Integrative Multiomics to Discover Insights for Rutin Biosynthesis in Wolfberry.

Comparative metabolite profiling of different wolfberry cultivars showed that rutin and ascorbic acid 2-glucoside (AA-2βG) accumulation were negatively correlated, with rutin predominantly enriched in leaves and green fruits where AA-2βG was absent. Transcriptomic and proteomic analyses showed expression styles of key enzymes in the rutin biosynthetic pathway. Weighted gene coexpression network analysis and phylogenetic analysis identified five UDP-dependent glycosyltransferases (UGTs) that catalyze two glycosylation steps of rutin, including a highly conserved sterol UGT. Enzymatic assays confirmed that LbUGT41 catalyzes the first glucosylation from quercetin to isoquercitrin, while LbUGT60/80 catalyzes the subsequent rhamnosylation to rutin. LbUGT41 exhibited UDP-dependent hydrolytic activity toward isoquercitrin. Site-directed mutagenesis by AlphaFold-assisted detection indicated that H18 and D365 confer hydrolytic function to LbUGT41, whereas G174 and F298 are essential for LbUGT80-mediated rhamnosylation. These findings uncover a distinct rutin biosynthetic mechanism involving a bifunctional glycosyltransferase/hydrolase in wolfberry, offering molecular insights for cultivar improvement and whole-plant utilization.