'Kadainou R-1' (Vitis Ficifolia Var. Ganebu

Influence of Temperature on Berry Composition of Interspecific Hybrid Wine Grape ‘Kadainou R-1’ (Vitis ficifolia var. ganebu × V. vinifera ‘Muscat of Alexandria’)

Puspa Raj Poudel1,2, Ryosuke Mochioka1*, Kenji Beppu3 and Ikuo Kataoka3

1University Farm, Faculty of Agriculture, Kagawa University, Sanuki 769-2304, Japan 2United Graduate School of Agricultural Sciences, Ehime University, Matsuyama 790-8566, Japan 3Faculty of Agriculture, Kagawa University, Miki 761-0795, Japan

High temperature affects berry composition, especially titratable acidity, total soluble solids, and anthocyanin content. Vitis ficifolia var. ganebu, a wild grape of sub-tropical origin with a low chilling trait, develops good coloration in its natural habitat, where daytime and nighttime temperatures are high during the berry ripening stage, while V. vinifera ‘Muscat of Alexandria’, a table grape, has large berries and high sugar content, hence, it was hypothesized that hybridizing V. ficifolia var. genebu with V. vinifera would improve the sugar content and reduce titratable acidity compared to V. ficifolia var. ganebu and retain berry color even under high temperature conditions. The aim of this study was to investigate the influence of temperature on the berry composition of ‘Kadainou R-1’, an interspecific hybrid wine grape derived from V. ficifolia var. ganebu × V. vinifera ‘Muscat of Alexandria’. Potted ‘Kadainou R-1’ vines with their ownroots were subjected to continuous temperatures of 20, 25, and 30°C in a phytotron during the berry-softening stage. Berries were harvested 15, 30, and 37 days after the vines were placed in the phytotron. Titratable acidity was lower at 30°C than at 20°C, while total soluble solids were highest at 25°C. Accumulations of glucose and fructose were higher at low rather than high temperatures. Total anthocyanin content (mg·g−1 of fresh weight) was significantly reduced at 20 and 30°C but not at 25°C. Flavonol content was highest at 25°C. The present study revealed that ‘Kadainou R-1’ grapes can produce optimum berry quality in locations where the night temperature reaches 25°C without significant loss in berry quality.

Key Words: anthocyanin, flavonols, ‘Kadainou R-1’, temperature effect, wine grape.

never drops below 10°C (National Astronomical Introduction Observatory, 2006). This species develops good High temperatures affect photosynthesis, thereby coloration even in its natural habitat (Nakagawa et al., decreasing berry growth of grapes (Kliewer, 1977b; 1991), where considerably high night temperatures of Kobayashi et al., 1965c). High temperature conditions more than 25°C have been reported during the berry are considered to contribute to lower total soluble solids growth and ripening stages (Japan Metrological Agency, and titratable acidity (Buttrose et al., 1971; Kobayashi 2007). In addition, high anthocyanin in its berry skins et al., 1965b). High temperature also inhibits anthocyanin and a lack of chilling requirement have been reported formation and alters anthocyanin composition (Mori et previously (Poudel et al., 2007a, 2007b). Unlike al., 2005; Mori et al., 2007; Yamane et al., 2006). V. ficifolia var. ganebu, several wine and table grapes Vitis ficifolia var. ganebu is native to subtropical have been reported to develop poor coloration under regions of Japan and is found in the Ryukyu Islands of similar conditions (Kataoka et al., 1984; Mori et al., the Okinawa region (Nakagawa et al., 1991), where the 2005; Naito et al., 1986; Yamane et al., 2006). Recently, average annual temperature is approximately 23°C. Even global warming has been thought to inhibit the during the winter, the temperature averages 16°C and accumulation of anthocyanin, as well as flavor substances in grapes grown in temperate regions (Jones Received; March 19, 2008. Accepted; August 18, 2008. et al., 2005). Hence, it was postulated that certain traits * Corresponding author (E-mail: [email protected]). of V. ficifolia var. ganebu could be useful for developing

