Transcript Sauvignon blanc Biochemistry and Molecular Biology: The

The Effects of Ultraviolet
Radiation and Canopy
Shading on Grape Berry
Biochemistry & Molecular
Biology
Professor Brian Jordan
Professor of Plant Biotechnology
Agriculture and Life Sciences Faculty
Lincoln University
Responses of Plants to Light
Photosynthesis
Light
Information
Sugars
other organic
compounds
Small amounts
of light
leaf growth
stem growth
germination, etc.
Daily duration
of light
flowering
dormancy
plant habit, etc.
Direction of
light
direction of
growth
UV-B
UV-A
Spectral irradiance (relative units)
Plants
Blue
Red & far red
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
300
400
500
600
700
800
Wavelength (nm)
900
1000
Ultraviolet Penetration through the Stratospheric Ozone Layer
PAR
700nm – 380nm
UV-A
380-315nm
UV-B
315-280nm
UV-C
<280nm
O3layer
100%
Earth’s surface
0%
Photoperception to gene
expression
Photoperception
Signal
Transduction
Gene
Expression
UV-B Photoreceptor
UV-B
Specific
Photoreceptor
Non-Specific
Via ROS
Signal Transduction
Changes to gene expression
Via DNA damage
Signal Transduction Pathways
UV-B
Peroxidase
NADPH oxidase
Receptor
NOS
?
O 2-
NO
Ca2+/CaM
H2O2
JA
Phosphorylation
Chloroplast signal,
electron transport/
photophosphorylation
Ethylene
H2O2
SA
Transcription factors
Chs
PDF1.2
PR genes
Photosynthetic genes
Role of UV/Light in Grape Development
and Wine Quality
•
Effect on “ageing” of white wines in New Zealand
•
Changes to polyphenolic compounds
•
Changes to amino acids/protein content
•
Impact on aroma/flavour (methoxypyrazines)
•
Lipoxygenase as an example of molecular approach
Vineyard experiments
•
•
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UVA+, UVB+ screen
UVA+, UVB- screen
UV- screen
No frame
No leaf removal, no frame
% Transmission
100
80
UV+
60
UVA+
40
UV-
20
0
250
275
300
325
Wavelength nm
350
375
400
UV-B Damage
No UV-B Damage
UV-absorbing compounds
Integrated area @ 352 nm
Total peak area
4000
3000
2000
1000
0
Lo UV
UV-A
UV-A/B
All UV
Amino Acid Metabolism and Implications for
Wine Industry
UV
(and PAR)
NITROGEN
(Uptake and assimilation)
Valine, isoleucine,
leucine
Methoxypyrazines:
amino acids as
precursors to
flavour and aroma
compounds
AMINO ACIDS
Phenylalanine,
tyrosine, tryptophan
Phenolics: amino
acids as
precursors –
implicated in
ageing and
bitterness in white
wine
Cysteine,
glutamate, glycine
All amino acids
except proline
Amino acid
composition and
implications for
fermentation
bouquet and
yeast assimilable
nitrogen
Glutathione:
implicated in the
prevention of
browning
process
Amino Acid Composition
Chardonnay
Sauvignon
blanc
Glutamine
Arginine
Proline
Proline
Arginine
Alanine
Increasing
Amounts
Glutamine
Alanine
Serine
Threonine
Glutamate
Serine
Light regulation of nitrogen
metabolism
• Light regulates the conversion of
glutamate into glutamine in the chloroplast
• This involves the GOGAT pathway and
requires ATP
Glutamate
Glutamine
• This assimilation of nitrogen then provides
amino acids/amines to the fruit
Amino acids
Glutamine
120
% of no-frame
100
80
60
40
20
0
Lo UV
UV-A
All UV
Amino acids
90
Glutamic acid
80
70
µM
60
50
40
30
20
10
0
No pluck
Lo UV
UV-A
All UV
No frame
Major aroma chemicals
• 3-mercaptohexanol/3mercaptohexanal
acetate
– Tropical fruit and
Citrus aromas
• Methoxypyrazines
– Green/green-pepper
or capsicum aromas
Present Understanding:
Synthesis of Thiol Precursors
Grape Metabolism through Berry Development and in
Response to the Environment
Lipids and
Fatty Acids
in Cell
Membranes
Hard
Solid
Berry
LOX
HPL
etc
5/6
Carbon
Backbone
eg, s-3(hexan-1-ol)GSTs Glutathione
VERAISON
‘Membrane Turnover’
Non
Volatile
s-cysteine
Conjugate
Precursor
Soft
Berry at
Harvest
Changes
during Must
Fermentation
Release
of Aroma
Volatiles
Primarily
by Yeast
LOX-HPL pathway
Biological membranes
Free fatty acids
Storage lipids
HOOC
CH3
a-linolenic acid
13-LOX
9-LOX
OOH
HOO
COOH
COOH
13(S)-HPOT
9(S)-HPOT
HPL
HPL
COOH
OHC
CHO
(3Z,6Z)-nonadienal
CHO
9-oxononanoic acid
ADH
ADH
IF
CHO
(2E,6Z)-nonadienal
IF
OHC
LOX?
