Functional characterization of terpene synthases SmTPS1 involved in floral scent formation in Salvia miltiorrhiza

Plants attract beneficial insects and promote pollination by releasing floral scents. Salvia miltiorrhiza, as an insectpollinated flowering plant, which has been less studied for its floral aroma substances. This study revealed that S. miltiorrhiza flowers produce various volatile terpenoids, including five monoterpenes and ten sesquiterpenes, with the sesquiterpene compound (E)-fl-caryophyllene being the most abundant, accounting for 28.1% of the total volatile terpenoids. Y -tube olfactometer experiments were conducted on the primary pollinator of S. miltiorrhiza, the Apis ceranas. The results indicated that (E)-fl-caryophyllene compound had an attractive effect on the Apis ceranas. By comparing the homologous sequences with the genes of (E)-fl-caryophyllene terpene synthases in other plants, the SmTPS1 gene was selected for further experiment. Subcellular localization experiments showed SmTPS1 localized in the cytoplasm, and its in vitro enzyme assay revealed that it could catalyze FPP into fl-Elemene, (E)-fl-caryophyllene and a-Humulene. Overexpression of SmTPS1 in S. miltiorrhiza resulted in a 5.29-fold increase in gene expression. The GC-MS analysis revealed a significant increase in the concentration of (E)-fl-caryophyllene in the transgenic plants, with levels 2.47-fold higher compared to the empty vector plants. Furthermore, Y -tube olfactometer experiments showed that the transgenic plants were significantly more attractive to Apis ceranas compared to the empty vector plants. Co -expression analysis suggested that four SmMYCs (SmMYC1, SmMYC5, SmMYC10, and SmMYC11) may be involved in the transcriptional regulation of SmTPS1. The yeast one-hybrid screen and the Dual luciferase assay indicated that SmMYC10 positively regulates the expression of SmTPS1. In conclusion, this study lays a foundation for the functional analysis and transcriptional regulation of terpene synthase genes in S. miltiorrhiza.