Abstract
Removing leaves around the grape cluster of Muscat Bailey A [Vitis × labruscana (Bailey) and Vitis vinifera (Muscat Hamburg)] cultivated in pergola style at the start of veraison increased the photosynthetically active radiation value by more than approximately 60- and 30-fold compared with those of the control and grape cluster from vines grown on ground covered with reflective film (reflective-film-treated grape cluster), respectively. The improved light exposure caused by leaf removal increased total anthocyanin concentration and changed the ratios of anthocyanin derivatives in the grape skins. Total anthocyanin concentration in the leaf-removal-treated grape skins 10 weeks after veraison increased by approximately 6.5-fold compared with that of the control. In addition, delphinidin-based anthocyanin concentrations in the leaf-removal-treated grape skins increased by approximately 7-fold compared with those of the control. Leaf removal up-regulated anthocyanin-synthesis-related genes in grape skins, such as CHS, F3′H, F3′,5′H, and UFGT. In particular, the overexpression of F3′,5′H in the leaf-removal-treated grape skins suggested that leaf removal contributed to the accumulation of delphinidin-based anthocyanins in grape skin. These findings are expected to improve viticultural practices with the aim of producing dark-colored red wine made from cultivars grown in pergola style.
Introduction
Anthocyanins impart red/blue color to fruits, vegetables, and plants, and are classified into six types: pelargonidin, cyanidin, peonidin, delphinidin, petunidin, and malvidin, based on the number of hydroxyl groups on the B-ring. All of the above-mentioned anthocyanins except pelargonidin are present in grape berries and are divided into cyanidin-based anthocyanins and delphinidin-based anthocyanins (Nyman and Kumpulainen, 2001). The cyanidin-based anthocyanins consist of cyanidin and peonidin. On the other hand, the delphinidin-based anthocyanins are composed of delphinidin, petunidin, and malvidin. The color of anthocyanins is sensitive to pH. In wine that has a pH of 4 or lower, the cyanidin-based anthocyanins produce a red color, whereas the delphinidin-based anthocyanins produce a blue color. The anthocyanin composition ratio in grape skin is affected by the difference in expression levels of flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′,5′H) (Bogs et al., 2006; Castellarin and Gaspero, 2007; Castellarin et al., 2006; Jeong et al., 2006). Anthocyanin concentration is determined by the expression of UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) (Kobayashi et al., 2001). Therefore, the transcription levels of F3′H, F3′,5′H, and UFGT in grape skins during the growing season influence skin color and consequently affect the appearance of red wine.
Muscat Bailey A is a cross-hybridized grape cultivar [Vitis × labruscana (Bailey) and Vitis vinifera (Muscat Hamburg)] that was developed in Japan in 1927. Muscat Bailey A is cultivated for table and wine use and is one of the most largely produced Japanese red wines. Muscat Bailey A grapes have certain characteristics that distinguish them from European grapes (V. vinifera L.) generally used for red wine making. Muscat Bailey A grapevine is cultivated in pergola style and fresh berry weight at the time of harvest is more than twice those of Merlot, Cabernet Franc, and Cabernet Sauvignon. Therefore, Muscat Bailey A has a low skin-to-juice ratio compared with the other grape cultivars used for winemaking. Moreover, the amount of proanthocyanidins in Muscat Bailey A wine is significantly less than in red wines made from other V. vinifera grape cultivars, such as Merlot, Cabernet Sauvignon, and Zweigeltrebe (Ichikawa et al., 2011). The relationships among viticultural conditions, anthocyanin composition, and transcription profiles of anthocyanin-synthesis-related genes of Muscat Bailey A grape have not been clarified, although environmental conditions, including air temperature (Mori et al., 2007), sunlight exposure (Chorti et al., 2010), and water deficit (Castellarin et al., 2007), were found to affect anthocyanin composition in grape skins. Matus et al. (2009) reported that exposure to sunlight induces anthocyanin-synthesis-related genes and affects anthocyanin composition in grape skin. In the present study, with the aim of improving the color of Muscat Bailey A wine, we investigated the effect of leaf removal, which enhances exposure to sunlight, on anthocyanin content and composition in Muscat Bailey A grape skins.
