Flower color is an essential biological and ornamental trait in plants. Paeonia rockii (flare tree peony, FTP) exhibits diverse flower colors, characterized by a distinctive basal flare in petals, which enhances its ornamental and ecological value. However, while previous research has mainly focused on flare formation, the regulatory mechanisms controlling the background color of petals remain unclear. This study identifies a novel regulatory module governing petal background coloration in FTP. Within this module, PrMYB75a acts as the central regulator to promote anthocyanin accumulation, as evidenced by stable transformation in Arabidopsis thaliana and tobacco (Nicotiana tabacum), as well as virus-induced gene silencing in FTP. Furthermore, yeast one-hybrid, dual-luciferase reporter, and electrophoretic mobility shift assays collectively demonstrated that PrMYB75a directly activates two key anthocyanin structural genes, PrCHS1 and PrANS, by interacting with MYB-binding sites nearest to the ATG start codon in their promoters. Additionally, we identified an upstream regulator, PrFRS2, which activates both PrMYB75a and PrANS by binding to the FAR1/FHY3-binding sites in their promoters. Modulation of PrFRS2 expression levels through gene silencing and overexpression resulted in alterations in flower pigmentation in both FTP and tobacco. In summary, within the PrFRS2-PrMYB75a module, PrFRS2 indirectly activates PrCHS1 and PrANS by regulating PrMYB75a, or directly activates PrANS, leading to anthocyanin accumulation in FTP purple petals. This module represents a novel regulatory mechanism of petal background coloration in FTP, providing new perspectives on color variation in flowering plants and offering genetic resources for the improvement of the flower color trait in tree peonies.

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