Identification of UDP-rhamnosyltransferases and UDP-galactosyltransferase involved in flavonol glycosylation inMorella rubra

Abstract

Flavonol glycosides are health-promoting phytochemicals important for human nutrition and plant defence against environmental stresses. Glycosylation modification greatly enriches the diversity of flavonols. Morella rubra, a member of the Myricaceae, contains high amount of myricetin 3-O-rhamnoside (M3Rha), quercetin 3-O-rhamnoside (Q3Rha), and quercetin 3-O-galactoside (Q3Gal). In the present study, MrUGT78R1 and MrUGT78R2 were identified as two functional UDP-rhamnosyltransferases, while MrUGT78W1 was identified as a UDP-galactosyltransferase. Site-directed mutagenesis identified Pro143 and Asn386 as important residues for rhamnosyl transfer activity of MrUGT78R1 while these two corresponding positions in MrUGT78W1 (i.e. Ser147 and Asn370) also play important role for galactosyl transfer activity. Transient expression data for these three MrUGTs in Nicotiana benthamiana tested the function of MrUGT78R1 and MrUGT78R2 as rhamnosyltransferases and MrUGT78W1 as a galactosyltransferase in glycosylation of flavonols. This work enriches the diversity of UDP-rhamnosyltransferase in planta and identifies two amino acid positions important for both rhamnosyltransferase and galactosyltransferase.

Genome-wide analysis of UDP-glycosyltransferase gene family and identification of members involved in flavonoid glucosylation in Chinese bayberry (Morella rubra)

“…PpUGT78A2 in group F was identified as a flavonoid 3-O-glycosyltransferase involved in different glycosylation of anthocyanin and flavonol in P. persica (Cheng et al, 2014;Xie et al, 2022). And in M. rubra, four UGT members in group F, i.e., MrUGT78R1, MrUGT78R2, MrUGT78W1, and MrUFGT in the present study, were identified as flavonoid 3-O-glycosyltransferases involved in accumulation of diverse flavonoid glycosides (Ren et al, 2022).…”

“…Enzymatic activity assay was carried out as reported with some modifications ( Ren et al, 2022 ). Reactions were performed in a total volume of 100 μl mixture containing 0.1 M Tris–HCl buffer (pH 7.5), 1 mM sugar donors (UDP-Glc/UDP-Gal/UDP-Rha), 60 μM sugar acceptors (Q/C), and 1–2 μg recombinant proteins at 30°C for 20 min.…”

“…Enzyme products were detected at 350 nm for flavonol glycosides and at 520 nm for anthocyanins. Enzyme products were analyzed by LC-MS/MS as reported ( Ren et al, 2022 ).…”

“…Four-week-old N. benthamiana leaves were infiltrated with different combination mixtures. Flavonoid glycosides were analyzed by LC-MS/MS after 5 days infiltration as previous reported ( Ren et al, 2022 ). Data were collected from at least three independent N. benthamiana plants.…”

“…Among the UV-B induced MrUGTs, only four members belong to group F, i.e., MrUGT78R1, MrUGT78R2, MrUGT78W1, and MrUFGT (Supplementary Table 8). Recently, MrUGT78R1 and MrUGT78R2 were identified as UDP-rhamnosyltransferases while MrUGT78W1 was identified as UDP-galactosyltransferase involved in flavonol glycosylation in M. rubra by our group (Ren et al, 2022). Therefore, MrUFGT was chosen as one of potential candidate UGTs for flavonoid glucosylation.…”