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EFFECTS OF TEMPERATURE AND WATER REGIMES


ON FLAVONOID CONTENTS
AND COMPOSITION IN THE SKIN OF RED-WINE GRAPES
Nami GOTO-YAMAMOTO1*, Kentaro MORI1,2,
Mineyo NUMATA1, Kazuya KOYAMA1 and Masahiko KITAYAMA2
1 : National Research Institute of Brewing, 3-7-1 Kagamiyama,
Higashi-Hiroshima, 739-0046, Japan
2 : Institute of Life Science, Ehime Womens College, 421 Ibuki-cho Baba,
Uwajima, 798-0025, Japan

Abstract

Rsum

Aims: This study aimed to determine what effects temperature and water
regimes have on flavonoid concentrations and composition in grape berry
skins.

Objectif: Cette tude a pour but de dterminer les effets de la temprature


et du rgime hydrique sur les concentrations des flavonodes et la
composition des pellicules des baies de raisins.

Methods and results: Potted vines of Vitis vinifera cv. Cabernet-Sauvignon


were cultivated in high (35C in daytime/25 C in night time) and low
(25C in daytime/20 C in night time) temperatures with dry, medium,
and wet regimes after vraison. The high temperature significantly reduced
the anthocyanin concentrations in the berry skins and modified the
anthocyanin composition. The dry regimes significantly increased
anthocyanin concentrations per skin weight. The high temperature after
vraison decreased proanthocyanidin and quercetin concentrations in
the berry skins, but the decreased rates were much smaller than that of
anthocyanin. Also, water regimes showed little or no effects on
proanthocyanidin and quercetin concentrations.

Mthodes et rsultats: Des vignes en pot de Vitis vinifera cv. CabernetSauvignon ont t cultives temprature leve (35 C, le jour et 25 C
la nuit) et basse temprature (25 C et 20 C la nuit) avec trois types de
rgime: sec, moyen et humide aprs la vraison. La temprature leve
a rduit significativement les concentrations en anthocyanes des pellicules
des baies et a modifi la composition anthocyanique. Le rgime sec a
augment significativement les concentrations en anthocyanes par poids
des pellicules. La temprature leve aprs la vraison a diminu les
concentrations en proanthocyanidines et en querctine dans les pellicules
des baies, mais les taux de diminution taient plus petits que ceux des
anthocyanines. Le rgime hydrique a peu ou pas dincidence sur les
concentrations en proanthocyanidines et en querctine.

Conclusion: The both temperature and water regimes had significant


effects on anthocyanin concentration. In addition, the high temperature
moderately reduced proanthocyanidin and quercetin concentrations in the
berry skins.

Conclusion: La temprature et le rgime hydrique ont des effets significatifs


sur la concentration en anthocyanes. De plus, la temprature leve rduit
modrment les concentrations en proanthocyanidines et en querctine
des pellicules des baies.

Significance and impact of study: This study showed that the temperature
and water regimes after vraison had different influences on each group
of flavonoids in the grape berry skins.

Signification et impact de ltude: Cette tude a montr que la temprature


et les rgimes hydriques aprs vraison ont des influences diffrentes sur
chaque groupe de flavonodes des pellicules des baies de raisins.

Key words: anthocyanin, proanthocyanidin, quercetin, Vitis vinifera

Mots-cls: anthocyanine, proanthocyanidine, quercetine, Vitis vinifera

manuscript received the 10th September 2008 - revised manuscript received the 7th May 2009

*Corresponding author : gotoh_n@nrib.go.jp

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Nami GOTO-YAMAMOTO et al.

INTRODUCTION

regimes have on the concentration and composition of


not only anthocyanin but also the other flavonoids,
proanthocyanidin and flavonol, in grape berry skins.

In Japan, red-wine grapes often do not accumulate


anthocyanin sufficiently. Anthocyanin accumulation in
grape berry skins is known to be inhibited by high
temperature, excess water, excess nitrogen fertilizer, and
so on, and the first two of these factors are believed to be
the major causes for low coloration in Japan. Also, high
temperature is becoming a serious issue of wine industries
in the world because of the global warming.

MATERIALS AND METHODS

1. Culture and sampling methods

Four-year-old vines of Cabernet-Sauvignon grafted


on F3309 were cultivated in 60 L pots containing 90%
(v/v) of sand and 10% (v/v) of bark compost in a phytotron
TP (Espec Mic, Oguchi, Japan) with two culture rooms.
Vines were trained on a simple Guyot trellising system.
After vraison, the temperature of one room was set high
(35C in daytime, 8 a.m. - 6 p.m./30C in night time), and
the other room was set low (25C in daytime/20C in
night time). In each room, the vines were grouped into
three water regimes: wet, medium and dry. The potted
vines were watered daily to maintain the lowest soil
moisture tension of -50 to -55 kPa for dry, -20 to -25 kPa
for medium, and ca -10 kPa for wet regimes using an
automatic watering timer with watering tubes. One, two
or four watering tubes were inserted into a pot of dry,
medium and wet regimes, respectively. Watering duration
was adjusted to maintain the same soil moisture range in
different temperature regimes. Each experimental regime
consisted of six vines, and each vine had 2.5 bunches in
average. One or two bunches were sampled with three
replications at vraison and 2, 4, and six weeks after
vraison. Randomly selected 30-40 berries from each
sample were used for analysis. The day when over 50%
of berries in a bunch softened was considered as vraison
in this study, and the date of vraison of each bunch was
recorded. The berries were peeled, and the skin samples
were frozen in liquid nitrogen and stored at -80C until
use.

