micro-environmental changes that alter berry phenolic and aromatic - PowerPoint PPT Presentation

Grape color variation involves genetic and micro-environmental changes that alter berry phenolic and aromatic composition Pablo Carbonell-Bejerano, M Rodríguez-Lorenzo, C Royo, N Mauri, G Hilbert, C Renaud, JL Rambla, G Diretto, A Granell, S Delrot and JM Martínez-Zapater

Primary aroma of wine Red wine White wine

Primary aroma of wine Red wine White wine • Wine making process • Specific varietal attributes • Berry color variation

Ripening: synthesis of anthocyanins Veraison Ripe Anthocyanidins MybA1 UFGT MybA2 Ripening Anthocyanins

Biological role of grape anthocyanins Veraison Ripe Anthocyanidins MybA1 UFGT MybA2 Ripening Anthocyanins Anthocyanins protect embryo from: SUNLIGHT Ripe seeds ROS embryo

Situation in white grapes Veraison Ripe Anthocyanidins MybA1 UFGT MybA2 Ripening Anthocyanins Absence of anthocyanins SUNLIGHT ROS

Effect of berry color variation? Veraison Ripe Anthocyanidins MybA1 UFGT MybA2 Ripening Anthocyanins Absence of anthocyanins SUNLIGHT Berry composition effects? 1. Controled by MYBA genes. 2. Side effects due to changes in berry microclimate . ROS

Experimental system Berry color somatic variants

Experimental system Tempranillo Tinto Garnacha Tinta Somatic Somatic Structural Variation Structural Variation Tempranillo Blanco Garnacha Blanca MybA MybA Chromos. 2 Chr. 2 • Hemizygous for 1 Mb / 24 genes Chr. 5 (Royo et al., in progress ) • Hemizygous for 8 Mb / 313 genes (Carbonell-Bejerano et al., Plant Physiol 2017)

Experimental set up 1. Microclimate: Temperature 2. Metabolites: 2.1 Sugars and Organic acids: Enzymatically 2.2 Amino acids : AQC-tag UHPLC 2.3 Flavonols : UHPLC-DAD (only in Skin) 2.4 No polar compounds : LC-DAD-APCI-HRMS APCI-MS (only at M) 2.5 Semipolar compounds : LC-ESI(+)-HRMS LC-MS (only at M) 2.6 Volatiles: HS-SPME GC-MS (only at M) 3. Transcriptome: Illumina RNA-seq (PV, V and PM) Sampling TT TB Density sorting Skin (S) and at four stages: Flesh (F) PV V PM M dissection GT GB

Color effect on berry microclimate BERRY TEMPERATURE Tempranillo Tempranillo Tinto Blanco Average of 10 ripening days

Color effect on berry metabolites TEMPRANILLO GARNACHA Sugars (g/l) Sugars (g/l) 300 300 200 200 100 100 0 0 PV V PM M PV V PM M Black White Black White Malic (g/l) Malic (g/l juice) 20 15 10 10 5 0 0 PV V PM M PV V PM M black White Black White • No significant effect in technological ripening

Color effect on berry metabolites Non polar carotenoids and chlorophylls Maturity, Skin and Flesh PCA PCA 6 TB3S 5 TB1S TB2S 4 3 Flesh F2 (15.7 %) 2 Tempranillo TT2F TT1F TB3F TT3F skin 1 TB2F TB1F GT2F GT1F GT3F 0 GB1F GB2S GB2F GB3F -1 GB1S TT2S TT3S GB3S Garnacha -2 TT1S skin -3 GT2S GT3S GT1S -4 -6 -4 -2 0 2 4 6 8 F1 (52.9 %)

Color effect on berry metabolites Non polar carotenoids and chlorophylls Maturity, Skin and Flesh PLS-DA PCA Color variable (VIP >1) 6 TB3S 0.5 5 TB1S TB2S 4 0.4 pyropheophorbide b pyropheophorbide a lutein-like structure violaxanthin esters 3 0.3 Flesh F2 (15.7 %) 2 Tempranillo chlorophyll a TT2F TT1F TB3F TT3F zeaxanthin skin α -carotene 1 TB2F TB1F 0.2 GT2F Standardized coefficients GT1F GT3F 0 GB1F GB2S 0.1 GB2F GB3F -1 GB1S TT2S TT3S GB3S Garnacha -2 0 TT1S pheophytin b pheophytin a-like lutein 5,6-epoxide skin -3 GT2S GT3S GT1S -0.1 -4 -6 -4 -2 0 2 4 6 8 F1 (52.9 %) -0.2 -0.3 -0.4

Color effect on berry metabolites Semipolar precursors: terpenoids , phenolics and amino acids Maturity, Skin and Flesh PLS-DA PCA Color vector (VIP >1) 8 OH- β -damascenone-arabinofuranose 0.4 Z-linalool oxide-Rhamnopyr.-glucose Tempranillo skin Z-linalool oxide-rhamnopyranose TB3S OH- β -cyclocitral-rhamnopyranose 6 TB1S TT3S 0.3 4 TT2S TB2S Flesh F2 (16.5 %) Leucine-Isoleucine Phenylacetaldehyde TT1S 2 TB3F 0.2 GB2F TB1F TB2F Phenylalanine Coumaric acid Standardized coefficients Cinnamic acid GB1F 0 GB3F GT1F 0.1 GT2F GT3F TT1F -2 TT3F TT2F GT3S GB2S GB1S Garnacha GT2S GB3S 0 OH- β -cyclocitral-glucoside Caffeoyl alcohol Coniferyl aldehyde Sinapyl aldehyde Benzoic acid -4 GT1S skin -0.1 -6 -6 -4 -2 0 2 4 6 8 F1 (50.3 %) -0.2 -0.3 -0.4

