Functional divergence of cshalcone synthase genes RcCHS in regulating anthocyanin biosynthesis and stress responses...

RcCHSa is the primary driver of anthocyanin accumulation in rose petals, while RcCHSb and RcCHSd are stress‑inducible paralogs expressed in vegetative tissues. RcCHSc contributes to both anthocyanin and flavonol biosynthesis, revealing functional divergence among the four rose CHS genes. Rose (Rosa spp.) flower color is primarily determined by anthocyanins, but the research on enzymes related to anthocyanin biosynthesis in roses remains insufficient. The rose genome contains four chalcone synthase genes (RcCHSa, RcCHSb, RcCHSc, and RcCHSd). Expression analysis revealed that RcCHSa is significantly highly expressed in red petals, whereas RcCHSb and RcCHSd primarily function in vegetative organs. RcCHSa, RcCHSb, and RcCHSd are highly linked on the chromosome and share high amino acid sequence identity. RcCHSc shows sequence divergence but conserves the critical catalytic residues. Functional validation demonstrated that transient overexpression of any RcCHS gene promoted anthocyanin accumulation, with RcCHSa being the most effective. Virus-induced gene silencing (VIGS) further confirmed that RcCHSa is the major contributor to petal anthocyanin accumulation. Metabolic analysis indicated that RcCHSa significantly enhances anthocyanin synthesis, while RcCHSc strongly promotes both anthocyanin and flavonol metabolism. Stress response studies showed that RcCHSa, RcCHSb, and RcCHSd are significantly induced by various stresses, with RcCHSd exhibiting the strongest response. In contrast, RcCHSc shows almost no response, potentially associated with the presence of numerous enhancer elements in the RcCHSd promoter and potential methylation regions in the RcCHSc promoter. In conclusion, this study reveals distinct functional divergence among the four RcCHS genes in rose, concerning anthocyanin biosynthesis and stress responses.