grape color variation involves genetic and ... -...

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

0

100

200

300

PV V PM M

Sugars (g/l)

Black White

0

100

200

300

PV V PM M

Sugars (g/l)

Black White

0

10

20

PV V PM M

Malic (g/l)

black White

0

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


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

0

1

2

3

4

5

6

-6 -4 -2 0 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

0

1

2

3

4

5

6

-6 -4 -2 0 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

zeax

anth

in

pyr

op

he

op

ho

rbid

e a

pyr

op

he

op

ho

rbid

e b

lute

in-l

ike

stru

ctu

re

vio

laxa

nth

in e

ster

s

chlo

rop

hyl

l a

α-c

aro

ten

e lu

tein

5,6

-ep

oxi

de

ph

eo

ph

ytin

a-l

ike

p

he

op

hyt

in b

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

Sta

nd

ard

ized

co

eff

icie

nts


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

0

2

4

6

8

-6 -4 -2 0 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-c

yclo

citr

al-r

ham

no

pyr

ano

se

Ph

enyl

acet

ald

ehyd

e

Z-lin

alo

ol o

xid

e-R

ham

no

pyr

.-gl

uco

se

OH

-β-d

amas

cen

on

e-a

rab

ino

fura

no

se

Cin

nam

ic a

cid

Z-lin

alo

ol o

xid

e-r

ham

no

pyr

ano

se

Leu

cin

e-I

sole

uci

ne

Co

um

aric

aci

d

Ph

en

ylal

anin

e

Ben

zoic

aci

d

Sin

apyl

ald

ehyd

e

Caf

feo

yl a

lco

ho

l O

H-β

-cyc

loci

tral

-glu

cosi

de

Co

nif

eryl

ald

ehyd

e

-0.4

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

Sta

nd

ard

ize

d c

oeff

icie

nts

PCA


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

0

1

2

3

4

-8 -6 -4 -2 0 2 4 6 8 10

F2 (

12

.7 %

)

F1 (48.4 %)

Volatile compounds Maturity, Skin and Flesh

PCA

(E)-

linal

oo

l oxi

de

α

-ter

pin

eol

β-c

yclo

citr

al

Lin

alo

ol

3-c

aren

e

α-p

inen

e n

-0.15

-0.1

-0.05

0

0.05

0.1

0.15

Sta

nd

ard

ize

d c

oeff

icie

nts

Tempranillo skin

Garnacha skin

Flesh

PLS-DA Color vector (VIP >0.5)

MONOTERPENES

Tropical / Floral

Resin Pine


Flavonols Skin (PV, V, PM, M)

• Absence of tri-hydroxylated flavonols in white variants

0

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 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


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

FPK

M

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


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

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 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.

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

0

1

2

3

-3 -2 -1 0 1 2 3 4

F2 (

27

.2 %

)

F1 (50.5 %)


grape color variation involves genetic and ... -...

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