## 2 Results
### Functional Characterization of SrTPSs  
To elucidate the exact function of SrTPSs, full-length ORFs of _SrTPSs_ were expressed as recombinant proteins in _Escherichia coli_ C41 (DE3) cells. Purified recombinant proteins were then tested for activity against geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP), and products were analyzed by GC-MS. Figure 4 shows that SrTPS2-5 reacted only with FPP to produce sesquiterpenoids, whereas SrTPS1 reacted with GPP alone to produce a monoterenoid. These results were consistent with our prediction on the potential function of SrTPSs based on phylogenetic analysis and subcellular localization experiments. SrTPS1, as a member of the TPS-b family, utilized only GPP to synthesize \(\alpha\)-terpineol (Figure 4A). The major product of SrTPS2 was \(\beta\)-caryophyllene, along with minor amounts of \(\alpha\)-caryophyllene (Figure 4B). SrTPS3 produced \(\beta\)-farnesene as a single product, which was the most abundant volatile compound in stems (Figure 4C and Table 1). SrTPS4 catalyzed the formation of three compounds, \(\gamma\)-curcumene, zingiberene, and \(\beta\)-sesquiphellandrene (Figure 4D). SrTPS5 predominantly formed himachalol, \(\beta\)-himachalene, and \(\alpha\)-longipinene, along with minor amounts of \(\alpha\)-himachalene, \(\gamma\)-himachalene, and 10s,11s-himachala-3(12),4-diene from FPP (Figure 4E,F). No product was observed from the heat-inactivated SrTPSs, which served as negative controls (Supplementary Figure S5). The products produced by SrTPSs were verified using authentic standards and essential oils that contain the terpenes of our interest. The terpenoid profiles of SrTPSs obtained by in vitro analysis were verified by transiently expressing them in _N. benthamiana_ leaves using _Agrobacteriumium_-mediated infiltration. The Arabidopsis _3-hydroxy-3-methylglutural coenzyme A reductase_ (_HMGR_) was co-expressed with sesqui-TPSs to increase heterologous sesquiterpenoid production in _N. benthamiana_, and its effects have been confirmed before [20]. GC-MS analysis of leaves expressing _SrTPS2_ and _SrTPS3_ revealed the presence of \(\alpha\)- and \(\beta\)-caryophyllene and \(\beta\)-farnesene, respectively, mimicking the in vitro results (Figure 5A,B). However, SrTPS4 and SrTPS5 yielded slightly different products in vivo compared to their corresponding in vitro assays. Plants expressing _SrTPS4_ produced zingiberenol (peak 14), in addition to three compounds identified from in vitro assay (Figure 5C). On the other hand, only \(\alpha\)-longipinene and himachalol could be detected from _N. benthamiana_ expressing _SrTPS5_ (Figure 5D). Although we identified SrTPS1 as \(\alpha\)-terpineol synthase by in vitro assay, \(\alpha\)-terpineol could not be detected from _N. benthamiana_ plants transiently overexpressing _SrTPS1_, suggesting the extreme instability of \(\alpha\)-terpineol in mesophyll cells of _N. benthamiana_ or possible further metabolism in _N. benthamiana_ plants. Figure 3: Subcellular localization of Stevia terpene synthases (TPSs). Subcellular localization of SrTPSs fused with YFP in _N. benthamiana_ leaves. The _Agrobacteriumium_-infiltrated leaves were visualized using confocal microscopy. Auto, chlorophyll auto-fluorescence; YFP, YFP channel image; Merged, merged image of Auto and YFP. Scale bars, 20 \(\upmu\)m.  
Figure 4: In vitro characterization of five Stevia TPSs. Gas chromatograms of products obtained from in vitro enzymatic assay of (**A**) SrTPS1, (**B**) SrTPS2, (**C**) SrTPS3, (**D**) SrTPS4, and (**E**) SrTPS5. (**F**) Alignment of peak 13 with himachalol from the essential oil of _Cedrus atlantica_. Purified recombinant proteins were incubated with the substrates geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). Peaks 1, 2, 3, 4, and 8 were identified using authentic standards.