and has been the subject of additional investigations by our group (2,5).

Materials And Methods

All studies employed maize (Zea Mavs L, cv. Tx 5855); seeds were surface sterilized, germinated and selected for uniformity as previously described (2,13). For studies of physical impedance,S unemerged seedlings with 35± 2 mm long roots were placed in fritted clay wet with 0.1 mM Ca02 or test solution [13]. The fritted clay particulate medium filled a tube-shaped membrane and was enclosed in a triaxial cell which could be pressurized to compress the medium particles against the seedlings while the gas phase around the seedlings remained at atmospheric pressure [13]. Continuous. air flow passed through the medium and subsequently through an ethylene collection system attached to a gas chromatograph equipped with an alumina column and flame ionization detector (7). Tests were conducted to insure that ethylene did not occur in the air supply or arise from components of the triaxial cells or flow system. Effects on elongation and radial enlargement were determined by removing seedlings from triaxial cells at selected times and measuring organs as previously described [13J.

232

J. C. Pech et al. (eds.). Cellular and Molecular Aspects of the Plalll Hormone Ethylene, 232-237. © 1993 Kluwer Academic Publishers.

233

For studies of nutrient withdrawal, seedlings were grown in control nutrient solution for 11 days after which -N or -P deficiency was imposed [2]. Adventitious roots rather than primary roots were studied. Aerenchyma were examined in transverse sections by light and scanning electron microscopy [5].

ACC and MACC were extracted from root tips and assayed by conversion to ethylene [9,15]. In vivo ACC synthase was estimated based on differences in ACC levels with or without a one hour period of anoxia [15]; in vivo ACC oxidase activity was estimated by supplying tissue with surplus ACC and assaying ethylene production [2,15].

Results

PHYSICAL IMPEDANCE At pressures of 25 and 100 kPa, root elongation rates and diameters were not

different after two hours, but after four hours elongation was inhibited and diameter had increased compared to controls [13]. In contrast, ethylene production had increased in all pressures tested except 25 kPa after one hour, and at 25 kPa the effect was evident at 2 hours (Fig. 1 ). In roots ACC and MACC levels rose significantly in 1 hour at both 25 and 100 kPa (Fig. 2). ACC synthase activity increased over 5 fold after 1 hour at 25 or 100 kPa, and differences in ACC oxidase were also evident at 1 hour with the higher pressure while at 25 kPa the effect was evident at 2 hours (Table 1). The time course of exogenous ethylene effects on elongation and radial expansion was not determined, but effects qualitatively similar to pressure were obtained by applying ethylene [13]. The combination of AVG (10mM) and STS (lmM) restored root elongation to 90% of the unimpeded control and reduced radial expansion to 45% of that resulting from impedance.

250 '0 .. .c <5 • 'i .i. .!.

200 T i Q ~ 40 T T T /1 40 Iii 1'!--r--rI E 4D 150 E .!!! '0 .!!! 30 T / •• _- J. 30 r.7- i--r-- L

• E • • .e: II '0

'0 20 20 :Ii :Ii I A--~ c

4D c

{?o:i;=-~/ .. = U U 10 U c 50 10 C

CD U 0-0---0---0--0-0 :Ii

>. c .c 0 0

iii 0 10 0 2 4 6 8 10 0 2 4 6 8

Time (h) Tim. (h)

Figure 1 (left). Time course of ethylene evolution from intact maize seedlings growing under physical impedance simulated by application of pressures of 0, 25, 50, 75, or 100 kPa on the growing medium. Vertical bars, SD (reference 13, used by permission).

Figure 2 (right). Time course of ACC and MACC accumulation in roots of maize seedlings growing under physical impedance simulated by application of pressure on the growing medium. Vertical bars, SD (redrawn from reference 15).

234

The question of whether inhibitors of ethylene synthesis and action could prevent occurrence of symptoms of physical impedance was tested by applying the inhibitors and then examining ethylene production rates and root growth after five hours at 50 kPa (Table 2).

Table 1. In vivo ACC Synthase and ACC Oxidase activities in maize seedling roots subjected to physical impedance for times indicated.

Enzyme Hours

ACC Synthase 1

ACC Oxidase 1 2

Applied Pressure (kPa) Q 25 100

(n moles (g fw) .lh·l) 4.1 ± 1.2 25.0 ± 2.3 28.6 ± 2.9

2.0 ± 0.21 2.1 ± 0.13 2.5 ± 0.15 2.1 ± 0.09 2.5 ± 0.15 2.8 ± 0.14

Table 2 Effect of inhibitors of ethylene synthesis and action on growth of roots of maize seedlings under physical impedance for 5 hours.

Treatments Elongation mm

Control 5.1 ± 1.0

Diameter Increase urn

o ± 20

Pressure 50 kPa No Inhibitor 2.6 ± 0.5 235 ± 20 AVG + STS 3.8 ± 0.6 108 ± 23

When pressure to the medium was alternately applied and released hourly for 10 hours, ethylene production rates followed in an up and down but generally rising pattern [13]. The conversion of ACC to MACC was linear over a wide range of ACC concentrations, but the conversion of ACC to ethylene increased only when ACC exceeded a threshold of about 12 n moles (gram fresh weight)"l. Direct application of atmospheric pressure to seedlings did not alter root elongation, radial enlargement or ethylene production until pressures reached the 500 to 800 kPa range [14].

NUTRIENT STARVATION Nutrient starvation for 4 days increased aerenchyma formation markedly in the

case of N03· and NH4 + (Fig. 3). Surprisingly, both treatments reduced ethylene production [2]. Exposure to -N lowered ethylene production by about 50% after 24 hours; reductions in ACC and MACC content and ACC oxidase (EFE) activity were observed after two to three days and later ACC synthase activity was reduced [2].

Despite reduced ethylene production, both Ag+ and AVG reduced formation of aerenchyma in adventitious roots of -N and -P plants [2]. When ethylene was bubbled

235

through nutrient solutions, -N treatment increased sensitivity to ethylene from 50 to 100 fold, compared to controls (Fig. 4 ).

Formation of aerenchyma by transient nitrogen deficiency, hypoxia and ethylene was associated with a 4 to 10- fold increase in cellulase activity in the apical 10mm of maize roots (Fig. 5). AVG prevented induction of cellulase by both -N and hypoxia (Table 3). Hypoxia caused ACC synthase to rise in less than 24 hours while cellulase activity did not increase in these roots until the third day (data not given).

35 -c::.tl_,. .. lIIIpr"

~ 30 .! CJ 0 ••• p. co ••

0 25 ~T.t.I "' ''II C. II . " '0 20 e

a 15 · · 10 · "$.

0 Control · N . p

Figure 3 (above left). Aerenchyma formation in adventitious root tips of maize seedlings deprived of N03- and NH4+ (-N) or phosphate (-P) for 4 days. Vertical bars, SD (reference 2, used by permission of the publisher).

Figure 4 (above right). Changes in induction

45

40 ~ J5 ~ 1:: 0 JO u

~ 25

20 "0 15 0

~ 10 0 .. 5

0

-5 Control -N - p Control - N - p

IU, 0.05 jJJ ,_' ethylene 45

40 ~ J5 G 1:: 8 JO

" 25

e 20 "0 15 0

~ '0 .. 0

- 5 Contfor -N - P Control - N - P

, pi ,- 'et"'ylene 5~ , - 'eU1yhm8

of aerenchyma in maize roots by exogenous ethylene in response to withdrawing nutrients (reference 5, used by permission of the publisher).

Figure 5 (below). Induction of cellulase activity in maize root segments by nutrient (-N) removal, hypoxia (4% 02) or ethylene (1ppm) for the durations indicated. Vertical bars, SD.

80 0 .. 70

i -0- 4 % 0 , lid)

~ 60 .... !! 50 C .:. ?: 40

~ ;; 30 II .. .. II 20 2 'i 0 10 --

0 5 25 45 65 85 105 125 145

Distance Irom tip (mm)

236

Table 3. Reversal of ethylene-dependent cellulase activity in maize seedling roots by AVG (10mM) and restoration by ethylene (lppm) after 4 days treatment.

Main Treatments Inhibitors Control -N 4% 02

[units (g fw)·l] x 103

Control 9.0 ± Ll 20.1 ± 1.4 26.1 ± 2.1

AVG 7.1 ± 1.0 9.3 ± 0.9 ILl ± 1.2

AVG+Eth 30.2 ± 2.1 35.2 ± 2.3 33.5 ± 2.1

Discussion

In our experimental system for physical impedance, ethylene is measured in near real time [13], but since the gas must diffuse from tissue before it can be carried in the air stream used in the collection system, our time courses would somewhat under estimate how soon ethylene production rises. However, increases in ethylene production rates, ACC and MACC levels, ACC synthase activity and, possibly, ACC oxidase activity precede the inhibition of elongation and promotion of radial expansion. This time course, plus the findings that the effects of stress are largely reversed by inhibitors of ethylene synthesis and action and mimicked by exogenous ethylene, seem to clearly establish a role of stress ethylene in the response to physical impedance of growth [13,15]. It is important to emphasize that the system does not impose an absolute barrier to growth and that as resistance increases, physical effects on turgor-driven cell expansion would presumably become more significant, perhaps over riding regulatory biochemical effects.

The absence of sensitivity of root growth and ethylene production to atmospheric pressure [14] suggests that roots are sensing physical pressure, presumably at the surface of the organ. That on/off increases of impedance are followed by rapid increases and decreases in ethylene production [13] suggest a highly reversible perception mechanism. These considerations have led us to the hypothesis that perception of physical impedance stress involves stretch activated ion channels in epidermal cells. Experiments are under way to test this hypothesis. In the maize root system the conversion of ACC to MACC appears to serve a regulatory role because this reaction is linearly related to ACC concentrations, while conversion of ACC to ethylene does not accelerate until a threshold concentration is exceeded [15]. Thus, a marginal stimulation of ACC production could be diverted to MACC without an effect on the ethylene synthesis rate; this possibility is under investigation.

Induction of aerenchyma by nutrient starvation represents an unusual situation where stress increases sensitivity to ethylene while decreasing production of the gas. This contrasts with induction by hypoxia, in which there is a marked stimulation of ethylene biosynthesis [3]. The ability of Ag+ and A VG to prevent formation of aerenchyma and the rise in cellulase activity with -N treatment indicates that ethylene is involved [5]. A simple hypothesis is that the nutrient deficiency stress increases receptor number or

237

affinity. Hall's group [11,12] has demonstrated that two different ethylene binding proteins occur in all plants tested, and that they vary in the time necessary to bind and release ethylene. Thus the effect of -N could be to alter the proportion of the two binding agents so as to increase sensitivity. Other explanations are possible, and this phenomenon is also being studied currently in our laboratory.

References

1. Barley, KP (1963) Influence of soil strength on roots. Soil Sci. 96:175-180. 2. Drew, Me, He, CJ, Morgan, PW (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. '

3. Drew, Me, Jackson, MB, Giffard, S (1979) Ethylene - promoted 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.

4. Goeschl, JD, Rappaport L, Pratt, EX (1966) Ethylene as a factor regulating the growth of pea epicotyls subjected to physical stress. Plant Physiol. 41:877·884.

5. He, CJ, Morgan, PW, Drew Me (1992) Enhanced sensitivity to ethylene in nitrogen·or phosphate-starved roots of Zea mays L during aerenchyma formation. Plant Physiol. 98:137-142.

6. Kawase, M (1981) Anatomical and morphological adaption of plants to waterlogging. HortSci.16:30-34.

7. Kays, 81, Nicklow, CW, Simons, DH (1974) Ethylene in relation to the response of roots to physical impedance. Plant Soil 40:565-571.

8. Konings, H, Verschuren, G (1980) Formation of roots of Zea mays in aerated solutions, and its relation to nutrient supply. Plant Physiol. 49:265-270.

9. Uzada, Mcc, Yang, SF (1979) A simple and sensitive assay for 1- aminocydopropane -1-carboxylic acid. Anal. Biochem. 100:140-145.

10. Moss, GI, Hall, Ke, Jackson, MB (1988) Ethylene and the response of roots of maize (Zea mays L) to physical impedance. New Phytol. 105:303-311.

11. Sanders,lO, Harpham, NVJ, Raskin, I, Smith, AR, Hall, MA (1991) Ethylene binding in wild type and mutant Arabidopsis thaliana (L.) Heynh. Ann. Bot. 68:97-103.

12. Sanders,lO, Ishfzawa, K, Smith, AR, HaD, MA (1990) Ethylene binding and action in rice seedlings. Plant Cell Physiol. 31:1091-1099.

13. Sarquis, JI, Jordan, WR, Morgan, PW (1991) Ethylene evolution from maize (Zea mays L) seedling roots and shoots in response to mechanical impedance. Plant Physiol. 96: 1171· 1177.

14. Sarquis, JI, Jordan W.R., Morgan, P.W. (1992) Effect of atmospheric pressure on maize root growth and ethylene production. Plant Physiol. (In Press).

15. Sarquis, JI, Morgan, PW, Jordan, WR (1992) Metabolism of 1- aminocydopropane -1-carboxylic acid in etiolated maize seedlings grown under mechanical impedance. Plant Physiol. 98: 1342-1348.

16. Taylor, HM, Ratliff, LF (1969) Root elongation rates of cotton and peanuts as a function of soil strength and soil water content. Soil Sci. 108:113-119.

17. Whalen, Me (1988) The effect of mechanical impedance on ethylene production by maize roots. Can. J. Bot. 66:2139-2142.

HEAVY METAL INDUCTION OF ETHYLENE PRODUCTION AND STRESS ENZYMES. I. KINETICS OF THE RESPONSES.

J. WECKX, J. VANGRONSVELD AND H. CLIJSTERS Lab. Plant Physiology, Dept. SBG Limburg.l· U niversitair Centrum U niversitaire Campus B-3590 Diepenbeek Belgium

In higher plants metal toxicity affects several physiological and biochemical processes. However, the primary target of metal action at the cellular level might be the plasmamembrane since this is the first functional structure eneountered by a metal when it penetrates into the cell. Progressive membrane desintegration, leading to enhanced permeability and to loss of membrane function, is observed during senescence (Thompson 1988). Increasing evidence becomes available that various stress factors (drought, chilling, injury, ozone, UV-8, heavy metals, salinity, etc.) induce responses at the cellular level, analogous to those observed during senescence (McKersie et a!. 1988, Noodcn 1988). Membrane destabilization always appears to be involved. It is attributed to lipid peroxidation, due to an enhanced production of highly toxic oxygen free radieals. Metals (Cu, Fe, Hg, etc.), which perform one electron oxidoreduction reactions in solutions, easily produce free radicals. These radicals arc also involved in the lipoxygenase (LOX) mediated oxidation of polyunsaturated fatty acids. Oxygen free radicals and hydroperoxides, generated by LOX, might stimulate the conversion of ACC into ethylene. There is at least indirect evidence that ethylene may facilitate the peroxidative pathway leading to membrane destabilization (Thompson et a!. 1982).

In this paper we compare the effects of the application of Cu or Zn to the roots on membrane integrity in primary leaves of bean seedlings. The latter metal does not perform one electron oxidoreduction reactions. For Cu a slightly (50 ppm) and a more toxic (100 ppm) concentration were tested. Zn was applied at a severely toxic concentration of 200 ppm.

Treatment with 50 ppm Cu had no effect on LOX capacity in the leaves during the period investigated. In contrast 100 ppm Cu induced a transient stimulation of LOX capacity from 7 until 48h after metal application. Ethylene production was already stimulated 2h after treatment with both Cu concentrations. Already 2h after application of 200 ppm Zn to the roots the LOX capacity was significantly stimulated in the leaves, while ethylene production was enhanced only after 48h.

Increased activities of enzymes involved in oxygen detoxification may be considered as circumstantial evidence for enhanced production of oxygen free radicals. Catalase (CAT) and peroxidase (POD) transform peroxides into non-toxic species. A transient stimulation of CAT capacity followed by an inhibition was observed for Cu as well as for Zn. This stimulatory effeet was already visible after 2h and can therefore be considered as a very quick defense response of the plant. POD capacity, measured with guaiacol (G) as substrate, was stimulated in leaves 48h after treatment with 50 ppm Cu. It seems that the role of CAT is taken over by POD in these stress conditions. There is a good resemblance between the results obtained with the higher Cu concentration and with those observed for Zn. In both cases, there was an immediate and transient stimulation of GPOD capacity from 2 to 48h. During the 2 following days, there was no significant eilect anymore but a second induction of GPOD capacity occurcd starting from day 8

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1. C. Pech et al. (eds.!, Cellular and Molecular Aspects of the Plant Honnone Ethylene, 238-239. © 1993 Kluwer Academic Publishers.

239

after metal application. It is tempting to postulate that in these conditions of metal stress, at first both CAT and POD arc involved in scavenging hydrogen peroxide and/or organic peroxides formed during lipid pcroxidation and that later on only POD intervenes.

The thiobarbituric acid (TBA) assay can be regarded as a reliable method for evaluating the degree of lipid oxidation (Kosugi and Kikugawa 1989). We analyzed TBA-reactive metabolites (rm) and ethane, another end product of lipid peroxidation. For 50 ppm Cu, the TBA-rm content in the leaves was higher than in the control from 6 until 72h. For 100 ppm Cu, the TBA-rm concentration was enhanced from 2 until 96h. These results suggest that after membrane peroxidation during a limited period the repair mechanism is effective in the given conditions of Cu stress. Measurements of ethane production confilm this conclusion since there was no significantly higher ethane production after both Cu treatments. In Zn plants, the TBA-rm content was higher than in control plants after 24h and throughout the investigated period a steady increase was observed. Ethane production also increased from 96h on. Defense mechanisms seem not to be suHicient to overcome the toxicity caused by this Zn treatment.

The first effects observed after Cu addition to the roots of the bean seedlings were stimulation of ethylene production, of CAT capacity and with the highest Cu dosis also of POD capacity. The induction of thc~~e scavenging systems for oxygen active species suggest that the enhanced ethylene production could be due to oxygen free radicals, chemically formed by Cu, that stimulate the conversion into ethylene of ACC present in excess. Treatment of the plants with 100 ppm Cu also resulted in a stimulatory eflect on LOX capacity, but this response was observed after the induction of ethylene production. This c~U1 be ,Ul indication for the possible interference of ethylene with the peroxidative pathway. The first clTccts observed after Zn application were stimulation of LOX, CAT and POD capacity. Induction of ethylene production was a later effect. The strong stimulation of the LOX capacity by Zn could increase the production of oxygen free radicals. Part of these radicals might escape the enzymatic scavenging system (CAT, POD) and might therefore stimulate the conversion of ACC into ethylene. However a possible direct interaction of Zn with the methionine pathway of ethylene biosynthesis cannot be excluded.

The authors th~mk Mrs. C. Bogaert- Yanherle for skilful technical assistance. This study was supported by the' lnstituut voor de Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw '(J.W.O.NL), BrusscJs, Belgium.

Kosugi, H. and Kikugawa, K. (1989) 'Potential thiobarbituric acid-reactive substances in peroxidized lipids', Free Radical Biology & Medicine 7, 205-207.

McKersie, B.D., Senaratna, T., Walker, M.A., Kendall, E.1. and Hetherington, P.R. (1988) 'Deterioration of membranes during aging in plants: evidence for free radical mediation', in Nooden, L.D. and Leopold, A.c. (cds.), Senescence and Aging in Plants, Academic Press, San Diego, pp. 441-464.

Nooden, L.D. (1988) 'The phenomena of senescence and aging', in Nooden, L.D. and Leopold, A.C. (cds.), Senescence and Aging in Plants, Academic Press, San Diego, pp. 1-50.

Thompson, J.E., Mayak, S., Shinitzky, M. and Halevy, A.H. (1982) 'Acceleration of membrane senescence in cut camation 110wers by treatment with ethylene', Plant Physiol. 69, 859-863.

Thompson, J.E. (1988) 'The molecular basis for membrane deterioration during senescence', in Nooden, L.D. and Leopold, A.C. (cds.), Senescence and Aging in Plants, Academic Press, San Diego, pp. 51-83.

HEAVY METAL INDUCTION OF ETHYLENE PRODUCTION AND STRESS ENZYMES: II. IS ETHYLENE INVOLVED IN THE SIGNAL TRANSDUCTION FROM STRESS PERCEPTION TO STRESS RESPONSES?

ABSTRACf

J. V ANGRONSVELD, J. WECKX, M. KUBACKA-ZEBALSKA AND H. CLIJSTERS Limburgs Universitair Centrum. Dep. SBG Universitaire Campus B-3590 Diepenbeek Belgium

The potential involvement of ethylene in the metal induced effects on membranes and enzymes was evaluated. Exogenous ethylene increased membrane permeability and the capacity of several enzymes. Kinetical comparison of the responses of ethylene production, membrane permeability, membrane degradation products and enzyme capacities on toxic metal concentrations delivered arguments against a mediator role of ethylene. After addition of a toxic zinc concentration to intact dwarf bean (Phaseolus vulgaris L. cV. Limburgse vroege) seedlings, the increase in ethylene production was preceeded by several of the other responses. Additional arguments against a mediator role of ethylene in these responses was provided by the effects of exogenous ethylene on the isoperoxidase patterns. Ethylene treatment intensified the staining of the isoperoxidases present in both control and metal treated plants, but was not able to induce metal specific Dl and D2 isozymes when no metals were supplied. Obviously, ethylene has a consolidating effect on the responses, but is not the triggering factor.

1. Introduction

Several reports show that toxic concentrations of various metals stimulate ethylene production in plants (Lau and Yang 1976; Goren and Siegel 1976; Hogsett et al. 1981; Rodecap et al. 1981; Fuhrer 1982; Gora and Clijsters 1989). Toxic metal concentrations also affect the activity of several enzymes. Depending on the enzyme considered, inhibition (e.g. enzymes related to photosynthesis) as well as increase in capacity of other enzymes (e.g. peroxidase, enzymes of the secundary metabolism) were described (for a review, see Van Assche and Oijsters 1990). The induction of the capacity of a particular group of enzymes (POD, CAT, SOD, NAD(P)+ -reducing enzymes is considered to play an important role in the cellular defence strategy against oxidative stress, caused by toxic metal concentrations. Toxic amounts of metals in the substrate result in the appearance of specific isoperoxidases (Van Assche and Oijsters 1990). The primary target for metal action at the cellular level might be the plasmamembrane since this barrier (including its associated proteins) is the first functional structure encountered by a metal ion penetrating into the cell. According to the nature of the metal it interferes with the membrane proteins and/or lipids and induces membrane destabilization. This effect generally is attributed to lipid peroxidation caused by an increased production of toxic oxygen free radicals (Kellog and Fridovich 1975; Mayak et al. 1983). Metals as copper and mercury which perform one electron oxidoreduction reactions easily induce free radical formation. For non redox active metals (e.g. zinc), free radicals can be formed by mediation of lipoxygenase (LOX). LOX mediates polyunsaturated fatty acid oxidation and produces free radicals; in their tum these free radicals cause membrane destruction. Free radicals appear to be involved in both membrane deterioration and ethylene biosynthesis. They should interfere with the conversion of ACC to ethylene (McRae et al. 1982). It was also

240


241

suggested that LOX could be involved in this reaction (Bousquet and Thimann 1984; Kacperska and Kubacka-Zebalska 1985). Ethylene stimulates the activity of peroxidase (Morgan and Fowler 1972; Miller et al. 1985; Hagege et al. 1988, Abeles et al. 1988, 1989). Several authors suggested that ethylene acts on membrane permeability (Hanson and Kende 1975; Mayak et al. 1977) and modifies membrane lipid composition (Thompson et al. 1982). Paulin et al. (1985) demonstrated that application of AOA, an inhibitor of ethylene biosynthesis, delayed the efflux of electrolytes from petals of cut carnations. The close relationship between these metal and ethylene responses leads to the speculation about the sequence of events. It is tempting to develop the hypothesis that ethylene, which generally shows a quick response to metal treatment, possibly plays a key role in the signal transduction chain from the metal (the 'elicitor') to a series of responses. Several experiments were performed to test the validity of this hypothesis. In intact bean seedlings, kinetics of responses after metal application on a time course basis as well as the effects of exogenously applied ethylene were investigated for ethylene production; enzyme capacities and membrane permeability. The time course experiments after metal application are described by Weckx et al. (1993). In this paper, effects of a 72 h lasting exogenous ethylene (1 ppm) application were studied on morphology, membranes (permeability and peroxidation products), enzymes capacities (peroxidase, NAD(P)H-producing enzymes) and isoperoxidase patterns in dwarf beans. From these isoperoxidase patterns essential information can be obtained about the role of ethylene in enzyme induction.

2. Material and methods

2.1. TEST PLANTS, METAL AND ETHYLENE TREATMENTS

Dwarf beans (Phaseolus vulgaris L. cv. Limburgse vroege) were grown on vermiculite under controlled environmental conditions (12h light, photosynthetic active radiation = 160J.Ullol m-2 s-l, 25°C, 65 % relative humidity; 12h darkness, 20°C, 65 % relative humidity). For control plants, the substrate was watered with a nutrient solution before sowing. Metals were supplied at the moment of sowing as zinc or copper sulphate in the nutrient solution. The metal concentrations cited in the text are final concentrations (v/v) in vermiculite. Exogenous ethylene was applied to 10 days old seedlings for 72 h. Plants were placed in glass cuvettes and supplied with a synthetic air mixture (N2:02, 80:20, enriched with 300 ppm C02 and Ippm ethylene). Control plants received synthetic air without addition of ethylene.

2.2. K+ -LEAKAGE AND TBA REACTIVE COMPOUNDS

To determine membrane permeability, discs (10 mm diameter) from primary leaves of 13 days old plants were rinsed in destilled water, thoroughly blotted and incubated for 3 h in Millipore water. Potassium content of the incubation medium was determined using flame AAS. For determination of TBA reactive compounds, plant material (1g) was homogenized with 5 ml TCA (trichloroacetic acid). The homogenate was centrifugated (15000 rpm, 10 min). 1 ml of the supernatant was added to 4 ml 0.5 % TBA (thiobarbituric acid) dissolved in 20 % TCA. This reaction mixture was heated to 95°C during 30 min, then cooled in ice and centrifugated (15000 rpm, 10 min). The difference in absorbance was measured between 532 and 600 nm.

2.3. (ISO-)ENZYME ANALYSIS

Frozen tissue was homogenized in an ice-cooled mortar in 0.1 M tris-HCl buffer at pH 7.8, containing ImM dithiotreitol and ImM EDT A. The homogenate was filtered through a nylon mesh and centrifugated for 10 min at 10.000 g and 4°C. On the supernatant (crude extract), the capacity (the potential activity measured in vitro under non-limiting reaction conditions) of the enzymes was measured spectrophotometrically as described by Van Assche et al. (1988). Enzyme capacity was

242

expressed in m U per gram fresh weight. Anionic peroxidases were separated by polyacrylamide gel electrophoresis on a 7.5 - 20 % gradient slab gel.

3. Results

3.1. MORPHOLOGICAL EFFECTS

Presence of metals in the substrate results in a significant growth inhibition compared to the control (Fig. 1). As expected, a 72 h application of 1 ppm ethylene to control plants also induces growth retardation. On a metal containing substrate, ethylene treatment causes reduction of the shoot elongation additional to the effect obtained with metals alone. Other mOIphological parameters (e.g. biomass of shoots, ... ) show similar responses (data not shown).

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Figure 1: Shoot length (cm) of 13 days old Phaseolus vulgaris grown on control, copper (100 ppm) and zinc (200 ppm) containing substrates after a 72 h treatment with exogenous ethylene (1 ppm). Given are the means for 20 seedlings of each treatment. (l = control; 2 = control + ethylene; 3 = copper; 4 = copper + ethylene; 5 = zinc; 6 = zinc + ethylene)

Presence of copper in the substrate results in an increased K + -leakage from leaf discs taken from the primary leaves (Fig. 2). Ethylene treatment of control plants shows a similar effect. A nearly additive response is obtained after combined copper and ethylene treatment. Zinc on the contrary has no effect on membrane permeability, but a combined zinc and ethylene treatment results in more than doubling of the K+ -leakage. This effect of ethylene, exogenously applied to intact plants, on membrane permeability is also observed in short term experiments on isolated leaf discs. Treatment of these discs from 12 days old bean seedlings with 1 ppm ethylene results in a more than 5-fold increase in K-leakage already after 24 h (data not shown). These results are fully confirmed by the levels of membrane peroxidation products in primary leaves after the treatments described (Fig. 3). Separate ethylene and copper application induce an increased level of TBA reactive products. Zinc only exerts a limited effect. Ethylene intensifies the copper effect and an additive response is observed, while combination of zinc and ethylene causes a much higher level of membrane peroxidation products.

3.3. EFFECTS ON ENZYME CAPACITIES

Presentation of results on enzyme capacities will be limited to a few representative examples for both primary leaves and roots. It was already reported that in an intact seedling zinc exerts its major stimulative effect on peroxidase at the level of the leaves, while copper mainly induces the stress peroxidases in the root (Van Assche and Clijsters 1990). In the primary leaves, G-POD (guaiacol peroxidase) capacity strongly increases after ethylene treatment of control plants (Fig. 4). As expected, copper has only a very limited effect on G-POD, while zinc induces a 100 % increase of the capacity of this enzyme.

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Figures 2 - 7: Responses of 13 days old Phaseolus vulgaris grown on control, copper (100 ppm) and zinc (200 ppm) containing substrates after a 72 h treatment with exogenous ethylene (I ppm). (I = control; 2 = control + ethylene; 3 = copper; 4 = copper + ethylene; 5 = zinc; 6 = zinc + ethylene). Fig. 2: Kleakage (mg/I) in primary leaves; Fig. 3: TBA- reactive products (!J.Ng FW) in primary leaves; Fig. 4: G-POD capacity (mU/g FW) in primary leaves; Fig. 5: G-POD capacity (mU/g FW) in roots; Fig. 6: G6PDH capacity (mU/ g FW) in primary leaves; Fig. 7: GIDH capacity (mU/g FW) in roots. Given are means ± SD of 3 (Fig. 2 and 3) or 5 measurements (other figures).

244

After ethylene treatment of plants grown on a copper containing substrate, G-POD capacity reaches the same levels as after ethylene treatment alone. As already observed for the effects on K-Ieakage and TBA reactive compounds, ethylene treatment of zinc grown plants results in a massive increase of the G-POD capacity. In the roots, the response is different from what was observed in the primary leaves (Fig. 5). Roots of plants grown on a copper containing substrate show in increase of G-POD capacity of more than 50 % as compared to the control. Zinc has only a very slight effect. As was observed in the primary leaves, treatment of control plants with exogenous ethylene again results in an important increase of the G-POD capacity in the roots. However, in contrast to the leaves, ethylene treatment of copper plants induces a much higher increase of enzyme capacity in the roots than could be observed for plants grown on the zinc containing substrate. Although in the latter plants the increase induced by ethylene is significant, this difference in response to ethylene between leaves and roots of zinc and copper treated plants suggests an organ specific reaction. As examples for responses of NAD(P)+ -reducing enzymes, results. are shown for glucose-6-phosphate dehydrogenase (G6PDH) capacities in the primary leaves (Fig. 6) and glutamatedehydrogenase (GlDH) in the roots (Fig. 7). Responses are very similar to those of GPOD.

primary leaf iso-POD root iso-POD

type C C* Cu Cu* Zn Zn* C C* Cu Cu* Zn Zn*

Al + ++ + ++ + ++ + ++ + ++ + ++

C1 + ++ + ++ + ++ + ++ + +++ ++ C3 -/+ + -/+ + -/+ ++ -/+ + -/+ + -/+ +

D1 + ++ + +++ D2 + ++ + +++

E1 + ++ + ++ + ++ E2 + + + + + +

F1 + ++ ++ +++ ++ ++ F2 + ++ ++ +++ ++ ++

Table 1: Summary of the patterns of the anionic isoperoxidases present in primary leaves and roots of 13 days old Phaseolus vulgaris seedlings after growing on a copper or zinc containing substrate and after a 72 h ethylene treatment. C = control; Cu = 100 ppm Cu; Zn = 200 ppm Zn; * = 100 ppm ethylene treatment during 72 h. +++ = very strong staining intensity; ++ = strong staining intensity; + = detected; +/- = detected with poor staining intensity; - = not detected.

3.4. EFFECTS ON ISOPEROXIDASE PATTERNS

A total of 9 different anionic isoperoxidases were studied and the results are summarized in table 1. POD isozyme profiles are different for primary leaves and roots. In the primary leaves of control plants, only 3 different isozymes are detected (AI, CI, C3). Ethylene treatment of these plants causes an important increase in staining intensity of these 3 isoperoxidases, confirming the increase in capacity described above. Moreover a new isozyme appears (E2). For plants grown on the

245

copper containing substrate, the isoperoxidase pattern is comparable to that of the control. Ethylene intensifies the bands already present and induces the E2 band. The primary leaves of seedlings grown on the zinc containing substrate however contain 2 supplementary metal induced D-type isoperoxidases. Ethylene treatment of the latter plants again results in an important increase in staining intensity of all isozyme types already present and, once again, in the appearance of the E2-type peroxidase. However, the metal induced D-band isoperoxidases do never appear after ethylene treatment only. A very similar response was found for the root isozyme patterns. The only remarkable difference is that in this organ not zinc but copper induces the synthesis of D-type isoperoxidases. Ethylene again intensifies the staining intensity of the different isoperoxidase bands and induces the appearance of an E2-type. However, application of ethylene without copper never induces the Dband isozymes.

4. Discussion

Two different observations demonstrate that both copper and ethylene separately induce modifications at the membrane level. In the primary leaves, membrane permeability (Fig. 2) and the content ofTBA reactive compounds (Fig. 3) both signficantly increase after ethylene treatment of control plants and in plants grown on a copper containing substrate. Combined copper and ethylene application results in an additive stimulation of membrane permeability and peroxidation. Zinc however does not affect the membranes, but a combined treatment of the seedlings with zinc and ethylene has a highly synergistic effect on both membrane parameters. It is clear that ethylene has a stimulatory effect on several stress enzymes in primary leaves and in roots. The capacities of G-POD (Fig. 4 and 5) and G6PDH (Fig. 6 and 7) are strongly increased after ethylene treatment. The results obtained for the effects on membrane destabilization and enzyme capacity do not exclude a role of ethylene as a mediator in the signal transduction chain. This hypothesis however is contradicted by the data of the kinetical comparison of the responses and by the isoperoxidase patterns. Kinetics of the responses (Weckx et al. 1993) deliver arguments against a causal relationship between the effects of metal toxicity on ethylene production, membrane destabilization and enzyme induction. After copper supply, a very quick increase of ethylene release was observed, but application of zinc, increased ethylene production only 48 h after metal supply. This increase did neither coincide with nor preceded the appearance of the responses at the membrane or enzymes level. Several of these responses already occured before stimulation of ethylene production. Arguments against a possible mediator role of ethylene in signal transduction after metal toxicity are also provided by the study of the isoperoxidase patterns (Table 1). Ethylene treatment intensifies the staining of the isoperoxidases present in the control and in metal treated plants. but is not able to mimic the effects of metals on the isozymes patterns. The metal induced Dl and D2 isoperoxidases found in the primary leaves after zinc toxicity and in the roots of copper treated plants are not induced by ethylene only, even after a 72 h treatment with the gas. When these isozymes are induced by the metal, ethylene is able to increase their intensity, but additional factors seem to be indispensable for the induction itself. Ethylene does not trigger the process, but has an consolidating effect on it. The E2 isoperoxidase band normally only appears in the primary leaves of older plants. Its induction by ethylene confirms the role of this compound in accelerating plant senescence. Ethylene and metal stress induce similar changes at the membrane and enzyme level. However, the presented results suggest that the metal effects are not mediated by metal stimulated ethylene production.

Acknowledgement: The authors thank Mrs. C. Gielen-Put and S. Claes for skilful technical assistance.

246

s. References

Abeles, F.B., Dunn, LJ., Morgens, P., Callahan, A., Dinterman, R.E., Schmidt, J. (1998) Induction of 33 kD and 60 kD peroxidases during ethylene-induced senescence of cucumber cotyledons. Plant Physioi. 87, 609-619.

Bousquet, J.F. and Thimann, K. (1984) Lipid peroxidation forms ethylene from 1-aminocyclopropane-1-carboxylic acid and may operate in leaf senescence. Proc. Natl. Acad. Sci. USA 81, 1742-1747.

Dhindsa, R., Plumb-Dhindsa, P., Thorpe, T. (1981) Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 126,93-101.

Fuhrer, J. (1982) Ethylene biosynthesis and cadmium toxicity in leaf tissue of beans (Phaseolus vulgaris L.). Plant Physioi. 70, 162-167.

Gora, L. and Clijsters, H. (1989) Effect of copper and zinc on the ethylene metabolism in Phaseolus vulgaris L. In: Clijsters et al. (eds.) Biochemical and physiological aspects of ethylene production in lower and higher plants. Kluwer Academic Publishers, Dordrecht, pp. 219-228.

Goren, R. and Siegel, S.M. (1976) Mercury-induced ethylene formation and abcission in Citrus and Coleus explants. Plant Physioi. 57,628-631.

Hagege, D., Kevers, c., Boucaud, J., Gaspar, T. (1988) Activites peroxydasiques, production d'ethylene, lignification et limitation de croissance chez Suaeda maritima cultive en I'absence de NaCI. Plant Physioi. Biochem 26, 609-614.

Hanson, A.D. and Kende, H. (1975) Ethylene enhanced ion and sucrose efflux in morning glory flower tissue. Plant Physioi. 55, 663-669.

Hogsett, W.E., Raba, R.M., Tingey, D.T. (1981) Biosynthesis of stress ethylene in soybean seedlings: similarities to endogenous ethylene biosynthesis. Plant Physioi. 53, 307-314.

Kacperska, A. and Kubacka-Zebalska, M. (1985) Is Iipoxygenase involved in the formation of ethylene from ACC? Physioi. Plant. 64, 333-338.

Kellogg, E.W. and Fridovich, 1. (1975) Superoxide, hydrogen peroxide and singlet oxygen in lipid peroxidation by a xanthine oxidase system. J. BioI. Chern. 250, 8812-8817.

Lau, O. and Yang, S.F. (1976) Stimulation of ethylene production in mung bean hypocotyls by cupric ion, calcium and kinetin. Plant Physioi. 57, 88-92.

Mayak, S., Legge, R.L., Thompson, J.E. (1983) Superoxide radical production by microsomal membranes from senescing carnation flowers: an effect of membrane fluidity. Phytochemistry 22, 1375-1380.

Mayak, S., Vaadia, Y., Dilley, D.R. (1977) Regulation of senescence in carnation (Dianthus caryophyllus) by ethylene: mode of action. Plant Physioi. 59, 591-593.

Mc Rae, D., Baker, J .E., Thompson, J .E. (1982) Evidence for involvement of the superoxide radical in the conversion of l-aminocyclopropane-I-carboxylic acid to ethylene by pea microsomal membranes. Plant Cell Physioi. 23, 375-383.

Miller, A.R., Crawford, D.L., Roberts, L.W. (1985) Lignification and xylogenesis in Lactuca pith explants cultured in vitro in the presence of auxin and cytokinin: a role for endogenous ethylene. J. Exp. Bot. 36, 110-118.

Morgan, P.W. and Fowler, J.L. (1972) Ethylene modification of peroxidase activity and isozyme complement in cotton (Gossypium hirsutum L.). Plant Cell Physiol. 13,727-736.

Paulin, A., Bureau, J.M., DroiJIard, MJ. (1985) Influence de I'acide oxyaminoacetique sur la senescence de I'oeillet coupe. C.R. Acad. Sci. Paris Sec. III 300, 301-304.

Rodecap, K.D., Tingey, D.T., Tibbs, J.H. (1981) Cadmium-induced ethylene production in bean plants. Z. Pflanzenphysiol. 105,65-74.

Thompson, J.E., Mayak, S., Shimitzky, M., Halevy, A.H. (1982) Acceleration of membrane senescence in cut carnation flowers by treatment with ethylene. Plant Physiol. 69, 859-863.

Van Assche, F. and Clijsters, H. (1990) Effects of metals on enzyme activity in plants. Plant, Cell and Environment 13, 195-206.

Weckx, J., Vangronsveld, J. and Clijsters H. (1993) Heavy metal induction of ethylene production and stress enzymes: 1. Kinectics of the responses. In: Cellular and molecular aspects of the plant hormone ethylene (eds. J.C. Pech, A. Latche and C. Balague). Kluwer Academic Pyblishers Dordrecht.

FLOODING RESISTANCE AND ETHYLENE. I. AN ECOPHYSIOLOGICAL APPROACH , WITH RUMEX AS A MODEL

J.G. RIJNDERS', L.A.C.J. VOESENEK, A.J.M. VAN DER SMAN and C.W.P.M. BLOM Department of Ecology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, the Netherlands.

As an ecophysiological research group, we are concerned with the occurrence and distribution of plant species in riparian areas of the Rhine delta in the Netherlands. The downstream flood plains are characterized by differences in elevation, leading to a distinct flooding gradient and a typical plant zonation. In order to elucidate this zonation, investigations are in progress with different species of the genus Rumex, each having a specific growth location within the flooding gradient. Rumex maritimus, and R. palustris occur at the lowest elevation level in the river foreland. R. thyrsiflorus and R. acetosa are found on higher and rarely flooded grounds while R. crispus and R. obtusifolius take an intermediate position. The central hypothesis is that the observed Rumex zonation is mainly determined by the degree of resistance to flooding [2].

Adaptations of Rumex species towards waterlogging and submergence are: (i) the development of new adventitious roots, (ii) aerenchyma formation [2,4] and (iii) enhanced shoot elongation [1,5]. Ethylene plays a central role in the initiation and regulation of these adaptive responses. An example of the petiole elongation upon total submergence of four Rumex species is given in fig. 1. Petioles of the flooding-tolerant R. maritimus and R. palustris elongate most, R. crispus shows moderate elongation, while R. acetosa shows no elongation.

Petiole growth :--____________ ---,

('.5 of air contrOl)

Fig. 1. The elongation of the youngest petioles of four Rumex species, as percentage of the growth in air (± 1 SE; n=10/12). Plants were submerged in tap water or exposed to 0.5 Pa ethylene for 4 days. Mean actual length (cm) in air and leaf number (between brackets): R. maritimus=2.1 (6) R. palustris=2.2 (5); R. crispus=2.5 (4); R. acetosa=7.6 (4).

247

J. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plant Honnone Ethylene. 247-248. © 1993 Kluwer Academic Publishers.

248

The submergence response can be mimicked by treatment with 0.5 Pa ethylene (fig. 1). The reduction in response to ethylene treatment compared with submergence, is most likely due to an absence of CO2 in the ethylene/air mixture. All Rumex species in fig. 1 show an elevated internal ethylene concentration during submergence compared to the air control.

Experiments, both in the field and under laboratory situation, are conducted in all life stages of the plants. Upon submergence during the vegetative stage, the petioles of the rosette leaves of the flooding resistant species elongate the most. When submerged at the fullgrown rosette stage, plants of R. palustris postpone their flowering and elongate the petioles of the rosette leaves, while R. maritimus plants commence bolting and elongate the stem internodes, appearing to be obligated to flower once fullgrown [3]. Differences in response not only occur between species or life stages but even within the development of the individual leaf. Petioles of young, furled leaves of R. palustris elongate more than petioles of fully expanded leaves.

These results indicate an ethylene response, regulated by sensitivity towards this hormone, whether direct or via other plant growth substances.

Tabel 1. Length (mm) of Rumex palustris petioles after 96 h treatment with or without paclobutrazol. Plants where watered four days in advance with 1.0 JlM and submerged in 0.1 JlM paclobutrazol (± 1 SE; n=9/14).

treatment

shoot in air submerged 0.5 Pa ethylene submerged + 5 JlM GA3

paclobutrazol +

11.1 (0.8) 39.1 (0.7) 31.5 (2.1)

11.8 (0.9) 29.8 (0.6) 23.9 (1.0) 39.8 (0.9)

Present research is focussed on the involvement and timing of action of auxin and gibberellin in the elongation of the rosette petioles. The elongation of petioles of R. palustris upon submergence reduces when plants are treated with paclobutrazol, a inhibitor of gibberellin synthesis, and can be restored by supplementation of 5 IA-M GA3 (tabel 1). In addition, investigations with respect to the kinetics of the ethylene production [1] and the formation of adventitious roots and aerenchyma are in progress [4].

1. Banga, M., Voesenek, L.AC.J. and Blom, C.W.P.M. (1992) 'The role of ethylene in shoot elongation of Rumex plants in respons to flooding', Proceedings of this symposium.

2. Blom, C.W.P.M., Bogemann, G.M., Laan, P., Van der Sman, AJ.M., Van der Steeg, H.M. and Voesenek, L.AC.J. (1985) 'Adaptations to flooding in plants from river areas', Aquat. Bot. 38, 29-47.

3. Van der Sman, AJ.M., Voesenek, L.ACJ., Blom, C.W.P.M., Harren, FJ.M. and Reuss, J. (1991) 'The role of ethylene in shoot elongation with respect to survival and seed output of flooded Rumex maritimus L. plants', Func. Ecol. 5, 304-313.

4. Visser, E.J.W., Voesenek, L.AC.J., Harren, FJ.M. and Blom, C.W.P.M. (1992) 'Application of an advanced laser-driven photoacoustic technique in ethylene measurements on flooded Rumex plants', Proceedings of this symposium.

5. Voesenek, L.AC.l, Harren, F.J.M., 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.

FLOODING RESISTANCE AND ETHYLENE. II. APPLICATION OF AN ADVANCED LASER-DRIVEN PHOTOACOUSTIC TECHNIQUE IN ETHYLENE MEASUREMENTS ON FLOODED RUMEX PLANTS.

E.J.W. Visser\ L.A.C.J. Voesenek\ F.J.M. Harren2 and C.W.P.M. Blom!. Departments of Ecolog/ and Molecular and Laser Physic;, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, The Netherlands

Flooding is an extremely severe stress condition for many higher plants. Species which occur in frequently submerged or waterlogged habitats often show a number of morphological and anatomical adaptations, which avoid the hazard of hypoxia or anoxia during a prolonged period of flooding. Common examples of these adaptations are petiole and stem elongation [5], which enable the submerged leaves to reach the water surface, and the development of aerenchymatous tissues, through which atmospheric oxygen can be transported from the emerging leaf tips to the submerged parts of the plant [2]. In some species of the genus Rumex the accelerated growth of submerged petioles starts within hours after the onset of flooding (fig. lA). Ethylene is found to accumulate in the shoot during submergence [1,2] and has a strong promotive effect on petiole elongation (fig. IB, [5]).

30

20

10

Growth of petiole (mm) A

s merg

control

°0~~~5~--~10~--~15~--~2~0----~2~5----~30

Time (h)

30

20

10

ethylene submergence

Treatment

Fig. lA. Time course of elongation of young petioles of R. palustris upon submergence. Upper lines: submerged; lower line: drained control. Age of the plants was 26 days; B. Elongation of young petioles of R. palustris upon submergence or exposure. to 5 ppm ethylene for 6 days. Age of the plants was 26 days; n=9; bars represent SE.

Oxygen levels in the flooded soil decrease within a few hours to hypoxic or even anoxic levels, mainly as a result of the high oxygen consumption rate of microorganisms and plant roots. In response to this limited soil oxygen availability, flooding-adapted Rumex species develop a large number of highly porous, adventitious roots [2], which start to emerge from the cortex of the tap root within one or two days after the onset of flooding (fig. 2A). Besides other hormones,

249

1. C. Pech et al. (eds.), Cellular and Molecular Aspects of the Plant HomlOne Ethylene, 249-250. © 1993 Kluwer Academic Publishers.

