uv radiation-and-molecular-effects
TRANSCRIPT
The Effects of Ultraviolet Radiation and Canopy
Shading on Grape Berry Biochemistry & Molecular
BiologyProfessor Brian Jordan
Professor of Plant Biotechnology
Agriculture and Life Sciences Faculty
Lincoln University
Responses of Plants to Light
Light
Photosynthesis Sugarsother organiccompounds
Information
leaf growthstem growthgermination, etc.
floweringdormancyplant habit, etc.
direction ofgrowth
Small amountsof light
Daily durationof light
Direction oflight
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
300 400 500 600 700 800
Wavelength (nm)
Sp
ectr
al i
rrad
ian
ce (
rela
tive
un
its)
900 1000
Plants
Red & far redBlueUV
-AU
V-B
Ultraviolet Penetration through the Stratospheric Ozone Layer
UV-A380-315nm
UV-B315-280nm
UV-C<280nm
O3layer
0%100%
Earth’s surface
PAR700nm – 380nm
Photoperception to gene expression
Photoperception Signal Transduction Gene
Expression
UV-B Photoreceptor
UV-B
Specific
Photoreceptor
Signal Transduction
Non-Specific
Via ROS Via DNA damage
Changes to gene expression
H2O2
PR genes
JA
O2-
PDF1.2
Ethylene
SA
Transcription factors
Photosynthetic genes
H2O2
Chloroplast signal, electron transport/photophosphorylation
UV-B
Peroxidase NADPH oxidaseReceptor
Signal Transduction Pathways
?
NO
Ca2+/CaM
Phosphorylation
NOS
Chs
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• UVA+, UVB+ screen• UVA+, UVB- screen• UV- screen• No frame• No leaf removal, no frame
0
20
40
60
80
100
250 275 300 325 350 375 400
Wavelength nm
% T
rans
mis
sion UV
+ UVA+
UV-
UV-B Damage No UV-B Damage
UV-absorbing compounds
0
1000
2000
3000
4000
Lo UV UV-A UV-A/B All UV
Total peak areaIn
tegr
ated
are
a @
352
nm
0
1000
2000
3000
4000
Lo UV UV-A UV-A/B All UV
Total peak area
0
1000
2000
3000
4000
Lo UV UV-A UV-A/B All UV
Total peak areaIn
tegr
ated
are
a @
352
nm
Amino Acid Metabolism and Implications for Wine Industry
UV(and PAR)
NITROGEN(Uptake and assimilation)
AMINO ACIDS
Methoxypyrazines: amino acids as precursors to
flavour and aroma compounds Phenolics: amino
acids as precursors – implicated in ageing and
bitterness in white wine
Amino acid composition and implications for
fermentation bouquet and
yeast assimilable nitrogen
Glutathione: implicated in the
prevention of browning process
Valine, isoleucine, leucine
Phenylalanine, tyrosine, tryptophan
All amino acids except proline
Cysteine, glutamate, glycine
Amino Acid Composition
Glutamine
Proline
Arginine
Alanine
Serine
Glutamate
Arginine
Proline
Glutamine
Alanine
Threonine
Serine
Increasing Amounts
ChardonnayChardonnay Sauvignon Sauvignon blancblanc
Light regulation of nitrogen metabolism
• Light regulates the conversion of glutamate into glutamine in the chloroplast
• This involves the GOGAT pathway and requires ATP
• This assimilation of nitrogen then provides amino acids/amines to the fruit
Glutamate Glutamine
Amino acids
Glutamine
0
20
40
60
80
100
120
Lo UV UV-A All UV
% o
f n
o-f
ram
e
Amino acids
Glutamic acid
0
10
20
30
40
50
60
70
80
90
No pluck Lo UV UV-A All UV No frame
µM
Major aroma chemicals
• 3-mercaptohexanol/3-mercaptohexanal acetate– Tropical fruit and
Citrus aromas
• Methoxypyrazines– Green/green-pepper
or capsicum aromas
Present Understanding: Synthesis of Thiol Precursors
Lipids and Fatty Acids
in Cell Membranes
5/6Carbon
Backbone
eg, s-3-(hexan-1-ol)-Glutathione
LOXHPLetc
Non Volatile
s-cysteine Conjugate Precursor
Grape Metabolism through Berry Development and in Response to the Environment
Changes during Must
Fermentation
Release of Aroma Volatiles Primarily by Yeast
VERAISON
Hard Solid Berry
Soft Berry
at Harve
st‘Membrane Turnover’
GSTs
COOH
OOH
13(S)-HPOT
CHO
(3Z)-hexenal
COOHOHC
(9Z)-12-oxododec-9-enoic acid
CHO
OH
COOHOHC
COOHOH
COOHHOOC
OH
CHO
O(O)H
Traumatin
(9Z)-12-hydroxy-9-dodecenoic acid
Traumatic acid
(3Z)-hexen-1-ol
(2E)-hexenal
(2E)-4-hydro(pero)xy-2-hexenal
(2E)-hexen-1-ol
HPL
IF
ADH
ADH
ADH
IF
LOX?
