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

Ripe

Ripening: synthesis of anthocyanins

Anthocyanidins Anthocyanins UFGT MybA1 MybA2 Veraison Ripening

Ripe Veraison

Anthocyanins protect embryo from:

embryo Ripe seeds SUNLIGHT

ROS

Biological role of grape anthocyanins

Anthocyanidins Anthocyanins UFGT MybA1 MybA2 Ripening

Ripe

Situation in white grapes

SUNLIGHT

ROS

Absence of anthocyanins

Anthocyanidins Anthocyanins UFGT MybA1 MybA2 Veraison Ripening

Ripe

Effect of berry color variation?

SUNLIGHT

ROS

Absence of anthocyanins

Anthocyanidins Anthocyanins UFGT MybA1 MybA2

Berry composition effects?

• 1. Controled by MYBA genes.
• 2. Side effects due to

changes in berry microclimate.

Veraison Ripening

Berry color somatic variants

Experimental system

Tempranillo Blanco

• Chromos. 2

Somatic Structural Variation Tempranillo Tinto Garnacha Blanca Garnacha Tinta Somatic Structural Variation

• Hemizygous for 8 Mb / 313 genes

(Carbonell-Bejerano et al., Plant Physiol 2017) MybA MybA

• Chr. 5
• Chr. 2
• Hemizygous for 1 Mb / 24 genes

(Royo et al., in progress)

Experimental system

Experimental set up

Density sorting at four stages: PV V PM M Skin (S) and Flesh (F) dissection TB TT GB GT

• 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

BERRY TEMPERATURE

Average of 10 ripening days

Color effect on berry microclimate

Tempranillo Blanco Tempranillo Tinto

Color effect on berry metabolites

100 200 300 PV V PM M

Sugars (g/l)

Black White 100 200 300 PV V PM M

Sugars (g/l)

Black White 10 20 PV V PM M

Malic (g/l)

black White

5 10 15 PV V PM M

Malic (g/l juice)

Black White

TEMPRANILLO GARNACHA

• No significant effect in technological ripening

Non polar carotenoids and chlorophylls

Maturity, Skin and Flesh PCA

Color effect on berry metabolites

TB1S TB2S TB3S TB1F TB2F TB3F GB1S GB2S GB3S GB1F GB2F GB3F TT1S TT2S TT3S TT1F TT2F TT3F GT1S GT2S GT3S GT1F GT2F GT3F

• 4
• 3
• 2
• 1

1 2 3 4 5 6

• 6
• 4
• 2

2 4 6 8

F2 (15.7 %) F1 (52.9 %)

PCA

Flesh

Tempranillo skin Garnacha skin

TB1S TB2S TB3S TB1F TB2F TB3F GB1S GB2S GB3S GB1F GB2F GB3F TT1S TT2S TT3S TT1F TT2F TT3F GT1S GT2S GT3S GT1F GT2F GT3F

• 4
• 3
• 2
• 1

1 2 3 4 5 6

• 6
• 4
• 2

2 4 6 8

F2 (15.7 %) F1 (52.9 %)

Non polar carotenoids and chlorophylls

Maturity, Skin and Flesh PLS-DA Color variable (VIP >1) PCA

Flesh

Tempranillo skin Garnacha skin

zeaxanthin pyropheophorbide a pyropheophorbide b lutein-like structure violaxanthin esters chlorophyll a α-carotene lutein 5,6-epoxide pheophytin a-like pheophytin b

• 0.4
• 0.3
• 0.2
• 0.1

0.1 0.2 0.3 0.4 0.5

Standardized coefficients

Color effect on berry metabolites

TT1F TT2F TT3F GT1F GT2F GT3F TT1S TT2S TT3S GT1S GT2S GT3S TB1F TB2F TB3F GB1F GB2F GB3F TB1S TB2S TB3S GB1S GB2S GB3S

• 6
• 4
• 2

2 4 6 8

• 6
• 4
• 2

2 4 6 8

F2 (16.5 %) F1 (50.3 %)

Semipolar precursors: terpenoids, phenolics and amino acids

Maturity, Skin and Flesh PLS-DA Color vector (VIP >1)

Tempranillo skin Garnacha skin Flesh

OH-β-cyclocitral-rhamnopyranose Phenylacetaldehyde Z-linalool oxide-Rhamnopyr.-glucose OH-β-damascenone-arabinofuranose Cinnamic acid Z-linalool oxide-rhamnopyranose

Leucine-Isoleucine

Coumaric acid

Phenylalanine

Benzoic acid Sinapyl aldehyde Caffeoyl alcohol

OH-β-cyclocitral-glucoside

Coniferyl aldehyde

• 0.4
• 0.3
• 0.2
• 0.1

0.1 0.2 0.3 0.4

Standardized coefficients

PCA

Color effect on berry metabolites

TB1S TB2S TB3S GB1S GB2S GB3S TB1F TB2F TB3F GB1F GB2F GB3F TT1S TT2S TT3S GT1S GT2S GT3S TT1F TT2F TT3F GT1F GT2F GT3F

• 6
• 5
• 4
• 3
• 2
• 1

1 2 3 4

• 8
• 6
• 4
• 2

2 4 6 8 10

F2 (12.7 %) F1 (48.4 %)

Volatile compounds

Maturity, Skin and Flesh PCA (E)-linalool oxide α-terpineol β-cyclocitral Linalool 3-carene α-pinene n

