LAUSR

Abstract The volatile oil extracted from Curcuma wenyujin has various benefits for human health, which can be attributed to large amounts of bioactive terpenoids. Although more than 80 terpenoids have been reported from this species, their biosynthetic pathway remains largely unexplored. In this study, coexpression networks were used to identify key genes involved in terpenoid biosynthesis (TBS) through combining throughput transcriptome and metabolome analyses. Headspace GC–MS analysis firstly identified a total of 250 terpenoids in tissues (flower, leaf, rhizome, and tuber), and revealed a distinct tissue-specific feature in the terpenoid accumulation. Secondly, coexpression networks identified a strong gene module containing 26 “signature” pathway genes involved in TBS, including genes encoding mevalonate kinases, farnesyl pyrophosphate synthases, terpene synthase, and cytochrome P450 enzymes, as well as 12 hub transcription factors. These identified genes made up a tight “terpenoids-genes” TBS network, in which 87 main terpenoids were directionally synthesized by the gene module. Finally, the TBS pathway originated from the cytosolic mevalonate was proposed based on the link between gene module and terpenoids, and the validation of subcellular localization for candidate genes. Two terpene genes (CwTPS04 and CwTPS09) were confirmed to be active both in vitro and in Nicotiana benthamiana, with trans-nerolidol as a major product. Collectively, this study provides new insights into understanding TBS in C. wenyujin, and elite candidate genes for metabolic engineering of the TBS pathway in novel hosts. This workflow adopted in this study will facilitate research on elucidating other unexplored specialized metabolite pathways in non-model plants.