Abstract: Casbene and neocembrene are casbene-type macrocyclic diterpenes; their derivatives play significant roles in plant defense and have pharmaceutical applications. We had previously characterized a casbene synthase, TERPENE SYNTHASE 28 (OsTPS28), in rice (Oryza sativa). However, the mechanism of neocembrene biosynthesis in rice remained unclear. In this study, we identified two genes of the TPS-a1 subfamily, OsTPS2 and OsTPS10, encoding a neocembrene synthase and sesquiterpene synthase, respectively, as supported by enzyme activity assays and determination of subcellular localization. Metabolic profiling of rice lines overexpressing either TPS confirmed the catalytic functions of OsTPS2 and OsTPS10, and suggested that OsTPS10 enhances resistance to rice bacterial blight. An evolutionary analysis revealed that OsTPS10 is conserved in monocots and first appeared in wild rice, whereas OsTPS2 and OsTPS28 sequentially evolved through gene duplication, transit peptide recruitment, and mutation of key amino acids such as H362R. In summary, this study not only deepens our understanding of the metabolic pathways and evolutionary history governing the biosynthesis of casbene-type diterpenoids in rice, representing parallel and divergent evolution within the gene family, and offers gene resources for the improvement of rice.

Key words: diterpenoid, divergent evolution, neocembrene, plant pathogen defense