Activity-based profiling of a physiologic aglycone library reveals sugar acceptor promiscuity of family 1 UDP-glucosyltransferases from grape

Abstract

Monoterpenols serve various biological functions and accumulate in grape (Vitis vinifera), where a major fraction occurs as nonvolatile glycosides. We have screened the grape genome for sequences with similarity to terpene URIDINE DIPHOSPHATE GLYCOSYLTRANSFERASES (UGTs) from Arabidopsis (Arabidopsis thaliana). A ripening-related expression pattern was shown for three candidates by spatial and temporal expression analyses in five grape cultivars. Transcript accumulation correlated with the production of monoterpenyl β-d-glucosides in grape exocarp during ripening and was low in vegetative tissue. Targeted functional screening of the recombinant UGTs for their biological substrates was performed by activity-based metabolite profiling (ABMP) employing a physiologic library of aglycones built from glycosides isolated from grape. This approach led to the identification of two UDP-glucose:monoterpenol β-d-glucosyltransferases. Whereas VvGT14a glucosylated geraniol, R,S-citronellol, and nerol with similar efficiency, the three allelic forms VvGT15a, VvGT15b, and VvGT15c preferred geraniol over nerol. Kinetic resolution of R,S-citronellol and R,S-linalool was shown for VvGT15a and VvGT14a, respectively. ABMP revealed geraniol as the major biological substrate but also disclosed that these UGTs may add to the production of further glycoconjugates in planta. ABMP of aglycone libraries provides a versatile tool to uncover novel biologically relevant substrates of small-molecule glycosyltransferases that often show broad sugar acceptor promiscuity.

Figure 1.

Grapes of ‘Muscat’ (A) and major monoterpenols found in grape (B). Photographs were taken every 2 weeks between week 4 and week 18 after flowering. The width of the photographs is 10 cm.

Figure 2.

Phylogenetic tree of glucosyltransferase protein sequences. Protein sequences from Arabidopsis (AtUGT) with known glucosyltransferase activity toward terpenes are also shown. VvGT14 to VvGT20 were investigated in this study. Glucosyltransferase subgroup assignment is shown in the boxes at right.

Figure 3.

Gene expression analysis of VvGTs by GeXP in nonberry tissues. The relative expression was quantified in ‘Gewurztraminer 11-18 Gm’ (black bars) and ‘White Riesling 239-34 Gm’ (gray bars). Sampled tissues were inflorescences at 4 weeks (I1) and 2 weeks (I2) before flowering and at full bloom (I3), leaves at the ages of approximately 1 week (L1), 3 weeks (L2), and 5 weeks (L3), and roots (R). Mean values + sd of three independent experiments are shown. o.o.r., Out of range.

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 skins (exocarp) of five different cultivars and clones. Mean values ± sd of three independent experiments are shown. o.o.r., Out of range.

Figure 5.

Relative specific activity (%) of VvGT14a (A), VvGT15a to VvGT15c (B), and VvGT16 (C) proteins from grape toward putative substrates as determined by radiochemical analysis with UDP-[¹⁴C]Glc. Relative activities refer to the highest levels of extractable radioactivity that were measured for the conversion of geraniol (100%) in the case of VvGT14a and VvGT15a to VvGT15c (order of columns is 15c, 15b, and 15a) and benzyl alcohol (100%) in the case of VvGT16. Data for two biological and two technical replicates are shown. Black and white bars represent monoterpenoids and nonmonoterpenoids, respectively.

Figure 6.

Detection of monoterpenyl β-d-glucosides as products of VvGT14a, VvGT15a, and VvGT16 by ESI-HPLC-MS/MS. HPLC-MS/MS analysis of citronellyl β-d-glucoside (A), geranyl β-d-glucoside (B), neryl β-d-glucoside (C), and linaloyl β-d-glucoside (D) formed by VvGT14a, VvGT15a, and VvGT16 and a mixture of synthesized monoterpenyl β-d-glucosides (E) is shown. Chromatograms display an overlay of single-product measurements, and traces show the total ion current of the characteristic transitions (see “Materials and Methods”). Gaussian smoothing was partly applied. The poor peak shapes of geranyl glucoside (B) and neryl glucoside (C) are probably caused by overloading of the chromatographic column.

Figure 7.

Enantioselectivity of VvGT14a and VvGT15a determined by chiral-phase SPME-GC-MS analysis of citronellol and linalool. A, A racemic mixture of R,S-citronellol was used as a substrate for VvGT14a and VvGT15a, and enantiomerically pure R-citronellol was used as a reference. B and C, Racemic citronellol is released by acid-catalyzed hydrolysis of citronellyl β-d-glucoside formed by VvGT14a and VvGT15a. Signals labeled with × are hydrolysis by-products. Chromatograms are shown in selected ion monitoring mode by using the characteristic ion traces m/z 69, 81, and 123 for citronellol. D, A racemic mixture of R,S-linalool was used as a substrate for VvGT14a. E, Enantiomerically pure R-linalool was used as reference material. F, A slight preference for R-linalool is revealed after enzymatic hydrolysis with AR 2000. Chromatograms are shown in selected ion monitoring mode (m/z 71 and 93).

Figure 8.

Targeted functional screening of small-molecule glucosyltransferases by means of aglycone libraries prepared from different plant tissues.

Figure 9.

Functional screening of VvGT14a, VvGT15a, and VvGT16. A, Radio-TLC analysis of products formed by VvGT14a (lanes 1–3), VvGT15a (lanes 4–6), and VvGT16 (lanes 7–9) after incubation with the aglycone library obtained from grape berries of ‘Gewurztraminer 11-18 Gm’ (lanes 1, 4, and 7). Positive control was geraniol (lanes 2, 5, and 8) and negative control was no acceptor molecule (lanes 3 and 6); UDP [¹⁴C]Glc (lane 9) was approximately 3,000 dpm. The plates were analyzed by digital autoradiography. The products formed from citronellol, geraniol, and nerol were verified by LC-MS analysis. B, GC-MS analysis (total ion chromatogram) of volatiles that were enzymatically released from glucosides that were formed by incubation of an aglycone library obtained from grape berries of ‘Gewurztraminer 11-18 Gm’ with UDP-Glc and VvGT14 (gray) or heat-inactivated VvGT14a (black) as a control. Peaks are as follows: 1, linalool; 2, citronellol; 3, nerol; 4, geraniol; 5, benzyl alcohol; and 6, phenylethanol. For response factors of volatiles (0.52–1.90) toward geraniol, see Supplemental Table S4. Thus, even when response factors are taken into account, geraniol is the major product that is released.