The PmRDUF1-PmMYB30-PmLAS Regulatory Module Is Involved in Terpenoid Biosynthesis of Pinus massoniana.

Abstract

The resin of masson pine (Pinus massoniana L.) exhibits considerable economic value and primarily consists of diterpenoid resin acids. However, the upstream regulation of the key diterpene biosynthetic gene, PmLAS, remains largely unclear. In the present study, through yeast one-hybrid screening, three PmMYB transcription factors were identified: PmMYB30, PmMYB68, and PmMYB74. Subsequent verification through dual-luciferase assays substantiated that these transcription factors interact with the PmLAS promoter, thereby facilitating its transcriptional activation. Subcellular localization analysis indicated that all three PmMYBs are localized in the nucleus, and transcriptional activation assays in yeast further revealed that their C-terminal regions are responsible for the transcriptional activation activity. Meanwhile, the transcript levels of three PmMYBs exhibited a significant positive correlation with resin yield in the xylem of 12-year-old masson pine trees. Moreover, the heterologous overexpression of these genes in tobacco resulted in a growth-inhibiting phenotype, a marked increase in leaf glandular trichome numbers, and an elevated proportion of diterpenoids compared to wild-type tobacco. To further elucidate the molecular regulation of the PmLAS gene by the transcription factor PmMYB30, yeast two-hybrid library screening was performed to screen its interacting proteins. A RING-type E3 ubiquitin ligase was identified from the screening results and designated PmRDUF1. Subsequent luciferase complementation imaging assays provided further evidence supporting the interaction between PmRDUF1 and PmMYB30. Furthermore, dual-luciferase reporter assays demonstrated that this interaction suppresses the ability of PmMYB30 to activate the expression of PmLAS, forming a PmRDUF1-PmMYB30-PmLAS regulatory module. Collectively, our findings suggest that a complex transcriptional network governs diterpenoid biosynthesis in masson pine, offering new perspectives on the regulation of terpenoid metabolism.