Journal of Experimental Botany, Vol. 58, No. 8, pp. 1935–1945, 2007
doi:10.1093/jxb/erm055 Advance Access publication 23 April, 2007
RESEARCH PAPER
Loss of anthocyanins in red-wine grape under high
temperature
Kentaro Mori1,2,*,†, Nami Goto-Yamamoto1, Masahiko Kitayama2 and Katsumi Hashizume1
1
National Research Institute of Brewing, Higashi-Hiroshima, Hiroshima 739-0046, Japan
2
Institute of Life Science, Ehime Women’s College, Uwajima, Ehime 798-0025, Japan
Received 30 December 2006; Revised 26 February 2007; Accepted 27 February 2007
Abstract
To determine the mechanism of inhibition of anthocyanin accumulation in the skin of grape berries due to
high temperature, the effects of high temperature on
anthocyanin composition and the responses in terms
of gene transcript levels were examined using Vitis
vinifera L. cv. Cabernet Sauvignon. High temperature
(maximum 35 C) reduced the total anthocyanin content to less than half of that in the control berries
(maximum 25 C). HPLC analysis showed that the
concentrations of anthocyanins, with the exception
of malvidin derivatives (3-glucoside, 3-acetylglucoside,
and 3-p-coumaroylglucoside), decreased considerably
in the berries grown under high temperature as compared with the control. However, Affymetrix Vitis
GeneChip microarray analysis indicated that the
anthocyanin biosynthetic genes were not strongly downregulated at high temperature. A quantitative real time
PCR analysis confirmed this finding. To demonstrate
the possibility that high temperature increases anthocyanin degradation in grape skin, stable isotopelabelled tracer experiments were carried out. Softened
green berries of Cabernet Sauvignon were cut and
aseptically incubated on filter paper with 1 mM aqueous L-[1-13C]phenylalanine solution for 1 week. Thereafter, the changes in 13C-labelled anthocyanins were
examined under different temperatures (15, 25, and
35 C). In the berries cultured at 35 C, the content of
total 13C-labelled anthocyanins that were produced
before exposure to high temperature was markedly
reduced as compared with those cultured at 15 C and
25 C. These data suggest that the decrease in anthocyanin accumulation under high temperature results
from factors such as anthocyanin degradation as well
as the inhibition of mRNA transcription of the anthocyanin biosynthetic genes.
Key words: Anthocyanin, degradation, gene transcription,
grape, high temperature.
Introduction
Anthocyanins are plant secondary metabolites that are
responsible for the characteristic red, blue, and purple
colour of plant tissues. Anthocyanins play an important
role in plant reproduction, by attracting pollinators and
seed dispersers, and also in protection from stress including photo-oxidative stress (Winkel-Shirley, 2002).
In grapes, the berry skin accumulates large amounts of
anthocyanins, which contribute to the sensory attributes of
wine. Furthermore, considerable attention has been paid
recently to the health benefits of anthocyanins, since epidemiological investigations have indicated that the moderate consumption of anthocyanin products such as red
wine is associated with a lower risk of cardiovascular
disease (Hou, 2003). In hot regions, however, anthocyanin
accumulation is inhibited in the skins of red and black
grapes (Winkler et al., 1962). In addition, any global
atmospheric warming trend may affect grape berry
ripening in the future. Jones et al. (2005) suggested that,
in regions that produce high-quality grapes on the margins
of their climatic limits, future climate change would
exceed a threshold, as a result of which the balanced fruit
ripening required for existing varieties and wine styles
would become progressively more difficult. Although the
effects of temperature on the content of anthocyanins in
* To whom correspondence should be addressed. E-mail: [email protected]
y
Present address: Institut des Sciences de la Vigne et du Vin (ISVV), UMR Ecophysiologie et Génomique Fonctionnelle de la Vigne, Domaine de la
Grande Ferrade, INRA, BP 81, 33883 Villenave d’Ornon, France.
ª The Author [2007]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
1936 Mori et al.
grape berry skins have also been studied intensively
(Kliewer, 1970; Buttrose et al., 1971; Kliewer and Torres,
1972; Spayd et al., 2002; Mori et al., 2005b; Yamane
et al., 2006), the mechanisms responsible for the poor
coloration of berry skin at high temperatures have not
been completely understood.
