Flavonoids in grapes Simon Robinson, Mandy Walker, Rachel Kilmister and Mark Downey 11 June 2014 PLANT INDUSTRY Grapes to wine – a 2 metabolic zoo • Grape variety • Soil, climate etc • Vineyard management • Seasonal influences • Harvest timing • Transport & storage • Processing Grapevines • Fermentation • Pressing • Added tannin, oak etc Hundreds of different • Ageing metabolites determine Grapes ‘Wine Quality’ Many of these compounds are synthesised in the grape berry Wine Consumer FlavonoidsGrape Flavonoids in grapes • 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 4000 3000 2000 1000 0 Flavonols Tannins Anthocyanins 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 Coumaroyl-CoA CHS Chalcones Good knowledge of this pathway in grapes CHI Flavanones F3H F3’5’H F3’H FLS FGT Dihydroflavonols Dihydroflavonols Flavonols DFR DFR LAR Leucoanthocyanidin Leucoanthocyanidin Catechin LDOX LDOX Tannins ANR ? Anthocyanidin Anthocyanidin Epicatechin UFGT UFGT Anthocyanins Anthocyanins Malvidin-3-glucoside 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 RegulatoryRegulatory ‘Master Genes Genes’ Control control the Pathway the pathway Regulatory Genes Structural (bHLH & MYB) Pathway Genes TANNINS Enzymes ANTHOCYANINS FLAVONOLS MYBA controls colour in grapes Red White grape seedlings Test for DNA marker Predicts fruit colour MYBA controls anthocyanin synthesis ‘Black’ Chardonnay – gain of VvMYBA gene expression Chardonnay MYBA 35S promoter promoter ‘Pink’ and ‘White’ Shiraz – reduction of VvMYBA gene expression Shiraz Pink White Formation of tannin polymers Leucocyanidin LAR Catechin LDOX ? Tannins ANR Anthocyanidin Epicatechin Catechin Leucocyanidin Flavan-3-ols Epicatechin TanninFormation Synthesis of -tannin the First polymers Steps Extension Extension unit units Terminal Terminal unit unit Monomers Dimer Trimer Tannin Polymer Catechin N=3-200 Leucocyanidin Flavan-3-ols Epicatechin Tannins ain’t tannins ! OH 3' OH 4' 8 B HO 7 O 5' R 2 1 6 A C n 5 4 3 OR2 OH OH Extension Subunit OH HO O R1 OR2 OH OH OH HO O R1 Leucocyanidin OR2 OH OH OH LAR Interflavan Bond HO O Catechin / Epicatechin Terminal Subunit OR2 OH • 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 Tannin composition Sunraysia Shiraz 2008 Sunraysia Cabernet Sauvignon 2008 Epicatechin Epicatechin gallate gallate Epigallotatechin Epicatechin Epicatechin Epigallotatechin Catechin Catechin Sunraysia Shiraz 2009 Glenrowan Shiraz 2009 Epicatechin Epicatechin gallate gallate Epigallotatechin Epigallotatechin Epicatechin Epicatechin Catechin Catechin 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 No obvious phenotype ! LAR Leucoanthocyanidin Catechin LDOX Tannins ANR ? Anthocyanidin Epicatechin UFGT Anthocyanins Silence the grape ANR gene Silencing tannin genes VvANR Si leaves More cyanidin Anthocyanin roots normal berries More cyanidin leaves More flavonols Flavonol seeds Lots of flavonols skins More flavonols skins Less tannin Tannin seeds Same tannin ? leaves Less tannin Shiraz VvANR Si • 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? 