A recently popular Japanese yellow-green-skin table grape, 'Shine Muscat' (Vitis labruscana Bailey × V. vinifera L.), has the problem of berry skin browning, which occurs at the maturation stage just before harvest. Tiny reddish-brown blotches appear on the surface of berries and considerably decrease the grape's market value. Although the mechanisms and factors for browning are unknown, we hypothesized the involvement of polyphenol compounds and their oxidation reactions. In this study, the gene expressions of polyphenol oxidase (PPO), stilbene synthase (STS), and chalcone synthase (CHS), which are key enzymatic genes related to the metabolic pathway for polyphenols, were analyzed during berry maturation to examine the molecular basis for browning. Skin browning occurred on several berries in a bunch of 'Shine Muscat' from 80 days after full bloom (DAFB), after which the number of berries with skin browning increased, and the browned area spread on the berry surfaces with maturation. Increases in the expression of VvPPO2, VvSTS type B, and VvCHS1 were associated with skin browning, and the trans-resveratrol content also increased in the browning skin, suggesting that biosynthesis and metabolic pathways for phenolic compounds were activated at the time of browning. In terms of VvPPO genes, specific up-regulation of VvPPO2 expression was observed compared with the VvPPO1 gene. The promoter sequence of VvPPO2 contains more Myb binding motifs and W-box motifs than does VvPPO1. The specific up-regulation of VvPPO2 gene expression will play a crucial role in understanding and managing the skin-browning mechanism in the grape berries of 'Shine Muscat'.

Abscisic acid (ABA) and ethylene are well-known phytohormones that are involved in the maturation of grape berries and other fruits. However, the process of yellow-green skinned grape berry maturation is not well understood due to difficulties in determining grape maturity from changes in skin color. Skin browning during maturation is a major commercial problem in some yellow-green skinned grape cultivars including 'Shine Muscat'. To resolve this issue, a better understanding of the mechanisms involved in grape maturation and skin-browning is needed. We treated 'Shine Muscat' grape clusters at the veraison stage (45-50 DAFB) with spray applications of ABA or ethephon. These treatments produced darker colors and increased the transresveratrol and flavonol contents of berry skins. The ABA and ethephon treatments significantly increased the severity of skin browning. Changes in the expression of genes involved in polyphenol biosynthesis and oxidation were consistent with increases in polyphenols and the severity of browning in berry skins. The expression of VvACO2 and VvYUC1 genes, which are involved in ethylene and auxin biosynthesis, respectively, were upregulated in berries with brown skins. Although ABA treatment also increased the size of the berries, the effect of ethephon treatment on berry maturation was similar to, or greater than, that of ABA treatment. In berry skins, the expression of VvACO3, which is involved in phytohormone biosynthesis, increased significantly in response to ABA treatment. Overall, the changes in gene expression produced by ABA and ethephon treatments differed. Therefore, different mechanisms may regulate the physiological responses to ABA and ethephon, although both treatments accelerate berry maturation.

i. Title: The potential aroma and flavor compounds in Vitis sp. cv. Koshu and V. vinifera L. cv. Chardonnay under different environmental conditions ii. Running title: Potential aroma and flavor compounds in non-aromatic wine grape cultivars under different environmental conditions iii. Sharon Marie Bahena-Garrido1, Tomoko Ohama1, Yuka Suehiro2, Yuko Hata1, Atsuko Isogai1, Kazuhiro Iwashita1, Nami Goto-Yamamoto1 and Kazuya Koyama1 iv.v. ABSTRACT BACKGROUND: Koshu, a hybrid of Vitis vinifera L. and V. davidii Foex, is the most popular indigenous cultivar for wine production in Japan. However, little is known about the potential aroma compounds it contains and how environmental factors affect these. In this study, we obtained comprehensive profiles of the volatile (both glycosidically bound and free) and phenolic compounds that occur in Koshu berries, and compared these with similar profiles for V. vinifera cv. Chardonnay. We then compared the response of these two cultivars to bunch shading and the ripening-related phytohormone abscisic acid (ABA). RESULTS:Koshu berries contained significantly higher concentrations of phenolic compounds, hydroxycinnamic acid derivatives, and some volatile phenols, such as 4-vinyl guaiacol and eugenol, than Chardonnay berries, which are thought to contribute to the characteristics of Koshu wine. In addition, Koshu berries had a distinctly different terpenoid composition from Chardonnay berries. Shading reduced the concentrations of norisoprenoid in both cultivars, as well as several phenolic compounds particularly their volatile derivatives in Koshu. The exogenous application of ABA induced ripening and increased the concentrations of lipid derivatives, such ashexanol, octanol, nonanol, and 1-octen-3-ol. Linear discriminant analysis showed that the aromatic potential could be discriminated clearly based on cultivar, bunch shading, and ABA application. CONCLUSION:The unique secondary metabolite profiles of Koshu and their different responses to environmental factors could be valuable for developing various styles of Koshu wines and new cultivars with improved quality and cultural characteristics.

