Enzymatic basis for stepwise <i>C</i> ‐glycosylation in the formation of flavonoid di‐ <i>C</i> ‐glycosides in sacred lotus ( <i>Nelumbo nucifera</i> Gaertn.)

Lotus plumule, the embryo of seed sacred lotus (Nelumbo nucifera), contains a high accumulation secondary metabolites including flavonoids and possesses important pharmaceutical value. Flavonoid C-glycosides, which accumulate exclusively in have attracted considerable attention recent decades due to their unique chemical structure special bioactivities. As well as mono-C-glycosides, plumule also accumulates various kinds di-C-glycosides by mechanisms are yet unclear. In this study we identified two C-glycosyltransferase (CGT) genes mining genome data provide vitro planta evidence that these enzymes (NnCGT1 NnCGT2, designated UGT708N1 UGT708N2, respectively) exhibit CGT activity. Recombinant UGT708N2 can C-glycosylate 2-hydroxyflavanones 2-hydroxynaringenin C-glucoside, forming flavone mono-C-glycosides di-C-glycosides, respectively, after dehydration. addition, above reactions were successfully catalysed cell-free extracts from tobacco leaves transiently expressing NnCGT1 or NnCGT2. Finally, enzyme assays using suggested (vicenin-1, vicenin-3, schaftoside isoschaftoside) biosynthesized through sequentially C-glucosylating C-arabinosylating/C-xylosylating 2-hydroxynaringenin. Taken together, our results novel insights into biosynthesis flavonoid proposing new biosynthetic pathway for C-glycosides N. nucifera identifying uridine diphosphate-glycosyltransferase (UGT708N2) specifically catalyses second glycsosylation, C-arabinosylating C-xylosylating C-glucoside.