Materials and Methods
Plant materials and experimental design
The vineyards cultivating Muscat Bailey A grapevines examined in this study were located in the Ichimiya region (latitude, 35°64′76″N; longitude, 138°69′83″E; elevation, 348 m), Yamanashi Prefecture, Japan. The grapevines were approximately 20 years old and grown by overhead trellis (pergola-style) cultivation. The average yield of each grapevine was adjusted to 300 clusters per grapevine. Leaf removal was carried out by removing approximately five leaves around a grape cluster on August 4, 2011 (start of veraison) (Fig. 1C). On the same day, a reflective film (Poly-shine; Hitachi AIC Inc., Tochigi, Japan) was spread on the ground to reflect sunlight onto each grape cluster (Fig. 1B). In this study, we focused on three grapevines and used approximately 20–30 grape clusters from 10–15 young shoots for each examination. Approximately 100 grape berries were randomly sampled from the grape clusters. Sampling was conducted five times from August 10 to October 8 (10 weeks after veraison) in the 2011 growing season. All plant materials were frozen immediately in liquid nitrogen and stored at −80°C until use.
Photosynthetically active radiation (PAR) and illuminance around the grape clusters subjected to each cultivation condition were measured. LI-190 SA and LI-210 SA sensors (LI-COR Inc., Lincoln, NE, USA) were used for the measurement of PAR and illuminance, respectively. In order to avoid the influence of climatic conditions on the measurements of PAR and illuminance, measurements were carried out around 11:00 am on the day following the start of veraison, which had fair weather.
Analysis of grape composition at harvest
Approximately 100 fresh grape berries were randomly sampled seven weeks after veraison (start of harvest phase) in the 2011 growing season. The grapes were divided into six groups, each composed of approximately 15 to 20 grape berries. After weighing the berries of each group, grape juice was obtained as described below. Briefly, the berries were pressed to 60% of total berry weight using a micropress (Quick juicer; Chibakogyo, Chiba, Japan). Total soluble solids (TSS) in juice were measured with a refractometer (Pocket PAL-1; Atago, Tokyo, Japan) and expressed as Brix. pH of juice was measured with a pH meter (MH-60S; Toakogyo, Tokyo, Japan). Total acidity (TA) in juice was determined by neutralization titration with 0.1 N NaOH to pH 7.0 of 10 mL juice diluted with 10 mL distilled water, and expressed as g tartaric acid/L.
Quantification of anthocyanin derivatives and total anthocyanins
Anthocyanin derivatives (cyanidin, peonidin, delphinidin, petunidin, and malvidin) and total anthicyanins in grape skins were analyzed by reversed-phase high-performance liquid chromatography (HPLC; LC-10Avp; Shimadzu, Kyoto, Japan) as described previously (Baranowski and Nagel, 1981; Liang et al., 2008; Wu and Prior, 2005) with slight modifications. Briefly, to collect the grape skin, approximately 100 fresh grape berries were randomly sampled five times during the grape-growing season. The skins of 10–15 berries were peeled off with tweezers, and each small piece of skin was pulverized in liquid nitrogen. One gram of pulverized skin was macerated in 10 mL of 0.1% HCl-methanol for 4 h at room temperature in the dark and the mixture was filtered through a 0.45 μm cellulose acetate filter (Advantec Toyo, Tokyo, Japan). Finally, these extract solutions were appropriately diluted using methanol before analysis. The conditions for HPLC (SCL-20Avp; Shimadzu) were as follows: CAPCELLPAC C18UG120, 5.0 μm, 4.6 × 250 nm column (Shiseido, Tokyo, Japan); column oven temperature, 40°C; mobile phase A [distilled water containing 0.4% (v/v) phosphoric acid]; mobile phase B [80% (v/v) acetonitrile + 20% mobile phase A]; and flow rate, 1.0 mL·min−1. Linear gradient programs were set from 10% of mobile phase B to 60% of mobile phase B over a 65 min period and absorbance was detected at 525 nm.
