The ZF-HD1/9 target CdSQS to promote squalene biosynthesis in Camellia drupifera

Camellia drupifera is an important economic oil crop and its quality is closely linked to its triterpenoid content, notably squalene, which exhibits significant potential in pharmaceutical applications. To elucidate the regulatory mechanism of squalene biosynthesis in C. drupifera with a view to improving its quality and efficiency of production, we constructed a methyl-jasmonate-induced cDNA yeast library. Using the promoter of squalene synthase (SQS) as bait, two zinc finger-homeodomain transcription factors, ZF-HD1 and ZF-HD9, were identified. Under methyl-jasmonate treatment, the expression of ZF-HD1/9, MYC2, and SQS was up-regulated, promoting squalene synthesis. Dual-luciferase reporter and EMSAs confirmed that ZF-HD1/9 directly bind to and activate the SQS promoter. Nanoparticle-mediated dsRNAi demonstrated that knock down of ZF-HD1/9 down-regulates key structural genes and significantly reduces the levels of squalene, 2,3-oxidosqualene, and teasaponins. By establishing an Agrobacterium rhizogenes-mediated hairy-root transformation system in C. drupifera, we confirmed the role of ZF-HD1/9 as positive regulators of squalene synthesis. This study is the first to identify ZF-HD1/9 as novel transcription factors involved in the complex regulatory network of squalene biosynthesis in C. drupifera, and expands the functional scope of this transcription factor family in plants. The hairy-root induction system that we have developed also provides a foundational platform for gene functional studies and high-quality germplasm development in this species.