169 170 P. R. Poudel, R. Mochioka, K. Beppu and I. Kataoka cultivars with a low chilling trait that would produce Total soluble solids (TSS), titratable acidity (TA) and anthocyanins without a significant reduction under sugar analysis higher day and night temperature conditions. To improve Approximately 10–115 berries were finger pressed to anthocyanin accumulation in grapes, chemical treat- extract the juice and the juice was filtered through two ments, e.g., ethylene (Chervin et al., 2004) and abscisic layers of cheesecloth. The expressed juice was analyzed acid (Hiratsuka et al., 2001; Jeong et al., 2004), have for TSS and TA. Juice extracted from 10–15 berries was been studied previously. Genetic improvement by taken as a replicate and the experiment was replicated crossing with high anthocyanin accumulation species/ at least three times. The same juice was used for TSS varieties is also one possible way to improve anthocyanin and TA analysis. TSS was measured as °Brix using a accumulation. ‘Kadainou R-1’, a new wine grape digital refractometer (Atago Co., Ltd., Japan), while TA cultivar, is a selection of hybridization between was determined as tartaric acid content by titrating the V. ficifolia var. ganebu and V. vinifera ‘Muscat of juice with 0.1 N NaOH to an endpoint of pH 8.1. Alexandria’. V. ficifolia var. ganebu has small black For sugar analyses, five grams of flesh were taken berries and low total soluble solids (TSS), while ‘Muscat from ten berries and homogenized (Ultra-Turrax T25; of Alexandria’, a white grape, has large berries and Ika-Labortechnik, Germany) with MilliQ water at contains high TSS. ‘Kadainou R-1’ was also found to 24,000 rpm for one minute. The mixture was incubated be rich in anthocyanin content, with a low chilling trait at 60°C for 30 minutes and centrifuged at 3,500 rpm for and short dormancy period (Poudel et al., 2007a, 2007b). ten minutes. The supernatant was collected, and the ‘Kadainou R-1’ has a berry size and composition of its residual tissue was re-extracted following the same parents, although its adaptability and berry composition procedure. The extracts were mixed and filtered through under high temperature conditions, which are the traits a 0.47 μm filter (Waters Co., USA). The filtrate was expected to be inherited from its female parent, are yet further purified by passing through a Sep-Pak C18 be known. Hence, the aim of this study was to determine cartridge (Waters Co.). The Sep-Pak was preconditioned the effect of high temperatures on the composition of with 5 mL of 75% acetonitrile and washed with 5 mL the ‘Kadainou R-1’ grape. Sugars and organic acid of distilled water before use. The eluent was diluted to composition determine the edibility of grape berries and appropriate concentrations using 75% acetonitrile and the final alcohol content of unfortified wine (Kliewer, was used for sugar analyses by high-performance liquid 1967), while phenolic compounds determine the taste chromatography (HPLC; pump: PU 980; detector: RI- and quality of wine (Arnold and Noble, 1978); hence, 930, Jasco Inc., Tokyo, Japan). Sugar was eluted using we determined all these constituents in order to estimate a Shodex Asahipak NH2-504E (Showa Denko Co., Ltd., the overall berry quality of ‘Kadainou R-1’ under Japan) column with acetonitrile-water (75 : 25) at a flow different temperature treatments. rate of 1 mL·min−1. The column was maintained at 30°C. Sugar constituents were identified based on their order Materials and Methods of elution and retention time of the standard compounds; Plant materials and experimental settings quantification was performed using the external standard Self-rooted ‘Kadainou R-1’ vines grown in 10 L pots method (Lòpez-Tamames et al., 1996). were used in the present experiments. Two clusters were maintained for each plant and each cluster contained Sample preparation for anthocyanin and phenolics approximately 100 berries. The vines were trained using analyses the vertical trellis system and transferred to a phytotron The selected berries were finger-pressed to remove on August 24, 2006 when the berries reached the juice and pulp. Seeds and skins were separated and softening stage, a high-temperature-sensitive stage washed several times with distilled water; moisture was (Yamane et al., 2006); this continued until final harvest absorbed with blotting paper. The skin samples (1 g each) (37 days after veraison). The high temperature limit for were blended for one minute at 24,000 rpm in a blender metabolic processes in grapevines is thought to be around (Ultra-Turrax T25; Ika-Labortechnik) with 10 mL of 30°C (Coombe, 1987) while night temperatures below acidified methanol (1 : 99 v/v, HCl : MeOH). The 20°C are reported to result in inferior berry coloration homogenate was incubated for 12 hours at 4°C in the (Mori et al., 2004), hence, three temperatures were dark before filtering with Whatman no. 1 filter paper maintained in the phytotron: low (20°C), intermediate (Whatman, UK) and centrifuging at 3,500 rpm for ten (25°C), and high (30°C). The day and night temperatures minutes. Once the extract was separated, the residual were kept the same under natural sunlight conditions. tissue was subjected to two subsequent extractions, Approximately 100 berries (15–25 berries from each following the same procedure, in 5 mL of acidified cluster, two clusters per vine, three plants per treatment) methanol each time. All extracts were combined, mixed were harvested randomly at 0, 15, 30, and 37 days after thoroughly and used for anthocyanin and phenolics veraison (DAV) for composition analysis. The sampled analyses. berries were counted, graded and normal size berries were used for further experiments. J. Japan. Soc. Hort. Sci. 78 (2): 169–174. 2009. 171

