Comprehensive investigation of the C2H2-type zinc finger protein transcription factor family in Aquilaria sinensis reveals the involvement of AsZFP9 in sesquiterpene biosynthesis.

Agarwood, a valuable resin produced by Aquilaria trees in response to environmental stresses, is characterized by its rich sesquiterpene content. However, the regulatory mechanisms underlying the sesquiterpene biosynthesis remain poorly understood. C2H2-type zinc finger proteins (C2H2-ZFPs) are key transcriptional regulators involved in plant growth, stress responses, and secondary metabolism. In this study, we identified 56 AsC2H2-ZFP genes in Aquilaria sinensis and systematically analyzed their subcellular localization, evolutionary relationships, conserved motifs and gene structures. Transcriptomic analysis revealed differential expression of 12 AsC2H2-ZFPs under agarwood inducer treatment, with their expression patterns significantly correlated with sesquiterpene biosynthesis-related genes. Notably, AsZFP9 was characterized as a nuclear-localized transcriptional activator, whose expression was markedly induced by wound and methyl jasmonate treatments. Transient overexpression of AsZFP9 in the stems of A. sinensis significantly increased sesquiterpene production and upregulated key biosynthetic genes. Investigations utilizing yeast one-hybrid, electrophoretic mobility shift assays, and dual-luciferase assays demonstrated that AsZFP9 directly binds to and activates the promoters of AsHMGR1 and AsTPS1, two critical genes in sesquiterpene biosynthesis. Our findings provide novel insights into the role of C2H2-ZFPs in A. sinensis and establish AsZFP9 as a key regulator of sesquiterpene accumulation and agarwood formation.