Loss of anthocyanins in red-wine grape under high temperature

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1 Journal of Experimental Botany Advance Access published April 3, 7 Journal of Experimental Botany, Page 1 of 11 doi:1.193/jxb/erm RESEARCH PAPER Loss of anthocyanins in red-wine grape under high temperature Kentaro Mori 1,, *,, Nami Goto-Yamamoto 1, Masahiko Kitayama and Katsumi Hashizume 1 1 National Research Institute of Brewing, Higashi-Hiroshima, Hiroshima 739-6, Japan Institute of Life Science, Ehime Women s College, Uwajima, Ehime 798-, Japan Received 3 December 6; Revised 6 February 7; Accepted 7 February 7 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 3 C) reduced the total anthocyanin content to less than half of that in the control berries (maximum 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-[1-13 C]phenylalanine solution for 1 week. Thereafter, the changes in 13 C-labelled anthocyanins were examined under different temperatures (1,, and 3 C). In the berries cultured at 3 C, the content of total 13 C-labelled anthocyanins that were produced before exposure to high temperature was markedly reduced as compared with those cultured at 1 C and 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, ). 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, 3). In hot regions, however, anthocyanin accumulation is inhibited in the skins of red and black grapes (Winkler et al., 196). In addition, any global atmospheric warming trend may affect grape berry ripening in the future. Jones et al. () 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 Downloaded from at Pennsylvania State University on April 8, 1 * To whom correspondence should be addressed. moriken7@gmail.com 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, Villenave d Ornon, France. ª The Author [7]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please journals.permissions@oxfordjournals.org

2 of11 Mori et al. grape berry skins have also been studied intensively (Kliewer, 197; Buttrose et al., 1971; Kliewer and Torres, 197; Spayd et al., ; Mori et al., b; Yamane et al., 6), 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., 6), Arabidopsis (Leyva et al., 199), grape (Mori et al., b; Yamane et al., 6), maize (Christie et al., 199), petunia (Shvarts et al., 1997), red orange (Lo Piero et al., ), and rose (Dela et al., 3). 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. () 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., 199; Yokotsuka and Singleton, 1997; Romero and Bakker, ; Morais et al.,, and references therein). Elevated temperature of storage decreased the concentration of anthocyanins in a model solution (Romero and Bakker, ; Morais et al., ). 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 SO (Selection Oppenheim No. ) grown in a phytotron. Vines were trained on a Guyot trellising system and each vine carried 1 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 (6.. h) temperature (max. 3 C) and a control (max. C). Under both conditions, the night-time (. 6. h) temperature was C. The photoperiod corresponded to natural day length. Twenty-five per cent of the berries (1 berries) in each cluster was sampled at random from each temperature treatment at 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 8 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., a). 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 6 Nano Assay on the Agilent 1 Bioanalyzer (Agilent Technologies). Total RNA ( lg) was used to synthesize crna, which was hybridized to an Affymetrix Vitis vinifera GeneChip Ò 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.) described by Solfanelli et al. (). A global scaling factor of, a normalization value of 1, and a default parameter setting for the Vitis vinifera GeneChip Ò were used. Signal values and detection call values were generated using the GCOS 1. 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. 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. 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 -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., 3). Quantitative real-time PCR analysis The transcript levels of anthocyanin biosynthetic genes were determined as described by Jeong et al. (6). The PCR mixture contained 1 ll of the cdna template, 1 ll of 3 Quantitect SYBR Ò Green PCR Master Mix (Qiagen), and. lm of the forward and reverse primers for each gene. Reactions were run on the GeneAmp 7 sequence detection system (Applied Biosystems, Foster City, CA, USA). The Q-PCR was performed under the following conditions: 9 C for 1 min, followed by cycles at 9 C for 1 s, at the annealing temperature of 6 C ( C for VvmybA1) for s and 7 C for s. The Q-PCR was carried out on four replicates per prepared cdna sample, and the transcript Downloaded from at Pennsylvania State University on April 8, 1

