A R2R3-MYB, VwMYB1, regulates anthocyanin biosynthesis in pansy petal blotch via transcriptional activation of VwF3’5’H

Anthocyanins play a pivotal role in determining the visual characteristics of ornamental plants. To elucidate the molecular basis of the distinctive “blotch” formation in pansy ( Viola × wittrockiana ), this study employed an integrated approach that combines targeted metabolomics and comparative transcriptomics. Comparative analysis of blotched versus non-blotched petal regions identified VwMYB1 , a differentially expressed R2R3-MYB transcription factor, as a key candidate regulator. Functional validation revealed that the heterologous overexpression of VwMYB1 in tobacco enhanced floral pigmentation, while its transient overexpression in pansy petals induced localized dark blotches, thereby confirming its role in promoting pigmentation. Targeted metabolomic profiling demonstrated that the blotch phenotype is primarily attributed to the substantial and specific accumulation of delphinidin-type anthocyanins. Given that F3’5’H is the pivotal branch-point enzyme for delphinidin biosynthesis, the regulatory interaction between VwMYB1 and VwF3’5’H was further examined. Yeast one-hybrid and dual-luciferase reporter assays confirmed that VwMYB1 directly binds to the promoter of VwF3’5’H and activates its expression. This study not only illustrates a streamlined pipeline from transcriptome-based gene discovery to functional and metabolic validation but also uncovers the core regulatory mechanism by which VwMYB1 governs delphinidin-based blotch formation through the direct transcriptional activation of VwF3’5’H , thereby offering both mechanistic insight and a practical genetic tool for precision breeding in flower color modification.