Molecular biology biotechnology of the grapevine

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 1
Softcover reprint of the hardcover 1st edition 200 1

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 1
MOLECULAR BIOLOGY OF SUGAR AND ANTHOCYANIN
ACCUMULA TION IN GRAPE BERRIES

1

P.K. Boss and C. Davies
1. Introduction
2. The Molecular biology of sugar transport and accumulation in grape

1
2

2.1. Grape sucrose transporters
2.2. Grape monosaccharide transporters
2.3. Grape invertases
2.4. Future directions
3. Anthocyanins

5
6
9
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
4. Summary

13
14
14
14
17
18
18
18
21
24

24
25
27

Acknowledgments

28

References

28

Chapter 2
GRAPE BERRY ACIDITY
N. Terrier and C. Romieu

35

1. Introduction

35

2. 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
3. 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 3
NITROGEN ASSIMILATION IN GRAPEVINE
K.A Loulakakis and K.A Roubelakis-Angelakis

59

1. Introduction

59

2. 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
3. Regulation of ammonia assimilating enzymes in grapevine by nitrogen
source

60
60
63
65

68
71
77

4. Future perspectives

80

References

80

Chapter 4
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

2. Amino acid composition of grape berries

87
87

3. The influence of grape berry proline on fermentation

91

1. Introduction

xi
4. Proline accumulation in plants

91

5. 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
6. Vvp5cs gene expression during grape berry development

92
92
94
94
97

7. 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
8. 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

1. Introduction

109

2. Biosynthesis of polyamines

110

3. 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
4. ADC enzyme activity and transcript levels in developing grapevine organs

112
ll2

116
118
119

5. 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
6. 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 6
PHYSIOLOGICAL ROLE AND MOLECULAR ASPECTS OF GRAPEVINE
STILBENIC COMPOUNDS

153
L. Bavaresco and C. Fregoni

1. Introduction

153

2. Plant disease resistance mechanisms

153

3. Phytoalexins and biotic/abiotic elicitors

154

4. 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
5. 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

6. Grapevine constitutive stilbenes

169

7. Stilbenes in the wine

170

8. Molecular and biotechnological aspects of stilbene synthesis in grapevine

171


xiii
8.1. Grapevine stilbene synthesis
8.2. Transfer of stsy genes
8.3. Grapevine breeding and fingerprinting based upon molecular aspects of
stilbene synthesis
Acknowledgements

171
173

References

176

174
176

Chapter 7
PATHOGENESIS RELATED PROTEINS-THEIR ACCUMULA TION IN
GRAPES DURING BERRY GROWTH AND THEIR INVOLVEMENT IN

WHITE WINE HEAT INSTABILITY. CURRENT KNOWLEDGE AND
FUTURE PERSPECTIVES IN RELATION TO WINEMAKING
PRACTICES

183

D.B. Tattersall, K.F. Pocock, Y. Hayasaka, K. Adams, R. van Heeswijck,
EJ. Waters and P.B. H¢j
1. Introduction

183

2. The nature of unstable wine proteins

185

3. The major wine haze forming proteins are PR-like proteins

185

4. The synthesis of haze-forming PR-like proteins in grape berries is regulated
in a developmental and tissue specific manner

187

5. The regulatory elements controlling PR-like protein synthesis at veraison
are not known

188

6. Grape PR-like proteins show antifungal activity in vitro

190

7. The contribution of growing and harvesting methods to wine protein
instability

191

8. Preventing visible haze formation with haze protective factors

192

9. The use of proteolytic enzymes to prevent protein haze formation

193

10. Use of PR-like proteins for varietal identification

194

11. Conclusions

195

Acknowledgements

196

References

196

Chapter 8
ALCOHOL DEHYDROGENASE: A MOLECULAR MARKER IN
GRAPEVINE

203


XIV

C. Tesniere and C. Verries
1. Introduction

203

2. Expression of Adhs in grape tissues

204

2.1. In developing fruit
2.1.1. ADH enzyme activity
2.1.2. ADH isoforms and biochemical properties
2.1.3. Adh gene expression
2.2. In response to an abiotic stress: anaerobiosis
2.3. In different tissues
3. Molecular characterisation of Adh genes from V. vinifera L.

