Effects Partial Solar Radiation on the Grapevine

Johann Martinez-Luscher, Christopher Chen, Luca Brillante, Monica Cooper and S. Kaan Kurtural* Department of Viticulture and Enology Flavonoids in berry • Properties • Color • Co-pigmentation • Astringency (tactile) • Bitterness (taste) • Health-promoting effects • Important antioxidant capacity Berry Anatomy Flesh (pulp) - juice - hydroxycinnamates

Seed - tannins (bitter) - flavan-3-ols - hydroxybenzoic acids

Skin - color pigments - tannins (astringent) - flavan-3-ols - Illustration by Jordan Koutroumanidis, Winetitles hdb d Phenylalanine (phenyl propanoid pathway) Flavonoid biosynthesis

Tannins Constitution of flavonol and anthocyanin profiles

3’4’ substituted 4’ substituted 3’4’5’ substituted

Shikimate

eriodictyol narigenin pentahydroxyflavone

F3H F3H F3H F3’H F3’5’H dihydroxyquercetin dihydroxykaempferol dihydroxymyricetin

O H

DFR FLS FLS O H FLS DFR O H O H leucocyanidin leucodelphidin H O O H O O H O O O H O H LDOX LDOX O H O H O H

O H O O H O cyanidin C H delphidin O H O 3 O

UFGT OMT OMT O H UFGT O H cyanidin 3-o-glucoside H O O delphidin 3-o-glucoside O H O H H O O O H O + O H H O O C H O H O H 3 O H + H O O O H O O H O H O C H IGlucose 3 OMT O O H I O H Glucose OMT syringetin O H H O O

O peonidin 3-o-glucoside OMT OMT O H H C 3 O H

+ H O O O H O petunidin 3-o-glucoside malvidin 3-o-glucoside H C H O C 3 3 O O H C 3 GlucoseF O H O H

O H

+ + H O O H O O Flavonols O H O

H C 3 Anthocyanins I I Glucose Glucose

O H O H

Abbreviations: F3’H: flavonoid 3’-hydroxylase; F3’5’H: flavonoid 3’5’-hydroxylase; FLS: flavonol synthase; DFR: dihydroflavonol reductase; LDOX: leucocyanidin dioxygenase; UFGT: UDP-glucose flavonoid 3-O-glucosyltransferase; OMT: O-methyltransferase Effect of water deficit on profiles

3’4’ substituted Shikimate 3’4’5’ substituted

eriodictyol narigenin pentahydroxyflavone

F3H F3H F3H F3’H F3’5’H dihydroxyquercetin dihydroxykaempferol dihydroxymyricetin

O H FLS DFR DFR FLS O H FLS O H O H leucocyanidin quercetin kaempferol myricetin leucodelphidin H O O H O O H O O O H O H LDOX LDOX O H O H O H …but flavonols? O H O O H O cyanidin C H delphidin O H O 3 O UFGT UFGT OMT OMT O H O H cyanidin 3-o-glucoside laricitrin H O O delphidin 3-o-glucoside O H isorhamnetin O H H O O O H O + O H H O O H O H O H C 3 O H + H O O O H O O H O H O C H IGlucose 3 OMT O O H I O H Glucose OMT syringetin O H H O O

O peonidin 3-o-glucoside OMT OMT O H H C 3 O H

+ petunidin 3-o-glucoside malvidin 3-o-glucoside H O O O H O H C H O C 3 3 O O H C 3 GlucoseF O H O H

O H

+ + H O O H O O

O H O

3’4’5’ substituted predominate with water C H 3 I deficit as F3’5’H expression levels increase I Glucose Glucose O H O H after veraison

Castellarin et al., 2007 (Planta; BMC Plant Biology; Plant Cell and Environment) 3’4’5’ 3’4’4’ substitutedsubstituted substituted Anthocyanins Flavonol homologues Cyanidins F3’H HOH Pelargonidins 3’ O H 2’ 4’ OH Uncommon in Vitis vinifera + H O O B 5’ HOH Delphidins F3’5’H Myricetins A C 6’ O

O H O H O

O H

O H O Flavonols impact on wine mouthfeel

Ferrer-Gallego et al., 2016 Proanthocyanidins

Important due to their astringent properties Role in long term color stability Grape based proanthocyanidins • (+)-catechin (C) • (-)-epicatechin (EC) • (-)-epicatechin-3-O-gallate (ECG) • (-)-epigallocatechin (EGC) Skin vs. seed proanthocyanidins • Skins contain EGC • Greater degree of polymerization • Lower proportion of galloylated subunits

9 20/06/2017 How can we relate this information to tactile and taste sensation?

