Environmental stress enhances biosynthesis of flavor precursors, S-3-(hexan-1-ol)-glutathione and S-3-(hexan-1-ol)-L-cysteine, in grapevine through glutathione S-transferase activation

Abstract

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.

Fig. 1.

Proposed pathway leading to the biosynthesis of the glutathionylated pro-precursor (3MH-S-glut) and the cysteinylated precursor (3MH-S-cys) in grapevine. The production of 3-mercaptohexan-1-ol (3MH) occurred during alcoholic fermentation.

Fig. 2.

Measurement of 3MH-S-glut, 3MH-S-cys, and GSH contents in grape leaves of seedlings exposed to stress conditions. (A) 3MH-S-glut, (B) 3MH-S-cys, (C) GSH. Chardonnay, Koshu, and Merlot seedlings were used and incubated under stress conditions as described in the Materials and methods. Data are shown as means ±SDs of triplicate experiments.

Fig. 3.

Induction of GST and GGT in UV-C-irradiated grape leaves and berries. (A) The expression of VvGST genes (VvGST1, VvGST2, VvGST3, VvGST4, and VvGST5) and 3MH-S-glut content. (B) GGT gene expression and 3MH-S-cys content. Chardonnay seedlings were exposed to UV-C irradiation and cultivated for the indicated periods as described in the Materials and methods. For gene expression analyses, 18S rRNA was used as the internal control. Data were calculated as gene expression relative to 18S rRNA gene expression. Data are shown as means ±SDs of triplicate experiments. (C) GST enzyme activity. (D) GGT enzyme activity. Sauvignon blanc bunches were exposed to UV-C irradiation and incubated for the indicated periods. GST and GGT enzyme activities were measured and calculated as described in the Materials and methods. Data are shown as means ±SDs of triplicate experiments. Control, no treatment; UV, UV irradiation. *P <0.05 as compared with control.

Fig. 4.

Measurement of 3MH-S-glut, 3MH-S-cys, and GSH contents in grape berries exposed to stress conditions. (A) 3MH-S-glut, (B) 3MH-S-cys, (C) GSH. Grape berries were collected at 16 weeks post-flowering and exposed to each stress. Data are shown as means ±SDs of triplicate experiments.

Fig. 5.

Activity of recombinant VvGSTs in the in vitro synthesis of 3MH-S-glut. (A) The recombinant proteins VvGST1, VvGST3, and VvGST4, in SDS–PAGE. Escherichia coli sonicates are shown in the left panel. His-purified proteins (15 μl of purified solution) electrophoresed on a 12.5% SDS–polyacrylamide gel (right). M, protein marker. Asterisks indicate recombinant VvGST proteins. Vector, pCold I vector as negative control. (B) VvGST3 and VvGST4 mediate 3MH-S-glut synthesis in vitro. Data are shown as means ±SDs of triplicate experiments. n.d., not detected. CGST, commercial GST from equine liver.

Fig. 6.

Hypothetical pathway for the biosynthesis of 3MH-S-glut and 3MH-S-cys in grapevine exposed to environmental stress conditions.