204

204
207
208
209
210
211

3.1. Adh gene cloning
3.2. Structural organisation of V. vinifera Adh genes
3.3. Analysis of putative regulatory sequences
3.3.1. Initiation and transcription sites
3.3.2. Translation-initiation site selection
3.3.3. Processing sequences in 3'-ends
3.3.4. Anaerobic response elements (ARE)
3.3.5. Comparison of the encoded ADH polypeptides
4. Evolution of Adh mUltigene family

211
212
212
213
214
214
215
215
216

4.1. Among Adh from other species
4.2. Among other Vitis species
5. Conclusions

216
218
218

References

219

Chapter 9
ENHANCEMENT OF AROMA IN GRAPES AND WINES:
BIOTECHNOLODICAL APPROACHES
O. Shoseyov and B. Bravdo

225

1. Free and glycosidic ally bound aroma compounds in grapes and wines

225

2. The role of terpenes as aroma compounds in must and wines

226

3. Terpenes cycle in leaves and fruit and their

227

effect on aroma formation

4. Applications of glycosidases to enhance aroma of wines

229

5. Cloning and expression of recombinant A. niger beta-glucosidase in yeast

232

6. Purification of A. niger B-glucosidase

233

7. Proteolysis and N-terminal sequences of A. niger Bl B-glucosidase

234

8. Cloning of bgll cDNA and genomic gene

234

9. Expression ofbgll cDNA in Saccharomyces cerevisiae and Pichia pastoris

236


xv
References

237

Chapter 10
WA TER TRANSPORT AND AQUAPORINS IN GRAPEVINE

241

S. Delrot, S. Picaud and J.P. Gaudillere
1. Introduction

241

2. SoilfPlantlAtmosphere continuum in grapevine
2.1. Soil root conductivity
2.2. Radial root conductivity
2.3. Xylem conductivity
2.4. Stomatal control of transpiration
2.5. Water use by grapevine in the vineyard
3. Water management and grape quality

242
242
242
243
244
244
245

4. Phloem contribution to water traffic

246

5. Water traffic and aquaporins
5.1. Aq uaporins
5.2. Plant Aquaporins
5.2.1. TIPs
5.2.2. PIPs
5.3. Grapevine aquaporins
6. Summary

248
248
250
251
252
253
257

Acknow ledgments

257

References

257

Chapter 11
PLANT ORGANIZATION BASED ON SOURCE-SINK
RELATIONSHIPS: NEW FINDINGS ON DEVELOPMENTAL,
BIOCHEMICAL AND MOLECULAR RESPONSES TO ENVIRONMENT
A. Carbonneau and A. Deloire

263

1. Introduction

263

2. General biological model
2.1. A general basic biological organization exists, which assures
functioning at each level
2.1.1. Classical model
2.1.2. Triptych model
2.2. A biological system is a complex network of triptychs and not only a
complex association of the basic elements of the triptychs

263
264
264
264
265


XVI

2.3. Three modalities of connections between triptychs exist, which reveal
the biological concepts of nutrition or "source-sink" relationships,
growth and development
2.4. Water constraint does not equate precisely to water "limitation"
2.5. Feed back mechanism
2.6. Plant aging
2.7. Strategies of adaptation

2.8. Polyvalence
2.9. The role of genes
3. Recent developments of molecular biology applied to grapevine physiology

266
267
267
267
268
268
268
270

3.1. Genes involved in general berry development and maturation
3.2. Pathogenesis related proteins
3.3. Phenolic compounds
3.4. Biochemical and molecular responses to biotic stress
3.5. Biochemical and molecular responses to abiotic stress
References

270
271
273
274
274
278

Chapter 12
IN VITRO CULTURE AND PROPAGATION OF GRAPEVINE

281

L. Torregrosa, A. Bouquet and P.G. Goussard

1. Introduction

281

2. Conditions of in vitro culture establishment

282

2.1. Choice of explants
2.2. Handling of stock plants
2.3. Production of sterile explants
2.4. Culture media and hormone requircmcnts
2.5. Browning of explants
3. Conditions of propagation and regeneration
3.1. Nodal and meristem tip culture
3.2. Axillary bud proliferation
3.3. Regenerative procedures
4. Physiological characteristics of in vitro cultures