Increase Tannin Molecular Size increase astringency/ chalky Sun et al, J. Agric. Food Chem. 2013, 61: 939-946 Vidal et al, J Sci Food Agric. 2003, 83: 564–573

Increase %ECG increase drying and chalkiness Vidal et al, J Sci Food Agric. 2003, 83: 564–573

Increase %EGC lower coarseness Vidal et al, J Sci Food Agric. 2003, 83: 564–573

Increase Color Incorporation less astringent Vidal et al, Analytica Chimica Acta. 2004, 513: 57-65 Varietal differences and effects of management practices

Anthocyanin profile composition

Effect of controlling vegetation

Bobeica et al., 2015 Effect of water deficit on wine hue

Cassasa et al., 2015 Changing environment in California

• Less cloud coverage, increasing temperatures • Yield loss: • Direct: Berry water content, shriveling • Indirect: Acidity, astringency, color hue • Shifts in: • Phenology • Compositional shifts in flavonoid profile

13 20/06/2017 Optimum light environment in the fruit zone during ripening Maximize diffuse or indirect sunlight within the canopy interior Minimize exposure of clusters to direct sunlight – particularly in warm climates B C F E

D H

A

G Radiation Effects on Whole Canopy

10 6% reflected 9

8 /vine/s) 2 7 100% incident 6 mol CO

µ LLN#1 5 4 10 % transmitted 3 2 LLN#2 Net Canopy Pn ( ( Pn Canopy Net 1 0 1% transmitted 0 750 1000 1250 1500 1750 2000 2250 2500 PPFD (µmol/m2/s) LLN#3

Kurtural et al. 2003; Dami et al. 2005; Kurtural et al. 2005; 2006 0.1% transmitted Plant UV-B photoreception : UVR8

Heijde and Ulm 2013 Trends Plant Sci. Experiment at Oakville Experiment Station

Vineyard description • Four year old producing vineyard CS clone 7/C3309. • Spacing: 6’ x 8’, NW-SE orientation • Bi-lateral cordon, relaxed vertical shoot-positioned training system Experimental design • Five colored shade nets (and untreated control) • Two applied water amounts • Arranged factorially in a split-plot design with four blocks

18 20/06/2017 Colored shade nets

• Five polyethylene cloths with ~ 80% transmissivity of visible light • Blue • Pearl • Aluminet • Red • Black • Untreated control

19 20/06/2017 Applied water amounts

• Well watered (SDI): 65% of estimated ETc and applied to maintain a mid-day leaf water potential ψl of -1.2 MPa, from fruit set to harvest. • Deficit watered (RDI): applied at the same ψl with SDI soon after bud break but was decreased to 25% ETc from fruit set to veraison with a ψl of -1.4 MPa and then reinstated to SDI from veraison until harvest.

20 20/06/2017 RESULTS

21 20/06/2017 What do the these nets actually do? Transmittance of wavelengths by net color

WL range % Tr Black % Tr White % Tr Silver % Tr Red % Tr Blue UV-B 76.31 71.72 60.63 58.41 49.27 UV-A 79.64 72.75 63.74 67.00 44.52 Violet 79.52 75.81 67.51 67.18 51.36 Blue 88.40 91.54 88.02 79.75 74.55 Green 99.99 99.96 99.98 99.96 99.99 Yellow 99.99 99.96 99.98 99.96 98.65 Red 97.69 99.93 98.65 99.93 75.86 Far red 83.18 86.48 78.14 91.79 59.94 Fr/R 85.15 86.54 79.21 91.85 79.02

22 6/20/2017 140 NE side (Morning exposed) Physical conditions50 of the berry 120

40 100

30 80 5 Open field Solar radiation Temperature (°F) Temperature Control (°C) Temperature 20 Blue 60 4 Pearl -1 10 Uncovered

s Black Shaded 40 -2 Red Ambient 3 0 140 Aluminet SW side (Evening exposed)

molm 2 50 120 

1 40 100

0 30 80 300 400 500 600 700 800 900 1000 Temperature (°F) Temperature Wavelength(°C) Temperature 20 (nm) 60

10 Uncovered Shaded Ambient 40 0 6:00 8:00 10:00 12:00 14:00 16:00 18:00 20:00 23 6/20/2017 Components of yield Water applied Net Cover Berry Mass Cluster mass (g) Yield (kg/vine) (g Berry-1) SDI Control 1.15 214 8.0 Blue 1.29 209 7.9 Pearl 1.20 190 7.7 Black 1.32 196 8.1 Red 1.16 203 7.2 Aluminet 1.10 214 6.4* RDI Control 1.13 196 8.1 Blue 1.20 202 7.6 Pearl 1.17 196 7.6 Black 1.20 205 7.5 Red 1.24 202 7.0 Aluminet 1.19 213 8.5

