The RcMYB308-RcEGL1 module regulates cyanidin accumulation by targeting the RcF3’H promoter in pink roses

BACKGROUND: Rose accounts for over one-third of the global cut flower industry, with pink varieties dominating the market. However, research on the mechanism for pink petal formation in rose is still insufficient. RESULTS: To elucidate the molecular mechanism of rose petal pigmentation, we performed metabolome and transcriptomics analyses of two rose progenies derived from the hybridization of Rosa chinensis var. Spontanea and 'Old Blush', which exhibit light pink (LP) and dark pink (DP) flowers, respectively. Metabolome analysis identified pelargonidin derivatives as major anthocyanins in DP, while cyanidin 3-O-coumaroylglucoside-5-O-glucoside (Cy3cG5G), as a key anthocyanins, was exclusively detected at S3 in DP, perfectly matching the formation timing of the dark pink phenotype. Transcriptomics data revealed differentially expressed genes involved in flavonoid and anthocyanin biosynthesis, corresponding to varying anthocyanin levels in petals, between the two roses. In addition, integrated analysis uncovered molecular networks regulating petal pigmentation and identified transcription factors RcMYB308 and RcMYB1b as key players in the accumulation of anthocyanins and flavonol derivatives. Here, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BIFC) assays demonstrated that these proteins form MYB-bHLH complexes with RcEGL1 to activate anthocyanin biosynthesis genes. The dual-luciferase (Dual-LUC) assays demonstrated that RcMYB308 activated RcF3'H, thereby bolstering anthocyanin accumulation, while RcMYB1b could bind to the RcF3'H promoter and exert a negative regulatory effect on RcF3'H. The overexpression and silencing of RcMYB308 in petals revealed that it facilitated anthocyanin accumulation by specifically regulating RcF3'H-mediated cyanidin synthesis, resulting in dark pink pigmentation. CONCLUSION: Collectively, our analysis of two pink rose progenies reveals that the RcMYB308-RcEGL1 module regulates petal color divergence during development via direct targeting of the RcF3'H promoter. These findings clarify key elements of the gene-metabolism network regulating rose petal coloration and provide a theoretical basis for rose flower color genetic improvement.