Phytohormones play major roles in the berry maturation process. Gibberellic acid (GA) and cytokinin (CK) are phytohormones used in seedless table grape production. Several studies have been conducted on the effects of GA and CK application on berry development. However, the detailed mechanisms underlying their physiological effects on berry maturation after the veraison stage have not been clarified. Skin browning during maturation is a major commercial problem in yellow-green skinned grape cultivars including 'Shine Muscat', and expanding our knowledge of these mechanisms is a necessary step towards addressing this problem. In this study, we investigated the effects of GA and CK treatments from the veraison stage to the subsequent developmental stages of this grape berry. Both treatments resulted in enlarged berries and the suppression of increases in sugar content. Chlorophyll in the berry skin was less decomposed after GA/CK treatment, and the occurrence of skin browning in the maturation stage was reduced, as expression of the VvPP2Cs gene decreased. GA/CK treatment at the veraison stage (45-50 DAFB) reduced the expression levels of phytohormone-related genes, particularly those of VvGID1 and VvCHKs, which are involved in GA and CK signaling, respectively. These similar changes in gene expression patterns suggest phytohormonal crosstalk and a common expressional regulatory mechanism. VvACO2 and VvYUC1 expressions were significantly increased in skin browning samples, regardless of treatment, indicating involvement of the ethylene and auxin biosynthesis pathways in skin browning. Therefore, GA/CK treatment at the veraison stage may broadly affect phytohormone biosynthesis and signaling pathways in subsequent developmental stages, although the effect size greatly differs depending on the experimental conditions, including year and plant.

BACKGROUND Koshu, a hybrid of Vitis vinifera L. and V. davidii Foex, is the most popular indigenous cultivar for wine production in Japan. However, little is known about the potential aroma compounds it contains and how environmental factors affect these. In this study, we obtained comprehensive profiles of the volatile (both glycosidically bound and free) and phenolic compounds that occur in koshu berries, and compared these with similar profiles for V. vinifera cv. chardonnay. We then compared the response of these two cultivars to bunch shading and the ripening‐related phytohormone abscisic acid (ABA). RESULTS Koshu berries contained significantly higher concentrations of phenolic compounds, such as hydroxycinnamic acid derivatives, and some volatile phenols, such as 4‐vinyl guaiacol and eugenol, than chardonnay berries, which are thought to contribute to the characteristics of koshu wine. In addition, koshu berries had a distinctly different terpenoid composition from chardonnay berries. Shading reduced the concentration of norisoprenoid in both cultivars, as well as several phenolic compounds, particularly their volatile derivatives in koshu berries. The exogenous application of ABA induced ripening and increased the concentrations of lipid derivatives, such as hexanol, octanol, 1‐nonanol, and 1‐octen‐3‐ol. Multivariate and discriminant analyses showed that the potential aroma and flavor compounds in the berries could be discriminated clearly based on cultivar and environmental cues, such as light exposure. CONCLUSION The unique secondary metabolite profiles of koshu and their different responses to environmental factors could be valuable for developing various types of koshu wines and new cultivars with improved quality and cultural characteristics. © 2018 Society of Chemical Industry

Polyphenol oxidases (PPOs) catalyze browning reactions in various plant organs, therefore controlling the reactions is important for the food industry. PPOs have been assumed to be involved in skin browning of white grape cultivars; however, the molecular mechanism underlying PPO-mediated browning process remains elusive. We have recently identified a new PPO gene named VvPPO2 from "Shine Muscat" (Vitis labruscana Bailey × V. vinifera L.), and have shown that the gene is transcribed at a higher level than the previously identified VvPPO1 in browning, physiologically disordered berry skins at the maturation stage. In this study, we expressed VvPPO2 in Escherichia coli and, using the purified preparation, revealed unique physicochemical characteristics of the enzyme. Our study opens up a way to not only understand the berry skin browning process but also to elucidate the enzymatic maturation process of grape PPOs.

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