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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., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background and Aims: The tree peony (Paeonia suffruticosa Andr.) has been widely cultivated as a field plant, and petal blotch is one of its important traits, which not only promotes proliferation but also confers high ornamental value. However, the regulatory network controlling blotch formation remains elusive owing to the functional differences and limited conservation of transcriptional regulators in dicots., Methods: We performed phylogenetic analysis to identify MYB44-like transcription factors in P. suffruticosa blotched cultivar 'High noon' petals. A candidate MYB44-like transcription factor, PsMYB44, was analysed via expression pattern analysis, subcellular localization, target gene identification, gene silencing in P. suffruticosa petals and heterologous overexpression in tobacco., Key Results: A blotch formation-related MYB44-like transcription factor, PsMYB44, was cloned. The C-terminal of the PsMYB44 amino acid sequence had a complete C2 motif that affects anthocyanin biosynthesis, and PsMYB44 was clustered in the MYB44-like transcriptional repressor branch. PsMYB44 was located in the nucleus, and its spatial and temporal expression patterns were negatively correlated with blotch formation. Furthermore, a yeast one-hybrid assay showed that PsMYB44 could target the promoter of the late anthocyanin biosynthesis-related dihydroflavonol-4-reductase (DFR) gene, and a dual-luciferase assay demonstrated that PsMYB44 could repress PsDFR promoter activity. On the one hand, overexpression of PsMYB44 significantly faded the red colour of tobacco flowers and decreased the anthocyanin content by 42.3 % by downregulating the expression level of the tobacco NtDFR gene. On the other hand, PsMYB44-silenced P. suffruticosa petals had a redder blotch colour, which was attributed to the fact that silencing PsMYB44 redirected metabolic flux to the anthocyanin biosynthesis branch, thereby promoting more anthocyanin accumulation in the petal base., Conclusion: These results demonstrated that PsMYB44 negatively regulated the biosynthesis of anthocyanin by directly binding to the PsDFR promoter and subsequently inhibiting blotch formation, which helped to elucidate the molecular regulatory network of anthocyanin-mediated blotch formation in plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Petal blotches are commonly observed in many angiosperm families and not only influence plant-pollinator interactions but also confer high ornamental value. Tree peony (Paeonia suffruticosa Andr.) is an important cut flower worldwide, but few studies have focused on its blotch formation. In this study, anthocyanins were found to be the pigment basis for blotch formation of P. suffruticosa, and peonidin-3,5-di-O-glucoside (Pn3G5G) was the most important component of anthocyanins, while the dihydroflavonol-4-reductase gene was the key factor contributing to blotch formation. Then, the R2R3-myeloblastosis (MYB) transcription factor PsMYB30 belonging to subgroup 1 was proven as a positive anthocyanin regulator with transcriptional activation and nuclear expression. Furthermore, silencing PsMYB30 in P. suffruticosa petals reduced blotch size by 37.9%, faded blotch color and decreased anthocyanin and Pn3G5G content by 23.6% and 32.9%, respectively. Overexpressing PsMYB30 increased anthocyanin content by 14.5-fold in tobacco petals. In addition, yeast one-hybrid assays, dual-luciferase assays and electrophoretic mobility shift assays confirmed that PsMYB30 could bind to the promoter of the anthocyanin synthase (ANS) gene and enhance its expression. Altogether, a novel MYB transcription factor, PsMYB30, was identified to promote petal blotch formation by activating the expression of PsANS involved in anthocyanin biosynthesis, which provide new insights for petal blotch formation in plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)