sugar accumulation in the developing grape berry

311 Pages·2016·14.77 MB·English

by  Spencer C. Brown

SUGAR ACCUMULATION IN THE DEVELOPING GRAPE BERRY
by
Spencer C. Brown, B.Ag.Sc. (Ade1;)
Department of Plant Physiology
Waite Agricultural Research Institute
The Univcrsity of Âdclai<lc
South Australia
Thesis submitted for the Degree of
Doctor of Phi losophy
January,1981
Frontispiece: T.A. Bramley in Finean et aL. (1978) with permission.
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TABLE OF CONTENTS
Page
SUMMARY x
STATEMENT xl11
xiv
PREFACE
LIST OF FIGURES XV
LIST OF TABLES xx
LIST OF SYMBOLS AND MISCELLANEOUS ABBREVIATIONS xxiii
LIST OF ABBREVIATIONS (CHEMICALS) xxiv
1.
CHAPTER DEVELOPMENT OF THE GRAPE BERRY
The grape inflorescence
1 1 1
) Designation of developnent
1 3
1 3 Ontogeny of the pericarp 4
1 4 The cuticle 6
1 5 Vascular connections 7
1 6 Constituents of the bqcry 7
a. Introduction
7
b. Watcr and carbohydratcs
B
c. Organic acids 10
d. Minerals
11
e. Nitrogen
L2
f.
Adenylates 12
g.
Phenolics
T2
h.
Enzymes 13
L.7 Studies of gaseous exchange L4
1.8 Malate, its enzymes and gluconeogenesis L7
1.9 Hormones 18
1. 10 Interactions of other factors 20
CHAPTER 2. THE PROCESS OF VERAISON
2.L What regulates ripening? 27
l4C
2.2 studies 23
2.3 Does the route of solute transfer change? 25
2.4 Specific studies of veraison
25
2.5 Water and osnotic relationships 28
2.6 Summary 30
CHAPTER 5. METABOLISM OF SUCROSE, GLUCOSE AND FRUCTOSE
3.1 Introduction 32
3.2 Structures 32
5.3 Metabolisrtl s4
3.3a Starch biosynthesis and degradation
34
3.3b Sucrose synthesis
35
3.4 Pathways of Carbon Metabolisn
36
3.4a Regulation
36
3.4b Examples of integration
37
3.4c funbiquitous enzfrnes 37
CHAPTER 4. TRANSPORT IN HIGHER PLANTS : CONCEPTS AND TERMINOLOGY
4.1 Introduction
43
4.2 Membrane Structure 45
4.3 Mernbrane Flux 45
4.4 Mediated Processes 47
4.5 Active Transport
48
4.6 Other Transport Processes 51
CHAPTER 5. TRANSPORT PROCESSES IN HIGHER PLANTS : SPECIFIC EXAMPLES
5.1 Cotransport of Sugars 53
5.2 Transport Metabolites
54
5.3 Group Transport of Sugars 54
5.4 Sugar Accumulation in Sugarcanc 57
5.5 Integration of Proccsses by Cotransport
5B
5.6 In Vitto Diffusion
5B
5.7 Superimposed Processes ? 59
5.8 Vesicular Transport 60
CFTAPTER 6. TTIE USE OF PROTOPLASTS IN TRANSPORT STUDIES
6.1 Protoplasts as a Tool in Fruit Physiology
61
6.2 Is the Physiology of the Protoplast Representative?
65
6.3 Pcrturbed Metabolism
63
6.4 Electrical Properties
64
6.5 rrRecoveredrr Protoplasts
65
6.6 The Literature
66
6.7 Protoplasts in Uptake Studies
66
CHAPTER 7. TI{E VACUOLE OF HIGIIER PLANTS AND TRANSPORT AT THE TONOPLAST
7.L Introduction
72
7.2 Tonoplast
73
i
7.3 Functions of the Vacuofie
7S
.3a Reg,ulatory function
7 75
7.3b Accumulative function
77
7.3c Lytic function
79
7.3d Defence function
80
v].