• 0.15
• 0.1
• 0.05

0.05 0.1 0.15

Standardized coefficients

Tempranillo skin Garnacha skin Flesh

PLS-DA Color vector (VIP >0.5) MONOTERPENES

Tropical / Floral Resin Pine

Color effect on berry metabolites

Flavonols

Skin (PV, V, PM, M)

• Absence of tri-hydroxylated flavonols in white variants

2 4 6 PV V PM M

Quercetin (µg/mg DW)

TT TB * * * 0.0 2.0 4.0 6.0 PV V PM M

Quercetin (µg/mg DW)

Black White 0.0 0.5 1.0 1.5 PV V PM M

Myricetin (µg/mg DW)

Black White * * * 0.0 0.5 1.0 PV V PM M

Myricetin (µg/mg DW)

Black White * * *

TEMPRANILLO GARNACHA

Di-hydroxylated Tri-hydroxylated

Color effect on berry metabolites

Color effect on berry transcriptome

RNA-seq

DEGs White vs Black ripening series

FPKM >1, FDR <5% in maSigPro, FC > 1.75 PV V PM PV V PM

vs Tempranillo Skin: 1220 genes Tempranillo Flesh: 1047 genes Garnacha Skin: 579 genes Garnacha Flesh: 399 genes

Color effect on berry transcriptome

RNA-seq

Tempranillo Skin: 1220 genes Tempranillo Flesh: 1047 genes Garnacha Skin: 579 genes Garnacha Flesh: 399 genes

DEGs

PV V PM

Tempranillo Tinto Up (Skin and Flesh) White up (Skin) Black Up (Skin)

• Genes on

chromosomes 2 and 5

• Photosynthesis
• Terpenoid biosynthesis
• Flavonoid

biosynthesis FPKM

White vs Black ripening series

FPKM >1, FDR <5% in maSigPro, FC > 1.75 PV V PM PV V PM White

Black

White

Black

White

Black

PV V PM PV V PM

vs

TT Up:

33 genes

TT Up:

37 genes Tempranillo Skin and Flesh DEGs

• Gene copy number affects expression levels

Color effect on berry transcriptome

RNA-seq

White vs Black ripening series

FPKM >1, FDR <5% in maSigPro, FC > 1.75 PV V PM PV V PM

vs

F3’5’H (10)

Phenylalanine Chalcones Flavonones Dihydroflavonols Leucoanthocyanidins Anthocyanidins

Anthocyanins

Trihydroxylated flavonols

FLS

CHS (6) CHI (3) F3H (3) DFR LDOX

UFGT

Direct effect on flavonoid pathway

Trihydroxylated dihydroflavonols

MYBAs

MYBPA1

Skin

Black skin-upregulated genes

F3’5’H (10)

Phenylalanine Chalcones Flavonones Dihydroflavonols Leucoanthocyanidins Anthocyanidins

Anthocyanins

Trihydroxylated flavonols

FLS

CHS (6) CHI (3) F3H (3) DFR LDOX

UFGT

Direct effect on flavonoid pathway

Trihydroxylated dihydroflavonols

MYBAs

MYBPA1

Skin

Black skin-upregulated genes

Skin Flavonol Synthase TPSs: Monoterpenes

Indirect effect on light responses

White skin-upregulated genes

Linalool synthase (3) Myrcene synthase (1) 1,8 cineole synthase (4) Flavonols MybF1 Hy5-1 Hy5-2 Violaxanthin de-epoxidase Zeaxanthin

Skin Flavonol Synthase TPSs: Monoterpenes

Indirect effect on light responses

White skin-upregulated genes

Linalool synthase (3) Myrcene synthase (1) 1,8 cineole synthase (4) Flavonols MybF1 Hy5-1 Hy5-2 Violaxanthin de-epoxidase Zeaxanthin Rinaldo et al., Plant Physiol (2015) Massonnet et al., Plant Physiol (2017)

Concluding remarks

1. Anthocyanins prevent the accumulation

• f light-inducible compounds.

2. Alternative photoprotectants and antioxidants accumulate in white grapes. 3. MYBA genes increase flavonoid backbone biosynthesis and trigger flavonol tri-hydroxylation.

J.L. Rambla Antonio Granell

Acknowledgements

Maite Rodríguez-Lorenzo Carolina Royo Nuria Mauri José M. Martínez-Zapater Ghislaine Hilbert Christel Renaud Serge Delrot Gianfranco Diretto

Anthocyanins

Color effect on berry microclimate

Tempranillo Tinto Garnacha Tinta

Flavonols

Skin (PV, V, PM, M) PCA

TBPE1 TBPE2 TBPE3 GBPE1 GBPE2 GBPE3 TBE1 TBE2 TBE3 GBE1 GBE2 GBE3 TBPM1 TBPM2 TBPM3 GBPM1 GBPM2 GBPM3 TBM1 TBM2 TBM3 GBM1 GBM2 GBM3 TTPE1 TTPE2 TTPE3 GTPE1 GTPE2 GTPE3 TTE1 TTE2 TTE3 GTE1 GTE2 GTE3 TTPM1 TTPM2 TTPM3 GTPM1 GTPM2 GTPM3 TTM1 TTM2 TTM3 GTM1 GTM2 GTM3

• 3
• 2
• 1

1 2 3

• 3
• 2
• 1

1 2 3 4

F2 (27.2 %) F1 (50.5 %)

Color effect on berry metabolites