Flavonoids in grapes

PLANT INDUSTRY

Simon Robinson, Mandy Walker, Rachel Kilmister and Mark Downey

11 June 2014

Grapes to wine – a 2 metabolic zoo

Grapevines

Grapes

• Grape variety

• Soil, climate etc

• Vineyard management

• Seasonal influences

• Harvest timing

Wine

• Transport & storage

• Processing

• Fermentation

• Pressing

• Added tannin, oak etc

• Ageing

Consumer

Hundreds of different metabolites determine

‘Wine Quality’

Many of these compounds are synthesised in the grape berry

• Flavonoids are important for the colour & taste of wine • Tannins are the major flavonoid present in grapes and wine • Tannins plus anthocyanins form pigmented polymers

Grape Flavonoids

Anthocyanins

Red Grapes

0

1000

2000

3000

4000

Flavonols Tannins

mg/kg

Flavonoids in grapes

‘Moon’ series carnations : on sale since 1996

Flavonoids – a diverse range of compounds

Basic anthocyanin structure

• Complex metabolic pathway • 1,000’s of different flavonoids • Pathway can be manipulated

FLS Flavonols

Coumaroyl-CoA

Chalcones

Flavanones

Dihydroflavonols

DFR

CHI

F3H

LDOX

Leucoanthocyanidin

Anthocyanidin

CHS

UFGT

Anthocyanins

Catechin LAR

Flavonoid biosynthetic pathway in grape

Epicatechin ANR

Tannins

FGT F3’H

Dihydroflavonols

DFR

LDOX

Leucoanthocyanidin

Anthocyanidin

UFGT

Anthocyanins

F3’5’H

Malvidin-3-glucoside

?

Good knowledge of this

pathway in grapes

Colour & tannin during development

• Anthocyanins are synthesised after veraison

•Tannins and flavonols are made between flowering & veraison

• Tannin maturation occurs from veraison to harvest

Tannin synthesis and maturation

Regulatory Genes Control the Pathway

ANTHOCYANINS

Structural Pathway Genes

Enzymes

Regulatory Genes

(bHLH & MYB) TANNINS

FLAVONOLS

Regulatory ‘Master Genes’ control the pathway

Red White

grape seedlings

Predicts fruit colour

Test for DNA marker

MYBA controls colour in grapes

Chardonnay MYBA promoter

35S promoter

‘Black’ Chardonnay – gain of VvMYBA gene expression

Shiraz Pink White

‘Pink’ and ‘White’ Shiraz – reduction of VvMYBA gene expression

MYBA controls anthocyanin synthesis

Formation of tannin polymers

Leucocyanidin Catechin

Epicatechin Flavan-3-ols

LDOX

Leucocyanidin

Anthocyanidin

Catechin LAR

Epicatechin ANR

Tannins ?

Tannin Synthesis - the First Steps Formation of tannin polymers

Leucocyanidin Catechin

Epicatechin Flavan-3-ols

Monomers Dimer

Terminal

unit

Extension

unit

Terminal

unit

Extension

units

Trimer

Tannin Polymer

N=3-200

We need to know about tannin composition, as well as the total amount

OHO

OH

OR2

OH

OH

R1

OHO

OH

OR2

OH

OH

R1

OHO

OH

OR2

OH

OH

R1

OHO

OH

OH

OH

OR2

A C

B

n

Terminal Subunit

Extension Subunit

Interflavan Bond

2

345

6

78

3'

4'

5'

LAR

Leucocyanidin

Catechin / Epicatechin

Tannins ain’t tannins !

• Seed tannins mdp 5 units • Skin tannins mdp 25 units • Different polymer composition

Tannin composition

Epigallotatechin

Catechin

Epicatechin

Epicatechin gallate

Sunraysia Shiraz 2008

Epigallotatechin

Catechin

Epicatechin

Epicatechin gallate

Sunraysia Shiraz 2009

Epigallotatechin

Catechin

Epicatechin

Epicatechin gallate

Sunraysia Cabernet Sauvignon 2008

Epigallotatechin

Catechin

Epicatechin

Epicatechin gallate

Glenrowan Shiraz 2009

Role of ANR & LAR in tannin synthesis using transgenic grapevines

LDOX

Leucoanthocyanidin

Anthocyanidin

UFGT

Anthocyanins

Catechin LAR

Epicatechin ANR

Tannins ?

Silence the grape ANR gene

Silence the grape LAR gene

No obvious phenotype !

Silence the genes for ANR & LAR to decrease their enzyme activity and determine the effect on tannin synthesis and grape tannin composition

VvANR Si

leaves More cyanidin

roots normal

berries More cyanidin

leaves More flavonols

seeds Lots of flavonols

skins More flavonols

skins Less tannin

seeds Same tannin ?

leaves Less tannin

Anthocyanin

Flavonol

Tannin

Silencing tannin genes

• Less tannin in leaves & grape skins from ANR Si lines (10-30% Controls)

• Changes in composition – extension units have less epicatechin, more catechin & epigallocatechin

• Seed tannin slightly lower (70-85% Controls)

• Impact on wine?

Shiraz VvANR Si

How do you manage colour in the vineyard?

Where are you now? Where do you want to be?

• More grape colour ? • Better colour extraction & stability ? • Better anthocyanin composition ?

Based on grape or wine observations?

• Season & site are the biggest drivers of variation • Day and night temperatures • Practices that effect colour include:

– Yield / vigour / vine balance

– Rootstocks

– Irrigation

– Canopy management / bunch exposure

– Vineyard variability

How do you manage tannin in the vineyard?

Where are you now? Where do you want to be?

• More tannin

• Less tannin

• Different tannin

• Better tannin • Smooth tannin

• Soft tannin

• Persistent tannin

Based on grape or wine observations?

