Biochemistry of fruit colour in apples (Malus pumila Mill.)

268 Pages·2010·10.56 MB·English

by  Carolyn Elizabeth Lister

BIOCHEMISTRY OF FRUIT COLOUR IN APPLES
(Malus pumila Mill.)
A thesis submitted in
partial fulfilment of
the requirements for the degree
of
Doctor of Philosophy in Biochemistry
at the
University of Canterbury
by
Carolyn Elizabeth Lister
University of Canterbury (1994)
Christchurch, New Zealand
ii
CONTENTS
Page
Ab breviations v
Key to trivial names of apple flavonoids vii
List of figures viii
List of plates x
List of tables xi
ABSTRACT 1
Section 1 • INTRODUCTION
1.1 Fruit colour 3
1.2 The flavonoids 6
1.2.1 Anthocyanins 10
1.2.2 Flavonols 12
1.2.3 Proanthocyanidins and related compounds 13
1.2.4 Other flavonoids 14
1.3 Apple cultivation and improvement 17
1.4 Biological significance of the flavonoids 20
1.5 Experimental strategy and outline 21
Section 2· IDENTIFICATION OF THE FLAVONOIDS IN APPLE SKIN
2.1 Introduction
2.1.1 Anthocyanins 22
2.1.2 Flavonols 25
2.1.3 Proanthocyanidins 28
2.1.4 Other flavonoids 30
2.1.5 Distribution of the flavonoids 31
2.1.6 HPLC 32
2.2 Materials and methods 34
2.3 Results 39
2.4 Discussion 55
2.5 Conclusions 64
III
Section 3 - FACTORS AFFECTING APPLE FRUIT COLOUR
3.1 Introduction
3.1.1 Pigments in fruit 65
3.1.2 Anthocyanins and fruit colour 66
3.1.3 Factors in the expression of colour 67
3.1.3.1 pH 67
3.1.3.2 In vivo stabilization of colour 68
3.1.3.3 Pigment distribution 71
3.1.3.4 Other factors 72
3.1.4 Influence of plastid pigments 72
3.1.5 Measurement of colour 76
3.2 Materials and methods 78
3.3 Results 81
3.4 Discussion 94
3.5 Conclusions 99
Section 4 - DEVELOPMENTAL CHANGES IN PIGMENT COMPOSITION
AND LEVELS
4.1 Introduction
4.1.1 Developmental changes in pigment composition 100
4.1.1.1 The flavonoids 100
4.1.1.2 Plastid pigments 105
4.1.2 Apple fruit development 106
4.2 Materials and methods 107
4.3 Results 109
4.4 Discussion 119
4.5 Conclusions 123
Section 5 - BIOSYNTHESIS OF THE FLAV ONOIDS
5.1 Introduction
5.1.1 The flavonoid biosynthetic pathway 124
5.1.2 Regulation of flavonoid biosynthesis 131
5.1.3 Organisation of flavonoid biosynthesis 132
5.2 Developmental regulation of flavonoid enzyme activities
5.2.1 Introduction
5.2.1.1 Phenylalanine ammonia-lyase 136
5.2.1.2 Chalcone isomerase 139
iv
5.2.1.3 Flavonoid glycosyltransferases 140
5.2.1.4 Extraction of enzymes 142
5.2.2 Materials and methods 143
5.2.3 Results 149
5.2.4 Discussion 161
5.3 Further elucidation of the regulation of flavonoid biosynthesis
5.3.1 Introduction
5.3.1.1 Elucidation of flavonoid biosynthesis by the
use of mutants 172
5.3.1.2 UV induction of flavonoids 175
5.3.2 Materials and methods 176
5.3.3 Results 177
5.3.4 Discussion 182
5.4 Conclusions 185
Section 6 - CONCLUDING DISCUSSION 186
Acknowledgements 194
References 195
Appendix A Source and description of apple genotypes and species 223
Appendix B Concentrations of flavonoids in apple genotypes and species 228
Appendix C Percentage composition of flavonoids in apple genotypes and species 235
Appendix D Papers published in relation to this thesis 242
v
ABBREVIATIONS
ANOVA analysis of variance
BAW butanol:acetic acid:water
BSA bovine serum albumin
octadecylsilica
CIS
eDNA complementary DNA
CHI chalcone isomerase
CHS chalcone synthase
CIE Commission Internationale de I'Eclairage
CoA coenzyme A
Cv(s) cultivar(s)
DHFR dihydroflavonol reductase
DHK dihydrokaempferol
DHQ dihydroquercetin
DNA deoxyribonucleic acid
DTE dithioerythritol
DTT dithiothrei tol
EC Enzyme Commission
EDTA ethylene-diaminetetraacetic acid
FW fresh weight
GT glycosyltransferase
HPLC high-performance liquid chromatography
ISCC-NBS Inter-Society Color Council - National Bureau of Standards
Kat Katal
Km Michaelis constant
LAB L'a'b' colour space coordinates
L-Phe L-pheny lalanine
LSD least significant difference
MES 2(N-morpholino )ethanesulfonic acid
mRNA messenger ribonucleic acid
MS mass spectroscopy
NADPH nicotinamide adenine dinucleotide phosphate, reduced form
NMR nuclear magnetic resonance
N.S. not significant at 5% rejection level
PAL phenylalanine ammonia-lyase
vi
PC paper chromatography
PVP polyvinylpyrrolidone
PVPP polyvinylpolypyrrolidone (PolyClar AT)
RP reversed phase
TBA t-butanol:acetic acid:water
TCA trichloroacetic acid
TFA trifluoroacetic acid
THF tetrahydrofuran
TLC thin-layer chromatography
Tris tris(hydroxylmethyl)aminomethane
UDP uri dine diphosphate
UV ultraviolet
A, wavelength