Temperature is an important factor that affects anthocyanin biosynthesis in plants. The expression of the
anthocyanin biosynthetic genes has been induced by low
temperature and repressed by high temperature in various
plants, such as apple (Ubi et al., 2006), Arabidopsis
(Leyva et al., 1995), grape (Mori et al., 2005b; Yamane
et al., 2006), maize (Christie et al., 1994), petunia (Shvarts
et al., 1997), red orange (Lo Piero et al., 2005), and rose
(Dela et al., 2003). Thus, it has already been established
that gene expression of the enzymes involved in anthocyanin biosynthesis is affected by temperature, but temperature would affect diverse metabolisms in plants as well as
anthocyanin biosynthesis. For example, Shaked-Sachray
et al. (2002) speculated that temperature might affect
not only the synthesis but also the stability and that,
therefore, the decrease in anthocyanin concentration at
elevated temperatures might result from both a decrease in
synthesis and an increase in degradation. However, very
little is known about the catabolism of anthocyanins in
plant tissue. Many studies about anthocyanin degradation
have been conducted in extracted pigments, wine, and
grape juice (Sarni et al., 1995; Yokotsuka and Singleton,
1997; Romero and Bakker, 2000; Morais et al., 2002, and
references therein). Elevated temperature of storage decreased the concentration of anthocyanins in a model
solution (Romero and Bakker, 2000; Morais et al., 2002).
However, as far as is known, no report has demonstrated
the enhancement of anthocyanin degradation due to high
temperature in plant tissue.
Here, the effects of high temperature on the biosynthesis
and stability of anthocyanin were investigated in the skin
of Vitis vinifera L. cv. Cabernet Sauvignon grape berries
in order to understand comprehensively the mechanisms
of the inhibition of anthocyanin accumulation due to high
temperature.
Materials and methods
Plant material and temperature treatments
The experiment was conducted using four 11-year-old potted
grapevines of V. vinifera L. cv. Cabernet Sauvignon grafted on
SO4 (Selection Oppenheim No. 4) grown in a phytotron. Vines
were trained on a Guyot trellising system and each vine carried
4–10 clusters of grapes. The experiment started approximately
1 week before veraison, when berry softening started, and continued
to fruit maturity. The two temperature regimes consisted of a high
day (06.00–20.00 h) temperature (max. 35 C) and a control (max.
25 C). Under both conditions, the night-time (20.00–06.00 h)
temperature was 20 C. The photoperiod corresponded to natural
day length. Twenty-five per cent of the berries (15–20 berries) in
each cluster was sampled at random from each temperature
treatment at 2 week intervals after veraison, and the berries were
pooled to produce two samples for each vine. Each sample pool was
derived from the different clusters. The berries were manually
peeled with a scalpel to eliminate any flesh. The berry skin was
frozen immediately in liquid nitrogen and stored at –80 C until use.
Anthocyanin extraction and HPLC analysis
The anthocyanin contents of grape berry skins were determined
using HPLC as described previously (Mori et al., 2005a). Analyses
were carried out on four biological replicates.
RNA isolation
Total RNA was extracted from 1 mg of mixtures of two pooled
berry skins as described by Geuna et al. (1998), and treated with
RNase-free DNase I (Takara, Otsu, Japan) and further purified using
RNeasy mini column (Qiagen, Valencia, CA, USA) following the
manufacturers’ specifications.
GeneChip analysis
The quality of the total RNA was examined with the RNA 6000
Nano Assay on the Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA (5 lg) was used to synthesize cRNA, which was
hybridized to an Affymetrix Vitis vinifera GeneChip microarray
(Affymetrix, Santa Clara, CA, USA). Two independent biological
replicate microarray hybridizations were performed for all samples.
Synthesis of cRNA, hybridization to the Vitis GeneChips, and
scanning were performed using an Affymetrix-recommended protocol. The hybridization data were analysed using GeneChip
Operating Software (GCOS 1.2) described by Solfanelli et al.
(2005). A global scaling factor of 500, a normalization value of 1,
and a default parameter setting for the Vitis vinifera GeneChip
were used. Signal values and detection call values were generated
using the GCOS 1.2 software. Probe pair sets (genes) called
‘Absent’ in control and high-temperature conditions were removed
from subsequent analyses. Furthermore, genes with ‘Absent’ for the
detection value in the control and ‘Decrease’ for the change call
were excluded from the list. Similarly, genes with ‘Absent’ for the
detection call in the experimental data and ‘Increase’ for the change
value were also excluded from the list. Differences in transcript
abundance, expressed as the signal log ratio, were calculated using
the GCOS 1.2 software change algorithm. The signal log ratio was
assumed to be correct only if the corresponding change call
indicated a significant change (‘Increase’ or ‘Decrease’ generated
using the GCOS 1.2 software). Expression data were filtered to
select only genes showing a coinciding change call in the two
biological replicate samples for each experimental treatment. Differentially expressed genes were selected on the basis of 2-fold changes
as compared with the control and further analysed using KMC
algorithms with a Euclidian distance metric as implemented in TIGR
MeV (Saeed et al., 2003).