.
7.4 Technique for Isolation 81
7.5 Transport at the Tonoplast B7
7.6 ATPase 88
7.7 Conments on Sugar Accumulating Mechanisms 89
CHAPTER B. EFFLUX STUDIES OF HEXOSE COMPARTMENTATION IN TIIE
DEVELOPING GRAPE BERRY AND TTIE EFFECTS OF SHADING
8.1 Introduction 94
8.2 Materials and Methods 95
8.5 Results 99
8.4 Discussion 106
8.4.1 Sugar in the developing grape bcrry
1.06
8.4.2 Effect of shading 108
8.5 Conclusions t72
CTI,APTER 9. ISOLATION OF PROTOPLASTS AND VACUOLES
9.1 Protop lasts 174
9.1.1 Initial protocol for protoplast preparation It4
I .I.2 Losses 115
9. 1.3 Alternative osmotica 115
9.1.4 A nannitol series 115
9.1.5 Protoplasts from berries in the first growth
cycle
TL7
9.1.6 Effects of ions and other additives 118
9.7.7 Geometry of the incubation vessel 720
9.1.8 Cell-wal1 degrading enzymes L20
9.1.9 Summary and protocol L2L
9.2 Protoplast yield and agglutination L22
9.2,I Chlorophyll as an index of yield L22
9.2.2 Concanavalin A with protoplasts L26
9.3 Vacuoles 127
9.3.1, Vacuole isolation L27
9 .3.2 Counts and yields t27
9.3.3 Key variables of the protocol L27
9.4 Microscopy L29
9.4.1 Observations of lysis t29
9.4.2 Micrographs and fluorescence 131
9.4.5 Electronmicrographs r32
v11
.
10.
CIIAPTER PURIFICATION OF PROTOPLASTS AND VACUOLES
10. 1 Introduction L48
t0.2 Fi ltration 148
10. 3 Phase Distribution L50
10.4 Centri fugation 1s1
10.4. 1 Materials 1s1
10.4.2 Differential pelleting Fi col t52
1
10.4.3
Metrizarnide 155
10.4.4 Ludox and Percoll 7s7
10.5 Discussion 159
C}IAPTER 11 . MEMBRANE TRANSPORT STUDIES WITH ISOLATED PROTOPLASTS AND
VACUOLES
11.1 Introduction 161
1,t.2 Mâterials and Methods 163
11.3 Results and Discussion 168
11.3.1 Protoplasts : Uptake of glucose and sucrose 168
11.3.2 Cornpeti.tion by deoxyglucose but not raffinose 169
11.3.3 Alternative glucose radionuclides with
protopl asts L72
11.3.4 Interaction of glucose and fructose L74
11.3.5 Effect of pH t74
11.3.6 Effect of pressure upon uptake by protoplasts 178
11.3.7 Vacuoles : Uptake of glucose and sucrose 180
L1.3.8 Variability between experiments t82
11.3.9 Uptake by vacuoles from stored berries? 186
tl.4
General Discussion 188
CtL\PTIllì 12. SUBSTIìATD DEPENDENCY OF SUCAR UPTAKD BY PIìOTOPLASTS ANI)
ISOLATI]D VACUOLES
12.t Introduction
191
12.2 Materials and Methods 191
12.3 Results and Discussion : Protoplasts 193
12.3.1 Data from protoplasts
193
L2 .3 .2 Ef f lux 193
12.3.3 The nature of rrzero-tine uptake" 196
L2.3.4 Algebraic description of accumulation 198
t2.3.5 And the plrysicaI basis? 200
v]-11.