250

ethylene appears to be involved in the regulation of adventitious root formation, since application of ethylene to aerated root systems of plants on hydroculture caused adventitious root formation to a comparable extent as under low oxygen concentrations (fig. 2B).

120

100

80

40

20

Number of adventi tious roots A

°0~~~===1~------~2--------~3--------~4-1 Time after onset of waterlogging (days)

ethylene 1 % oxygen

Treatment

Fig. 2A. Time course of adventitious root formation of R. palustris upon waterlogging. Age of the plants was 70 days; n= 4; bars represent SE; B. Adventitious root formation of R. palustris in hydroculture after 8 days of exposure to hypoxic conditions (1 % oxygen) or ethylene (10.0 ppm). Age of the plants was 49 days; n= 4; bars represent SE.

The current methods of ethylene detection all depend on accumulation of the gas, which gives only a global insight into the timing of the mentioned ethylene mediated processes in Rumex. Therefore, a technique is required which can detect the very low concentrations of ethylene, released by a plant in the gas flow of a flow-through system. Furthermore, measurements should succeed each other within minutes or even seconds to get a most accurate view of the timing of a process. A technique, which depends on the photoacoustic principle has shown to overcome both problems [3]. The photoacoustic effect occurs when a frequently interrupted light beam, in this case a laser beam, hits a gas sample. At an appropriate laser wave length, ethylene molecules will be excitated by the light. Due to this excitation, the kinetic energy of the gas molecules increases during every light pulse, and decreases during the interruption of the beam, thus creating small pressure changes. These pressure changes are recorded as acoustic waves. The concentration of ethylene in the sample flow is reflected in the amplitude of the signal. In this way, we have achieved a sensitivity of 0.03 ppb ethylene under average experimental conditions. In addition, measurements can be made every minute. This method made it possible to establish the exact time course of ethylene evolution from the shoot during de-submergence, while at present ethylene production of flooded roots is under investigation.

1. Banga, M., Blom, C.W.P.M., Van der Sman, A.J.M., Voesenek, L.A.C.J. and Harren, F.J.M. (1992) 'flooding resistance and ethylene. III. The role of ethylene in shoot elongation of Rumex plants in response to flooding', this book.

2. Blom, C.W.P.M., B6gemann, G.M., Laan, P., Van der Sman, A.J.M., Van de Steeg, H.M. and Voesenek, L.A.C.J. (1990) 'Adaptations to flooding in plants from river areas', Aquatic Botany 38, 29-47.

3. Harren, FJ.M., Bijnen, F.G.C., Reuss, 1., Voesenek, L.A.CJ. and Blom, C.W.P.M. (1990) 'Sensitive intracavity photoacoustic measurements with a CO, waveguide laser', Applied Physics B 50,137-144.

4. Rijnders, J.G., Voesenek, L.A.c.J., Van der Sman, J.M. and Blom, C.W.P.M. (1992) 'Flooding resistance and ethylene. I. An ecophysiological approach with Rumex as a model', this book.

5. Voesenek, L.A.c.J. and Blom C.W.P.M. (1989) 'Growth responses of Rumex species in relation to submergence and ethylene', Plant, Cell and Environment 12, 433-439.

FLOODING RESISTANCE AND EmYLENE. III. mE ROLE OF EmYLENE IN SHOOT ELONGATION OF RUMEX PLANTS IN RESPONSE TO FWODING.

M. Banga', L.A.C.J. Voesenek and C.W.P.M.Blom Department of Ecology, University of Nijmegen, Toernooiveld, 6525 ED Nijmegen, the Netherlands.

In river forelands in the Netherlands several Rumex species occur. R. palustris is found in lower areas that are frequently flooded during the growing season, R. acetosa and R. thyrsiflorus occur on higher, seldomly inundated places such as dykes [1], while R. acetosella is restricted to dry sandy soils (e.g. slopes of terraces) that remain unflooded.

III

8

Q) 4 rn o Q)

Q) '-

Q) 2 C Q)

>. ..r:: ..... W

o

Onset of treatment

25 26 27 28

Plant age (days)

- Control ............... Waterlogged -- Submerged

29 30 31

Figure 1. Kinetics of ethylene release of R. palustris in response to three flooding treatments, measured by the laser-driven photoacoustic detection system.

251


252

R. palustris is resistant to prolonged periods of flooding. This resistance can be attributed to several adaptations. One of these is the ability to enhance elongation growth of rosette leaves during partial or total submergence. This phenomenon enables the plant to restore contact with the atmosphere. The elongation process is regulated by the gaseous hormone ethylene [2,4]. The ethylene physiology of R. palustris was compared with that of R. acetosella, which does not show any enhanced growth upon flooding. The release of ethylene in both Rumex species was continuously measured for a week with a laser-driven photoacoustic detection system [3,5]. At the start of the experiment the plants just developed their fifth leaf. Control plants were kept drained throughout the experiment and treated plants were either waterlogged or totally submerged. A representative ethylene release pattern of R. palustris is shown in figure 1. In contrast to the control treatment both flooding treatments cause a substantial release of ethylene, but totally submerged plants have a somewhat lower ethylene production than waterlogged plants. The internal ethylene concentration of totally submerged plants is nevertheless much higher than that of waterlogged plants. This is caused by the fact that the release of ethylene into a water phase is very slow compared with air, resulting in an accumulation of the hormone in totally submerged plants. As a consequence, submerged plants show the highest shoot elongation rates. Preliminary results indicate that R. acetosella plants hardly produce ethylene. Therefore, high internal ethylene concentrations probably do not occur in this species. Further investigations will focus on the internal concentration and the sensitivity towards this hormone in both Rumex species.

1. Rijnders, J.G., Voesenek, L.A.C.J.,Sman, A.J.M. van der and BJorn, C.W.P.M. (1992) 'Flooding resistance and ethylene. I. An ecophysiological approach with Rumex as a model', Proceedings of this symposium.

2. Sman, A.1.M. van der, Voesenek, L.A.C.J.,BJom, C.W.P.M.,Harren, F.J.M. and Reuss, 1. (1991) 'The role of ethylene in shoot elongation with respect to respect to survival and seed output of flooded Rumex maritimus L. plants', Funct. Ecol. 5,304-313.

3. Visser, E.J.W., Voesenek, L.A.C.l.,Harren, F.l.M. and BJorn, C.W.P.M.(1992) 'Flooding resistance and ethylene. II. Application of an advanced laser-driven photoacoustic technique in ethylene measurements on flooded Rumex plants', Proceedings of this symposium.

4. Voesenek, L.A.C.J. and BJom, C.W.P.M. (1989) 'Growth responses of Rumex species in relation to submergence and ethylene', Plant Cell Environ. 12,433-439.

5. Voesenek, L.A.C.l.,Harren, F.l.M., Bogemann, G.M., BJorn, C.W.P.M. and Reuss, 1. (1990) 'Ethylene production and petiole growth in Rumex plants induced by soil waterlogging. The application of a continuous flow system and a laser driven intracavity photoacoustic detection system' , Plant. Physiol. 94,1071-1077.

EFFECT OF SALINE STRESS ON GROWTH OF lYCOPERSICON ESCUlENTUM PLANTS AND ITS RELATION WITH ENDOGENOUS ETHYLENE METABOLISM.

F. BOTEllA; J.A. DEL RIO; A. ORTUNO. Dpto. Biolog'a Vegetal, Univ. de Murcia, Espinardo, 30071 Murcia, Spain.

Salinity is known to affect tomato production in arid and semi arid land. Considerable genetic variation in salt tolerance exists among tomato cultivars and many efforts have been made to improve this tolerance by conventional means. There have been very few satisfactory results along this lines and there is and urgent need for developing physiological or biochemical methods for rapid detection of salt tolerant genotypes. Ethylene has been shown to be involved in plant growth and its metabolism may be strongly influenced by stress. For this reason we consider a study of the possible role of ethylene in the control of tomato plant growth in saline conditions to be of scientific and commercial interest.

Plants of lycopersicon esculentum were grown in a semihydroponic culture system (silica sand as substrate) and submitted to salt stress with two different NaCl concentrations in the nutrient solution (80 and 160 mM). At different ages, stem length of control and treated plants was measured. At the same time, cross sections of stem were used to determine the characteristics of cell growth in the different tissues according to [1] and [2]. The whole stem of control and treated plants was cut in 0.5 cm sections and a random sample was selected and inmediately enclosed in a glass vial to determine ethylene production during 5h by GC. EFE activity was estimated as described in [3]. Free ACC was extracted [4], a fraction of this extract was hydrolysed [5] and in the two extracts total ACC was determined [6]. The plants growing with 80 mM NaCl only showed a delay in the stem growth rate up to six days after treatment. The final stem length is similar to control plants. The higher saline level lead to a strong reduction in plant growth (Figure 1). On the other hand, the reduction in stem tissues thickness is mainly due to reduct ions in the vascu 1 art issues and ina 1 esse r deg ree to the cortex tissue (data not shown). An inc rease in ethy 1 ene product i on was detected in st ressed plants when compared with the control ones (Figure 2). After the 5th day of treatment with 80 mM NaCl solution, production of

253


254

endogenous ethylene decreased to control levels. The ini tial increase in ethylene production could explain the slower growth rate detected (see Figure 1). The increase of NaCl concentration (160 mM) in the nutrient solution, produced a new and larger increment in ethylene production over control plants (Figure 2). This increment could explain the inhibition of growth detected.

Figure 1. TIme couree of stem growth in control and atreeHd plante. Bare denote SE when larger than aymbola (n-3).

Figure 2. Ethylene production of stem of control aiicl stl"8llHd plante. Bare denote SE when larger

than symbols (n-3). OAr----------------------,

46.0 0-0_ e-elOmU A-AllOmU

3 4 II I 7 I I 10 11 12 13 Days after treatment

\.. 0..

I i 0.4

The increment of ethylene production observed 24 h after the begining of the 80 mM NaCl treatment (Figure 2) correlated with a high EFE activity and a low total ACC content (Table 1). The application of 160 mM NaCl did not produce a new increment of EFE activity probably due to adaptation phenomena. In this case only free ACC was detected. A conversion of conjugated ACC to free ACC could explain these results.

Table 1.- ErE activity and ACC content in to.ato stel. 80 III treataent las started IIhen plants lIere 60 d old. 160 II! treataent las started one leek later. The SE is indicated (n=3).

Plant Age Treatlent EFE Total ACC Free ACC Conjugated ACC (days) (naol g-lFlI) (nlol g-lFIl) (i) (i)

61 Control 1.9 ± 0.1 10.9 ± 1.2 58.8 41.2 80 II! lIaCl 2.6 ± 0.2 2.9 ! 0.1 11.0. 100.0

68 Control 6.9 ± 0.3 9.2 ± 0.2 34.6 65.3 80 II! NaCl 7.7! 0.1 9.6 ! 0.3 29.9 70.1

160 III NaC! 7.3 ± 0.2 5.6 ± 0.1 100.0 R.D.

References 1.-0rtuDo A., SAnchez-Bravo J., Acosta II., Sabater F. 1988. BioI. Plant 33: 81-90. 2.-0rtufio A., Sanchez-Bravo J., Moral JR., Acosta II., Sabater F. 1990. Physiol. Plant. 78:211-217. 3.-0rtuiio A., Del Rio JA., Casas JL., Serrano II., Acosta II., Sanchez-Bravo J. 1991. BioI. Plant. 33:81-90. 4.-Atta-Aly II.A., Saltveit 11K., Bobson GK. 1987. Plant Physiol., 83, 44-48. 5.-Bofflan U., Yang SF., IIcKeon T. 1982. Biochel. Biophys. Res. Couun., 104, 765-770. 6.-Lizada IIC, Yang S.Y. 1979. Anal. Biochel.,100:140-145.

ETHYLENE BIOSYNfHESIS IN "HAYWARD" KIWIFRUIT INFECfED BY BOTRYTIS CINEREA

N. NIKLlS*, E. SFAKIOTAKIS* and C. C. THANASSOULOPOULOS**. * Laboratory of Pomology, and **Laboratory of Plant Pathology,

Aristotle University Thessaloniki 54006, Greece.

ABSTRACf. It has been found from other studies that kiwifruit did not produce ethylene, if the fruit were stored below a critical range of temperature (1F-14.8°C), and this was attributed to the limitted ACC production. In this study, "Hayward" kiwifruit inoculated with BotIytis cinerea and stored at low temperatures (0° and lO°C) were found to produce considerable amount of ethylene and accumulated ACC which were found to be closely associated with the mycelial growth in the Botrytis infected tissues.

Materials and methods

Kiwifruit (Actinidia deJidosa) cv Hayward harvested from a commercial orchard were inoculated, with an isolate from kiwifruit of Botrytis cinerea maintained on PDA. The fruit were inoculated with a piece of agar (c. 5 mm) on which mycelium of B. . cinerea was grown, in two parallel punctures in the stemend area. The infected and the control (punctured and non inoculated) fruit were placed into 5 I jars each containing 15 fruits and representing one plot. The jars were placed in waterbaths adjusted to 0° or lOoC and a continues aeration (100 ml/min) was used. Ethylene concentration was measured in the gas phase in periodic intervals (1-4 days). Nine fruit samples were removed from each jar for measurements of fungus growth rate, C2li4 and ACC. Infected rotted flesh area was measured by ruler from the inoculation point to petal end in mm. From each fruit a tissue plunger (10 rom diameter X 65 mm length) taken longitudinally from the infected area and ehtylene production and ACC accumulation were measured by deviding the plunger in three sections. (A=0-15 rom, B=15-30 rom and C=30-45 mm).

Fig.!. Diagramatic scetch of a kiwifruit showing the points of inoculation, the infected area with BotIytis and the tissue area removed (with a plunger) devided in sections A, B and C for measurements of C2li4 and ACC production.


The results showed that: (1) The non inoculated kiwifruit did not produce ethylene and ACC at temperatures 0° and lOoC (Fig. 2A-I, 2A-II). (2) The infected fruits with B. cinerea at lOOC produced considerable amount of C2li4 55 days after inoculation (Fig. 2B-n), while those kept at O°C for 82 days (Fig. 2B-I) produced reduced amount of

255

J. C. Pech et af. (eds.), Cellular alld Molecular Aspects o/the Plant Honnone Ethylene, 255-256. © 1993 Kluwer Academic Publishers.

256

ethylene. Ethylene production was also high at O°C in the tissue plungers taken from the infected tissue and this production was associated with the fungus front (Fig. 2C-n). Reduced production but significantly above the control tissue was found in the infected fruit kept 55 days at O°C. Measurements of ACC showed accumulatlon of ACC at the front area of fungus in tissue plungers taken from infected fruit kept in both temperature (00 and lO°C). This finding gives evidence that the fungus is inducing ethylene biosynthesis in the infected kiwifruit throum the methionine pathway.

A-I O°C

i= ///?--;![; ~ o ....... ..,

o 20

2.0 B-1

~ 1.5

~ co

40 60

• ~ :. . ~ .. •

80

-.

100

::l 1.0 .a, .,. i!;i 0.5 o

\ Infected fruit

~ .. 0.00

1.5 C-I

~ . co ••

20 40 60 80 100

::> •• ! 1.0 ! .', ;- ____ .. Front C ~ : -', / +.' : ...... // ,a. Front B CD'" ........ / .,. .... ..... ~ 0.5 ... .;.~:.j.' Front A· ........

~ ! .ff\-.::.~:::= ......... :~.~.~.~.~.:~.~.~.~.~.~.~.~~:: Control

OQ~ ~ 40 60 00 100

1.0 0-1

0.8 ,. Front A , Ci

" ,

Ql :,'.'-"'--- .f_.-· ... Front B '0 0.6 ...... ., .. ~:;# /~ Front C E .s .

.. ..... - .. /-: .... # , · 0.4 · , . / 0 · ., . ..-' //

/ 0 · · < 0.2 · .'

,: .~ . .- ..... .-. """,J '"'.~.:::.:::."'.::: ........ .,: ......... ContrOl

0.00 20 40 60 80 100

A-II

20 30 40 50 10 B-II '. ,: ...... , 8 .. \ ... . ..

• It ~ \ ~fected fruit : -, : \

6

4 : -..

2 . ' ... , " Control ........... .

10 20 30 40 50

40

20

10 20 30 40 50

30 0-11

, .... , 20

10

---

.,. °'0, C Front

.,' .- __ °'-T;::::.::t , ~ ............ A Front

.,. ( ... ~ ... -"'" '0, . .' ~ '. ~ .. #' ~ ... ., .. , B Front

." / ,'rI' / ......... ' , Control

00 10 20 30 40 50 DAYS (after inoculation) DAYS (after-infection)

Fig. 2. Growth of Botrytis cinerea (A), ethylene production (B) in the infected and non infected fruits kept at 0° C for 55 days and at lOoC for 82 days, ethylene prodUction rates (C) and ACC accumulation (D) in tissue plungers taken from control and infected fruit (kept at 0° and 10°C) after 1 hour incubation in 50 ml erlenmeyer flasks.

ETHYLENE,STRESS AND ENZYMATIC ACTIVITIES IN HEVEA LATEX: THE DIVERSITY OF RESPONSES

J.C. PREVOT, A. CLEMENT, V. PUJADE-RENAUD, SISWANTO and J.L. JACOB IRCAICIRAD B.P.5035 34032 MONTPELLIER Cedex 1 FRANCE

Latex is a fluid cytoplasm[1] which contains high quantity of rubber (30 to 50% of its fresh weight). It is expeled by tapping from articulated laticiferous cells located in Hevea phloem. Ethrel (Ethephon), an ethylene releaser is used on a large scale in Hevea brasiliensis as a latex production stimulant[2]. So, 25 mg of this product mixed with 750 mg of palm oil are applied on the tapping cut of rubber tree, 48 hours before tapping. The treatment causes the extension of latex flow time and the activation of latex regeneration in laticiferous cells between two tappings. The activation is able to cause considerable and transitory modifications in the laticifers metabolism[3]. Study of the mechanisms involved in the modifications has shown that although certain biologically important enzymatic reactions are not (or) little influenced, others may be activated or slowed. An exemple of each case is described and replaced in its physiological context.

Pyrophosphate : fructose-6-phosphate I-phosphotransferase (PP-PFK, EC 2.7.1.90) catalyses reversibly the following reactions:

F-6-P + PPi ++ F-l,6-dP + Pi Ethylene treatment causes significant decreases, in presence or not of activator: F2,6P,

of its potential activity and its specific activity as the cytosol protein content remains unchanged.

TABLE 1. Influence of ethylene on PP-PFK potential activity

Activities of PP-PFK nKat. ml-1 cytosol

Clones Before stimulation After stimulation

GT 1 PB 235

without F2,6P

4.2 ± 0.4 4.5 ± 0.9

with F2,6P

10.4 ± 0.5 7.9 ± 0.7

without F2,6P

2.2 ± 0.7 2.5 ± 0.8

with F2,6P

6.1 ± 0.7 6.0 ± 0.9

The activity of 6-phosphofructokinase (ATP-PFK, EC 2.7.1.11) a key enzyme in glycolysis[4] which catalyses irreversibly the phosphorylation of F-6-P into F-l,6-P in the presence of A TP, display no significant change before and after ethylene treatment. The values for the two clones GT 1 and PB 235 are between 2.4 ± 0.3 and 2.6 ± 0.4 nKat. ml-1 cytosol.

PP-PFK is essentially functionning to gluconeogenic pathway in latex[4] and acts as a glycolysis brake; so the ratio PP-PFKlATP-PFK activities after stimulation is decreased and can be one of the reasons of glucidic catabolism activation observed in situ[3].

The potential and specific activity of cytosolic glutamine synthetase (GS1, EC 6.3.1.2) 257

J. C. Pech et al. (eds.), Cellular and Molecular Aspects o/the Plant Honnone Ethylene, 257-258. © 1993 Kluwer Academic Publishers.

258

key enzyme for ammonium fixation and initial major step for N metabolism is significantly increased after ethylene treatment [Figure 1]. From the physiological point of view, this phenomenum, which may cause an acceleration in in situ N~ fixation and amino acids synthesis, is logically part of overall metabolic activation process already mentionned. In addition, it may be one of the causes of the decrease in glutathion (GSH) content observed[4] at the same time insofar as GS1 and the synthesis pathway of GSH possess glutamate and ATP as common substrates.

GT 1 CLONE 7 35

E 6-

trear~ - 30

:l L Gl

25 fI) 5---t{- Act. Tern. 0

1~ E

E ..... --\j- Act. Stirn.

..... 4- 20 1 -:;;

.:.! 0)

..5 c ---It}- Prot. Tern. fI) 3 ~V 15 ~

-!!! 0 .... L

-8- Prot. Stirn . . ~ ~-.~ a.

-0 2 &::::--e-e--~~~ 10 « (j)

1 5 C}

0 ..L- 0 0 1 2 3 4 5 6 7 8

TAPPINGS Figure 1. Influence of ethylene on GS1 activity.

References

[1] Dickenson, P.B. (1969) 'Electron microscopical studies of latex vessel system of Hevea brasiliensis', J. Rubb. Res. Inst Malaya 21,543-559.

[2] d'Auzac, J. and Ribaillier, D. (1969) 'L'ethyhlne nouvel agent stimulant la production de latex chez l'Hevea brasiliensis', C.R. Acad. Sci. Paris 268, 3046-3049.

[3] Coupe, M. and Chrestin, H. (1989) 'Physical-chemical and biochemical mechanisms of hormonal (ethylene) stimulation', in J. d'Auzac, J.L. Jacob and H. Chrestin (eds.) , Physiology of Rubber Tree Latex, CRC Press, Boca Raton, pp. 296-319.

[4] Jacob, J.L., Prev6t, J.C., Clement-Vidal, A., L'Huillier, L. and d'Auzac, J. (1990) 'Pyrophosphate fructose-6-phosphate phosphotransferase: an enzyme for regulation of laticiferous metabolism. In situ function and influence of stimulation', in Proc. IRRDB, Symposium Physiology and Exploitation of Hevea brasiliensis, Kunming, Chine, P.W. Allen and M.E. Crunin (eds.), IRRDB, Brickendonbury, pp. 1-10.

MOLECULAR AND PHYSIOLOGICAL CHARACTERISATION OF THE ROLE OF ETHYLENE DURING PATHOGEN ATTACK OF TOMATO FRUIT

W.Cooperl, M. Bouzayen\ C. Barri, AJ. Hamilton2, S. RossalF and D. Grierson2

IDept. Plant physiology, 2AFRC Plant gene regulation group, School of Agriculture, Nottingham university, Sutton Bonington, Leics LE12 5RD. 3 ENS AT, 145 Av. Muret, 31076 Toulouse, France.

We are investigating the induction of ethylene in tomato fruit in response to infection with the post harvest pathogen, Colletotrichum gloeosporioides. C. gloeosporioides is a post harvest pathogen which infects a wide range of fruit and vegetable crops including tomato (Lycopersicon esculentum) fruit. It is incapable of causing aggressive infection on the immature green fruit and undergoes a period of quiescence until the fruit ripens. An increase in levels of ethylene biosynthesis in host tissue in response to pathogen recognition has been shown to occur very early on in colonisation and is thought to act as a trigger to induce other defense-related genes within the host plant (Stahman et al., 1966). Inoculation of red-ripe tomato fruit (var. Ailsa craig) with an isolate of C. gloeosporioides resulted in a large increase in ethylene biosynthesis as measured by gas chromatography. The induction of ethylene occurred within 24h of inoculation at which stage infection is barely detectable visually. This rose to a maximum of 23 nllgfwtlh at approximately 48hr post inoculation. Levels remained high until approximately day 6 by which stage the fruit were severely infected. Perhaps surprisingly, disease progression was retaraed in red-ripe fruit from tomato plants in which the synthesis of ethylene during ripening had been inhibited with an EFE antisense gene (Hamilton et al., 1990). Ethylene measurements in the infected fruit revealed that there was still an induction of biosynthesis although this was to a much reduced level with the maximum induction no more than 1.5 nl/gfwt/h by day 6 post inoculation. It seems likely that in terms of infection, the incomplete ripening of these of these fruit is of greater significance than their ability to produce only small amounts of ethylene.

It has recently been shown that genes homologous to the tomato eDNA, pTOM13, encode the ethylene-forming enzyme (EFE) in tomato (Hamilton et al., 1990; Hamilton et al., 1991; Spanu et al ., 1991). We used this eDNA to characterise at the molecular level the host's response to colonisation by C. gloeosporioides. The expression pattern showed that induction of EFE gene expression occurred very early on while symptoms were barely visible. The transcript level rose to a maximum at approximately 10% surface area infection after which it dropped slightly but remained stable until late infection.

Previous studies had indicated that in tomato, EFE was encoded by a small multigene family of at least three distinct genes and genomic clones of three of these have been isolated (Holdsworth et al., 1988; Kock et al., 1991). These three genes, ethl, eth2 and eth3 display negligible sequence homology in their 3'-untranslated regions and we have cloned these after amplification by PCR. Slot blot hybridisations revealed no cross hybridisation indicating that the

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probes were gene specific. Northern analysis using an ethl specific probe revealed a pattern of expression in the infected fruit which correlated almost exactly with that shown using the full length pTOM13 cDNA. Further analysis using eth2 and eth3 specific probes revealed no detec.table levels of expression in infected fruit. These results suggest that in red-ripe tomato fruit ethl is the pathogen induced EFE gene. Further analysis is underway to determine how soon after inoculation induction at the mRNA level can be detected and of the three genes, which is induced in a tomato cell suspension culture when exposed to fungal elicitors.

References. Hamilton, AJ., Lycett, G.W., Grierson, D. (1990) Antisense gene that inhibits synthesis of the hormone ethylene in transgenic plants. Nature 346, 284-287. Hamilton, AJ. Bouzayen. M., 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. Holdsworth, M J., Schuch, W., Grierson, D. (1988) Organisation and expression of a wound !ripening-related small multigene family from tomato. Plant Mol. BioI. 11, 81-88. Kock, M., Hamilton, AJ., Grierson, D. (1991) eth 1, a gene involved in ethylene synthesis in tomato. Plant Mol. BioI. 17, 141-142. Spanu, P., Reinhardt, D. and Boller, T. (1991) Analysis and cloning of the ethylene-forming enzyme from tomato by functional expression of its messenger-RNA in Xenopus Laevis oocytes. EMBO 1. 10, 2007-20l3.

THE USE OF ANTISENSE TRANSGENIC TOMATO PLANTS TO STUDY THE ROLE OF ETHYLENE IN RESPONSES TO WATERLOGGING.

P.J. ENGLISH, G.W. LYCETI, J.A. ROBERTS, IK.C. HALL AND IM.B. JACKSON. Department of Physiology and Environmental Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK and lUniversity of Bristol, Department of Agricultural Sciences, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol, BS189AF, UK.

Fruit of tomato plants transformed with an antisense construct to the ripening-related cDNA clone, pTOM13, demonstrate a reduced capacity to produce ethylene. Recent work shows that this is because the clone encodes for one form of the ethylene forming enzyme (EFE) (Hamilton et al.,1991). Ethylene is thought to mediate in several developmental effects, such as petiole epinasty and adventitious root formation, when plants are exposed to waterlogged conditions (Jackson, 1985). This hypothesis would be supported if the expected reduced capacity for ethylene production in vegetative tissue of antisense plants was associated with a diminution of the physiological responses to waterlogging. Thus, the pTOM13 antisense transformants may serve as a powerful tool for investigating ethylene action in waterlogged plants. We have studied the extent to which epinasty, ethylene production and the accumulation of ACC and bound (putative malonyl) ACC in waterlogged plants is affected by pTOMl3 antisense transformation of the tomato cultivar 'Ailsa Craig'.

Plants were grown for 5 weeks in 8.2 cm pots under summer glasshouse conditions before waterlogging up to the cotyledonary node. Measurements of leaf epinastic curvature were made for 72 h. The angle between the base of the petiole and the stem was measured using a protractor. A more detailed time-course of epinastic curvature was obtained using an auxonometer to measure epinastic movement every 10 min. Ethylene production rates by petioles excised from the second and third oldest leaves from both waterlogged and well-drained plants was measured using a gas chromatograph fitted with a flame ionization detector. An estimation of ethylene production in intact plants ('pre-wound production') was made by measuring the ethylene produced by 4 x 2 cm petiole sections, during the first 30 min. Subsequent woundinduced ethylene production was then monitored at 90 min intervals. Vials were aerated after each measurement. A gas chromatograph, fitted with a nitrogen-phosphorous detector, was used to determine amounts of ACC and bound ACC after derivatizing to N-benzoyl n-propyl ACC and purification by HPLC (Hall, Else and Jackson, in preparation).

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Waterlogging of wild type plants resulted in an epinastic response within 12 h. The pTOM13 antisense plants reacted more slowly, showing a delay of a further 4 h before epinastic curvature commenced. The subsequent rate of curvature was also reduced and the duration of curvature shorter. This resulted in a final epinastic curvature which was considerably reduced relative to wild type plants.

The transformation made no impact on the basal (i.e. 'pre-wound') ethylene levels in welldrained plants. 'Pre-wound' ethylene production was stimulated in response to waterlogging in both the wild type and transformed plants, but to a lesser extent in transformed plants. Woundinduced ethylene was not markedly affected in well-drained plants by the transformation. However, the pattern of responses to wounding differed between wild type and transformed plants. In the wild type, the effects of flooding and wounding· were additive, whilst in transformants the contribution by flooding was negated by wounding. The latter suggests that a ceiling on ethylene production is imposed by the antisense transformation which limits the total amount of ethylene synthesised.

Preliminary ACC measurements indicate little difference between the wild type and transformed plants, or between waterlogged and well-drained plants. However, larger amounts of bound (putative malonyl) ACC were present in wild type petioles, although waterlogging failed to change the levels significantly.

The expected marked reduction in ethylene production resulting from the antisense transformation to annul EFE activity was not seen. Instead, only a modest attenuation of waterlogging-induced ethylene production was observed. This presumably explains the reduced but not eliminated epinastic effect. This possibility will be tested further. The patterns of woundinduced ethylene suggest a lower capacity for ethylene production in the transformed plants.

The expected accumulation of ACC or bound ACC arising from the blocking of ACC oxidation by the pTOM13 antisense transformation was not observed. This suggests there is still a considerable amount of ACC oxidation occurring in the transformed plants and that antisense clones to all the EFE genes may be required to transform tomato plants in order to generate a genuinely ethylene deficient mutant.

Hamilton, A.J., Bouzayen, M. and Grierson, D. (1991).'Identification of a tomato gene for the ethylene-forming enzyme by expression in yeast.' Proceedings of the National Academy of Sciences USA 88, 7434-7437.

Jackson, M.B. (1985). 'Ethylene and responses of plants to soil waterlogging and submergence.' Annual Review of Plant Physiology 36, 145-147.

RESEARCH ON THE DIURNAL COURSES OF ABSCISSIC ACID, I-AMINOCYCLOPROPANE CARBOXYLIC ACID AND ITS MALONYL CONJUGATE CONTENTS IN NEEDLES OF DAMAGED AND UNDAMAGED SPRUCES.

C. YANG, A. WESSLER and A. WILD. Institute of General Botany, Johannes Gutenberg University, SaarstrafJe 21, 6500 Mainz, Germany

Ethylene regulates many aspects of plant growth, development and senescence. The immediate precursor of ethylene was identified as 1-aminocyclopropane carboxylic acid (ACC) [7]. Amrhein et aI. [1] discovered that ACC supplied exogenously was efficiently converted into ACC malonyl conjugate (MACC) along with ethylene production.

This paper investigates diurnal courses of ACC and MACC contents and discussed the relationship between ACC and MACC contents, ACC and ABA contents in the needles of damaged and undamaged spruce trees.

Two-year-old needles were harvested from one pair of damaged and undamaged spruce trees at september 7th 1989 in HunsrUck mountains in SW-Germany, and frozen immediatly in liquid nitrogen and stored at -80°C. The site was described in detail by Wild et aI. [5]. 80 % (v/v) methanol was applied to one gram of sample at 4°C for 2 hours, 4 %(v/v) unsoluble PVP was used to remove phenolic substances. The process was repeated once more. After centrifugation, the supernatants were collected and evaporated in a vacuum at 38°C to dryness, then dissolved in I ml chloroform.ACC and MACC were extracted with 2 ml of water. The measurement was conducted according to Hoffinan et aI. [2]. The data of ABA contents were cited from Wessler [4].

Figure I shows that ACC and MACC contents in both undamaged and damaged trees tended to decrease in the morning, and increase in the afternoon, whereas ABA contents changed in an opposite way. Both ACC and MACC contents in the needles of the damaged trees

25

A , 20

I ~ ~ 0 15 I 0

~ 0>

I :::, 0 0

2 10

~\j 2

70

60

~ 50

40

30

20

10

2·

~1.5~ g ~ I o E s:;: 0,5

o oL-----------------61891J111213141516111819 6789101112131415161718

Middle European Summer Time (h1

Fig. I The diurnal course of ACC (A), MACC(B) and ABA (C) contents in the needles of damaged (e) and undamaged (0) spruces. Data represent the average of 4 measurements.

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were significantly higher as compared with those of the undamaged ones thourought the day. ACC and MACC contents fluctuated more intensely in needles of the damaged trees than in the undamaged ones during the day

The diurnal courses of ACC and ABA contents show obviously that the ACC contents responded quite well to the ABA contents one hour before. Formula 1 shows the relantionship of them observed in this experiment.

[ACC] = 3.95*[ABAr3.17 (I)

R2 = 0.80

The relationship between ABA and ACC in our result is perhaps a phenomenom of the restraint of ABA on ACC synthesis discovered with wheat leaves by Wright [6] and McKeon et al. [3]. The mediation of ABA to ACC synthesis is perhaps also a mechanism of stress adaptation which prevents the plants from converting too much ACC to ethylene under stress conditions.

Many works suggest that MACC accumulated in plant tissues under stress conditions [7]. This steady accumulation of MACC was not observed in the spruce trees in this short term experiment. MACC contents change along with that of ACC in the damaged and undamaged spruce as well. The significance of the MACC fluctuation in this short term is not clear. More experiments are needed to elucidate the metabolism and the physiological function of MACC in the whole plant system.

We are gratefully acknowledge Prof. Amrhein, ETH Zurich, for providing us with the standard MACC.

(l) Amrhein N, Schneebeck D, Skorupka H, Tophof S (1981) Identification of a major metabolite of the ethylene precursor l-aminocyclopropane-l-carboxylic acid in higher plants. Naturwissenschaften 67:619-620. (2) Hoffinan N.E., Yu L. and Yang S.F. (1983) Changes in l-(malonylamino)-cyclopropane-lcarboxylic acid content in wilted wheat leaves in relation to their ethylene production rates and 1-aminocyclopropane-l-carboxylic acid content. Planta 157, 518-523. (3) 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 stresssed wheat leaves", Planta 155,437-443. (4) Wessler A. (1991) "Hormonphysiologische Untersuchungen zum gehalt an Indol-3-essigsaure une Abscisincsaure an Nadeln von Fichten verschiedener Standorte", thesis. (5) Wild A., Forschner W and Schmitt V. (1990) "Physiologische, biochemische und cytopmorphologische Unteruchungen an immissionsbelasteten Fichten. Forschungsendbereicht zum FE Vorharben Nr. 10803046/16, im Auyftrag des Umweltbundesamtes berlin (6) Wright S.T.C. (1980). The effect of plant growth regulator treatment on the levels of ethylene emanating from eexdised turgid and wilted rwheat leaves. Planta 148, 381-388. (7) Yang S.F. & Hoffinan N.E., (1984). Ethylene biosynthesis and its regulation in higher plants. Ann. Rev. Plant. Physiol. , 35: 155-189.

HORMONAL AND TISSUE-SPECIFIC REGULATION OF CELLULASE GENE EXPRESSION IN ABSCISSION

M. L. TUCKER·, G. L. MATTERS, S. M. KOEHLER, E. C. KEMMERER and S. L. BAIRD USDA, ARS, PSI Plant Molecular Biology Lab Beltsville, Maryland, 20705, USA R.SEXTON Dept. Biological Molecular Science Stirling University Stirling FK9 4LA, Scotland, U.K.

Abstract. Cellulase (endo-I,4-/3-D-glucanase) is one of several cell wall hydrolases playing a critical role in many plant developmental processes. We have identified cDNA and genomic clones encoding a cellulase associated with bean leaf abscission. The tissue- and cell-specific accumulation of cellulase mRNA was examined using RNA gel blots and in situ hybridization. In situ hybridization indicates that all cells in the abscission fracture plane, regardless of cell type, accumulate cellulase mRNA. Experiments with 2,5-norbomadiene, a competitive inhibitor of ethylene action, show that ethylene is required not only to initiate cellulase gene expression in abscission but also to maintain its expression. Auxin, in the presence of 5 JlL/L ethylene, inhibits the accumulation of cellulase mRNA. Deletions through the 5' upstream region of the bean cellulase gene were fused to a /3-glucuronidase (GUS) reporter gene. These promoter constructs are being analyzed in bean using a particle bombardment transient assay and in stably transformed transgenic tomato plants.

Introduction

Abscission is the process by which plants shed organs. Leaf abscission in bean (Phaseolus vulgaris L.) has been used as a model for study of abscission (16). When the fracture line is examined in sections of a weakened abscission zone it appears that only two or three rows of cells are weakened in what is called the "separation layer" (23). Immediately adjacent cells in the pulvinus and the petiole appear completely nonnal. When the fracture surfaces are examined with the SEM (12) they are covered with turgid rounded cells. Light microscope studies (23) and analysis of wall polysaccharides (11) all implicate wall degradation during cell separation. Although it is likely that many cell wall hydrolases are expressed during abscission (11), cellulase (endo-(I,4)-/3-D-glucanase), as assayed by degradation of carboxymethylcellulose, has been a focal point for the study of leaf abscission in bean. .

Sexton et al. (17) used a cellulase-specific antiserum to immunocytochemically localize cellulase in the abscission zone. They showed the enzyme to be localized in a two or three cell wide cortical separation layer. The enzyme's distribution in the stele (vascular tissue) was masked by non-specific staining; however, direct assay of cellulase activity in small fragments dissected from the abscission zones indicated high levels of activity in the stele as far away as 4 mm distal to the fracture. Del Campillo et al. (4) used tissue printing to immunolocalize cellulase protein transferred onto nitrocellulose membranes and observed a similar distribution of the

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J. C. Pech etal. (eds.), Cellular and Molecular Aspects of the Plallt Han/zone Ethylene, 265-271. © 1993 Kluwer Academic Publishers.

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enzyme. The immunologically identified cellulase appears to be associated with many cell types in the abscission zone; however, since cellulase is secreted into the cell wall (1), immunological data cannot distinguish if all the abscission zone cells are synthesizing cellulase or whether extracellular cellulase diffuses along the fracture from a secretory tissue, perhaps in the stele.

Jackson and Osborne (7) reported that an increase in endogenous levels of ethylene correlates with the onset of abscission. More recently, several laboratories have shown that inhibitors of ethylene synthesis or action can delay abscission (2,19). The addition of auxin to debladed petioles prior to ethylene exposure stops weakening and the accumulation of cellulase (1,13). Originally it was thought that auxin addition inhibited abscission only if administered before weakening commenced (1,16); however, Osborne et al. (13) showed that auxin negatively regulates cellulase production throughout the abscission process.

An abscission zone cellulase cDNA clone from bean has been identified (22) and sequenced in this laboratory (21). We have used the bean abscission cellulase clone (21) to study the hormonal and tissue-specific expression of cellulase mRNA in bean leaf abscission. In addition, we have begun experiments to locate regulatory elements in the promoter of this cellulase gene.

Materials and methods

Plant material. Bean seeds (cv. Red Kidney from W.A. Burpee Co., Westminster, PA, USA) were germinated and grown in the greenhouse until the primary leaves were fully expanded at approx. 12 d. Explants were prepared by cutting the stem below the cotyledon and then removing the cotyledons, leaf blades, and apical bud. Explants were surface sterilized by submersion in 0.5% hypochlorite (lO-fold dilution of commercial bleach) for 3 min and rinsed with distilled water. Explants were placed in beakers with water and put into a 250C chamber where 25 )J.l..1L ethylene in air was passed through at a flow rate of 2 L/min.

Light microscopy & in situ hybridization. Abscission zones were fixed in 2% glutaraldehyde in 50 roM sodium cacodylate buffer, pH 7.2, for 6-8 h. The abscission zones were then embedded with JB4 methacrylate resin (polysciences, Warrington, PA, USA), thin sectioned, and stained with toluidine blue as described by Sexton (18).

In situ hybridization was carried out essentially as described by Cox and Goldberg (5). Sections (2 mm either side of the fracture) were cut from 48 h ethylene treated explants and fixed with I % glutaraldehyde, dehydrated, and embedded with Paraplast Plus (Lancer, St. Louis, MO, USA). For more detail see Tucker et al. (20).

Northern and dot blot hybridizations. All RNA samples were prepared from polysomal RNA fractions (6). For details of experiments see Results, Figure legends, and ref. 27.

Particle gun bombardment. A helium charged particle delivery system from E.I. Du Pont de Nemours & Co. (Biotechnology Systems Division, Wilmington, DE, USA) was used for transformation of bean tissues. Gold particles (1.6 J.l.m) were coated with DNA as described in the instrument manual and shot at 1550 psi. Intact leaves were shot and kept in air at 25° for 48 h prior to histochemical staining for GUS activity (8). Abscission zones (4 mm) were taken from 24 h ethylene treated explants, cut in half longitudinally, shot, placed on agar plates and put back in the ethylene chamber for another 24 h before histochemical staining.

Results

Light microscopic observations of the abscission zone. Fig. IA is a bright field image of a toluidine blue stained longitudinal thin section from an ethylene-induced abscission zone. The fracture line seen in Fig. lA passes across the petiole at the juncture of the petiole and pulvinus

267

just below the leaf blade. The weakening process clearly involves only two or three layers of cells. The fracture surface cells are rounded and swollen (Fig. lA). This indicates a loss in the integrity of the cell wall and middle lamella allowing the separation of intact cells. The pulvinus on the distal side of the fracture appears shrunken compared with the proximal petiolar tissues (Fig. 1 A). This is thought to be because of a loss of turgor in the pulvinus resulting from tylosic blockages of the xylem and subsequent organ separation. The reduction of the diameter of the pu1vinus relative to that of the adjacent petiole may create tensions in the abscission zone that facilitate rupture of the xylem and cuticle (14).

Figure 1. Detection of a cellulase mRNA in the abSCission of bean leaves by in situ hybridization. (A) Bright field micrograph of a toluidine stained thin longitudinal section from an ethylene-induced bean leaf abscission zone. m = vascular bundle.

(8) Dark field micrograph of thin longitudinal section from the upper abscission zone after 48 h exposure to 25 J.LLlL ethylene in air. Section was hybridized to 35S-labeled antisense (complementary) strand RNA transcripts of a bean abscission cellulase eDNA. Hybridization signal is seen as bright. light-reflecting silver grains.

In situ hybridization. Northern blot results showed that cellulase mRNA is virtually absent from uninduced abscission zones but increases as the zones weaken in response to ethylene treatment (Fig. 2 and ref. 20). To identify which cells in the weakening abscission region are responsible for cellulase production we have employed in situ hybridization (5). Figure 1 shows two longitudinal sections through an abscising laminar abscission zone. The section shown in Fig. 1B was hybridized to 35S-labeled antisense strand transcripts. The hybridization signal in dark field image of the autoradiograph is apparent by bright, light-reflecting silver grains. The antisense strand-specific RNA transcripts hybridize to nucleic acids in one or two layers of cells on either side of the fracture (Fig. 1B, arrows) and also to cells in the vascular bundles running through the petiole and pulvinus. The hybridization signal to sense strand transcripts was uniform across the tissue indicating non-specific hybridization (not shown). The signal for cellulase mRNA-specific hybridization is seen in all cell types along the fracture line and is not restricted to either surface.

268

C2H4 C2 H4 + then

Air IAA N8D~~

Figure 2. Northern blots showing the effects of ethylene, auxin (IAA), and NBD on the accumulation of polysomal RNA hybridizing to a cellulase cDNA clone, pBAC1. Each lane contains 10 Jlg of polysomal RNA. Ethylene was given at a concentration of 5 JlUl in air except where noted below. IAA (5 x 10-6 M in lanolin) was applied to the petiolar stump 4 h prior to a 48 h exposure to ethylene. In the three lanes on the far right, explants were exposed to ethylene for 31 h, a fraction of the explants harvested (labeled 31), and the remainder treated for an additional 24 h with 1000 JlUl NBD (labeled 55-) or 1000 JlUl NBD plus 100 JlUl ethylene (labeled 55+).

Northern and dot blot analyses of hormonal regulation. Experiments were undertaken to examine whether the continued presence of ethylene was necessary after cellulase mRNA accumulation had commenced. Earlier results showed that removal of explants from ethylene to air slowed the increase in cellulase activity but did not stop it (15). Continued increase in cellulase activity after removal of exogenous ethylene could be caused by endogenously synthesized ethylene. To test this hypothesis we first treated explants with 5 J,JLIL ethylene for 31 h to initiate abscission and cellulase expression and then, instead of transferring the explants to air, explants were placed in an atmosphere containing 1000 JlLlL 2,5 norbomadiene (NBD) or 1000 J,JLIL NBD plus 100 JlLIL ethylene (Fig. 2). NBD, a competitive inhibitor of ethylene action, was used to inhibit the action of endogenous ethylene (19). At the end of a 24 h exposure to NBD, cellulase mRNA had declined to undetectable levels (Fig. 2). The results show that ethylene is necessary not only to initiate but also to maintain expression of cellulase mRNA.

1000

800

~ 600 o 400

200

o

A B

04872 72 Hours of Ethylene

Figure 3. Scintillation counting of dot blots to quantify ethylene-induced cellulase mRNA levels in explants treated with auxin analogs and actinomycin D. (A) Cellulase mRNA levels in explants not receiving a 2 Jll treatment. (8) Cellulase mRNA levels in explants receiving 2 Jll of 50 mg/ml mannitol alone (Control) or 50 mglml mannitol plus 10-5 M aNAA, 10-5 M ~NAA, 10-4 M 2,4-D, or 1 mg/ml actinomycin D. The 2 Jll treatments were applied directly on the fracture surface cells 48 h after ethylene exposure began.

269

Application of auxin to the distal end of explant petioles, prior to exposure to ethylene, inhibits abscission and accumulation of cellulase. IAA at 5 x 10-6 M in lanolin applied 4 h prior to a 48 h exposure to ethylene completely inhibited the accumulation of cellulase mRNA. To determine if auxin could reverse the ethylene induced increase in cellulase mRNA when applied several hours after ethylene exposure began, we used a modification of the procedure described by Osborne et al. (13). Explants were placed in 5 IJi.IL ethylene for 48 h to induce separation. The distal and proximal tissues were separated from each other and each portion placed in 1.5% agar plates with the fracture surface facing up. Two microliters of various treatments described below in 50 mg/mL mannitol as an osmoticum was applied directly onto the fracture surface of the explants and the explants placed back into 5 J.1LIL ethylene for another 24 h (Fig. 3). Treatment with 10-5 M IAA had no effect on cellulase mRNA levels measured 24 h after treatment (data not shown). However, treatments containing 10-5 M nNAA (a-naphthaleneacetic acid) and 10-4 M 2,4 D (2,4-dichlorophenoxyacetic acid) reduced cellulase mRNA levels nearly as much as 1 mg/mL actinomycin D which should have inhibited all new mRNA transcription. Applying 2 J.1L 10-5 M J3NAA, an inactive auxin analog, or 50 mg/mL mannitol alone (Control in Fig. 3B) had a slight inhibitory effect on cellulase mRNA levels compared to explants not receiving 2 J.1L of any treatment (Fig. 3A). Inhibition of cellulase mRNA observed in control or J3NAA treated explants could be due to rupture of sensitive fracture surface cells or inhibition of oxygen diffusion into the liquid soaked cells. Nevertheless, the data suggest that auxin acts as an antagonist of ethylene induced cellulase mRNA accumulation throughout the abscission process.