Quantitative real-time PCR analysis
The transcript levels of anthocyanin biosynthetic genes were
determined as described by Jeong et al. (2006). The PCR mixture
contained 1 ll of the cDNA template, 10 ll of 23 Quantitect
SYBR Green PCR Master Mix (Qiagen), and 0.25 lM of the
forward and reverse primers for each gene. Reactions were run on
the GeneAmp 5700 sequence detection system (Applied Biosystems, Foster City, CA, USA). The Q-PCR was performed under the
following conditions: 95 C for 15 min, followed by 40 cycles at
95 C for 15 s, at the annealing temperature of 56 C (52 C for
VvmybA1) for 20 s and 72 C for 20 s. The Q-PCR was carried out
on four replicates per prepared cDNA sample, and the transcript
Decrease of anthocyanins in grape skin 1937
levels of each gene were normalized to the VvUbiquitin1 (Fujita
et al., 2005) control gene. The data were presented as the mean
value of two vines.
Enzyme assay
The extraction was performed according to the method of Ozeki
et al. (1987) with some modifications. The following procedures for
protein extraction were conducted at 4 C. Grape skin (3 g) was
ground with a mortar and pestle in liquid nitrogen until a fine powder
was obtained. The skin powder was homogenized with 15 ml of
a 250 mM TRIS–HCl buffer (pH 7.5) containing 10 mM polyethylene glycol 3400, 20 mM Na-diethyldithiocarbamate, 2 mM
dithiothreitol, and 14 mM of 2-mercaptoethanol. After centrifugation
of the homogenate at 9400 g for 20 min, 1 g of Dowex 134 (HClform, equilibrated with the same buffer as above) was added to the
supernatant. The supernatant was incubated for 20 min on ice with
gentle stirring and centrifuged again at 4900 g for 5 min. Solid
ammonium sulphate was added to the supernatant to achieve 30%
saturation, and the mixture was centrifuged at 9400 g for 10 min.
Protein was precipitated from the supernatant by adding ammonium
sulphate to 70% final saturation, and the sample was centrifuged at
9400 g for 30 min. The protein pellet was resuspended in 2.5 ml of
a 25 mM TRIS–HCl buffer (pH 7.5). The extract was passed through
a PD10 column (Sephadex G-25, GE Healthcare, Amersham, Bucks,
UK) equilibrated with a 25 mM TRIS–HCl buffer (pH 7.5). The
desalted crude extract was used as the enzyme solution in the
following enzyme assay. The method of Ford et al. (1998) was
employed with some modifications for the analysis of UFGT activity.
The reaction mixture consisted of 100 ll of a 100 mM TRIS–HCl
buffer (pH 8.0), 10 mM polyethylene glycol 3400, 20 mM
Na-diethyldithiocarbamate, 2 mM dithiothreitol, and 14 mM of
2-mercaptoethanol, 0.1 mM cyanidin chloride, 10 mM UDP-glucose,
and 100 ll of an enzyme solution. The assay mixture was incubated
for 6 min at 30 C. The reaction was terminated by adding 150 ll of
5% HCl. The quantity of the product, namely cyanidin 3-glucoside
was measured using HPLC at 520 nm. One unit of UFGT was
defined as the production of 1 mol of cyanidin 3-glucoside per
second, and UFGT activity was expressed as kat g1 protein. The
protein concentration was determined using the Bio-Rad Quick Start
kit (Bio-Rad, Hercules, CA, USA) based on the Bradford technique.
Berry culture and 13C stable isotope tracer experiment
At veraison, softened green berries (V. vinifera L. cv. Cabernet
Sauvignon) were excised from the rachis, and sterilized with a dilute
solution of sodium hypochlorite (1%), then with ethanol (70%), and
finally rinsed twice with deionized water. The sterilized berries were
cut around the peduncle and aseptically incubated on filter paper in
a Petri dish. A 4.5 ml filter-sterilized aqueous [13C]phenylalanine
solution (0.3 M sucrose, 1 mM L-[1-13C]phenylalanine) was applied
to the berries, and the berries were then incubated at 25 C under
fluorescent light at approximately 40 lmol m2 s1. After 1 week
of culture, the berries were placed on a new Petri dish containing
a 0.3 M sucrose solution. Thereafter, the berries were cultured
without phenylalanine at 15, 25, and 35 C under fluorescent light
at approximately 40 lmol m2 s1. After 0, 2, 5, and 7 d of
temperature treatment, the berries from each dish were collected. As
stated above, the berries were peeled, frozen immediately in liquid
nitrogen and stored at 80 C until use. Experiments were
triplicated, with each replicate consisting of six or seven berries
in a Petri dish.
