MOLECULAR BIOLOGY & BIOTECHNOLOGY OF THE GRAPEVINE MOLECULAR BIOLOGY & BIOTECHNOLOGY OF THE GRAPEVINE edited by KALLIOPI A ROUBELAKIS-ANGELAKIS Professor of Plant Physiology and Biotechnology, Department of Biology, University of Crete, Heraklion, Greece and President of the Federation of European Societies of Plant Physiology SPRINGER-SCIENCE+BUSINESS MEDIA, B.V A C.I.P Catalogue record for this book is available from the Library of Congress ISBN 978-94-017-2310-7 ISBN 978-94-017-2308-4 (eBook) DOI 10.1007/978-94-017-2308-4 Printed an acid-free paper An Rights Reserved © 2001 Springer SciencetBusiness Media Dordrecht Originally published by Kluwer Academic Publishers in 200 Softcover reprint of the hardcover 1st edition 200 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 permis sion from the copyright owner To the Memory of my Parents Apostolos and Maria Roubelakis PROLEGOMENA Research in Plant Biology, in the pre-molecular era, dealt mostly with work at the organismallevel The molecular era has opened new avenues in our understanding of the secrets of life Molecular Biology and Biotechnology have emerged as the crossing-point of basic biological sciences, such as Biochemistry, Cellular Biology, Genetics, Microbiology, and Physiology The use of molecular techniques and other analytical instrumentation has increasingly contributed to further understanding' how, when and where' physiological phenomena occur in organisms Non-molecular plant biotechnological methods, such as the plant tissue culture techniques, have been developed during the past decades whereas the advances in Plant Molecular Biology have been used for the development of molecular biotechnological application; they have been based upon the non-molecular developments Grapevine is one of the most widely cultivated plant woody species As with most wooc(y plant species, and also some cereals and legumes, Molecular Biology and Biotechnology have had progressed at a slower pace, due to several obstacles, which have had to be overcome In any case, it is now that substantial progress has been made and useful information has been accumulated in the literature During the last decade, more than 100 genes have been characterized from grapevine and several genomic and chloroplastic microsatellite sequences have been deposited in the Genbanks These genes encode for enzymes mediating synthesis and transport of sugars, polyphenols and pigments, organic acids, amino acids and polyamines, as well as for proteins related to biotic and abiotic stresses and to cell wall structure Furthermore, protocols for non-molecular and molecular biotechnol-ogical applications for grapevine have been published In an effort to collect and present the available information on Grapevine Molecular Biology and Biotechnology, 51 scientists from 10 countries jointly worked for the preparation of this Book It is intended to be used as a reference-book by researchers, graduate and undergraduate students, viticulturists, biotechnological companies and any scientist, who is interested in the Molecular Biology and Biotechnology of Grapevine Sincere thanks are due to all worldWide-leading scientists in their field, who have contributed and especially for their impeccable collaboration during the preparation Vlll of this Volume; to Mrs Mary Papadakis-Savvopoulos for editorial assistance; to Miss Maria Mandelenakis for secretarial assistance; to Mr Nikolaos Papadoyannakis for his endless and devoted work during the preparation of the ready-to-camera material; to Kluwer Academic Publishers for the publication of the Book Last but lot least to my husband, Andreas Angelakis, for his continuous encouragement and patience Herak/ion, Crete, Greece January 2001 Kalliopi A Roubelakis-Angelakis University of Crete, Greece CONTENTS Contributing Authors xxi Chapter MOLECULAR BIOLOGY OF SUGAR AND ANTHOCYANIN ACCUMULA TION IN GRAPE BERRIES P.K Boss and C Davies Introduction The Molecular biology of sugar transport and accumulation in grape 2.1 Grape sucrose transporters 2.2 Grape monosaccharide transporters 2.3 Grape invertases 2.4 Future directions Anthocyanins 12 13 3.1 Grape anthocyanins 3.2 The anthocyanin biosynthesis pathway 3.2.1 Introduction 3.2.2 The structural genes 3.2.3 Genes involved in pathway regulation 3.3 Grape anthocyanin gene expression 3.3.1 Anthocyanin gene expression in grapevine seedlings 3.3.2 Anthocyanin gene expression in berry skins during development 3.3.3 Anthocyanin gene expression in red and white grapes 3.5 ManipUlating grapevine anthocyanins 3.5.1 Total anthocyanins 3.5.2 Specific anthocyanins Summary 13 14 14 14 17 18 18 18 21 24 24 25 27 Acknowledgments 28 References 28 Chapter GRAPE BERRY ACIDITY N Terrier and C Romieu 35 Introduction 35 Changes in acidity during berry development 36 2.1 Evolution pattern of berry composition 2.2 Organic acid metabolic pathways in grape berries 36 38 x 2.2.1 Organic acid synthesis 2.2.2 The induction of malate respiration during ripening 2.2.3 Aerobic fermentation and malate breakdown Compartmentation of organic acids in grape berries 3.1 Vacuolar proton pumps 3.1.1 Molecular structure 3.1.2 Thermodynamic properties 3.1.3 Enzymic properties 3.1.4 Two pumps on the same membrane 3.2 Organic acid accumulation 3.3 