Flavonoids in grapes. Simon Robinson, Mandy Walker, Rachel Kilmister and Mark Downey. 11 June 2014 PLANT INDUSTRY

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1 Flavonoids in grapes Simon Robinson, Mandy Walker, Rachel Kilmister and Mark Downey 11 June 2014 PLANT INDUSTRY

Grapes to wine a 2 metabolic zoo Grapevines Hundreds of

Soil, climate etc Vineyard management Seasonal influences

Fermentation Pressing Added tannin, oak etc Ageing Many of

2 Grapes to wine a 2 metabolic zoo Grapevines Hundreds of different metabolites determine Wine Quality Grape variety Soil, climate etc Vineyard management Seasonal influences Harvest timing Grapes Transport & storage Processing Fermentation Pressing Added tannin, oak etc Ageing Many of these compounds are synthesised in the grape berry Consumer Wine

3 Flavonoids in grapes Grape Flavonoids 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 mg/kg Red Grapes Flavonols Tannins Anthocyanins

1,000 s of different flavonoids Pathway can be

4 Flavonoids a diverse range of compounds Basic anthocyanin structure Complex metabolic pathway 1,000 s of different flavonoids Pathway can be manipulated Moon series carnations : on sale since 1996

Flavonoid biosynthetic pathway in grape Dihydroflavonols DFR Leucoanthocyanidin LDOX Anthocyanidin UFGT F3 5 H Anthocyanins Malvidin-3-glucoside Coumaroyl-CoA CHS Chalcones CHI

5 Flavonoid biosynthetic pathway in grape Dihydroflavonols DFR Leucoanthocyanidin LDOX Anthocyanidin UFGT F3 5 H Anthocyanins Malvidin-3-glucoside Coumaroyl-CoA CHS Chalcones CHI Flavanones F3H F3 H Dihydroflavonols DFR Leucoanthocyanidin LDOX Anthocyanidin UFGT Anthocyanins FLS LAR ANR Good knowledge of this pathway in grapes FGT Catechin Epicatechin Flavonols? Tannins

6 Tannin synthesis and maturation Colour & tannin during development Anthocyanins are synthesised after veraison Tannins and flavonols are made between flowering & veraison Tannin maturation occurs from veraison to harvest

7 Regulatory Master Genes control the pathway Regulatory Genes Control the Pathway Regulatory Genes (bhlh & MYB) Structural Pathway Genes TANNINS Enzymes ANTHOCYANINS MYBA controls colour in grapes FLAVONOLS Red White grape seedlings Test for DNA marker Predicts fruit colour

8 MYBA controls anthocyanin synthesis Black Chardonnay gain of VvMYBA gene expression Chardonnay MYBA promoter 35S promoter Pink and White Shiraz reduction of VvMYBA gene expression Shiraz Pink White

9 Formation of tannin polymers Leucocyanidin LDOX Anthocyanidin LAR ANR Catechin Epicatechin? Tannins Leucocyanidin Catechin Epicatechin Flavan-3-ols

10 Tannin Synthesis - the First Steps Formation of tannin polymers Extension unit Extension units Monomers Dimer Terminal unit Trimer Terminal unit Tannin Polymer Leucocyanidin Catechin Epicatechin Flavan-3-ols N=3-200

11 Tannins ain t tannins! HO A 5 OH 4 O C OH 3' OH B 4' 5' R OR 2 OH OH n Extension Subunit HO O R 1 OH OR 2 OH OH HO O R 1 Interflavan Bond OH HO OR 2 O OH OH OH OR 2 Terminal Subunit Seed tannins mdp 5 units Skin tannins mdp 25 units Different polymer composition We need to know about tannin composition, as well as the total amount

12 Tannin composition Sunraysia Shiraz 2008 Epicatechin gallate Sunraysia Cabernet Sauvignon 2008 Epicatechin gallate Epicatechin Epigallotatechin Epicatechin Epigallotatechin Epicatechin gallate Catechin Sunraysia Shiraz 2009 Catechin Glenrowan Shiraz 2009 Epicatechin gallate Epigallotatechin Epigallotatechin Epicatechin Epicatechin Catechin Catechin

13 Role of ANR & LAR in tannin synthesis using transgenic grapevines Silence the genes for ANR & LAR to decrease their enzyme activity and determine the effect on tannin synthesis and grape tannin composition Silence the grape LAR gene Leucoanthocyanidin LDOX Anthocyanidin LAR ANR Catechin Epicatechin No obvious phenotype!? Tannins UFGT Anthocyanins Silence the grape ANR gene

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

14 Silencing tannin genes Anthocyanin Flavonol leaves roots berries leaves seeds skins VvANR Si More cyanidin normal More cyanidin More flavonols Lots of flavonols More flavonols Tannin skins Less tannin seeds Same tannin? Shiraz VvANR Si leaves Less tannin 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?

