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

As an important economic oil crop, the quality of Camellia drupifera (C. drupifera) 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 and to support its quality improvement and efficient production, this study constructed a MeJA-induced cDNA yeast library of C. drupifera. Using the promoter of squalene synthase (CdSQS) as bait, two zinc finger-homeodomain transcription factors, ZF-HD1 and ZF-HD9, were identified. Under MeJA treatment, the expression of ZF-HD1/9, MYC2, and SQS was upregulated, promoting squalene synthesis. Dual-luciferase reporter (DLR) and electrophoretic mobility shift assays (EMSA) confirmed that ZF-HD1/9 directly bind to and activate the CdSQS promoter. Using nanoparticle-mediated dsRNAi transient silencing, we demonstrated that knockdown of ZF-HD1/9 downregulates key structural genes(SQS, SQE, bAS, CYP716A17, and UGT73B2) and significantly reduces the levels of squalene, 2,3-oxidosqualene, and teasaponins. Furthermore, by establishing an Agrobacterium rhizogenes-mediated hairy root transformation system in C. drupifera, we conducted functional analyses of ZF-HD1/9, confirming their role 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, expanding the functional scope of this transcription factor family in plants. The hairy root induction system developed herein also provides a foundational platform for gene functional studies and high-quality germplasm development in C. drupifera.