Total anthocyanin content Ciocalteu’s colorimetric assay (Singleton and Rossi, The total anthocyanin content was measured from a 1965). A 0.5 mL aliquot of the prepared extract was calibration curve using malvidin 3,5-diglucoside as an diluted five times, after which a 100 μL aliquot was internal standard. A 0.5 mL aliquot of the sample was taken for further analysis. The 100 μL aliquot was mixed diluted 10 times, and absorbance was read at 537 nm with 1 mL phenol reagent, 1 mL 10% sodium using a UV/VIS spectrophotometer (Shimadzu Co., Ltd., bicarbonate, and 4 mL distilled water. The mixture was Japan). Results were expressed as malvidin 3,5- allowed to stand in the dark for one hour. Absorbance diglucoside equivalent (ME) against the fresh weight of was read at 760 nm, and the total phenolic content was the samples (mg·g−1). calculated from the calibration curve, using gallic acid as a standard. The results are expressed as the gallic acid Procyanidin monomers (Flavan-3-ols) equivalent (GAE) against the fresh weight of the samples The flavan-3-ols content was determined following (mg·g−1). the procedure described by Arnous et al. (2001). Briefly, a sample (0.2 mL) diluted 1 : 100 with MeOH was placed Data analysis in a 1.5 mL Eppendorf tube (Eppendorf, Germany), and Differences between means were calculated by 1 mL of p-dimethylaminocinnamaldehyde (DMACA; Tukey’s test at 5% level of significance. Computations 0.1% in 1 N HCl-MeOH) solution was added. The sample were performed using Statistical Package for the Social was vortexed and stood for ten minutes at room Sciences (SPSS) for windows (Version 13.0). temperature. Absorbance was recorded at 640 nm. The Results and Discussion concentration of total flavanols was determined from a calibration curve constructed by plotting absorption at TSS increased gradually for all temperature treat- 640 nm (r2 = 0.9997) against known concentrations of ments; the highest TSS was recorded at 25°C at final catechin. The results are expressed as the catechin harvest on 37 DAV (Fig. 1). This study revealed that equivalent (CE) against the fresh weight of the sample 25°C was the optimum temperature for the highest TSS (mg·g−1). accumulation, with a tendency for lower values at 20 and 30°C (Fig. 1). The TSS accumulation rate was faster Total flavonols at 30°C than at 20°C until 30 DAV and then leveled Total flavonols were determined following the off; at 20°C, however, slower but steadily increasing procedure described by Mazza et al. (1999) and TSS accumulation was observed until final harvest. expressed as quercetin equivalent (QE) against fresh Kobayashi et al. (1965a) reported that when ‘Delaware’ weight (mg·g−1). Briefly, the sample was diluted with vines were subjected to a range of night temperatures 10% ethanol. A 0.25 mL sample or standard was taken from 15 to 35°C during the ripening stage (August to in a test tube, and 0.25 mL 0.1% HCl in 95% ethanol September), the highest TSS value occurred at 28°C and 4.55 mL 2% HCl were added. The solution was when berries were harvested 21 days after treatment thoroughly mixed and allowed to stand for approximately during late August. However when the berries were 15 minutes before reading absorbance at 360 nm with a harvested during the first week of September 34 days spectrophotometer. Quercetin dissolved in 95% ethanol after treatment, a low TSS value at the same temperature was used as the standard. was recorded compared to at 22°C and 15°C. The results indicated that the TSS at low temperature accumulates Total flavonoids gradually. Similar to the results mentioned in Kobayashi The flavonoids were determined following the et al. (1965a), the present study also revealed that the procedure described by Kim et al. (2003). A 1 mL aliquot of a sample diluted ten times was planced in a 10 mL capacity tube containing 4 mL double-distilled water. A 0.3 mL sample of 5% NaNO2, 0.3 mL of 10% AlCl3 and 2 mL of 1 N NaOH were added to the mixture at zero, five and six minutes, respectively. Immediately, 2.4 mL of double-distilled water was added to the reaction mixture and thoroughly mixed. Absorbance of the mixture was then measured at 510 nm versus a prepared water blank. Total flavonoid content was determined from calibration curve using catechin as the standard and expressed as the catechin equivalent (CE) mg·g−1 of the fresh weight of the sample. Fig. 1. Changes in TA and TSS during ripening of ‘Kadainou R-1’ at different temperatures. Vertical bars represent standard error. Juice extracted from 15 berries was taken as a single replicate Total phenolic content and the experiments were repeated at least three times. The same Total phenolics content was determined using Folin- juice was used for TSS and TA analysis. 172 P. R. Poudel, R. Mochioka, K. Beppu and I. Kataoka