283
283
284
284
284
285
286

286
287
289

5. Factors affecting success in producing plants

293
293
293
294
295
297

5.1. Stage I: In vitro culture establishment
5.2. Stage II: Regeneration and multiplication
5.3. Stage III: Pretransplantation
5.4. Stage IV: Transplant to soil
6. In vitro culture for grapevine improvement
6.1. Virus sanitation
6.2. Establishment of genetic repositories

297
300


xvii

6.3. In vitro embryo rescue
6.4. Haploid plant production and mutation breeding
6.5. Somaclonal variation

7. Other applications of in vitro culture
7.1. Callus culture
7.2. Cell culture
7.3. Organ culture
8. Conclusions
References

303
305
306

309
309
310
311
312
313

Chapter 13
SOMA TIC EMBRYOGENESIS IN GRAPEVINE
L. Martinelli and I. Gribaudo

327

1. Introduction

327

2. Protocols for somatic embryogenesis in grape

328

2.1. Induction and culture of embryogenic callus
2.2. Long-term embryogenic cultures
2.3. Somatic embryogenesis from embryonic tissues
3. Embryo teratology and low conversion rate

329
332
332
334

3.1. Somatic embryo teratology
3.2. Plant development
3.2.1. Dormancy
3.2.2. Morphological and physiological alterations
3.2.3. Culture conditions
3.2.4. Germination treatments
4. Towards a better understanding of grape somatic embryogenesis

334
336
336
337
338
340

340

4.1. Ontogenesis and differentiation of somatic embryogenesis

4.2. Molecular markers for somatic embryogenesis characterization
5. Conclusions

341
343

Abbreviations

346

Acknowledgments

346

References

346

345

Chapter 14
PROTOPLAST TECHNOLOGY IN GRAPEVINE
A. Papadakis, G. Reustle and K.A. Roubelakis-Angelakis
1. Introduction

353

353

XVIll

2. Recalcitrance

354

2.1. Plasma membrane functioning
2.2. Oxidative stress
2.2.1. Generation of Active Oxygen Species
2.2.2. Scavenging of active oxygen species
2.3. The role of polyamines
3. Isolation of grapevine protoplasts

355
356
357
362
369
369

3.l. Donor plant material
3.2. The isolation method
3.2.1. Enzymic treatment
3.2.2. Purification
3.2.3. Assessment of protoplast quality
3.2.4. Culture conditions
4. Progress in grapevine protoplast technology

370
373

373
375
376
376
381

5. Applications of protoplast technology

382

5.1. SomacIonal variation
5.2. In vitro selection
5.3. Somatic hybridization
5.4. Genetic transformation
5.5. Protoplasts as test system
5.6. Prospects
Acknowledgements

382
383
383
384
385
385
386

References

386

Chapter 15
GRAPEVINE GENETIC ENGINEERING

393

J.R. Kikkert, M.R. Thomas and B.!. Reisch
1. Introduction

393

2. Application of Genetic Engineering to Grapevine Breeding and Genetics

394

3. Historical development of grapevine transformation systems

395

3.1. Early transformation work
3.2. Importance of embryogenic cultures
3.3. Successful transformation methods
3.3.l. Agrobacterium
3.3.2. Biolistics
3.4. Methods for selection and evaluation of transformants
4. Current status of grapevine transformation