Regulatory analysis 0/5' upstream sequences. Two overlapping bean genomic clones containing the entire abscission cellulase coding region and flanking DNA were isolated. The transcript start site was determined by S 1 nuclease protection and primer extension, and putative TAT A (-25) and CAAT (-180) boxes identified. Approximately 1.2 kbp of upstream genomic DNA has been sequenced (sequence not shown). The bean abscission cellulase upstream sequence was compared to upstream sequences of other ethylene-induced genes. The most notable sequence similarity was found between the bean abscission cellulase and bean chitinase (3). Three regions of similarity were found (Fig. 4). Two of these regions (-561 to -554 and -585 to -579) are similar to sequences that are perfectly conserved between the chitinase CHSA and CH5B genes which were shown to be required for ethylene-responsive chitinase gene expression (3).

Figure 4. Sequence similarity between bean cellulase and chitinase promoters.

Bean cellulase

Bean chitinase

-683 -673 CTAACATTTTTCT IIIII II IIII CTAACTTTCTTCT

-365 -353

-585 -579 GATTCTGGT I IIIIIII GGTTCTGGT

-263 -255

-561 -554 GCTTGGGAAG I I I I I II I I I

GCTTGGGAAG -220 -211

We prepared six deletion constructs of the cellulase promoter ligated to a J3-glucuronidase (GUS) reporter gene (8). We are currently analyzing these gene constructs by measuring GUS expression in stably transformed tomato plants and in bean tissue transiently transformed by particle gun bombardment. The results from these experiments are very preliminary; however, some of our transient expression results may be of interest in the context of this report.

Our initial experiments with transient expression of GUS activity in bombarded bean tissues has focused on counting the number of spots (blue) that histochemically stain for the presence of GUS activity. Histochemical staining for GUS activity, although not highly quantitative, is easily scored and useful for optimizing the transformation protocol and providing a qualitative measure of GUS expression from our promoter constructs. Somewhat surprising to us, we got GUS expression from our longest cellulase promoter construct (-2100 bp) in not only bean abscission zones but also stems, petioles, and leaves. It's possible that expression was higher in and around

270

the abscission zones but a more definitive conclusion awaits a quantitative measure of GUS activity and confirmation in transgenic plants. Nevertheless, expression in leaves makes it possible for us to perform some of the promoter analysis in leaf tissue which is easier to collect, transform, and assay. It is possible that expression from the bean cellulase promoter in bombarded abscission zones and leaves may be in part a response to the wounding caused by the particle striking the tissue. It is noteworthy that the average number of blue spots per 3 half abscission zones decreased between constructs 1-1 and 2-22 and then again between 2-9 and the TATA constructs (Table 1). Interestingly, in leaves, the average number of spots per leaf decreased between constructs 2-22 and 4-11 (Table 1). The difference between abscission zones and leaves points to regions that will need to be examined in greater detail in future experiments.

Table 1. Number of blue spots (GUS-positive cells) in bean abscission zones and leaves bombarded with cellulase and 35S CaM V promoter/GUS constructs. Numbers in parenthesis indicate length in base pairs of cellulase sequence 5' from the start of mRNA transcription.

Construct (bp) 1-1 (-2100) 2-22 (-881) 4-11 (-665) 2-9 (-211) TATA (-40) 35S CaMV No DNA

Discussion

Blue Spots per 3 half absc. z 53.5 23.2 18.8 24.2 5.0

53.5 0.0

Blue Spots per leaf 20.7 19.3 3.7 3.3 0.3

56.7 0.0

A cellulase with a pI of 9.5 has long been known to increase during leaf abscission in bean (9). Immunological localizations have confirmed that the enzyme is located in the separation layer and also in vascular bundles up to a few millimeters from the fracture (4,17). We have used both tissue printing (20) and in situ hybridization (Fig. 1 and ref. 20) to localize a cellulase mRNA in the abscission zones of bean leaves. The in situ hybridization (Fig. 1) demonstrates that a cellulase mRNA is synthesized in all living cell classes of the separation layer as well as in the parenchyma cells of the adjacent stele. The earlier concern that cellulase protein detected in the stele several millimeters away from the separation layer diffused there apoplastically is less likely in light of the evidence that cellulase mRNA is also present in these distant cells. The in situ localization indicates a very precise positional specificity which is somewhat unusual in that it is not delimited to a morphologically recognizable class of cell prior to the onset of abscission.

Earlier studies have focused on the hormonal control of bean abscission and the associated change in cellulase enzyme activity (1,7,13,16). We have extended these studies to examine the hormonal control of cellulase mRNA accumulation during abscission. In abscission zones at the base of the lamina and petiole, cellulase gene expression appears to be induced by ethylene and repressed by auxin. The stimulatory and inhibitory effects of ethylene and auxin, respectively, on cellulase expression in abscission remain effective up to and after organ separation has occurred.

Understanding the hormonal regulation and tissue-specificity of the accumulation of cellulase transcripts is an important prerequisite to our goal of identifying cis-acting elements in the abscission cellulase gene promoter. Working toward this goal, we have identified overlapping genomic clones for the abscission cellulase gene, determined the start of transcription and prepared several gene fusions with the GUS reporter gene. Transient expression assays using particle gun bombardment of bean tissues show that the bean promoter does promote the expression of the GUS reporter gene (Table 1). We hope to be able to report more detailed analysis of the bean abscission cellulase promoter soon.

271

References

1. Abeles. F.B .• Leather. G.R. (1971) Abscission: Control of cellulase secretion by ethylene. Planta 97. 87-91

2. Baird. L.M .• Reid. M.S .• Webster. B.D. (1984) Anatomical and physiological effects of silver thiosulfate on ethylene-induced abscission in Coleus. J. Plant Growth. Regul. 3. 217-225

3. Broglie KE. Biddle P .• Cressman. R.. 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

4. del Campillo. E .• Reid. P.D .• Sexton. R.. Lewis. L.N. (1990) Occurrence and localization of 9.5 cellulase in abscising and nonabscising tissues. Plant Cell 2,245-254

5. Cox. K.H .• Goldberg. R.B. (1988) Analysis of plant gene expression. In Plant Molecular Biology: A Practical approach. pp. 1-35. Shaw. C.H.. ed. IRL Press. Eynsham. Oxford. England

6. Jackson A.O .• Larkins. B.A (1976) Influence of ionic strength. pH. and chelation of divalent metals on isolation of polyribosomes from tobacco leaves. Plant Physiol. 57. 5-10

7. Jackson M.B .• Osborne. DJ. (1970) Ethylene. the natural regulator of leaf abscission. Nature 225. 1019-1022

8. Jefferson R.A .• Kavanagh. T.A. Bevan. M.W. (1987) GUS fusions: ~-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO 6: 3901-3907

9. Koehler. D.E .• Lewis. L.N .• Shannon. L.M .• Durbin. M.L. (1981) Purification of a cellulase from Kidney bean abscission zones. Phytochemistry 20. 409-412

10. Lewis. L.N .• Varner. J.E. (1970)- Synthesis of cellulase during abscission of Phaseolus vulgaris leaf explants. Plant Physiol. 46, 194-199

11. Mom:. D.J. (1968) Cell wall dissolution and enzyme secretion during leaf abscission. Plant Physiol. 43, 1545-1559

12. Osborne. DJ. (1984) Abscission in agriculture. Outlook on Agriculture 13,97-103 13. Osborne. D.J .• McManus. M.T .• Webb. J. (1985) Target cells for ethylene action. In

Ethylene and plant development. pp. 197-212. Roberts. J.A .• Tucker. G.A .• eds .• Butterworths.London

14. Scott. P.C .• Miller. L.W .• Webster. B.D .• Leopold. AC. (1967) Structural changes during bean leaf abscission. Amer. J. Bot. 54, 730-734

15. Sexton R.. Lewis. L.N •. Trewavas. A.J •. Kelly •. P (1985) Ethylene and abscission. In Roberts JA. GA Tucker. eds. Ethylene and Plant Development. Butterworths. London pp 173-196

16. Sexton. R.. Roberts. J.A. (1982) Cell biology of abscission. Annu. Rev. Plant Physiol. 33, 133-162

17. Sexton. R.. Durbin. M.L.. Lewis. L.N .• 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

18. Sexton. R. (1979) Spatial and temporal aspects of cell separation in the foliar abscission zones of Impatiens sultani Hook. Protoplasma 99, 53-66

19. Sisler E.C.. Goren. R.. Huberman. M. (1985) Effect of 2.5-norbornadiene on abscission and ethylene production in citrus leaf explants. Physiol. Plant. 63. 114-120

20. Tucker. M.L.. Baird. S.L.. Sexton. R. (1991) Bean leaf abscission: Tissue-specific accumulation of a cellulase mRNA Planta 186. 52-57

21. Tucker. M.L.. Milligan. S.B. (1991) Sequence analysis and comparison of avocado fruit and bean abscission cellulases. Plant Physiol. 95, 928-933

22. Tucker. M.L .• Sexton. R .• del Campillo. E .• Lewis. L.N. (1988) Bean abscission: Characterization of a cDNA clone and regulation of gene expression by ethylene and auxin. Plant Physiol. 88,1257-1262

23. Wright. M .• Osborne. D.l (1974) Abscission in Phaseolus vulgaris. The positional differentiation and ethylene-induced expansion growth of specialized cells. Planta 120. 163-170

CHANGES IN GENE EXPRESSION DURING LEAF ABSCISSION

J. A ROBERTS, J. E. TAYLOR, S. A COUPE, N. HARRIS, S. T. J. WEBB Department of Physiology and Environmental Science Faculty of Agricultural and Food Sciences University of Nottingham Sutton Bonington Campus Loughborough Leics. LE12 5RD UK

ABSTRACT. Abscission of the leaflets of Sambucus nigra is stimulated by ethylene. Associated with cell separation is an increase in the activity of ~ 1,4 glucanase (cellulase) and pOlygalacturonase (PG) and this rise is restricted to the cells comprising the abscission zone. A cDNA library has.been generated from mRNA extracted from separating abscission zone cells and screened using a heterologous ~ 1,4 glucanase probe from Phaseolus vulgaris. A number of positive cDNAs have been isolated from the S. nigra library and the largest clone designated plETl has a size of 1.7Kb. plETl has been sequenced and exhibits over 70% deduced amino acid homology with the protein encoded by the P. vulgaris cDNA. Using a differential screening strategy over 20 abscission-related cDNAs have been isolated from the library. None of these hybridizes to plETl and their role in abscission remains to be determined.

1. Introduction

Abscission is a widespread phenomenon which causes the shedding of a range of plant organs including leaves, flowers and fruit. The process is precipitated by the breakdown of the cell to cell cohesion at a precise site known as the abscission zone and there is convincing evidence that the progress of cell separation can be regulated by ethylene (Sexton and Roberts, 1982). It has been proposed that the timing of abscission is strongly influenced in vivo by ethylene produced by tissue distal to the site of abscission. The production of this gaseous plant growth regulator has been shown to increase during the senescence of leaves and flowers and the ripening of climacteric fruit. The ability of ethylene to influence the time course of abscission has been used to study the biochemical and cellular events which accompany cell separation. If excised abscission zones or explants from leaves, flowers or fruit are incubated in an atmosphere of ethylene cell separation can be observed to take place within hours of exposure to the gas.

Associated with wall breakdown is an increase in the activity of several hydrolytic enzymes including p 1,4 glucanase and polygalacturonase (Tucker, Schindler and Roberts, 1984; Roberts et al. 1989) . In general, the increase in activity is restricted to the cells comprising the abscission zone. Recent studies indicate that the increase in activity is the result of an elevated accumulation of the mRNA encoding these cell wall degrading

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enzymes (Tucker et al. 1988; Tucker, Baird and Sexton, 1991). Other proteins have also been demonstrated to increase during abscission and whilst some of these may share homology with pathogenesis-related polypeptides their function and regulation of expression are unknown (del Campillo and Lewis, 1992).

One difficulty of studying the biochemical and molecular changes that accompany abscission is that the key events may be restricted to those rows of cells which actually undergo separation. In some systems this may be as few as one or two rows. The leaf abscission zone of the herbaceous shrub S. nigra is an excellent system on which to study the molecular and biochemical events associated with abscission because the zone is made up of approximately 30 rows of cells (Taylor et al. 1992; Webb et al. 1992). In this paper we report some of the changes which are associated with wall breakdown in this system and the changes in gene expression which may expedite them.

2. Materials and Mdbods

2.1 PLANT MATERIAL

Leaves from S. nigra were collected locally and explants were excised to include 20mm of rachis tissue containing a leaflet abscission zone. The basal ends of these pieces of tissue were then embedded into 1% plain agar and incubated in 101111-1 ethylene in continuous light. Abscission was determined at intervals after treatment as described in Roberts, Schindler and Tucker (1984) and immediately prior to harvesting zone and non zone tissue into liquid nitrogen. When necessary, tissue was stored at -80"C before enzyme or nucleic acid extraction.

2.2 ENZYME EXTRAcrION AND ASSAY

Frozen tissue was extracted as described in Webb et al. (1992). Protein extracts from zone and non zone tissue were assayed as outlined for p 1,4 glucanase (Webb et al. 1992) or polygalacturonase (Taylor et al. 1992).

2.3 NUCLEIC ACID EXTRAcrION

2.3.1 Isolation of total RNA. RNA was extracted from abscission zones of leaf explants as described in Taylor et al. (1990).

2.3.2 Isolation of poly(A)-containing RNA. Poly (A)-containing RNA was purified from total RNA using oligo(dt) chromatography. The RNA concentration was calculated from the A260nm.

2.4 cDNA LIBRARY GENERATION 5j.tg of poly (A)-containing RNA extracted from abscission zone tissue was used to generate a cDNA library using the i..ZAP-cDNA synthesis kit (Stratagene). cDNA was ligated into prepared Uni-Zap arms for 3 days at 4°C and then packaged into Gigapack high efficiency packing extracts (Stratagene). This resulted in the production of a library containing approximately 500,000 recombinants.

274

2.5 SCREENING OF cDNA LIBRARY

2.5.1 UsingpBACIO. Replicate nitrocellulose lifts taken from platings of the library were probed with a 32P-CfP-labelied clone designated pBAClO which was obtained from Dr Mark Tucker. These were hybridized overnight at 6SOC and then washed, the final wash being 1 X SSPE, 0.1 %SDS, at 65°C for 20min. Plaques which hybridized to pBAClO were isolated and the cDNA inserts amplified by PCR.

2.5.2 Differential screening. Replicate lifts were probed with a 32P-CfP-labelied first strand cDNA probe made from either zone or non zone derived poly(A)-containing RNA These were hybridized overnight at 6SOC and then washed, the final wash being 0.1 X SSPE, 0.1 %SDS, at 65°C for 20min. Plaques which differentially hybridized to the zone cDNA probe were isolated and the cDNA inserts amplified by PCR. .

2.6 SEQUENCING OF CLONES Sequencing of cDNA was carried out using the Sequenase Version 2.0 protocol (USB, USA).

3. Results

3.1 TIME COURSE OF ENZYME ACTIVITY

When leaflet explants of S. nigra were exposed to ethylene, separation was apparent within 18h of treatment and was complete by 36h. Associated with abscission was an increase in activity of both P 1,4 glucanase and polygalacturonase and this rise in activity was primarily restricted to the abscission zone tissue (Figure lA & B).

80.------------,

o o 12 18 24 36 Duration of ethylene treatment (h)

A

~ 2.-------:---'-----, c:

~ Q.

I~ 1.5 E

~ a. I 1

! :g as 0.5 o '2 e i ~ 0 o 12 18 24 36

Duration of ethylene treatment (h)

B

Figure 1. Time course of A) ~ 1,4 glucanase and B) polygalacturonase activity in ethylene treated explants from leaflets of S. nigra.

275

3.2 cDNA LIBRARY GENERATION AND SCREENING

A ZAP cDNA library was generated using mRNA extracted from separating abscission zone tissue. The library contained over 500,000 recombinants. Aliquots of the library of approximately 50,000 pfu were probed with the cDNA pBAClO which has been reported to encode the p 1,4 glucanase from the leaf abscission zone of P. vulgaris (Tucker and Milligan, 1991). About 10 positive clones were isolated from each plate and taken through a second round of screening before phage containing the insert was in vivo excised resulting in the generation of plasmid. After purification of the plasmid, the insert was amplified by PCR and then size fractionated on an agarose gel (Figure 2a). The PCR product was transferred on to a nitrocellulose membrane and reprobed with pBAClO (Figure 2b). The largest insert hybridising to the heterologous probe had a molecular size of approximately t.7kB and was designated pJETt. .

A

B

a c

Figure 2. (A) cDNA inserts amplified by PCR from clones which hybridized to pBAClO through two rounds of screening. PCR products were separated on a 1 % TBE agarose gel at BOV for 40min. a: 2ng pBAClO insert; b,c,d, 4J.d PCR product. (B) Southern blot of the gel described in (A). The membrane was probed with pBAClO labelled with 32p-dCTP. Hybridization was carried out at 45°C for 16h, and the final wash was 0.1XSSPE, 0.1% SDS at 65°C, 30min.

3.3 SEQUENCING OF pJETl Plasmid containing the insert pJETt was extracted and purified using PEG 8000 for sequencing purposes. The insert was sequenced using the standard Sequenase Version 2.0 procedure and primers were designed to complete the sequencing of pJETt. Although the largest open reading frame contained an ATG codon 46 nucleotides from the start of the cDNA, by comparing the sequence with pBAClO it would appear that the insert is not quite full length (Figure 3). The sequence of pJETt has a close similarity to pBAClO with over 70% homology over the first 250 amino acids. The clone is also significantly homologous (>50%) to a cDNA which has been shown to encode p 1,4 gIucanase from ripe avocado fruit. The cysteine residues in the mature polypeptides from S. nigra and P. vulgaris align throughout the sequences. Although no glycosylation concensus sequences have been found in bean there are at least 3 in the the pJET1 sequence.

276

3.4 ISOLATION OF ABSCISSION-RELATED CLONES Portions of the library were probed with 32P-Iabelled first strand cDNA synthesized using mRNA from either ethylene-treated zone or non zone tissue. Over 20 ab~cission-related cDNAs were processed through a second round of differential screening and were found to contain representatives of a number of different families of clones. None of the members of these families cross hybridise with pJETl.

ildG.... M v G G A K E D E G L Y S A T T Y D Y 1 1 1

.IIUD.... A M M D D G K L T S S S G P P N Y D Y

K D A L T K A L F F E G Q R S G K L 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

A D A L A K A L F F E G Q R S G K L

P T N Q R V K W R G D S A L S D G K P 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 p s s Q R V K W R E D S A L S D G 'K L

H N V N L T G G Y Y D A G D N V K F G 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Q N V N L M G G Y Y D A G D N V K F G

W P M A Y T V S L L S W A A V E Y L Q 1 1 1 1 1 1 1 1 1 1 1 1 1 1 w P M A F S T S L L S W A A v .11 y E S

E S P T N ~ L R Y L ~ T A R W G T 1 1 1 1 1 1 1 1 1 I

E S S V N Q L G Y L Q s A R W G A

N Y I L R A H T S S T T L Y T Q v G D I 1 1.1 1 I 1 I I I I I I I I

D F M L R A H T S P T T L Y T Q v G D

G N S D H Q c W E R P E D M D T P R T I I 1 I I 1 1 1 I 1 I I I 1 1 I I

G N A D H N C W E R P E D M D T P R T

L Y K T S T S P G T E V A A D N A A I 1 1 1 I 1 1 1 I 1 I I

v y K D A N S P G T E V A A E Y A A

A L A A A S I V F Q E V D S N Y S A K I I I I I I I I I I I A L S A A 5 I V F K K I D A K Y S S T

L L R H S K L L F Q F A D Q Y R G S Y I I I I I I I I I I I I I I L L S H S K S L F D F A D K N R G S Y

Figure 3. Partial amino acid sequence of pJETl eDNA from S. nigra (elder) and pBAClO cDNA from P. vulgaris (bean).

4 Discussion

Leaflet abscission in explants from S. nigra can be observed within 18h of exposure to ethylene. Associated with abscission is a rise in activity of the hydrolytic enzymes p 1,4 glucanase and polygalacturonase and this elevated activity is primarily restricted to the site of cell separation. These observations are in accord with those reported for ethylene promoted abscission of leaves (Sexton et ai, 1984), flowers (Tucker et ai, 1984) and fruit (Rascio et al. 1985).

A cDNA encoding a p 1,4 glucanase enzyme isolated from abscission zones of P. vulgaris (Tucker and Milligan, 1991) has been used in this study as a heterologous probe to identify an equivalent clone from S. nigra. This approach has proved successful and a putative cellulase cDNA termed pJET1 has been identified from our abscission zone library.

277

Although it would appear that this clone is not quite full length, the open reading frame that we have sequenced encodes a protein of 493 amino acids and this is only 2 less than the pedicted bean sequence. Comparison of the nucleotide and deduced amino acid sequences for the elder and bean proteins show close homology. At the amino acid level this represents in excess of 70%. The homology is slightly less when comparing the sequence with that from avocado fruit. Similarities in secondary and tertiary structures are suggested by alignment of the cysteine residues in the bean and elder sequences. One significant difference between the two proteins is that the elder protein has glycosylation sites whereas the mature protein from bean does not appear to be glycosylated. Interestingly, the cellulase from avocado is a glycoprotein. '

REFERENCES del Campillo, E. and Lewis, L.N. (1992) 'Identification and kinetics of 'accumulation of proteins

induced by ethylene in bean abscission zones', Plant Physiology 98, 955-%1. Rascio, N., Casadoro, G., Ramina, A and Masia, A (1985) 'Structural and biochemical aspects of

peach fruit abscission (Prunus persica L. Ba,tsch)', Planta 164, 1-11. Roberts, J.A, Schindler, c.B. and Tucker, G.A (1984) 'Ethylene-promoted tomato flower abscission

and the possible involvement of an inhibitor', Planta 160, 164-167. Roberts, J.A, Taylor, J.E., Lasslett, Y.V. and Tucker, G.A (1989) 'Changes in gene

expression during ethylene-induced leaf abscission', in D.J. Osborne and M.B. Jackson (eds.), Signals for cell separation, NATO Advanced Workshop, Springer-Verlag, Wien, pp. 61-68.

Sexton, R., Burdon, J.N., Reid, J.S.G., Durbin, M.L. and Lewis, L.N. (1984) 'Cell wall breakdown and abscission', in W.M. Dugger and S. Barnicki-Garcia (eds), Structure, function, and biosynthesis of plant cell walls, Waverly Press, Maryland, pp. 195-221.

Sexton, R. and Roberts, J.A (1982) 'Cell biology of abscission', Annual Review of Plant Physiology 33, 133-162.

Taylor, J.E., Tucker, G.A, Lasslett, Y., Smith, c.J.S., Arnold, C.M., Watson, C.F., Schuch, W., Grierson, D. and Roberts, J.A (1990) 'Polygalacturonase expression during leaf abscission of normal and transgenic tomato plants', Planta 183, 133-138.

Taylor, J.E., Webb, S.T.J., Coupe, S.A, Tucker, G.A and Roberts, J.A (1992) 'Changes in polygalacturonase activity and solubility of polyuronides during ethylene-stimulated leaf abscission in Sambucus nigra', Journal of Experimental Botany (In Press).

Tucker, G.A, Schindler, C.B. and Roberts, J.A (1984) 'Flower abscission in mutant tomato plants', Planta 160, 164-167.

Tucker, M.L., Baird, S.L. and Sexton, R. (1991) 'Bean leaf abscission: Tissue-specific accumulation of a cellulase mRNA', Planta 186, 52-57.

Tucker, M.L. and Milligan, S.B. (1991) 'Sequence analysis and comparison of avocado fruit and bean abscission cellulases', Plant Physiology 95, 928-933.

Tucker, M.L., Sexton, R., Del Campillo, E. and Lewis, L.N. (1988) 'Bean abscission cellulase. Characterisation of a cDNA clone and regulation of gene expression by ethylene and auxin', Plant Physiology 88, 1257-1262.

Webb, S.T.J., Taylor, J.E., Coupe, S.A, Ferrarese, L. and Roberts, J.A (1992) 'Purification of ~ 1,4 glucanase from ethylene-treated leaflet abscission zones of Sambucus nigra', Plant, Cell and Environment (In Press).

RAPID ETHYLENE-INDUCED GENE EXPRESSION DURING PETAL ABSCISSION.

K.B. EVENSEN*, D. G. CLARK, AND A. SINGH. Penn State University Department of Horticulture University Park, PA 16802 USA

ABSTRACT. Ethylene induces petal abscission in diploid geranium flowers. If the flowers are exposed to 1 Ill/L ethylene for 1 hour, petal abscission begins after about 60 minutes and is complete by 90 minutes from the start of the ethylene treatment. This response is inhibited by Actinomycin D, a transcriptional inhibitor, and by cycloheximide, an inhibitor of translation. The amount of total protein in abscission zone tissue doubles during the ethylene treatment. SDS gel electrophoresis of petal abscission zone proteins shows that there are some individual protein species which appear and some which qisappear during ethylene treatment. Our hypothesis is that ethylene induces gene activation, and possibly repression, in abscission zone cells.

Introduction

Understanding the mechanisms by which ethylene regulates gene expression depends upon the comparison of many different response systems. Ethylene-induced gene expression is being analyzed in several systems, including ethylene-induced abscission in bean leaves (Tucker and Milligan, 1991; Tucker et al, 1988), flower senescence (Raghothama et al, 1991; Woodson and Lawton, 1988), and host responses to pathogens (Broglie et al, 1989). Comparison of ethyleneresponsive elements and gene expression in systems which vary in their responsiveness, rapidity of response, organ and tissue specificity, and interaction with other controlling factors will eventually help unravel the mystery of ethylene's complex role in plant development and senescence.

Ethylene-induced gene expression in leaves and fruit of tomato has been shown to involve differential regulation of transcriptional and post-transcriptional processes for different genes (Lincoln and Fischer, 1988). DNA sequences flanking the 5' region of the transcriptional units which appear to confer ethylene responsiveness have been identified in several ethylene-regulated genes, including the E-8 gene associated with tomato fruit ripening (Cordes, et al .. 1989; Deikman and Fischer, 1988), a cellulase associated with avocado fruit ripening (Cass et al, 1990), bean chitinase (Broglie et al, 1989) and a gene of unknown function expressed during carnation flower senescence (Raghothama et al, 1991). Some of these sequences are homologous. Still unknown are the means by which ethylene activates these genes, the identity of protein or other intermediaries, and the conditions which confer the tissue-specificity and varying responsiveness found in different plant tissues and organs.

We are studying the ethylene response in a system which is highly sensitive to ethylene: petal abscission in Pelargonium X hortorum. This response is very rapid, with petals beginning to abscise about 1 hour after commencement of a 1 IllIL ethylene treatment. Investigation of hormone action on gene expression in this system should allow us to study the ethylene responsive elements of genes and to examine the means of control of gene expression by ethylene. In this paper, we provide evidence for ethylene-induced gene expression during petal abscission in P. X hortorum.

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Plants of a Pelargonium X hortorum (geranium) line descended from the cultivar 'Nittany Lion Red' were used for these experiments. This diploid line has a single-whorl corolla (5 petals) and was obtained from Dr. Richard Craig, Penn State University. Cultivars with 7-20 petals (semidoubles and doubles) make up most of the horticultural trade; these may be diploid or, more typically, tetraploid, and are less responsive to ethylene or pollination (S. Wallner and R. Craig, unpublished data).

Individual flowers were harvested from greenhouse-grown plants. The flower pedicels were placed in eppendorf tubes containing water or treatment solutions. Flowers which were receptive to pollination, i.e. their stigmatic lobes had just separated, were used for most experiments. For inhibitor experiments, the flower pedicels were held in solutions of 10 l1g/ml cycloheximide or Actinomycin D for 2 hours at 22°C prior to ethylene treatment. Ethylene treatments consisted of holding the flowers in sealed containers fitted with serum stoppers and adding ethylene to give the desired final concentration. Ethylene concentrations were checked by gas chromotagraphy. Flowers at various stages of development were tested for ethylene response. "Open" flowers had reached anthesis, but the style had not elongated above the anthers. In flowers described as "prereceptive", the style had elongated above the anthers, but the stigmatic lobes had not separated. In "receptive" flowers, the stigmatic lobes had separated but were not fully reflexed. In "postreceptive" flowers, the stigmatic lobes were fully reflexed and some or all of the anthers had been shed. All ethylene treatments were performed at 22°C.

For protein extractions, abscission zone tissue was collected from ethylene treated and untreated flowers by harvesting petals and cutting 1-1.5 mm of tissue from the attachment point. Only the distal part of the abscission zone was collected. The abscission zone tissue was quickly frozen using dry ice and liquid N2 and held at -70°C until use.

Proteins were extracted from 1.0 g of abscission zones collected from control and treated (1 J.llIL ethylene for 60 minutes at 22°C) flowers. The frozen tissue was ground to a fine powder with a mortar and pestle cooled by liquid N2. Next, 2.0 ml of a high-salt extraction buffer (3 mM EDTA, 1% PVP, 500 mM NaCI and 20 mMTris at pH 8.1) was added to the ground tissue. The homogenate was centrifuged for 30 min at 6000 x g. The proteins in the supernatant were precipitated overnight with 5X volumes of cold acetone. After centrifugation at 6000 x g for 30 min, the pellet was redissolved in 100111 of 100 mM Na-phosphate buffer (pH 7.0). The extract was subjected to electrophoresis on a 12.5% SDS-polyacrylamide gel using a discontinuous buffer system as described by Laemmeli et al. (1970). The gel was silver-stained using a kit from ICN. Protein concentration in the extracts was determined using the Bradford procedure (Bio-Rad Protein Assay).


Petals of P. X hortorum abscise within 2 hours after commencement of ethylene treatment, and the first petals begin to fall after about 60 minutes. More mature flowers require lower concentrations of ethylene to elicit this response (Figure 1). Post-receptive flowers showed substantial abscission of untreated controls. To induce petal abscission, the flowers must be exposed to ethylene for at least 40-50 minutes (Figure 2), and less mature flowers required longer ethylene treatments to induce the abscission response. Flowers at the "receptive" stage of development were used for subsequent experiments, since these showed a strong ethylene response, but very little abscission in untreated controls.

Petal abscission in P. X hortorum is similar to that reported for P. X domesticum (Evensen, 1991). Both Pelargonium species display an exceptionally rapid ethylene response. The only other modern report of a similarly timed response is that of Sexton (1983) describing petal abscission in Geranium robertianum, which began after 2.25 hours of ethylene exposure. Even more rapid petal abscission was reported by Fitting (1911), who found that several species of Geranium, Pelargonium and Erodium (all members of the Geraniaceae) lost their petals within 1.5

280

minutes to 3 hours of exposure to coal gas. Very rapid petal abscission (i.e. in 1.5-30 minutes) in response to what was probably ethylene in the coal gas has never been confirmed.

c:: .s:l '" '" .() '" .r:> -< ~

•

Figure 1. Effect of ethylene concentration and duration on petal abscission in flowers of P. X hortorum treated at different stages of development. "Open" flowers had reached anthesis, but the styles had not elongated above the anthers. "Pre-receptive" flowers had elongated styles, but the stigmatic lobes had not opened. On "Receptive" flowers the stigmatic lobes were partially open. "Post-receptive" flowers had fully reflexed stigmatic lobes and some or all of the anthers had been shed. For ethylene concentration treatments, the ethylene treatments were 60 minutes in duration. For the ethylene duration treatments, flowers were treated with 1 j.ll/L ethylene.

Open A Pre-receptive D Post-receptive

100 100 • Open 0 0

0 Receptive 0 0 0 •

80 c:: 80 • .s:l 0 0

'" 0 .~ 60 u 60 0 •

~ '" .r:> • 40 -< 00 000

~ 40 • 0 00 • •

20 20 00 0 • 0 0

A 00

0.0 0.5 1.0 1.5 2.0 0 20 40 60 80 100 120 Ethylene concentration (ul/L) Duration of ethylene treatment (min)

A few other plant families demonstrate ethylene-induced petal abscission. In addition to members of Geraniaceae, these include species in Labiatae, Ranunculaceae, Rosaceae, and Scrophulariaceae (Woltering and Van Doom, 1988). The timing of the ethylene response for some species in these families has been reported, and in all cases petal or corolla abscission occurs more slowly than in P. X hortorum.

P. X hortorum has several advantages for the study of ethylene-regulated gene expression. The rapidity and sensitivity of the response make it likely that ethylene is a natural regulator of petal abscission in Pelargonium (Deneke et al., 1990; Evensen, 1991). Since the response is so rapid, the experiments are short, reducing the possibility of confusion between events involved in the abscission response and unrelated developmental changes. It is easy to grow, and it can be vegetatively propagated to provide a continuous supply of clonal material. More is known about the genetics of Pelargonium X hortorum than about the other plants demonstrating a similar response, with the exception of Antirrhinum majus, which has a much slower response. The chromosome number of the diploid species is 2N=18 (Craig, 1982); the genome size is unknown.

Our hypothesis is that ethylene induces petal abscission by gene activation. Ethylene-induced abscission is inhibited by cycloheximide, an inhibitor of protein synthesis, and by Actinomycin D, an inhibitor ofmRNA synthesis (Table 1). Cycloheximide inhibition is typical of many abscission systems (see Sexton et al, 1985). Actinomycin D usually inhibits leaf abscission (Sexton et aI, 1985). However, previous investigators studying flower abscission have shown no inhibition (Henry et al., 1974) or only a slight inhibition (Hanisch Ten Cate et al., 1975) with Actinomycin D. In our previous work with P. X domesticum, petal abscission was not affected by Actinomycin D (unpublished results). It is possible that transport of Actinomycin D to the site of action is less efficient than that of other inhibitors in flowers, or that some inhibitor studies produce artefactual results unrelated to the intended effect.

281

Table 1. Effect of cycloheximide, an inhibitor of protein synthesis, and actinomycin D, an inhibitor of transcription, on petal abscission of Peiargonium Xhortorum. Excised florets were incubated in the inhibitor solution or water for 2 hours, then treated with 1 j.il/L ethylene for 1 hour at 22°C. Abscission was scored 1 hour after completion of the ethylene treatment.

Inhibitor

Cycloheximide (10 Ilg Iml)

Actinomycin D (10 Ilg Iml)

Water control

Petals abscised (%)

o 16.7

78.3

The endoplasmic reticulum and ribosomes are very prominent in transmission electron micrographs of ethylene treated abscission zones (Evensen et al, 1992), indicating active protein synthesis. Indeed, the amount of total protein approximately doubles during ethylene treatment of abscission zones, from 28 Ilg/g fresh wt to 57 Ilg/g fresh wt (unpublished results). SDS-PAGE analysis of abscission zone extracts shows several protein bands that appear, or increase in amount, after ethylene treatment. These include double bands at approximately 60 kD and 23 kD, and a single band at about 18 kD, that are particularly prominent after ethylene treatment (arrows in Figure 2). Also, some bands are no longer detectable following ethylene treatment, in particular a number of bands below 20 kD and a number of bands with molecular weights larger than 70 kD.

Figure 2. SDS-PAGE analysis of abscission zone extracts from untreated and ethylenetreated tissue. The proteins in the crude extract were separated on a 12.5 % SDS-PAGE gel and visualized using a silver stain. Left lane (C) shows proteins in untreated tissues, right lane (E) shows proteins in ethylene-treated tissues.

C E

--

282

The genes most likely to be synthesized or activated in response to ethylene are the cell-wall degrading enzymes. Sexton et al (1983) have provided evidence of cell wall breakdown in petal abscission zones of Geranium robertianum. Our transmission and scanning electron micrographs show dissolution of the middle lamella in petal abscission zones of P. X hortorum after ethylene treatment (Evensen et aI, 1992). The anatomical evidence is therefore consistent with the view that cell wall degradation precedes petal abscission in a manner similar to that in stem tissues, despite the fact that leaf abscission takes 12-48 hours and petal abscission in G. robertianum and P. X hortorum takes only 1-3 hours. In ethylene-treated geranium flowers, the attachment force of the petals began to decline after about 45 minutes, indicating that the enzymes active in cell wall hydrolysis had become effective by that time (Evensen et al, 1992).

If petal abscission does, indeed, involve gene activation, then this ethylene response would be an excellent example of rapid hormone-induced gene expression. Rapid hormonal responses have been demonstrated in a few other plant systems. Auxin induced mRNAs were shown to appear as early as 10 minutes after addition of IAA (an auxin) to pea epicotyl tissue (Theologis, 1989) and within 2.5 minutes of auxin application to soybean hypocotyls (McClure and Guilfoyle, 1987). Comparison of the events occurring during signal transduction in these and other ethylene responsive systems will eventually allow us to construct a model of ethylene action in plants.

References

Broglie, KE, P Biddle, R Cressman and R Broglie (1989) Functional analysis of DNA sequences responsible for ethylene regulation of a bean chitinase gene in transgenic tobacco. Plant Cell 1 :599-607.

Cass, LG, KA Kirven, and RE Christoffersen (1990) Isolation and characterization of a cellulase gene family member expressed during avocado fruit ripening. Mol Gen Genet 223:76-86.

Cordes, S, J Deikman, L Margossian and RL Fischer (1989) Interaction of a developmentally regulated DNA-binding factor with sites flanking two different fruit-ripening genes from tomato. Plant Cell 1: 1025-1034.

Craig, R (1982) 'Chromosomes, genes, and cultivar improvement', in: JW Mastalerz and EJ Holcomb (eds), Geraniums, Third Edition, PA Flower Growers, pp. 380-411.

Deikman, J and RL Fischer (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.

Deneke, CF, KB Evensen and H Craig (1990) Regulation of petal abscission in Pelargonium Xdomesticum. Hort Science 25: 937-940.

Evensen, K.B. (1991) Ethylene responsiveness changes in Pelargonium Xdomesticum florets. Physiol Plant 82: 409-412.

Evensen, KB, AD Stead, and A Page (1992) Anatomy of petal abscission in Pelargonium Xhortorum. Ann Bot, in press.

Fitting, H (1911) Untersuchungen uber die vorzeitiger Entblatterung von Bluten. Jahrb fur wiss Bot 49:187-263.

Hanisch Ten Cate, ChH, J Van Netten, IF Dortland, and J Bruinsma (1975) Cell wall solubilization in pedicel abscission of Begonia flower buds. Physiol Plant 33:276-279.

Henry, EW, JG Valdovinos, and TE Jensen (1974) Peroxidases in tobacco abscission zone tissue. Plant PhysioI54:192-196.

Laemmli, UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685.

McClure, BA and TJ Guilfoyle (1987) Characterization of a class of small auxin-inducible soybean polyadenylated RNAs. Plant Molec BioI 9:611-623.

283

Raghothama, KG, KA Lawton, PB Goldsbrough and WR Woodson (1991) Characterization of an ethylene-regulated flower senescence-related gene from carnation. Plant Molec BioI17:61-71.

Sexton, R, WA Struthers and LN Lewis (1983) Some observations on the very rapid abscission of the petals of Geranium robertianum L. Protoplasma 116: 179-186.

Sexton, R, LN Lewis, AJ Trewavas and P Kelly (1985) 'Ethylene and abscission', in: JA Roberts and GA Tucker, (eds), Ethylene and Plant Development, Butterworths, London. pp. 173-196.

Theologis, A (1989) 'Auxin-regulated gene expression in plants', in: S King and CJ Arntzen, (eds), Plant Biotechnology, Butterworths, Stoneham, MA, pp. 229-243.

Tucker, ML, and SB Milligan (1991) Sequence analysis and comparison of avocado fruit and bean abscission cellulases. Plant PhysioI95:928-933.

Tucker, ML, R Sexton, E del Campillo, and LN Lewis (1988) Bean abscission cellulase. Plant PhysioI88:1257-1262.

Woodson, WR and KA Lawton (1988) Ethylene-induced gene expression in carnation petals. Plant Physiol 87:498-503.

ABSCISSION STUDIES IN A NEW MUTANT OF NAVEL ORANGES

L ZACARIAS, ER. TADEO, R. BONO & E. PRIMO-MILLO Instituto Vaienciano de Investigaciones Agrarias 46113 Moncada, Valencia Spain

ABSTRACT. 'Ricalate' is a spontaneous bud mutation of 'Washington Navel' oranges with a delay in fruit coloration, high fruit firmess and high attachment of mature fruits to the peduncle. The pattern of abscission of flowers and young fruits in leafly inflorencences of both genotypes was not different. In leafless inflorescences abscission rate of Ricalate fritlets was lower. The retention force of mature fruits remained constant as long as July. Explants of abscission zone from leaves at different developmental stages displayed a gradual reduction in the sensitivity to the abscission with the age of the leaf, either in presence of high ethylene concentrations. Abscission zone explants from mature leaf did not abscise after prolonged periods of incubation. Mature tissues of Ricalate, however, showed normal ethylene responses and production. Protein profile during abscission of mature leaf explants was similar in both genotypes. Two new polypeptides (27 and 12.5 kDa) appeared during the course of the abscission. The unusual ascission behavior of Ricalate, either in frutis and leaves, was more patent with the development of the tissue, being highly resistant in later stages of development. Different mechanisms to explain the altered abscission in Ricalate are evaluated and discussed.

Introduction

Preharvest drop of mature fruits is the main restriction delaying the harvesting season of Citrus fruits. The main harvesting period of oranges in Spain is November to February. Having new cultivars with late harvesting and extending the commercial period of this crop would be therefore of great interest. Because of the difficulties of the genetics in Citrus, many of the new Citrus cultivars have been found by the isolation and characterization of spontaneous bud mutations. Recently, in a commercial population of Navel trees (RIbera de Cabanes, Castellon) a new spontaneous bud mutation, named Ricalate, has been found with a delayed fruit coloration. In addition, the mutation displayes a high peel firmness and, importantly, high attachment of the fruit to the peduncle. These characteristics allow to harvest mature fruits as late as July (Bono et al. (1992)). The unusual abscission behavior of Ricalate provides, therefore, a interesting experimental system to understand the physiological and biochemical mechanisms involved in the regulation of abscission in Citrus.

Abscission mutants have been described in a number of species. In tomato, the mutant jointiess fail to shed flowers and fruits by deficient differentiation of the separation zone. The mutant is also fails to abscise in response to ethylene and the mutation appears to be related to structural modifications (Roberts at al. (1987)). The rate of flower abscission in the ripening

284

1. C. Pech et at. (eds.), Cellular and Molecular Aspects o/the Plant Honnone Ethylene, 284-290. © J 993 Kluwer Academic Publishers.

285

mutant Nr was delayed in response to ethylene, and it was associated to a reduction in the activity of the hydrolytic enzymes: pOlygalacturonase (PO) and B-I-4 glucanhydrolase (cellulase). In the mutant Abs- of Lupinus tifolium the reduction in leaf and pod abscission was also related to an suppression on the cellulase activity (Osborne and Thompson (1991».

In this work we describe a comparative study of the abscission behavior of the mutant Ricalate and the parental line W. Navel. The pattern of natural abscission of flowers and fruits, and ethylene-induced abscission in leaf abscission zone explant at different developmental stages have been studied. Possible differences in the ethylene response, biosynthesis and protein content of abscission zone cells are evaluated and discussed.


The different plant materials were harvested from adult trees of 'Washington Navel' (Citrus sinensis L. Osbeck) and the mutant 'Ricalate' ,both grafted on sour oranges (Citrus aurantium L.). located in the same experimental orchard and under the same cultural conditions. Abscission rates of flowers and young fruits were determined on leafy and leafless inflorescences, by tagging population of flowers before anthesis on different trees. Explants of leaf abscission zone were generated by cutting 5-10 mm section of lamina and petiole from leaves at the following developmental stages: Stage I and II, 10 and 40 mm long young expanding leaves, respectively; stage III, young fully-expanded leaves; stage IV, 3 month-old mature leaves, and stage V, more that 8 to 12 month-old leaves. The explants were inserted into 1 % plain agar find incubated in desiccators at 25 0 C in a ethylene-free atmosphere or with 12 /J.II·1 ethylene. At intervals abscission rates was determined by counting explants that separated by gentle touching. For the different experiments 12 to 20 explants were used, and each assay was repeated at least twice.

Fruit retention force was determined monthly from November to July in mature fruits of both cultivars with a Chantillon dynamometer. Color index of mature fruit rind incubated with or without ethylene was determined using mxHunterlab D25 P-2 colorimeter (JimenezCuesta et al. (1981)).

Albedo discs (5 mm diameter) excised from mature oranges and adult leaf discs (10

E c 0 'iii .. '0 .. .0 «

100

75

50

25

A

-2 0 2 4 6 8 10 12 -2 0 2 4 6 8 10 12

Weeks after onthesis

Figure 1. Abscission of flowers and young fruits from leafy (A) and leafless (B) inflorescences of 'W. Navel' and 'Ricalate'

286

mm) were incubated as the explants. Rates of ethylene production by the different tissues were determined by gas chromatography.

Total protein was extracted on frozen lyophilized 2 mm abscission zone explants from mature leaves in 50 mM acetate-Na (pH 5.0), 2 mM B-mercaptoethanol, 0.4 M NaCI and 0.5 mM PMSF. After centrifugation at 20,000 g for 20 min, the clear supernatant was prepared for fractionation by SDS-PAGE (Kanellis et al. (1989)). The protein content was determined by the method of Bradford (1979) and SDS-PAGE was performed according to Conejero and Semancik (1977) in 14% polyacrylamide gels.


ABSCISSION OF FLOWERS AND FRUITS

100

75

50

25

o

OJfMAt.CYJNJl

Month

Figure 2. Fruit retention force during the ripening of 'W. Navel' and 'Ricalate' oranges.

Accumulative rate of abscission of W. Navel and Ricalate flowers and fruitlets during the setting period was recorded in leafy (Fig. lA) and leafless (Fig 1 B) inflorescences. During the first weeks of fruit development no significant differences in the abscission of flowers and ovaries from both inflorescences were detected. In later stages of development Ricalate displayed a lower rale of fruit drop. specially in leafless inflorescences, where massive fruit drop occurred. Final fruit set!ing percentage in both oranges was similar. Preharvest drop of mature fruit was substantially different. Whereas in W. Navel the fruit retention force declined progressively after December, Ricalate mature fruits were highly attached to the peduncle until July (Fig. 2). From these results it appears that the unusual abscission behavior of Ricalate fruits is becoming more patent with the development of the fruits, to reach achieve almost complete insensitivity at latter development stages.

ABSCISSION OF LEA YES

The rate of shedding of abscission zone explants (lamina-petiole) from leaf of W. Navel and Ricalale at different developmental stages was also examined. Explants from young expanding leaves (stage I and II) of both plants showed higher rate of abscission, but in Ricalate it was slightly delayed (Fig. 3). With the age of the leaf, abscission of zone explant were delayed. In W. Navel expJants at stage III, IV and V, 50% abscission was produced after about 59, 62 and 67 h of incubation, respectively. In Ricalate, however, abscission rate declined gradually and in zone explanL.~ at stage III and IV 50% abscission occurred after 93 and 143 h of incubation, respectively. In explants of older leaves, more that 12 months-old, abscission was almost completely suppressed even after prolonged periods of incubation. These explants only showed a partial separation of the outer cens of the bark at the edge of the groove, being the vascular cylinder and the pith still attached. In explants from leaves at stage IV a small rind of callus surrounding thc abscission zone was formed after 48 h of incubation. In explants of older leaves callus formation was apparent after 4 days and increased with the time.

