A UDP-Glucose:Monoterpenol Glucosyltransferase Adds to the Chemical Diversity of the Grapevine Metabolome

Abstract

Terpenoids represent one of the major classes of natural products and serve different biological functions. In grape (Vitis vinifera), a large fraction of these compounds is present as nonvolatile terpene glycosides. We have extracted putative glycosyltransferase (GT) sequences from the grape genome database that show similarity to Arabidopsis (Arabidopsis thaliana) GTs whose encoded proteins glucosylate a diversity of terpenes. Spatial and temporal expression levels of the potential VvGT genes were determined in five different grapevine varieties. Heterologous expression and biochemical assays of candidate genes led to the identification of a UDP-glucose:monoterpenol β-d-glucosyltransferase (VvGT7). The VvGT7 gene was expressed in various tissues in accordance with monoterpenyl glucoside accumulation in grape cultivars. Twelve allelic VvGT7 genes were isolated from five cultivars, and their encoded proteins were biochemically analyzed. They varied in substrate preference and catalytic activity. Three amino acids, which corresponded to none of the determinants previously identified for other plant GTs, were found to be important for enzymatic catalysis. Site-specific mutagenesis along with the analysis of allelic proteins also revealed amino acids that impact catalytic activity and substrate tolerance. These results demonstrate that VvGT7 may contribute to the production of geranyl and neryl glucoside during grape ripening.

Figure 1.

Structural formulae of selected monoterpenes and their glycosylated conjugates found in grapes and wines.

Figure 2.

Phylogenetic tree of GT protein sequences. Protein sequences are from Arabidopsis (AtUGT), E. perriniana (EPGT1 and EPGT2), and S. bicolor (SORBI) with known glucosyltransferase activity toward terpenes and biochemically characterized proteins from Vitis spp. (Vitis vinifera [Vv] and Vitis labrusca [Vl]). VvGT7 to VvGT13 were investigated in this study. For methods used to construct the phylogeny, see “Materials and Methods.” GT subgroup assignments are shown in the boxes.

Figure 3.

Gene expression analysis of VvGTs by GeXP in nonberry tissues. The relative expression was quantified in cv Gewurztraminer 11-18 Gm (black bars) and cv White Riesling 239-34 Gm (gray bars). Sampled tissues were as follows: inflorescences 4 weeks (I1) and 2 weeks (I2) before flowering and at full bloom (I3); leaves at approximate ages of 1 week (L1), 3 weeks (L2), and 5 weeks (L3); and roots (R). Mean values and se of three independent experiments (biological replicates) are shown.

Figure 4.

Gene expression analysis of VvGTs by GeXP. Different stages of berry development are given as weeks after flowering. Expression was determined in berry exocarp of five different varieties and clones. Mean values and se of three independent experiments (biological replicates) are shown. Cultivars are as follows: black circles, cv Gewurztraminer 11-18 Gm; white triangles, cv Gewurztraminer FR 46-107; black squares, cv Muscat FR 90; white diamonds, cv White Riesling 239-34 Gm; and black triangles, cv White Riesling 24-196 Gm.

Figure 5.

Amounts of free monoterpenes and monoterpenyl β-d-glucosides in grape exocarp of different cultivars during ripening. Note that graphs have different scales. Grape exocarp was peeled and extracted. Free (black bars) and glycosidically bound (gray bars) monoterpenes were isolated by solid-phase extraction. Free monoterpenes were measured by GC-MS and monoterpenyl β-d-glucosides by HPLC-MS/MS (n = 2).

Figure 6.

Detection of monoterpenyl β-d-glucosides as products of VvGT7 by HPLC-electrospray ionization-MS/MS. A, HPLC-MS/MS analysis of linaloyl-β-d-glucoside (peak 1), neryl-β-d-glucoside (peak 2), geranyl-β-d-glucoside (peak 3), and citronellyl-β-d-glucoside (peak 4) of 0.1 mg mL⁻¹. B to D, Neryl-β-d-glucoside (B), citronellyl-β-d-glucoside (C), and geranyl-β-d-glucoside (D) produced by VvGT7. Traces display the total ion current of the characteristic transitions (see “Materials and Methods”). Gaussian smoothing was applied.

Figure 7.

Ribbon diagram of the calculated 3D structure of VvGT7b with UDP-Glc (orange) and nerol (green; A) and amino acid sequence alignment of VvGT7g and five plant GTs that have been crystallized and their 3D structures solved (B). UDP-Glc and nerol are shown as sticks and marked with arrows. β-Strands and α-helices are shown in gray. The residues at positions 186, 210, and 318, which differentiated the active from the inactive forms, are shown as sticks and are denominated. The alignment was performed using ClustalX. The plant GTs include M. truncatula UGT71G1 (protein data bank identification 2ACW), UGT78G1 (3HBF), and UGT85H2 (2PQ6), grape VvGT1 (2C1Z), and Arabidopsis UGT72B1 (2VCE). A consensus sequence logo is shown in the top traces, conserved amino acids are displayed as dots, and amino acids within 5 Å to the acceptor and sugar donor molecules (UDP-Glc) are marked in yellow and blue, respectively. Overlaps are shown in green (blue + yellow) and black (blue + red). The plant secondary glucosyltransferase box is confined by the amino acids marked in red.

Figure 8.

Enantioselectivity of VvGT7a determined by chiral phase GC-MS analysis of citronellol. A, A racemic mixture of R,S-citronellol was used as a substrate for VvGT7a. B, Residual citronellol is depleted in R-citronellol. C, R-Citronellol is released by acid-catalyzed hydrolysis from citronellyl β-d-glucoside. Signals labeled with x are by-products of the hydrolysis. D, Enantiomerically pure R-citronellol was used as reference material. Traces in A, C, and D were smoothed.