Functional characterization identified five sesquiterpene synthases that confer terpenoid diversity in Nardostachys...

Terpenoids constitute the principal bioactive and aromatic components of Nardostachys jatamansi, an edible spice plant native to the alpine Himalayas. However, their biosynthetic pathways in this species remain largely unexplored. Through analysis of an updated reference transcriptome and genome, we successfully amplified 16 full-length terpene synthase (TPS) genes in N. jatamansi. Subsequent biochemical characterization of the encoded enzymes revealed that five TPSs are functionally active in sesquiterpene biosynthesis. Specifically, NjTPS2 catalyzed the formation of fourteen sesquiterpenes: aciphyllene, cyclohexane, aristolene, (+)-calarene, α-panasinsene, γ-maaliene, aromadendrene, alloaromadendrene, selina-5,11-diene, valerena-4,7(11)-diene, β-caryophyllene, β-guaiene, (+)-ledene, and β-cyclogermacrane. NjTPS3 produced two distinct sesquiterpenes, γ-selinene and valerena-4,7(11)-diene. NjTPS6 generated α-gurjunene, β-maaliene, and β-cyclogermacrane. NjTPS7 synthesized a suite of sesquiterpenoids, including β-patchoulene, α-guaiene, α-patchoulene, γ-gurjunene, humulene, Δ-guaiene, α-selinine, (-)-7-epi-α-selinene, patchouli alcohol, and pogostole. NjTPS11 was characterized as a β-bergamotene synthase. In addition, NjTPS10 utilized geranyl diphosphate (GPP) to produce the monoterpene alcohol linalool. Tissue-specific gene expression analysis revealed that, with the exception of NjTPS11, which was highly expressed in leaves, the remaining four genes were all highly expressed in roots and rhizomes. This expression profile aligns with the predominant accumulation of sesquiterpenes in the underground parts of N. jatamansi, providing molecular insight into the spatial regulation of its characteristic aroma and bioactive compound synthesis.