Application of ethylene accelerated the abscission of the different explants of W. Navel to a similar extent, as well as abscising explants of Ricalate (Fig. 4). Explants of older leaves

g c 0

'iii III '0 III .D ..:

100

75

50

25

a

a 2 3 4 5 678

Days

Figure 3. Rate of abscission of explants from leaves or'w. Navel' (open symbols) and 'Ricalate' (fiIJed symhols) at different developmenWI stages: I (triangle). II (square), III (reverse triangle), IV (diamond) and V (circle). Description of each stage in material and methods.

287

of Ricalate, however, also failed to abscise, either in presence of high ethylene concentrations. Callus formation was also accelerated by ethylene in explants from leaves at stage IV, but not significantly in those from older leaves (data not shown). Thus, during development of W . Navel leaves their abscission zone became less sensitive to the abscission, whereas in Ricalate decreased nearly to suppression.

ETHYLENE RESPONSES AND PRODUCTION

Since ethylene has been implicated in the regulation of the abscission process in citrus fruits and leaves

(Sagee et al. (1980), Goren (1983)), the possibility that the mutation in Ricalate may affect in latter stages of development either the response to or the production of ethylene was further investigated. Exogenous ethylene accelerated in a similar percentage the rate of fruit coloration in mature fruits in both plants (Tab. lA). Stimulation in leaf discs and autoinhibition of ethylene production in albedo tissue was also coincident in both plants. The rates of ethylene production and ACC-dependent ethylene production in leaves and abscission-zone explants were also similar in both cases (Tab. 1 B). These results indicate that the unusual abscission behavior of Ricalate is not the result of impairing the response to or the production of ethylene.

PROTEIN CHANGES

Changes in the polypeptide pattern resolved by SDS-PAGE during the abscission of old leaf explants of W. Navel and Ricalate are summarized in Tab. 2. Protein profile of abscission-zone at the time of excision were very similar in both genotypes. The major difference was the high abundance of a 15 kDa polypeptide in Ricalate abscission-zones. During the course of the abscission, several polypeptides decreased in abundance (42, 38, 31, 29 and 23 kDa), whereas one of 24 kDa increased. A polypeptide of21 kDa decreased in the course of the abscission and after 72 h disnppeared. Other two polypeptidc..~ of 27 and 12.5 kDa were not present before the induction of the abscission and appeared after 48 and 24 h of incubation, respectively. These pattern of changes were similnr in both genotypes, excepting the polypeptide of 15 kDa that was in an abundance about 5 times higher in Ricalate that in W. Navel. This polypeptide was present at the incubation time in both genotypes and decreased during the abscission of W. Navel explant.~, whereas in Ricalate was at high level throughout the process. Ethylene-induced abscission accelerated the changes in the protein profile associated to the process, and not specific polypeptides were detected (data not shown). Changes in the patterns of proteins during the abscission process have also been studied in other species. Translation products of abscission zone of Phaseolus vulgaris revealed the presence of three polypeptides of 32, 27 and 17 kDa, and a 42 kDa product induced by ethylene (Kelly et aL (1987)). The 32 kDa polypeptide was further identified as chitinase, that appears to be ubiquitous in this plant and not related to the abscission process (Gomez el aL (1987). McManus and Osborne (199Oa, 1990b) have identified several polypeptides (34. 32 and 28 kDa in S. nigra and 68, 45 and 36

288

kDa in P. vulgaru) apparently abscission-zone specific. The identity of the polypeptides identified in this study is still unkown. Two polypeptides of 27 and 12.5 kDa appeared during the course of the abscission and in presence of ethylene. The identity of these proteins is also unknown but they may be polypeptides associated to the abscission process in Citrus.

From the results of the abscission rates of fruits and leaf explants, it appears that the unusual abscission behavior of Ricalate is more patent during the development of either fruits and leaf abscission-zones, suggesting that the mutation may have affected a developmental regulated process leading to insensitivity, either to the stimulus or to the responses associated to the abscission process. Because Ricalate mature tissues responded to ethylene, and displayed normal rates of ethylene production, it seems more likely that the mutation has affected specific anatomical or biochemical features associated

90

~ 60 'iii .. . ~ 45 Jl o

~ 30 If)

15

/ ... -0

r III IV V

Developmental stage

Figure 4. Effect of ethylene on the abscission rate of explants from leaves of 'W. Navel' (open symbols) and 'Ricalate' (filled symbols) a t different developmental stages.

with abscission. A possible explanation to the abscission behavior of Ricalate would take into account a progressive reduction of the synthesis or activity of the hydrolytic enzymes involved in the breakdown of the cell walls. However. the transition to a non-abscising stage of the different abscission-zones of Ricalate is a comparable phenomenon to that of the abscissionzone A (pedicel-stem) of young citrus fruits. This zone lost the ability to abscise 5 to 8 weeks after flowering, even though cellulase ad PG activity increased by ethylene treatment

TABLE 1. Effect of ethylene (15 J.'l )-1) on fruit color (72. h) and ethylene production by albedo and leaf discs (24 h) of 'W. Navel' and 'Ricalate' (A). ACC-dependent ethylene production by leaf discs and explant of 'W. Navel' and 'Ricalate'.

A Fruit color Ethylene production

Leaf Albedo

B Leaf discs (nllh g)

Control ACC

Leaf explants (nIl h 5 exp.) Control

ACC

W.N. Ricalate

(% air contrOl) 271.6 298.8

550.8 533.1 12.8 9.9

1.2 ± 0.1 47.8 ± 1.2

1.1 ± 0.1 9.4 ± 1.9

0.9 ± 0.1 51.1 ± 3.1

0.8 ± 0.1 10.1 ± 2.8

289

TABLE 2. Qualitative changes in abundant polypeptides during abscission of 'W. Navel' and 'Ricalate' rmlture leaf abscissioll-zone explallts.

W.N. Ricalate

66 53 42 38 31

MW (lcDa)

29 27

+ +

24

l' l'

23 21 15 125 9

+ +

(Huberman et al. (1983)). The inability of this abscission-zone to abscise appears to be related to structural modifications of the ceIl walls by secondary thickness or progressive lignification (Sexton and Roberts (1982). Osborne (1989)). Therefore, it seems likely that the mutation may have affected important structural modifications of the cell walls in the different abscissionzones, similar to those producted only in the abscission-zone A of the parental line, resulting in a progressive and generalized lost of sensitivity to the abscission with the development of the abscission-zones.

References

Bono, R, Fernandez de Cordova, L., Soler, J. and Segui, M.V. (1992) 'Characteristics of Ricalate, a late variety of the Navel group', Proc. Int. Soc. Citric., paper 117, (In press).

Bradford, M.M. (1979) 'A rapid and sensitive method for the quantitation of micrograms quantities of protein utilizing the principle of protein-dye binding', Anal. Biochem. 72, 248-254.

Conejero, V. and Semancik, J.S. (1977) 'Analysis of the protein in crude plant extracts by polyacrilamide slab gel electrophoresis', Phytopathology 67, 1424-1427.

Gomez Lim, M.A, Kelly, P., Sexton, Rand Trewavas, A.J. 'Identification of chitinase mRNA in abscission zones from bean (Phaseolus vulgaris Red Kidney) during ethylene-induced abscission', Plant Cell & Environ. 10,741-746.

Goren, R (1983) 'Physiological aspects of abscission in citrus', Acta Hortic. 137, 15-27.

Huberman, M., Goren, Rand Zamski, E. (1893) 'Anatomical aspects of hormonal regulation of abscission in citrus-the shoot-peduncle abscission zone in the non-abscising stage', Physiol. Plant. 59, 445-454.

Jimenez-Cuesta, M., CuquereIla, J. and Martinez-Javega, J.M. (1981) 'Determination of a color index for citrus fruit degreening', Proc. lnt. Soc. Citric. 2, 750-753.

KaneIIis, A.K, Solomos, T. and Mattoo, A.K (1989) 'Hydrolytic enzyme activities and protein pattern of avocado fruit ripened in air and low oxygen, with and without ethylene', Plant Physio!. 90, 257-266.

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Kelly, P., Trewavas, AJ., Lewis, LN., Durbin, M.L and Sexton, R (1987) 'Translatable mRNA changes in ethylene induced abscission zones of Ph as eo Ius vulgaris (Red Kidney)" Plant Cell & Environ. 10, 11-16.

McManus, M.T. and Osborne, D.J. (1990a) 'Identification of polypeptides specific to rachis abscission zone cells of Sambucus nigra', Physiol. Plant. 79, 471-478.

McManus, M.T. and Osborne, D.J. (1990b) , Evidence for the preferential expression of particular polypeptides in leaf abscission zones of the bean Phaseolus vulgaris L.', J. Plant Physiol. 136, 391-397.

Osborne, D.J. (1989) 'Abscission', Crit. Rev. Plant Sci. 8, 103-129 ..

Osborne, D.J. and Thompson, D.S. (1991) 'Target or non-target: hormonal signal perception and response in the determination of cell performance', in C.M. Karssen, LC. van Loon and D. Vreugdenhil (eds.), Progress in Plant Growth Regulation, Kluwer Academic Pub., Dordrech, pp. 237-247.

Roberts, J.A, Grierson, D. and Tucker, G.A (1987) 'Genetic variants to examine the significance of ethylene on development', in G.V. Hoad, J.R Lenton, M.B. Jackson and R K Atkin (eds. ), Hormone action in plant development: a critical appraisal, Butterworth, Bodmin, pp. 107-118.

Sagee, 0., Goren, Rand Riov, J. (1980) 'Abscission in citrus leaf explants. Interrelationships of abscisic acid, ethylene and hydrolytic enzymes', Plant Physiol. 66, 750-753.

Sexton, R and Roberts, J.A(1982) 'Cell biology of the abscission'. Ann. Rev. Plant Physiol. 33, 133-152

Tucker, G.A, Schindler, c.B. and Roberts, J.A (1984) 'Flower abscission in mutant tomato plants', Planta 160, 164-167.

ETHYLENE REGULA nON AND FUNCTION OF FLOWER SENESCENCERELATED GENES

W.R. WOODSON, A.S. BRANDT, H. ITZHAKI, I.M. MAXSON, H. WANG, K.Y. PARK AND P.B. LARSEN Purdue University Department of Horticulture West Lafayette, IN 47907-1165

ABSTRACT. The senescence of carnation (Dianthus caryophyllus L.) flower petals is associated with increased synthesis of the phytohonnone ethylene. This ethylene serves to initiate and regulate the processes of programmed organ death. We have isolated and cloned mRNAs which encode the ethylene biosynthetic pathway enzymes 1-aminocyclopropane-1-carboxylate (ACC) synthase and ACC oxidase. These cDNAs have been used to study the regulation of ethylene biosynthesis during flower petal senescence. The increase in ethylene production and onset of petal senescence are associated with dramatic changes in gene expression. Several senescence-related (SR) mRNAs have been isolated and used to study specific changes in gene expression during flower petal senescence. In several cases, the increase in SR mRNA abundance occurs concomitant with the ethylene climacteric. Indeed, treatment of flowers with inhibitors of ethylene synthesis or action prevents petal senescence and the accumulation of SR mRNAs. Sequence analysis of SR mRNAs has revealed homologies with other known proteins, which in some cases points to putative roles of these genes in the regulation of petal senescence. In order to elucidate the molecular mechanisms involved in the regulation of SR gene expression, we have initiated an analysis of the cis-elements responsible for developmental and honnonal gene regulation during floral senescence.

1. Introduction

The senescence of carnation flower petals is associated with a dramatic increase in the synthesis of ethylene. In presenescent flower petals ethylene production is limited by low activities of both ACC synthase and ACC oxidase [1], which catalyze the conversion of SAM to ACC and ACC to ethylene respectively. An increase in activities of these enzymes leads to the climacteric-like production of ethylene during petal senescence. The developmental signals which lead to a concomitant increase in ACC synthase and ACC oxidase activities during petal senescence are unknown; however, it is clear ethylene plays a role in stimulating both enzymes through an autocatalytic mechanism. Consistent with autocatalytic regulation, treatment of presenescent flowers with exogenous ethylene stimulates both ACC synthase and ACC oxidase activities, whereas interruption of ethylene action with the competitive inhibitor 2,5-norbomadiene (NBD) inhibits these enzymes and reduces ethylene production in senescing petals [9].

291


292

It is clear that the developmental expression of increased ethylene production in aging flower petals is responsible for inducing many of the biochemical processes leading to programmed organ death, including an activation of senescence-related gene expression [1,4,11]. We previously reported the molecular cloning of several mRNAs expressed in senescing petals of carnation flowers [4]. The expression of these mRNAs during the developmental program of flower senescence appears to be coupled to the increase in ethylene production [4,11]. Indeed, treatment with inhibitors of ethylene action prevents their expression. Nuclear run-on transcription studies indicate ethylene regulates SR genes at the transcriptional level [4]. It is reasonable to suggest that at least some of these mRNAs encode proteins functionally important to the biochemical alterations that occur during programmed organ death.

In our lab we are interested in the molecular mechanisms involved in the developmental regulation of ethylene biosynthesis and its role in the regulation of flower petal senescence. In this paper we summarize our recent work on the molecular cloning and expression of ethylene biosynthetic pathway transcripts, and the regulation of senescence-related genes during the programmed death of flower petals.


2.1 CLONING AND EXPRESSION OF ETHYLENE BIOSYNTHETIC PA THW A Y mRNAs

We recently described the isolation of cDNA clones from carnations for three enzymes in the ethylene biosynthetic pathway including SAM synthetase [3], ACC synthase [7] and ACC oxidase [10]. To relate the temporal pattern of ethylene production with the amount of transcripts for ethylene biosynthetic pathway enzymes, we subjected total RNA extracted from flower petals to gel blot analysis using cDNA probes for SAM synthetase (PSAM2), ACC synthase (pCARACC3) and ACC oxidase (PSRI20). The increase in ethylene production which accompanies petal senescence was apparent 5 days after harvest at which time a dramatic increase in the levels of both ACC synthase and ACC oxidase rnRNAs was apparent (Fig. 1). In contrast, the level of SAM synthetase mRNA decreased during petal senescence. Treatment of flowers with 2,5-norbornadiene, a competitive inhibitor of ethylene action, prevented the increase in ethylene and the accumulation of ACC synthase and ACC oxidase mRNAs (Fig. 2). Consistent with its role in induction of autocatalytic ethylene production and petal senescence, treatment of flowers at anthesis with 2 ~ ethylene resulted in an increase in ACC oxidase transcripts within 3 hand ACC synthase mRNA within 6 h of treatment (Fig.3). These results are consistent with the de novo synthesis of ACC synthase and ACC oxidase leading to the increase in ethylene production in senescing petals [1]. The dramatic increase in ACC synthase and ACC oxidase rnRNAs during the ethylene climacteric suggests that transcription of these genes likely leads to the increase in ethylene biosynthesis; however, a role for mRNA stability cannot be ruled out by these data. In addition, from these data we can conclude that the increase in ethylene production does not require an increase in SAM synthetase mRNA. In relation to the requirement of SAM as a methyl donor in a variety of reactions, ethylene biosynthesis does not likely represent a significant demand for SAM [12].

Days After Harvest

02456 7

pSAM2 • • pCARACC3

pSR120

Figure 1. RNA gel blot analysis of ethylene biosynthetic pathway mRNAs in carnation flower petals during senescence.

Air NBD

o 6 6

pSAM2

pCARACC::

pSR120

Figure 2. Effect of the ethylene action inhibitor 2,5-norbomadiene on the abundance of ethylene biosynthetic pathway transcripts in carnation flower petals. Flowers were held in an atmosphere of air or 2,000 f.,ll/L NBD for 6 days after harvest.

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Ethylene Exposure (h)

o 0.5 1 3 6 12

pSAM2

pCARACC3

pSR120 • Figure 3. Accumulation of ethylene biosynthetic pathway transcripts in presenescent flower petals following exposure to 2 J..ll/L ethylene for various times.

2.2 ORGANIZATION AND STRUCTURE OF THE SR ACC SYNTHASE GENE

It is clear from the data presented that pCARACC3 represents a SR ACC synthase mRNA. In order to begin to elucidate the developmental and hormonal signals that regulate the expression of its corresponding gene we have isolated a region of the carnation genome which contains the SR ACC synthase gene. A library of carnation nuclear DNA fragments was screened with pCARACC3 and three recombinant phage were identified as potentially representing the SR ACC synthase gene. Restriction maps revealed all three genomic clones represented the same region of the carnation genome (Fig. 4A). Extensive sequence analysis of these genomic clones revealed they contained the entire transcription unit of the mRNA represented by pCARACC3 in 5 exons interrupted by 4 introns (Fig. 4B). We have designated this gene ACC]. The introns exhibit splice junction sequences similar to other plant genes (data not shown). The positions of introns 1,2 and 3 are identical to those described for LE-ACC2 [8] from tomato and CP-ACC]A [2] from zucchini squash. LE-ACC2 does not contain a 4th intron, while CP-ACCIA has a 4th intron in a similar but not identical position as that of ACC] from carnation. The cloning of the SR ACC synthase will allow us to address the cis-elements and trans-acting factors involved in the regulation of its expression during petal development.

2.3 SENESCENCE-RELATED mRNAs

To understand the molecular mechanisms that regulate programmed organ death in response to the increase in ethylene synthesis, we have cloned mRNAs which are

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expressed specifically in senescing flower petals [4]. A cDNA library was prepared from poly(A)+RNA isolated from senescing carnation petals during the ethylene climacteric [4]. This library was screened by differential hybridization using cDNA synthesized from poly(A)+RNA isolated from fresh and senescing flower petals. Using this approach we have, to date, isolated 9 unique cDNA clones which represent SR mRNAs (Fig. 5). Several of these cDNAs have been sequenced and found to exhibit significant homology with known proteins. The predicted protein of pSR8 shares extensive homology with glutathione s-transferases (GST) from a number of organisms [6] . GST catalyzes the conjugation of glutathione to a number of electrophilic compounds and aids in detoxification of many xenobiotics in mammals. We are interested in the possible substrates for the carnation SR GST since little information is available on natural endogenous substrates for these enzymes in plants. The predicted protein of another cDNA clone, pSR132, was found to share significant homology with an enzyme from Streptomyces, carboxyphosphonoenolpyruvate mutase (H. Wang, A. Brandt and W.R. Woodson, unpublished results). This enzyme is involved in the formation of phosphono (C-P) bonds in the biosynthetic pathway of bialaphos, an antibiotic synthesized in Streptomyces which is used commonly as a herbicide. The formation of C-P bonds or the analysis of compounds containing these bonds has not been investigated in plants.

A

H H E ).ACC· ' 0 I I I

).ACC -2 0

E ).ACC-l0 0 I

B

HE

-+~ • • ATG

E I

H I

H I

H I

E I

E I

E I

HE I

HE I

HE II

S !

E I

E I

E I

S I 0

S I 0

S I 0

~

( :r-TAA

Figure 4. Organization and structure of the senescence-related ACC synthase gene in carnation. (A). Restriction endonuclease maps of genomic clones representing the SR ACC synthase gene. Area of hybridization as determined by Southern blot analysis is shown as shaded box. (B), Transcription unit of ACC synthase gene. Introns are shown as open areas and exons as shaded areas.

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pSRS

pSR8

pSR12

pSR103

pSRl18

pSRl20

pSR123 .' • pSR132

pSR139

2.4 ETHYLENE AND SR GENE EXPRESSION

ure 5. Expression of senescence-related lscripts in vegetative organs, and petals I styles from flowers at anthesis (A) or escence (S). Ten f..Ig of total RNA was 'jccted to northern blot analysis and uidized with labelled SR cDNA clones.

The patterns of expression of several of the cloned SR mRNAs have been extensively characterized [4] and in several cases the increase in ethylene associated with petal senescence has been shown to be essential for the transcription of these genes [5]. In order to understand the developmental and hormonal processes that regulate these SR genes. we have begun to characterize the DNA sequences responsible for their expression. Recently. we have focused on the gene encoding the SR GST represented by the cDNA clone pSR8 [6], A cloned region of the genome which shared extensive homology with pSR8 was recently found to contain two tandemly arranged genes encoding GST. The first gene in this cluster contained the entire transcription unit of the SR GST represented by pSR8 in 10 exons interrupted by 9 introns. The 5' flanking DNA of this gene has recently been used to construct a chimeric gene with the coding region of the reporter gene ~-glucuronidase (GUS). In transient gene expression assays following delivery of this chimeric gene into carnation petals by particle bombardment. a 1400 bp GST promoter was sufficient to confer ethylene-responsiveness to GUS expression (H. Itzhaki and W.R. Woodson. unpublished results). These preliminary experiments put us in a position to begin to more critically define the cis-elements involved in the ethylene-induced transcription of GST in senescing carnation petals.

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3. Acknowledgements

This research was supported by grants to W.R.W. from the National Science Foundation (DCB-8911205 and ffiN-9206729) and the United States-Israel Binational Agricultural Research and Development Fund (US-1876-90R).

4. References

1. Borochov, A. and Woodson, W.R. (1989) Physiology and biochemistry of flower petal senescence. Hortic Rev 11,15-43.

2. Huang, P., Parks, J.E., Rottman, W.H. and Theologis, A. (1991) Two genes encoding l-aminocyclopropane-l-carboxylate synthase in zucchini (Cucurbita pepo) are clustered and similar but differentially regulated. Proc Natl Acad Sci USA 88,7021-7025.

3. Larsen, P.B. and Woodson, W.R. (1991) Cloning and nucleotide sequence of a sadenosylmethionine synthetase cDNA from carnation. Plant Physiol 96,997-999.

4. Lawton, K.A., 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.

5. Lawton, K.A., Raghothama, K.G., Goldsbrough, P.B. and Woodson, W.R. (1990) Regulation of senescence-related gene expression in carnation flower petals by ethylene. Plant Physiol 93,1370-1375.

6. Meyer, R.C., Goldsbrough, P.B. and Woodson, W.R. (1991) An ethyleneresponsive flower senescence-related mRNA encodes a protein which shares homology with glutathione s-transferases. Plant Mol BioI 17,277-281.

7. Park, K.Y., Drory, A. and Woodson, W.R. (1992) Molecular cloning of an 1-aminocyclopropane-l-carboxy late synthase from senescing carnation flower petals. Plant Mol BioI 18,377-386.

8. Rottmann, W.H., Peters, G.F., Oeller, P.W., Keller, J.A., Shen, N.F., Nagy, B.P., Taylor, L.P., Campbell, A.D. and Theologis, A. (1991) 1-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.

9. Wang, H. and Woodson, W.R. (1989) Reversible inhibition of ethylene action and interruption of petal senescence in carnation flowers by norbornadiene. Plant Physiol 89,434-438.

10. Wang, H. and Woodson, W.R. (1991) A flower senescence-related mRNA from carnation shares sequence similarity with fruit ripening-related mRNAs involved in ethylene biosynthesis. Plant Physiol 96,1000-1001.

11. Woodson, W.R. and Lawton, K.A. (1988) Ethylene-induced gene expression in carnation petals. Relationship to autocatalytic ethylene production and senescence. Plant Physiol 87,498-503~

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

CLONING OF ETHYLENE BIOSYNTHETIC GENES INVOLVED IN PETAL SENESCENCE OF CARNATION AND PETUNIA, AND THEIR ANTISENSE EXPRESSION IN TRANSGENIC PLANTS.

M.Z. MICHAEL*, K.W. SAVIN, S.c. BAUDINE1TE, M.W. GRAHAM, S.F. CHANDLER, C-Y. LU, C. CAESAR, I. GAUTRAIS, R. YOUNG, G.D. NUGENT, K.R. STEVENSON, E.L-J. O'CONNOR, C.S. COBBETT* and E.C.CORNISH Calgene Pacific Pty. Ltd. * Genetics Department. 16 Gipps St., Collingwood, The UniversityofMelboume. Victoria, Australia, 3066 Parkville, Victoria, Australia, 3052

ABSTRACT. Ethylene regulates the processes of petal senescence in both carnation and petunia. To investigate the relationship between transcriptional regulation of ethylene production and petal senescence, we have cloned genes involved in ethylene biosynthesis from the petals of these species. Using the polymerase chain reaction we have isolated a cDNA clone encoding l-aminocyclopropaneI-carboxylic acid (ACC) synthase from petunia. Heterologous screening of a senescing petal cDNA library provided an ACC synthase enCOding clone from carnation. Increased levels of the mRNAs hybridizing to these cDNA clones is observed in climacteric petals and can be correlated with the onset of senescence. A cDNA clone encoding ACC oxidase was isolated by differential screening of a senescing carnation petal library. Antisense expression of ACC oxidase in transgenic carnation plants inhibits petal inrolling, increasing the post harvest life of the flowers.

Plant senescence is a normal developmental process resulting from endogenously controlled deteriorative changes. Rather than being a passive, chaotic collapse of cellular functions, it involves a tightly regulated sequence of catabolic events.

Flower-life is terminated either by abscission or petal senescence. In species where petal senescence is the primary determinant of flower life-span, the senescence process is generally rapid and predictable. These flowers provide an excellent model system for the molecular characterization of senescence events. Many of the physiological and biochemical changes associated with petal senescence in carnation have been defined. The most dramatic of these being increased ethylene production and biochemical changes in the lipid and protein composition of membranes. While many phytohormones appear to be involved in the regulation of petal senescence in carnation and petunia, ethylene plays a pivotal role in promoting the degradative pathways characteristic of the process (review Cook and Van Staden, 1988). The climacteric nature of carnation and petunia petal senescence is defined by the increased respiration and ethylene production associated with irreversible petal in-rolling or wilting (Joss of turgor). The typical triphasic pattern of ethylene production by petals involves an initially low steady state level, followed by a second phase of accelerated synthesis, with ethylene production reaching a climacteric peak, and finally a third phase in which production declines. The timing of enhanced ethylene production can be correlated to flower longevity. Treatments that inhibit or delay ethylene biosynthesis or action prolong the life of flowers. These include silver ions which appear to inhibit ethylene binding to its receptor (Veen, 1986),and rhizobitoxine and some of its analogues such as aminoethoxyvinylglycine (AVG) and aminooxyacetic acid (AOA) which inhibit production of the ethylene precursor 1-aminocyclopropane-l-carboxylic acid (ACC) (Borochov et al.,1982).

Genetic control over the process of senescence has been reported for petals from a variety of plant species, including carnation (Woodson, 1987). De novo synthesis of RNA in the senescing petals

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J. C. Pech et al. (eds. J. Cellular and Molecular Aspects of the Plant Honnone Ethylene. 298-303. © 1993 Kluwer Academic Publishers.

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of carnation, and ethylene induced changes in gene expression, imply that the catabolic processes responsible for senescence are activated at the level of transcription.

The biosynthetic pathway for ethylene production was determined in 1979 by Adams and Yang. S-adenosylmethionine is converted to l-aminocyclopropane-l-carboxylic acid by ACC synthase. The Ethylene Forming Enzyme (ACC oxidase) acts to oxidize ACC releasing ethylene. The genes encoding both enzymes of the ethylene biosynthetic pathway have been isolated from a variety of sources. ACC synthase was first purified and cloned from zucchini (Sato and Theologis, 1989) and then tomato (Van der Straeten et ai., 1990). Hamilton et al. (1990), showed that antisense expression of a tomato ripening-associated cDNA clone, pTom 13 in transgenic plants resulted in decreased ethylene production, making fruit more resistant to overripening. Subsequent analysis of pTom 13 confirmed that it encodes ACC oxidase.

An understanding of the temporal and spatial regulation of genes involved in ethylene biosynthesis in flowers should offer greater insight into the hormonal control of petal senescence. To undertake such a study, we have isolated cDNA clones which are expressed in the senescing petals of carnation and petunia and encode enzymes involved in ethylene biosynthesis. The antisense expression of one of the carnation clones, pCGP320, which encodes ACC oxidase, in transgenic carnation plants has greatly reduced floral production of ethylene and extended the post harvest life of the flower.

ACC SYNTHASE

Clones encoding ACC synthase have been isolated from petunia (Petunia hybrida cv. Old Glory Blue) and carnation (Dianthus caryophyllus cv. Scania) cDNA libraries using a probe generated by the polymerase chain reaction,together with oligonucleotide primers based on the COding region of the tomato ACC synthase and template cDNA from senescing petunia petal RNA. The carnation cDNA clone, pCGP214, contains a 1.82kb insert which hybridizes with a 2kb mRNA on a Northern blot. The hybridizing transcript is seen only in samples from petals producing elevated levels of ethylene and to a lesser extent from mature ovaries and receptacles. Sequence analysis ofpCGP214 and alignment of the predicted translation product with other sequences in the protein databases suggests that it contains the entire ACC synthase coding region and Ilbp of 5' untranslated region. This clone is 133bp shorter than the published carnation ACC synthase sequence of Park et ai, (1991) and contains several nucleotide differences leading to two amino acid changes (when compared to updated sequence in Genpept library) and an added threonine at pOSition 130. Nucleotide identity between the two sequences is 99.2%.

The petunia cDNA clone, pCGP213, contains an insert of 475bp and hybridizes to a 1.8kb mRNA which has, to date, only been detected in climacteric petals. The Rapid Amplification of cDNA Ends (RACE) strategy of Frohman et al (1988) facilitated isolation of a full length petunia ACC synthase cDNA clone, pCGP217. Sequence analysis of the full length petunia clone indicates that it contains an open reading frame enCOding a polypeptide with an approximate molecular weight of 55 kDalton. Comparison of the predicted pCGP217 product with other sequences in the EMBL and Gcnpept protein databases shows that it displays 84%, 65% and 65% homology with the published ACC synthase amino acid sequences of tomato (Van Der Straeten et ai, 1990), zucchini (Sato et ai, 1991) winter squash (Nakajima et ai, 1990), respectively. It also displays 66% identity to the amino acid sequences derived from carnation clone pCGP214 and the published carnation ACC synthase sequence of Park et ai, (1991).

Translational fusion constructs annealing the petunia and carnation ACC synthase sequences to the B-galactosidase gene of pBluescript (Stratagene) have enabled synthesis of the two active ACC synthase enzymes in Escherichia coli. (XLI-blue). TranSCription of the fusion gene was placed under control of the lac Z promoter. An assay for ACC content in bacterial culture medium has shown that while E. coli, untransformed or containing only the pBluescript vector, produce no ACC, bacteria containing the fusion constructs synthesize detectable amounts of ACe. This synthesis is further enhanced by induction of the lac Z promoter with isopropyl B-Dthiogalactopyranoside. This observed activity of ACC synthesis in E. coli, together with DNA sequence homology data and the expression patterns of hybridizing RNA, indicates that clones pCGP214 and pCGP217 encode ACC synthase enzymes.

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Transgenic petunia and carnation plants expressing their respective ACC synthase sequences in the antisense orientation under the control of the cauliflower mosaic virus 35S and MAC (Calgene Inc.) promoters are presently being generated.

ACC OXIDASE

A cDNA library was created using RNA from the senescing petals of carnation (Dianthus caryophyllus cv.Scania). This library was differentially screened to isolate clones representing mRNAs which were induced with senescence. One of the clones characterised, pCGP320, hybridizes to an approximate 1.3kb mRNA only present in inrolling petals (which are producing elevated levels of ethylene), although it is also seen, to a lesser extent, in other mature tissues. The DNA sequence ofpCGP320 was determined and found to encode a predicted protein of 321 amino acids. Comparison of this translation product with proteins in the EMBL and Genbank databases found that it displayed 68% homology with the tomato fruit ripening-associated cDNA clone, pTom13 later found to encode ACC oxidase (Hamilton et ai, 1990). It was inferred from this homology and the ethylene-associated expression in petals that pCGP320 encodes the carnation ACC oxidase enzyme. A similar clone from Dianthus caryophyllus cv. White Sim has also been identified (Wang and Woodson, 1991).

ANTISENSE EXPRESSION OF ACC OXIDASE IN CARNATION

The cDNA fragment from clone pCGP320 was inserted into a binary expression vector in reverse orientation under the regulation of the constitutive MAC promoter thus generating the plasmid pCGP407. The T-DNA portion of pCGP407 was transferred into the carnation genome using Agrobacterium tumefaciens strain AgIO and transgenic plants were regenerated after selection on kanamycin (Lu et ai, 1991). While most of the transgenic plants generated showed no detectable alteration in phenotype, one transformation event yielded plant #705 which displayed a greater than 90% reduction in ethylene production by its flowers through the period when peak synthesis would be expected (figure 1). Analysis of ethylene generated by leaf wounding showed that this was also greatly reduced in the #705 plant (figure 2). The petals of this plant did not undergo the imolling phenomenon characteristic of the senescence process in control plants and the vase-life of its flowers extended to almost double that of nontransgenic carnation (cv. Scania) plants grown under similar conditions. The flowers of the plant also appear to contain less petals than is normally expected and these petals show a slight decrease in pigmentation. Southern analysis of the #705 plant indicates that it may contain five copies of the introduced DNA.

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CONCLUSIONS

cDNA clones encoding ACC synthase have been isolated from carnation and petunia. Steady state levels of the mRNA hybridizing to these clones is dramatically increased in petals which are synthesizing greatly elevated levels of ethylene correlating with the onset of petal senescence. Such findings support the notion that regulation of ethylene biosynthesis in these tissues is at the level of transcription, although post transcriptional and post translational control of the ACC synthase may also occur. Antisense expression of ACC synthase in transgenic tomato has inhibited ethylene biosynthesis in the fruit, thus delaying ripening(Oeller et ai, 1991). It is hoped that similar experiments in carnation and petunia will also affect ethylene production in petals.

Antisense expression of a carnation ACC oxidase clone in transgenic plants has suppressed ethylene production in mature petals and wounded leaves. The petals of these carnations also fail to display the in-rolling response, a characteristic of climacteric senescence in carnation petals. The normal processes of senescence in the petals therefor appear to have been perturbed, presumably by the failure to oxidize ACC and produce ethylene. This finding confirms the results of physiological experiments carried out by many other laboratories in which petal senescence was delayed by exogenously applied inhibitors of ethylene biosynthesis.

60 ethylene -0-- control (n=9)

nllg/hr • 705-1 50 • 705-2

• 705-3 40 0 578

--0- 791 30 ,. 790

A 802A

20 0 723A

10

0 0 2 4 6 8

Day post harvest

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ethylene nllg/hr (3hr)

30

20

10

o

plants

Figure 2. Ethylene produced by wounded leaf samples from control (con 1,2,3,4) and pCGP407 transgenic carnation plants, over a 3 hour period.

LITERA TURE CITED:

Borochov, A, Mayak, S.and Brown, R., (1982) 'The involvement of water stress and ethylene in senescence of cut carnation flower~', J. Exp. Bot. 33: 1202-1209

Cook, E.L. and Van Staden, (1988) 'The carnation as a model for hormonal studies in flower senescence', Plant Physiol. Biochem. 26: 793-807

Frohman, M.A., Dush, M.K. and Martin G.R., (1988) 'Rapid production of full-length cDNAs from rare transcripts: Amplification using a single gene-specific oligonucleotide primer', Proc. Nat. Acad. Sci. ,USA 85: 8998-9001

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

Lu, C-Y., Nugent, G., Wardley-Richardson, T., Chandler, S.F., Young, R., and Dalling, M..T. (1991) 'Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.)', Biotechnology 9: 846-868

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

Sato, T. and Theologis, A, (1989) 'ClOning the mRNA encoding l-aminocyclopropane-lcarboxylate synthase, the key enzyme for ethylene biosynthesis in plants', Proc. Natl. Acad. Sci., USA, 86: 6621-6625

Van Der Straeten, D., Van Wiemeersch, L., Goodman, H.M. and Van Montagu, M., (1990) 'Cloning and sequence of two different cDNAs encoding l-aminocyclopropane-lcarboxylate synthase in tomato', Proc. Nat. Acad. Sci., USA, 87: 4859-4863

Veen H. (1986) 'A theoretical model for anti-ethylene effects of silver thiosulphate and 2,5-norbornadiene', Acta Horticulturae 181: 129-134

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Wang, H., and Woodson, W.R., (1991) 'A flower senescence-related mRNA from carnation shares sequence similarity with fruit ripening-related mRNAs involved in ethylene biosynthesis', Plant Physiology 96: 1000-1001

Woodson, W.R., (1987) 'Changes in protein and mRNA populations during the senescence of carnation petals', Physiologia Plantarum 71: 495-502

INTERORGAN REGULATION OF POST-POLLINATION EVENTS IN ORCHID FLOWERS

J.A. Nadeau, A.Q. Bui, X. Zhang and S.D. O'Neill ~prurunentofBowmy University of California, Davis Davis, CA 95616

ABSTRACT Pollination of many flowers initiates a sequence of precisely regulated developmental events that include senescence of the perianth and development of the ovary. The plant hormones ethylene and auxin are known to play key roles in regulating the biochemical and anatomical changes that constitute the pollination syndrome. The Phaiaenopsis orchid flower provides a unique system to study pollination events because the pollination syndrome is especially well developed and does not overlap temporally with age-related senescence programs. We have chosen to begin study of the pollination syndrome by examining the spatial and temporal location of ethylene biosynthesis within the orchid flower as it relates to the observed developmental events, and how this biosynthesis is regulated by factors that influence expression of genes that encode two major enzymes in the ethylene biosynthesis pathway. This information leads to a model of how ethylene production is modulated within the flower to control the pollination syndrome.

1. Introduction

It has been recognized for many years that the pollination of flowers greatly accelerates senescence of nonessential floral organs, in addition to triggering development of the ovary. Pollination of the flower results in a climacteric rise in ethylene production very similar to that in ripening fruit, which is thought to regulate the senescence process. Treatment of flowers with ethylene accelerates senescence (Borochov and Woodson, 1989), while senescence of the perianth can be reversibly inhibited by the competitive inhibitor of ethylene action, norbornadiene (Wang and Woodson, 1989). This suggests that ethylene is both sufficient and necessary for normal pollination-induced senescence of the perianth. This conclusion is complicated, however, by the observation in some systems that sensitivity of the perianth to ethylene increases after pollination (Halevy et aI., 1984, Whitehead and Halevy, 1989. and Stead and Moore, 1979).

The post-pollination syndrome is fundamentally different in several important ways, however, from that of age-related senescence though both processes are mediated through ethylene production. In orchid flowers, cells of the column swell so that the organ becomes enlarged and the stigmatic cavity is closed around the pollinia, ovule development is stimulated, and the ovary begins to enlarge and differentiate (Curtis, 1943). Treatment of flowers with ethylene does not stimulate these developmental events to occur, though treatment of pollinated flowers with norbornadiene does inhibit these events (O'Neill, unpublished observations). This suggests that ethylene is necessary, but not sufficient to

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initiate these processes. With this in mind, the question remains as to how the pollination event is initially

perceived by the flower, and how the signal is communicated to other parts of the flower such that they are induced to fulfill an appropriate developmental fate. Advances in our understanding of the genetic basis of ethylene biosynthesis will, for the first time, allow us delve deeper into the process of post-pollination development to understand its basis at the level of gene expression. This should allow a dissection of the physiological control of this process that has been elusive up to this point. Following is a brief summary of the advances we have made in understanding the regulation of the post-pollination syndrome of Phalaenopsis orchids in relation to previous physiological work.

2. Initial Stimulus

2.1 AUXIN

Early investigators proposed that pollen contained a substance which triggered the postpollination syndrome in Phaiaenopsis orchids (Fitting, 1909). Later it was suggested that auxin, which is present in pollen, might be the "pollenhormon" (Muller, 1953). These suggestions agreed well with the observation that auxin can induce ethylene production in many tissues (Abeles, 1966), leading various investigators to suggest that auxin deposited on the stigma was the primary signal perceived by the gynoecium, which stimulated ethylene production (Burg and Dijkman, 1967). On the other hand, in Petunia, pollination with incompatible or heat-killed pollen does not elicit premature senescence, which argues against the suggestion that auxin diffusing out of pollen onto the stigma is the primary inducing stimulus (Oilissen, 1977; Singh et aI, 1992).

In order to examine the role of auxin as the primary stimulus, we have examined the effect of auxin treatment on the abundance of ACC synthase (AS) and ACC oxidase (AO) transcripts in the flower. Neither AS nor AO mRNA is detectable in unpollinated flowers, however, within 24 hours of harvest AO mRNA is present at low levels in the stigma. Treatment of the stigma with 151lg NAA results in a dramatic increase in both AS and AO mRNAs in the stigma, perianth and labellum. This increase in gene expression is abolished by pretreatment of the stigma with 100 nmoles of the ACC synthase inhibitor aminoethoxyvinylglycine (A VO), indicating that auxin induction must be mediated through ethylene (data not shown). It is still possible, however, that a component of expression of AS and AO is induced directly by auxin contained in the pollen, but that it is below the level of detection by RNA gel blot hybridization analysis. Experiments using Polymerase Chain Reaction (PCR) technology are underway to determine if auxin can directly induce an AS or AO gene during the initial recognition of pollination by the stigma.

2.2 ACC

Whitehead et al. (1984) and Reid et al. (1984) have reported that significant ethylene production is observed when ACC is applied to the stigmas of Petunia or carnation. Since ACC, the precursor to ethylene, is present in most types of pollen at physiologically significant levels (Whitehead et aI, 1983) it was suggested that ACC may serve as the pollination signal by diffusing out of the pollen to the stigma where it is converted to ethylene (Whitehead et aI, 1984). This would then induce senescence of the flower, or more likely, autocatalytic ethylene prOduction. Hoekstra and Weges (1986) have found, however, that pollen ACC is not converted to ethylene on the stigma of Petunia plants, andthe work of Pech et al. (1987) reinforces this finding by suggesting that the osmotic environment of the Petunia stigma is not conducive to diffusion of ACC out of the pollen.

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On the other hand, Singh et al. (1992) have observed a rapid initial peak in ethylene production after compatible and incompatible pollinations of Petunia that is followed by a second, larger peak in ethylene production by compatible pollinations only. They reason that the first peak is due to stylar conversion of pollen ACC, since it is not inhibited by the ACC synthase inhibitor aminooxyacetic acid (AOA).

Work in our laboratory demonstrates that in Phalaenopsis orchids, application of ACC to the stigma causes the production of ethylene by the flower and ultimately results in complete senescence of the flower without the full suite of events normally associated with the post-pollination syndrome, in a manner analogous to that of exogenously applied ethylene. Application of ACC to the stigma dramatically increases AS and AO gene expression in all organs of the flower above those of unpollinated flowers. This increase in gene expression can be abolished by treatment with A VG or norbornadiene (NBD), which suggests that the observed induction is autocatalytic ally induced by ethylene produced by the enzymatic oxidation of ACC (data not shown). PrelimitJary measurement of the quantity of ACC in Phalaenopsis pollinia suggest that there is little or no ACC present. This, coupled with the fact that ACC cannot mimic the range of effects observed after pollination, has lead us to conclude that pollen-born ACC alone is not the initial stimulus in the orchid system.

2.3 WOUNDING

In Petunia, Gilissen (1977) and Whitehead et al. (1984) have suggested that pollen tube penetration of the stigma and style is equivalent to wounding, and as in other wounded tissues, evokes ethylene synthesis. The results of Singh et al. (1992) suggest that this cannot be the only factor signalling pollination because premature senescence is not induced by incompatible pollen tubes that penetrate the style before aborting in Petunia.

Wounding can also take the form of emasculation which is a natural consequence of pollinator visitation in orchid systems. The work of Woltering and Harren (1989) has demonstrated that desiccation of the rostellum that is uncovered during emasculation is the trigger for ethylene production in Cymbidium that ultimately results in senescence of the flower. Emasculation does not, however, lead to the full spectrum of post-pollination events.

In the Phalaenopsis system, wounding of the stigma and emasculation that involves wounding both result in ethylene production and complete senescence of the flower without the complete post-pollination syndrome. Both treatments result in dramatic increases in AS and AO mRNA in all organs of the flower that normally express these genes. This suggests that emasculation may serve as part of the pollination stimulus, though it clearly cannot be responsible for all of the observed changes. Additionally, pollen tube penetration of the stigmatic surface plays no role in Phalaenopsis, since most of the events described take place before pollen tubes emerge from the grains (data not shown). This does not, however, preclude the possibility that polysaccharides are released from the hydrated pollen grains that effect a wound-like response.

3. Propagation of the Response

3.1 ACC

After the pollination event a signal must be transmitted from the stigma, the initial site of perception, to the other floral organs where appropriate developmental events are triggered. It has been proposed that ACC synthesized by the stigma might serve as the transmitted stimulus in flowers. Woltering (1990) infers that the stimulus that leads to coloration of the

307

lip and eventual senescence of Cymbidium orchids is ACC translocated from the emasculated column. Separation of the floral parts results in a rapid decline in ethylene production, presumably because they have been isolated from the source of ACC. Furthermore, lip coloration is still induced in flowers treated with A VG and emasculated after they are treated with ACC. Finally, Reid et a1. (1984) have demonstrated that 14C_ ACC applied to the stigma is translocated to the petals of carnation flowers, where it is released as ethylene during senescence.

Our work with Phaiaenopsis orchids suggests that ACC may indeed be translocated from the gynoecium, where the ACC level is very high, to the perianth, where ACe standing pools are very low. Blot hybridization shows that AS mRNA is absent or expressed at undetectable levels in the perianth, which strongly suggests that this tissue is incapable of ACC synthesis (data not shown). It still remains a possibility, however, that AS normally expressed in the perianth has low sequence homology to genes we have cloned from the gynoecium such that it is not detected. Experiments to determine whether AS activity is found in the perianth are underway.

3.2 ETHYLENE

Woltering (1990) has suggested that ethylene is translocated within the flower after emasculation in order to stimulate lip coloration and later senescence of the flower. Application of ethephon to the stigma of an A VG treated flower results in pigmentation of the lip, implicating ethylene itself as the transmitted stimulus.

From our work with Phaiaenopsis orchids, it is apparent that pollination results in increased levels of AS and AO in the floral organs, but this accumulation can be abolished by treatment of pollinated flowers with NBD (data not shown). Flowers treated in this way also do not senesce normally. This indicates that ethylene production stimulated by pollination is responsible for autocatalytic ethylene biosynthesis in the flower.

Additionally, it is apparent that translocated ACC acts only to actuate a response induced by ethylene perceived by other parts of the flower. Application of ACC to the stigma results in dramatic increases in AS in the stigma and labellum and AO mRNA levels in the stigma, perianth and labellum, as stated previously. When these flowers are treated with NBD, however, AS and AO mRNA levels are equivalent to control flowers. These results indicate that ethylene must induce AO gene expression in the perianth before ACC can be oxidized to ethylene in the perianth. We would predict that later, the process of AO gene expression becomes autocatalytic ally self-sustaining within this organ. Thus, ACC should not be thought of as the transmissible signal that communicates to other parts of the flower that pollination has taken place, but as the actuator of the response. The primary signal, we believe, is ethylene produced by the column.

3.3 AUXIN

Strauss and Arditti (1982) and Reid et a1. (1984) have demonstrated that auxin movement is too slow within the flower to account for the transmission of the pollination stimulus to other organs. None the less, it is obvious that the ovary receives a signal that induces development, rather than senescence, upon pollination. This observation is born out by the fact that in Phaiaenopsis, mRNA levels of both AS and AO decrease in abundance in the ovary after both normal pollination and application of NAA to the stigma. Flowers treated with NAA and 10ppm ethylene demonstrate that NAA has an antagonistic effect on the ethylene-regulated induction of these genes in the ovary (data not shown). We speculate that repression of AS and AO in the ovary is the normal prerequisite of further development of this tissue rather than senescence and ultimate abscission.

