The biosynthesis of S-(3-hexan-1-ol)-glutathione (3MH-S-glut) and S-(3-hexan-l-ol)-L-cysteine (3MH-S-cys), which act as flavour precursors in wines, in Vitis vinifera grapes exposed to various environmental stress conditions is reported here. Ultraviolet (UV-C) irradiation, water deficit, and biological stimulation up-regulated 3MH-S-glut and 3MH-S-cys biosynthesis in grape leaves. 3MH-S-glut and 3MH-S-cys contents in grape berries were increased by cold shock, heat shock, UV-C irradiation, and biological stimulation. The results suggest that environmental stress enhances the biosynthesis of both flavour precursors in grapevine. The transcription of VvGST1, VvGST3, VvGST4, and GGT in grapevine exposed to the stress conditions was increased markedly compared with that in control grapevine. Also, UV irradiation increased GST (glutathione S-transferase) and GGT (γ-glutamyl transferase) enzyme activities in grape berries. Recombinant VvGST3 and VvGST4, but not VvGST1, mediated the synthesis of 3MH-S-glut from reduced glutathione and trans-2-hexenal in vitro. The enzymatic mediation of flavour precursor production is a novel function of plant GSTs and may result in the detoxification of damaged grape cells under stress conditions.

Bacillus subtilis KS1 was isolated from grape berry skin as a biological control agent against grapevine fungal diseases. KS1 was identified as a new strain of B. subtilis according to morphological, biochemical, and genetic analyses. In vitro bioassay demonstrated that KS1 suppressed the growth of Botrytis cinerea (the casual agent of grape grey mold) and Colletotrichum gloeosporioides (the casual agent of grape ripe rot). The biocontrol activity of KS1 against grapevine fungal diseases in vineyards was evaluated over a 3-year span (from 2007 to 2009). Downy mildew, caused by Plasmopara viticola, was reduced on berry skins and leaves by treatment with KS1. The KS1 genome possesses ituD and lpa-14 genes, both of which play a role in iturin A production followed by iturin A production in the culture. In contrast, mutants lacking both genes lost the antagonistic activity against B. cinerea and C. gloeosporioides and the activity in iturin A production, suggesting that the antagonistic activity of KS1 against grapevine fungal pathogens may depend on iturin A production. As KS1 showed tolerance to various chemical pesticides, chemical pesticides could be applied before and/or after KS1 treatment in vineyards. Due to its potential as a biological control agent against grape downy mildew, KS1 is expected to contribute to the further improvement of integrated pest management systems and to potentially reduce the amount of chemical fungicides applied in vineyards.

(−)-Rotundone is a potent odorant molecule with a characteristic spicy aroma existing in various plants including grapevines (Vitis vinifera). It is considered to be a significant compound in wines and grapes because of its low sensory threshold and aroma properties. (−)-Rotundone was first identified in red wine made from the grape cultivar Syrah and here we report the identification of VvSTO2 as a α-guaiene 2-oxidase which can transform α-guaiene to (−)-rotundone in the grape cultivar Syrah. It is a cytochrome P450 (CYP) enzyme belonging to the CYP 71BE subfamily, which overlaps with the very large CYP71D family and, to the best of our knowledge, this is the first functional characterization of an enzyme from this family. VvSTO2 was expressed at a higher level in the Syrah grape exocarp (skin) in accord with the localization of (−)-rotundone accumulation in grape berries. α-Guaiene was also detected in the Syrah grape exocarp at an extremely high concentration. These findings suggest that (−)-rotundone accumulation is regulated by the VvSTO2 expression along with the availability of α-guaiene as a precursor. VvSTO2 expression during grape maturation was considerably higher in Syrah grape exocarp compared to Merlot grape exocarp, consistent with the patterns of α-guaiene and (−)-rotundone accumulation. On the basis of these findings, we propose that VvSTO2 may be a key enzyme in the biosynthesis of (−)-rotundone in grapevines by acting as a α-guaiene 2-oxidase.

As the use of chemical fungicides has raised environmental concerns, biological control agents have attracted interest as an alternative to chemical fungicides for plant-disease control. In this study, we attempted to explore biological control agents for three fungal phytopathogens causing downy mildew, gray mold, and ripe rot in grapevines, which are derived from shoot xylem of grapevines. KOF112, which was isolated from the Japanese indigenous wine grape Vitis sp. cv. Koshu, inhibited mycelial growth of Botrytis cinerea, Colletotrichum gloeosporioides, and Phytophthora infestans. The KOF112-inhibited mycelial tips were swollen or ruptured, suggesting that KOF112 produces antifungal substances. Analysis of the 16S rDNA sequence revealed that KOF112 is a strain of Bacillus velezensis. Comparative genome analysis indicated significant differences in the synthesis of non-ribosomal synthesized antimicrobial peptides and polyketides between KOF112 and the antagonistic B. velezensis FZB42. KOF112 showed biocontrol activities against gray mold caused by B. cinerea, anthracnose by C. gloeosporioides, and downy mildew by Plasmopara viticola. In the KOF112–P. viticola interaction, KOF112 inhibited zoospore release from P. viticola zoosporangia but not zoospore germination. In addition, KOF112 drastically upregulated the expression of genes encoding class IV chitinase and β-1,3-glucanase in grape leaves, suggesting that KOF112 also works as a biotic elicitor in grapevine. Because it is considered that endophytic KOF112 can colonize well in and/or on grapevine, KOF112 may contribute to pest-management strategies in viticulture and potentially reduce the frequency of chemical fungicide application.

Misfolded protein aggregation in mammalian cells is one of the cellular responses to environmental stresses. However, the aggregation of misfolded proteins in plant cells exposed to environmental stresses is still poorly understood. Here, we report the misfolded protein aggregation in plant cells in response to environmental stresses, including ultraviolet (UV) radiation, heat stress and cold stress. Treatment of grape and tobacco cultured cells with MG-132, a proteasome inhibitor, induced misfolded protein aggregation. All of the environmental stresses examined induced the endoplasmic reticulum (ER) stress response in the cells. The cells under ER stress showed aggregation of misfolded proteins. The misfolded protein aggregation was completely inhibited by treatment of the cells with trichostatin A or colchicine, suggesting that the misfolded proteins might be aggregated in plant cells in a microtubule-dependent manner. Detected aggregates were initially observed immediately after exposure to the environmental stresses (1 min after UV radiation, 5 min after heat stress exposure, and 15 min after cold stress exposure). Based on these findings, we hypothesize that environmental stresses induce misfolded protein aggregation in plant cells in a microtubule-dependent manner.