395
396
399
399

400
401
402

5. Environmental release/commercialisation
5.1. Regulatory issues

403
403


xix

5.1.1. Europe
5.1.2 Australia
5.1.3 United States
5.2. Patenting
5.3. Naming
5.4. Public perception
6. Acknowledgments

403
404
404
405
405
406
406

References

407

Chapter 16
GENETICALLY ENGINEERED GRAPE FOR DISEASE AND STRESS
TOLERANCE

411

V. Colova-Tsolova, A. Perl, S. Krastanova, J. Tsvetkov and A. Atanassov
1. Introduction

411

2. Basic terms in genetics of host/pathogen interaction

412

3. Advantages and limitations of genetic transformation

414

4. Gene transfer in Grape for improved tolerance toward biotic and abiotic
stress
4.1. Viruses
4.2. Fungal pathogens
4.3. Bacteria
4.4. Nematodes and insects
4.5. Abiotic stress
5. Co-transformation as an advanced approach for integration of multiple

genes to confer for disease tolerance in grape

417
417
421
423
424
425
425

6. Concluding remarks

427

Acknowledgements

427

References

427

Chapter 17
MICROSATELLITE MARKERS FOR GRAPEVINE:
A STATE OF THE ART

433

K.M. Sefc, F. Lefort, M.S. Grando, K.D. Scott, H. Steinkellner
and M.R. Thomas

1. Introduction

433

2. What are micro satellites ?

436


xx
3. Development of microsatellite markers in Vilis

436

4. EST derived microsatellite markers: a new strategy

438

5. Identification of cultivars of Vitis vinifera and rootstocks from Vitis species
5.1 Source and quality of DNA used for PCR amplification
5.2. Analysis methods available and comparison between them
5.3. Identification of grapevine cultivars and rootstocks by using nuclear
SSRS
5.4. Synonyms
5.5. Clonal lines and somatic mutants
5.6. Pedigree reconstruction
5.6.1 Methodology
5.6.2. Examples for the reconstruction of grapevine crosses
6. Genetic studies of the european Vitis vinifera germplasm

439
439
439
440
443
445
445
445
447
449

7. Chloroplast SSR markers

451

8. Use of SSR markers for genetic mapping of Vitis vinifera in combination
with other markers

451

9. Computer programs for micro satellite data analysis
9.1. Introduction
9.2. Identity 1.0
9.2.1. Management of germplasm collections
9.2.2. Evaluation of micro satellite markers
9.3. Popgene
9.3.1. Evaluation of microsatellite markers
9.3.2. Characterisation of grapevine gene pools
9.3.3. Cluster analysis
9.4. Other computer programs

9.4.1. Other programs for cluster analysis
9.4.2. Other programs for the characterisation of grapevine gene pools
10. Genetic databases of SSR profiles

452
452
453
453
453
453
453
454
454
454
454
454
454

11. On the way to commercial certification of cultivars

455

12. Conclusion and prospects for the future

456

Acknowledgments

457

References

457

Author Index

465

Subject Index

467


CONTRIBUTING AUTHORS
K. Adams

V. Colova-Tsolova

Department of Horticulture,
Viticulture & Oenology, Waite Campus,
University of Adelaide, PMB 1,
Glen Osmond, South Australia 5064, Australia.

Center for Viticultural Science,
College of Engineering Sciences,
Technology and Agriculture,
Florida Agricultural and Mechanical University,
Tallahassee, FL 32307, USA.

A. Atanassov

Institute of Genetic Engineering, 2232
Kostinbrod-2, Bulgaria.

A.Aziz
Laboratory of Plant Biology and Physiology,
UPRES EA 2069 URVVC,
University ofReims, B.P. 1039,
F-5l687 Reims Cedex 2, France.

L. Bavaresco
Institute ofPomology and Viticulture,
Sacred Heart Catholic University,
Via Emilia Parmense 84,
29100 Piacenza, Italy.

P.K. Boss
Commonwealth Scientific and Industrial
Research Organisation, Plant Industry,
Horticulture Research Unit, P.O. Box 350,
Glen Osmond, South Australia 5064, Australia.

A. Bouquet
UMR Diversity and Genomes of Cultivated
Plants, INRA, Grape Breeding Experimental
Station "Le Chapitre",
34751 Villeneuve iI':s Maguelone, France.

B. Bravdo
The Hebrew University of Jerusalem,
Faculty of Agriculture,

Institute of Plant Sciences and Genetics,
The Kennedy-Leigh Center for Horticultural
Research, Jerusalem, Israel.

A. Carbonneau
Institut Superieur de la Vigne et du Vin, AGRO
Montpellier-Viticulture, 2 Place P. Viala F,
34060 Montpellier Cedex 1, France.