Vacuolar transport and pH variation 3.3.1 Proton pumps 3.3.2 Secondary transport 3.3.3 Vacuolar content efflux References 38 38 40 41 41 41 42 43 44 46 49 49 51 51 52 Chapter NITROGEN ASSIMILATION IN GRAPEVINE K.A Loulakakis and K.A Roubelakis-Angelakis 59 Introduction 59 Nitrogen assimilation 2.1 Reduction of nitrate 2.2 Ammonium assimilation 2.2.1 Glutamine synthetase 2.2.2 Glutamate synthase 2.2.3.Glutamate dehydrogenase Regulation of ammonia assimilating enzymes in grapevine by nitrogen source 60 60 63 65 68 71 77 Future perspectives 80 References 80 Chapter MOLECULAR BIOLOGY AND BIOCHEMISTRY OF PROLINE ACCUMULATION IN DEVELOPING GRAPE BERRIES 87 R van Heeswijck, AP Stines, J Grubb, I Skrumsager Mpller and P.B Hpj Amino acid composition of grape berries 87 87 The influence of grape berry proline on fermentation 91 Introduction xi Proline accumulation in plants 91 Pathways of proline biosynthesis 5.1 The glutamate pathway of proline biosynthesis 5.2 The Ornithine pathway of proline biosynthesis 5.3 Genes encoding P5CS and OAT are expressed in grape berry tissue Vvp5cs gene expression during grape berry development 92 92 94 94 97 Other factors which could affect proline accumulation in grape berries 7.1 Ammonium and glutamine metabolism 7.2 Arginine metabolism and regulation of OAT 7.3 Proline degradation 7.4 Protein accumulation Conclusions 99 99 100 101 102 103 Acknowledgments 104 References 104 ChapterS POLYAMINES IN GRAPEVINE K.A Paschalidis, A Aziz, L Geny, N.! Primikirios and 109 K.A Roubelakis-Angelakis Introduction 109 Biosynthesis of polyamines 110 Endogenous polyamines in grapevine organs 3.1 Polyamines in various grapevine organs 3.2 Spatial and temporal free and conjugated polyamine distribution in grapevine leaves 3.3 Polyamines and berry development 3.3.1 Polyamine oxidase activities and diaminopropane contents during floral development in grapevine 3.3.2 Hydroxycinnamic acid amines in flowers and berries of grapevine ADC enzyme activity and transcript levels in developing grapevine organs 112 ll2 116 118 119 Polyamines and disorders of grape berry development 5.1 Polyamines and fruit set 5.2 Polyamines and abnormal development of berry (shot grape berries) 5.3 Polyamine metabolism in relation to flower and fruitlet abscission 5.3.1 Polyamines and abscission potential 5.3.2 Polyamines counteract abscission 5.3.3 Polyamine biosynthesis and abscission 5.3.4 Polyamine catabolism and abscission 121 121 122 122 124 125 128 129 ll2 116 xii 5.3.5 Photodependance of polyamine levels and abscission 5.3.6 Modulation of carbohydrate and amino acid levels by polyamines Polyamines and stress 6.1 Free polyamines, ADC enzyme activity and transcript levels in grapevine cell suspension cultures under different treatments 6.2 Free polyamine titers and stress adaptation 6.3 Polyamines and potassium nutrition 6.3 Polyamines and biotic stress (Botrytis cinerea) References 130 130 133 133 136 140 142 144 Chapter PHYSIOLOGICAL ROLE AND MOLECULAR ASPECTS OF GRAPEVINE STILBENIC COMPOUNDS 153 L Bavaresco and C Fregoni Introduction 153 Plant disease resistance mechanisms 153 Phytoalexins and biotic/abiotic elicitors 154 Grapevine induced stilbenes 4.1 First evidence of stilbenes in grapevine 4.2 Biotic elicitors 4.2.1 Botrytis cinerea 4.2.2 Plasmopara viticola 4.2.3 Phomopsis viticola 4.2.4 Rhizopus stolonifer 4.2.5 Bacteria 4.3 Abiotic elicitors 4.3.1 UV irradiation 4.3.2 Aluminum chloride 4.3.3 Ozone 4.3.4 Wounding 4.3.5 Fosetyl-Al 4.3.6 Other chemicals 4.4 Stilbene glycosides in Vitis 4.5 Cultural factors affecting induced stilbene synthesis 4.5.1 Fertilizer supply 4.5.2 Rootstock Stilbenes in soft tissues of field grow grapevines 155 155 157 158 161 162 162 163 163 163 165 165 165 166 166 166 167 167 168 168 Grapevine constitutive stilbenes 169 Stilbenes in the wine 170 Molecular and biotechnological aspects of stilbene synthesis in grapevine 171 K.M SEFC et al 458 172 Botta, R., Scott, N.S., Eynard, I., and M.R Thomas (1995) Evaluation of micro satellite sequence-tagged site markers for characterizing Vilis vinifera cultivars Vitis 34(2): 99-102 Bourquin, l-C, Otten, L., and B Walter (1991) Identification of grapevine root-stocks by RFLP c.-R Acad Sci Paris, ser 1II, 312: 593-598 Bourquin, J.-c., Otten, L., and B Walter (1995) PCR-RFLP analysis of Vilis, Ampelopsis and Parlhenocissus and its application to the identification of rootstocks Vitis 34(2): 103-108 Bourquin, J.-c., Sonko, A., Otten, L., and B Walter (1993) Restriction fragment length polymorphism and molecular taxonomy in Vilis vinifera L Theor App! 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Kyvclos, C.J., and KA Roubelakis-Angelakis (2000c) Genetic relationships within the Greek cultivars of Vilis vinifera assessed by microsatellite markers 6'h Intern Symposium on Grapevine Physiology and Biotechnology, Hcraklion, Crete, Greece, 11-15 June 2000 Book of Abstracts, p 199 (http://www.biology.uch.gr/meetings/book.pd/) Visual poster at http://www.biology.uch.gr/gvd Lefort, F and K.A Roubelakis-Angelakis (2000a) The Greek Vilis Database, a multimedia web-backed genetic database for germplasm managemcnt of Vilis resources in Greece J Wine Res 11 (3): 233-242 Lefort, F and KA Roubelakis-Angelakis (2000b) Ll.1UHoupyia Tp(xm,1;Ui; m.'1PO