Color effect on berry metabolites Volatile compounds Maturity, Skin and Flesh PLS-DA PCA Color vector (VIP >0.5) 4 MONOTERPENES GB3S GB1S GT3S GT1F 3 0.15 GB2S GB1F GB2F Tropical / Floral (E)-linalool oxide 2 GT2S GT1S TT1F GT2F Garnacha skin 1 0.1 TT2F TB1F β -cyclocitral α -terpineol TB2F GB3F F2 (12.7 %) 0 GT3F Standardized coefficients Linalool -1 0.05 TT3F -2 TB3F Tempranillo skin Flesh -3 0 3-carene α -pinene n TB2S TT1S -4 TT2S TT3S TB1S -5 TB3S -0.05 -6 -8 -6 -4 -2 0 2 4 6 8 10 F1 (48.4 %) -0.1 Resin Pine -0.15

Color effect on berry metabolites Flavonols Skin (PV, V, PM, M) TEMPRANILLO GARNACHA Quercetin (µg/mg DW) Quercetin (µg/mg DW) Di-hydroxylated 6 6.0 * 4 4.0 * 2 2.0 * 0 0.0 PV V PM M PV V PM M TT TB Black White Myricetin (µg/mg DW) Myricetin (µg/mg DW) Tri-hydroxylated 1.5 1.0 * 1.0 * * * 0.5 0.5 * * 0.0 0.0 PV V PM M PV V PM M Black White Black White • Absence of tri-hydroxylated flavonols in white variants

Color effect on berry transcriptome RNA-seq PV V PM PV V PM vs White vs Black ripening series FPKM >1, FDR <5% in maSigPro, FC > 1.75 Tempranillo Skin : 1220 genes Tempranillo Flesh : 1047 genes DEGs Garnacha Skin : 579 genes Garnacha Flesh : 399 genes

Color effect on berry transcriptome RNA-seq PV V PM PV V PM vs White vs Black ripening series FPKM >1, FDR <5% in maSigPro, FC > 1.75 Tempranillo Skin : 1220 genes Tempranillo Flesh : 1047 genes DEGs Garnacha Skin : 579 genes Garnacha Flesh : 399 genes Tempranillo Tinto Up Black Up (Skin) White up (Skin) (Skin and Flesh) Black White Black FPKM White Black White PV V PM PV V PM PV V PM • Flavonoid • Photosynthesis • Genes on biosynthesis • Terpenoid biosynthesis chromosomes 2 and 5

Color effect on berry transcriptome RNA-seq PV V PM PV V PM vs White vs Black ripening series FPKM >1, FDR <5% in maSigPro, FC > 1.75 Tempranillo Skin and Flesh DEGs TT Up: TT Up: 33 genes 37 genes • Gene copy number affects expression levels

Direct effect on flavonoid pathway Black skin-upregulated genes Skin Phenylalanine CHS (6) Trihydroxylated Chalcones flavonols CHI (3) FLS Flavonones F3H (3) Trihydroxylated Dihydroflavonols dihydroflavonols F3’5’H (10) DFR Leucoanthocyanidins LDOX MYBPA1 Anthocyanidins MYBAs UFGT Anthocyanins

Direct effect on flavonoid pathway Black skin-upregulated genes Skin Phenylalanine CHS (6) Trihydroxylated Chalcones flavonols CHI (3) FLS Flavonones F3H (3) Trihydroxylated Dihydroflavonols dihydroflavonols F3’5’H (10) DFR Leucoanthocyanidins LDOX MYBPA1 Anthocyanidins MYBAs UFGT Anthocyanins

Indirect effect on light responses White skin-upregulated genes Skin Flavonol Synthase Flavonols MybF1 Hy5-1 TPSs: Linalool synthase (3) Hy5-2 Myrcene synthase (1) 1,8 cineole synthase (4) Monoterpenes Violaxanthin de-epoxidase Zeaxanthin

Indirect effect on light responses White skin-upregulated genes Skin Flavonol Synthase Flavonols MybF1 Hy5-1 TPSs: Linalool synthase (3) Hy5-2 Myrcene synthase (1) 1,8 cineole synthase (4) Monoterpenes Violaxanthin de-epoxidase Zeaxanthin Rinaldo et al., Plant Physiol (2015) Massonnet et al., Plant Physiol (2017)

Concluding remarks 1. Anthocyanins prevent the accumulation of light-inducible compounds . 2. Alternative photoprotectants and antioxidants accumulate in white grapes. 3. MYBA genes increase flavonoid backbone biosynthesis and trigger flavonol tri-hydroxylation .

Acknowledgements Ghislaine Hilbert Christel Renaud Maite Rodríguez-Lorenzo Serge Delrot Carolina Royo Nuria Mauri José M. Martínez-Zapater J.L. Rambla Antonio Granell Gianfranco Diretto

Color effect on berry microclimate Anthocyanins Tempranillo Garnacha Tinto Tinta

Color effect on berry metabolites Flavonols Skin (PV, V, PM, M) PCA 3 TBPE1 GBPM1 GTPM3 2 GBPE1 TBPM2 TBPE3 GTM2 GBPE3 TBM3 GTM3 TBM2 GBM1 1 GTM1 TBPM1 TBPE2 F2 (27.2 %) TBPM3 GTPM2 TBM1 GBM2 GBE1 GBPM3 0 TTM1 TTE2 GTE1 GTE2 GTPE1 TTE3 GBPM2 TBE2 GTPM1 TTPM1 TTE1 TTM2 GTPE2 TTPE1 TTPM3 -1 TBE1 TTM3 GTE3 GTPE3 TTPE3 TTPM2 GBE3 TBE3 GBM3 GBPE2 GBE2 -2 TTPE2 -3 -3 -2 -1 0 1 2 3 4 F1 (50.5 %)