308

3.4 SENSITIVITY FACTORS

Studies of several systems such as Cyclamen (Halevy et aI, 1984) and Digitalis (Stead and Moore, 1979) have suggested that pollination induces sensitivity of floral tissues to ethylene. In both cases, exposure to exogenous ethylene does not promote normal corolla abscission of unpollinated flowers. In Digitalis, auxin applied to the stigma cannot replace the effect of pollination by allowing the corolla to respond to ethylene by abscising. Whitehead and Halevy (1989) have suggested that short chain fatty acids may be synthesized in the stigma and transported to the corolla of Petunia in order to promote sensitivity to ethylene.Work with PhalaelWpsis orchids indicates that pollination may cause changes in perianth sensitivity to ethylene. When senescence of the perianth is scored after treatment of the flower with ethylene at discrete intervals after pollination, it is apparent that sensitivity to ethylene increases with time. Pre-treatment of flowers with AOA does not abolish this change in sensitivity, indicating that the increase ih sensitivity to exogenous ethylene is not dependent on the production of ethylene by the flower (Porat et aI, in preparation).

OVARY

G SAM

T a1'>S ort? ACC

ethylene 0

Figure 1. Model of the regulation of pollination-induced ethylene production that brings about senescence oJ the perianth and which is involved in regulating other developmental events of the post-pollination syndrome of Phaiaenopsis flowers. Abbreviations: stigma, (s), petal (p) and ovary (0).

4. Conclusion

In conclusion, analysis of the spatial and temporal regulation of genes involved in ethylene biosynthesis has allowed us to dissect the complex control of the post-pollination syndrome in Phalaenopsis orchids (Figure 1). In the orchid system, pollen-born auxin induces the expression of genes encoding enzymes involved in ethylene biosynthesis. Ethylene, however, is required for both auxin-induced induction of ACC synthase and ACC oxidase gene expression and the full spectrum of pollination-induced developmental events. It has not yet been resolved how this apparent "Catch-22" can be resolved, but our future efforts

309

will focus on the process of early gene induction by pollination (auxin) in order to explain the co-dependency of these events on both ethylene and auxin. We believe that pollen-born ACC is unlikely to playa role in the initial stimulus, but that ACC produced by the column is translocated to the perianth were it serves to actuate the process of ethylene evolution by these organs which, in turn, regulates their senescence. In the orchid system, it is also possible that wounding due to emasculation or other substances released by the pollen may playa role in the initial pollination stimulus. Our research suggests that the transmitted signal that acts to propagate the pollination response throughout the flower is ethylene produced by the gynoecium. This ethylene induces ACC oxidase gene expression in the perianth which results in competence of the tissue to produce ethylene. Other transmissible signals are undoubtedly involved in regulating ovary development and sensitivity or response of tissues to ethylene.

5. References

Abeles, F.B. (1966) Auxin stimulation of ethylene evolution. Plant Physio!. 41:585-588. Borochov, A and Woodson, W.R. (1989) Physiology and biochemistry of flower petal senescence. Hort.

Rev. 11:15-43. Burg, S.P. and Dijkroan, MJ. (1967) Ethylene and auxin participation in pollen induced fading oWanda

orchid blossoms. Plant Physio!. 42:1648-1650 Curtis, J.T. (1943) An unusual pollen reaction in Phaiaenopsis. Amer. Orchid Soc. Bull. 21: 98-100. Fitting, H. (1909) Die beeinflussung den Orchideenbluten durch die bestaubung und durch andere umstande.

Zeit. Bot. 1: 1-86. Gilissen, LJ.W. (1977) Style-controlled wilting of the flower. Planta 133:275-280. Gilissen, L.J.W. and Hoekstra, F.A. (1984) Pollination-induced corolla wilting in Petunia hybrida rapid

transfer through the style of a wilt-inducing substance. Plant Physio!. 75:496-498. Halevy, A.H., Whitehead, C. S., and Kofranek, A.M. (1984) Does pollination induce corolla abscission in

Cyclamen flowers by promoting ethylene production? Plant Physio!. 75:1090-1093. Hoekstra, F.A. and Weges, R. (1986) Lack of control by early pistillate ethylene of the accelerated wilting

of Petunia hybrida flowers. Plant Physio!. 80:403-408. Muller, R. (1953) Zur quantitatinen Bestimmung von Indolylessigsaure mittels Papierchromatographie and

Papierelektrophorese. Beitrage zur Bio!. der Pflanzen 30: I -32. Pech, J-c., Latche, A., Larrigaudiere, C., and Reid, M.S. (1987) Control of early ethylene synthesis in

pollinated petunia flowers. Plant Biochem. 25:431-437. Reid, M.S., Fujino, D.W., Hoffman, N.F., and Whitehead, C.S. (1984) l-aminocyclopropane-l-carboxylic

acid: the transmitted stimulus in pollinated flowers? J. Plant Growth Reg. 3:189-196. Singh, A., Evensen, K.B., and Kao, T. (1992) Ethylene synthesis and floral senescence following

compatible and incompatible pollinations in Petunia inflata. Plant Physio!. 99:38-45. Stead, A.D. and Moore, K.G. (1979) Studies on flower longevity in Digitalis. Planta 146:409-414. Strauss, M. and Arditti, J. (1982) Post-pollination phenomena in orchid flowers. X. Transport and fate of

auxin. Bot. Gaz. 143:286-293. Wang, H. and Woodson,W.R. (1989) Reversible inhibition of ethylene action and interruption of petal

senescence in carnation flowers by norbomadiene. Plant Physio!. 89:434-438. Whitehead, C.S. and Halevy, AH. (1989) Ethylene sensitivity: the role of short chain saturated fatty acids

in pollination-induced senescence of Petunia hybrida flowers. Plant Growth Reg. 8:41-54. Whitehead, C.S., Halevy, AH., and Reid, M.S. (1984) Roles of ethylene and l-aminocyclopropane-l

carboxylic acid in pollination and wound-induced senescence of Petunia hybrida flowers. Physio!. Plant. 61:643-648.

Whitehead, C.S., Fujino, D.W., and Reid, M.S.(1983) Identification of the ethylene precursor, ACC, in pollen. Sci. Hort. 21:291-287.

Woltering. EJ. (1990) Interorgan translocation of l-aminocylopropane-l-carboxylic acid and ethylene coordinates senescence in emasculated Cymbidium flowers. Plant Physio!. 92:837-845.

Woltering, EJ. and Harren, F. (1989) Role of rostellum desiccation in emasculation-induced phenomena in orchid flowers. J. Exp. Bot. 40(217):907-912.

ROLES OF ETHYLENE, ACC AND SHORT-CHAIN SATURATED FATTY ACIDS IN INTER-ORGAN COMMUNICATION DURING SENESCENCE OF CYMBIDIUM FLOWERS

ERNST J. WOLTERING Agrotechrwlogical Research Institute (ATO-DLO) P.O. Box 17. 6700 AA Wageningen. The Netherlilnds

ABSTRACT. In Cymbidium flowers. emasculation leads to a transient increase in ethylene production. In intact flowers, the major part of the ethylene is derived from the perianth (lip and petals), whereas an increase in the level of l-aminocyclopropane-l-carboxylic acid (ACC) and Nmalonyl-ACC is only observed in the upper part of the central column (gynostemium). In perianth parts, excised at different times after emasculation, the production of ethylene ceases within 10 min after excision. Application of ACC to the top of the central column leads to the production of ethylene by the petals. These results indicate that both endogenously produced as well as applied ACC is translocated within the flower.

Following emasculation the ethylene concentration in the internal gas phase of the central column showed an increase up to 4 J.1L L·'. In the perianth parts the concentration amounted to 0.7 J.1L L·'. Treatment of the central column with ethylene or ethephon leads to an ethylene response in the lip and petals, while no increase in ACC in any of the flower parts is observed. This indicates that ethylene, too, is translocated within these flowers.

Application of octanoic and decanoic acids to the stigma had no effect on flower life. Treatment of isolated lips with these acids had no effect on red coloration or the sensitivity to exogenous ethylene. The roles of ACC, ethylene and short-chain saturated fatty acids in the coordination of flower senescence is discussed.

1. Introduction

In an early stage of senescence, transport of substances between the floral parts may play a role in the initiation and coordination of the senescence processes. This has been studied in pollinated flowers. Pollination generally has a dramatic effect on flower life and it has been suggested that mobile wilting factor(s), present in the pollen or produced in the stigma after pollination, playa role. Gilissen (1976; 1977) noticed that, in Petunia, pollen tube growth or mechanical wounding of the stigma and style induced premature wilting of the flower and argued that the stigma may produce a signal that moves to the other flower parts. This mobile wilting stimulus was produced within 4 to 6 h after pollination or wounding of the stigma.

Circumstantial evidence has accumulated over the years that in pollinated flowers, the direct precursor of ethylene, ACC, may play a role in the coordination of the senescence process (Nichols et al. 1983; Hoekstra and Weges, 1986). Reid et al. (1984) provided direct proof for the translocation of ACC between flower parts. These authors treated the carnation stigma with ACC and were able to recover a burst of ethylene from the petals. In addition, petals produced radiolabeled ethylene from stigma-applied radiolabeled ACC. It has been suggested that besides ACC, other wilting factors (Le. sensitivity factors) may also be translocated in pollinated flowers. In Cyclamen flowers, pollination-induced corolla abscission was ascribed to the action of a pollination-induced ethylene-sensitivity factor as abscission in pollinated flowers could be prevented by silver thiosulphate, whereas it could not be induced by ethylene or ACC in non-pollinated flowers (Halevy et al., 1984).

Eluates from pollinated styles possess wilt-inducing properties when re-applied to the stigma of fresh flowers while these eluates did not contain any detectable ACC (Gilissen and Hoekstra. 1984). GC-MS analysis of the composition of the Petunia style eluate showed that a number of short-chain saturated fatty acids were present (Whitehead and

310

J. C. Pech et al. (eds.), Cellular alld Molecular Aspects of the Plant H0171lolle Ethylene, 310-316. © 1993 Kluwer Academic Publishers.

311

Halevy, 1989a). These fatty acids seemed to have wilt-inducing properties and it was suggested that they were produced in the pollinated style and subsequently transported to the corolla. In the corolla, they are thought to render the tissue more sensitive to ethylene thereby causing an acceleration of the wilting process (Whitehead and Halevy, 1989a, 1989b).

In Cymbidium flowers, senescence can be induced by emasculation, i.e. removal of the pollinia and the anther cap (Fig. I). Desiccation of the tissue, fonnedy covered by the anther cap (i.e. the rostellum) was found to be the primary trigger of the emasculation-induced senescence process. Localized desiccation induces a small peak in ethylene production and red coloration of the labellum (lip). The latter can be regarded as the first visible sign of senesa:nce (Woltering and Harren, 1989; Figure 1. Emasculation treatment. Woltenng and Somhorst, 1990).

This paper discusses the roles of ACC, ethylene and short-chain fatty acids and their translocation during emasculation-induced senescence in Cymbidium flowers.

2. Materials and Methods The experiments were carried out with Cymbidium cv Jacobi obtained from a commercial grower. Individual flowers or isolated lips, cut from the middle region of the spike, were used. Generally, the flowers were placed with their stems in water and chemical treatments were carried out by pipeting a fixed amount of the chemical in an aqueous solution onto the rostellum or the stigma. Treatment of the central column with gases was carried out by flushing the gases through a 7 mm wide tube that was fitted over the upper half of the column. When isolated lips were used, they were placed with their cut base in aqueous solutions of the chemicals. In vitro ethylene production was measured by enclosing excised flower parts in a small glass vial after which samples of the headspace were analysed by GC. In vivo ethylene production was measured using a flow-through system coupled to a laserphotoacoustic detector.

ACC was measured by the method described by Lizada and Yang (1979) with internal standardization. Internal ethylene levels were measured by the method described by Beyer and Morgan (1970). Measurements of the production of radioactive ethylene from column applied radiolabeled ACC were carried out in a flow-through system. The produced ethylene was trapped in mercuric perchlorate and radioactivity measured by liquid scintillation counting.

For details on the experimental procedures, see Woltering et al., 1988; Woltering, 1 990a; Woltering, 1990b.

3. Results and Discussion 3.1. EVIDENCE FOR ACC TRANSLOCATION Following emasculation, pink. coloration of the lip is generally visible within 24 h. In one extra day the color of the lip is dark-red (Woltering and Somhorst, 1990). Coloration of isolated lips was shown to be an effect of endogenously produced or applied ethylene (Woltering, 1989). It may therefore be concluded that the factor responsible for lip coloration and perianth senescence is related to ethylene e.g. ethylene or ethylene sensitivity or production inducing factors.

Measurements of the ethylene production in different flower parts, isolated at different

312

times after emasculation, revealed that the ethylene is exclusively produced by the central column (Fig. 2). The perianth parts (5 petals and the lip) produced less than 10% of the total amount of ethylene produced by the flower during this experiment. These results are in line with the data on ACC and MACC levels in different flower parts (Fig. 3 and 4). Only in the upper part of the central column (approximately 1/3 of the length) an increase in ACC and MACC was detected; in the lower part of the column and in the perianth parts, no significant changes were found. Similarly, after treatment of the central column with ACC, the ethylene produced was exclusively derived from the central column, whereas the lip showed rapid coloration (data not shown). These data seem to rule out the possibility that ethylene production in the lip is responsible for the observed coloration. In addition, translocation of ACC seems unlikely. Rather, the lip may become more sensitive to ethylene following emasculation due to the involvement of mobile ethylene sensitivity factor(s).

To further characterize the presumed translocated factor, the central column was treated with different chemicals (Table 1). In emasculated flowers, lip coloration was effectively inhibited by treatment with A VG, whereas treatment with air allowed coloration. Treatment of an A VG-treated flower with ACC restored the normal response. These data indicate that diffusion of ethylene out of the column and, through the air, into the lip does not play a role. Contrary to what may be hypothesized from the ethylene production measurements in isolated organs (Fig. 2), the effect of A VG strongly suggests that ACC is the translocated factor.

Similar conflicting data were reported by Hoekstra and Weges (1986) who studied the ethylene production of different flower portions of Petunia, isolated at different times after pollination. During the early pollination-induced increase in ethylene production, the wilting factor was shown to be translocated to the corolla, whereas no extra ethylene could be detected in this organ. In this case, the majority of the ethylene was produced by the gynoecium. They also found that A VG applied to the stigma, was a very effective inhibitor of pollination-induced senescence, even when the entire style was removed before the A VG could have reached the corolla. This made Hoekstra and Weges (1986) conclude that, although direct proof was lacking, ACC may be the transported wilting factor in Petunia.

To further explore the possible role of ACC translocation in emasculation-induced senescence we measured the in vivo ethylene production of the central cohnnn and the perianth following emasculation. A simultaneous increase in ethylene production was apparent in the different flower parts (Fig. 5). The perianth started to produce ethylene within 3 h of emasculation. In this experiment, the perianth produced over 80 % of the total amount of ethylene. This is in marked contrast to the results obtained from in vitro ethylene production measurements (Fig. 2). Following application of radiolabeled ACC to the rostellar surface of an emasculated A VG-treated flower, both the column and the perianth produced substantial amounts of radiolabeled ethylene (Table 2).

These data show that in the intact flower, all the different organs produce significant amounts of ethylene following emasculation. This ethylene may well be derived from ACC, which is translocated from the site of production/application to the other parts of the flower. Experimental data on ethylene production in isolated plant parts (in vitro) may easily lead to erroneous conclusions concerning the in vivo processes. Excision of a plant part blocks the influx of ACC, resulting in immediate cessation of ethylene production. In figure 6 it is shown that lips, at the time of excision from an emasculated flower, indeed show a high ethylene production. Within about 10 min ethylene production ceases.

3.2. EVIDENCE FOR ETHYLENE TRANSLOCATION Measurements of internal ethylene levels in different flower parts revealed that, in

particular in the central column, the concentrations may be quite high (Fig. 7). The ethylene response in the perianth may therefore partly be due to the diffusion of ethylene from the column. The possibility of ethylene translocation within the flower was therefore investiga-

:2 ,": 2.0 " " ....

...J

" -1,5 " o 't; -61,0 o 0..

o 10 15 20 25 30

time Ihl

15

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313

upper part column

perianth and lower part column

of=~~~~~:=~ o 5 10 15 20 25 30

time Ihl

Figure 2 (left). In vitro ethylene production of different flower parts following emasculation at t ::;; O. Flower parts were excised at different times after emasculation and enclosed in a small vial. Ethylene was measured by GC after 1 to 2 h incubation. Figure 3 (right). ACC content in different flower parts following emasculation at t::;; O.

80

~ u-C> 60 .... '0 E ..s 40 () ()

upper part column

lower part column

:;; 20 r~===~:7"'~---...---__ 1

perianth

o+---,---,---,----.---,---,~

o 5 10 15 20 25 30

time Ihl

:2 ,~ 1.2

" .... ...J ..s " ,20.8 U " -0 o 0.. ",0.4

" '" -;;. .s:: Q;

column

0.0 +----r----,--,-----,---r--,---'

o 5 10 15 20 25 30

time (hI

Figure 4 (left). MACC content in different flower parts following emasculation at t ::;; O. Figure 5 (right). In vivo ethylene production of the central column and the perianth following emasculation at t ::;; O. Ethylene release from column and perianth was continuously monitored with a laser photoacoustic detector.

~2.0 .... ...J

" " 1.5 o ';:; o

" -g 1.0 0..

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lips excised from emasculated flower

Q; control lips

0.0 o 5 10 15

5 ::; .... ...J

4 2-

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0 0 5 10 15

time Iminl time (hI

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20 25 30

Figure 6 (left). Ethylene production of lips excised from emasculated flowers at t ::;; 15 h and of lips excised from non-emasculated flowers (control). Figure 7 (right). Ethylene concentration of the internal gas phase of different flower parts following emasculation at t ::;; O.

314

Table 1. Effect of different chemicals on lip coloration in Cymbidium. The gases were applied to the upper part of the central column; A VG, ACC and ethephon were applied onto the rostellum of an emasculated flower. In A VG-treated flowers any additional chemicals were applied 16 h after the AVG treatment.

Treatment Emasculation Time to lip (+/-) coloration (days)

Air - 9 C2H4 (4 J.1L L-t ) - 1.5 Air + 1.5 A VG (100 nmo1) + 12 A VG + ACC (5 nmol) + 1.5 AVG + C;H4 + 2 A VG + ethephon (2 Ilg) + 1.5

Table 2. Radioactivity in ethylene traps 20 h after application of radiolabeled ACC to the rostellum of an emasculated A VG-treated Cymbidium flower.

Source of activity Activity (Bq) % of total

Ethylene from column 2901 42

Ethylene from perianth 3953 5

Table 3. Effects of octanoic and decanoic acids on time to lip coloration (color score = 2) The chemicals were either applied to the stigma of intact flowers (pooled data from 2, 10 and 20 nmol application) or they were applied via the cut base of isolated lips (0.1 roM) in a 7 mM citrate buffer (PH 4).

flower parts Chemical treatment Ethylene treatment Time to lip colora-(J.1L/L) tion (days)

Whole flowers water 0 8.5

octanoic acid 0 8.5

decanoic acid 0 9.5

Isolated lips buffer 0 > 25 0.4 1.7

octanoic acid 0 > 25 0.4 1.7

decanoic acid 0 > 25 0.4 2.0

315

ted. When the column of a non-emasculated flower is treated with ethylene, the lip shows

coloration (Table 1). Similarly, application of ethylene or ethephon to an emasculated AVG-treated flower leads to lip coloration (Table 1). This indicates that ethylene does not act through the induction of ACC synthase in the column and subsequent translocation of ACC. ACC content in the columns did not show an increase during the treatment (data not shown) which confirms this view. The transport of ethephon is thought to be slow (Beaudry and Kays, 1988) and lips excised from ethephon-treated flowers, in contrast to the columns, did not show an increased ethylene production (data not shown). This indicates that no ethephon was present in the lip and that ethylene externally applied or internally derived from ethephon may be translocated within the flower.

Although the translocation of ACC was shown to be quite fast (Fig. 5), ethylene translocation appeared to be much slower (data not shown). Ethylene, therefore, can only play an additional role in inter-organ communication during senescence of Cymbidium flowers.

3.3. ROLE OF SHORT-CHAIN SATURATED FATTY AODS In a recent paper, Whitehead and Halevy (l989b) described the nature of a presumed

pollination-induced ethylene-sensitivity factor in Petunia flowers. These authors showed that following pollination, but also during aging in unpollinated flowers, there is an increase in the levels of, among others, the short-chain saturated fatty acids octanoic and decanoic acids in the corolla. It was argued that, following pollination, these fatty acids were produced in the style and transported to the corolla. Like pollination, treatment of the stigma with these subtances was shown to increase sensitivity of the flower to ethylene.

In our experiments, the fatty acid solutions were applied onto the stigma of intact Cymbidium flowers. The solutions were prepared and applied in a similar way as was described by Whitehead and Halevy (1989b). Even after treatment with five or ten times higher amounts than used by these authors, no effect on flower life was observed (Table 3; Woltering et al., 1992). Our data seem in disagreement with the data presented by Whitehead and Halevy. However, these authors always studied the effects of fatty acids in the presence of exogenous ethylene whereas our experiments with intact flowers were performed under natural conditions. This leaves open the possibility that the senescence-promoting effect of fatty acids depends on the presence of ethylene.

Therefore, an additional study was carried out with isolated lips. These flower parts produce negligible amounts of ethylene and may stay fresh for more than a month when placed with their cut bases in water. Isolated lips are responsive to treatment with ethylene. This causes a rapid increase in phenylalanine ammonia-lyase activity and a change in color, which is easy to assess and quantify by using color prints (Woltering, 1989; Woltering and Somhorst, 1990). Treatment of the lips with octanoic and decanoic acids, applied at pH4, had no appreciable effect on coloration and ethylene sensitivity (Table 3). It may, therefore, be concluded that these fatty acids apparently play no pivotal role in senescence of Cymbidium flowers. Their senescence-promoting effect may be restricted to certain species or developmental stages.

4. Conclusion Perianth senescence (including lip coloration) in emasculated Cymbidium flowers is a result of ACC translocation from the site of production (the rostellum) to the other flower organs. In addition, accumulation of ethylene in the central column results in translocation of the gas to other parts of the flower. The translocation of ACC is much faster than that of ethylene and the latter may therefore only play a minor role in the coordination of the senescence process. No indications for the involvement of short-chain saturated fatty acids in inter-organ communication were found.

316

References

Beaudry, R.M. and Kays, S.l., 1988. Flux of ethylene from leaves treated with a polar or non-polar ethylene-releasing compound. l. Amer. Soc. Hort. Sci. 113:784-789.

Beyer, E.M. and Morgan, P.W., 1970. A method for detennining the concentration of ethylene in the gas phase of vegetative plant tissues. Plant Physiol. 46:352-354.

Gilissen, L.J.W., 1976. The role of the style as a sense organ in relation to wilting of the flower. Planta 131:201-202.

Gilissen, L.l.W., 1977. Style-controlled wilting of the flower. Planta 133:275-280. Gilissen, L.l.W. and Hoekstra, F.A., 1984. Pollination-induced corolla wilting in Petunia

hybrida. Rapid transfer through the style of a wilting-inducing substance. Plant Physiol. 75:496-498.

Halevy, A.H., Whitehead, C.S. and Kofranek, A.M., 1984. Does pollination induce corolla abscission of cyclamen flowers by promoting ethylene production? Plant Pbysiol. 75: 1090-1093.

Hoekstra, F.A. and Weges, R., 1986. Lack of control by early pistillate ethylene of the accelerated wilting in petunia hybrida flowers. Plant Physiol. 80:403-408.

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.

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. l. Plant Growth Regul. 2:1-8.

Reid, M.S., Fujino, D.W., Hoffman, N.E., and Whitehead, C.S., 1984. l-aminocydopropane-l-carboxylic acid (ACC) -- The transmitted stimulus in pollinated flowers. l. Plant Growth Regul. 3:189-196.

Whitehead, C.S. and Halevy, A.H., 1989a. The role of octanoic acid and decanoic acid in ethylene sensitivity during pollination-induced senescence of Petunia hybrida flowers. Acta Hortic. 261:151-156.

Whitehead, C.S. and Halevy, A.H., 1989b. Ethylene sensitivity: The role of short-chain saturated fatty acids in pollination-induced senescence of Petunia hybrida flowers. Plant Growth Regul. 8:41-54.

Woltering, E.l., Harren, F. and Boerrigter, H.A.M., 1988. Use of a laser-driven photoacoustic detection system for measurement of ethylene production in Cymbidium flowers. Plant Physiol. 88:506-510.

Woltering, E.l., 1989. Lip coloration in Cymbidium flowers by emasculation and by liipproduced ethylene. Acta Hortic. 261:145-150.

Woltering E.J. and Harren, F., 1989. Role of rostellum desiccation in emasculation-induced phenomena in orchid flowers. l. Exp. Bot. 40:907-912.

Woltering, E.J. and Somhorst, D., 1990. Regulation of anthocyanin synthesis in Cymbidium flowers: Effects of emasculation and ethylene. l. Plant Physiol. 136:295-299.

Woltering, E.l., 1990a. Interrelationship between the different flower parts during emasculation-induced senescence in Cymbidium flowers. l. Exp. Bot. 41:1021-1024.

Woltering, E.l., 1990b. Interorgan translocation of l-aminocyclopropane-l-carboxylic acid and ethylene coordinates senescence in emasculated Cymbidium flowers. Plant Physiol. 91:837-845.

Woltering, E.l., Van Hout, M., Somhorst, D., and Harren, F., 1992. Roles of pollination and short-chain saturated fatty acids in flower senescence. Plant Growth Regul. (in press).

THE ROLE OF ETHYLENE IN THE ABSCISSION AND RIPENING OF RED RASPBERRY FRUIT Rubus idaeus cv Glen Clova

R. SEXTON, IN. BURDON and lM. BOWMER. Department of Biological and Molecular Sciences, Stirling University Stirling FK9 4LA Scotland

ABSTRACT. During the development of raspberry flower buds into ripe fruit there are two phases of elevated ethylene production. The first coincides with the opening and abscission of the petals and the second the ripening of the fruit. Aminoethoxyvinylglycine has been used to inhibit these rises in ethylene production in detached developing flowers and fruit. The treatment slows but does not prevent abscission of petals and drupelets. Similar results are obtained using silver thiosulphate. Ethylene levels above 0.25-0.5 ulll accelerate abscission and ripening of mature green fruit. Measurements of ethylene in naturally ripening fruit indicate concentrations greater than this threshold are found in some large green fruit and virtually all fruit where abscission and ripening are naturally in progress. The data suggests that increased ethylene production is involved in the acceleration and coordination of abscission.

1. Introduction.

A great deal of evidence has accumulated which implicates ethylene in the senescence, ripening and abscission of plant organs (Roberts and Tucker, 1985). During a study of the development of young flower buds into ripe raspberries two periods of elevated ethylene production have been identified (Fig. I). The first coincides with petal opening and abscission and the second with ripening and abscission of the fruit. Using aminoethoxyvinylglycine (A VG) an inhibitor of ethylene synthesis (Yu and Yang, 1979) and silver thiosulphate (STS) an inhibitor of ethylene action (Veen. 1983) we have examined the importance of increased ethylene production rates (EPR) on abscission and other ethylene induced processes. Parts of this experimental programme have been described in more detail elsewhere (Burdon and Sexton, 1989; 1990a).

2. Materials and Methods.

The growth of raspberries (Rubus idaeus cv Glen Clova). ripeness stages. measurement of ethylene production rates. and extraction of internal gas atmospheres are described by Burdon and Sexton (1990a). The methods used to expose green fruit to exogenous ethylene and 100 uM aminoethoxyvinylglycine (A YG) and measurement of fruit removal forces are also detailed in the same paper. The effects of silver thiosulphate on abscission of detached green fruit was investigated by allowing individual fruit to transpire lOOul of 2mM STS before transferring them to water for a total of 48h. Anthocyanins were

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extracted in acidified ethanol (Deubert. 1978) and quantified according to Fuleki and Francis. (1968). Chlorophyll was assayed using the method of Wintermans and de Mots (1965). Skin strengths were measured by driving a O.8mm diameter blunt ended probe at 8mmlmin into the fruit and measuring the force necessary to rupture the skin using a JJ tensile testing machine with a 5N load cell. Raspberry flowers were harvested and categorized into stages as described previously (Burdon and Sexton, 1989). The measurement of EPRs of detached and freshly harvested flowers is described in the same paper.

3. Results

3.1 ETHYLENE PRODUCTION RATES AT DIFFERENT STAGES OF FLOWER AND FRUIT DEVELOPMENT.

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Closed Bud Petal Drupe'et development

bud opening 1088

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Fruit tra.h walght (g)

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Figure 1. The EPRs nllglh of individual flowers and fruit arranged as a developmental spectrum from unopened buds on the left through to over ripe fruit on the right. The closed buds are ordered by increasing size. The stages of bud opening involve the progressive rej7exing of sepals followed by various degrees of petal opening. There is then a sequential loss of petals followed by the early stages of drupelet development which are characterized by the gradual senescence of the anthers and styles and the swe,lling of the drupelets. Fruit development is ordered according to weight for green fruit between 0.3 - 4.5g and then by the extent of abscission or decline in fruit removal force (fruit retention strength) from 7.0 - 0.25N. This contrasts with the normal visual assessment of pigment changes.

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It is apparent from Fig.l that the EPRs of closed buds are low but there is a large increase as the petal opening and abscission occurs. Once all the petals have been shed the EPR declines and remains low during the growth of· the green fruit. The second increase in ethylene production coincides with fruit ripening and abscission.

3.2 THE INCREASE IN ETHYLENE PRODUCTION RATE DURING FLOWERING.

Raspberry flower buds that are just about to open will complete the process and shed their petals when kept transpiring water in the laboratory. Ethylene production rises to a peak just before petal shedding then declines again, a pattern similar to that of field harvested flowers (Fig 1) except that the EPR peak of laboratory held flowers is 10 fold greater (Burdon and Sexton, 1989).

The role of the rise in EPR of detached flowers was examined by inhibiting ethylene production by allowing flowers to transpire O.4mM A VG. This prevented the increase in EPR but it did not stop flower opening and abscission (Table 1). Although the A VG treated flowers lost their petals it occurred over a much longer period (72h) compared with the controls (3-4h). The normal rate of petal abscission was reinstated in A VG treated plants if they were simultaneously treated with 4Oul/l ethylene. The ethylene action inhibitor 0.2mM STS fed in the transpiration stream was rather more effective but again merely slowed the loss of petals (Table 1). Both A VG and STS appeared to disrupt the coordination of abscission which normally ensures the petals in a single flower are shed fairly synchronously. The treatments also inhibited the senescent browning of petal bases (Table 1).

Table J Effect ofO.4mM AVe or O.2mM STS on the abscission of petals from detached flowers

treatment % flowers % flowers % petals lost some petals lost all petals senescent

44h water 80 54 70 44h AVG 28 14 1 44h STS 18 0 -

68h water 100 93 92 68h AVG 80 14 -68h STS 26 0 5

3.3. THE EFFECT OF ETHYLENE ON LARGE GREEN FRUIT.

If large green fruit with low EPRs are selected and exposed to 4Oul/l ethylene for 24-48h, ripening changes like anthocyanin accumulation and skin strength reduction are accelerated (Table 2). Abscission as measured by declining FRF is also faster than in air held controls (Table 2). The slower changes in FRF and pigmentation in control fruit may be associated with the increase in their EPRs which occurs during this 48h period of detachment (see section 3.5).

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Table 2 The effect of exposing large green fruit to either ethylene or air for various periods. 40ull/ ethylene was used in the experiments with pigments and fruit removal force (FRF) , 8 ull/ for the skin strength investigations.

,---------_.-

I Treatment Anthocyanins Chlorophyll Skin FRF

ug/g ug/g Strength N N

Period 48h 48h 32h 48h

Initial 32 18.9 1.24 >7.3 Air I 64 7.8 1.10 4.28

Ethylene I 183 4.1 0.57 1.79 ! L-. _________ . _._ 1

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The minimum level of ethylene necessary to accelerate the abscission of these mature green fruit was found to be between 0.25 - 0.5 ul/l (Burdon and Sexton 1990a).

3.4 THE CONCENTRATION OF ETHYLENE IN DRUPELETS AND RECEPTACLES OF DIFFERENT MATURITIES.

Having established that 0.25 - 0.5 ul/l ethylene is necessary to accelerate abscission of green fruit, the ethylene concentrations in berries at different stages of development were examined to see if this threshold was exceeded during the period of natural fruit removal force decline (Table 3).

Table 3 Internal ethylene concentrations and fruit removal forces of fruit of different maturities.

Stage of berry FRF ethylene concentration ul/l development N whole fruit receptacle drupelets

green <0.8g > 7.35 0.1 - -greel). 1.5g > 7.35 0.05 - -

mottled 5.0 0.55 1.7 0.4 red ripe 1.5 3.8 7.4 2.3

purple ripe <0.5 7.6 15.1 4.6

The ethylene concentrations in green fruit are lower than the threshold, the latter being exceeded in mottled and ripe fruit. The receptacles (whose surfaces are covered with the abscission zones) have higher levels of ethylene than the drupelets. If ethylene is supplied to detached fruit at various stages of development it stimulates drupelet abscission in green fruit but has little effect on the decline in FRF in mottled or ripe fruit (Burdon and Sexton, 1990a). This result is consistent with the ethylene concentrations in Table 3 which suggest the weakening fruit contain physiologically active levels of the gas. The ethylene releasing spray Ethrel will also accelerate FRF decline in large green fruit still attached to the plant.

3.5 THE EFFECfS OF AVG AND STS ON ABSCISSION OF DETACHED GREEN FRUIT.

The EPR of detached large green fruit allowed to transpire water increases from 0.1 nl/g/h to

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4.1 nl/g/h after 48h. This rise is accompanied by the start of abscission and a reduction in the FRF from >7.35N to 4.28N. If the detached fruit are allowed to transpire 100 ul of l00uM A VG it keeps the increase in EPR down to 0.11 nl/g/h after 48h. This slows abscission and the decline in FRF is inhibited by 20%. If ethylene is added to the A VG treated fruit, it completely reverses the inhibition. Similarly 100u1 of 2mM STS also fed by the transpiration stream, inhibits the reduction in FRF by 66%. As with the petals the treatments slow down but do not prevent abscission.

4. Discussion.

During the ripening of raspberry fruit there is a dramatic increase· in ethylene production (Burdon and Sexton,1990a; Perkins-Veazie and Nonnecke, 1992). Our data suggest that the levels of ethylene that develop within fruit from the mottled stage onwards are physiologically significant since they will accelerate abscission of large green fruit. Detached green fruit also develop increased EPRs if they are left transpiring water. Inhibiting this increase with A VG or interfering with ethylene action using STS slows down but does not prevent associated abscission zone weakening. These data strongly support the view that ethylene acts as an abscission accelerator. Further evidence comes from the comparison of different raspberry cultivars (Burdon and Sexton. 1990b). In general those cultivars with the lowest FRF when ripe have the highest EPRs. This can be related to a difference in the time of increased EPR relative to drupelet pigmentation, with an earlier increase in EPR resulting in a lower FRF.

In Glen Clova fruit there is a very strong correlation between the rise in EPR and both the induction of abscission and pigment and textural changes. Analysis of individual fruit (data not presented) has shown that 66% of the very largest green fruit that do not yet show any decline in FRF have ethylene levels above the 0.25 ul/l threshold. Ethylene increases which precede an apparent response like ripening or abscission have been used to support the notion that ethylene not only accelerates but also induces the process. The hazards of over interpreting such correlations are well illustrated in this case since other cultivars do not initiate increases in EPR until well after FRF and pigment changes have begun (Sexton and Burdon 1990b).

Our data also implicate increased EPRs in the acceleration and coordination of petal abscission, An accelerated programme of petal abscission is common after the pollination of flowers presumably to prevent unnecessary visits from scarce pollinators (Burdon and Sexton, 1989). Similarly the ethylene mediated acceleration of abscission might have evolved to promote the rapid loss of organs subjected to pathogen attack, excessive dehydration, herbivore damage etc where speed is all important.

Inside the raspberry fruit there are about 70 abscission zones, one at the base qf each drupelet. These must weaken synchronously to allow the cap of drupelets to become detached from the central receptacle. The increase in ethylene concentration may help orchestrate the process. Perhaps ethylene is similarly involved in the coordinated shedding of the 5 petals in the flower since inhibiting the increase in EPR in detatched flowers appears to disorganize the process. Coordination is also required within a single abscission zone to ensure that cells across the entire width of the zone degrade their walls together.

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This study provides no evidence to support the commonly held view that ethylene induces abscission. Experiments which purport to have completely stopped abscission either by reducing ethylene levels or interfering with ethylene action can often be criticised for being too short term to distinguish between slowing the process (ie demonstrating acceleration) and completely blocking it (ie showing induction). It is pertinent to recall that IAA will prevent abscission of debladed leaves for weeks, even in the presence of ethylene, and its potential role as a regulator should not be overlooked.

5. References

Burdon, J.N. and Sexton, R. (1989) 'The role of ethylene in petal abscission in red raspberry', in DJ. Osborne and M.B. Jackson (eds.), Cell Separation in Plants, Springer-Verlag, Berlin, pp 371-6

Burdon, J.N. and Sexton, R. (1990a) 'The role of ethylene in the shedding of red raspberry fruit', Ann. Bot. 66, 111-120.

Burdon, IN. and Sexton, R. (1990b) 'Fruit abscission and ethylene production in red raspberry cultivars', Sci. Hort. 43, 95-102.

Deubert, K.H. (1978) 'A rapid method for the extraction and quantitation of total anthocyanin in cranberry fruit' . J. Agri. Food Chern., 26, 1452-1453.

Fuleki, T. and Francis, FJ. (1968) 'Quantitative methods for anthocyan ins I' J. Food Sci. 33, 72-77

Perkins-Veazie, P. and Nonnecke, G. (1992) 'Physiological changes during raspberry fruit ripening'. HortScience 27, 331-333.

Roberts, J.A. and Tucker. G. (1985) 'Ethylene in plant development', Butterwoiths, London.

Veen" H. (1983) 'Silver thiosulphate: an experimental tool in plant science'. Sci. Hort. 20, 211-224.

Wintermans. J.F.G.M. and de Mots, A. (1965) 'Spectrophotometric characteristics of chlorophylls a and b and their pheophytins in ethanol'. Biochim. Biophys. Acta 109,448-453.

Yu, Y-B., and Yang, S.F. (1979) 'Auxin induced ethylene production and its inhibition by AVG and C02+'. Plant Physiol. 64. 1074-1077.

EXPRESSION OF TWO ACC SYNTHASE mRNAs IN CARNATION FLOWER PARTS DURING AGING AND FOLLOWING TREATMENT WITH ETHYLENE

HANS HENSKENS, DIANNE SOMHORST AND ERNST I. WOLTERING Agrotechnological Research Institute (ATO-DLO) P.O. Box 17, 6700 AA Wageningen, The Netherlands

ABSTRACf. Synthetic oligonucleotides homologous to conselVed sequences of ACC synthase were used to prime the synthesis and amplification of fragments of about 1000 base pairs by PCR in samples of cDNA to RNA isolated from senescing carnation flowers. Two putative ACC synthase PCR clones were isolated of which one was identical to the sequence of a carnation ACC synthase cDNA clone (CARACC3) recently isolated by Park et al. (plant Mol. BioI. 18:377-386, 1992), the other clone (CARASl) was approximately 66% homologous to CARACC3. For both ACC synthase clones, specific oligonucleotides were synthesized. Using PCR we were now able to distinguish between the two ACC synthase mRNAs in samples of total RNA isolated from different flower parts. The occurrance of both messengers was stimulated by aging and by treatment with ethylene in a tissue-specific way.

1. Introduction The phytohormone ethylene (C;H,J regulates many aspects of plant growth and development including ripening in climacteric fruits and petal senescence in a variety of flowers. In carnation flowers, a huge increase in ethylene production occurs at the onset of senescence. This increased production is autocatalytic in nature i.e. the produced ethylene stimulates the activity of enzymes involved in its production (Borochov and Woodson, 1989).

In plants, ethylene is synthesized from S-adenosylmethionine by means of the intermediate l-aminocyclopropane-l-carboxylic acid (ACC). The synthesis of ACC is catalyzed by the enzyme ACC synthase which was shown to be the rate-limiting step in many systems (Mattoo and Suttle, 1991). To study the regulation of ethylene synthesis at the molecular level we focused our research on the isolation and characterization of ACC synthase genes in the carnation flower.

2. Materials and Methods Carnation (Dianthus caryophyllus) cv White Sim flowers were obtained from a commercial grower and transported dry to the laboratory. The stems were recut to a length of approximately 40 cm and either allowed to senescence in water or treated for 20 h with 10 J.1L1L ethylene. Ethylene production of different flower parts was measured by enclosing them for 1 h in a small glass vial after which the ethylene concentration in the headspace was measured by gaschromatography.

Total RNA was isolated from frozen tissue. First strand cDNA was made using reverse transcriptase and a degenerate 3' ACC synthase primer. cDNA was used as a template for PCR amplification using degenerate oligonucleotide primers homologous to conselVed nucleotide sequences of published ACC synthases (Van der Straeten, 1990). PCR products were separated by gel electrophoresis and the band with the expected length of approximately 1000 base pairs was isolated and the fragments cloned into a vector to facilitate DNA sequencing.

Specific oligonucleotide primers for two different carnation ACC synthases together with a degenerate 3' primer were used to prime the synthesis of short fragments by PCR in samples of cDNA to RNA. Digoxigenin-II-dUTP (DIG) was added to the reaction mixture. Twenty cycles of denaturation (94°C, I') annealing (40°C, 1'20") and Taq polymerase mediated extension (nOC, 1'30") were used to amplify the two different fragments.

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Following gel electrophoresis and blotting the DIG-labeled fragments were detected by autoradiography.

3. Results and Discussion The ethylene production of the different flower parts and leaves is shown in table 1. Both during aging in the vase and after treatment with ethylene, an increase in production was apparent in all the flower parts. The greatest stimulation was obServed in ethylene treated flowers while ovaries and styles generally produced more ethylene (on a fresh basis) than the petals. Ethylene production in leaves was not affected by aging and slightly inhibited by the ethylene treatment.

The amino acid sequence of the ACC synthase PCR clone we isolated (CARAS 1; EMBL X66605) showed 66% homology to a carnation ACC synthase cDNA (CARACC3; EMBL M66619) recently isolated by Pm et al. (1992). For both carnation ACC synthases, specific oligonucleotide primers (18 bp) were synthesized. The oligos, together with a 27 bp 3' primer to a conserved region, were used to prime the synthesis of a 525 and a 472 bp fragment by PCR in samples of cDNA. Nucleotide sequencing revealed that the 525 and 472 bp fragments were indeed identical to the sequences of the homologous regions of CARASI and CARACC3, respectively. In this way we were able to distinguish between the two ACC synthase messengers in RNA samples from different flower parts.

Southern blots of PCR fragments revealed that the two. messengers were of low abundance in RNA from fresh tissues. Both aging and ethylene stimulated the occurance of these messengers in a tissue-specific way. CARACC3 was more abundantly present in the petals while CARAS 1 was more abundantly present in the styles. Despite the high ethylene production observed in ovaries (Table 1), the level of both messengers was low. This may be explained by assuming the existance of a third ACC synthase gene that may be specifically expressed in the ovary. In other systems, e.g. Cymbidium flowers, ACC was shown to be translocated from the site of production to other flower parts. Alternatively, the ethylene produced in the carnation ovary may, therefore, be a result of ACC influx from other parts of the flower. No expression of neither CARASI nor CARACC3 was observed in the leaves, which is in line with the low ethylene production in these organs.

References Borochov, A. and Woodson, W.R., 1989. Hort. Rev. 11:15-43. Mattoo, A.K. and Suttle, C.S., 1991. The Plant Hormone Ethylene. CRC Press, Boston. Pm, K.Y. et al., 1992. Plant Mol. BioI. 18:377-386. Van der Straeten, D. et al., 1990. Proc. Natl. Acad. Sci. 87:4859-4863. Woltering, EJ., 1990. Plant Physiol. 92:837-845.

Table 1

Flower part

Flower Petals Ovary Styles Leaves

Ethylene production in isolated flower parts and leaves measured in fresh flowers, after 7 days vase life and after treatment of fresh flowers with ethylene (10 J.Ll../L; 20 h).

Ethylene production (nLJgFW.h)

Fresh 7 Days Ethylene

0.1 13 43 0.1 21 36 1.5 30 55 1.0 84 70 0.12 0.12 0.05

PROMOTING THE ACTIVITY OF ARGININE DECARBOXYLASE AND ORNITHINE DECARBOXYLASE BY ETHYLENE AND ITS SIGNIFICANCE TO THE CONTROL OF ABSCISSION IN CITRUS LEAF EXPLANTS

R. GOREN, M. HUBERMAN, N. LEVIN, AND A. ALTMAN Department of Horticulture Kennedy-Leigh Centre for Horticultural Research Hebrew University of Jerusalem Rehovot Israel, 76100

It iswell accepted that auxin delays and ethylene accelerates abscission (5). During the abscission process cells in the abscission zone (AZ) pass from a juvenile stage, characterized by cell division and enlargement, to a senescence stage leading to cell wall degradation and organ separation. Extensive evidence links polyamines (PA) to the control of cell division perceives them as "antisenescence" agents (l,4,8), while ethylene, is considered a senescence-promoting hormone (1). The antisenescence properties of exogenously applied PA may result from both an inhibition of ethylene biosynthesis and promotion of endogenous P A biosynthesis (8). Many studies dealt with the interactions between auxin and ethylene in the control of abscission (5). However, the biosynthetic linkage between P A and ethylene via S-adenosylmethionine as a common intermediate, and the fact that PA directly antagonize many ethylene-mediated responses (1,8), raised the question whether PA are involved in the control of abscission. The linkage between PA and ethylene was previously studied both at the biosynthetic and at the physiological levels, and mainly in relation to senescence and fruit ripening (1). Most of the reports dealt with the effects of exogenous PA on several aspects of ethylene production and biosynthesis, and only a few examined how exogenous ethylene interferes with PA levels and biosynthesis 0,8). The interactions between plant hormones and PA can be summarized by saying that in general, plant hormones that promote growth induce an increase in the biosynthesis of PA while plant hormones that retard growth decrease PA biosynthesis (for detailed references see reviews 1,4,8). Exogenous ethylene was reported to decrease the activity of arginine decarboxylase (ADC) and Sadenosylmethionine decarboxylase, and to increase the activity of ornithine decarboxylase (ODC) and lysine decarboxylase (2,6). In the following we refer to a study on the effects of ethylene on abscission of citrus leaf explants; PA biosynthesis was examined to establish the kind of relationships existing between the two regulators in the process of abscission.

Leaf explants, consisting of 10 mm of the petiole and 10 mm of the midrib tissues proximal and distal to the AZ, were prepared from 6 to 8 months old leaves of'Shamouti' (Citrus sinensis [L.] Osbeck) orange trees and used for abscission studies as previously described (7). The activity of ADC and ODC, and the endogenous titer of putrescine (Put) and spermidine (Spd) were tested according to Palavan et al. (6) and F10res et al. (3), respectively. After determining the optimum conditions for enzymatic reactions, the enzymatic activity was investigated in the AZ, midrib and leaf blade. PA titer was analyzed only in the AZ.