LC-MS analysis of 13C-labelled anthocyanin
Anthocyanin extracts were quantified by LC-MS (LCQ Advantage,
Thermo Finnigan, San Jose, CA, USA) with a Zorbax SB-C18
column (5 lm, 4.6 mm3250 mm; Agilent Technologies). Solvent A
consisted of water/formic acid (95:5, v/v) and solvent B was
methanol/acetonitrile/water (33:60:70, by vol). The solvent system
initially consisted of 80% A and 20% B with the following changes:
10 min, 35% B; 30 min, 52% B; 35 min, 60% B; 45 min, 60% B;
50 min, 75% B; and 55–60 min, 20% B. The flow rate was 0.3 ml
min1, and the sample volume injected was 5 ll. The unsplit eluent
entered the ESI-interface through a fused silica capillary. The ion
spray voltage was +2.5 kV; the capillary temperature was 280 C.
The mass spectrometer was operated in a selected ion monitoring
mode (SIM) detecting positive ions. The levels of 13C-labelled
anthocyanins were calculated from peak areas of 12C (A12) and
13
C (A13) using the following formula: L¼[A13/(A12+A13)–
R]*(A12+A13), where R is the isotope abundance A13/(A12+A13) of
unlabelled anthocyanins. Since each anthocyanin naturally contains
1.1% of the stable isotope 13C, the isotope abundances of unlabelled
anthocyanins were subtracted from the isotope abundances of 13Clabelled anthocyanins. The amounts of anthocyanins were expressed
as the external standard equivalent (malvidin 3-glucoside) from the
calibration curve.
Results
Effects of high temperature on anthocyanin content
and composition
The total anthocyanin content in skins of Cabernet
Sauvignon berries increased after veraison and peaked at
4 weeks after veraison (WAV) under control conditions
(Fig. 1A). However, high temperature reduced the total
anthocyanin content to less than half of that in the control
berries at 4 WAV. HPLC analysis showed that the major
anthocyanins in the skin of Cabernet Sauvignon berries
were the 3-monoglucoside, 3-acetylglucoside, and 3-pcoumaroylglucoside derivatives of delphinidin, cyanidin,
petunidin, peonidin, and malvidin. The composition of
anthocyanin varied in response to high temperature (Fig.
1B, C). The content of individual anthocyanins, with the
exception of malvidin derivatives (3-glucoside, 3-acetylglucoside, and 3-p-coumaroylglucoside), decreased considerably under high temperature as compared with the
control.
Effects of high temperature on the patterns of global
gene transcription and anthocyanin biosynthesis
To examine the mechanisms responsible for the reduction
of anthocyanin accumulation in the skin of berries under
high temperature, the effects of high temperature on gene
transcription in the skin were investigated using a highdensity oligonucleotide microarray (Affymetrix GeneChip). A total of 405 genes that were differentially
transcribed by at least 2-fold between the berry skins
grown under high temperature (see Materials and methods) were identified and subjected to k-means clustering
(KMC) analysis with the Euclidian distance metric (Fig. 2).
The list of 405 genes is shown in Supplementary Table
S1 available at JXB online. Cluster 1 (16 genes) decreased
at an early stage (2 WAV) and increased at a later stage
1938 Mori et al.
A
Anthocyanin (mg g-1 Skin FW)
22
20
18
16
14
12
10
8
6
4
Control
2
High temp
0
0
2
4
6
Weeks after veraison
Anthocyanin (mg g-1 Skin FW)
B
2 WAV
4 WAV
6.0
6 WAV
4.0
2.0
0.0
Anthocyanin (mg g-1 Skin FW)
C
2 WAV
4 WAV
6.0
6 WAV
4.0
2.0
0.0
Dp-
Cy-
Pt-
Pn-
Mv-
Dp-
Cy-
Pt-
Pn-
Mv-
Dp-
Cy-
Pt-
Pn-
Mv-
3G
3G
3G
3G
3G
3G-
3G-
3G- 3G-
3G-
3G-
3G-
3G-
3G-
3G-
Ac
Ac
Ac
Ac
pC
pC
pC
pC
pC
Ac
Fig. 1. Effects of high temperature on anthocyanin accumulation in the skin of Vitis vinifera L. cv. Cabernet Sauvignon grape berries. (A) Changes
in total anthocyanin accumulation in the skin of berries grown under control (25 C; closed circles) and high temperature (35 C; open circles). (B, C)
Changes in individual anthocyanin accumulation in the skin of berries grown under control (B) and high temperature (C). Values are expressed on
a skin fresh weight (FW) basis. Vertical bars indicate the standard deviation of the mean (n¼4 biological replicates). Abbreviations: Dp, delphinidin;
Cy, cyanidin; Pt, petunidin; Pn, peonidin; Mv, malvidin; 3G, 3-glucoside; Ac, acetate; pC, p-coumarate.
