Molecular Insights into RrMYB5 Promote Flavonoid Accumulation in Rosa roxburghii

Rosa roxburghii Tratt is characterized by its abundant flavonoid content. However, the mechanisms underlying MYB-mediated regulation of flavonoid biosynthesis in R. roxburghii remain largely unknown. In this study, we found that flavonoid accumulation was markedly higher in fruits. By WGCNA of differentially expressed genes (DEGs) with flavonoid accumulation profiles, we identified RrMYB5 as a key regulatory factor in flavonoid biosynthesis of R. roxburghii. RrMYB5 contained characteristic R2R3 domains and a conserved PA1-type motif YEEYLQALL. It was localized exclusively to the nucleus. The qRT-PCR analysis showed that RrMYB5 was constitutively expressed, with peak expression occurring at the rapid fruit expansion stage. The total soluble flavonoid accumulation in R. roxburghii calli was substantially increased by overexpression of RrMYB5. Further LC-MS-based metabolomic analysis revealed significant enrichment of flavonols and proanthocyanidins in RrMYB5-OE calli. Consistently, the transcript levels of RrLAR (Rr404249) and RrANR (Rr300417) were markedly elevated in RrMYB5-overexpressing calli. Moreover, DAP-seq analysis suggested that RrMYB5 might directly bind the promoters of flavonoid structural genes. Subsequent yeast one-hybrid and dual-luciferase assays confirmed that RrFLS (Rr101307) and RrF3H (Rr306546) were direct downstream targets of RrMYB5. These findings indicated that RrMYB5 promoted the expression of flavonol and flavanol biosynthetic genes through different regulatory routes. Thus, our study elucidates the regulatory role of RrMYB5 in flavonoid biosynthesis and provides a valuable molecular target for improving the quality and utilization of R. roxburghii.