The highest level of activity of ADC was recorded in the midrib, whereas the activity of ODC in the AZ was somewhat higher than in the other sites. These data indicated that the activity of the

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enzymes is not specific to the AZ and, therefore, that these enzymes do not meet one of the required characteristics of enzymes involved in abscission, namely, specific response to any signal causing abscission at the AZ. Ethylene caused a marked increase in the activity of both enzymes, the course of which was similar to that of abscission. It took 12 hand 72 h before any significant ethyleneinduced activity of ADC and ODC, respectively, was detected. The existence of a lag time before the observed increase in ADC and OAC, when explants were exposed to ethylene, indicated that probably new synthesis of the protein of the enzymes is involved. However,further study is required to establish whether the effect of ethylene on the increase in both enzymes activity is due to a new synthesis of the enzymes, or a result of the activation of existing enzymes. The maximum effect of ethylene on ADC activity was obtained with 100 J.1lIL ethylene; however, 2 J.IlIL were enough to induce a significant increase in ADC activity, while 10 J.1lIL ethylene were required to induce a significant increase in abscission. 2,4-D retarded abscission with a slight increase in the activity of ADC. The fact that 2,4-D did not show a clear parallel effect on the activity of ADC and abscission raise doubts whether the activity of the enzymes at the AZ is related to the control mechanism of abscission. The two inhibitors of putrescine biosynthesis, a-difluoromethylarginine (DFMA) and a-difluoromethylornithine (DFMO), inhibited ethylene-induced activity of ADC and ODC, the fonner more than the latter. In both cases no effect on ethylene-induced abscission was observed. Ethylene-induced activity of ADC and ODC was accompanied by a marked increase in spennidine in the AZ. The differences between air control and ethylene treatment were even more evident in the course of abscission. The titer of putrescine in the AZ of ethylene-treated explants was higher than air control, but the effect of ethylene was weaker than its effect on spennidine ..

In conclusion, the results of the present study clearly indicate that ethylene treatment increased the activity of ADC and ODC as well as the endogenous titer of spennidine and putrescine in the AZ of citrus leaf explants, and that this effect became even stronger in the course of abscission. However, the findings that DFMA and DFMO inhibited the rise in ADC and ODC activity, respectively, while no effect was recorded on ethylene-induced abscission suggest that the effects of ethylene on polyamine biosynthesis and on abscission are two independent phenomena.

1. Altman, A (1989) 'Polyamines and plant hormones' in U. Bachrach and M. Heimer (eds.), The Physiology ofPolyamines, CRC Press Inc. Boca Raton, Florida, Vol. II, pp. 121-145.

2. Apelbaum, A (1990) 'Interrelationship between polyamines and ethylene and its implication for plant growth and fruit ripening', in H. E. Flores, R. N. Arteca and J. C. Shannon, Polyamine and Ethylene: Biochemistry, Physiology, and Interactions, Proc. 5th Ann. Pen State Univ. Am. Soc. Plant Physiol. Publications, pp. 287-294.

3. Flores, H. E. and Galston, AW. (1982) 'Analysis of polyamines in higher plants by high perfonnance liquid chromatography' Plant Physiol. 69:701-706.

4. Galston, A W. (1983) 'Polyamines as modulators of Plant development, Bioscience, 33: 382-388.

5. Osborne, D.J (1989) 'Abscision' CRC Critical Reviews in Plant Science Vol. 8 Issue 2. pp. 103-129.

6. Palavan, N. Goren R. and Galston A.W. (1984) 'Effects of some growth regulators on polyamines biosynthetic enzymes in etiolated pea seedlings', Plant Cell Physiol. 25:541-546.

7. Ratner, A Goren, R. and Monselise, S.P. (1969) 'Activity of pectinesterase and cellulase in abscission zone of Citrus leaf explants' Plant Physiol. 44: 1717-1723.

8. Smith. T. A. (1985) 'Polyamines' Ann. Rev. Plant Physiol. 36: 117-143.

EXPRESSION OF EFE ANTISENSE RNA IN TOMATO CAUSES RETARDATION OF LEAF SENESCENCE AND MOST FRUIT RIPENING CHARACTERISTICS

A. J. MURRAya, G. E. HOBSONa , W. SCHUCHb, C. R. BIRDb Horticulture Research International, Worthing Road, Littlehampton, West Sussex, BN17 6LP, Englanda and ICI Seeds, Jealotts Hill Research Station, Bracknell, Berkshire, RG12 6EY, Englandb.

The rise in ethylene production is one of the earliest indicators of the onset of tomato fruit ripening. The rate of ethylene synthesis continues to increase during early ripening, reaching a peak at the mid-ripening stage (Grierson and Kader, 1986). Up till now it has not been clear whether sustained, high concentrations of the hormone are required to maintain the normal rates of ripening. Tomatoes from plants expressing antisense RNA for the ethylene-forming enzyme (EFE) , which produce 3-4% of normal ethylene, do ripen, but at a slower rate than unmodified tomatoes (Hamilton et al., 1990). In this study, the ripening characteristics, including response to exogenous ethylene, of EFE antisense fruit are studied in detail. Additionally, the effect of the antisense gene on leaf senescence is considered.

Tomato plants (cv. Ailsa Craig) homozygous for the EFE antisense gene were produced by Hamilton et al. (1990). Plants were grown with wild-type controls in two successive trials. Fruit ripening on the plants and in storage, and the response of post-harvest fruit to exogenous ethylene (150 ppm for 3 days) were investigated. Fruit survivability was assessed, and the tomato colour index (TCI) and fruit compression were determined as described in Schuch et a1. (1991). The tomatoes were frozen at -18cC. Samples were later thawed, and the sap extracted. The sap was assessed for titratab1e acidity as described by Hobson and Kilby (1985) using an auto-titrator, and reducing sugar content as described by Bittner and Manning (1967). In a further investigation, plants of the two lines were grown in independent glasshouse compartments during the last quarter of 1991, and the rate and degree of leaf senescence was observed. In each trial it was confirmed that ethylene production in the antisense fruit was 3-4% that of the control fruit.

The survivability of the EFE antisense fruit ripened on the plants and in storage was improved, i.e. splitting and subsequent infection were reduced. Likewise, the TCI was reduced. Antisense fruit softened at the same rate as the controls, but the continued softening of ripe fruit was reduced. The acidity of the controls declined more rapidly during ripening than that of antisense tomatoes. Antisense fruit contained 17-21% less sugar than control samples at all ripening

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stages. Antisense tomatoes showed a greater response to ethylene than controls when treated at mature green, breaker and 4 days post-breaker (dpb). No response was shown by the control fruit at 4 dpb. The antisense and control plants grown in isolation showed a sharp distinction in the rate of leaf senescence. The leaves of the control plants senesced to some considerable extent, whilst those on the antisense plants remained relatively green and healthy.

These results demonstrate that modified tomato plants with specific inhibition of ethylene biosynthesis are a valuable tool for studying the co-ordination of the ripening process. All aspects of ripening that have been studied are initiated in fruit that synthesise ethylene at 3-4% of the normal rate. However, higher levels of ethylene synthesis are required to maintain the rates of colouration and decline in acidity, and to attain the sugar content of the wildtype, but are not required for fruit softening. The retardation of softening of over-ripe fruit together with enhanced resistance to damage may provide opportunities for reducing post-harvest losses.

We wish to thank Professor D. Grierson and Mr A. Hamilton of Nottingham University for supplying seed from EFE-antisense fruit, and Mr R.N. Edmondson of Horticulture Research International for expert advice on the statistical analysis of these trials.

Bittner, D. L. and Manning, J. (1967) glucose method: critical factors and Technicon Symposium 1966, Automation 1, pp. 33-36.

'Automated neocuproine normal values', in in analytical chemistry,

Grierson, D. and Kader, A. A. (1986) 'Fruit ripening and quality', in J. G. Atherton and J. Rudich (eds.), The Tomato Crop, Chapman and Hall, London/New York, pp. 241-280.

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.

Hobson, G. E. and Kilby, P. (1985) 'Methods for tomato fruit analysis as indicators of consumer acceptability', Ann. Rpt. Glasshouse Crops Res. Inst. for 1984, pp. 129-136.

Schuch, W., Kanczler, J., Robertson, D., Hobson, G., Tucker, G., Grierson, D., Bright, S. and Bird, C. (1991) 'Fruit quality characteristics of transgenic tomato fruit with altered polygalacturonase activity', HortScience 26, 1517-1520.

mE ROLE OF EfHYLENE IN REGULATING GROWfll OF DEEPWATER RICE

H. KENDE, S. HOFFMANN-BENNING AND M. SAUTER MSU-DOE Plant Research Laboratory Michigan State University East Lansing, Michigan 48824, USA

ABSTRACT. Submergence induces rapid elongation of rice coleoptiles (Oryza sativa, L) and of deepwater rice internodes. This adaptive feature helps rice to grow out of the water and to survive flooding. The growth response of submerged deepwater rice plants is mediated by ethylene and gibberellin (GA). Ethylene promotes growth, at least in part, by increasing the responsiveness of the internodal tissue to GA We examined the possibility that increased responsiveness to GA was based on a reduction in endogenous abscisic acid (ABA) levels. Submergence and treatment with ethylene led, within 3 h, to a 75% reduction in the level of ABA in the intercalary meristem and the growing zone of deepwater rice internodes. Our results indicate that the growth rate of deepwater rice internodes is determined by the ratio of an endogenous growth promoter (GA) and a growth inhibitor (ABA). The growth response in deepwater rice internodes is based on an increased cell production rate in the intercalary meristem and on increased cell elongation. Our investigations were aimed at establishing the temporal sequence of these GA-regulated processes. Treatment with GA promoted cell elongation in the intercalary meristem within 2 h. After 4 h of treatment with GA, the fraction of meristematic cells in the G2 phase had declined, indicating that cells in the G2 phase had entered mitosis. Subsequent activation of DNA replication led to an overall increase in the cell production rate. An increase in the final cell length contributed to the growth response after 7 h of GA application. Our results are consistent with the hypothesis that GA promotes first cell elongation in the intercalary meristem and that cell division is stimulated as a result of cell growth.

I. Introduction

Rice (Oryza sativa L) has a number of physiological and metabolic adaptations that enhance its chances for survival under conditions of temporary flooding. One of these is the capacity of plants to elongate rapidly when they become submerged. This feature helps rice plants to emerge from the water and to avoid drowning. In seedlings, submergence promotes coleoptile growth and in adult deepwater rice plants, elongation of the internode. In both instances, the plants respond to the altered gas composition of their submerged organs, namely to reduced partial pressure of O:z, to increased partial pressure of CO:z, and to the accumulation of ethylene (for a review see Jackson and Pearce 1991). Research described below was directed towards elucidating the chain of events leading from submergence to the growth response and towards understanding the cellular basis of internodal elongation.

329

J. C. Pech et at. (eds.), Cellular and Molecular Aspects o/the Plant Honnone Ethylene, 329-334. © 1993 Kluwer Academic Publishers.

330


Seeds of deepwater rice. (Oryza sativa L, cv. Habiganj Aman II and Pin Gaew 56) were obtained from the Bangladesh Rice Research Institute, Dacca, Bangladesh, and the International Rice Research Institute, Los Banos, Philippines. Methods of growing and treating plants were as descnbed (Hoffmann-Benning and Kende 1992; Sauter and Kende 1992). Abscisic acid (ABA) was extracted and assayed according to Walker-Simmons (1987). Flow cytometry, incorporation of [3H]thymidine into DNA, and cell size determinations were according to Sauter and Kende (1992). The in vivo assay of l-aminocyclopropane-lcarboxylate (ACC) synthase was as described by Cohen and Kende (1987).

3. Results

3.1. THE HORMONAL BASIS OF TIlE RESPONSE TO SUBMERGENCE

The response of deepwater rice to submergence can be mimicked by exposing internodes to a gas atmosphere resembling that found in submerged plants, namely to 3% 020 6% CO2 and 1 ",1/1 ethylene (Raskin and Kende 1984a). Either reduced partial pressures of O2 or 1 ",1/1 of ethylene in air also induce internodal elongation. Based on these results, we suggest that the environmental signal for enhanced elongation is the reduced O2 tension in the submerged internodes. In submerged plants or under low O2 pressure, the rate of ethylene formation is greatly increased (Raskin and Kende 1984a). Low oxygen levels do not promote conversion of ACC to ethylene but enhance the activity of ACC synthase (Cohen and Kende 1987).

Ethylene promotes growth, at least in part, by increasing the responsiveness of the internodal tissue to GA (Raskin and Kende 1984b). We investigated one possible mechanism by which ethylene may modulate the responsiveness of deepwater rice internodes to GA Zeevaart (1983) found that applied ethylene reduced the level of ABA in leaves ofXanthium stnunarium. Thus, increased responsiveness to GA in deepwater rice may be based on an ethylene-mediated reduction in the level of endogenous ABA, a potent inlubitor of growth in rice (Hoffmann-Benning and Kende 1992). To examine this possibility, we measured the level of ABA in the intercalary meristem and the growing zone of deepwater rice plants that had been induced to grow rapidly by submergence or treatment with ethylene (Fig. 1). Plants were immersed in 300-liter plastic tanks filled with water or were kept in air in the same growth chamber. Within 1 h of submergence, the ABA level in the intercalary meristem and the cell elongation zone above it decreased by more than 50% (Fig. lA). After 3 h, it was reduced by 75% and decreased further during the subsequent 21 h. In air-grown control plants, the ABA content remained at its original level. These data were subjected to analysis of variance for a randomized complete block design with three replicates. The differences in ABA levels between different treatments were highly significant (F-test; df = 7, 13: p < 0.(05) with a highly significant effect of submergence (F-test; df = 1, 13; P < 0.(05).

In a second set of experiments, deepwater rice plants were placed in plastic cylinders through which ethylene-free air or air containing 3 to 5 ",1/1 ethylene was passed. After 3 h of treatment with ethylene, the ABA level in the intercalary meristem and the cell elongation zone was 75% lower than that in the corresponding control plants at the same time (Fig. IB). This difference in the ABA concentrations was maintained over the subsequent 21 h. Again, the data were evaluated by analysis ofvariance for a randomized complete block design with four replicates. There were significant differences in ABA content between different

331

treatments (F-test; df = 5, 15; P < 0.025) with a highly significant effect of ethylene (F-test; df = 1, 15; P < 0.005).

25

30 B 20 25

~ 15 .:: S!' CJ)

E <l: 10 (IJ

<l:

~ 20 I Air-grown I .:: S!'

-'0'

15 '0'-

1 CJ)

1 E <l: (IJ

<l: 10

5 Ethylene-treated

5 2 ~ 0

0 3 6 12 15 18 21 24

OLL_L--'----''---'---'--'---'---'-' o 3 6 9 12 15 18 21 24

Duration of Treatment (h) Duration of Treatment (h)

Fig. 1. ABA levels in submerged and ethylene-treated internodes of deepwater rice. A Plants were submerged in 3OO-liter tanks filled with water (IJ) or kept in air as control (Y). One-cm portions containing the intercalary meristem and part of the cell elongation zone above it were excised from the internodes at the times indicated. Each point represents the mean of three independent experiments ± SE. B. Plants were grown in plastic cylinders through which ethylene-free air (Y) or air containing 3-5 1'1/1 ethylene (IJ) was circulated at a flow rate of400 mUmin. One-cm portions containing the intercalary meristem and part of the cell elongation zone above it were excised from the internodes at the times indicated. Each point represents the mean of four independent t:xperiments ± SE. (From HoffmannBenning and Kende, 1992.)

We also determined the amount of the endogenous GAl and GA20 in the intercalary meristem and internodal elongation zone of deepwater rice plants which had been submerged for 0, 1,3, and 24 h. The level of GAb the biologically active GA in rice, increased fourfold from 15.2 to 62.3 ng/g dry weight within 3 h of submergence. The level of GA2O> which is the immediate precursor of GAb also increased, but more slowly than that of GAl' After 24 h of submergence, it was three times above the original value (from 12.5 to 40.9 ng/g dry weight).

3.2. THE CELLULAR BASIS OF TIlE GROwrH RESPONSE IN DEEPWATER RICE INTERNODES

Based on cell-size analysis and autoradiography of tissue labeled with PH]thymidine, three zones of internodal development could be distinguished in deepwater rice (Bleecker et al. 1986; Metraux and Kende 1984; Sauter and Kende 1992): the intercalary meristem, 2 to 3 mm in length, at the base of the internode, where cells divide and elongate; the elongation zone, where cells reach their final length; and the differentiation zone, where secondary wall and xylem formation take place. During the phase of most rapid growth, internodes of submerged plants elongate at rates up to 4.5 mm h-l. These high growth rates are achieved by a threefold increase in the cell production rate in the intercalary meristem and a three- to

332

fourfold increase in the final length of cells that have reached the top of the elongaQon zone (Bleecker et aL 1986). As shown above, ethylene regulates elongation of deepwater rice via GA Therefore, GA is the immediate growth-promoting hormone in this plant To identify the sequence of events that contnbute to GA-induced growth, we undertook a detailed temporal analysis of cell division and cell elongation in the intercalary meristem and the growing zone of deepwater rice internodes.

The average size of meristematic cells increased within 2 h after application of GA, and elongation of these cells continued up to 16 h with GA treatment (Fig. 2). Cells that emerged from the intercalary meristem following the start of GA treatment attained an up to threefold greater length than did cells in control internodes (Sauter and Kende 1992). Flow cytometry was used to determine the cell cycle status of nuclei isOlated from the meristematic cells. Before induction of growth, approximately 7% of the meristematic cells contained nuclei in the G2 phase (Fig. 2). During the first 4 h of GA treatment the fraction of nuclei in G2 dropped to 4.5% but started to increase after 7 h. Incorporation of PH]thymidine into DNA did not significantly increase until after 4 h of treatment with GA (Fig. 2), when the growth rate had at least doubled (Sauter and Kende 1992).

E

::~ .3 Cell Size Q)

6 N

iii Qi b u

~

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'" Q)'<:

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!!! Qi

0 U 3 0 5 10 15 20 25

Treatment with GA [hI

Fig. 2 Size of cells, DNA syntheSis as determined by PH]thymidine incorporation, and percent of nuclei in the G2 phase in the intercalary meristem of deepwater rice internodes treated with GA

4. DISCUSSION

We asked whether, in internodes of deepwater rice, ethylene may cause a reduction in endogenous ABA levels as it does in Xanthiwn leaves (Zeevaart 1983), and whether responsiveness to GA (Raskin and Kende 1984b) may be a function of ABA content Our data support this possibility. The level of ABA decreased in the intercalary meristem and cell elongation zone of submerged and ethylene-treated internodes by 75% within 3 h (Fig. 1). At the same time, the level of GAb which is the active GA in rice, increased threefold. Based on the results reported above, we can postulate two new links in the chain of events that leads from submergence to accelerated growth in deepwater rice (Fig. 3). The reduced O2

333

tension in submerged internodes promotes ethylene synthesis by enhancing the activity of ACC synthase. In addition, ethylene is physically trapped in submerged internodes because of its low rate of diffusion in water. Ethylene enhances the responsiveness of deepwater rice internodes to GA, at least in part because of a reduction in endogenous ABA content While the level of ABA decreases in submerged internodes, that of GA increases. Thus, rapid internodal growth of deepwater rice may result from an altered balance between growthpromoting (GA) and growth-inhibiting (ABA) hormones.

SUBMERGENCE

~ REDUCED OXYGEN TENSION

~ INCREASED ETHYLENE BIOSYNTHESIS

REDUCED ABA LEVELS INCREASED GA LEVELS

INCREASED RESPONSIVENESS TO GA / INTERNODAL GROWTH

Fig. 3. The chain of events linking submergence of deepwater rice plants to accelerated growth.

Regarding the cellular basis of accelerated growth in,deepwater rice, we postulate that GA first induces cell elongation in the intercalary meristem followed by a round of cell divisions, predominantly of those cells that had already duplicated their DNA and were in the G2 phase of the cell division cycle. A further rise in the cell production rate was evident after 7 h of GA treatment when DNA synthesis, as measured by PH]thymidine incorporation, had increased (Fig. 2). We propose that the increase in cell size promotes mitosis in the intercalary meristem. Cells that are displaced from the meristem during GA treatment elongate to approximately three times their normal length. Therefore, the high growth rates observed in deepwater rice internodes are achieved by an increase in the formation of new cells that are competent to repond to GA by increased elongation.

S. ACKNOWLEDGEMENTS

This work was supported by grant No. DCB 9103747 from the National Science Foundation and grant No. DE-FG02-90ER20021 from the Department of Energy. M.S. was the recipient of a fellowship from the Deutsche Forschungsgemeinschaft

334

6. REFERENCES

Bleecker, A B., Schuette, J. L, and Kende, H. (1986) 'Anatomical analysis of growth and developmental patterns of deepwater rice', Planta 169,490-497.

Cohen, E. and Kende, H. (1987) 'In vivo 1-aminocyclopropane-1-carboxylate synthase activity in internodes of deepwater rice', Plant Physiol. 84, 282-286.

Hoffmann-Benning, S. and Kende H. (1992) 'On the role of abscisic acid and gibberellin in the regulation of growth in rice', Plant Physiol. 99, 1156-1161.

Jackson, M. B. and Pearce, D. M. E. (1991) 'Hormones and morphological adaptation to aeration stress', in M. B. Jackson and D. D. Davis (eds.), Plant Life under Oxygen Deprivation, SPB Academic Publishing, The Hague, Netherlands, pp. 47-67.

Metraux, J.-P. and Kende, H. (1984) 'The cellular basis of the elongation response in submerged deep-water rice', Planta 160, 73-77.

Raskin, I. and Kende, H. (1984a) 'Regulation of growth in stem sections of deepwater rice', Planta 160,66-72.

Raskin, I. and Kende, H. (l984b) 'Role of gtbberellin in the growth response of deepwater rice', Plant Physiol. 76, 947-950.

Sauter, M. and Kende, H. (1992) 'Gibberellin-induced growth and regulation of the cell division cycle in deepwater rice', Planta, in press.

Walker-Simmons, M. (1987) 'ABA levels and sensitivity in developing wheat embryos of sprouting resistant and susceptible cultivars', Plant Physiol. 84, 61-66.

Zeevaart, J. A D. (1983) 'Metabolism of abscisic acid and its regulation inXanthium leaves during and after water stress', Plant Physiol. 71, 477-481.

GRAVITY DEPENDENT ETHYLENE ACTION

1S. P. BURG AND 2B. G. KANG 1VacuFresh Division of Gelco Internacional S.A. 3770 Kent Court, Miami, FL 33133, USA

2Department of Biology, Yonsei University, Seoul 120-749, Korea

ABSTRACT. Ethylene inhibits 5- 3 H-IAA gravitransport in the subapex of horizontal etiolated pea epicotyls within 30 min, stopping upward curvature, and in addition the gas inverts gravitransport at the hook/subpical juncture, promotingdowncurving. Phototropic IAA transport is not effected. Inverted (-) gravitransport is as intense in >1 ~l/l ethylene as (+) gravitransport is in air, with a compensation point at 0.2 ~l/l. Seedlings. nutate when ethylene-induced changes in gravitransport amplify the natural subapical backbending caused by an auxin assymetry imposed on the subapex by the shorter transport path through the concave side of the hook. Ethylene also inverts 5-3 H-IAA gravitransport in tomato petioles, without altering epinastic transport, stimulating curvature of upright petioles, and inhibiting curvature of inverted petioles. The gas causes IAA to accumulate in the petiole base, and stimulates adaxial basal cells to elongate in response to IAA.

1. Introduction

Between 1878 and 1939 an extensive literature on ethylene and illuminating gas developed the concepts that ethylene diminishes geo-sensitivity, that the direction of the tropistic curving which results is autonomically determined by epinastic forces expressed when gravitropism is weakened, and that the equilibrium position is gravity dependent [2,8]. This view of the interrelation between gravity and tropistic responses to ethylene is reexamined to determine if it is compatible with modern concepts of ethylene action.


2.1. SEVEN DAY OLD ETIOLATED PISUM SATIVUM CV ALASKA SEEDLINGS

Lanolin paste containing 3.6 ~M 5-3 H-indole-3-acetic acid (100 ~C/g) was applied either to the apical cut end of 1 cm subapical segments or to explants comprised of the 3rd internode, 2nd node, and in some instances

335

J. C. Pech et al. (eds.J, Cellular and Molecular Aspects of the Plant Hormone Ethylene, 335-340. © 1993 Kluwer Academic Publishers.

336

the hook elbow. The basal end was inserted in 1% agar in a vertically positioned petri dish, orienting the stems and the plane of the hook elbows horizontally. After 4 h in darkness, a 7 mm subapical piece was split into middle half and upper and lower quarters; the zones were weighed, and assayed by scintillation counting in Bray's solution.

Subapical 1 cm sections were floated in darkness without shaking on 50 mM K-phosphate buffer (pH 6.8) with 3.6 nM 5-3 H-IAA (1 ~C/ml) to assay transport; with 1 ~M IAA to assay curvature. The segments were blotted dry; the central 8 mm split into upper quarter, middle half, and lower quarter; and the quarters weighed before counting radioactivity.

2.2. SEVEN WEEK OLD LYCOPERSICUM ESCULATUM CV HOMESTEAD 24 PLANTS

Explants from greenhouse tomato plants, selected with an axillary angle 1.5 cm The

of 50° to 60° at the 3rd node, were prepared by severing the stem above and below the 3rd node, and the petiole 2 cm from the node. basal or apical stem end was inserted in 1% agar in a petri dish, the distal cut petiolar surface covered with lanolin paste containing 100 ~C/g of 3.6 ~M 5- 3 H-IAA (0.6 ~g/g) plus varying amounts of unlabeled IAA, and the dishes placed in 250 mm desiccators. Stoppers were ommited, except when ethylene was injected. After 18 h, 5 mm segments excised from the petiole base'were split into middle half and upper and lower quarters, the zones weighed, and radioactivity counted. Results are expressed as dpm/mg dry wgt to correct for tissue assymetry. Curvature was measured from shadowgraphs made initially and after 18 h.


3.1. GRAVITY DEPENDENT HORIZONTAL NUTATION IN ETIOLATED PEA SEEDLINGS

Only certain seedlings with plumular hooks nutate in ethylene [8], always bending away from the hook, so that eventually the hook is held uppermost regardless of the seedling's initial spatial orientation [2]. Etiolated pea epicotyls grow at an oblique angle in 0.1 ~l/l ethylene [3]; decline by at most 70° to 85° in 0.2 to 0.8 ~l/l [2,3]; and swell at the tip, but barely nutate in 1 to 10 DI/I [2].

The longer transport path through the hook's convex side creates a lateral IAA gradient in the etiolated pea subapex because the diffusible IAA content is halved in each successive 2.5 mm interval as IAA moves basipetally [10]. When 5-3 H-IAA was applied to the cut stump of upright (or inverted [4]) explants with deb laded hooks, 4 h later the side/side and inner/outer S.A. ratios (inner = facing the hook) were I.03! .14 and 1.53! .11 respectively. This imbalance causes curving in the expected direction during horizontal clinostat rotation [8], and in subapical segments floated with continuous shaking to compensate gravity [1]. As ethylene prevents these tropisms [1,8], they cannot be due to induced ethylene production [7]. In upright seedlings, the backbending is corrected by (-) geotropism (Weisner, 1878 [8]), but in ethylene it intensifies when the correction is prevented (Richter, 1906 [8]).

337

Within 30 min (figure 1, RIGHT), ethylene inhibited gravitransport in subapical segments to the extent previously reported [1], confirming a report that 'geosensitivity' persists in ethylene for a short time, and then rapidly decreases within 1 h (von Guttenberg, 1910 [8]). When the experiment was repeated with epicotyls lacking hooks, upward transport was detected, and if the hook elbow was left intact, a few ul/l ethylene caused 5- 3 H-IAA to move upward as intensely as it moves downward in air, with a compensation point at 0.2 ul/l (figure 1, LEFT). In 4 h, these epicotyls bent upward by 51! 3.6 0 in air, and inspite of a reduced growth rate, downward by 13 ~ 4.50 in 100 )11/1 ethylene. Since the 'ageotropic' hook has already responded to its high endogenous ethylene content [3], and as inversion occurred without an attached hook, but not in subapical segments, only the transition zone (the 'neck') joining the hook to the subapex could have developed inverted transport. Ethylene may invert gravitropic transport by inhibiting gravitational adaptation, overstimulating and reversing the transport system [4].

Ethylene did not alter phototropic lateral transport in etiolated pea epicotyls, or gravitransport in light grown tomato stems and etiolated black bean hypocotyls, and it does not effect gravitransport in other tissues which do not respond tropistically to ethylene [1,4].

a: ~ 2.0 0.. ::0 .,. a: w :s: 1.5 o ..J

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Figure 1. (LEFT) Effect of ethylene on 5- 3 H-IAA gravitransport in pea explants during 4 h. (RIGHT) Response of etiolated subapical segments to 10 pl/l ethylene. Change in (upper S.A.)/(lower S.A.) in each interval.

Oblique growth occurs in 0.1 pl/l ethylene because at that angle the increased gravity vector perpendicular to the stem causes weakened (+) gravitransport to offset the hook-imposed subapical auxin assymetry. Progressively as the ethylene concentration is raised to 0.8 ul/l, the subapical IAA content decreases and radial cell expansion is promoted, inhibiting growth and elongation in the inner and outer subapical

338

surfaces [1]. Increased (-) gravitransport in the 'neck', and decreased (+) gravitransport in the subapex [1], amplify the hook-imposed auxin assymetry, concentrating IAA in the upper side of the subapex [6]. The resultant overgrowth increases the gravitional vector perpendicular to the 'neck', promoting additional upward IAA movement and downward curving, until the subapex declines below the horizontal plane and the gravitional vector begins to decrease. The lengthened transport path through the upper subapical surface, augmented by residual (+) gravitransport, is abnormally effective in decreasing IAA delivery to the upper side of the sub tending epicotyl, because in ethylene the diffusible IAA content decreases at a faster rate per unit length due to an inhibition of polar IAA transport [1]. These tendencies balance when the epicotyl is slightly upslanted and the subapex visibly downbent [3], with the gravity sensing 'neck' tilted below the horizontal plane.

3.2. GRAVITY DEPENDENT LEAF EPINASTY IN LIGHT GROWN TOMATO PLANTS

Leaves on the underside of horizontally positioned tomato plants develop intense epinasty in ethylene, but those on the upper side hardly respond, whereas in air, leaves on the upper side undergo an intense epinastic response because they are in an inverted position, while leaves on the lower side do not bend [2]. In air and ethylene, upright and inverted petiole explants curved in the expected manner (table 1).

Table 1. Effect of 10 ul/l Ethylene on the Polar and Gravitropic Transport of 5-3H-IAA in Tomato Petiole Explants. 18 h incubation.

U~right Inverted Air Ethylene Air Ethylene

S.A. Ratio (L/U) 0.6 ug/g IAA 0.95 (.17) 0.54 (.12) 2.14 (.42) 1.10 (.17) 10 1.12 (.08) 0.49 ( .11) 2.24 ( .37) 1.01 (.14) 10,000 1.16 (.16) 1.18 (.16) 0.89 ( .07) 0.89 ( .07)

Epinastic Curvature (degrees) o ug/g IAA 2 (3) 12 (6) 14 (11) 24 (23) 1 o (2) 33 (12) 24 (15) 33 (18)

10 9 (6) 73 (22) 41 (15) 45 (21) 100 24 (12) 77 (12) 52 (20) 62 (23) 1000 40 (20) 66 (27) 52 (20) 65 (24)

Transport to the Basal 5 mm (dpm/mg dry wgt) 0.6 ug/g IAA 153 (48) 306 (82) 150 (48) 332 (44) 10 96 (20) 130 (24) 80 (19) 121 (43) 10,000 11.1 {3.42 16.3 ~4.22 12.2 ~0.32 16.8 ~6.22

(L/U) = (lower specific activity/upper S.A.) relative to gravity. (S.D.)

The petiole base contained 0.023, 0.22 and 29.6 uM 3H-IAA respectively, 18 h after feeding 0.6, 10 and 10,000 ug/g 3H-IAA (table 1, air). Only 29.6 uM IAA markedly stimulated ethylene production in basal segments (table 3). In air, upright petioles transported 0.6 and 10 ug/g IAA with an L/U ratio close to unity because epinastic adaxial transport is balanced by (+) gravitransport (table 1). When petioles are inverted,

339

epinastic transport and (+) gravitransport cooperate to increase the L/U ratio to 2.2. In ethylene, upright petioles transported IAA toward the upper surface because epinastic transport and (-) gravitransport acted in the same direction, whereas inverted petioles developed no lateral gradient because (-)gravitransport offset epinastic transport. The data indicate that ethylene did not alter epinastic transport.

The intensity of curving depended on the amount of 5- 3 H-IAA which reached the adaxial surface at the petiolar base. Because ethylene promoted IAA transport to the base (table 1), labeling in the adaxial surface and the curving of inverted petioles was the same in air and ethylene, even though inverted explants developed a large L/U IAA gradient in air, and none in ethylene. When 10,000 pg/g 5- 3 H-IAA was fed, L/U gradients did not develop, and curvature depended only on the amount of IAA transported to the base. In a study claiming to show that IAA fails to redistribute laterally in Coleus during ethylene-induced epinasty [9], 42,200 Pg/g 1 4 C-IAA was applied to the petiolar stump.

TABLE 2. Effect of Hypobaric Treatment Enriched in Carbon Dioxide on the Epinastic Response of Tomato Petiole Explants.

IAA (ug/g) o 1

10 100

1,000 10,000

Hypo = a flowing .112 atm O2 ; .02

Curvature Upri-ght

Air Hypo 2 (3) 2 (3) o (3) 5 (4) 9 (6) 7 (6)

24 (12) 11 (7) 40 (20) 15 (10)

108 (27) 29 (8) mixture containing .028 atm CO2; .16 atm. total

(degrees) Inverted

Air 14 (11) 24 (13) 41 (15) 52 (20) 52 (20)

Hypo 34 (25) 46 (7) 53 (22) 56 (21) 55 (21)

atm water vapor (100% rh); pressure at 23°C. (S.D.)

TABLE 3. Curvature Development and Ethylene Production in 1 cm Basal Tomato Petiole Segments Floated With Continuous Shaking for 18 h.

IAA Conc. o

0.1 1

10 100

Segments were containing 2%

Curvature (degrees) Ethylene Production Air Ethylene (10 pI/I) (nl/g.h)

11.5 (2.2) 13.5 (3.5) 0.40 13.5 (3.3) 19.0 (5.8) 1.10 21.5 (6.4) 25.0 (6.1) 2.70 30.5 (6.9) 30.5 (7.8) 4.00 11.0 (3.6) 10.5 (4.3) 7.75

incubated in 50 rnM potassium phosphate buffer (pH 6.8) sucrose and various IAA concentrations. (S.D.)

Hypobaric conditions should decrease curving in upright explants and increase it in inverted explants if endogenous ethylene influences epinasty by inducing (-) gravitransport, whereas an ethylene-induced stimulation of adaxial growth would be inhibited in both upright and inverted explants. The data (table 2) prove the first alternative without excluding participation of the second.

340

Ethylene interacted synergistically with 0.1 to 1 nM IAA to promote adaxial growth in petiole segments (table 3) under gravity compensated conditions, possibly by inhibiting the adaptation process which reduces the IAA sensitivity of cells [5]. The response would have been greater if 0.1 to 1 }lM IAA had not slightly stimulated ethylene production. Petioles exposed to more than 10 nM IAA produced enough ethylene to preclude any effect of applied gas on curvature [1].

Labeled IAA reached the petiole base in 150 min, and subsequently if endogenous ethylene was present, a large L/U ratio would not have developed in air (table 1). Apparently a short exposure to transient ethylene production, induced when tomato explants are inverted in air, stimulated cells to grow for an extended time in response to IAA [7].

In summary, the classical view of gravity-dependent ethylene action, combined with modern concepts of ethylene action, adequately accounts for ethylene-induced, gravity dependent tropistic responses.

References

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

2. Crocker, W., Zimmerman, P~ and Hitchcock, A. (1932) 'Ethylene induced epinasty in leaves and the relation of gravity to it', Contrib. Boyce Thompson Inst. 4, 177-218.

3. Goeschl, J. and Pratt, H. (1968) 'Regulatory roles of ethylene in the etiolated growth habit of Pisum sativum', in F. Wightman and G. Setterfield (eds.), Biochemistry and Physiology of Plant Growth Substances, Runge Press Ltd., Ottawa, Canada, pp. 1229-1242.

4. Kang, G. and Burg, S. (1974) 'Ethylene action on lateral auxin transport in tropic responses, leaf epinasty, and horizontal nutation', in Plant Growth Substances, Tokyo, Hirokawa, 1090-1094.

5. Kang, B., Park, W., Nam, M. and Hertel, R. (1991) 'Ethylene-induced increase of sensitivity to auxin in Ranunculus petioles and its implications regarding ethylene action on adaptation', in C. Karssen, L. van Loon, and D. Vreugdenhil (eds.), Progress in Plant Growth Regulation, Kluwer Academic Publ., Netherlands, pp. 248-253.

6. Laan, P. (1934) 'Der einfluss von aethylen auf die wuchstoffbildung bei Avena und Vicia', Rec. Trav. Bot. Neer. 31, 691-742.

7. Leather, G., Forrence, L. and Abeles, F. (1972) 'Increased ethylene production during clinostat experiments may cause leaf epinasty', Plant Physiol. 40, 183-186.

8. Neljubow, D. (1911) 'Geotropismus in der Laboratoriumsluft', Ber. Deutsch. Bot. Ges. 29, 97-112.

9. Palmer, J. (1976) 'Failure of ethylene to change the distribution of indoleacetic acid in the petiole of Coleus blumei X frederici during epinasty', Plant Physiol. 58, 513-515.

10. Varder, Y. and Kaldewey, H. 'Auxin transport and apical dominance in Pisum sativum', in H. Kaldewey and Y. Varder (eds.), Hormonal Regulation in Plant Growth and Development, Verlag Chemie GmbH, Weinheim/Bergstr, pp. 401-411.

ETHYLENE AND THE GROWTH OF ETIOLATED SEEDLINGS OF Lupinus ~L.

J.L. CASAS1, M.B. ARNA02, G. GARRID02, M. ACOSTA2, J. SANCHEZ-BRAV02 . 1Department of Environmental Sciences and Natural Resources. University of A1icante. P.O.Box 99. E-03080 ALICANTE (SPAIN). 2Department of Plant Biology. University of Murcia. Santo Cristo, 1. E-30001 MURCIA (SPAIN).

ABSTRACT. The possible influence of ethylene on growth was investigated in etiolated lupin (Lupinus albus L.) hypocotyls. The treatment of lupin seeds with either l-aminocyclopropane-I-carboxylic acid (ACC) or ethephon led to a shorter and thicker seedling, ethephon being the most effective in causing this effect. In both cases, the effects on growth could be drastically prevented by pretreating the seeds with silver thiosulphate, a potent inhibitor of ethylene action. Furthermore, when lupin seeds were incubated in silver thiosulphate a transient reduction in both length and hypocotyl diameter was also observed. The resul ts suggest that ethylene is implicated in the establishment of the final length and size of the lupin hypocotyl.

1. Introduction

Since the discovery of ethylene as a phytohormone in 1901 by Neljubow, it has been shown that plants treated with ethylene modify their growth pattern. However, as Reid (1987) stated, there is no agreement concerning the involvement of ethylene in the control of normal vegetative growth. In some cases a good correlation between ethylene production and the growth pattern of certain plant organs has been demonstrated (Schierle and Schaark, 1988). However, in other cases the appl ication of a known strong inhibitor of ethylene action such as Ag+, does not affect growth (Cameron and Reid, 1983). Over the last four years, we have investigated this topic in our laboratory, using etiolated lupin hypocotyls as plant material. Previous works carried out on this plant model indicated the possible involvement of IAA in the regulation of longitudinal growth (Sanchez-Bravo et al., 1986; Ortufio et al., 1990). The observed pattern of growth of different cell types is similar and suggests that once the period of organ growth is concluded, apical cells are larger with lower diameter and thicker cell walls than cells located in the

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1. C. Pech et at. (eds.!, Cellular and Molecular Aspects of the Plallt Honnone Ethylene, 341-346. © 1993 Kluwer Academic Publishers.

342

basal region of the plant (Sanchez-Bravo et al., 1992). This longitudinal gradient in size and shape of the cells suggests that ethylene, besides IAA might contribute to the hormonal regulation of the cellular growth of this organ.

In this paper we discuss the possible involvement of ethylene in etiolated lupin hypocotyl growth.


2.1 GROWTH OF PLANTS

Lupin seeds (Lupinus albus L. cv. Multolupa) were imbibed for 24 h in water or, alternatively, in an aqueous solution of ACC or ethephon (commercial Ethrel, 48% l-chloroethyl phosphonic acid) and germinated in damp vermiculite at 25°C in darkness.

2.2 GROWTH MEASUREMENTS

The length and diameter of the hypocotyls of control and treated plants were measured on a five-plant uniform sample from 3-days old seedlings.


Some evidence has recently suggested that ethylene, besides IAA, may be acting as an additional control of growth in etiolated lupin hypocotyls. Firstly, lupin hypocotyl was shown to produce ethylene during the growth period (PerezGi I abert et al., 1991). But, interest ingly the younger the hypocotyls were, the more ethylene they produced. Furthermore, if ethylene production was analyzed not in the whole hypocotyl but in different zones, which are known to show different growth kinetics (Ortufio et al., 1988), the results showed again that younger and actively growing zones produced more ethylene and showed a higher EFE activity than older zones (Perez-Gilabert et al., 1991).

To obtain more information about the possible role of ethylene on lupin growth, some experiments were conducted in which lupin seeds incubated in ethylene-releasing compounds were germinated and grown in the dark. Two ethylene-releasing compounds were used: ACC, the physiological precursor of this plant hormone, and ethephon.

When lupin seeds were submerged before germination in different concentrations of these chemicals, a variation with respect to the growh pattern of untreated plants was obtained (Figs. lA and IB). Ethephon was shown to be more effective than ACC in reducing hypocotyl length. Thus, 1 mM ACC showed almost no effect on growth, but treatment of seeds with 5 mM ACC or 0.66 mM ethephon caused a reduction of around 15% in

343

........ ~ A B .... c: 0 0 100 00"000-00 -0 •• ~ I A .. AA-AAA ,-

'-' OA A • .c .... 70 '/ • 01 , c: t·-· .!! A ••• ~ 40 . \ . " 0 ., .. 0 0 ... " Q. >- 10 ::I: 0 5 10 15 20 25 0 5 10 15 20 25

Age (days) Age (days)

Figure 1. Effect of lupin seed treatment with ACC (A) or ethephon (8) on hypocotyl length, expressed as percentage of control untreated plants. Concentrations used were: 1 roM ACC (0); 5 roM ACC (~); 0.66 roM ethephon (e) and 3.3 roM ethephon (.A.) •

the final length of the hypocotyl. Incubation with 3.3 roM ethephon delayed the enlargement of the hypocotyl during two weeks, the growth subsequently restarting until a final length of 40% of untreated plants is reached.

The reduction in the enlargement of the organ observed after the treatment of the lupin seeds with either ACC or ethephon was shown to be accompanied in all cases by a progressive increase in the diameter of the organ (Figs. 2A and 28).

Therefore, treatment by exogenously produced ethylene, by submersion of lupin seeds in either ACC or ethephon solutions led to a shorter but thicker hypocotyl. Similar effects were obtained when spraying lupin seedlings with ACC of ethephon solutions (Ortufio et al., 1991). To ensure that the observed effects were due to ethylene, further experiments were carried out in which seeds were pre-incubated in an Ag+ solution before being incubated in the chemicals. Ag+ almost completely nullified and significantly reduced the effects of ACC and ethephon, respectively, on both the elongation and thickening of the organ. These results may suggest that the alterations observed in hypocotyl growth after treatment with

344

ACC or ethephon might be attributed to the ethylene generated by these compounds .

......... e .... c 0 260 0 -0

~ 220 ......, L.. Q) 180 .... Q)

E 0 140 :g

~ 100 o o 0..

~

A B

5 10 15 20 25 0 5 10 15 20 25 Age (days) Age ( days)

Figure 2. Effect of lupin seed treatment with ACC (A) and ethephon (B) on hypocotyl diameter, expressed as percentage of control untreated plants. Concentrations used were: 1 roM ACC (0); 5 roM ACC (/:;.); 0.66 roM ethephon ( .) and 3.3 roM ethephon (A).

If exogenously added or produced ethylene is able to induce appreciable growth responses, endogenous ethylene might also be expected to be involved in the normal development of lupin growth. To test this, a group of lupin seeds were incubated in Ag+ solutions and grown as usual. The results obtained showed that Ag+ also modified the growth pattern within the first week, increasing the length by 30% (Fig. 3A) and significantly reducing the diameter of the hypocotyl (Fig. 3B). It was interesting to notice that Ag+ only showed some physiological effect during the first days of growth, when the hypocotyl is producing the highest amount of ethylene.

These results support others obtained from experiments with IAA-induced growth (Perez-Gilabert et al., 1991). In these experiments, subapical sections (with young cells) of lupin hypocotyls treated with Co2+ totally suspended ethylene production and exhibited a concomitant increase in IAAinduced growth of about 30%

In view of the results obtained, it can be deduced that ethylene affected the establishment of the final length and size of the lupin hypocotyls. In an effort to investigate the mechanisms by which this process takes place, we recently carried out some experiments that showed that different lupin tissues exhibit different responses to ethylene, the epidermis being the least sensitive tissue to ethylene (Sanchez-Bravo et al., 1992). Besides reducing length and increasing cell diameter, ethylene was shown to provoke an

345

....... ....... e A

e B +' +' I: I: 140 0 0 O· 140 Q Q - ./0 -0 0

~ 120 ~ \ 120 ~ '-'

~ ..c: .~~ 00 .s ....

.-e,lt:% at 100 .... , " 100

G) I: ~~O-O E .!! 0

~ ~-~ I ... ~

0 80 80 ~ Q 0 0 Q. Q

~ 60 0 60 Q.

0 5 10 15 20 25 0 5 10 15 20 25 ~ Age (days) Age (days)

Figure 3. Effect of lupin seed treatment with Ag+ on hypocotyl length (A) and diameter (B) both expressed as percentage of control untreated plants. Concentrations used were: 0.5 mM Ag+ (t::.. , .&) and 1 mM Ag+ (0,.).

increase in the thickening of cell walls. Electron microscopy revealed that cells in treated plants

showed a higher cytoplasmic density, with an increase in RER and dictyosomes being the most significant changes (SanchezBravo et al., 1992). Furthermore, the number of membrane vesicles that were near or fusing with cell walls was higher in ethephon-treated plants. All these results suggest that ethylene stimulates the secretion of cell wall materials.

To summarize, it seems clear that ethylene actively participates in the growth pattern of etiolated lupin hypocotyls. Exogenously produced ethylene, by submersion of the seeds in either ACC or ethephon, results in a shortening and thickening of the hypocotyl. Conversely, inhibition of the ethylene action by Ag+ provokes a transient increase in length and a reduction in the diameter of the hypocotyl.

4. Acknowledgements

This work has been supported by Comisi6n Interministerial de Ciencia y Tecnologia (CICYT). Project ALI89-0293.

5. References

Cameron, A.C. and Reid, M.S. (1983) 'The use of thiosulfate to prevent flower abscission from plants' Sci. Hortic. 19, 373-378

silver potted

346

Ortufio, A., Sanchez-Bravo, J., Acosta, M. and Sabater, F. (1988) 'Evolution and distribution of growth in etiolated hypocoty1s of Lupinus albus' BioI. Plant. 30(4), 268-274.

Ortufio, A., Sanchez-Bravo, J., Moral, J.R., Acosta, M. and Sabater, F. (1990) 'Changes in the concentration of indole-3-acetic acid during the growth of etiolated lupin hypocotyls' Physiol. Plant. 78, 211-217.

Ortufio, A., Del Rio, J.A., Casas, J.L., Serrano, M., Acosta, M. and Sanchez-Bravo, J. (1991) 'Influence of ACC and ethephon on cell growth in etiolated lupin hypocotyls. Dependence on cell growth state' BioI. Plant. 33, 81-90.