(6 WAV). By contrast, cluster 7 (23 genes) increased at an
early stage and decreased at a later one. Clusters 2 and
3 showed patterns of early induction by high temperature
and contained 8 and 61 genes, respectively. In these
clusters, some heat-shock proteins and genes related
to photosynthesis were included. Clusters 5 and 6 were
groups that were increased at a later stage and contained
94 and 55 genes, respectively. These two clusters included
Decrease of anthocyanins in grape skin 1939
Fig. 2. Cluster analysis of 405 genes differentially expressed in the skin of Cabernet Sauvignon berries under control (25 C) and high (35 C)
temperature conditions. Temporal patterns of transcription were visualized using k–means clustering with a Euclidian distance. The genes of each
cluster are listed in Supplementary Table S1 available at JXB online.
most of the genes differentially transcribed by high
temperature. In particular, a number of defence-related
genes like genes encoding resveratrol synthase, chitinase,
and the pathogen-related protein, were induced. Genes
included in clusters 4 and 8 were continually induced by
high temperature. As with clusters 2 and 3, some genes of
the heat-shock protein were highly induced and the
chlorophyll-binding protein was also induced. Only 58
genes (cluster 9) were continually repressed by high
temperature.
However, GeneChip microarray analysis indicated that
the transcript levels of anthocyanin biosynthetic genes
were not changed more than 2-fold in response to high
temperature, with the exception of caffeoyl-CoA Omethyltransferase (1614643_at), although most of the
genes were slightly repressed by high temperature
(Table 1). A quantitative real-time PCR analysis confirmed this finding. The mRNA levels of most anthocyanin biosynthetic genes that were exposed to high
temperature increased transiently at 2 WAV and then
decreased at 4 WAV; however, the difference in mRNA
levels between the high temperature condition and the
control was smaller than the difference in the total
anthocyanin content, with the exception of flavanone 3hydroxylase (F3H2) and dihydroflavonol 4-reductase
(DFR) (Fig. 3).
To confirm that the mRNAs of the anthocyanin biosynthetic genes produce functionally active protein under
high temperature, the enzyme activity of UFGT, a key
enzyme of the anthocyanin biosynthetic pathway, was
assayed. At the assay temperature of 30 C, the UFGT
activity under high temperature was no different from that
of the control (Fig. 4A). To examine the UFGT activities
at the temperature in each condition, the control sample
was assayed at 25 C and the high-temperature sample at
35 C (Fig. 4B). In this case, UFGT activity in the skin of
berries grown under high temperature was higher than that
of the control. These results indicated that the berries
grown under high temperature had active UFGT enzymes.
13
C isotope tracer experiment for anthocyanin stability
in grape skin
To examine the effect of temperature on the turnover of
anthocyanin in the skin, stable isotope-labelled tracer
experiments were carried out. Softened green berries of
Cabernet Sauvignon were treated with a 1 mM aqueous
13
L-[1- C]phenylalanine solution for 1 week and, thereafter,
were cultured without phenylalanine at 15, 25, and 35 C.
The sums of unlabelled (12C) and labelled (13C) anthocyanin contents are shown in Fig. 5. The total anthocyanin
content was highest in berries cultured at 25 C and
lowest at 35 C (Fig. 5A). The contents of delphinidin
A: CHS3
30
Relative abundance of mRNA
Relative abundance of mRNA
1940 Mori et al.
25
20
15
10
Control
High Temp
5
0
2
4
25
20
15
10
5
2
4
6
4
6
4
6
D: LDOX
Relative abundance of mRNA
C: DFR
16
0
6
14
12
10
8
6
4
2
20
15
10
5
0
0
2
4
E: UFGT
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0
2
0
6
Relative abundance of mRNA
Relative abundance of mRNA
Relative abundance of mRNA
30
0
0
0
B: F3H2
35
4
6
Weeks after veraison
2
F: VvmybA1
0.16
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
2
Weeks after veraison
Fig. 3. Changes in transcription levels of anthocyanin biosynthetic genes in the skin of Vitis vinifera L. cv. Cabernet Sauvignon grape berries grown
under control (25 C; closed circles) and high temperature (35 C; open circles): (A) CHS3; (B) F3H2; (C) DFR; (D) LDOX; (E) UFGT; (F)
VvmybA1. Transcript levels of each gene are expressed relative to the internal control VvUbiquitin1 gene. Vertical bars indicate the standard deviation
(n¼2 biological replicates).
3-glucoside, cyanidin 3-glucoside, and petunidin 3-glucoside were highest at 15 C, while changes in other
derivatives of peonidin and malvidin were similar to the
total anthocyanin (Fig. 5B–F). When the contents of
individual anthocyanins were compared among three
temperatures, the differences in malvidin 3-glucoside
p-coumarate were small (Fig. 5B–J).
