The RrMYB4-RrTPS31 module regulates the synthesis of sesquiterpenoids in Rosarugosa

Rosa rugosa (R. rugosa) is an economically important plant valued for its vibrant floral coloration, intense fragrance, and diverse bioactive compounds, exhibiting significant ornamental, edible, aromatic, and medicinal applications. Terpene compounds are the main components of the rose fragrance. Therefore, terpene synthases (TPSs) plays an important role in the synthesis of the rose fragrance. In this study, we identified 53 RrTPSs through genome-wide analysis in R. rugosa and functionally characterized those with high tissue-specific expression. Among these, we confirmed three monoterpene synthases (RrTPS17, RrTPS25, and RrTPS29), a bifunctional synthase (RrTPS38), and a sesquiterpene synthase (RrTPS31). Subcellular localization revealed that RrTPS31, which is predominantly expressed in flowers, localizes to the cytoplasm. Promoter cis-element analysis identified RrMYB4 as a putative regulator of RrTPS31. and subsequent EMSA, Y1H, and Dual-LUC assays demonstrated that RrMYB4 directly binds to the RrTPS31 promoter and activates its expression. Furthermore, VIGS-mediated silencing of RrMYB4 in rose plants resulted in significantly reduced sesquiterpenoid content and correspondingly decreased RrTPS31 expression compared to controls. These findings provide new insights into the biosynthesis and transcriptional regulation of volatile terpenoids in R. rugosa.