3 levels of each gene were normalized to the VvUbiquitin1 (Fujita et al., ) 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 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 1 ml of a mm TRIS HCl buffer (ph 7.) containing 1 mm polyethylene glycol 3, mm Na-diethyldithiocarbamate, mm dithiothreitol, and 1 mm of -mercaptoethanol. After centrifugation of the homogenate at 9 g for min, 1 g of Dowex 13 (HClform, equilibrated with the same buffer as above) was added to the supernatant. The supernatant was incubated for min on ice with gentle stirring and centrifuged again at 9 g for min. Solid ammonium sulphate was added to the supernatant to achieve 3% saturation, and the mixture was centrifuged at 9 g for 1 min. Protein was precipitated from the supernatant by adding ammonium sulphate to 7% final saturation, and the sample was centrifuged at 9 g for 3 min. The protein pellet was resuspended in. ml of a mm TRIS HCl buffer (ph 7.). The extract was passed through a PD1 column (Sephadex G-, GE Healthcare, Amersham, Bucks, UK) equilibrated with a mm TRIS HCl buffer (ph 7.). 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 1 ll of a 1 mm TRIS HCl buffer (ph 8.), 1 mm polyethylene glycol 3, mm Na-diethyldithiocarbamate, mm dithiothreitol, and 1 mm of -mercaptoethanol,.1 mm cyanidin chloride, 1 mm UDP-glucose, and 1 ll of an enzyme solution. The assay mixture was incubated for 6 min at 3 C. The reaction was terminated by adding 1 ll of % HCl. The quantity of the product, namely cyanidin 3-glucoside was measured using HPLC at 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 g 1 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 13 C 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 (7%), 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. ml filter-sterilized aqueous [ 13 C]phenylalanine solution (.3 M sucrose, 1 mm L-[1-13 C]phenylalanine) was applied to the berries, and the berries were then incubated at C under fluorescent light at approximately lmol m s 1. After 1 week of culture, the berries were placed on a new Petri dish containing a.3 M sucrose solution. Thereafter, the berries were cultured without phenylalanine at 1,, and 3 C under fluorescent light at approximately lmol m s 1. After,,, 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 8 C until use. Experiments were triplicated, with each replicate consisting of six or seven berries in a Petri dish. LC-MS analysis of 13 C-labelled anthocyanin Anthocyanin extracts were quantified by LC-MS (LCQ Advantage, Thermo Finnigan, San Jose, CA, USA) with a Zorbax SB-C18 column ( lm,.6 mm3 mm; Agilent Technologies). Solvent A consisted of water/formic acid (9:, v/v) and solvent B was methanol/acetonitrile/water (33:6:7, by vol). The solvent system initially consisted of 8% A and % B with the following changes: 1 min, 3% B; 3 min, % B; 3 min, 6% B; min, 6% B; min, 7% B; and 6 min, % B. The flow rate was.3 ml min 1, and the sample volume injected was ll. The unsplit eluent entered the ESI-interface through a fused silica capillary. The ion spray voltage was +. kv; the capillary temperature was 8 C. The mass spectrometer was operated in a selected ion monitoring mode (SIM) detecting positive ions. The levels of 13 C-labelled anthocyanins were calculated from peak areas of 1 C (A 1 ) and 13 C (A 13 ) using the following formula: L¼[A 13 /(A 1 +A 13 ) R]*(A 1 +A 13 ), where R is the isotope abundance A 13 /(A 1 +A 13 )of unlabelled anthocyanins. Since each anthocyanin naturally contains 1.1% of the stable isotope 13 C, the isotope abundances of unlabelled anthocyanins were subtracted from the isotope abundances of 13 C- labelled anthocyanins. The amounts of anthocyanins were expressed as the external standard equivalent (malvidin 3-glucoside) from the calibration curve. Results Decrease of anthocyanins in grape skin 3of11 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 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 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 Gene- Chip Ò ). A total of genes that were differentially transcribed by at least -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. ). The list of genes is shown in Supplementary Table S1 available at JXB online. Cluster 1 (16 genes) decreased at an early stage ( WAV) and increased at a later stage Downloaded from at Pennsylvania State University on April 8, 1