C. Davies
Commonwealth Scientific and Industrial
Research Organisation, Plant Industry,
Horticulture Research Unit, P.O. Box 350,
Glen Osmond, South Australia 5064, Australia.

A. Deloire
Institut Superieur de la Vigne et du Vin, AGRO
Montpellier-Viticulture, 2 Place P. Viala,
34060 MontpelIier Cedex 1, France.

S. Delrot
UMR CNRS 6161, Laboratoire de Physiologie
et Biochimie Vegetales, University of Poi tiers,
40 Avenue du Recteur Pineau,
86022 Poitiers Cedex, France.

C. Fregoni
Institute ofPomology and Viticulture,
Sacred Heart Catholic University,
Via Emilia Parmense 84, 29100 Piacenza, Italy.

J.P. Gaudillere
Unite d'Agronomie, BP 81, INRA,
33883 Villenave d'Omon, France.

L. Geny
Faculty of Enology, University Victor Segalen
Bordeaux II, 33405 Talence, France.

P.G. Goussard
Department of Viticulture and Oenology,
University of Stellen bosch Private Bag Xl, 7602
Matieland (Stellenbosch), South Africa.

M.S. Grando
Istituto Agrario, Lab. Biologia Molecolare,
Via Mach I, 38010 San Michele all'Adige, Italy.


XXII

I. Gribaudo

K.A. Paschalidis

Centro Miglioramento Genetico e Biologia del1a
Vite - CNR, via Leonardo da Vinci 44,
10095 Grugliasco, Italy.

Department of Biology, University of Crete,

P.O.Box 2208,71409 Heraklion, Crete, Greece.

J. Grubb
Cooperative Research Centre for Viticulture,
Glen Osmond, South Australia 5064, Australia.

Y. Hayasaka
The Australian Wine Research Institute,
P.O. Box 197, Glen Osmond,
South Australia 5064, Australia.

P.B. Hoj
Department of Horticulture, Viticulture &
Oenology, Waite Campus,
University of Adelaide, PMB I,
Glen Osmond, South Australia 5064, Australia.

A. Perl
Department of Fruit Tree Breeding and
Molecular Genetics, Institute of Horticulture,
Agricultural Research Organization,
The Volcani Center, P.O. Box 6,
50250 Bet-Dagan, Israel.

S. Picaud
UMR CNRS 6161, Laboratoire de Physiologie
et Biochimie Vegetales, University of Poitiers,
40 Avenue du Recteur Pineau,
86022 Poi tiers Cedex, France.

K.F. Pocock

J.R. Kikkert

The Australian Wine Research Institute,
P.O. Box 197, Glen Osmond,
South Australia 5064, Australia.

Cornell University, New York State Agricultural
Experimental Station, Department of
Horticultural Sciences, Geneva,
NY 14456, USA.

Department of Biology, University of Crete,
P.O.Box 2208, 714 09 Heraklion, Crete, Greece.

N.I. Primikirios

S. Krastanova

B.I. Reisch

Cornell University, New York State
Agricultural Experimental Station,
Department of Plant Pathology, Geneva,
NY 14456, USA.

Cornell University, New York State
Agricultural Experimental Station,
Department of Horticultural Sciences, Geneva,

NY 14456, USA.

F. Lefort

G. Reustle

Department of Biology, University of Crete,
P.O.Box 2208,71409 Hcraklion, Crete, Grecce.

2 Centrum Grtine Gentechnik, SLFA Neustadt,
Breitenweg 71, D67435 NeustadtlWeinstrasse,
Germany.

K.A. Loulakakis
Department of Horticulture,
Technological Educational Institution of Crete,
71500 HerakJion, Crete, Greece.

C. Romieu

L. Martinelli

INRA, Unite de Recherche des Produits de la
Vigne, Institut National de la Recherche
Agronomique, 2 place Viala,
34060 Montpcllier Cedex 01, France.

Laboratorio Biotecnologie, Istituto Agrario,
38010 San Michele all'Adige (TN), Italy.

K.A. Roubelakis-Angelakis

A. Papadakis

Department of Biology, Univcrsity of Crete,
P.O.Box 2208,71409 Heraklion, Crete, Greece.