9(S)-HPOT
COOH
HOO
HPL
COOHOHC
9-oxononanoic acid
CHO
(3Z,6Z)-nonadienal
CHOOH
(2E,6Z)-nonadienal(3Z,6Z)-nonadien-1-ol
IF
ADH
HOOC CH3
a-linolenic acid
Storage lipids
Biological m em branes
Free fatty acids
13-LOX 9-LOX
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;
HPL - hydroperoxide lyase;
LOX - lypoxygenase;
ADH - alcohol dehydrogenase;
IF - isomerization factor;
LOX-HPL pathway
13-LOXs Type I
9-LOXs Type I
Type II13-LOXs
LO X1 Gm 1LO X1 G m 2
LO X1 A h 1
LO X1 Ps 2
LOX 1 G m 6
LOX1 Gm 7
L O X1 G m 3
LO X1 Ps 3
LO X1 Lc 1
LOX
1 Gm
4L
OX
1 Gm
5L
OX1 C
s 1
LO
X1
Cs
2LO
X1
St 2
LO
XL
Vv
LO
X1
At 2
LO
X1
St
1L
OX
1 L
e 1
LOX
1 N
t 1
LOX
1 P
rd 1
LO
X1
At 1
LOX 1 C
a 1
L OXM V
v
LOX B Vv
L O XC V v
L OX 1 Hv 1
LOX 1 Z m 3
LO X1 O s 1
LO X1 Z m 1
L OX 2 Zm 6
LO XD V v
L OX 2 A t 2
LOX 2 A t 3L O X2 S t 2LOX O VvLO XR Vv
LOX2 A t 4
LOXP
Vv
LOX 2 O
s 1
LOX
2 Zm 1
LO
X2 H
v 1
LO
X2
Os
2
LO
X2
At 1
LO
X2
Bn
2
LO
X2
St
1
LO
X2
Pod
1
LO
X2
Po
d 2
LO
XJ
Vv
LOXK VvLOXA Vv
L OX E VvL OX F Vv
LO XG V v
LO X H V v
LOXI Vv
Phylogenetic analysis of grape LOXs and characterised LOXs from other plants
Proportional distribution of grape LOXs in different berry fractions
Relative expression of four berry expressed LOXs
SB berry expressed LOXs
0%
20%
40%
60%
80%
100%
VvLOXA VvLOXC VvLOXD VvLOXO
Pro
port
iona
l tra
nscr
ipt
abun
danc
e
Skin
Pulp
Seed
Relative gene expressions of berry expressed LOXs during development
Relative gene expressions of berry expressed LOXs during upon wounding
I – berries with obvious signs of infection, NI – berries closely located to the infected, Control – healthy berries distantly located from the infected.
Relative LOX gene expressions in SB berries infected with Botrytis
[FA substrate], µM
0 20 40
Rat
e, µ
mo
l•m
g-1
•min
-1
0
2
4
6
8
10
12
14
Parameter Value Std. Error
Vmax 16.0546 0.6008
Km 2.1092 0.3049
Recombinant VvLOXA Enzyme Kinetics Data
1 / [FA substrate], µM
0 0.2 0.4 0.6 0.8 1 1.2
1 /
Ra
te,
µm
ol•
mg
-1•m
in-1
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
0.22
0.24
0.26
Parameter Value Std. Error
Vmax 7.5836 0.1551
Km 0.8196 0.0981
Parameter Value Std. Error
Vmax 6.6200 0.0911
Km 0.5582 0.0482
LnA
LA
AA
LnA: LA: AA:
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/green-pepper aromas
UV responses & wine quality
+UV No leaf No No No UV removal frame UV-B
UV responses & wine quality
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
• Amino acid composition different in juice in response to light environment
• Regulation of proline biosynthesis important for fermentation
• Flavonoids accumulate with UV exposure: role of transcription factors
• Lipoxygenase pathway: complex gene family and expression pattern
Acknowledgements
Grape Biotechnology and UV Research• 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:• 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
