Objective The rhizomes and flowers of Hedychium coronarium are rich in terpenoid essential oils with diverse bioactivities, including anti-inflammatory, analgesic, and antimicrobial properties. Therefore, this study aimed to elucidate the roles of HcTPS9 and HcTPS6 in terpenoid biosynthesis and to uncover the mechanistic basis underlying their functional divergence, thereby providing a theoretical framework for understanding the metabolic network of essential oils in H. coronarium and exploiting functional terpene resources.

Method The HcTPS9 gene, a homolog of HcTPS6 with known enzyme catalytic function, was cloned. The catalytic activity of HcTPS9 was verified through prokaryotic expression and in vitro enzymatic assays. In addition, phylogenetic analysis, amino acid sequence alignment, subcellular localization, and gene expression analysis were performed to characterize HcTPS6 and HcTPS9.

Result Both genes cluster within the TPS-b subfamily and contain conserved motifs typical of monoterpene synthases, sharing 90.6% amino acid sequence identity. In vitro assays showed that HcTPS9 catalyzes the formation of α-pinene (38.0%), limonene (32.1%), and α-phellandrene (12.9%) from GPP, and β-bisabolene (61.3%) and β-farnesene (16.9%) from FPP. Subcellular localization revealed that HcTPS9 is targeted to plastids, whereas HcTPS6 is localized to the cytosol. Real-time quantitative PCR analysis indicated that HcTPS9 is constitutively expressed, while HcTPS6 is predominantly expressed in floral tissues.

Conclusion HcTPS9 plays a role in the biosynthesis of monoterpenes such as α-pinene and limonene, while HcTPS6 is involved in floral sesquiterpene production in H. coronarium. The functional divergence of HcTPS9 and HcTPS6 is primarily attributed to the differences in signal peptide retention, catalytic function, and gene expression pattern.