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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
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
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
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
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
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
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
Color effect on berry metabolites
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 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
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
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 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 %)
Color effect on berry metabolites