TSS accumulation rate was higher at 30°C than at 20°C anthocyanin biosynthetic pathway. Bakhshi and until 30 days after the onset of temperature treatments; Arakawa (2006) reported 24°C as the optimum however, TSS accumulation at both temperatures (20 temperature for anthocyanin biosynthesis in apples. Mori and 30°C) coincided at final harvest, 37 days, after et al. (2004) also reported the highest anthocyanin forcing (late ripening stage). accumulation at 25°C in ‘Kokuo’ (a bud mutation of TA decreased at the fastest rate at 30°C and the lowest ‘Kyoho’) grape berries compared to at very high (30°C) recorded value was under the highest temperature and cool (15°C) night temperatures. In addition, Mori (Fig. 1). TA fell at higher rates at high temperature (25 et al. (2005) reported reduced anthocyanin biosynthesis and 30°C) than at low temperature (20°C). This result in ‘Darkridge’ grape berries under high night was in general agreement with previous findings in temperatures (continuous 30°C) as compared to high day V. vinifera cultivars (Buttrose et al., 1971; Kobayashi et and cool night (30/15°C) temperatures. Higher temper- al., 1965b). Malic acid declines in berries following atures (above 32°C) have also been reported to inhibit veraison due to cellular respiration while tartaric acid anthocyanin accumulation (Kliewer, 1977a; Kliewer and remains nearly constant (Peynaud and Maurié, 1958); Torres, 1972). The female parent of ‘Kadainou R-1’ is hence, it is likely that the decline in TA of ‘Kadainou V. ficifolia var. ganebu, which is native to subtropical R-1’ grape berries at more than 25°C was attributed to regions; hence, it is likely that ‘Kadainou R-1’ grapes increased malic acid degradation because of increased can tolerate high night temperatures to some extent, respiration. resulting in a considerable amount of anthocyanin Glucose and fructose were the major sugars in production, even at 30°C temperature (Table 1). The ‘Kadainou R-1’ grapes. At low (20°C) and intermediate anthocyanin contents of ‘Kadainou R-1’ berry skins (25°C) temperatures, both sugars increased at a faster presented in this study were higher than those of ‘Kokuo’ rate than at high temperature (30°C) and leveled off at grapes (Mori et al., 2004) under similar night temperature upwards of 30 DAV (Fig. 2). At 30°C, both sugars conditions (25 and 30°C); however, it was hard to increased until harvest (Fig. 2C); however, both sugars compare our data with other known grape cultivars due were always lower than those recorded at 20 and 25°C to large differences in the experimental settings and (Figs. 2A and 2B). In general as the temperature analytical procedures used in the present study and in increased, the accumulation rates of both sugars (glucose the literature. and fructose) decreased. This finding was in agreement Total phenols and flavonols were found to be highest with Radler (1965), who reported reduced sugar at 25°C and differed significantly according to accumulation when clusters of ‘Sultana’ vines were exposed to day and night temperatures of 33°C as Table 1. Phenolic composition of the berry skin of ‘Kadainou R-1’ compared to that of clusters exposed to a normal grape at different temperatures 37 days after veraison. environment in Australia. Anthocyanin content was highest at 25°C and dropped Phenolic components Temperatures −1 significantly at low (20°C) or high (30°C) temperatures; (mg·g FW) 20°C25°C30°C however, anthocyanin content at 30°C was still higher Anthocyanin 1.60cz 6.00a 2.40b than that at 20°C (Table 1). The remarkable characteristic Phenols 29.33c 49.84a 31.69b of ‘Kadainou R-1’ grapes was significantly (P = 0.05) Procyanidin 2.57b 10.66a 2.95b reduced anthocyanin accumulation at 20°C as compared Flavonols 1.31b 1.65a 1.14c to that at 30°C; however, both values were still lower Flavonoids 10.58b 19.10a 12.65b than at 25°C. The highest anthocyanin content, at 25°C, z Same letter(s) within the same row indicate no significant differences was accompanied by the highest procyanidin monomers, among treatments by Tukey’s test at P < 0.05 level. Data represent a compound produced in an earlier step of the three replications per treatment.

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