We determined the concentrations of cyanidin-based anthocyanins [cyanidin 3-glucoside, cyanidin 3-(6″-acetyl)glucoside, cyanidin 3-(6″-coumaroyl) glucoside, peonidin 3-glucoside, peonidin 3-(6″-acetyl) glucoside, and peonidin 3-(6″-coumaroyl)glucoside] and delphinidin-based anthocyanins [delphinidin 3-glucoside, delphinidin 3-(6″-acetyl)glucoside, delphinidin 3-(6″-coumaroyl)glucoside, petunidin 3-glucoside, petunidin 3-(6″-acetyl)glucoside, petunidin 3-(6″-coumaroyl)glucoside, malvidin 3-glucoside, malvidin 3,5-diglucoside, malvidin 3-(6″-acetyl)glucoside, malvidin 3-(6″-coumaroyl)glucoside, and malvidin 3-(coumaroyl)-5-diglucoside]. Total anthocyanin concentrations in grape skins were determined by the amount of anthocyanin derivatives. Total anthocyanin and anthocyanin derivative concentrations in grape skin were expressed as mg malvidin-3-glucoside·g−1.
RNA extraction
One gram of skin was pulverized in liquid nitrogen. Total RNA was obtained from the pulverized samples as described previously (Kobayashi et al., 2009). Total RNA was purified with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) as described previously (Tesniere and Vayda, 1991).
Transcription analysis of anthocyanin-synthesis-related genes
Transcription analysis and selection of anthocyanin-synthesis-related genes were performed as described previously (Kobayashi et al., 2009) with slight modifications. Briefly, first-strand cDNA was synthesized from 500 ng total RNA using a PrimeScript RT Reagent Kit (TaKaRa-Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. Nucleotide sequences of the PCR primers used in this study were designed by Primer Express software version 3.0 (Applied Biosystems Japan Ltd., Tokyo, Japan), and were as follows: chalcone synthase (CHS) primers (5′-TCTGAGCGAGTATGGGAACATG-3′ and 5′-CT GTGCTGGCTTTCCCTTCT-3′, corresponding to bases 1469–1490 and 1563–1544 of VvCHS, GenBank accession no. AB015872, respectively), F3′H primers (5′-TATGGGCTGACCCTACAACGA-3′ and 5′-CCTG GGCAAACAACCTCATT-3′, corresponding to bases 2566–2586 and 2664–2645 of VvF3′H4, GenBank accession no. AB213605, respectively), F3′,5′H primers (5′-AGGGTCGGAGTCAAATGAGTTC-3′ and 5′-CGCTGGATCCCTTGGATGT-3′, corresponding to bases 639–660 and 758–740 of VvF3′,5′H, GenBank accession no. AB213606, respectively), UFGT primers (5′-CTTCTTCAGCACCAGCCAATC-3′ and 5′-AGG CACACCGTCGGAGATAT-3′, corresponding to bases 548–588 and 647–628 of VvUFGT, GenBank accession no. AB047099, respectively), and 18S rRNA primers (5′-CGAAAGCATTTGCCAAGGAT-3′ and 5′-CCTGGTCGGCATCGTTTATG-3′, corresponding to bases 522–541 and 625–606 of Vv18S ribosomal RNA, GenBank accession no. AF207053, respectively). Real-time quantitative reverse transcription PCR (real-time quantitative RT-PCR) was performed using an ABI PRISM 7300 Real-Time PCR System (Applied Biosystems) with SYBR Green detection modules according to the manufacturer’s instructions. Data were analyzed using the 7300 system software SDS 1.3.0 (Applied Biosystems). Expression levels were determined as the number of amplification cycles needed to reach a fixed threshold, as described previously (Pfaffl, 2001; Reid et al., 2006). Duplicate experiments of three samples (control, leaf-removal-treated, and reflective-film-treated) were performed in 96-well reaction plates.
Statistical analysis
The significant difference among the cultivation treatments was calculated by Tukey’s statistical analysis.
Results and Discussion
Light exposure conditions for each cultivation treatment
The values of PAR and illuminance for each cultivation condition are listed in Table 1. The PAR value of the leaf-removal-treated grape cluster was more than approximately 60-and 30-fold higher than those of control and reflective-film-treated grape clusters, respectively. The PAR value of the reflective-film-treated grape cluster was also increased by more than 2-fold compared with that of the control. In addition, the illuminance value of the leaf-removal-treated grape cluster was 45-and 25-fold higher than those of control and reflective-film-treated grape clusters, respectively. The illuminance value of the reflective-film-treated grape cluster was also increased by more than 1.5-fold compared with that of the control. These results suggested that our experiment was well designed.
