## 2 Results
### Morphological Characterization of Glandular and Non-Glandular Trichomes of T. sinensis
Among them, we identified two genes (_TsTPS1262_ and _TsTPS466_) that are highly similar to the _AtTPS21_ gene involved in sesquiterpene synthesis in Arabidopsis [23]. We further analyzed the expression profiles of _TsTPS1262_ and _TsTPS466_ in different tissue types of _T. sinensis_ (Figure 5b). We found that both genes were highly expressed in the leaves, _TsTPS1262_ mainly in the mature leaves and _TsTPS466_ in the young leaves (Figure 5c,d). We cloned and verified the full coding sequences of _TsTPS1262_ and _TsTPS466_ and performed a phylogenetic analysis of the TPS genes from _Arabidopsis_. Both _TsTPS1262_ and _TsTPS466_ are the most common among the _T. sinensis_ and _TsTPS466_ are the most common among the _T. _TsTPS466_ were closely related to _AtTPS21_ (Figure 6a), suggesting functional relevance in sesquiterpenoid biosynthesis. Further, through multiple sequences alignment, we showed that _TsTPS1262_ and _TsTPS466_ have a high degree of sequence similarity with _AtTPS21_, including \"DDXXD\", \"RRX8W\", and \"NST/DTE\" signatures in conserved catalytic motifs (Figure 6b). We constructed a _TsTPS1262_-GFP fusion expression vector to study cellular localization. By transforming _Arabidopsis thaliana_, we obtained a stable positive transgenic line. Through confocal observation, we found that the _TsTPS1262_-GFP signal was localized in chloroplasts in leaves, while the observation of root cells revealed that it was localized in the cytoplasm (Figure 6c). Ectopic Expression of TsTPS1262 and TsTPS466 Leads to Complex Sesquiterpene Biosynthesis in Tobacco and T. sinensis  
To investigate the function of secondary metabolism, we ectopically expressed the TsTPS1262 and TsTPS466 genes in tobacco. We showed that transient expression can effectively induce a high expression of candidate genes (Figure 7a,b). Based on this, we measured the content and composition of volatiles in transgenic leaves. Both the overexpression of TsTPS1262 and TsTPS466 resulted in a variety of terpene profiles  
Figure 6: Phylogenetic analysis and subcellular localization of TPS genes from _T. sinensis_. (**a**) The phylogenetic analysis of the TPS genes from _Arabidopsis thaliana_ and _T. sinensis_. (**b**) A multiple sequence comparison of terpene synthase (TPS) genes from _T. sinensis_, _Arabidopsis thaliana_, _Solanum lycopersicum_, _Oryza sativa_, and _Gossypium hirsutum_ was conducted. The red rectangles indicate the conserved structural domains. (**c**) Subcellular localization analysis of _TsTPS1262_ was conducted using leaf and root tip tissues from a positive line of transgenic _Arabidopsis thaliana_. (Table S3). We found that a variety of sesquiterpenes were present in the overexpression plants but not in the control (Figure 7c,d). Specifically, in the overexpression of TsTPS1262, we identified three sesquiterpene constituents; in the overexpression of TsTPS466, we identified two sesquiterpene constituents (Figure 7c,d). In order to further study the biological function, we established an overexpression system in _T. sinensis_ based on vacuum filtration. First, we tested the transformation efficiency through the RUBY reporter gene and found that more than half of the young leaves of a single transformant had a red signal expression, indicating that the overexpression was effective (Figure 8a). We analyzed the overexpression of _TsTPS1262_ and found that _TsTPS1262_ gene expression was up-regulated in three independent transformations, and two transformants were significantly up-regulated (Figure 8b). We detected the volatile components of the transformants by GC-MS (Table S4) and found that the sesquiterpene components of elemene and \(\gamma\)-elemene were significantly detected in three replicates (Figure 8c,d). Figure 7: Ectopic expression of _TsTPS1262_ and _TsTPS466_ in tobacco and the analysis of volatiles. Relative gene expression in tobacco leaves following the ectopic expression of _TsTPS1262_ (**) and _TsTPS466_ (**b**).