t2.4 Results and Discussion : Vacuoles 203
12.4.1 Data from vacuoles 203
12.4.2 The relative rates of accumulation 206
12.4.3 Zero-tirne uptake may have a different source
in
vacuoles 208
72.5 Conclusions 209
CHAPTER 13. RADIOCHEMICAL LABELLING FROM THE INCUBATION OF SKIN
SEGMENTS, PROTOPLASTS AND VACUOLES OF THE GRAPE BERRY
13. 1 Introduction 2LI
13.2 General Materials and Methods 2L2
13.3 Labelling patterns in skin segments 275
13.3. 1 Results 2rs
13.3.2
Discussion
220
(i) The distinctive nature of each fraction 220
(ii) X, Y,
Z 220
(iii) On specific activities 220
(iv)
Sucrose 222
(v) Glucose, fructose and isomerization
222
(vi)
Changes upon storage 225
13 .4 Electrophoresis of l\C-conpounds 225
Results and Discussion 225
(i) Mobility of standards in electrophoresis
and chromatography 225
(ii) xl, Yl,
7r 227
(iii) Labelling of galactose
232
13. 5 l4C labe1ling in protoplasts and vacuoles 234
Results and Discussion 234
(i) Metabolism of sucrose and isomerization of
glucose in protoplasts
234
(ii) Evidence for fructose-, glucose- and sucrose-
phosphate from protoplasts 238
(iii) Accurnulation of ZI is cytoplasrnic 238
(iv) Zl was not labelled from 3-3H-glucose, but
rapidly from U-taC-G6p 240
(v) Sucrose synthesis and hydrolysis by isolated
vacuoles 24r
13.6 General discussion 24s
lx.
14.
CHAPTER INTEGRATIVE DISCUSSION
t4.L Preamble 247
74.2 Mediated Transport 247
L4.3 Indeed, Group TransPort 248
.4 Sucrose Synthesizing Apparatus 249
1.4
14. s Hexose compartmentation in the developing grape berry 250
t4.6 -Inter a LLa 252
L4 .7 Conclusion 254
BIBLIOGRAPHY 256
X
SUMMARY
Sugar accumulation was examined in the berry of the grape, Vitis
1
vinifera cv. Muscat Gordo Blanco, at the inception of the ripening
stage of bemy developnent.
Reviews of the literature are presented pertaining to development of
2
the grape berry, menbrane transport of sugars in higher plants,
techniques for the isolation of vacuoles from plant naterial, and the
use of isolated protoplasts and vacuoles i.n uptake studies.
3 The working hypothesis describing the mechanisn of accumulation of
carbohydrates has been: unloading of sucrose from the phloem into
the apoplasm followed by inversion to hexoses and diffusion,
novement of hexoses across the plasnalernma, synthesis of sucrose or
sucrose phosphate in the cytoplasm with transport to the vacuole
where, finalI¡ inversion occurs.
4 Components of the hypothesis r^rere examined stepwise by analysis of
sugar compartmentation in vivo, and by in vitro manipulations of
excised pericarp tissue, and of protoplasts and vacuoles isolated
therefrom.
5 Compartmentation of hexoses in the pericarp of the developing grape
berry, proceeding from veraison, was exanined by efflux analysis, as
too the effect of shading upon this. The method indicated half the
hexoses to be diffusible at veraison, and 15 days later B0% (of a
larger total) were diffusj.ble. This suggests that the onset of rapid
sugar accu¡nulation in the grape berry results primarily from changes
in phloem unloading rather than from concentrative accumulation by
the celI. Nevertheless the skin of a pre-veraison berry is a glucose
accumulator, and, post-veraison, it becomes a fructose and glucose
accumulator.
6 Methods were developed for protoplast release frorn the skin of berries
by the action of cellulases, and for subsequent isolation of vacuoles
by DEAE-Dextran-induced lysis of these protoplasts. Protoplasts and
vacuoles were purified by filtration through coarse Sephadex and by
ultracentrifugation in metrizamicle. A method is presented for a
sequential, enzymatic assay of glucose, fructose and sucrose.