(3Z)-hexen-1-ol
COOH
(9Z)-12-oxododec-9-enoic acid
IF
OH
OH
OHC
(3Z)-hexenal
COOH
Traumatin
CHO
(3Z,6Z)-nonadien-1-ol
(2E)-hexenal
O(O)H
HO
9(S)-HPOT - (10E, 12Z, 15Z)-9-hydro(pero)xy-10,12,15-octadecatrienoic acid;
13(S)-HPOT - (9Z,11E,15Z)-13-hydro(pero)xy-9,11,15-octadecatrienoic acid;
CHO
(2E)-4-hydro(pero)xy-2-hexenal
COOH
ADH
(9Z)-12-hydroxy-9-dodecenoic acid
HPL - hydroperoxide lyase;
LOX - lypoxygenase;
OH
ADH - alcohol dehydrogenase;
IF - isomerization factor;
(2E)-hexen-1-ol
HOOC
COOH
Traumatic acid
ADH
Phylogenetic analysis of grape LOXs and characterised
LOXs from other plants
13-LOXs
Type I
Type II13LOXs
9-LOXs
Type I
SB berry expressed LOXs
Relative expression of four berry
expressed LOXs
Proportional transcript abundance
100%
80%
60%
Skin
Pulp
40%
Seed
20%
0%
VvLOXA
VvLOXC
VvLOXD
VvLOXO
Proportional distribution of grape LOXs in
different berry fractions
Relative gene expressions of berry expressed LOXs during
development
Relative gene expressions of berry expressed LOXs during
upon wounding
Relative LOX gene expressions in SB berries infected with
Botrytis
I – berries with obvious signs of infection, NI – berries closely located to the
infected, Control – healthy berries distantly located from the infected.
Recombinant VvLOXA Enzyme Kinetics Data
LnA
LA
12
AA
10
0.26
0.24
8
1 / Rate, µmol•mg-1•min-1
Rate, µmol•mg-1•min-1
14
6
4
2
0
0.22
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0
20
0
40
0.2
0.4
0.6
0.8
1
1.2
1 / [FA substrate], µM
[FA substrate], µM
LnA:
Parameter
Vmax
Km
Value
Std. Error
16.0546
2.1092
LA:
Parameter
Value
Std. Error
0.6008
Vmax
7.5836
0.3049
Km
0.8196
AA:
Parameter
Value
Std. Error
0.1551
Vmax
6.6200
0.0911
0.0981
Km
0.5582
0.0482
pH effect on recombinant VvLOXA
activity
pH effect on recombinant VvLOXO
activity
Methoxypyrazines
• Little is known about their
biosynthesis
– Thought to derive from amino
acid biosynthesis
• Accumulate up until veraison
• Degrade after veraison and
with exposure of grape
bunches to light
• At low concentrations (ng.L-1)
contribute to green/greenpepper aromas
UV responses & wine quality
UV responses & wine quality
+UV
No leaf
No
No
removal frame UV-B
No UV
Effects of UV and Leaf Removal on Wine
Quality
•
Methoxypyrazine levels low in juice at harvest, but high early in grape
development: control of gene expression from amino acid precursors
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Amino acid composition different in juice in response to light environment
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Regulation of proline biosynthesis important for fermentation
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Flavonoids accumulate with UV exposure: role of transcription factors
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Lipoxygenase pathway: complex gene family and expression pattern
Acknowledgements
Grape Biotechnology and UV Research
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Jason Wargent, Lancaster University, UK
Scott Gregan
Stephen Stilwell
Andriy Podolyan (Ph.D.)
Jim Shinkle, Trinity University, USA
Dr Rainer Hofmann
Dr Chris Winefield
Professor Brian Jordan (Programme Leader)
Support From:
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Foundation for Research, Science & Technology
NZ Royal Society/MoRST COST-ACTION 858
Marlborough Wine Research Centre, Auckland University
& Plant & Food Research
New Zealand Wine Industry
Lincoln University