Perez-Gilabert, M., Ortufio, A., Acosta, M. and Sanchez-Bravo, J. (1991) 'Variations in ethylene production rate, ethylene forming enzyme acti vi ty and 1-aminocyclopropane-1-carboxylic acid content during the growth of etiolated hypocotyls of Lupinus albus', Plant Physiol. Biochem. 29, 319-325.

Reid, M.S. (1987) 'Ethylene in plant growth, development and senescence', in P.J. Davies (ed.), Plant Hormones and Their Role in Plant Growth and Development, K1uwer Academic Publishers, New York, pp. 257-279.

Sanchez-Bravo, J., Ortufio, A., Acosta, M. and Sabater, F. (1986) 'Distribution of indole-3-acetic acid in relation to the growth of etiolated Lupinus albus hypocotyls' , Physiol. Plant. 66, 509-514.

Sanchez-Bravo, J., Ortufio, A., Perez-Gi1abert, M., Acosta, M. andSabater, F. (1992) 'Modification by ethylene of the cell growth pattern in different tissues of etiolated lupine hypocotyls', Plant Physiol. 98, 1121-1127.

Schierle, J. and Schaark, A. (1988) 'Asymmetric synthesis and concentrations of ethylene in the hypocotyl hook of Phaseolus vulgaris', J. Plant Physiol. 133, 325-331

VARIOUS CONDITIONS OF ILLUMINATION AND ETHYLENE EVOLUTION

V.I. KEFELI, T. Y.A RAKITINA, P.V. VLASOV, F. JALILOVA and AE. KALEVICH K. A Timiryazev Institute of Plant Physiology, Academy of Sciences, Russia, Botanicheskaya ul., 35, 127276 Moscow.

ABSTRACT The evolution of ethylene by seven-day green, etiolated, and red litght (650 run) -

irradiated pea seedlings (Pisum sativum L.) was studied. The production of ethylene was the greatest in green seedlings; illumination of etiolated seedlings by red light for 5 min increased the formation of ethylene in the leaves but decreased it in the stems. It is suggested that the changes in the evolution of ethylene are due to endogenous lAA, the content of which increased in the leaves during irradiation and decreased in the apical portion of the stem. Effect of UV -B (280-320 run) radiation on ethylene production by Arabidopsis mutants with different sensitivity to UV-B light was studied. Arabidopsis mutants were exposed to UV illumination under the constant integral

intensity of radiation (8-9 W/m2). Ethylene release from the resistant mutant line UCERGGAN after the treatment was higher than ethylene production by the wild type ENKHEIME. At the same time the lowest rate of ethylene production was observed in the sensitive mutant E. glabra. The highest rate of ethylene production by all three types of Arabidopsis was observed during the first two hours after UV treatment.

INTRODUCTION The growth of seedling during de-etiolation depends on the intensity and spectral

composition of the light. The participation of the phytochrome system in this process is confirmed by the reversibility of the effect (1) and by the negative correlation between the growth rate and the content of the physiologically active form of phytochrome (2,3). It has been suggested that ph}tochrome control of growth processes is implemented with the participation of phytohormones, in particular ethylene.

In certain cases exogenous ethylene can stimulate the action of light. For example, in etiolated pea seedlings, both light and ethylene slow down the elongation of the internodes (4). In other cases ligth and ethylene induce opposite reactions. Thus, light induces an opening of the apical loop in pea seedlings, while exogenous ethylene blocks this process (5). The formation of endogenous ethylene depends on the conditions of illumination. In isolated green leaves, light supresses its evolution but at increased concentrations of C02 it stimulates this process (6,7).

Some data indicate that intact green plants form virtually the same amount of ethylene both in light and in darkness (8,9). In etiolated seedlings light either suppresses (1O-l2) or, in a number of cases, enhances the liberation of ethylene (13,14). Such contradictory experimental data maintain

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J. C. Pech et al. (etis.), Cellular and Molecular Aspects olthe Plant Honnone Ethylene, 347-352. © 1993 Kluwer Academic Publishers.

348

the interest in the problem of participation of ethylene in the photodependent processes. The present work is devoted to the question of the participation of ethylene in the photoinduced change in growth in pea seedlings grown under various conditions of illumination.

MATERIALS AND METHODS In the experiments we used seven-day pea seedlings (Pisum sativum L.) of the Konservnyi

variety, grown in tap water under while light fluorescent lamps with a 16-h photoperiod and

illumination 8 W/m2 or in darkness. Begining with the fourth day, the etiolated plants were irradiated daily for 5 min with a red Philips lamp (France) through an interference filter with A =

650 ±5 nm. The illumination was 8 W/m2. All the experiments were conducted at 24°C. Ethylene evolution by whole plants was determined by incubating plants for 30-60 min in

hermetic glass vessels with a volume of 800 mi. The experiment was conducted 1 h after the last irradiation with red light. Green plants were incubated in white light, etiolated plants and plants irradiated with red light were incubated in darkness.

The dynamics of the evolution of ethylene by the leaves and apical portions of the stem (third internode, including the apex and the loop) was determined after a single irradiation of whole etiolated seedlings with red light. After various time intervals following irradiation, the leaves and stern were cut off and incubated in darkness in 15 mI glass vessels with hermetically closed lids. To avoid distortions of the results on account of the formation of wound ethylene, the site of the cut was rinsed with distilled. water (15), and ethylene was determined no later that 30 min after the cutting. Arabidopsis plants were exposed under UV-B light (280-320 nm), 6-8

W/m2 and then exposed on white light. Ethylene was analysed on a gas chromatograph with a flame-ionisation detector, using the concentrating system ofRakitin and al.(17), which permits a substantial increase in the sensitivity of the method through the use of large volumes of the investigated samples (in one case from 15 to 100 mI). It is known that the evolution of ethylene depends on the C02 ands 02 concentration (7, 18) ; therefore the level of these gases was

monitored during the experiment. For this purpose, at the end of the exposure 1 mI of air was collected from each vessel with a syringe and analysed by a gas chromatographic method (16). The C02 concentration in the vessels at the end of the experiment was 0.5-1.2 %.

The isolation and purification of IAA from plant tissues was performed by the method of extraction and liquid chromatography (19). Indolepropionic acid (IPA) was added to the plant tissue before homogenization as an internal standard. The purified samples were methylated with diazomethane, and IAA was determined quantitatively on a Hitachi M-70 chromato-mass spectrometer (Japan) in a system of detection according to three masses: 130 - indole, 180 -methyl ester ofIAA, and 203-methyl ester oflAA. The colunm was 2m X 3 mm, 3% OV-17 on Gazkhrom Q (80-100 mesh). The carrier gas was helium, velocity 40 mVmin, and the colunm temperature was programmed from 180 to 250 Co, 10 CO/min.

The tables and figures present the aritmethic means of three to four biological repetitions and their standard deviations. The analytical repetition was two to three times.

RESULTS Seven-day seedlings, grown under various conditions of illumination, differed

substantially morphologically and produced ethylene differently (table 1). The stem length of the green seedling growth on a 16-h photoperiod was one fifth as great and the weight of the leaves 5.5 times as great as in the etiolated seedlings. Even a 5 min daily red-light illumination of plants growth in darkness induced almost 30 % inhibition of stem growth and a threefold

349

increase in weigth of the leaves in comparison with non irradiated plants, i.e., led to the change in growth processes characteristic of de-etiolation. Moreover, the evolution of ethylene by whole plants was incrased by 27-28 % in comparison with the dark variant. The evolution of ethylene by green plants was twice as great on a per seedlings basis as that by plants grown in darkness, and three times as great on a per gram of fresh weight basis. (Table 1).

Table I : Evolution of ethylene, height of stem and weight of leaves of seedlings growth under various conditions of illumination.

Conditions Height of stem Weight of leaves nl.h-l g-1 fresh nl.h-1.seedlings em of one seedling weight

Darkness 11.6 ± 0.9 11 ±2 0.72..± 0.11 0.55 ± 0.07 Darkness + Red 8.3 ± 0.4 36±6 0.91 ± 0.04 0.70 ± 0.03 light (5 min daily) White light (16 h 1.9±0.4 81 + 10 2.43 ± 0.38 1.33 ± 0.21 daily)

* For the first 3 days the plants of all variant were grown in darkness

Thus, in the process of de-etjolation the evolution of ethylene by whole plants was increased, and in the process the growth of the leaves was enhanced, while growth of the stem was inhibited. If the photoinduced changes in the growth of these organs was mediated by ethylene, it should be assumed that the dynamic of the evolution of ethylene after a single irradiation would be different in the leaves and stem.

Both the leaves and the apical portion of the stem (third internode, including the loop and apex) liberated 50 % more ethylene during the first 30 min after a single irradiation of etiolated seedlings with red light in comparison with the leaves and stems of non irradiated plants (Fig. 1). In the leaves the evolution of the ethylene gradually decreased until the end of the experiment, but after 3.5 h it remain higher that in the dark variant. In the stem, ethylene production had already dropped to the level of the dark variant after 1 h, while by the end of the experiment the stems of the irradiated plants released 40 % less ethylene than the stems of non-irradiated plants. These data show that in the organs whose growth is enhanced under the action of light, the evolution of ethylene increased, while in the organs whose growth is inhibited after irradiation, after a brief rise, a stable decrease in the evolution of ethylene was observed, down to lower level that in plants growing in total darkness.

In a number of studies it has been shown that IAA can be an inducer of ethylene formation (see the survey (20». Possibly in our experiments also, the changes in the evolution of ethylene under the action of light are associated with a change in the content of endogenous IAA. To test this hypothesis, we determined the content of IAA in the leaves and third (from the top) internode of seddlings growth under various conditions of illumination.

From table 2 it is evident that the leaves of seedlings contain almost twice as much IAA calculated per gram of fresh weight in comparison with the leaves of etiolated seedlings and eight times as musch, calculated per seedling. In the apical portion of the stem of one green seedling, there was one fourth as much IAA as in the apical portion of the stem of one etiolated seedling. Calculated per gram of crude weight of the stem, the lAA content was negligibly decreased under the action of light. The maximun amount of IAA was detected in the fastest growing organs : green leaves and etiolated stems.

350

O~~--~~~~~~ ____ -J

o 0,5 1 1,5 2 2,5 3 3,5 4

time after irradiation, h

Figure 1 : Evolution of ethylene ofleaves (1,2) and the apical portion of the stem (3,4) of etiolated seedlings (1,3) and seedlings irradiated once (5min) with red light (2,4)

Table 2 : Content ofIAA in leaves an apical portion of the stem of seedlings gro",th under various conditions of illumination.

Conditions Content of lAA, ng of illumination Per gram of fresh \\1. Per seedling

Darkness Darkness + Red light (5 min daily)

22.7±1.2 33.3±3.4

LJ;.'AVE"l 0.3 ± 0.02 1.1 ± 0.1

White light (16 h daily) 43.3±5.2 2.6±O.3 APIC'AL PORTION OF THE STEM

Darkness Darkness + Red light (5 min daily) White light (16 h daily)

50.0±5.3 45.0±2.5

47.1+5.1

2.0±O.2 1.7±0.1

0.5+0.1

A 5 min-daily illumination of etiolated seedlings with red light induced an increase in the IAA content in the leaves and decrease in the IAA content in the apical portion of the stem in comparison with seedlings gro\\th in total darkness. Thus, during deetiolation the content of lAA in the leaves is increased and that in the third internode of the stem is decreased.

UV -light also activate ethylene evaluation. The resistent mutant of Arabidopsis produce more ethylene after UV-B illumination (Fig. 2).

IE6g1, sens·1

OL-~--~--~--~~--~

o 30 60 90 120 150 180

UV-B treatment time

Fig.2 : Evolution of ethylene by different Arabidopsis mutants

351

DISCUSSION During de-etiolation, intensive growth of the stem, necessary to carry the photosynthesing

organs to the surface of the soil, gives way to growth and development of leaves. A brief illumination with red light is sufficient to induce changes in the growth processes characteristics of de-etiolation. Our experiments show that during illumination the content of lAA in the stem decreased, but the amount in the leaves increased. After irradiation with red light there was either a displacement of lAA out of the apical portion of the stem into the leaves or a synthesis of IAA in the leaves and inactivation in the stem. Since lAA possesses attractant properties, the increase in its concentration in the leaves led to an increase in the attractant ability of the leaves and to their gro\\'th.

By activating ACC (l-aminocyclopropane-l-carboxylic acid) synthase, IAA can enhance the formation of ethylene in leaves (20). But formation of ethylene may can occur from ACC in the leaves. (21). It is known (22) that the initiation of many physiological processes is accompanied by an increase in ethylene formation. In a number of studies it has been shown that ethylene can activate an alternative pathway of respiration and thereby participate in the enhancement of growth processes (23). Moreover, ethylene can accelerate sugar transport (24).

It is extremely probable that in our experiments the increase in the evolution (and that means formation as well) of ethylene by leaves after irradiation with red or UV-B light is necessary to provide for the supplementary energy expenditures arising during the photoinduction of growth processes in the leaves.

REFERENCES l. Pike C.S., Richardson A.E., Weiss E.K et al., 1979. Short stem phytochrome control of oat coleoptile and pea epicotyl growth. Plant Physiol. 63. No.3, 440-442 2. Wall lK, Johnson C.B., 1981. Phytochrome action in light growth plants: the influence of light quality and influence rate on extension growth in Sinapsis alba L. Planta 53. No.2, 101-103. 3. Uematsu H., Hosoda H. and Furuya M., 1981. Bip~sic effect of red light on the growth of coloeptiles in etiolated barley seedlings. 1981. Bot. Mag. Tokyo. 94. No. 1035,273-278. 4. Goeschl lD. and Pratt H.K, 1968. Regulatory roles of ethylene in the etiolated growth habit of Pisum sativum, in : Biochemistry and Physiology of Plant Growth substances, Wightman F. and Setterfield G., eds., Runge Press, Ottawa. 1229-1232. 5. Burg S.P and Burg E.A., 1968. Auxin stimulated ethylene formation. Its relationship to auxininhibited growth, root geotropism and other plant processes. In : Biochemistry and Physiology of Plant Growth substances, Wightman F. and Setterfield G., eds., Runge Press, Ottawa. 1275-1280. 6. Grodzinski B., Boesel I., and Horton R.F. 1983. Light stimulation of ethylene release from leaves of Gomphrena g/obosa L. Plant Physiol. 71, No.3, 588. 7. Horton R.F., 1985. Carbon dioxide flux and ethylene production in leaves. Ethylene and plant development. Roberts lA., and Ticker G.A., eds., Butterworths, London, 37-40. 8. Bassi P.K. and Spencer M.S., 1983. Does light inhibit ethylene production in leaves? Plant. Physiol., 73. Noo 3, 758-760. 9. Rodecap KD. and Tinger D.T., 1983. The influence of light on ozone-induced 1-aminocyclopropane-I-carboxylic acid and ethylene production from intact plants. Z. Pflanzenphysiol., 1l0, No.5, 419-422. 10. Rohwer F. and Schierk l, 1984. Effect of red light on the s)nthesis of would ethylene in etiolated pea shoots, 1 Plant Physiol. 116, No., 1,31-36.

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11. Vangronsveld J., Van Genechten A., and Van Poucke M., 1985. The effect oflight on ethylene biosynthesis in Phaseolus vulgaris L. seedlings. Phytochrome-mediated effect in etiolated seedlings, Colloquia Pflanzenphysiologia, No.9, Light and Hormone interaction in plants, Bleiss W. and Goring H. eds., Humbolt Universitat, Berlin, 50-52. 12. Vangronsveld J., Clijster. H and Van Poucke M., 1988. Phytochrome-controlled ethylene biosynthesis of intact etiolated bean seedlings. Planta., 174, No. I, 19-22. 13. Rohwer F., and Schierle J., 1982. Effect of light on ethylene production : red light enhancement of l-aminocyclopropane-l-carboxylic acid concentration in etiolated pea shoots. Z. Pflanzenphysiol., 107, No.4, 295-297. 14. Jiao X.Z., Yip W.K. and Yang S.F., 1987. Effect of light and phytochrome on 1-aminocyclopropane-I-carboxylic acid metabolism in etiolated wheat seedlings leaves. Plant Physiol. 85, No.3, 643-648. 15. Saltveit M.E., Jr. and Dilley D.R., 1979. Studies of rapidly induced would ethylene synthesis by excised of etiolated Pisum Sativum L. cv. Alaska. Plant Physiol. 64, No.3, 417-420. 16. Zalilova F. Kh., Vlasov P.V., Mednik I.G., Kefeli V.I. 1991. Effect of UV-radiation on activity and content of some phytohormones. In: Ecological aspects of growth regulation and plant productivity., Jaroslavl, 97-101. 17. Rakitin V. Yu and Rakitin L. Yu. 1986. Determination of gas exchange and the content of ethylene, carbon dioxide, and oxygen in plant tissues, Fiziol. Rast. 33, No.2, 403-407. 18. Kao e.H. and Yang S.F., 1982. Light inhibition of the conversion of I-aminocyclopropane-lcarboxylic acid to ethylene in leaves mediated through carbon dioxide, Planta, 155, No.3, 261-264. 19. Rakitin V. Yu and Karyakin V.V.,1984. Gas chromatographic determination of 4-chlorophenoxycetic acid - a stimulator of fruit formation in the tomato. Fiziol. Rast. 31. No.6, 1191-1192. 20. McKeon T.A; and Yang S.F., 1987. Biosynthesis and metabolism of ethylene, in : Plant Hormone and their role in plant growth and development. Davies P.T. and Martinus Nijhoff. Dordrecht, 94-97. 21. Taylor J.E., Grosskopf D.G., McGaw W.A. et al. 1988. Apical localization of 1-aminocyclopropane-l-carboxylic acid and its conversion to ethylene in etiolated pea seedlings. Planta, 174, No.1, 112-114. 22. Reid M.S. Ethylene in plant growth, development and senescence, in : Plant Hormones and their role in plant growth and development. Davies P.T. and Martinus Nijhoff. Dordrecht, 257-259. 23. Palmer J.M., ed., 1984. The physiology and biochemistry of plant respiration. University Press, Cambridge, London. 24. Nikols R., and Ho L.e., 1975. An effect of ethylene on the distribution of C-sucrose from the petals to other flower parts in the senescent cut in inflorescence of Diamantus caryophyllus. Ann. Bot. 39, No. 161,433-444. 25. Kalevich A.E., 1991. Effect ofmonochromate UV on growth and photosynthesims of plant. In : Ecological aspects of growth regulation and plant productivity, Jaroslavl, 10 I-I 02.

ETHYLENE AND VITRIFICATION OF FRAXINUSEXPLANTS IN VITRO

F. LEFORESTIER(I),C. JOSEPfI{l,2) and D. COME.<2) (1) Universite d'Orleans Faculte des Sciences Laboratoire des Ligneux Forestiers B.P.6769 45067 Orleans Cedex 02 France

(2) Universite Pierre et Marie Curie (Paris 6) Laboratoire de Physiologie Vegetale Appliquee

Tour 53, ler etage 4, place lussieu 75252 Paris Cedex 05 France

ABSTRACT. In micropropagation of ash morphology of explants depended of the nature of the gelling agent. With agar they appeared vigourous whereas with Technogel@they were vitreous. In other respects, when transferred onto an: agar solidified medium, the abnormal plantlets formed normal new organs. With this system of culture, considered as a vitrification inducing system, the involvement of ethylene was studied. TechnogelS' enhanced production of ethylene. Moreover, AgNOJ without affecting ethylene release succeeded in a decrease of vitrification. Lastly, CoCQ greatly reduced both ethylene synthesis and vitrification. On agar medium, ACC or ethephon increased ethylene concentration in culture vessels but never induced vitrification. Differences in physiological states induced by using different gelling agents, could lead to differences in sensitivity to ethylene and thus to formation either of normal or vitreous explants.

In micropropagation, a particular process of abnormal growth called vitrification or hyperbydricity sometimes occurs. Morphology, anatomy and physiology of plants are affected. In industrial production such plants, the culture of which must be stopped, are eliminated. Yet, frequency and intensity of the phenomenon are completely random. It can affect any species (Debergh et al., 1981) and can result in losses upper than 60% (Navatel, 1982). Because of the apparently irrational appearance of the process, it is difficult to determine its causes and its regulation. To succeed, it would be advisable to dispose of a model in which induction and also elimination or inhibition of vitrification would be completely controlled. However, it is not very easy to perfect such a system, as shown by BOttcheret al. (1988) who compared the possibility to induce and to reverse vitrification with cultures of nodes of birch, carnation and artichocke. The culture in a liquid medium favoured the induction of vitrification for these different materials without leading systematically to it, whereas the culture on a solid medium did not always avoid the development of the phenomenon. However, we have observed in preliminary experiments performed with isolated ash nodes cultured on media solidified with various compounds that it was possible at pleasure to obtain normal or vitreous explants (Leforestier et aI., 1991). This

353


354

model made it possible to tackle the study of the intervention of ethylene in hyperhydricity. As recalled by Gaspar et aI. (1987), ethylene is effectively considered as one of the essential factors involved in vitrification.

After having briefly recalled some characteristics of vitrification, the experimental model perfected with ash will be described. Then, the results obtained concerning the likely ethylene involvement in vitrification of ash explants will be presented. In conclusion, prospects for further investigations will be proposed.

1. Characteristics of vitrification

A recent and highly complete review published by Ziv (1991) has presented all the morphological and anatomical characteristics of vitreous explants. The purpose here is not to list these data. Only two essential aspects will be recalled. First, vitreous tissues appear generally translucent. Second, leaves of vitreous explants have reduced and rolled limbs.

At a physiological level, vitreous plantlets have a bad rooting and their acclimatization is very difficult, even impossible. This abnormality of growth confers its damageable consequences when micropropagation is used at an industrial level.

Induction of vitrification seems to depend on several non exclusive factors among which the most frequently mentioned in literatur~ are ammonium ion, rigidity of the medium, exogenous cytokinins and ethylene. Concerning the intervention of ethylene, Kevers et al. (1984) showed that vitreous explants produce first much ethylene than normal ones, and then less ethylene. Moreover, Kevers and Gaspar (1985) observed with vitreous carnation an increase in ACC synthesis and a higher ability to convert ACC to ethylene. On the other hand, Phan (1991) suggested recently that ethylene is not an inductive factor of vitrification, and that the early increase in ethylene synthesis observed with vitreous explants must only be considered as a response to exogenous cytokinins.

In order to determine wether ethylene is involved in vitrification, a model for induction or elimination of vitrification has been perfected.

2. Materiel and methods

2.1. MICROPROPAGATION OF ASH

Micropropagation of ash was previously described (Leforestieret aI., 1991). During the stage of rhizogenesis the culture medium was solidified either with agar (6 g. r 1) or with Technogel® (1 g. t l). Technoge1!9 is a gelling agent obtained from Pseudomonas elodea. Its solidifying power is higher than that of agar. On Technoget® solidified rhizogenesis medium, elongation of ash explants was more important and the fonned roots were more numerous than these observed on agar, but vitrification appeared whereas it was never the case with agar. Moreover, the number of vitreous plants depended on the quantity of Technoget® added to the medium. The number of vitreous explants increased with decreasing concentration of TechnogefID. With 1 g of TechnogefID per liter, almost all explants were vitreous. Even after transfer to agar they remained vitreous, however the new differentiated organs became normal. Media solidified with 6 g. r 1 of agar or with 1 g. t I of Technoget® were used for studying the possible involvement of ethylene in vitrification.

355

2.2. MEASUREMENTS OF ETHYLENE RELEASE

Cultures were made in 850 ml jars containing 20 explants cultured on 150 ml of medium. Each jar was closed with a screw-lid on which was fixed a rubber plug which allowed to take a gazeous sample of the atmosphere with the help of a needle linked to a syringe. These samples were taken after 24 h during which the lid was hennetically fixed onto the jar with several thicknesses of ParafilniID.

Measurements of ethylene were made by gas chromatography. The column heated to 60°C contained alumina. Detection was perfonned by flame ionisation at 90°C. To investigate the role of ethylene, silver nitrate (5 and 10 mg. t 1) or cobalt chloride (5, 50 or 100 J.lM) were added to the medium to inhibit respectively effect or synthesis of ethylene. Ethephon (I, 10 or 100 mg. t 1) or ACC (0.1 or 1 J.lM) were also used for increasing ethylene production.

3. Results and discussion

3.1. EFFECT OF THE GELLING AGENT

.c

..,. 1.0 ~ c: ~

0.

~ ~ 0.5 c.

=..,. N

U

7 14

. 1

2 3 4

21 Days

Figure 1. Production of ethylene by ash explants duri~culture on different media 1, Technogel@ ; 2, agar; 3, Technogef.!9 + 5 /lM CoCll ; 4, Technoge~ + 50 J.lM CoCI2 ; 5, Technoge~ + 100 J.lM COC12

As shown in figure 1, ethylene production on agar and TechnogefID solidified media was not significantly different until the 7th day of culture. Then, ethylene emission on TechnogeiID became about two times higher than on agar. After 21 days of culture, 65% of explants were vitreous on TecbnogefID whereas no vitrification occurred on agar (fable I).

These results confinn those previously obtained by Kevers et al. (1984) who showed that vitreous explants release much ethylene than nonnal ones.

356

Table I. Percentages of vitreous explants obtained after 21 days of culture with or without CoCl2

Technogel® + 50 ~M CoCl2 I 45 Technogei® + 1 00 ~M CoCl2 I 20

I I

Table II. Percentages of vitreous explants obtained after 21 days of culture on Technoget® medium containing or not silver nitrate

Concentration of AgN03 I Vitreous explants I (m2.1-1) (%)

0

II 65

I 5 30

10 I 25

3.2. EFFECT OF COBALT CHLORIDE

In the presence of 5 ~M cobalt chloride in Technoget® medium, the rate of ethylene release was inhibited and became quite identical to that measured on agar (Fig. 1). However, vitrification remained high and concerned 65% of the population (Table I). At the concentrations of 50 and 100 ~M cobalt chloride inhibited ethylene production (Fig. 1) and reduced vitrification (Table I). Thus, development of vitrification decreased with increasing cobalt concentration, that is to say with decreasing ethylene synthesis.

Since ethylene release and vitrification paraUely evolved, a causal relationship could be considered between ethylene and vitrification. Nevertheless no vitrification was observed on agar solidified medium though ethylene production was relatively high.

3.3. EFFECT OF SIL VER NITRATE

Ethylene production on Technoge1® solidified medium was not significantly affected by the presence of silver ions (results not shown), which is not surprising. However, silver nitrate induced a decrease in vitrification of explants (Table II).

357

3.4. EFFECfS OF ETHEPHON AND ACC

Addition of 100 mg. t l of ethephon to the medium solidified with agar was toxic, and led to necrosis of plantlets. Lower concentrations (I or 10 mg. t I) enriched considerably the atmosphere in ethylene, but never induced vitrification (results not shown). Similar observations were made when ACe was added to the agar medium.

Thus exogenous ethylene does not seem to be able to induce vitrification in explants cultured on agar. The previous hypothesis concerning ethylene involvement in vitrification on TechnogeiID is therefore somewhat contentious. The suspected relation between ethylene and vitrification must not omit the possible role of the gelling agent Differences in sensitivity to ethylene of explants growing on different solidified media might be an explanation of the results. Indeed, when cultured on TechnogeiID explants would acquired some, physiological state and would produce ethylene to which they would be sensitive, inducing thus vitrification. When cultured on agar, explants, the physiological state of which would be different, would also produce ethylene but they would be insensitive or less sensitive to it So, in this case, ethylene would not lead to vitrification.

Conclusion

Results here presented allow us to propose a practical solution to the problem of vitrification of ash explants by using agar to solidify the medium. However, at a basis level, they do not give information on the mechanism of vitrification regulation.

Vitrification is a multifactorial phenomenon which does not appear as an inescapable consequence to an ethylene production, and in which the role played by ethylene remains still unknown.

The physiological state of the explants according to the gelling agent of a medium could determine the level of sensitivity to ethylene. In fact, according to the solidifying compound used, elements could be differently absorbed from the medium, confering thus a particular physiology to the explants. In order to verify this hypothesis it would be advisable to inveStigate the absotption of ammonium ions and cytokinins which, according to Daguin and Letouze (1986) and Phan (1991), might be involved in vitrification.

References

Bottcher, I., Zoglauer, K. and GOring, H. (1988) 1nduction and reversion of vitrification of plant cultured in vitrd, Physiol. Plant. 72, 560-564.

Daguin, F. and Letouze, R. (1986) 'Ammonium-induced vitrification in cultured tissues', Physiol. Plant. 66, 94-98.

Debergh, P., Harboui, Y. and Lemeur, R. (1981) 'Mass propagation of globe artichoke (Cynara sco/ymus) : evaluation of different hypothesis to overcome vitrification with special reference to water potential', Physiol. Plant. 53,181-187.

Gaspar, T., Kevers, C., Debergh, P., Maene, L., Paques, M. and Boxus, P. (1987) 'Vitrification: motphological, physiological and ecological aspects', in 1.M. Bonga and OJ.

Durzan (eds.), Cell and tissue culture in forestry, vol. 1, Martinus Nijhoff Publishers,

358

Dordrecht, pp. 152-166. Kevers, C., Coumans, M., Coumans-Gilles, M.F. and Gaspar, T. (1984) 'Morphological and

biochemical events leading to vitrification of plants cultured in vitro', Physio!. Plant. 61, 69-74.

Kevers, C. and Gaspar, T. (1985) 'Vitrification of carnation in vitro: changes in ethylene production, ACC level and capacity to convert ACC to ethylene', Plant Cell Tissue Organ Culture 4, 215-223.

Leforestier, F., Gras-Courtois, M. and Joseph, C. (1991) 'Etude comparative de la micropropagation de quelques especes de Fraxinus: Acta Horticulturae 289, 125.

Navatel, J.C. (1982) 'Problemes lies a la production de porte-greffes d'arbres fruitiers par multiplication in vitrd, Fruits 37, 331-336.

Phan, C.T. (1991) 'Vitreous state in vitro culture : ethylene versus cytokinin', Plant Cell Reports 9, 517-519.

Ziv, M. (1991) 'Vitrification: morphological and physiological disorders of in vitro plants', in P.C. Debergh and R.H. Zimmerman (eds.), Micropropagation technology and application, Kluwer Academic Publishers, Dordrecht, pp. 45-69.1

STIMULATION OF SOMATIC EMBRYOGENESIS IN CARROT BY ETHYLENE

P. NISSEN NL VF Cell & Tissue Culture Group Agricultural University of Norway P.O. Box 5014, N-1432 As Norway

ABSTRACT. In apparent contrast to findings of other workers, endogenous ethylene levels were suboptimal for somatic embryogenesis in a suspension culture of carrot. Low concentrations (~0.5 mg 1-1 ) of ethephon caused statistically significant increases in ethylene (~3 fold) and embryo formation (~50%) whereas higher concentrations increased ethylene still further and inhibited embryogenesis. 1-Aminocyclopropane-1-carboxylic acid (ACC) in most experiments stimulated embryo and plantlet formation (up to 4 fold) but was inhibitory above 50 /-LM. The hypothesis that 2,4-dichlorophenoxyacetic acid (2,4-D) and other auxins inhibit embryogenesis by stimulating the production of ethylene cannot hold in systems that are suboptimal with respect to ethylene. Consistent with this, difluoromethylornithine (DFMO), which counteracts the inhibition by 2,4-D of somatic embryogenesis in carrot, did not counteract the inhibition by high concentrations of ethephon or ACC.

INTRODUCTION

The development of somatic embryos from embryogenic cell clumps (proembryogenic masses) in carrot is inhibited by auxins (Michalczuk et al. 1992 and references therein). This development has also been found to be inhibited by ethephon/ethylene (Wochok and Wetherell 1971, Tisserat and Murashige 1977, Roustan et al. 1989a, b, 1990) but it was stimulated by 10 /-LM ACC (Verma and Tarka 1985). It has been proposed (Wochok and Wetherell 1971 J

Robie and Minocha 1989) that 2,4-D inhibits embryo development by inducing the production of ethylene. This and other hypotheses for the mechanism of action of 2,4-D in inhibiting embryogenesis have been tested (Nissen and Minocha 1991) by use of the finding (Robie and Minocha 1989) that DFMO, an irreversible inhibitor of ornithine decarboxylase, counteracts the inhibition by 2,4-D. (If 2,4-D inhibits by stimulating the production of a compound X, DFMO should counteract the inhibition caused by the addition of X in the absence of 2,4-D.)

As part of this study, the effects of a wide range of concentrations of ethephon and ACC on somatic embryogenesis and relative ethylene levels in a suspension culture of carrot have been determined, in part in the presence of DFMO.

359


360

MA TERIAL AND METHODS

A cell culture of domesticated carrot (Daucus carota L.) was maintained in suspension in B5 medium, pH 5.8, with 2% sucrose and 0.1 mg r1 2 ~ 4-D on a shaker at 150 rpm under a 16 h photoperiod of approx. 90 /AE S-l m- at 24°C.

To initiate embryogenesis, sieved cell clumps (100-250 /Am) were rinsed with B5 medium and transferred to experimental solutions to give 5 ml of suspensions (50-250 clumps ml- 1) in 50 mm Petri dishes which were sealed with Nescofilm and put on a shaker at 100 rpm at the above light and temperature conditions. All compounds used in experimental treatments were added to the medium as filter-sterilized solutions adjusted to pH 5.8.

Embryos at or past the torpedo stage O~0.5 mm) were counted. Fresh weights were determined by weighing cells, embryos and/ or plantlets scraped off Miracloth on which they were collected by vacuum filtration.

Ethylene in the head space of the Petri dishes was determined as described by Biddington and Robinson (1991). At specified times, a 1.0-ml sample of air was taken through a small hole made in the side of the base and the lid and injected into a gas chromatograph fitted with a flame ionization detector. The hole and the gap between the lid and the base were sealed with 4 layers of Nescofilm and were resealed with 2 more layers after sampling.

RESULTS

Data of Roustan et al.

Salicylic acid and acetylsalicylic acid (Roustan et al. 1989a) and C02+ and Ni2+

(Roustan et al. 1989b) stimulated somatic embryogenesis in carrot, presumably through inhibition of ethylene production. Embryogenesis decreased when the levels of ethylene were increased by the addition of ethephon (Roustan et al. 1989b, 1990). Figure 1 shows that the different treatments yielded consistent results and that ethylene was supraoptimal for embryogenesis at all levels. Embryos were still formed, however, at high ethylene concentrations (50% of control at a 150-fold increase; 20% at a 1000-fold increase).

° Figure 1. Relationship

A Roustan et al. 1989a between ethylene produc-g 300 o Roustan et al. 1989b tion and somatic embryo-c: o Roustan et al. 1990

genesis by a cell culture 0 ()

0 of carrot. Data: Roustan '0 0 et al. 1989a (acetylsali-200 0 il-!! "R cylic acid), Roustan et al. vi 1989b (COC1, NiCI2 , ethe-0 .c >- 0 phon) and oustan et al. ..c 100 a E D 1990 (ethephon). ill

ell

<S> 0

10 100 1000 10000 100000

Ethylene production, % of control

361

Effects of Ethephon

At 0.2 mg 1-1 , ethephon gave a consistent but nonsignificant stimulation of embryogenesis in the experiment in Figure 2. DFMO stimulated embryogenesis but did not counteract the inhibition by high ethephon concentrations.

In the experiment in Figure 3B, ethephon stimulated embrrogenesis by about 50% after 15 days at the optimal concentration of 0.5 mg 1-. Thissti-

en 0 ~ .0 E w

en o

100

80

60

40

20

0

15

10

5

/ f/ +DFMO

0 0.2 0.5 1 2

Ethephon, mg 1 -1

A 11 days

5

0.8

0.6

0.4 E

0.2 §:

~ 0 1-'-----,,/-'----'----'----'-----"-1 0 Q) t: Q) E

w

100 2

50

0~----,'/-~-~--L---L--1~.~0 0 o 0.05 0.1 0.2 0.5

Ethephon, mg 1-1

>,5 w

Figure 2. Effect of ethephon on somatic embryogenesis in carrot after 12 days in the absence and presence of 5 mM DFMO. Means ± SE (n = 4).

Figure 3. Effect of ethephon on ethylene levels and somatic embryogenesis in carrot after 11 and 15 days. Ethylene determined in head space of Petri dishes (cf. Materials and Methods). Means ± SE (n = 8). Significance of difference from control: * - 5% level, ** - 1% level, *** - 0.1% level.

362

mulation was statistically highly significant, as also evident by the gradual increase in stimulation with increasing ethephon concentration. The stimulation by ethephon was presumably caused by the increase in ethylene (3-fold at the optimal concentration of ethephon). The inhibition above 0.5 mg rl ethephon was probably caused by the ethylene concentration becoming too high. Similar but more erratic results were obtained after 11 days (Figure 3A). The dip in the ethylene curve for 0.2 mg rl ethephon was probably accidental, but the corresponding dip in number of embryos is a further indication that embryogenesis is indeed stimulated by ethylene in this system.

Effects of ACC

ACC in the range 5-50 JlM stimulated embryogenesis up to 4 fold in the experiment in Figure 4 and was completely inhibitory at 200 JlM. DFMO stimulated embryogenesis but did not counteract the inhibition by high ACC concentrations, in agreement with the finding for ethephon (Figure 2). Plantlet formation, as measured in this case by fresh weight after 10 weeks, was similarly stimulated by ACC but the optimum was shifted to somewhat higher concentrations (Figure 4) .

900

800 OJ E 700

~ 600 LL

500

4OO?O a

60

50

(J) 40 0 ~ 30 .D E ill 20

10

a

r: fr'

/

f

a "" 5

Figure 4. Effect of ACC on somatic embryogenesis in carrot in the absence and presence of 5 mM DFMO. Embryos counted after 15 days, fresh weights determined after 70 days. Means ± SE (n = 4).

In the less detailed experiment in Figure 5, embryogenesis was stimulated by 10-20 JlM ACC, both as measured by number of embryos and by plantlet mass. There was a slight, nonsignificant increase in ethylene at the stimulatory ACC concentrations and a steeper increase as ACC started to become inhibitory.

363

2

E 80 800 0. Figure 5. Effect of ACC 0. gs 60 600 Ol ethylene levels and ai E on c ~

~ somatic embryogenesis in Q) .D. >. E 40 400 u. carrot culture. Embryos .s:::. w W determined after 11 days,

20 200 ethylene after 12 days and 9-- fresh weights after 47

0 0 0 days. Means ± SE (n = 8 0 10 20 50 for control, n = 4 for

ACC, 11M treatments) .

DISCUSSION

The finding that ethylene can stimulate somatic embryogenesis in carrot appears to be new and is in marked contrast to the finding that ethylene is supraoptimal at all levels (Roustan et al. 1989a,b, 1990; cf. Figure 1). Ethylene appears in most cases to inhibit somatic embryogenesis (Biddington 1992). An exception is embryogenesis in citrus ovular callus which was increased almost 9 fold by 0.1 mg rl ethephon (Kochba et al. 1978).

In the present experiments, ACC has for the most part been somewhat more stimulatory that ethephon (Figure 4 vs. Figure 3). Both compounds have occasionally failed to stimulate embryogenesis, presumably because of uncontrolled differences in the carrot culture or experimental conditions. Whether the stimulation by ACC is due to ethylene remains to be determined.

The effects of ethylene may apparently vary greatly even within a species (and with experimental conditions), as also found for anther culture where its effects on microspore embryogenesis may depend on the cultivar (Biddington 1992) .

The finding that ethylene may be suboptimal for somatic embryogenesis in carrot seems to rule out the possibility that 2,4-D and other auxins inhibit embryo development by stimulating the production of ethylene. (Such stimulation should enhance rather than inhibit somatic embryogenesis.) The failure of DFMO (which counteracts the inhibition by 2,4-D) to counteract the inhibition by high concentrations of ethephon (Figure 2) constitutes further evidence against this hypothesis.

The role, if any, of ACC in the inhibition of embryogenesis by 2,4-D remains unclear. In the present experiments, however, the results for ACC parallel those for ethephon, i.e. stimulation of embryogenesis by low concentrations and no reversal by DFMO of the inhibition by high concentrations (Figure 4).

364

ACKNOWLEDGEMENTS. I thank Anne Guri MarlllY for able assistance. Dr. T . J. Cooke kindly provided the embryogenic carrot culture and the Marion Merrell Dow Research Institute, Strasbourg, France provided a generous supply of DFMO. This investigation was supported by the Agricultural Research Council of Norway.

REFERENCES

Biddington, N .L. (1992) 'The influence of ethylene in plant tissue culture', Plant Growth Regul. 11, 173-187.

Biddington, N .L. and Robinson, H. T. (1991) 'Ethylene production during anther culture of Brussels sprouts (Brassica oleracea var gemmifera) and its relationship with factors that affect embryo production', Plant Cell Tiss. Org. Cult. 25, 169-177.

Kochba, J., Spiegel-Roy, P., Neumann, H. and Saad, S. (1978) 'Stimulation of embryogenesis in citrus ovular callus by ABA, ethephon, CCC and Alar and its suppression by GA " z. Pflanzenphysiol. 89, 427-432.

Michalczuk, L., Cooke, T.J. and Cohen, J.D. (1992) 'Auxin levels at different stages of carrot somatic embryogenesis', Phytochemistry 31, 1097-1103.

Nissen, P. and Minocha, S.C. (1991) 'Inhibition by 2,4-D of somatic embryogenesis in carrot as explored by its reversal by DFMO', Physiol. Plant. 82, B6.

Robie, C.A. and Minocha, S.C. (1989) 'Polyamines and somatic embryogenesis in carrot. I. The effects of difluoromethylornithine and difluoro:-methylarginine', Plant Sci. 65, 45-54. .

Roustan, J.-P., Latche, A. and Fallot, J. (1989a) 'Effet de l'acide salicylique et de l'acide acetylsalicylique sur la production d'ethylE~ne et l'embryogenese somatique de suspensions cellulaires de carotte (Daucus carota L. )'. C. R. Acad. Sci. Paris 308, 395-399.

Roustan, J.-P., Latche, A. and Fallot, J. (1989b) 'Stimulation of Daucus carota somatic embryogenesis by inhibitors of ethylene synthesis: cobalt and nickel', Plant Cell Rep. 8, 182-185.

Roustan, J.-P., Latche, A. and Fallot, J. (1990) 'Control of carrot somatic embryogenesis by AgN03 , an inhibitor of ethylene action: effect on arginine decarboxylase activity', Plant Sci. 67, 89-95.

Tisserat, B. and Murashige, T. (1977) 'Effects of ethephon, ethylene, and 2,4-dichlorophenoxyacetic acid on asexual embryogenesis in vitro', Plant Physiol. 60, 437-439.

Verma, D.C. and Tarka, T. (1985) 'Influence of 1-aminocyclopropane carboxylic acid (ACC) and consequent ethylene biosynthesis on growth and somatic embryogenesis in wild carrot (Daucus carota L.) cell sus;" pensions', in M. Terzi, L. Pitto and Z. R. Sung (eds.), Somatic Embryogenesis of Carrots, IPRA, Roma, pp. 152-158.

Wochok, Z.S. and Wetherell, D.F. (1971) 'Suppression of organized growth in cultured wild carrot tissue by 2-chloroethylphosphonic acid', Plant Cell Physiol. 12, 771-774.

RELATIONSHIP BETWEEN ETHYLENE AND POLYAMINE SYNmESIS IN PLANT REGENERATION.

J.-P. ROUSTAN, K.M. CHRAffiI ., A. LATCHE and J.FALLOT Laboratoire de Biotechnologie-Amelioration des PI antes, ENSAT 145, Avenue de Muret. 31076 Toulouse Cedex, France.

We have previously shown that ethylene inhibits carrot somatic embryogenesis and sunflower organogenesis [1,2,3]. The role of ethylene in plant regeneration is still not clear. However, it has been suggested that polyamines play an important role in the process of somatic embryogenesis [4,5]. Also, in this study, the relationship between ethylene production, polyamine biosynthesis and plant regeneration was investigated in carrot cells and sunflower tissues.

Carrot cells was maintained in liquid Gamborg's B5 medium with 4.5 /lM 2,4-D (embryo induction phase). Embryogenesis expression was obtained by transfer of the cells to hormone-free medium. Aliquots of3-day-old embryogenic cells were incubated with 3,4-[l4C]methionine for 20 h. Sunflower cotyledon explants were cultured in liquid Murashige and Skoog's modified medium for 3 days then transferred on the same medium solidified by agar. Two-day-old explants were incubated for 16 h in the presence of [U-14C]arginine.

When carrot embryogenic cells, cultured in the presence of Ethephon, were incubated with 3,4-[l4C] methionine, the flux of radioactivity into speimidine and speimine was greatly inhibited (Table I). Similarly, the incorporation of radioactivity from [14C] arginine into putrescine, speimidine and spermine was highly reduced in sunflower cotyledons cultured in the presence of Ethephon (Table 2). The inhibition of polyamine formation by ethylene can be attributed at the reduction of ADC and SAMDC activities caused by this hormone in cultured tissues (Tables 1,2). Furthermore, the reduction of polyamine synthesis caused by Ethephon was followed by a strong reduction of carrot somatic embryogenesis and a complet inhibition of shoot organogenesis in sunflower tissues. In carrot cells, inhibition of ethylene production caused an enhancement oflabel from 3,4-[14C] methionine into speimidine and spermine, a stimulation of ADC and SAMDC activities, and an increase of the number of somatic embryos formed (Table 1). In sunflower, the addition of putrescine, spermidine or spermine at 0.1 mM to cotyledon explants treated by Ethephon allowed to restore the organogenesis potential, while the shoot formation was totaly inhibited in the presence ofEthephon alone (Table 3).

The results of the present study indicate that there is a close physiological link between ethylene production, polyamine biosynthesis and plant regeneration. It has

365

J. C. Pech et at. (eds.), Cellular and Molecular Aspects of the Plant Honnone Ethylene, 365-366. © 1993 Kluwer Academic Publishers.

366

been clearly demonstrated that ethylene inhibited the formation of polyamines in carrot cells and sunflower cotyledon explants, likely by reducing the activities of key regulatory enzymes for polyamine pathway. Furthermore, inhibition of polyamine synthesis is followed by a decrease of plant regeneration; addition of polyamines can restore organogenesis potential in sunflower. These observations seem to demonstrate that the decrease of polyamine synthesis caused by ethylene could give rise to the reduction of carrot somatic embryogenesis or sunflower organogenesis. However, the question remains as to whether or not ethylene inhibits plant regeneration only by affecting polyalI'ine metabolism.

Table 1. Effect of Ethephon and CoCl2 on the incorporation of3,4-[14C] methionine into spermidine and spermine in carrot cells, on the activities of arginine decarboxylase (me) and S-adenosylmethionine decarboxylase (SAMDC), and on the number of carrot somatic embryos formed.

Treatment [l4qSpermidine [l4qSpermine ADC SAMDC Embryos {KBq g-IFff; (pKat mg-1 protein) (N ml-I )

Control 0.39±O.05 (100) O. 76±O.11 (100) O.l1±O.06 0.20±0.01 25±3 (100) Ethephon 70 JIM 0.29±O.04 (73) 0.31±0.04 (42) 0.07±O.01 O.II±O.OI I0±2 (40) COC12 50 JIM 0.47±O.07 (120) 1.10±0.I4 (145) 0.19±O.02 0.30±0.02 58±5 (232) CoCl2 50 JIM + Ethephon 70 11M 0.28±O.03 (71) 0.33±O.07 (43) 0.07±O.OI 0.12±O.02 12±2 (48)

Table 2. Effect of Ethephon on the incorpordtion of [14C] arginine into putrescine, spermidine and spermine in swrllower cotyledons, and on the activity of arginine decarboxl'lase (ADe).