Wine Tannin = Grape Tannin x Extractability x Stability

• How much tannin is in the grapes ?

• How much of that tannin is extracted into must ?

• How stable is the tannin in the wine ?

If the tannin isn’t extracted during winemaking, there’s no point trying to get more into the grapes.

Grape tannin versus wine tannin

0.0

0.5

1.0

1.5

2.0

2.5

3.0

2003 2004 2005 2006 2007 2008 2009 2010

Tannin

(m

g/g

FW

T b

err

y)

Season effects Sunraysia Shiraz- whole berries at harvest

0.0

1.0

2.0

3.0

4.0

5.0

Carignane

(n=

5)

Barb

era

(n=

18)

Rubired

(n=

149)

Zin

fandel (n

=68)

Gre

nache (

n=

13)

Carn

elia

n (

n=

3)

Shiraz (

n=

73)

Ruby C

abern

et (n

=33)

Cabern

et S

auvig

non (

n=

77)

Merlot (n

=154)

Chard

onnay (

n=

3)

Durif(n

=8)

Tota

l T

annin

(m

g/g

fre

sh w

eig

ht w

hole

berr

y)

0.0

1.0

2.0

3.0

4.0

5.0

Carignane

(n=

5)

Barb

era

(n=

18)

Rubired

(n=

149)

Zin

fandel (n

=68)

Gre

nache (

n=

13)

Carn

elia

n (

n=

3)

Shiraz (

n=

73)

Ruby C

abern

et (n

=33)

Cabern

et S

auvig

non (

n=

77)

Merlot (n

=154)

Chard

onnay (

n=

3)

Durif(n

=8)

Tota

l T

annin

(m

g/g

fre

sh w

eig

ht w

hole

berr

y) ~600 grape samples

whole berry homogenate

Californian vineyards

Variation between varieties

0.0

1.0

2.0

3.0

4.0

5.0

6.0

SHIRAZ CABERNET SAUVIGNON

PINOT NOIR SHIRAZ CABERNET SAUVIGNON

PINOT NOIR SHIRAZ CABERNET SAUVIGNON

PINOT NOIR

Sunraysia 2003-2004 Yarra Valley 2003-2004 Mornington 2003-2004

Tan

nin

mg

/g f

wt

ber

ry

Varietal differences and site effects

Sunraysia Yarra Valley Mornington

Viticultural management

• Season is the biggest driver of variation

• Understanding other drivers creates management strategies

• Practices that effect tannins might include:

– Irrigation

– Canopy management

– Trellis design

– Nutrient status

– Pruning

– Harvest date

– Cover crop

– Mulching

– Rootstock

Yield

0.0

1.0

2.0

3.0

4.0

5.0

0.0 2.0 4.0 6.0 8.0 10.0

To

tal T

an

nin

(m

g/g

wh

ole

be

rry)

r2 = 0.2771

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.0 2.0 4.0 6.0 8.0 10.0

To

tal A

nth

ocya

nin

(m

g/g

who

le b

err

y)

r2 = 0.5724

California

Yield (kg/m2) Yield (kg/m2)

Anthocyanin Tannin

Sustained deficit irrigation

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

2004 2005 2006 2007

To

tal Tan

nin

(m

g/g

berr

y)

Cabernet Sauvignon 43% 52% 70% 100%

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

2004 2005 2006 2007

To

tal Tan

nin

(m

g/g

berr

y)

Shiraz 35% 45% 65% 100%

Sunraysia

• Large seasonal variation

• Irrigation has no major effect on tannin synthesis

• It may effect vigour, berry size and tannin extractability

Rootstocks

0

2

4

6

8

10

12

14

16

18

1103

PAULSEN

140

RUGGERI

DOGRIDGE FREEDOM MG5512 MG6262 MS1010 MS3769 RAMSEY

Ta

nn

in (

mg

/g f

wt

sk

in)

2008 2009

Sunraysia Shiraz

0.90 - 1.29

1.30 - 1.68

1.69 - 2.07

2.08 - 2.46

2.47 - 2.85

2.86 - 3.24

3.25 - 3.63

3.64 - 4.02

Tannins (mg/g berry CE)

0.35 - 0.39

0.40 - 0.43

0.44 - 0.47

0.48 - 0.51

0.52 - 0.55

0.56 - 0.59

0.60 - 0.63

0.64 - 0.67

Canopy Assessments (EAS)

Averaged Verasion to Harvest 2007

Canopy Assessments Tannins at Harvest 2007

0 50 100 150 20025Meters

Vine vigour Sunraysia Shiraz

Vine vigour

r² = 0.3741

r² = 0.4389

r² = 0.4284

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Phe

no

lics

(mg

/g b

err

y)

Canopy Assessment (EAS)

Anthocyanin

tannin

PhenolicsFeReact

Vineyard variability Sum of normalised Tannins

Lowest

Low

Moderate

High

Highest

Sum of normalised anthocyanins

Lowest

Low

Moderate

High

Highest

Sunraysia Shiraz

2006

2009 2008

2007

Tannins at Harvest

0.90 - 1.29

1.30 - 1.68

1.69 - 2.07

2.08 - 2.46

2.47 - 2.85

2.86 - 3.24

3.25 - 3.63

3.64 - 4.02

Tannins (mg/g berry CE)

0 50 100 150 200 25 Meters

Vineyard variability

Summary Tannin synthesis to veraison

Tannin maturation post-veraison

Measurement

Tannin composition & extractability

Season & site Yield / vigour / vine balance Irrigation Rootstocks Vineyard variability

Simon Robinson CSIRO Plant Industry Flavonoids in grapes

t +61 8 8303 8600 e simon.robinson@csiro.au w www.csiro.au

PLANT INDUSTRY

Thank you