Department of Biology, University of Crete,
P.O.Box 2208,71409 Heraklion, Crete, Greece.


xxiii
K.D. Scott

N. Terrier

Centre for Plant Conservation Genetics,
P.O. Box 157, Lismore NSW 2480,
Southern Cross University, Australia.

INRA Unite de Recherche des Produits de la
Vigne, Institut National de la Recherche
Agronomique, 2 place Viala,
34060 Montpellier Cedex 01, France.

K.M. Sefc
Zentrum flir Angewandte Genetik,
Universitat flir Bodenkultur,
Wien Muthgasse 18, A-II90 Vienna, Austria.

O. Shoseyov
The Hebrew University of Jerusalem,
Institute of Plant Sciences and Genetics,
The Kennedy-Leigh Center for Horticultural
Research, Jerusalem, Israel.

I. Skrumsager Moller

C. Tesniere
INRA, Unite de Recherche sur les Produits de la
Vigne, 2 Place Viala,
34060 Montpellier Cedex 1, France.

M.R. Thomas
CSIRO Plant Industry, P.O. Box 350,
Glen Osmond, South Australia 5064, Australia.

L. Torregrosa
UMR Biology of Development of Cultivated
Perennial Plants, ENSAM-INRA, 2 place Viala,
34060 Montpellier Cedex I, France.

Department of Horticulture, Viticulture and
Oenology, Waite Campus,
University of Adelaide, Glen Osmond,
South Australia 5064, Australia

I. Tsvetkov

H. Steinkellner

Institute of Genetic Engineering, 2232
Kostinbrod-2, Bulgaria.

Zentrum flir Angewandte Genetik,
Universitlit fUr Bodenkultur Wien
Muthgasse 18, A-I 190 Vienna, Austria.

R. van Heeswijck

A.P. Stines
Department of Horticulture, Viticulture and
Oenology, Waite Campus,
University of Adelaide, Glen Osmond,
South Australia 5064, Australia

D.B. Tattersall
Department of Horticulture,
Viticulture & Oenology, Waite Campus,
University of Adelaide, PMB 1,
Glen Osmond, South Australia 5064, Australia

Department of Horticulture, Viticulture and
Oenology, Waite Campus,
University of Adelaide, Glen Osmond,
South Australia 5064, Australia

C. Verries
INRA, Unite de Recherche sur les Produits de la
Vigne, 2 Place Viala, 34060 Montpellier Cedex

1, France.

E.J. Waters
The Australian Wine Research Institute,
P.O. Box 197, Glen Osmond,
South Australia 5064, Australia.


1
MOLECULAR BIOLOGY OF SUGAR AND ANTHOCYANIN
ACCUMULATION IN GRAPE BERRIES
P.K. Boss and C. Davies
Commonwealth Scientific and Industrial Research Organisation
Plant Industry, Horticulture Research Unit, P.O. Box 350
Glen Osmond, South Australia 5064, AUSTRALIA

1. INTRODUCTION
As grape berries develop, they change in size and composition. Grape berries exhibit a
double sigmoid pattern of growth (Coombe, 1992); the first rapid growth phase that occurs after fruit set is due to an increase in cell numbers and an expansion of existing
cells. Cell division in the pericarp is largely completed in the first few weeks of development (Harris et al., 1968). In most cultivars, the first expansion phase is followed by a
lag phase during which little or no growth occurs. The second growth phase, which occurs at the end of the lag phase, coincides with the onset of ripening. The French word
"wiraison", which describes the change in berry skin colour as ripening commences, has
been adopted as a useful tenn to describe the onset of ripening.
The most dramatic change in berry development occurs as the fruit enters into the ripening phase. During ripening, berries change from being small, finn and acidic with little
sugar, desirable flavours or aroma into larger, softened, sweet, highly flavoured, less
acidic and, in the case of some varieties, highly coloured fruit. The development of these
characteristics detennine the quality of the final product. The increase in berry size and
the reduction in firmness, that occur during ripening, are accompanied by changes to the
structure and composition of the berry cell wall (Nunan et al., 1998). The development
of flavour in grapes is due to both the acid/sugar balance (particularly important in table

K.A. Roubelakis-Angelakis (ed.), Molecular Biology & Biotechnology of the Grapevine, 1-33.
© 2001 Kluwer Academic Publishers.


