Functional characterization of multiple terpene synthases from Idesia polycarpa reveals diverse catalytic activities in vitro and in planta

Introduction Terpenes are an important and structurally diverse class of specialized metabolites. Volatile mono- and sesquiterpenes are often components of herbivore-induced volatile bouquets in perennial plants, including tree species of the Salicaceae family such as the Japanese orange cherry ( Idesia polycarpa ), which produces a wide array of volatile terpenes. However, the terpene synthases (TPSs) contributing to the formation of these herbivore-induced volatiles in I. polycarpa have not yet been characterized. In this study, we aimed to characterize the complement of TPSs involved in the formation of herbivore-induced terpenes in the I. polycarpa leaf volatile bouquet. Methods Using a previously generated de novo assembled I. polycarpa leaf transcriptome, we identified seven candidate TPS genes through comprehensive tBLASTn analysis. Subsequent in silico analyses, including phylogenetic analysis, subcellular localization prediction, and conserved motif identification, supported their assignment as TPS candidates. Results Reanalysis of the transcriptome revealed that six of the identified TPS genes were significantly upregulated in response to herbivory. Functional in vitro characterization confirmed their activity in producing monoterpenes such as ( E )-β-ocimene and 1,8-cineole, as well as sesquiterpenes including germacrene D and ( E , E )-α-farnesene, among other terpene compounds. Furthermore, heterologous overexpression of the identified IpTPS genes in Nicotiana benthamiana , followed by volatile analysis, validated their activity in planta . Discussion These findings provide insights into the TPS-mediated biosynthesis underlying herbivore-induced volatile emissions in a non-model woody species and highlight the coordinated activity of multiple TPS enzymes in plant defense.