Quantification of total anthocyanins and anthocyanin derivatives
The compositions of the treated grape berries at harvest are listed in Table 2. Although berry weight was not influenced by the treatments, TSS were higher in the leaf-removal-treated grape berries than in the control, whereas TA was lower. In addition, pH was higher in the leaf-removal-treated grape berries than in the control. The compositions of the reflective-film-treated grape berries were between those of the leaf-removal-treated grape berries and the control. These results suggested that the increase in PAR value with pergola-style cultivation affected the maturity of grape berries. Palliotti et al. (2011) reported that early leaf removal conducted before flowering affected gas exchange and chlorophyll fluorescence, resulting in increases in grape berry compositions, such as must soluble solids, total anthocyanins, and total phenolics. Taken together, these results indicated that grape maturity might be promoted by the enhanced photosynthetic efficiency caused by leaf removal. Total anthocyanin concentration in the leaf-removal-treated grape skins showed a marked increase 10 weeks after veraison compared with those in the control and reflective-film-treated grape skins (Fig. 2A). Cyanidin-based anthocyanin concentrations in the leaf-removal-treated grape skins showed a minimal increase or hardly changed compared with those in the control and reflective-film-treated grape skins (Fig. 2B).
On the other hand, delphinidin-based anthocyanin concentrations in the leaf-removal-treated grape skins were increased by more than 3-fold relative to those in the control and reflective-film-treated grape skins (Fig. 2C). These results indicated that the improved light exposure caused by leaf removal could contribute to the accumulation of delphinidin-based anthocyanins in grape skins, as reported previously (Matus et al., 2009). Increasing light exposure by leaf removal might improve anthocyanin composition in grape skins regardless of cultivation style. As a result, these changes in anthocyanin composition in grape skin might contribute to wine quality, such as wine hue. In addition to Muscat Bailey A, leaf removal should be applied to other table-use cultivars, such as Kyoho, Kaiji, and Pione, which are similarly grown by pergola-style cultivation for table use in Japan, with the aim of improving their skin color. In contrast, Downey et al. (2004) reported that bunch shading increased cyanidin-based anthocyanins. Controlling grape skin color by shading and/or leaf removal is expected to contribute to the enhancement of commodity value.
Transcription analysis of anthocyanin-synthesis-related genes
The transcription profiles of anthocyanin-synthesis-related genes in grape skins during the growing season are shown in Figure 3. The expression of four anthocyanin-synthesis-related genes, CHS, F3′H, F3′,5′H, and UFGT, in the leaf-removal-treated grape skins was the highest among the grapes tested throughout the grape-growing season (Fig. 3). Five weeks after veraison, leaf removal had up-regulated CHS expression by more than 4-and 1.5-fold relative to those of control and reflective-film-treated grape skins, respectively (Fig. 3B, CHS). The expression of UFGT, the key enzyme for anthocyanin synthesis, in the leaf-removal-treated grape skins was up-regulated by more than 7-fold compared with those of control and reflective-film-treated grape skins seven weeks after veraison (Fig. 3C, UFGT). In addition, F3′H and F3′,5′H expression was up-regulated by more than 2.5-and 4-fold compared with that of the control, respectively (Fig. 3C, F3′H and F3′,5′H). Therefore, in addition to the up-regulation of F3′,5′H, the change in anthocyanin composition in grape skin might be affected by the expression ratios of F3′H and F3′,5′H. These results supported previous reports (Castellarin et al., 2006; Matus et al., 2009) and well explained anthocyanin concentration and composition ratios in grape skins (Fig. 2). Consequently, increasing the exposure of grape clusters to sunlight improved anthocyanin composition in grape skins regardless of cultivation style and cultivar. Controlling sunlight exposure during the growing season might improve anthocyanin concentration and composition ratios in Muscat Bailey A grape skins. These findings could contribute to improving viticultural practices with the aim of producing dark-colored Muscat Bailey A wines. Further investigation to determine the optimum canopy management for pergola-style cultivation, such as leaf removal position and timing, is expected to improve anthocyanin composition.
Literature Cited
- Baranowski, J. D. and C. W. Nagel. 1981. Isolation and identification of the hydrocinnamic acid derivatives in white Riesling wine. Am. J. Enol. Vitic. 32: 5–13.
- Bogs, J., A. Ebadi, D. McDavid and S. P. Robinson. 2006. Identification of the flavonoid hydroxylases from grapevine and their regulation during fruit development. Plant Physiol. 140: 279–291.