2

P.K. BOSS and C. DAVIES

grapes) and the synthesis of flavour and aroma metabolites during the ripening phase.
Berry colour results from the synthesis and accumulation of a group of coloured secondary metabolites, called anthocyan ins.
Much of the basic physiology of grape berry ripening has been described but less is
known of the biochemical and molecular events involved. Grapevines provide particular
challenges for these studies as their tissues are rich in phenolic compounds, in particular,
tannins. This makes the extraction of nucleic acids somewhat difficult and the extraction
of proteins, especially active enzymes, even more problematic. In addition, the enzymes
that produce the secondary metabolites, responsible for quality factors such as flavour
may be present in low amounts further increasing the problems of studying them directly.
The striking change in gene expression that occurs at veraison (Davies and Robinson,
2000), indicates that much of the transition into ripening is driven by changes in gene
transcription. Both structural genes and the controlling genes that affect their transcription are involved. It is likely that altered levels of plant hormones, or altered sensitivity
to them, affect this dramatic change in gene expression. However, unlike climacteric
fruits, where ethylene affects ripening and the expression of ripening-related genes, the
hormone(s) controlling grape berry ripening are yet to be identified.
The use of molecular biology to study ripening related processes is therefore appropriate for two reasons. First, molecular biology techniques can be adapted to deal with
difficult tissues and have the sensitivity to detect small but important changes in gene
expression. Second, changes in gene expression are a crucial component of the berry
ripening process. The understanding of ripening thus gained, offers the potential to improve grape quality and productivity via modified cultural practices or plant breeding,
which includes the use of molecular markers and molecular breeding.
The development of colour in red and black grape varieties and the rate and level of
sugar accumulation in both white and coloured varieties are considered to be important

contributors to grape berry quality. The current state of knowledge of the molecular biology ofthese two processes will be outlined in this Chapter.

2. THE MOLECULAR BIOLOGY OF SUGAR TRANSPORT AND
ACCUMULATION IN GRAPE
In grapevines, carbohydrate produced by photosynthesis is exported from the leaf as
sucrose and is transported via the phloem to the berry (Swanson and Elshishiny, 1958).
The mechanisms of carbon partitioning and accumulation in grapevines are poorly understood. However, considerable progress has been made in this area in other plants and
this information can be used to aid our understanding of these processes in grapes. There
is convincing evidence that membrane-located sugar transporter proteins are heavily
involved in the active transport and redistribution of sugars between cells and tissues
(reviewed by KUhn et aI., 1999; Lalonde et al., 1999). In Figure 1.1 the possible routes
of sugar transport and the roles the various transporters might play are illustrated. Sugars


SUGARS AND ANTHOCYANINS IN GRAPE BERRlES

3

Photosynthesis

~Trlos. p
~sue

Gle.Fru

i/"'-

Symplastic
unloading

~

j Yi.fI§:M

Sue
Apoplastic
loading

Recovery

Symplastic
unloading

yi.~sue
Apoplastic
unloading

Gle + Fru

Figure 1.1. Schematic of the possible transport routes of photosynthate in grapevine. Sucrose
(Suc) produced in mesophyll cells is loaded into the phloem by either a symplastic or apoplastic
mechanism. Arrows indicate the direction of sugar movement and in the case of symporters the
direction ofW movement. Transporter proteins are shown as solid (anti porters) or hatchcd (symporters) circles. Plasmadesmata are indicated as channels through the cell wall. Sucrose loaded
into the phloem is then transported by mass flow (the movement of water into the phloem is not
shown for the sake of clarity) to the sink tissue, the berry. Along the phloem pathway sucrose
symporters may be active in the recovery ofleaked photosynthate. Unloading may be by two basic
methods. Symplastic unloading occurs via plasmadesmata. Sucrose may then be moved across the
tonoplast into the vacuole by a sucrose transporter protein. Cleavage by invertase, in this case
vacuolar invertase, is thought to account for the roughly equal amounts of glucose (Glc) and fructose (Fru) that are present in berries during ripening. In apoplastic unloading, sucrose transporters
release sucrose into the apoplast. It may then be transported as sucrose into the vacuole, where

cleavage by vacuolar invertase occurs. The alternative is that cleavage may occur in the apoplastic
space by an extracellular invertase. The resultant glucose and fructose are then imported across the
plasma membrane by a monosaccharide symporter and then across the tonoplast for storage in the
vacuole.