Changes in the content of 13C-labelled anthocyanins
that were produced before temperature treatment indicated
the loss of anthocyanins in response to high temperature
(Fig. 6A–J). In the berries cultured at 35 C, the total
content of 13C-labelled anthocyanin was markedly reduced, while there was no decrease in labelled anthocyanin content in the skin of berries at 15 C and 25 C (Fig.
6A). Similar to the accumulation patterns of the anthocyanin content, the 13C-labelled anthocyanin content of
malvidin 3-glucoside p-coumarate was not significantly
affected by temperature (Fig. 6B–J).
Decrease of anthocyanins in grape skin 1941
Table 1. Transcription levels of anthocyanin biosynthetic genes in the skin of Vitis vinifera L. cv. Cabernet Sauvignon grape berries
grown under control (25 C) and high temperature (35 C) using Affymetrix Vitis vinifera GeneChip microarray
Probe set ID
1606663_at
1607732_at
1607943_at
1615447_at
1617019_at
1615912_at
1607607_s_at
1607739_at
1620675_at
1607760_at
1611847_at
1609765_s_at
1617171_s_at
1614658_a_at
1615117_a_at
1616495_at
1619917_s_at
1611897_s_at
1614643_at
1616898_at
1620959_s_at
a
Public ID
CB981710
AF020709
CB971933
CF404765
X75969
CA813921
CF213943
CF415693
CB969894
CF515831
BM437829
CB007712
AF000371
CF518071
CB980625
CF517304
CF518071
CB347033
CF214966
BQ796057
CB915151
Gene title
log2 (high/control)
Chalcone synthase (CHS3)
Chalcone synthase
Chalcone synthase
Chalcone synthase (CHS3)
Chalcone synthase
Chalcone isomerase (CHI1)
Naringenin 3-dioxygenase [Arabidopsis thaliana] (F3H2)
Flavanone 3-hydroxylase (F3H1)
Dihydroflavonol reductase
Flavonoid-3#,5#–hydroxylase
Flavonoid-3#,5#-hydroxylase
Leucoanthocyanidin dioxygenase
UDP glucose:flavonoid 3-o-glucosyltransferase
Glutathione S-transferase (GST4)
Glutathione S-transferase (GST4)
Glutathione transferase [Arabidopsis thaliana]
Glutathione S-transferase (GST4)
S-adenosylmethionine-dependent methyltransferase [Arabidopsis thaliana]
Caffeoyl-CoA O-methyltransferase
S-adenosylmethionine-dependent methyltransferase [Arabidopsis thaliana]
VvMYBA1 myb-related transcription factor
2 WAVa
4 WAV
6 WAV
–0.04
–0.11
–0.02
–0.12
0.39
0.19
–0.20
–0.08
–0.56
0.06
0.28
0.04
0.25
–0.03
–0.04
–0.17
–0.04
0.17
0.75
0.10
0.15
–0.01
–0.27
0.62
–0.28
–0.68
0.15
–0.27
–0.30
–0.58
–0.12
–0.27
–0.20
0.01
–0.05
–0.01
0.12
0.03
0.41
1.96
0.00
–0.02
–0.24
–0.49
0.47
–0.40
0.33
0.21
–0.13
–0.51
–0.39
–0.37
–0.60
–0.38
–0.23
–0.20
–0.18
0.37
–0.14
0.80
2.51
–0.15
–0.19
Weeks after veraison.
4
3
2
1
Control
UFGT activity (kat g-1 protein)
UFGT activity (kat g-1 protein)
A
B
3
2
1
Control (25°C)
High temp (35°C)
High temp
0
0
2
4
6
Weeks after veraison
0
0
2
4
6
Weeks after veraison
Fig. 4. Changes in enzyme activities of UFGT in the skin of Vitis vinifera L. cv. Cabernet Sauvignon grape berries grown under control (25 C;
closed circles) and high temperature (35 C; open circles) at the same assay temperature (A; 30 C) and at a different assay temperature (B; 25 C and
35 C). Vertical bars represent the standard deviation of means (n¼3 assays).
In the cultured berries, most anthocyanin biosynthetic
genes were not strongly down-regulated at 35 C, with the
exception of F3H2 and DFR (data not shown).
Discussion
Despite many studies on the effects of temperature on
anthocyanin accumulation, most of the work has dealt
with the effects on the biosynthesis of anthocyanins. The
expression of anthocyanin biosynthetic genes is strongly
affected by temperature, with low temperature causing an
increase and high temperature causing a decrease in the
transcript levels of the genes (see Introduction). In the
Japanese red table grape Aki Queen, Yamane et al. (2006)
reported that high temperature reduced the endogenous
ABA level, which affected the expression of VvmybA1;
the product of VvmybA1 then controlled the expression
of the anthocyanin biosynthetic enzyme genes. In addition, these authors also discussed the possibility of the
contribution of another mechanism (e.g. anthocyanin
degradation) to the inhibitory effect of high temperature
on anthocyanin accumulation. However, no report has
demonstrated the enhancement of anthocyanin degradation due to high temperature in plant tissue.