With regard to the inhibitory effect of high


temperatures on anthocyanin accumulation in berry skins,
there have been many intensive researches (e.g.,
KLIEWER, 1970; KLIEWER and TORRES, 1972;
TARARA et al., 2008). SPAYD et al. (2002) reported
that the elevated bunch temperature was the cause of the
decreased total anthocyanin concentrations in berry skins
in the west-exposed side from a field experiment which
separated the effects of light and temperature. Thus,
importance of canopy management has been emphasized
to maintain adequate light exposure and bunch temperature
(BERGVIST et al., 2001; HASELGROVE et al., 2000).
For the inhibitory effect of high temperature on
anthocyanin accumulation, two mechanisms have been
reported. As one mechanism, it was reported that high
temperatures during maturation decreased the
concentration of abscisic acid, a plant hormone which
induces anthocyanin biosynthesis, in the berry skins and
also decreased the mRNA levels of anthocyanin
biosynthetic pathway genes (YAMANE et al., 2006). As
another mechanism, a tracer experiment with 13C-labeled
anthocyanin (MORI et al., 2007) revealed that high
temperatures decreased the concentration of anthocyanin
which was once biosynthesized. The total (labeled and
unlabeled) anthocyanin concentrations of the same grape
skin samples were almost stable, which means the
biosynthesis and decomposition of anthocyanin occurred
concurrently.

2. Analytical methods

At harvest stage (six weeks after vraison), the total


soluble solid contents (TSS (Brix)) of juice were
measured using a digital reflectometer (PR-101, Atago,
Tokyo, Japan). Total acid was measured by titration of
10 ml of juice with 0,1 N NaOH to pH 8.2 and expressed
as tartaric acid concentration (% w/v).

With regard to the effect of water status, moderate and


not severe water stress or drought stress has been reported
to increase anthocyanin concentrations (e.g., OJEDA et
al., 2002). Moderate water stress was reported to increase
the skin/pulp ratio by decreasing of berry size (e.g.,
KENNEDY et al., 2002) and to increase the anthocyanin
concentration even if the berry sizes were same as the
control (ROBY et al., 2004). It is also reported that water
stress increased the expression of anthocyanin biosynthetic
pathway genes; however, there were limited effects on
the biosyntheses of proanthocyanidin (condensed tannin)
and flavonol (CASTELLARIN et al., 2007).

Proanthocyanidin was extracted with 70% acetone


and analyzed with a reversed phase HPLC with detection
of absorbance at 280 nm after phloroglucinolysis based
on the method of KENNEDY and JONES (2001). (+)Catechin (Sigma-Aldrich Chemie, Steinheim, Germany),
(-)-epicatechin, (-)-epicatechin-3-O-gallate, and (-)epigallocatechin (Kurita Water Indutries, Tokyo, Japan)
were used for standards curves. The analysis was carried
out as described previously (KOYAMA et al., 2007).

There is, however, no report which combines


temperature and water status. Thus, the objective of this
study was to determine what effects temperature and water

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temperature, water regime, and time for flavonoid


concentrations were assessed using two-way and threeway ANOVA, respectively.

Anthocyanin was extracted from frozen berry skin


samples with 70% methanol containing 2% formic and
analyzed with a reversed phase HPLC with detection of
absorbance at 520 nm using malvidin 3-glucoside
(Extrasynthese, Genay, France) as a standard (KOYAMA
et al., 2007).

RESULTS AND DISCUSSION

1. Berry composition

The concentration of quercetin, the main flavonol


of grapes, was determined with a reversed phase HPLC
with detection of absorbance at 365 nm after acid
hydrolysis of the extract. The same extract as anthocyanin
analysis (1.4 ml) was combined with 0,1 ml of 0.42 mg/ml
morin (Merck, Darmstadt, Germany) as an internal
standard and 21 mg/ml of tert-butylhydroquinone (SigmaAldrich Chemie) as an antioxidant in 70% methanol, as
well as with 0.6 ml of 4.2 N HCl. The mixed solution in
a glass tube was kept at 85C for two hours with refluxing.
The HPLC condition was the same as a previous report
(FUJITA et al., 2006).

