Screening and first validation of MYB transcription factors influencing the biosynthesis of (−)-pulegone monoterpene constituents in <i>Schizonepeta tenuifolia</i> Briq.

This study investigates the secondary metabolism regulatory mechanism of the Labiatae medicinal plant Schizonepeta tenuifolia Briq. The monoterpene synthase StLS catalyzes the manufacture of (−)-pulegone, a distinctive monoterpene active component of this species, and its expression level directly impacts secondary metabolite accumulation. Due to the limited knowledge of the StLS transcriptional regulatory network, this study employed a multi-omics combined analysis to investigate the role of the R2R3-MYB transcription factor in plant metabolic regulation. It was discovered that StMYB13 and StMYB76 are regulatory elements after a deep analysis of the Arabidopsis thaliana transcriptome database using bioinformatics screening. The yeast one-hybrid system (Y1H) showed that StMYB13/StMYB76 could specifically bind CAACGG in the StLS promoter region from −1,234 to −1,274 bp. Virus-induced gene silencing (VIGS) and transient overexpression were used to unveil the StMYB transcription factor's monoterpene metabolic pathway regulatory network. The transcription factor controlled the monoterpene synthase StLS's transcriptional activity and downstream biosynthetic enzyme-encoding gene expression. The silencing of StMYB genes led to a dramatic reduction (p < 0.001) in the expression of the pivotal gene StIPD, whereas the overexpression system caused a substantial elevation in its mRNA levels. Further analysis demonstrated that this regulatory process exhibits a distinct expression-level-dependent pattern: (−)-pulegone content was significantly elevated in overexpression lines, whereas no statistically significant difference in (−)-pulegone accumulation was observed in silenced groups. These findings revealed StMYBs' transcriptional regulation mechanism and provided molecular evidence for the analysis of S. tenuifolia's volatile oil synthesis nodes. This discovery deepened the monoterpene biosynthesis pathway and laid the theoretical groundwork for metabolic engineering and molecular-assisted breeding systems of S. tenuifolia.