A: Total
8
7
15°C
25°C
35°C
6
5
4
3
2
1
0
0.20
0.15
0.10
0.05
0.00
2
4
6
1.0
0.8
0.6
0.4
0.2
0.0
0
2
4
6
2.0
1.5
1.0
0.5
0.0
0
2
4
6
Days after treatment
0.10
0.05
0.00
0
8
H: Mv3G
2.5
0.15
8
E: Pn3G
1.2
8
C: Cy3G
0.20
2
4
6
F: Pn3GAc
0.5
0.4
0.3
0.2
0.1
0.0
0
2
4
6
0.4
0.3
0.2
0.1
0.0
0
2
4
6
Days after treatment
0.20
0.15
0.10
0.05
0.00
0
8
2
4
6
8
6
8
6
8
G: Pn3GpC
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
8
I: Mv3GAc
0.5
D: Pt3G
0.25
8
Anthocyanin (mg g-1Skin FW)
0.25
Anthocyanin (mg g-1Skin FW)
8
Anthocyanin (mg g-1Skin FW)
6
0
Anthocyanin (mg g-1 Skin FW)
Anthocyanin (mg g-1 Skin FW)
4
B: Dp3G
0.30
0
Anthocyanin (mg g-1 Skin FW)
2
Anthocyanin (mg g-1Skin FW)
Anthocyanin (mg g-1Skin FW)
0
Anthocyanin (mg g-1 Skin FW)
Anthocyanin (mg g-1 Skin FW)
1942 Mori et al.
2
4
J: Mv3GpC
0.8
0.6
0.4
0.2
0.0
0
2
4
Days after treatment
Fig. 5. Changes in anthocyanin (unlabelled and 13C-labelled) accumulation in the skin of cultured berries of Cabernet Sauvignon at 15 C (open
triangles), 25 C (closed circles), and 35 C (open circles). Values are expressed on a skin fresh weight (FW) basis. Vertical bars indicate the standard
deviation of the mean (n¼3 biological replicates). (A) total anthocyanin; (B) delphinidin 3-glucoside; (C) cyanidin 3-glucoside; (D) petunidin
3-glucoside; (E) peonidin 3-glucoside; (F) peonidin 3-glucoside-acetate; (G) peonidin 3-glucoside-p-coumarate; (H) malvidin 3-glucoside; (I)
malvidin 3-glucoside-asetate; (J) malvidin 3-glucoside-p-coumarate.
This study showed the possibility that the mechanism of
decreases in anthocyanin accumulation due to high
temperature involves the loss of anthocyanin from the
following three points. The first is the change in the
anthocyanin composition in the skin of berries grown in
high temperatures. The contents of individual anthocya-
nins, with the exception of malvidin derivatives, decreased
considerably under high temperature as compared with the
control. This result is consistent with previous studies of
the grape berry and model solution (Romero and Bakker,
2000; Morais et al., 2002). In general, methoxylation,
glycosylation, and acylation lead to an increase in the
A: Total
350
300
250
200
150
100
15
25
35
50
0
10
8
6
4
2
0
2
4
6
8
E: Pn3G
100
80
60
40
20
0
0
70
2
4
6
50
40
30
20
10
0
0
2
4
6
Days after treatment
30
20
10
0
8
0
2
4
6
8
F: Pn3GAc
30
25
20
15
10
5
0
8
H: Mv3G
60
C: Cy3G
40
Anthocyanin (µg g-1 Skin FW)
12
Anthocyanin (µg g-1 Skin FW)
8
Anthocyanin (µg g-1 Skin FW)
6
0
2
4
6
8
I: Mv3GAc
25
Anthocyanin (µg g-1 Skin FW)
Anthocyanin (µg g-1 Skin FW)
4
B: Dp3G
14
0
Anthocyanin (µg g-1 Skin FW)
2
Anthocyanin (µg g-1 Skin FW)
Anthocyanin (µg g-1 Skin FW)
0
16
Anthocyanin (µg g-1 Skin FW)
Anthocyanin (µg g-1 Skin FW)
Decrease of anthocyanins in grape skin 1943
20
15
10
5
0
0
2
4
6
Days after treatment
8
D: Pt3G
14
12
10
8
6
4
2
0
0
2
4
6
8
6
8
6
8
G: Pn3GpC
50
40
30
20
10
0
0
2
4
J: Mv3GpC
30
25
20
15
10
5
0
0
2
4
Days after treatment
Fig. 6. Changes in 13C-labelled anthocyanin content in the skin of cultured berries of Cabernet Sauvignon at 15 C (open triangles), 25 C (closed
circles), and 35 C (open circles). Values are expressed on a skin fresh weight (FW) basis. Vertical bars indicate the standard deviation of the mean
(n¼3 biological replicates). For details see Fig. 5.