4 of11 Mori et al A Control High temp 6 Weeks after veraison B C WAV WAV 6 WAV WAV WAV 6 WAV Downloaded from at Pennsylvania State University on April 8, 1. Dp- 3G Cy- 3G Pt- 3G Pn- 3G Mv- 3G Dp- 3G- Ac Cy- 3G- Ac Pt- 3G- Ac Pn- 3G- Ac Mv- 3G- Ac Dp- 3GpC Cy- 3GpC Pt- 3GpC Pn- 3GpC Mv- 3GpC 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 ( C; closed circles) and high temperature (3 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 (n¼ 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 (3 genes) increased at an early stage and decreased at a later one. Clusters 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 and 6 were groups that were increased at a later stage and contained 9 and genes, respectively. These two clusters included

5 Decrease of anthocyanins in grape skin of11 Fig.. Cluster analysis of genes differentially expressed in the skin of Cabernet Sauvignon berries under control ( C) and high (3 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 and 8 were continually induced by high temperature. As with clusters and 3, some genes of the heat-shock protein were highly induced and the chlorophyll-binding protein was also induced. Only 8 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 -fold in response to high temperature, with the exception of caffeoyl-coa O- methyltransferase (16163_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 WAV and then decreased at 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 3- hydroxylase (F3H) and dihydroflavonol -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 3 C, the UFGT activity under high temperature was no different from that of the control (Fig. A). To examine the UFGT activities at the temperature in each condition, the control sample was assayed at C and the high-temperature sample at 3 C (Fig. B). 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 L-[1-13 C]phenylalanine solution for 1 week and, thereafter, were cultured without phenylalanine at 1,, and 3 C. The sums of unlabelled ( 1 C) and labelled ( 13 C) anthocyanin contents are shown in Fig.. The total anthocyanin content was highest in berries cultured at C and lowest at 3 C (Fig. A). The contents of delphinidin Downloaded from at Pennsylvania State University on April 8, 1

6 6of11 Mori et al A: CHS3 B: F3H Control High Temp C: DFR 6 6 Weeks after veraison D: LDOX 6 E: UFGT F: VvmybA1. 6 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 ( C; closed circles) and high temperature (3 C; open circles): (A) CHS3; (B) F3H; (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 (n¼ biological replicates). Downloaded from at Pennsylvania State University on April 8, 1 3-glucoside, cyanidin 3-glucoside, and petunidin 3-glucoside were highest at 1 C, while changes in other derivatives of peonidin and malvidin were similar to the total anthocyanin (Fig. B F). When the contents of individual anthocyanins were compared among three temperatures, the differences in malvidin 3-glucoside p-coumarate were small (Fig. B J). Changes in the content of 13 C-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 3 C, the total content of 13 C-labelled anthocyanin was markedly reduced, while there was no decrease in labelled anthocyanin content in the skin of berries at 1 C and C (Fig. 6A). Similar to the accumulation patterns of the anthocyanin content, the 13 C-labelled anthocyanin content of malvidin 3-glucoside p-coumarate was not significantly affected by temperature (Fig. 6B J).

