Geraniol contributes significantly to the floral scent of herbaceous peony (Paeonia lactiflora) and is abundant in fragrant cultivars. However, the regulatory mechanism of geraniol biosynthesis in herbaceous peony remains unclear. In this study, we identified a transcriptional regulatory complex (PlMYB73–PlMYB70–PlMYB108) that cooperatively regulated geraniol biosynthesis in herbaceous peony. The three MYB members were identified through correlation analysis between geraniol content and gene expression profiles in 17 herbaceous peony cultivars. Transient overexpression and gene silencing experiments revealed that PlMYB73, PlMYB108, and PlMYB70 positively regulated PlTPS1 expression and geraniol accumulation. PlMYB108 and PlMYB70 directly upregulate PlTPS1 by binding to the TAACCA and CAACTG motifs, respectively, as demonstrated by yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays. Although PlMYB73 did not directly bind to the PlTPS1 promoter, yeast two-hybrid, bimolecular fluorescence complementation, luciferase complementation imaging, and dual-luciferase assays revealed its interaction with PlMYB70 in the nucleus, resulting in synergistic activation of PlTPS1. PlMYB108 was also found to interact with PlMYB70. The three MYB transcription factors formed the PlMYB73–PlMYB70–PlMYB108 complex. Gene co-overexpression and co-silencing experiments demonstrated that the complex significantly enhanced geraniol biosynthesis. In conclusion, our research provides novel insights into the molecular mechanism by which transcription factors cooperatively regulate geraniol biosynthesis.

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