Identification of putative genes for caffeoylated flavonoid glycoside biosynthesis in Pseudognaphalium affine

Pseudognaphalium affine (D. Don) Anderberg, commonly found in East Asia, has extensive applications as both a traditional medicine and a vegetable in China. The caffeoylated flavonoid glycosides produced by P. affine exhibit remarkable anti-complement activities. Although these compounds have potential therapeutic value, the biosynthetic pathway responsible for their production remains largely unknown. To elucidate the key catalytic steps involved in caffeoylated flavonoid glycoside biosynthesis, we conducted a comprehensive analysis of the full-length transcriptome of P. affine. Further phylogenetic tree analysis predicted potential UDP glycosyltransferase (UGT) and BAHD acyltransferase (BAHD-AT) related with caffeoylated flavonoid glycoside biosynthesis. Subsequently, enzyme assay led to the discovery of PaUGT23 as a key enzyme responsible for the glycosylation of hydroxy groups in flavonoids, resulting in the formation of luteolin-4'-O-glucoside, luteolin-7-O-glucoside, quercetin-4'-O-glucoside, quercetin-7-O-glucoside, and apigenin-7-O-glucoside, while PaBAHD21 was found to catalyze the caffeoylation of flavonoid glycosides, resulting in the formation of luteolin 4'-O-β-D-(6″-E-caffeoyl)-glucopyranoside, quercetin 4'-O-β-D-(6″-E-caffeoyl)-glucopyranoside, apigenin 4'-O-β-D-(6″-E-caffeoyl)-glucopyranoside and apigenin 7-O-β-D-(6″-E-caffeoyl)-glucopyranoside. Moreover, their catalytic activities were verified in vivo by transient transfection experiment. This study presents the first comprehensive analysis of the full-length transcriptome in P. affine, providing significant insights into the biosynthesis and accumulation mechanisms of bioactive caffeoylated flavonoid glycosides.