7 Decrease of anthocyanins in grape skin Table 1. Transcription levels of anthocyanin biosynthetic genes in the skin of Vitis vinifera L. cv. Cabernet Sauvignon grape berries grown under control ( C) and high temperature (3 C) using Affymetrix Vitis vinifera GeneChip Ò microarray Probe set ID Public ID Gene title log (high/control) 7of11 WAV a WAV 6 WAV _at CB98171 Chalcone synthase (CHS3) _at AF79 Chalcone synthase _at CB Chalcone synthase _at CF76 Chalcone synthase (CHS3) _at X7969 Chalcone synthase _at CA81391 Chalcone isomerase (CHI1) _s_at CF1393 Naringenin 3-dioxygenase [Arabidopsis thaliana] (F3H) _at CF1693 Flavanone 3-hydroxylase (F3H1) _at CB96989 Dihydroflavonol reductase _at CF1831 Flavonoid-3#,# hydroxylase _at BM3789 Flavonoid-3#,#-hydroxylase _s_at CB771 Leucoanthocyanidin dioxygenase _s_at AF371 UDP glucose:flavonoid 3-o-glucosyltransferase _a_at CF1871 Glutathione S-transferase (GST) _a_at CB986 Glutathione S-transferase (GST) _at CF173 Glutathione transferase [Arabidopsis thaliana] _s_at CF1871 Glutathione S-transferase (GST) _s_at CB3733 S-adenosylmethionine-dependent methyltransferase [Arabidopsis thaliana] _at CF1966 Caffeoyl-CoA O-methyltransferase _at BQ7967 S-adenosylmethionine-dependent methyltransferase [Arabidopsis thaliana] _s_at CB9111 VvMYBA1 myb-related transcription factor a Weeks after veraison. UFGT activity (kat g -1 protein) 3 1 A Control High temp 6 Weeks after veraison UFGT activity (kat g -1 protein) 3 1 B Control ( C) High temp (3 C) 6 Weeks after veraison Fig.. Changes in enzyme activities of UFGT in the skin of Vitis vinifera L. cv. Cabernet Sauvignon grape berries grown under control ( C; closed circles) and high temperature (3 C; open circles) at the same assay temperature (A; 3 C) and at a different assay temperature (B; C and 3 C). Vertical bars represent the standard deviation of means (n¼3 assays). Downloaded from at Pennsylvania State University on April 8, 1 In the cultured berries, most anthocyanin biosynthetic genes were not strongly down-regulated at 3 C, with the exception of F3H 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. (6) 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.

8 8of11 Mori et al A: Total C C 3 C B: Dp3G. 1. E: Pn3G H: Mv3G C: Cy3G. F: Pn3GAc I: Mv3GAc. D: Pt3G G: Pn3GpC J: Mv3GpC Fig.. Changes in anthocyanin (unlabelled and 13 C-labelled) accumulation in the skin of cultured berries of Cabernet Sauvignon at 1 C (open triangles), C (closed circles), and 3 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. Downloaded from at Pennsylvania State University on April 8, 1 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 anthocyanins, 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, ; Morais et al., ). In general, methoxylation, glycosylation, and acylation lead to an increase in the

9 Decrease of anthocyanins in grape skin 9of11 Anthocyanin (µg g -1 Skin FW) Anthocyanin (µg g -1 Skin FW) Anthocyanin (µg g -1 Skin FW) Anthocyanin (µg g -1 Skin FW) A: Total B: Dp3G 1 E: Pn3G H: Mv3G Anthocyanin (µg g -1 Skin FW) Anthocyanin (µg g -1 Skin FW) Anthocyanin (µg g -1 Skin FW) C: Cy3G F: Pn3GAc 1 1 I: Mv3GAc 1 1 Anthocyanin (µg g -1 Skin FW) Anthocyanin (µg g -1 Skin FW) Anthocyanin (µg g -1 Skin FW) 1 D: Pt3G G: Pn3GpC J: Mv3GpC 1 1 Downloaded from at Pennsylvania State University on April 8, 1 Fig. 6. Changes in 13 C-labelled anthocyanin content in the skin of cultured berries of Cabernet Sauvignon at 1 C (open triangles), C (closed circles), and 3 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.. 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

10 1 of 11 Mori et al. temperature suppressed the expression of anthocyanin biosynthetic genes (Yamane et al., 6). 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 (197) 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., ), 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 13 C-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 13 C 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., ). Peroxidase in vacuoles has also been found in grape cells and would be involved in anthocyanin degradation in the presence of H O (Calderon et al., 199). H O 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. 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