Treatment

Control

[l4q Putrescine [14C] Spermidine [l4q Spermine (K8qg-l FW)

Ethephon (35 JIM) 939 ± 192 107 ± 11

40±5 13 ±2

33 ±4 16±2

ADC activity (pKat mg-lprotein)

0.42 ± 0.03 0.02 ±0.01

Table 3. Effects of Ethephon and polyantines on shoot regeneration from swrllower cotyledon explants lines F817 and F827.

Treatment

Control Ethephon (35 JI.M) Ethephon (35 JI.M) + Putrescine (0.1 roM) Ethephon (35 1lM) + Spennidine (0.1 mM) Ethephon (35 JI.M) + Spermine (0.1 roM)

F8I7

76.2 o

69.5 92.4 64.7

Organogenesis (%) F827

71.5 o

66.5 73.3 69.5

1 Roustan J.P., Latche A. and Fallot J. (1989) Plant Cell Rep. 8, 182-185. 2 Roustan J.P., Latche A and Fallot J. (1990) Plant Sci. 67, 89-95. 3 Chraibi B.M.K., Latche A, Roustan J.P. and Fallot J. (1990) Rep. 10,204-207. 4 Fienberg AA, Choi J.R., Lubich W.P. and Sung Z.R. (1984) Planta 162, 532-539. 5 Robie C.A. and Minocha S.C. (1989) Plant Sci. 65, 45-54.

ETHYLENE INHmITS THE MORPHOGENESIS OF VITIS VINIFERA CUTTINGS CULTURED IN VITRO.

O. SOULIE, J.-P. ROUST AN and J. F ALLOT Laboratoire de Biotechllologie-Amelioratioll des pi antes, ENSAT 145, Avenue de Muret, 31076 Toulouse Cedex, France.

The gazeous environment and especially ethylene accumulation in air-tight containers where tissues or plants are grown has been shown to affect growth and development of plants [1] and the regeneration from tissues or cells: maize [2], carrot [3] and sunflower [4]. In grapevine in vitro plants, the C02 content of atmosphere was studied [5], but ethylene production and effects of this hormone on the micropropagation have not be elucidated. In this study, the relationship between ethylene production, ethylene effect and opening of axillary buds and root formation was investigated in Vitis vinifera ill vitro plants.

Node-segments of Vitis vinifera cultivar Merlot are cultured on hormone-free halfstrength Murashige and Skoog's (MS) medium in 25 ml flasks, at 25°C, under a light flux of 40 J.lE.s-l.m-2 with a 16 h photoperiod. Each flask contains five cuttings. Aqueous solutions of Ethephon and AgN03 are sterilized by filtration and added to autoclaved medium at required concentrations. Ethylene measurements are performed on cuttings in flasks with cellulose caps. Each two or three days, a 1 ml gas sample is withdrawn with a sterile hypodermic syringe and the ethylene content determined by gas chromatography. The microcutting development is determined at the end of the culture by the weight of leaves and roots excised from 10 plants.

Evolution of ethylene accumulation in flask is characterized by a strong increase at 10th day (beginning of root formation) and an other enhancement less important at 17th day (beginning of bud opening) (fig. 1 ). Addition of Ethephon caused an increase of ethylene concentration which is followed by an inhibition of opening buds (fig.2) and a reduction of weight of leaves and roots (fig.3). In these cultures, the percentages of morphogenesis decreased with the increase in ethylene concentration. When silver nitrate is added at cuttings treated by Ethephon 10J.lM, the percentage of axillary buds opened is about 90 % while it was 60 % when the plantlets were cultured in the presence of Ethephon alone (fig.4). A similar profile is obtained for weight of roots.

These data demonstrate that the increase of ethylene concentration in the culture vessel by the addition of Ethephon, caused an inhibition of shoot and root formation. Furthermore, the inhibitor effect of ethylene can be reversed by an inhibitor of ethylene action, silver nitrate. Also, these observations suggest that development of cuttings of Vitis vinifera cultured in vitro are clearly affected by ethylene.

367

J. C. Pech et al. (etis.), Cellular and Molecular Aspects of the Plant Honnone Ethylene, 367-368. © 1993 Kluwer Academic Publishers.

368

198 .. .. ... ~ ..!. , l B8

" 15e !: !: 0 7e .2 to ., j 68 .. 0

I ~ J 5e 198 = "

~ 19 '" ~

= ~ 38

! " '!! ze ~ " 18 ~ .. ...

8 8 18 ZS 58 198 Etloephon concentration (~1 . 1-1l

m)( of uillary .... ds .. Eth>,/Iene cone .

Figure 1 : Ethylene production by cuttings of Vilis vinifera cv Merlot.

Figure 2: Effect ofEthephon on ethylene concentration and the opening of axillary buds of cuttings of Vilis vinifera cv Merlot.

8.5 .18

~ .08

~ .06

:.c ... j 8. .91

.c ~

~ .82 ... .08

Et/oephon concentntlon (~1.1- 1I

.. Le4ues (nI) .. Le4ues (l1li)

+ Roots (nI) ... Roots (DII)

Figure 3 : Effect of Ethephon on weight of leaves and roots produced by cuttings of Vitis vinifera cv Merlot.

~ ~ .. '" .. j

l

I 0

1 r ~ '!!

"

ConIrol Ethopboo AgN03 10 I'M + 10f1M Etbepbon 10 f1M

Em )( lOX IIlary...... • Roots (g)

Figure 4 : Effect ofEthephon and AgN030n the opening of axillary buds and on weight of roots of cuttings of Vilis vin~fera cv Merlot.

[I] Hussey G, and Stacey N.J. , (1981), Ann. Bot., 48, 787-796. [2] Vain p" Flament P. and Soudain P., (1989), I. Plant Physiol., 135, 537-540. [3] Roustan I.-P. , Latche A. and Fallot J., (1992), Plant Physiol. Biochem., 30(2), 201-205 [4] Chraibi B.MK, Latche A., Roustan I.-P. and Fallot I., Plant Cell Rep., (1991), 10, 204-207. [5] Fournioux I.e. and Bessis R., (1986), Can., J. Bot., 64, 2608-2616.

ENHANCED ETHYLENE PRODUCTION BY PRIMARY ROOTS OF ZEA MAYS L. IN RESPONSE TO SUB-AMBIENT PARTIAL PRESSURES OF OXYGEN.

R. BRAILSFORD, L.A.C.J. VOESENEK\ C.W.P.M. BLOM\ A.R. SMITH2, M.A. HALL2 AND M.B. JACKSON. Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol, BS189AF, UK. lDepartment of Ecology, University of Nijmegen, Toemooiveld, 6525 ED Nijmegen, The Netherlands. 2Department of Botany and Microbiology, University College of Wales Aberystwyth, Penglais, Aberystwyth, Dyfed, SY21 3DA, UK.

Flooding or soil waterlogging inhibits gaseous exchange between the soil and atmosphere. Flooded soils rapidly become oxygen deficient, whilst the products of plant and microbial respiration such as CO2 accumulate. Plants require metabolic and/or morphological adaptions to survive such conditions. Many of the latter are mediated by plant hormones, most notably ethylene. There is evidence of a causal relationship between the rate of ethylene biosynthesis, oxygen supply and physiological adaptions such as aerenchyma development. When maize roots are exposed to hypoxia or to exogenous ethylene, aerenchyma develops in the cortex. Jackson et ai. (1982; 1985) reported enhanced ethylene production in nodal roots of maize and barley under low oxygen partial pressures (3 - 12.5 kPa), whilst a similar response was observed in the stems of deep water rice by Metraux and Kende (1983). This enhancement of biosynthesis is controversial, since ethylene biosynthesis requires molecular oxygen. The techniques used by Jackson and co-workers and Metraux and Kende (Le. head space analysis of excised tissue enclosed in small incubation vials) may have resulted in artifacts, e.g. as a result of woundethylene production after excision. To overcome this, we employed a sensitive laser-driven photoacoustic technique (sensitivity limit: 0.041 pmol m 0

3) to measure ethylene production from individual roots of intact seedlings, thus minimising physical perturbations.

Single primary roots (20 - 25 mm long) of intact, three day-old maize seedlings (Zea mays L. cv LGll) were sealed into glass cuvettes with plaster of Paris. The root was isolated in the darkened lower chamber of the cuvette, through which a humidified gas stream passed (flow rate: 1 x 1003 m3 hoi). Roots were exposed to 6 h of air before treatment with 21 kPa (control), 12.5 kPa, 5 kPa, 3 kPa, 1 kPa or 0 kPa Oz (pure nitrogen). After 16 h, the gas was switched back to air for a further 6 h to monitor post-stress production. Before gas from the cuvettes entered the photoacoustic detector, it was scrubbed of CO2, water and ethanol. All experiments took place at 22°C, with continuous light (200 /Lmol m03 sol). Root length and morphology were recorded at the start, finish and before each gas change. Estimated fresh weight based on root

369


370

length was used during the experiment to express ethylene production as nl g.fw- I h-I •

Experiments were replicated 2 - 5 times. Representative data are presented.

Primary root extension was inhibited by partial pressures of oxygen (P0J below 21 kPa compared to aerobic controls; the level of inhibition increasing with decreasing p~. Almost no growth was observed in the absence of 02' Post-treatment root extension rates were similar to those during treatment, except after 16 h of 5 kPa O2 where extension recovered rapidly to approach that of air. Roots exposed to 3 or 5 kPa ~ were thickened, plagiotropic and possessed dense root hairs whilst, those treated with 12.5 and 1 kPa O2 resembled those of air-grown controls. The morphology of tissue produced after returning to air was similar to air-grown roots in all cases except 0 kPa 02' Returning roots to air after anoxia caused death of the meristem. There were clear trends in ethylene production (nl g.fw-1h-l) in response to different partial pressures of oxygen. Ethylene production during the preliminary 6 h acclimatization in air declined as the root recovered from the mild stress imposed during the transfer of plants. In aerobic controls and during and after treatment with 12.5 kPa °2, production continued to decline at a rate of approximately 0.15 nl h-I • In contrast, 5 kPa, 3 kPa and 1 kPa O2 caused ethylene production to increase. The most marked stimulation occurred in 3 kPa 02' Here the rate increased within the first hour oftreatment and remained at a high level (8 - 10 nl g.lh-l) for 16 h. Production rates returned rapidly to those of air controls when air was returned. Anoxia completely eliminated ethylene synthesis during and after treatment.

These results confirm that a stimulation of ethylene production occurs in roots of intact maize seedlings in response to hypoxia. Little is known about the mechanisms behind this stimulation or the effect of hypoxia on maize root ethylene forming enzyme. We are currently investigation whether l-aminocyclopropane-l-carboxylic acid is produced in an anoxic root core and diffuses to better oxygenated cortical regions where it is converted.

Jackson, M.B. (1982). 'Ethylene as a growth promoting hormone under flooded conditions.' In P.F. Wareing (ed.) Plant Growth Substances 1982, Academic Press, London, pp. 291-301.

Jackson, M.B., Fenning, T.M., Drew, M.e., and Saker, L.R. (1985). 'Stimulation of ethylene production and gas-space (aerenchyma) formation in adventitious roots ofZea mays

L. by small partial pressures of oxygen.' Planta 165, 486-492. Metraux, J.-P. and Kende, H. (1983). 'The role of ethylene in the growth response of

submerged deep water rice.' Plant Physiology 72, 441-446.

EIHYLENE AND PIKEPHORYLATION OF PEA EPIOOlYL PROTEINS

G.V.NOVIKOVA, I.E.MOSHKOV, A.R.SMITH* and M.A.HALL* Institute of Plant Physiology Acad.Sci.Russia, 35 Botanichestaya str., Moscow 127276, Russia * - Dept. of Biological Sciences, University College of Wales, AberystwYth, Dyfed SY23 3DA, U.K ..

The way that signals in the form of hormones, neurotransmitters and growth factors are translated into a diverse array of cellular function is a central question in cellular biology. There is convincing evidence demonstrating several molecular pathways through which individual regulatory agents produce specific biological responses frequently converge at the stage of protein phosphorylation. Phosphorylated proteins and protein kinases have been detected in all compartments of plant cells. Phosphorylation has been shown to be involved in the action of auxin, gibberellins [1], cytokinin and abscisic acid [2]. These data stimulated the investigation of a role of protein phosphorylation in res'ponse of plant cell to ethylene.

For investigation of this problem 5-day-old etiolated pea seedlings (Pisum sativum L., cv.Alaska) were used. Applied ethylene had a halfmaximum effect on 'triple response' at 0.1 ulll and maximum was reached between 1 and 10 ul/l. For monitoring of protein phosphorylation in vivo epicotyl tips (1.5 cm length) were incubated in a buffer in a dark for 1 or 20 hs at 230 C in the presence of 10 ulll of ethylene. 1 mCi [32-P]H3P04 was added for the last 30 min of the incubation. Membrane enriched fraction was isolated, and proteins were analyzed by SDS-PAGE. Protein phosphorylation in vitro was performed in a mixture (50 ul) containing 50 mM Tris-HCl, pH 7.3; 15 mM MgC12; 0.1% Triton X-100; 10 mM NaF; 20 nM ATP; 10-20 ug of protein (postmitochondrial or membrane fraction from ethylene treated tips) and 1 uCi [ -32-P]ATP. SBDlples were incubated for 10 min at 30°C and subjected to electrophoresis with SDS.

The [32-P] incorporation in vitro was found to be enhanced with in creasing of ethylene concentration reaching a maximum between 1 and 10 ul/l. In vivo labelling experiments showed that the phosphorylation level of membrane polypeptides was increased 6 times after 1 h and 3 times after 20 hs in comparison with uritreated tips. Thus, ethylene affected protein phosphorylation in vivo and in vitro. To demonstrate the specificity of ethylene effect 2,5-norbornadiene - the antagonist of ethylene action (3)- was used. As reported earlier [4}, 2,5-NBD at the concentration of 2,000 ulll inhibited ethylene binding by 80%. Our experiments have shown 2,5-NBD at this concentration did not change growth parameters of seedlings and did not affect in vitro phospho-

371


372

rylation of proteins. When they were applied together (2,000 ul/l 2,5-NED and 1 ul/l ethylene) elimination of ethylene effect including ethylene provoked protein phosphorylation was found. It should be noted that ethylene caused significant increasing of phosphorylation level of 15 kD polypeptide. The polypeptide corresponds electrophoretically to ethylene binding protein [5].

In order to study an influence of phosphorylation on ethylene binding activity, membrane enriched fraction was (1) defosphorylated in vitro with phosphatase, or (2) appropriate conditions for phosphorylation were created (AlP, inhibitors of phosphatases, temperature).

Effect of phosphorylation on in vitro ethylene binding with membrane enriched fraction from pea tips

Treatment

None Phosphatase NaF NaF + AlP

Specifically bound ethylene (pmol/g FW) ± S.E.

0.118 ± 0.012 0.239 ± 0.003 0.070.± 0.011 0.067 .± 0.010

The data of the table indicate that if membrane enriched fraction was preincubated in vitro with phosphatase and then ethylene binding assay was conducted the specific binding increased. The presence of NaF in the medium was accompanied by quite afficient fall of ethylene binding. If NaF was added with AlP to membrane fraction ethylene binding was decreased as well. Thus, dephosphorylation appears to be necessary for ethylene binding.

This report represents the first demonstration that some ethylene effects may be transduced via protein kinase(s). In particular, dephosphorylation of isolated membrane proteins promotes and phosphorylation diminishes ethylene binding with such preparation.

References 1. Ranjeva, R. and Boudet, A.M. (1987) 'Phosphorylation of proteins in

plants: Regulatopy effects and potential involvement in stimulus/ response coupling', Ann.Rev. Plant Physiol. 38, 73-93.

2. Kulaeva, O.N. (1988) 'The possible role of protein kinases in the plant cell response to phytohormones', in R.Pharis (ed.), Plant Growth Substances 1988, Springer-Verlag, Berlin, pp. 547-551.

3. Sisler, E.C. and Yang, S.F. (1984) 'Anti-ethylene effect of cis-2-butene and cyclic olefins', Phytochemistry 12, 2765-2768.

4. Sanders, 1.0., Ishizawa, K., Smith, A.R. and Hall, M.A. (1990) 'Ethylene binding and action in rice seedlings', Plant Cell Physiol. 31, 1091-1099.

5. Hall, M.A., Connern, C.P.K., Harpham, N.V.J. et al. (1990) 'EtQvlene: receptors and action', in J.Roberts, C.Kirk and M.Venis (eds.), Hormone perception and signal transduction in animals and plants, Cambridge, pp. 87-110.

KNOWLEDGE OF XYLEM SAP FWW RATE IS A PRE-REQUISITE FOR ACCURATE ESTIMATES OF HORMONE TRANSPORT FROM ROOTS TO SHOOTS.

M. A. ELSE, W. J. DAVIESl , K. C. HALL AND M. B. JACKSON. Department of Agricultural Sciences, University of Bristol, Long Ashton Research Station, Bristol BSl8 9AF, UK. IDivision of Biological Sciences, University of Lancaster, Lancaster, LAl 4YQ, UK.

Changes in the concentration or the amount of a hormone or precursor entering the shoots from the roots in the transpiration stream may modify shoot development (e.g. promote leaf epinasty and close stomata) when roots are stressed. A pre-requisite for testing the hypothesis is to estimate correctly the concentration and the delivery rates of hormone in the transpiration stream of whole plants. Measurements are usually made in sap bleeding from the root systems of detopped plants. However, these measurements could suffer from potentially confounding effects of the large (approx. 90%) decrease in sap flow which occurs when shoots are removed. We have examined the extent to which this is a problem and show that large errors in estimating hormone levels are likely unless sap is first made to flow at rates similar to those of whole-plant transpiration.

Tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) plants were de-topped just below the cotyledonary node, and a 20 mm length of rubber tubing was attached to the stump. The assembly was then placed in a pressure vessel designed to collect sap exuding from the cut stump. The pressure was raised by connecting the inlet pipe to a cylinder of compressed gas. Air was used to pressurise the root system of well-drained plants and oxygen-free nitrogen was used for the flooded root systems. Sap flow rate was varied by applying pressures to de-topped root systems to give a range of flows centred on whole-plant transpiration rates. Samples were collected in Eppendorf tubes, weighed, frozen in liquid nitrogen and stored at -30°C until assayed.

l-aminocyclopropane-l-carboxylic acid (ACC) was quantified in HPLC-purified leaf extracts and sap samples by a direct method using a capillary column gas chromatograph fitted with a standard nitrogen/phosphorous detector (Hall, Else and Jackson, manuscript in preparation). Results were statistically indistinguishable from GC-MS after N-benzoyl n-propyl derivatisation of ACC.

The concentration of ACC in xylem sap decreased in proportion to increased sap flow rate. Thus, the concentration in sap is a function of sap flow and therefore a poor guide to the amounts transported. However, the amount of hormone delivered from roots to shoots can be calculated

373


374

by multiplying concentration by the sap flow rate at which the measurements are made. The diluting effect of increased sap flow was in proportion to flow rate. Thus, the calculated delivery rates of the hormones from roots to shoots were similar over a wide range of flow rates.

Using measurements in sap flowing through de-topped roots at rates equivalent to transpiration, concentrations and delivery rates of ACC in sap of intact plants have been estimated. Flooding increased the concentration of ACC after 6 hours and this effect was enhanced with time up to 36 hours. Delivery rates showed similar increases, in association with epinastic leaf curvature. However, the magnitude of the effect was less marked because flooding also enhanced concentration by slowing transpiration. A significant export of ACC from well-drained roots (25 finol S-l) was also calculated. This has not been reported hitherto. This rate of supply is sufficient to account for the ethylene production rate of the whole shoot system, assuming a foliar production rate of 25 nl g-l h-1•

Our results show that ACC is diluted by increases in sap flow rate. Thus, if sap flow from detopped root systems is slower than whole-plant transpiration, concentration in this sap will overestimate that of sap of intact plants. This has important implications for studies of root stresses such as soil flooding where .transpiration or rates of sap flow from de-topped root systems may be slowed due to stomatal closure or increased hydraulic resistance. In these situations, any increase in xylem sap concentration may simply be a consequence of less dilution caused by slower transpiration or sap flow rather than of increased hormone delivery by roots. Therefore, estimates of hormone concentration alone are insufficient to establish a positive hormonal message i.e. increased hormone output from stressed roots. Additional information regarding sap flow rates is needed to allow calculation of hormone delivery rates, which must increase if a positive message is to be established unambiguously. These delivery rates are best derived using sap flowing at rates similar to whole-plant transpiration.

ETHYLENE SYNTHESIS BY FRUIT PLANTS CULTURED IN VITRO.

R.JONA. A.FRONDA. A.CATTRO. A.GALLO Isti tuto di Col tivazioni Arboree Uni vel'si ty of Turin, Facu1 ty of Agricu1 ture Via Pietro Giul'ia 15 10126 TORINO Italy

Several observation in our laboratory have demonstrated that ethylene accumulates into the vessels of some plants cultured in vitro and this is enhanced by ethylene released by some components of the media. mainly agar. while pectins. though is an inferior jellying agent is not releasing ethylene (Jona et a1 .. 1984.1987.1990,1992). Ethylene released by the medium. in turn enhances the production of the same gas by the plant itself. but not all species react in the same way: some plants reacts. as usual by producing themselves more ethylene, while other do not produce it at all (Jona et a1 .. 1992).

In order to gain a better insight of these phenomena. we devised an experiment which eliminated all interferences by the medium: for t~is purpose instead than proliferating plantlets. rooted ones have been employed and a mineral medium identical for all species have been used in order to avoid any difference between the plants which could be ascribed to the effect of a specific formulation of the medium.

Rooted plantlets of GF 677 peach rootstock. plum. cherry. strawberry have been grown on (Sorbarod) imbibed with MS mineral lacking any jellying agent.

(peach x almond hybrid) apple. grapevine and

small cellulose pads salts acqueous solution.

The plantlets were grown inside special screwcapped Erlenmeyers. which have been used to analyze the gaseous content of the vessel. The screwcaps made out of rigid plastic formed only a ring which pressed a rubber membrane which could be perforated for sampling the inner atmosphere and' analyze it by gas chromatography.

The cultures were initiated by stopcocks incompletely screwed. in order to gaseous exchanges with the environment. until w~re well established. At the moment of the

375

keeping the facilitate the the plantlets initiation of


376

the experiment, the sample was divided into two subsamples of 5 individuals each, which were either kept under tightly closed atmosphere or loosely closed flasks.

The eventual production of ethylene by the medium has been checked by running appropriate controls: some flasks containing the MS medium and the Sorbarod cellulose pads (but no plantlets) were prepared and monitored alongside those containing the plantlets.

Samples of 0.5 ml of the inner atmosphere were extracted at regular intervals for 30 days and analyzed by gas chromatographer for their ethylene content.

As it is clearly demonstrated by the following diagram, there are differences in production of ethylene between the various species, which the statistical analysis showed they are highly significant.

. :1251 .:1

::... I OJ .875-1 c:

J OJ

;,'I .85 1 .c: .. I OJ . 825 1

8!

o Apple

• GF x

P.do/lle~tica

+ Strawberry

~

P.auiu/Il

1 3 5 7 9 11 13 17 21 25 38 0

days Grapeuine

References Jona R., Webb J.K .. 1978. Callus and axillary bud

culture of Vitis vinifera 'Sylvaner Riesling'. Sci Hortic. 9: 55-60.

Jona R., Gribaudo I., Vigliocco R. 1984. Effects of naturally produced ethylene in tissue culture jars. In: Ethylene: Biochemical, Physiological and applied aspects, Y. Fuchs and E. Chalutz (eds.), M. Njihoff/Dr Junk Publ., The Hague (NL): 161-2

Jona R., Gribaudo I., Vigliocco R. 1987. Natural development of ethylene in air tight vessels of GF 677.Florizel 87, ArIon tB),: 61-6

Jona R., Gribaudo I. ,1990. Ethylene production in tissue culture of PeachxAlmond Hybrid,Tomato,Sweet Cherry.and Grape.Acta Hort 280: 445-6

Jona R. Fronda. Gallo A.and Cattro A. 1992. Ethylene accumulation inside the tissue culture vessels. In press.Symp.on Plant Growth Regulators in fruit plants.Jerusalem 14/18.6.92

Index of Authors

Acosta, M. 53, 341 Aguilar, M. 100 Aho, H.M. 168 Altman, A 325 Amoros, A 148 Ampunpong,C. 152 Anderson, lD. 197 Arnao, M.B. 53, 341 Asaf, U. 174· Auzac (d'), l 162,205 Avni, A 197 Ayub, R. 98

Bailey, B.A. 197 Bailly, e. 90 Baird, S.L. 92 Banga, M.L. 251 Barry, C. 76, 259 Barton, S. 82 Baudinette, S.e. 298 Ben Arie, R 150 Bennett, AB. 123 Berry, AW. 168 Bird, e.R. 327 Blankenship, S.M. 182 Blom; C.W. 247,249,251,369 Bono, R 284 Botella, F. 253 Botondi, R 154 Bottin, A 217

377

Bouteau, F. 205 Bouzayen, M. 71,76,259 Bowers, A 46 Bowmer, lM. 317 Brailsford, R 369 Brame, S. 94 Brandt, AS. 291 Sui, A.Q. 304 Bull, lH. 94 Burdon, IN. 317 Burg, S.P. 335 Burdmeister, D.M. 46

Caesar, e. 298 Callahan, A.M. 31 Casas, lL. 53,341 Castellano, lM. 53 Cattro, A 375 Chandler, S.F. 298 Chraibi, K.M. 365 Chrestin, H. 205 Christoffersen, RE. 65 Clark, DJ. 278 Clement, A 205, 257 Cleyet-Marc1, J.e. 39,96 Clijsters, H. 238, 240 Cobbett, C.S. 298 Come D., 90, 253 Cooper W. 76, 259 Corbineau, F. 90

378

Cornish, E.e. 298 Coupe, S.A. 272 Cowan, D.S. 168

Davies, WJ. 373 Dedonder, A. 24 Del Rio,1.L. 146,253 Dilley, D. 46 Dong,1.G. 59 Drew, M.e. 232 Dunn, L.J. 31 Dupille, E. 39

Else, M.A. 373 English, P.I. 261 Escribano, M.l. 156,160 Esquerre-Tugaye, M.T. 217 Evensen, K.B. 278

Fallot, J. 365, 367 Fearn,1.e. 182 Feiner, l. 174 Fernandez-Maculet, J.e. 53,59 Fishel. D. 31 Fischer, R.L. 100 Fournier, 1. 217 Fray, R. 82 Frimer, A.A. 174 Fronda, A. 375 Fusch, Y. 197

Gallo, A. 375 Garcia-Lindon, A. 146 Garcia-Puig, D. 146 Garrido, G. 341 Gautrais, 1. 298 Gladon, R.I. 144 Gobattoni, E. 154 Goldschmidt, E.E. 164 Goren, R. 325 Graham, MW. 298 Gran, e. 46 Gray, 1.E. 82 Grierson, D. 71, 76, 82, 92, 259 Guern,1. 162

Hall, K.c. 261,373 Hall, M.A. 168, 195,369,371

Hamilton, AJ. 7 L 76, 82, 259 Harpham, N.V. 168 Harren, M.J. 249 Harris, N. 272 Hassan, M. 117 Haynes, R. 182 He, CJ. 232 Henskens, H. 323 Hewett, E.W. 152 Hiatt, W.R. 106 Hobson, G.E. 327 Hoffinan-Benning, S. 329 Holland, M.G. 168 Honma, M. III Hubennan, M. 325

Imaseki, H. I, 7 Itzhari, H. 291

Jackson, M.B. 261,369,373 Jacob, .J.L. 162,205,257 Jalilova, F. 347 John, P. 33 Jona, R. 375 Jordan, W.R. 232 Joseph, e. 253

Kacp~rska, A. 211 Kalcvich, A.E. 347 Kanellis, A. 117, 166 Kang, B.G. 335 Kefeli, V. 347 Kemmerer, E.e. 265 Kende, H. 329 Koehler, S.M. 265 Kuai,1. 46 Kubacka-Zebalska, M. 211,240

Lacrotte, R. 205 Larrigaudiere, e. 13 6 Larsen, P. 291 Lashbrook, e.e. 123 Lasserre, E. 94 Latche, A. 39, 96, 98, 365 Leforestier, F. 253 Lelievre,1.M. 39,69,98 Leshem,Y.Y. 174 Levin, A. 150

Levin, N. 325 Li, N. 223 Liu, D. 223 Loulakis, KA. 117 Lu, C.Y. 298 Lycett, G. 261

Margossian, L. 100 Martinez, G.M. 148 Massantini, R 154 Matters, G. 265 Mattoo, A.K 197,223 Maxson, J.M. 291 McGarvey, DJ. 65 Mencarelli, F. 154 Merodio, e. 156, 158, 160 Metzidakis, J. 129 Michael, M.Z. 298 Ming-Wong, L. 19 Montero, L.M. 156, 160 Morgan, PW. 232 Mori, H. 1,7 Moser, O. 166 Moshkov, J.E. 168, 195,371 Murray, A.J. 327

Nadeau, J.A. 304 Nakagawa, N. 1 Niklis, N. 255 Nissen, P. 359 Nonnecke, GR 144 Novikova, G. 168, 195,371 Nugent, G.D. 298

O'Connor, E.L. 298 O'Neil, S.D. 304 Oeller, P.W. 19 Olson, D. 59 Ono, T. 1 Ortufio, A. 146,253

Park, KY. 291 Pcch, J.e. 39, 92, 94, 96, 98 Pekker, Y. 46 Pefiarrubia, L. 100 Perez, M.L. 146 Perkins-Veazie, P. 144 Perrot-Rcchenmann, C. 162

Pesis, E. 152 Petitprez, M. 96, 98 Picton, SJ. 82 Poneleit, L. 46 Porras, I. 146 Pouenat, M.L. 217 Pretel, M.T. 148 Prevot, J.e. 205,257 Primo-Millo, E. 284 Pujade-Renaud, V. 162,205,257

Rakitina, T. 347 Rapoport, D. 174 Reid, M.S. 188 Rickauer, M. 217 Rinjders, J.G. 247 Riov, J. 164 Riquelme, F. 148 Roberts, J.A. 261,272 Rodrigues-Pousada, R.A. 24 Rombaldi, C. 39,96,98 Romojaro, F. 148 Rona, J.P. 90, 205 Rossall, S. 259 Roubelakis-Angclakis, KA. 117 Rouge, P. 96 Roustan, J.P. 365, 367

Sabater, F. 146 Sancan, J.P. 217 Sanchez-Bravo, J. 53,341 Sarquis, J.l. 232 Satoh, S. 7 Sauter, M. 329 Savarese, P. 65 Savin, KW. 298 Schuch, W. 327 Serrano, M. 148 Sexton, R. 317, 265 SfiUciotakis,E. 129,142,255 Sharon, A. 197 Sheehy, RS. 106 Shusiri, B. 152 Singh, A. 278 Sisler, E.e. 182 Siswanto 257 Smith, A.R 168,195,369,371 Smith, H. 82

379

380

Smith, J.l 33 Somhorst, D. 327 Sonego, L. 150 Soulie, O. 367 Stavroulakis, G. 142 Stevenson, K.R. 298 Strul, G. 174

Tadeo, F.R. 284 Taylor, lE. 272 Taylor, R. 197 Thanassoulopoulos, C.C. 255 Theologis, A. 19 Trebitsh-Sitrit, T. 164 Tucker, M. L. 265 Tumer,A.92

Ursin, V. 106

Van der Pan, S. 106 Van Der Sman, A.J. 247 Van Der Straten, D. 24 Van Doom, W.G. 188 Van Montagu, M. 24 Vangronsveld, J. 238, 240 Vendrell, M. 136 Vioque, B. 53 Visser, EJ. 249 Vlasov, P. 347 Voesenek, L.A. 247,249,251,369

Wang,H.291 Watson, C.F. 92 Webb, S.T. 272 Weekx, l 238,240 Wessler, A. 263 Wild, A. 263 Wilson,I.D. 46 Woltering, EJ. 188,310,323 Woodson, W.R. 291 Wu, MJ. 188

Yamagishi, N. I Yang, C. 263 Yang, S.F. 13 Young, R. 298

Zacarias, L. 284

Zamorano, J.P. 156, 158, 160 Zegzouti, H. 76 Zhang, X. 304 Zhu Y. 46 Zutkhi, Y. 150

Index of Keywords

Abscissic acid 263, 329 Abscission 265, 272, 284, 317, 325 Abscission related cDNAs 272 ACC (I-aminocyclopropane-I-carboxylic

acid) analogs III degraradation to a-ketobutyratc 111 diurnal courses 263 free radicals 53 hydroxylation 714 oxidation 53 translocation 310 in xylem sap 373

ACC deaminase 19, 106, III AC£ oxidase

accumulation of RNA during ripening 59

activation by bicarbonate and CO2 33, 46,59

activity in ripening fruits 31, 33, 129, 136, 142, 144, 148, 150

activity in transgenic carnation 298 activity in transgenic grape cells 98 amino acid sequence 39,59,298 antisense genes 65, 261 antisense plants 39, 46, 65, 71, 96, antisense RNA 298, 327 effects of plasmolysis 39, 98 enzyme kinetics 46, 33, 39 expression of transgene 82 fusion polypeptide 39, 65, 96

381

gene expression 31, 46, 291, 304 glycosylation 39 immunocytolocalization 39,96,98 inhibition by carbonyl sulphide 33 inhibition by trinitrobenzene sulfonate

39 low molecular weight factor 33 multigene family 259 pathogene-induced gene 259 polyclonal antibodies 39 46 59 96

98,92 ' , , ,

promoter-GUS fusion 94 purification 39, 46, 59 recombinant polypeptide,(see fusion

polypeptide) regulation by calcium 90 ripening-related eDNA 59, 65, 82, 92 role of histidine in catalytic activity 65 signal peptide 71 stabilization by glycerol 33 stimulation by calcium 90 subcellular localization 39, 65 transient transformation 94 wound-induced eDNA 92 wounding 71

ACC synthase active site 13 activity in fruits 1, 7, 148 aminoacid sequence I, 13 antisense RNA 19,298 auxin-induced 1,19 C-terminal region 1, 223

382

cDNA fusion to ~-galactosidase gene 298

deletion of amino acid 1 expression in E.coli 223, 232 fusion protein 7 gel filtration chromatography 7 gene expression 1, 13, 19,24,31,223,

291,304, 323, gene organisation and structure 291 gene promoter 24 genomic clones 24 immunopurification 13 isozymes 1, 13 mechanism-based inactivation 7 monomeric and dimeric forms 1,7 multigene family 13,19, 24, 76, phosphorylation 223 ripening-induced I, 13 specific primers for two cDNAs 323 structural characteristics 1 structure-function relationship 223' temporal and spatial regulation 24 wound induced I, 13,24,223

Actinidia deliciosa 142,255 Adventitous roots 247,249 Aerenchyma 232,247 Agrobacterium tume!asciens 98,106 Alcohol dehydrogenase 117, 154 Amino oxyacetic acid 144 l-amino-2-ethylcyclopropane-l-carboxylic

acid (AEC) 71 2-aminoethoxyvinylglycine (AVG) 154,232,

317 Annona cherimola 148, 156, 160 Antisense expression 298 Antisense RNA 19, 82 Arabidopsis thaliana 24, 168,347 Arginine decarboxylase 325, 365 Ascorbate 33,46,59,65,71,100 Ascorbate oxidase 166 L ascorbic acid-6-palmitate 46 ATPase 205 Autocatal)-tic ethylene 129, 142,223, Auxin 1,247,304,359

Botritys cinerea 255 Botritys induced ethylene production 255 Browning of cherimoya 148

Buckminsterfullerene 174

Calmodulin 205 Carnation cultivars 188 Carotenoid 82 Catalase 238 Cell division 329 Cell elongation 329 Cellulase 117, 132, 129

activity 129, 232, 272 antisense construct 123 auxin effect on accumulation ofmRNA

265 fusion to GUS 265 gene expression 123, 265, 272 low oxygen action 117 in situ hybridization ofmRNA 265 sequence relationship to microbial

cellulases 123 sequence relationship to plant

cellulases 123 tissue specific regulation 265

Chimeric gene construct 106 Chitinase 162 Chlorophyllase 164 Citrus paradisis 146 Citrus sinensis 164,284,325 Climacteric rise in ethylene production 136

188 Clinostat rotation 335 CO2 effects on ethylene production 150

Cobalt chloride 353, 359 Cold-induced climacteric behaviour 136 Cold-induced ethylene 136 Collectotrichum 259 Cucumis melD 33,39,92,94, 166 Cucurbita maxima I, 7 Cx-cellulase (endo ~-1-4 glucanase, see

cellulase) Cycloheximide 278 Cymbidium 310

Daucus carota 359, 365 Deamination of D-serine III Delayed ripening 158 Dianthus caryophyllus 174, 182, 188,291,

298,323

Diazocyclopentadiene 182 2,4-dichlorophenoxyacetic acid (2.4-0) 359 Difluoromethylornithine 359 2,4 dinjtrophenol (DNP) 90 Deepwater rice 329 Dominant nuclear gene 197

2D-clectrophoresis .117, 156 Elicitors 197,217 Elongation (shoot) 251 Elongation (roots) 232, 247 Elongation (stem) 249 13-1-4 endoxylanase,(see xylanase) Enzyme purification 39, 46 59 E~cherichia coli I, 7, 39, 65, 7L92, 94, 96,

223,298 Ethephon 146,341,353,359,365,367 Ethanol 152,154 Ethanol effect on ethylene production 154 Ethrel 205, 257 Ethyl acetate 152 Ethylene

action inhibitors 182 binding 168, 182,188, 195, 371 cold-induction 136 defense induction 217 gene expression 278 growth 329,341 induction by heavy metals 238, 240 inhibition by antisense RNA 19 inhibition of release 174 insensitive mutants 168 overproduction 100 production (effect of illumination) 347 production of flower parts and leaves

323 production of fruits 317 production rates of flowers 317 proteins phosphorylation 168 (sensitivity to) 188 synthesis by fruit plants in vitro 375 translocation 310 vitrification 353

E8 protein 100 Ethylene Forming-Enzyme (see ACC

oxidase)

Fe(lI) super familly ascorbate requiring 65

Flavanone-3-hydroxylase 71 Flooding 247,249,251 Flower abscission (orange) 284 Flower emasculation 310 Flower senescence 291, 298, 310, 323 Flower senescence-related gene 291 Flow cytometry 329 Fraxinus 353 Fruit abscission 284, 3 17 Fruit ripening 2 L 71, 100, 123, 158 327

Gelling agents 353 Gibberellin 247,329 Glucose-6-phospate dehydrogenase 240 Glutamate dehydrogenase 240 Glutamine synthase 162, 257 Glycosylation 39 Gravitransport 335 Gravity dependent ethylene action 335 GUS fusion 24, 94, 265

Hanseanula saturnus III Helianthus annulls 90, 365 Hevea brasiliensis 162,205,257 Heveamines 205 Hevein 205 High CO2 treatment 152

Horizontal nutation 335 Hormone transport 373 Hydroperoxide 53 Hydroxyproline rich glycoproteins 162 3-Hydroxypropylamide 53 Hyoscyamine 6b-hydroxylase 100 Hypersensitive responses 197 Hypobaric treatment 335 Hypoxia 232, 369, 339

383

IAA content (in leaves and apical stem) 347 Immunodetection of chlorophyllase 164 Intercalary meristem 329 Interorgan communication 3 10 Interorgan regulation 304 Inverse PCR 94 Invertase 205 Iron 33,46,39,59,65, 71 Iron (II) dioxygenases 100 Isopenicillin N synthase 65, 100

384

Isotope competition technique 188

Jellying agents 375

K+ leakage 240

Latex 205, 257 Laticifers 205 Leaf abscission 278 Leaf albumin 325 Leaf epinasty 335 Leaf senescence 82, 327 Lipid peroxidation 211 Lipoperoxides 211 Liposomes 174 Lipoxygenase 211,238 Low and high 0 2 150

Low oxygen action of fruit ripening 117, 129

Lupinus albus 341 Lupin hypocotyl 341 Lutoid205 Lycopene 82 Lycopersicon esculentum 7, 13, 19,39, 71,

96, 100, 106, 123, 223, 253, 259, 327,373

Magnaporthe grisea 211 Malonyl-ACC 136, 232, 263 Malus domestica 33, 39, 46, 136, 150, 152,

375 Medicago sativa 217 Membrane degradation 240 Membrane depolarization 90 Membrane permeability 240 Membrane injury 211 4-methylpyrazole (4-MP) 154 Microprojectile bombardment 94 Micropropagation 353, 367 Morphogenesis 367 Multigene family 13 19,24, 76

N-(iodoacetamidoethyl)-aminonaphtalane sulfonic acid Ill,

Navel orange mutants 284 Nickel 359 Nicotiana tabaccum 197

Norbonadiene 31, 223, 265 Nutrient starvation 232

Octanoic and decanoic acids 310 Organogenesis 365 Ornithine decarboxylase 325 Oryza sativa 329

Pacobutrazol 247 Particle gun bombardment265 Pathogen attack 259 PCR 1, 13,24, 76, 94, 223, 298, 323 Peach rootstock 375 Pelargonium x hortorum 278 Penicillium citrinum III Peroxydase 53,238,240 Persea americana 65, 117, 129, 158 Petal inrolling 188 Petal abcission zone proteins 278 Petal abscission 278, 317 Petal senescence (see flower senescence) Petunia 298 Phalaenopsis 304 Phaseolus vulgaris 168,240,265,272 1,10 phenantroline 39,71 Phenyl hydrazine 211 Photoaccoustic technique 249, 251 Phot<;>lysis 182 Phototropic lateral transfert 335 Physical impedance 232 Phytochrome 347 Phytoene synthase 82 Phytophtora 217 Polar IAA transport 335 Pollination-induced ethylene 304 Polyamines 160,223,325,365 PolycIonal antibodies against ethylene

binding protein 168 Polygalacturonase 82, 123, 129, 272 Post pollination events 304 Postharvest shrivelling 82 Propylene induced ethylene biosynthesis 142 Protein pattern during abscission 284 Protein pattern during ripening 156 Protein phosphorylation 371 Protoplast 65,90 Prunus persica 31 375 Pseudomonas sp 19, 106, III

Pyrophosphate:fructose-6-phosphate-lphosphotransferase 257

Radial swelling 232,247 Raspberry 144 Reducing sugar 327 Relationship ethylene polyamines 160 Reverse genetic 19 Reverse transcription peR 24 Rhizobium 217 Rubus idaeus 317 Rumex avicum 375 Rumex 247, 249, 251 Rumex domestica 375

S-adenosylmethionine decarboxylase 365 Saccharomyces cerevisiae 71 Salicylic acid 197, 223 Saline stress 253 salicylic acid and acetylsalicylic acid 359 Sambricus nigra 272 Senescence 188 Senescence-related mRNAs 291 Sesquiterpene noot:ka.t<me 146 Short chain saturated fatty acids 310 Signal transduction 19, 240 Silver nitrate 353, 367 Silver thiosulfate (STS) 182, 232, 317, 341 Softening 31, 129, 142,158,160,317 Somatic embryogenesis 359, 365 Spatial and temporal location of ethylene biosynthesis 304 Spruce needles 263 Strawberry 375 Stress ethylene 205, 232, 257 Sub-ambient partial pressures of oxygen 369 Superoxide dismutase 162

Thiobarbituric acid 240 Tissue printing 197 Titrable acidity 327 Tomato yellow flesh mutant 82 Tonoplast ATPase 205 Transformed plants (see transgenic plants) Trangenic grape cells 98 Transgenic plants 82, 100, 106,261,298 Transgenic tomato fruits 96, 123 Translability ofPG mRNA 19

Trichoderma viride 197 Tryptophan synthase 111

UV illumination 347

Vanadate 90 Viciafaba 174 Vitis vinifera 98,367,375

Waterlogged 251 Waterlogging 261 Winter rape 211 Winter squash 1, .., Winter wheat 211 Wound ethylene 1,71,211,304

Xenopus laevis 71 Xylem sap flow 373 Xylanase 197

Zea mays 232, 369

385


1. HJ. Evans, PJ. Bottomley and W.E. Newton (eds.): Nitrogen Fixation Research Progress. Proceedings of the 6th International Symposium on Nitrogen Fixation (Corvallis, Oregon, 1985). 1985 ISBN 90-247-3255-7

2. RH. Zimmerman, RJ. Griesbach, F.A Hammerschlag and RH. Lawson (eds.): Tissue Culture as a Plant Production System for Horticultural Crops. Proceedings of a Conference (Beltsville, Maryland, 1985). 1986 ISBN 90-247-3378-2

3. D.P.S. Verma and N. Brisson (eds.): Molecular Genetics of Plant-microbe Interactions. Proceedings of the 3rd International Symposium on this subject (Montreal, Quebec, 1986). 1987 ISBN 90-247-3426-6

4. E.L. Civerolo, A Coli mer, RE. Davis and AG. Gillaspie (eds.): Plant Pathogenic Bacteria. Proceedings of the 6th International Conference on this subject (College Park, Maryland, 1985). 1987 ISBN 90-247-3476-2

5. RJ. Summerfield (ed.): World Crops: Cool Season Food Legumes. A Global Perspective of the Problems and Prospects for Crop Improvement in Pea, Lentil, Faba Bean and Chickpea. Proceedings of the International Food Legume Research Conference (Spokane, Washington, 1986). 1988 ISBN 90-247-3641-2

6. P. Gepts (ed.): Genetic Resources of Ph as eo Ius Beans. Their Maintenance, Domestica-tion, Evolution, and Utilization. 1988 ISBN 90"247-3685-4

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8. RS. Sangwan and B.S. Sangwan-Norreel (eds.): The Impact of Biotechnology in Agriculture. Proceedings of the International Conference 'The Meeting Point between Fundamental and Applied in vitro Culture Research' (Amiens, France, 1989). 1990.

ISBN 0-7923-0741-0

9. H.J.J. Nijkamp, L.H.W. van der Plas and J. van Aartrijk (eds.): Progress in Plant Cellular and Molecular Biology. Proceedings of the 8th International Congress on Plant Tissue and Cell Culture (Amsterdam, The Netherlands, 1990). 1990

ISBN 0-7923-0873-5

10. H. Hennecke and D.P.S. Verma (eds.): Advances in Molecular Genetics of Plant-Microbe Interactions. Volume 1. 1991 ISBN 0-7923-1082-9

11. J. Harding, F. Singh and J.N.M. Mol (eds.): Genetics and Breeding of Ornamental Species. 1991 ISBN 0-7923-1094-2

12. J. Prakash and R.L.M. Pi erik (eds.): Horticulture - New Technologies and Applications. Proceedings of the International Seminar on New Frontiers in Horticulture (Bangalore, India, 1990). 1991 ISBN 0-7923-1279-1

13. C.M. Karssen, L.c. van Loon and D. Vreugdenhil (eds.): Progress in Plant Growth Regulation. Proceedings of the 14th International Conference on Plant Growth Substances (Amsterdam, The Netherlands, 1991). 1992 ISBN 0-7923-1617-7

14. E.W. Nester and D.P.S. Verma (eds.): Advances in Molecular Genetics of Plant-Microbe Interactions. Volume 2. 1993 ISBN 0-7923-2045-X

15. c.B. You, Z.L. Chen and Y. Ding (eds.): Biotechnology in Agriculture. Proceedings of the First Asia-Pacific Conference on Agricultural Biotechnology (Beijing, China, 1992). 1993 ISBN 0-7923-2168-5


16. J.e. Pech, A. Latche and C. Balague (eds.): Cellular and Molecular Aspects of the Plant Hormone Ethylene. 1993 ISBN 0-7923-2169-3

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