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 Sugars other 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)

Spec

tral

irra

dian

ce (r

elat

ive

units

)

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 acidsGlutamine

0

20

40

60

80

100

120

Lo UV UV-A All UV

% o

f no-

fram

e

Amino acidsGlutamic 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)-hexenalCOOHOHC

(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

CHO OH

(2E,6Z)-nonadienal (3Z,6Z)-nonadien-1-ol

IF

ADH

HOOC CH3

a-linolenic acid

Storage lipidsBiological 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

L OX 1 G m 6

LOX 1 Gm 7

L O X1 G m 3

LO X1 Ps 3

LO X1 Lc 1

LOX1 Gm 4

LOX1 G

m 5

L OX1 Cs 1

LOX 1 C

s 2LO

X 1 St 2

L OX

L V vL O

X1 A

t 2

LOX1

St 1

L OX 1

Le

1LO

X1 N

t 1LO

X1 P

rd 1

LOX1 A

t 1

LOX 1 Ca 1

L OXM Vv

LOX B Vv

L O XC V vL OX 1 Hv 1

LOX 1 Zm 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 VvLOX2 A t 4

LOXP V v

LOX 2 Os 1

LOX2 Zm 1

LOX2 Hv 1

LOX2 O

s 2

L OX

2 At 1LO

X2 Bn 2

LOX

2 S

t 1

LOX 2

Pod

1

LOX2

Pod

2

LOXJ

Vv

LOXK VvLOXA VvL OX E VvL OX F Vv

LO XG V v

LOX 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

porti

onal

tran

scrip

t abu

ndan

ce

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

mol

•mg-

1•m

in-1

0

2

4

6

8

10

12

14

Parameter Value Std. Error

Vmax 16.0546 0.6008Km 2.1092 0.3049

Recombinant VvLOXA Enzyme Kinetics Data

1 / [FA substrate], µM0 0.2 0.4 0.6 0.8 1 1.2

1 / R

ate,

µm

ol•m

g-1•

min

-1

00.020.040.060.08

0.10.120.140.160.18

0.20.220.240.26

Parameter Value Std. Error

Vmax 7.5836 0.1551Km 0.8196 0.0981

Parameter Value Std. Error

Vmax 6.6200 0.0911Km 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

AcknowledgementsGrape 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