Characterization of three novel R2R3-MYB transcription factors PrMYBi(1–3) repressing the anthocyanin biosynthesis in tree peony

Petal blotch is a prevalent pigmentation pattern observed in the Xibei tree peony ( Paeonia rockii ), possessing significant aesthetic value and playing a crucial role in the species' reproduction and fitness. Despite years of research, deciphering the molecular mechanisms underlying blotch formation remains challenging. As is well known, floral pigmentation is frequently associated with the familiar R2R3-MYB transcription factors. The key MYB anthocyanin activators of P. rockii ‘Shu Sheng Peng Mo’ were previously reported in our preceding study. In this study, we identified and characterized three R2R3-MYBs, PrMYBi1, PrMYBi2, and PrMYBi3, which belong to subgroup 4 (SG4) and play repressor roles in anthocyanin biosynthesis. A quantitative real-time PCR (qRT-PCR) assay indicated that the expression of PrMYBi1 and PrMYBi3 gradually increased during flowering development and was substantially up-regulated in non-blotch compared to blotch. Yeast one-hybrid and dual-luciferase assays demonstrated that PrMYBi(1–3) directly target the anthocyanin structural genes and repress their transcription. The genetic transformation of tobacco demonstrated that the overexpression of PrMYBi(1–3) decreased anthocyanin accumulation in flowers, with PrMYBi1 serving as the most effective repressor. Our results revealed that SG4 R2R3-MYBs negatively regulate the anthocyanin pathway in P. rockii conservatively, and we provide the definite members. These findings will advance future research to unravel the mystery of blotch pattern formation.