that are ultimately stored in the cell vacuole must cross tonoplast and/or plasma membranes at some stage in their transport cycles whether the modes of loading and unloading are symplastic or apoplastic. Both sucrose and hexose transporters have been identified and may be important in sugar accumulation in grape berries.


4

P.K. BOSS and C. DAVIES

Until veraison, most of the sugar imported into the berry is metabolised and so there
is little storage. After veraison, there is a steady rise in the levels of stored sugars and the
mechanism of sugar uptake into the berry may alter at this time. Cultivars of V. vinifera
generally store little sucrose and accumulate sugars in the form of the hexoses glucose
and fructose in roughly equal amounts. However, the levels of sugars stored and the sucrose to hexose ratio can vary somewhat depending on the cultivar (Lott and Barrett,
1967; Hawker et aI., 1976). Some cultivars, in particular those derived from V. labrusca,
store considerable amounts of sucrose, suggesting that they accumulate sugars in a different manner to vinifera cultivars. The sucrose cleaving enzyme invertase is likely to be
involved in the control of the composition of stored sugars. This review will concentrate
on the situation as it pertains to vini{era. In this species, cleavage of the transported sucrose to release glucose and fructose occurs in the berry (Kliewer, 1965). The site of this
cleavage has not been experimentally demonstrated and could occur in the apoplast, the
cytoplasm or in the vacuole itself (Coombe, 1992).
Phloem loading may occur by two different routes. The symplastic route involves the
movement of assimilates from leaf mesophyll cells through plasmadesmata to the
phloem. The alternative, apoplastic route requires the movement of assimilate across
membranes. The determination of which route predominates in a particular species has
been based on anatomical features, the type of assimilate transported and the reaction to
inhibitors (Kuhn et aI., 1999). The movement of assimilates across membranes is thought
to be an energy requiring process conducted by specialised transport proteins located

within the membrane. Sucrose and monosaccharide transporters are members of a large
family of transporter proteins that share amino acid sequence similarity. These transporters also have similar structures as they contain 12 membrane-spanning regions. Members
of this family, known as the major facilitator superfamily, conduct the proton-coupled
transport of a wide range of molecules, including disaccharides and monsaccharides
(Marger and Saier, 1993). There are potentially two functional types of sugar transporters. Those that transport sugars in the direction of the proton gradient are symporters,
and those that transport sugar molecules against the proton gradient are antiporters. Proton-sucrose symporters localised to the plasma membrane conduct the import of sucrose
into the cell and have been isolated from a number of plant species (Sauer et aI., 1994;
Rentsch and Frommer, 1996). Some of these sucrose transporters have been localised
predominantly to the phloem (Riesmeier et aI., 1993; Stadler et aI., 1995; Truernit and
Sauer, 1995; Stadler and Sauer, 1996) and it is therefore proposed that these transporters
are involved in an apoplastic mechanism of phloem loading. The increased expression of
these transporter genes in source tissues supports this view as do the results of experiments using antisense technology to suppress sucrose transporter gene expression (Riesmeier et aI., 1994; Kuhn et aI., 1996; Lemoine et aI., 1996).
The mechanism of unloading sucrose from the phloem is less clear. In some species a
symplastic model for unloading is preferred (KUhn et aI., 1999). The alternative to this is
an apoplastic model where sucrose is downloaded by antiporter proteins. A number of
sucrose transporter genes are expressed in non-source tissues (Riesmeier et aI., 1993;
Stadler et aI., 1995; Truernit and Sauer, 1995; Gahrtz et al., 1996; Hirose et aI., 1997).