Four PpMYB genes govern alternative transcription of the coumarin biosynthesis branch in a synergistic manner

R2R3-type MYB transcription factors (TFs) play a crucial role in regulating plant secondary metabolism. However, the transcriptional regulatory mechanisms underlying coumarin biosynthesis in the medicinal industrial crop Peucedanum praeruptorum Dunn remain elusive. Here, we investigated the interaction of four MYB genes from P. praeruptorum and their interactions with eleven coumarin biosynthetic genes in vivo and in vitro . AlphaFold3 and molecular docking initially predicted the potential interactions between coumarin biosynthetic genes and main MYB TFs. Subcellular localization revealed that all PpMYB genes were localized in the nucleus, while key biosynthetic enzymes showed distinct distributions in the endoplasmic reticulum, plasma membrane, chloroplast and cytoplasm. Yeast one-hybrid assays revealed the physical interactions between four MYB TFs and the target genes. Electrophoretic mobility shift assays further confirmed the strong in vitro binding of PpMYB3 to S8H and PT-6 , PpMYB69 to F6H , PpMYB82 to PT-6 and F6H , and PpMYB103 to S8H . Dual luciferase reporter assay revealed that PpMYB103 significantly inhibited the transcription of S8H , while PpMYB3 and PpMYB82 activated the expression of PT-6 by binding to its promoter. HPLC-MS analysis revealed significant variations in coumarin content among the Arabidopsis transgenic lines with distinct accumulation patterns observed, where the total content of coumarins was consistently higher in all transgenic lines. The qRT-PCR results revealed that PpMYB3 overexpression upregulated F6H , COSY-1 and BMT but downregulated COSY-2 , PT-6 , PS and UGT-1 . PpMYB69 overexpression decreased transcript levels of COSY-2 , PT-6 , PS and UGT-1 . Overexpression of PpMYB82 enhanced most tested genes except UGT-1 , and PpMYB103 broadly activated the majority of biosynthetic genes with only UGT-1 being inhibited. This study establishes a comprehensive regulatory network of PpMYBs governing coumarin biosynthesis, providing valuable gene targets for quality improvement and molecular breeding of P. praeruptorum and related industrial medicinal crops. • AlphaFold3 and molecular docking predicted the potential interactions between coumarin biosynthetic genes and main PpMYB genes. • PpMYB3 activates the expression of S8H, PT-6, F6H, and BMT, while inhibiting PS and UGT-1. PpMYB69 activates F6H and BMT, but represses PT-6, PS, and UGT-1. • PpMYB82 and PpMYB103 both activate PT-6, F6H, BMT, and PS, while repressing UGT-1.