P(net) 0.043 0.2658 0.4519

P(water) 0.289 0.8403 0.2233

P(cnets x water) 0.347 0.6905 0.0059

24 6/20/2017 Berry composition

25 6/20/2017 Water Total Anth. Profile Titratable applied Net Cover Berry Mass Anthocyanins Hydroxylation TSS pH Acidity In vines without (g Berry-1) (mg Berry-1) 3'4'5'/3'4' (Brix°) (g L-1) nets (Control), well SDI Control 1.15 1.47 11.12 24.45 3.52 6.38 exposed berries did Blue 1.29 1.65 12.58 24.38 3.51 6.50 not make it to the Pearl 1.20 1.51 11.98 24.70 3.52 6.38 end of the Black 1.32 1.67 12.11 24.15 3.44 6.87 Red 1.16 1.35 11.88 24.23 3.48 6.50 experiment! Aluminet 1.10 1.57 10.80 24.40 3.52 6.28 RDI Control 1.13 1.74 12.44 24.15 3.47 6.65 Blue 1.20 1.48 13.85 23.90 3.47 6.65 Pearl 1.17 1.53 12.82 23.88 3.47 6.83 Black 1.20 1.36 12.94 23.85 3.48 6.40 Red 1.24 1.72 12.64 24.15 3.48 6.53 Aluminet 1.19 1.47 13.47 24.10 3.45 6.68

P(cloth) 0.043 0.192 0.454 0.337 0.087 0.943

P(water) 0.289 0.215 0.008 <0.001 0.004 0.113

P(cloth x water) 0.347 0.083 0.817 0.293 0.774 0.938 Kinetic development of anthocyanins

27 6/20/2017

UV Delphidins anthocyanins Cyanidins B and water deficit affected differently the anthocyanin groups anthocyanin the affected differently deficit andwater-B Total skin

3’4’5’’ substituted 3’4’ substituted Total skin anthocyanins -1 (mg g dry wt) -1 (mg g-1 dry wt) 10 3'4'5' substituted (mg g dry wt) 15 20 25 10 15 20 3'4' substituted 0 5 2 3 4 0 1 0 5 P P P P P P (UV-B) (WA) (UV-BxWA) (UV-BxWA) (WA) (UV-B) =0.605 =0.029 =0.005 =0.059 =0.404 =0.571 P P P (UV-BxWA) (WA) (UV-B) <0.001 =0.041 =0.634

Relative expression Relative expression 0.00 0.01 0.02 0.03 0.04 0.05 Relative expression 0.00 0.02 0.04 0.06 0.00 0.02 0.04 0.06 0.08 0.10 Gene expression one expression Gene week after veraison after week P P P P P P P P P (UV-B) (WA) (UV-BxWA) (UV-BxWA) (WA) (UV-B) (UV-BxWA) (WA) (UV-B) =0.624 =0.662 <0.001 =0.020 =0.027 =0.863 =0.704 =0.480 =0.280 F3’5’H UFGT F3’H F3'5'H UFGT F3'H

5.98 9.66 kJ m kJ 9.66 0 kJ m kJ m -2 d -2 -2

-1 d d -1 -1 Anthocyanin profile hydroxylation under higher light and water deficit Tot. Anth : 13.6 mg g-1 Cy: 17 % Tot. Anth: 16.2 mg g-1 Dp: 83 % Cy: 21 % -1 Dp: 79 % Tot. Anth : 13.2 mg g Cy: 9 % Dp: 91 %

Tot. Anth : 14.85 mg g-1 Cy: 10% Dp: 90 %

Delphidin (molar %) Water applied Net cover Skin mDP Seed mDP In vines without nets (Control), well SDI Control 52 6.9 exposed berries did Blue 46 7.1 not make it to the Pearl 49 7.1 end of the Black 50 6.7** Red 47 6.7** experiment! Aluminet 51 7.4 RDI Control 47 8.0 Blue 45 6.5** Pearl 47 7.4 Black 48 7.7 Red 50 7.7 Aluminet 51 7.7

P(nets) 0.5392 0.1390

P(water) 0.8590 0.0029

P(nets x water) 0.6126 0.0454

30 6/20/2017 How can we explain flavonol-anthocyanin profile behavior with transcript levels?

Total flavonols Total anthocyanins Methylated Flavonols Methylated Anthocyanins Quercetins Cyanidins Methylated Flavonols Methylated Anthocyanins Delphidins Myricetins Discussion • Whereas moderate solar radiation exposure can equilibrate berry acidity and promote anthocyanin biosynthesis; • extreme temperatures can lead to organic acids and anthocyanin degradation (Mori et al., 2007; Sweetman et al., 2014). • In the present study, the 3.5°C reduction in temperature in the moment of highest berry temperature (i.e. 15:00 Hrs in the SW side) was associated with ameliorated berry composition. • Greater anthocyanin content in berry • Uncovered grapevines often displayed a greater level of proportion of berries with sunburn.

32 6/20/2017 So far based on 2016 results… • Primary metabolism: • Yield reduction: Aluminet • RDI irrigation treatment: Greater Brix accumulation • Secondary metabolism: • Hydroxylation of anthocyanins: Greater with Blue +RDI • No measurable effect on skin mDP (tactile) • Seed mDP > Control +RDI => Greater bitternes (taste) • Seed mDP < Black, Red +SDI or Blue+RDI => Less bitterness (taste)

33 6/20/2017 Thank you for listening!