Anthocyanin accumulation underlies petal blotch coloration in Rhododendron rex and its medicinal potential

Rhododendrons are prominent indigenous plants recognized for their various applications owing to the ornamental, edible and medicinal properties. Among their diverse traits, the petal blotches significantly contribute to their ornamental value. However, the components and underlying molecular mechanisms regulating rhododendron blotch coloration remain poorly elucidated. In this study, we performed integrative metabolomic and transcriptomic analyses to elucidate the anthocyanin biosynthesis pathway in Rhododendron rex, which is differentially activated between the contrasting colored tissues of the petals (blotches versus backgrounds). Flavonoids were identified as the most abundant metabolites in R. rex petals; anthocyanins such as pelargonidin, cyanidin 3,5-di-O-glucoside (cyanin), cyanidin 3-O-glucoside (chrysanthemin), and delphinidin were significantly enriched in the blotch regions when compared to the background counterparts. These findings indicate that anthocyanins primarily contributed to blotch pigmentation. Notably, pelargonidin derivatives with antioxidant properties, which are rare in wild rhododendron flowers, were identified in R. rex petals, indicating the potential pharmaceutical value of R. rex. Transcriptomic data revealed that the upregulation of CHS, CHI, F3′5′H, and DFR, alongside the downregulation of HCT and FLS, promoted anthocyanin accumulation in the petal blotches. Furthermore, the combined regulation of flavonoid and lignin biosynthesis by CHS and HCT, as well as that of anthocyanin and flavonols by DFR and FLS, demonstrates that the metabolic balance is maintained through the regulation of the structural gene expression in response to competition for shared precursors involved in the biosynthesis of some secondary metabolites in plants. Moreover, key transcription factors, including bHLH, MYB, WRKY, and bZIP, are correlated with the regulation of anthocyanin biosynthesis. This study provides novel insights into the blotch formation of petals in woody plants by revealing unique metabolites and regulatory pathways in R. rex; moreover, these study outcomes establish a foundation for further research on the medicinal constituents and therapeutic potential of R. rex.