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 Based on grape or • Day and night temperatures wine observations? • 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 Based on grape or • Different tannin wine observations? • Better tannin • Smooth tannin • Soft tannin • Persistent tannin 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. Season effects 3.0 Sunraysia Shiraz- whole berries at harvest 2.5 2.0 1.5 1.0 Tannin (mg/g FWT (mg/g berry) FWT Tannin 0.5 0.0 2003 2004 2005 2006 2007 2008 2009 2010 Variation between varieties between Variation 5.0 4.0 3.0 2.0 1.0 Total Tannin (mg/g fresh weight whole berry) berry) whole whole weight weight fresh fresh (mg/g (mg/g Tannin Tannin Total Total 0.0 vineyards Californian berry whole homogenate grape ~600 samples (n=8) (n=8) (n=5) (n=5) (n=18) (n=18) (n=149) (n=149) Durif Durif Shiraz (n=73) (n=73) Shiraz Shiraz Merlot (n=154) (n=154) Merlot Merlot Barbera Barbera Carnelian (n=3) (n=3) Carnelian Carnelian Rubired Rubired Carignane Carignane Zinfandel (n=68) (n=68) Zinfandel Zinfandel Grenache (n=13) (n=13) Grenache Grenache Chardonnay (n=3) (n=3) Chardonnay Chardonnay Ruby Cabernet (n=33) (n=33) Cabernet Cabernet Ruby Ruby Cabernet Sauvignon (n=77) (n=77) Sauvignon Sauvignon Cabernet Cabernet Varietal differences and site effects 6.0 5.0 Sunraysia Yarra Valley Mornington 4.0 berry 3.0 fwt 2.0 Tannin mg/g Tannin 1.0 0.0 SHIRAZ CABERNET PINOT NOIR SHIRAZ CABERNET PINOT NOIR SHIRAZ CABERNET PINOT NOIR SAUVIGNON SAUVIGNON SAUVIGNON Sunraysia 2003-2004 Yarra Valley 2003-2004 Mornington 2003-2004 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 California 3.0 5.0 2 r2 = 0.5724 r = 0.2771 Anthocyanin Tannin 2.5 4.0 2.0 3.0 1.5 2.0 1.0 1.0 0.5 whole berry) TotalTannin (mg/g Total Anthocyanin (mg/g whole berry) 0.0 0.0 0.0 2.0 4.0 6.0 8.0 10.0 0.0 2.0 4.0 6.0 8.0 10.0 Yield (kg/m2) Yield (kg/m2) Sustained deficit irrigation Sunraysia 8.0 8.0 Cabernet Sauvignon 43% 52% 70% 100% Shiraz 35% 45% 65% 100% 7.0 7.0 6.0 6.0 5.0 5.0 4.0 4.0 3.0 3.0 2.0 2.0 Total Tannin (mg/g berry) (mg/g Tannin Total Total Tannin (mg/g berry) Tannin Total 1.0 1.0 0.0 0.0 2004 2005 2006 2007 2004 2005 2006 2007 • Large seasonal variation • Irrigation has no major effect on tannin synthesis • It may effect vigour, berry size and tannin extractability Rootstocks Sunraysia Shiraz 18 16 2008 2009 14 12 10 8 6 Tannin (mg/g fwt skin) fwt (mg/g Tannin 4 2 0 1103 140 DOGRIDGE FREEDOM MG5512 MG6262 MS1010 MS3769 RAMSEY PAULSEN RUGGERI Vine vigour Sunraysia Shiraz Averaged Verasion to Harvest 2007 Tannins at Harvest 2007 Canopy Assessments Canopy Assessments (EAS) Tannins (mg/g berry CE) 0.35 - 0.39 0.52 - 0.55 0.90 - 1.29 2.47 - 2.85 0.40 - 0.43 0.56 - 0.59 1.30 - 1.68 2.86 - 3.24 0.44 - 0.47 0.60 - 0.63 1.69 - 2.07 3.25 - 3.63 0.48 - 0.51 0.64 - 0.67 0 25 50 100 150 200 2.08 - 2.46 3.64 - 4.02 Meters Vine vigour 9.00 Anthocyanin 8.00 tannin 7.00 PhenolicsFeReact 6.00 r² = 0.4284 5.00 (mg/g berry) 4.00 3.00 r² = 0.4389 2.00 Phenolics r² = 0.3741 1.00 0.00 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Canopy Assessment (EAS) Vineyard variability Sunraysia Shiraz Sum of normalised Tannins Lowest Low Moderate High Highest Sum of normalised anthocyanins Lowest Low Moderate High Highest Tannins (mg/g berry CE) Vineyard variability 0.90 - 1.29 2.47 - 2.85 1.30 - 1.68 2.86 - 3.24 1.69 - 2.07 3.25 - 3.63 2.08 - 2.46 3.64 - 4.02 0 25 50 100 150 200 Meters 2006 2007 2008 2009 Tannins at Harvest Summary Tannin synthesis to veraison Tannin maturation post-veraison Measurement Tannin composition & extractability Season & site Yield / vigour / vine balance Irrigation Rootstocks Vineyard variability Thank you Simon Robinson CSIRO Plant Industry Flavonoids in grapes t +61 8 8303 8600 e [email protected] w www.csiro.au PLANT INDUSTRY .