The bZIP24-bZIP53 transcription factor complex regulates kaempferol 3-O-rutinoside biosynthesis in Rubus chingii by targeting 1,6-rhamnosyltransferase

Rubus chingii, a plant celebrated for its dual role as medicine and food, contains kaempferol 3-O-rutinoside (K3R) as a principal bioactive flavonol glycoside. Yet, the K3R biosynthetic pathway in this species, particularly the crucial final glycosylation step, remains poorly characterized. Our investigation identified 172 UGTs within the R. chingii genome, pinpointing 18 candidate UGTs potentially involved in K3R synthesis. Phylogenetic and expression analyses revealed that three flavonol 3-glucosyltransferases (RcUGT87, RcUGT89, RcUGT169) and eight flavonol 1,6-rhamnosyltransferases (RcUGT7, RcUGT13, RcUGT27, RcUGT72, RcUGT73, RcUGT97, RcUGT166, RcUGT160) exhibited high expression in green fruits. Enzyme activity assays confirmed RcUGT169 as the most active 3GlcT, efficiently converting kaempferol to K3G, and identified RcUGT27 as the key 1,6RhaT, synthesizing K3R from K3G. Molecular docking highlighted Asp-210, Glu-203, and Leu-204 as critical residues underpinning RcUGT27 activity. Y1H and dual luciferase assays demonstrated that transcription factors (TFs) RcWHY1 and RcbZIP24/44/53 directly bind the RcUGT27 promoter, driving its expression; notably, RcbZIP24 and RcbZIP53 form a complex to amplify this regulation. Hormone treatments showed ABA and MeJA significantly induce RcUGT27 expression through these TFs. This study establishes a comprehensive regulatory network governing K3R biosynthesis in R. chingii, offering valuable insights to guide future molecular breeding strategies and industrial production.