Abstract: INTRODUCTION: The tree peony (Paeonia × suffruticosa Andrews) is a renowned ornamental plant in China, whose floral color- a key determinant of its economic and aesthetic value- is predominantly governed by flavonoid pigments.  RATIONALE: To systematically elucidate the molecular basis of flavonoid regulation in tree peonies, this study focused on the R2R3-MYB transcription factor family, specifically members of the SG4, SG5, SG6, SG7, and SG20 subgroups, which are known to be involved in flavonoid biosynthesis. We integrated genomic resources from P. ostii and P. ludlowii to conduct a comprehensive analysis of their evolutionary relationships, amino acid sequence features, and intron insertion patterns.  RESULTS: The flavonoid-related R2R3-MYB members in tree peonies exhibited remarkable evolutionary diversity, which likely underpins their functional divergence. A transient expression assay in petunia petals revealed distinct regulatory roles for different subgroups. SG6 members primarily regulated the anthocyanin biosynthetic pathway centered on ANS; SG5 members were mainly involved in proanthocyanidin biosynthesis via ANR; and SG7 members predominantly influenced flavonol synthesis by modulating FLS expression. Notably, MYBs-SG4a, one member of the SG4 subgroup, retained the ancestral capacity to positively regulate flavonoid synthesis. Furthermore, we identified both competitive and collaborative interactions among different R2R3-MYB members, which collectively fine-tune the expression of structural genes in the flavonoid pathway.  CONCLUSION: This study provides novel insights into the evolutionary and functional diversification of the R2R3-MYB family in plants. The findings establish a foundational framework for understanding the complex regulatory network of flavonoid biosynthesis in tree peonies and offer valuable theoretical guidance for molecular breeding aimed at floral color improvement.

Key words: tree peony, R2R3-MYB, flavonoids, flower color