Identification and Functional Characterization of Two UDP-Glycosyltransferases Involved in Narcissoside Biosynthesis in <i>Anoectochilus roxburghii</i>

Flavonoid rutinosides, a group of bioactive compounds in <i>Anoectochilus roxburghii</i>, contribute greatly to the plant's beneficial effects on human health. However, the glycosylation mechanism of flavonoid rutinosides in <i>A. roxburghii</i> remains unclear. In this study, two efficient and selective glycosyltransferases, AUTG25 and AUTG23, involved in the biosynthesis of narcissoside, a major flavonoid rutinoside in <i>A. roxburghii</i>, were identified through transcriptome analysis and functional validation. AUTG25 could regioselectively catalyze 3-O-glucosylation of isorhamnetin to produce isorhamnetin 3-O-glucoside, while AUTG23 could further catalyze 6"-O-rhamnosylation to generate narcissoside. Both AUTG25 and AUTG23 exhibited high positional and sugar donor selectivities in the catalytic reaction. Homology modeling and site-directed mutagenesis showed that H20, E83, E385, and F143 in AUTG25 and E280, E89, D188, W327, D369, and Y191 in AUTG23 may be critical for their catalytic functions. Transient expression in <i><i>Nicotiana benthamiana</i></i> finally confirmed that AUTG25 possesses flavonol-3-O-glucosyltransferase activity and AUTG23 has flavonol-3-O-glucoside (1→6) rhamnosyltransferase activity. This study clarified and provided candidate UDP-dependent glycosyltransferase genes for narcissoside biosynthesis in <i>A. roxburghii</i>.