- Castellarin, S. D. and D. G. Gaspero. 2007. Transcriptional control of anthocyanin biosynthetic genes in extreme phenotypes for berry pigmentation of naturally occurring grapevines. BMC Plant Biol. 30: 46–55.
- Castellarin, S. D., G. D. Gaspero, R. Marconi, A. Nonis, E. Peterlunger, S. Paillard, A. F. Adam-Blondon and R. Testolin. 2006. Colour variation in red grapevines (Vitis vinifera L.): genomic organisation, expression of flavonoid 3′-hydroxylase, flavonoid 3′,5′-hydroxylase genes and related metabolite profiling of red cyanidin-/blue delphinidin-based anthocyanins in berry skin. BMC Genomics 7: 1471–1488.
- Castellarin, S. D., M. A. Matthews, G. D. Gaspero and G. A. Gambetta. 2007. Water deficits accelerate ripening and induce changes in gene expression regulating flavonoid biosynthesis in grape berry. Planta 227: 101–112.
- Chorti, E., S. Guidoni, A. Ferrandino and V. Novello. 2010. Effect of different cluster sunlight exposure levels on ripening and anthocyanin accumulation in Nebbiolo grapes. Am. J. Enol. Vitic. 61: 23–30.
- Downey, M. O., J. S. Harvey and S. P. Robinson. 2004. The effect of bunch shading on berry development and flavonoid accumulation in Shiraz grapes. Aus. J. Grape Wine Res. 10: 55–73.
- Ichikawa, M., K. Ono, M. Hisamoto, T. Matsudo and T. Okuda. 2011. Concentrations of BSA-binding proanthocyanidins in red wines produced in Japan. Food Sci. Technol. Res. 17: 335–339.
- Jeong, S. T., N. Goto-Yamamoto, K. Hashizume and M. Esaka. 2006. Expression of the flavonoid 3′-hydroxylase and 3′5′-hydroxylase genes and flavonoid composition in grape (Vitis vinifera). Plant Sci. 170: 61–69.
- Kobayashi, S., M. Ishimura, C. K. Ding, H. Yakushiji and N. Goto. 2001. Comparison of UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) gene sequences between white grapes (Vitis vinifera) and their sports with red skin. Plant Sci. 160: 543–550.
- Kobayashi, H., S. Suzuki, F. Tanzawa and T. Takayanagi. 2009. Low expression of flavonoid 3′,5′-hydroxylase (F3′,5′H) associated with cyanidin-based anthocyanins in grape leaf. Am. J. Enol. Vitic. 60: 362–367.
- Liang, Z., B. Wu, P. Fan, C. Yang, W. Duan and X. Zhen. 2008. Anthocyanin composition and content in grape berry skin in Vitis germplasm. Food Chem. 111: 837–844.
- Matus, J. T., R. Loyola, A. Vega, A. P. Neira, E. Bordeu, P. A. Johnson and J. A. Alcalde. 2009. Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera. J. Exp. Bot. 60: 853–867.
- Mori, K., N. Goto-Yamamoto, M. Kitayama and K. Hashizume. 2007. Effect of high temperature on anthocyanin composition and transcription of flavonoid hydroxylase genes in ‘Pinot noir’ grapes (Vitis vinifera). J. Hort. Sci. Biotechnol. 82: 199–206.
- Nyman, N. A. and J. T. Kumpulainen. 2001. Determination of anthocyanidins in berries and red wine by high-performance liquid chromatography. J. Agric. Food Chem. 49: 4183–4187.
- Palliotti, A., M. Gatti and S. Poni. 2011. Early leaf removal to improve efficiency: gas exchange, source-to-sink balance, and reserve storage responses. Am. J. Enol. Vitic. 62: 219–228.
- Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29: 2003–2007.
- Reid, K. E., N. Olsson, J. Schlosser, F. Peng and S. T. Lund. 2006. An optimized grapevine RNA isolation procedure and statistical determination of reference genes for real-time RT-PCR during berry development. BMC Plant Biol. 6: 27–37.
- Tesniere, C. and M. E. Vayda. 1991. Method for the isolation of high-quality RNA from grape berry tissues without contaminating tannins or carbohydrates. Plant Mol. Biol. Rep. 9: 242–251.
- Wu, X. and R. L. Prior. 2005. Systematic identification and characterization of anthocyanins by HPLC-ESI-MS/MS in common food in the United States: fruits and berries. J. Agric. Food Chem. 53: 2589–2599.