thermal stability of anthocyanin (Jackman and Smith,
1996). If high temperature increases the degradation rate
of anthocyanin, it is reasonable that only malvidin
derivatives, which are highly methylated anthocyanins,
accumulated and other anthocyanins decreased under high
temperature. Besides, these changes in anthocyanin composition due to high temperature also have significant
implications on grape and wine quality, since it suggests
a change in the hue of the grape colour in addition to the
change in intensity.
The second is that mRNA accumulations of anthocyanin biosynthetic genes and enzyme activity of UFGT were
not inhibited under high temperature. These results
suggest that the ability of anthocyanin biosynthesis was
kept under high temperature. However, this is not in
agreement with a previous study reporting that high
1944 Mori et al.
temperature suppressed the expression of anthocyanin
biosynthetic genes (Yamane et al., 2006). Although there
is no experimental evidence to explain this disagreement,
it is likely that a difference between varieties is involved.
Black skin cultivars, such as Cabernet Sauvignon, would
have a stronger ability to biosynthesize anthocyanin than
red skin cultivars, such as Aki Queen. Kliewer and Torres
(1972) reported that Tokay (a red-skin cultivar) was the
least tolerant of high temperature and Pinot noir and
Cabernet Sauvignon were the most tolerant. While the
loss of pigmentation in white cultivars of V. vinifera is
caused by the mutation in VvmybA1 (Kobayashi et al.,
2004), the molecular bases of the determination of red or
black skin colour remain unknown. The difference of pigmentation between red and black cultivars may explain the
high temperature tolerance of anthocyanin biosynthesis in
Cabernet Sauvignon.
The third is that 13C-labelled anthocyanins, which were
synthesized before they were exposed to high temperature,
significantly decreased after high temperature treatment.
This is definitive evidence of the loss of anthocyanins
in the skin of grape berries due to high temperature.
Furthermore, a 13C tracer experiment showed that acylated
anthocyanins, especially malvidin 3-glucoside p-coumarate,
were more stable under high temperature. This result
demonstrates that the stability of anthocyanins depending
on its structure is important in the skin as well as in wine
and juice (see Introduction). So far, there have been few
studies on the loss of anthocyanin in living plant tissue,
including grapes. It has been assumed that the turnover of
anthocyanins includes various processes: chemical degradation, enzymatic degradation, and polymerization with
proanthocyanidin (Sipiora and Gutiérrez-Granda, 1998).
The chemical degradation is affected by pH, temperature,
light, oxygen, and structure of anthocyanins (Jackman and
Smith, 1996). The degradation rate of anthocyanins increases as the temperature rises. Therefore, it is possible
that anthocyanins in grape skins are chemically degraded
in response to high temperature. In addition, the enzymatic degradation may be involved in the decrease of
anthocyanins in grape skins. Recently, it was reported that
peroxidase is involved in the active anthocyanin degradation of Brunfelsia calycina flowers among candidates for
anthocyanin degradation enzymes, such as polyphenol
oxidase and peroxidase (Vaknin et al., 2005). Peroxidase
in vacuoles has also been found in grape cells and would
be involved in anthocyanin degradation in the presence of
H2O2 (Calderon et al., 1992). H2O2 levels in plant tissues
have been shown to increase in response to heat stress
(Dat et al., 1998). In the present study, a GeneChip microarry analysis showed that grape berries grown under high
temperature would receive oxidative stress since genes
encoding peroxidase and some oxidoreduction enzymes
were induced (see Supplementary Table S1 at JXB
online). Therefore, it is suggested that high temperature
gives oxidative stress to grape berries and induces
peroxidase, thereby degrading anthocyanins in the skin.
The polymerization with proanthocyanidin in living plant
tissue remains unknown at present. However, further
investigation into the degradation pathway and degradative products may resolve this issue.
In conclusion, the decrease of anthocyanins in grape
skins under high temperature could be caused by many
factors, such as chemical and/or enzymatic degradation,
not just the inhibition of anthocyanin biosynthesis.
Supplementary data
Supplementary data can be found at JXB online.
Table S1. Representative transcripts differentially expressed under high temperature condition.
Acknowledgement
We would like to thank Ms M Numata, National Research Institute
of Brewing, for sample preparation.
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