At the harvest stage, berries grown in the high


temperature showed significantly lower total soluble
solids, lower total acid, and higher pH than those in the
low temperature. Water regimes had a slight effect on the
berry weight (Table 1).
2. Effects of temperature and water regimes on
flavonoid

The p values of three-way ANOVA (Table 2) showed


that the temperature regimes had significant effects on
anthocyanin, proanthocyanidin and quercetin concentrations. Also, the time factor showed significant effects on
anthocyanin, proanthocyanidin (per berry and per berry
weight) and quercetin (per berry and per berry weight)
concentrations. Significant interactions among factors

3. Statistics

Significance of the effects of temperature and water


regimes for berry weight and composition and that of

Table 1 - Effects of temperature and water regimes on berry weight and composition at harvest
(six weeks after vraison).

Figure 1 Effects of temperature and water regimes on anthocyanin concentrations in the berry skins.
G, glucoside.

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Nami GOTO-YAMAMOTO et al.

berry skins under high temperatures. Thus, the difference


of the stability can explain at least a part of the change of
anthocyanin composition.

were observed only between temperature and time for


anthocyanin concentrations.
The high temperature significantly reduced the
anthocyanin concentrations (figure 1). In the high
temperature, the anthocyanin concentrations were almost
stable from two to six weeks after vraison, while they
increased in the low temperature. In addition, the high
temperature reduced the proportion of cyanidin and
peonidin 3-glucosides and increased the proportion of
acylated anthocyanins. Most of the anthocyanins in the
berry skins grown in the high temperature were
3-glucoside, 3-acetylglucoside and 3-p-coumaroylglucoside of malvidin, and proportion of acylated forms
increased. These results were inconsistence with other
reports (MORI et al., 2007; TARARA et al., 2008). MORI
et al. (2007) reported that the 3-p-coumaroylglucoside of
malvidin was most stable among the anthocyanins in the

The high temperature decreased the concentrations


of proanthocyanidin (figure 2) and quercetin (figure 3),
even though the decreased rates were much smaller than
that of anthocyanin. Composition of proanthocyanidin
was not influenced by temperature regimes significantly.
It has been reported that a large part of proanthocyanidins
had already accumulated prior to vraison (DOWNEY
et al., 2003a; FUJITA et al., 2007), which is a probable
reason that the proanthocyanidin concentration was more
stable than the anthocyanin concentration under the
different temperature regimes after vraison. Recently,
COHEN et al. (2008) also reported that the temperature
regimes after vraison had little effect to the
proanthocyanidin concentration and composition.

Table 1 - Effects of temperature and water regimes on berry weight and composition at harvest
(six weeks after vraison).

Figure 2 Effects of temperature and water regimes on proanthocyanidin concentrations in the berry skins.
P, Phloroglucinol adduct.

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Figure 3 Effects of temperature and water regimes on quercetin concentrations in the berry skins.

The inhibitory effect of high temperatures on flavonol


was also reported in a pair of comparison reported by
SPAYD et al. (2002) (in Table 4 of their report, sun
exposure samples of 1999), although the enhancement
by light was much more significant. It is reported that the
expression of flavonol synthase genes was induced by
light significantly (DOWNEY et al., 2004; FUJITA et al.,
2006). Flavonols were reported to be biosynthesized
around flowering time and after vraison (DOWNEY
et al., 2003b). The temperature regimes after vraison,
however, showed less effect on the quercetin concentration
than that on the anthocyanin concentration.

The lowest soil moisture of dry regime in this study


was -50 to -55 kPa, while soil moisture between -40 to
-60 kPa and -100 to -400 kPa were reported to deficit
available water (NICHOLAS, 2004). Thus, the dry regime
in this study was rather mild. Nevertheless, the
enhancement of anthocyanin accumulation by dry regimes
was observed particularly in the low temperature. Other
studies showed that the water stress after vraison and
also before vraison enhanced anthocyanin concentration
(OJEDA et al., 2002; CASTELLARIN et al., 2007). Now,
we are planning to determine the effect of water regimes
before vraison under high temperature on flavonoid
concentrations and composition.

In this study, difference between high and low


temperatures (10C through the day) was relatively large
compared to other studies (SPAYD et al., 2002; TARARA
et al., 2008; COHEN et al., 2008). In addition, the
temperature of culture room atmosphere, not that of bunch
surface, was controlled in this study. These experimental
methods are possible reasons to reveal the moderate
inhibitory effect of high temperatures on proanthocyanidin
and quercetin concentrations.

CONCLUSION
The both temperature and water regimes had
significant effects on the anthocyanin concentration. In
addition, the high temperature moderately reduced
proanthocyanidin and quercetin concentrations in the
berry skins. Thus, this study showed that the temperature
and water regimes after vraison has different influences
on each group of flavonoids in grape berry skins.

Thus, this study showed that the temperature regimes


after vraison have different influences on each flavonoid
group in grape berry skins, even though the three flavonoid
groups: anthocyanin, proanthocyanidin and flavonol, share
main part of their biosynthetic pathway, and both
anthocyanin and flavonol accumulate after vraison. The
decreasing effect of high temperature on the amount of
biosynthesized anthocyanin (MORI et al., 2007) possibly
contributes to this difference, although we have no
information if high temperatures decrease the amount
of biosynthesized proanthocyanidin or flavonol.

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