Synergistic actions of 3 MYB transcription factors underpin blotch formation in tree peony.

Blotches in floral organs attract pollinators and promote pollination success. Tree peony (Paeonia suffruticosa Andr.) is an internationally renowned cut flower with extremely high ornamental and economic value. Blotch formation on P. suffruticosa petals is predominantly attributed to anthocyanin accumulation. However, the endogenous regulation of blotch formation in P. suffruticosa remains elusive. Here, we identified the regulatory modules governing anthocyanin-mediated blotch formation in P. suffruticosa petals, which involves the transcription factors PsMYB308, PsMYBPA2, and PsMYB21. PsMYBPA2 activated PsF3H expression to provide sufficient precursor substrate for anthocyanin biosynthesis. PsMYB21 activated both PsF3H and PsFLS expressions and promoted flavonol biosynthesis. The significantly high expression of PsMYB21 in nonblotch regions inhibited blotch formation by competing for anthocyanin biosynthesis substrates, while conversely, its low expression in the blotch region promoted blotch formation. PsMYB308 inhibited PsDFR and PsMYBPA2 expressions to directly prevent anthocyanin-mediated blotch formation. Notably, a smaller blotch area, decreased anthocyanin content, and inhibition of anthocyanin structural gene expression were observed in PsMYBPA2-silenced petals, while the opposite phenotypes were observed in PsMYB308-silenced and PsMYB21-silenced petals. Additionally, PsMYBPA2 and PsMYB308 interacted with PsbHLH1-3, and their regulatory intensity on target genes was synergistically regulated by the PsMYBPA2-PsbHLH1-3 and PsMYB308-PsbHLH1-3 complexes. PsMYB308 also competitively bound to PsbHLH1-3 with PsMYBPA2 to fine-tune the regulatory network to prevent overaccumulation of anthocyanin in blotch regions. Overall, our study uncovers a complex R2R3-MYB transcriptional regulatory network that governs anthocyanin-mediated blotch formation in P. suffruticosa petals, providing insights into the molecular mechanisms underlying blotch formation in P. suffruticosa.