Multi-omics analysis elucidates sesquiterpenoid compounds and identifies IrTPS3 as a key multifunctional terpene synthase in roots and rhizomes of Inula racemosa

BACKGROUND AND AIMS: Inula racemosa is a rare and endangered medicinal plant, the therapeutic value of which is largely attributed to its sesquiterpenoid compounds. Terpene synthases (TPSs) play a central role in the biosynthesis of these metabolites. However, until now the diversity of sesquiterpenoids present in I. racemosa, as well as the specific IrTPS enzymes driving their formation, had not been clearly defined. METHODS: A comprehensive metabolomic analysis was conducted to profile the volatile constituents across five distinct tissue types - roots, rhizomes, stems, leaves and flowers - of I. racemosa. The full-length transcriptome and next-generation transcriptome were employed to identify the IrTPS genes and their expression patterns. In vivo and in vitro enzymatic assays were performed to characterize the functional properties of IrTPS. KEY RESULTS: A total of 33 sesquiterpenoid compounds were identified, the majority being newly reported in this species. Notably, these compounds exhibited preferential accumulation in roots and rhizomes, suggesting that the IrTPS genes responsible for their biosynthesis might also show tissue-specific expression patterns. Transcriptomic data supported this hypothesis, revealing that several IrTPS genes, including IrTPS3 and IrTPS4, were predominantly expressed in root and rhizome tissues. These genes were introduced into a microbial host engineered to produce high levels of farnesyl pyrophosphate (FPP), the key precursor for sesquiterpenoid synthesis. Functional assays demonstrated that only IrTPS3 was capable of converting FPP into a range of sesquiterpenoid products, whereas IrTPS4 showed no catalytic activity under the same conditions. CONCLUSIONS: These findings support the idea that IrTPS3 is likely the principal enzyme involved in sesquiterpenoid biosynthesis in I. racemosa, providing a foundation for further studies aimed at metabolic engineering and sustainable production of these valuable compounds.