"-max wavelength of maximum light absorption
vii
KEY TO TRIVIAL NAMES OF APPLE FLAV ONOIDS
Afzelin kaempferol-3-rhamnoside
Astragalin kaempferol-3-g1ucoside
Avicularin quercetin-3-0:-L-arabinofuranoside
Guaijaverin quercetin-3-0:-L-arabinopyranoside
Hyperin quercetin-3-galactoside
Idaein cyanidin-3-galactoside
Isoquercitrin quercetin-3-g1ucoside
Meratin quercetin diglucoside
Quercitrin quercetin-3-rhamnoside
Reynoutrin quercetin-3-xy los ide
Rutin quercetin-3-rutinoside [L-rhamnose( 0:1-76)D-glucose]
Taxifolin dihydroquercetin
viii
LIST OF FIGURES
Figure Title Page
Figure 1.1 Structure and numbering of the flavonoid nucleus 8
Figure 1.2 Overall pathway to the main flavonoid groups 9
Figure 1.3 Structures of the common anthocyanidins 11
Figure 1.4 Structures of the common flavonols 12
Figure 1.5 Structures of the natural flavan-3-0Is, (+ )-catechin and (-)-epicatechin 15
Figure 1.6 Structure of the procyanidins Bl, B2 and Cl 15
Figure 1.7 Structure of the dihydrochalcone phloretin 16
Figure 1.8 Schematic diagram of a hypothetical biosynthetic pathway 19
Figure 2.1 Structure of cyanidin-3-galactoside 22
Figure 2.2 Structure of quercetin-3-galactoside 27
Figure 23 Structure of phloridzin 31
Figure 2.4 TLC of apple skin anthocyanins 40
Figure 2.5 Typical HPLC trace for the anthocyanins extracted from
a standard sample of apple skin 41
Figure 2.6 TLC of apple skin flavonols 43
Figure 2.7 Typical HPLC trace for the flavonols extracted from
a standard sample of apple skin 44
Figure 2.8 TLC of apple skin proanthocyanidins 47
Figure 2.9 Typical HPLC trace for the proanthocyanidins extracted from
a standard sample of apple skin 48
Figure 2.10 Proposed biosynthetic relationship of the flavonoids in apples 60
Figure 3.1 The structural transformations of anthocyanins in aqueous solutions at
room temperature at varying pH 67
Figure 3.2 Mechanisms of anthocyanin stabilization in vivo 69
Figure 3.3 Solid L*a*b* colour chart 76
Figure 3.4 Hue sequence and hue angle orientation on a CIELAB diagram
with ISCC-NBS colour names 92
Figure 3.5 Chart of modifiers related to colour tones 92
Figure 4.1 Developmental changes in concentrations of the three flavonoid groups
in the skin of three apple cultivars 110
Figure 4.2 Developmental changes in the amounts of the flavonoid groups per apple
for three apple cultivars 112
IX
Figure 4.3 Developmental changes in individual quercetin glycosides for two
apple cultivars 113
Figure 4.4 Developmental changes in individual proanthocyanidins for two
apple cultivars 115
Figure 4.5 Developmental changes in chlorophyll levels for three apple cultivars 117
Figure 4.6 Developmental changes in carotenoid levels for three apple cultivars 118
Figure 5.1 Biosynthetic origin of the flavonoid precursors 124
Figure 5.2 Putative biosynthetic pathway for the flavonoids in apples 127
Figure 5.3 Integrated and parallel pathways to flavonoid end products 133
Figure 5.4 Hypothetical model of a membrane associated CIS sequence capable
of accumulating cyanidin-3-glucoside 134
Figure 5.5 Deamination of L-phenylalanine, the reaction catalysed by PAL 136
Figure 5.6 General outline of phenylpropanoid metabolism 138
Figure 5.7 Synthesis of naringenin via chalcone synthase (CHS) and
chalcone isomerase (CHI) 140
Figure 5.8 Glycosylation of the flavonoids catalysed by UDP-flavonoid
glycosy ltransferase 141
Figure 5.9 Changes in enzyme activities and flavonoid concentrations in
'Splendour' apples during development 153
Figure 5.10 Changes in enzyme activities and flavonoid concentrations in
'Granny Smith' apples during development 154
Figure 5.11 Changes in enzyme activities and flavonoid amount per apple in
'Splendour' apples during development 156
Figure 5.12 Changes in enzyme activities and flavonoid amount per apple in
'Granny Smith' apples during development 157
Figure 5.13 Absorption spectra for flavonol complexes with Naturstoffreagenz A 159
Figure 5.14 Balanced relationship between protein metabolism and
phenylpropanoid accumulation 169
Figure 5.15 Quantification of flavonoid groups in chimeral stripes of four
apple cultivars 178
Figure 5.16 Quantification of the flavonoid groups in control, tree-reddened
and UV-induced apples 180
x
LIST OF PLATES
Plate Title Page
Plate 1.1 Range of colours displayed by apple fruit 5
Plate 3.1 Anthocyanin distribution in apple skin 73
Plate 3.2 Apple genotypes placed on a L"a'b' grid 90
Plate 5.1 Examples of chimeral stripes on apple skins 174