15 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? 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 Based on grape or wine observations?

16 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?

17 Grape tannin versus wine tannin How much tannin is in the grapes? How much of that tannin is extracted into must? How stable is the tannin in the wine? Wine Tannin = Grape Tannin x Extractability x Stability If the tannin isn t extracted during winemaking, there s no point trying to get more into the grapes.

18 Tannin (mg/g FWT berry) Season effects 3.0 Sunraysia Shiraz- whole berries at harvest

19 Carignane (n=5) Barbera (n=18) Rubired (n=149) Zinfandel (n=68) Grenache (n=13) Carnelian (n=3) Durif (n=8) Shiraz (n=73) Ruby Cabernet (n=33) Cabernet Sauvignon (n=77) Merlot (n=154) Chardonnay (n=3) Total Tannin (mg/g fresh weight whole berry) Variation between varieties 5.0 ~600 grape samples 4.0 whole berry homogenate 3.0 Californian vineyards

20 Tannin mg/g fwt berry Varietal differences and site effects Sunraysia Yarra Valley Mornington SHIRAZ CABERNET SAUVIGNON PINOT NOIR SHIRAZ CABERNET SAUVIGNON PINOT NOIR SHIRAZ CABERNET SAUVIGNON Sunraysia Yarra Valley Mornington PINOT NOIR

Viticultural management Season is the biggest driver of variation Understanding other drivers creates management strategies Practices that

21 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

22 Total Anthocyanin (mg/g whole berry) Total Tannin (mg/g whole berry) Yield California Anthocyanin r 2 = Tannin r 2 = Yield (kg/m 2 ) Yield (kg/m 2 )

23 Total Tannin (mg/g berry) Total Tannin (mg/g berry) Sustained deficit irrigation Sunraysia Cabernet Sauvignon 43% 52% 70% 100% Shiraz 35% 45% 65% 100% Large seasonal variation Irrigation has no major effect on tannin synthesis It may effect vigour, berry size and tannin extractability

24 Tannin (mg/g fwt skin) Rootstocks Sunraysia Shiraz PAULSEN 140 RUGGERI DOGRIDGE FREEDOM MG5512 MG6262 MS1010 MS3769 RAMSEY

25 Vine vigour Sunraysia Shiraz Averaged Verasion to Harvest 2007 Canopy Assessments Tannins at Harvest 2007 Canopy Assessments (EAS) Tannins (mg/g berry CE) Meters

26 Phenolics (mg/g berry) Vine vigour Anthocyanin tannin PhenolicsFeReact r² = r² = r² = Canopy Assessment (EAS)

27 Vineyard variability Sunraysia Shiraz Sum of normalised Tannins Lowest Low Moderate High Highest Sum of normalised anthocyanins Lowest Low Moderate High Highest

28 Vineyard variability Tannins (mg/g berry CE) Meters Tannins at Harvest

29 Summary Tannin synthesis to veraison Tannin maturation post-veraison Measurement Tannin composition & extractability Season & site Yield / vigour / vine balance Irrigation Rootstocks Vineyard variability

30 Thank you Simon Robinson CSIRO Plant Industry Flavonoids in grapes t e simon.robinson@csiro.au w PLANT INDUSTRY

Flavonoids in grapes. Simon Robinson, Mandy Walker, Rachel Kilmister and Mark Downey. ASVO SEMINAR : MILDURA, 24 July 2014 AGRICULTURE FLAGSHIP

Flavonoids in grapes. Simon Robinson, Mandy Walker, Rachel Kilmister and Mark Downey. ASVO SEMINAR : MILDURA, 24 July 2014 AGRICULTURE FLAGSHIP Flavonoids in grapes Simon Robinson, Mandy Walker, Rachel Kilmister and Mark Downey ASVO SEMINAR : MILDURA, 24 July 2014 AGRICULTURE FLAGSHIP Flavonoids in grapes Grape Flavonoids Flavonoids are important