## 3 Discussion  
Most of the studies using Stevia have focused on diterpenoid SGs due to its commercial importance, while no research has investigated the biosynthesis of other terpenoids in Stevia. From our VOC analysis, we found the following results regarding terpenoids biosynthesis in Stevia. (1) Over 90% of the total VOCs of Stevia leaves, flowers, stems, and roots were composed of terpenoids. (2) Diterpenoids were mainly accumulated in Stevia flowers and leaves. (3) Sesquiterpenoids were the major VOCs of roots and stems. (4) Many terpenoids were differentially produced in Stevia tissues. (5) Most terpenoids identified from roots were root-specific (Figure 1 and Table 1). However, information to uncover biosynthetic pathway genes for terpenoids other than SGs was limited in Stevia until this study. Through the search for full-length mono- and sesqui-TPSs and biochemical assays, we were able to characterize five SrTPSs. SrTPS1 was a mono-TPS catalyzing the formation of \(\alpha\)-terpineol from GPP. Terpineol, a mixture of four isomers, \(\alpha\)-, \(\beta\)-, \(\gamma\)-terpineol, and terpinen-4-ol, is known for its pleasant fragrance. Some terpineol synthases can produce multiple enantiomeric forms [26; 27] while others produce a single product similar to SrTPS1 [28; 29]. _SrTPS1_ was highly responsive to most phytohormones and stresses in leaves and roots (Figure 6). Since \(\alpha\)-terpineol is shown to be toxic to insects and pests [30], SrTPS1 may play a crucial role in plants' defenses. TPS in Stevia. SrTPS3 may protect Stevia stems from aphids as \(\beta\)-farresene is the major component of the aphid alarm pheromone [36]. SrTPS4 showing the highest homology (74% identity) with (Z)-\(\gamma\)-bisabolene synthase from _Helianthus annulus_ catalyzed the formation of multiple sesquiterpenoids, \(\gamma\)-curcumene, zingiberene, and \(\beta\)-sesquiphellandrene, upon reaction with FPP (Figure 4D). These reaction products could also be found among the compounds produced in vitro by zingiberene synthases in many plants, including tomato and rice, suggesting functional similarity [37; 38]. As zingiberene and \(\beta\)-sesquiphellandrene were the most abundant terpen in ginger and turmeric, they are among the most important sesquiterpenoids for the defense in roots [37; 39]. Although zingiberenol was not produced by SrTPS4 in vitro, the transient expression of _SrTPS4_ in _N. benthamiana_ formed zingiberenol in addition to \(\gamma\)-curcumene, zingiberene, and \(\beta\)-sesquiphellandrene (Figure 5C). Moreover, we also detected zingiberenol among Stevia root VOCs (Table 1), suggesting a possible further modification of zingerberene in Stevia and _N. benthamiana_ by enzymes, such as cytochrome P450s. Zingiberene, \(\gamma\)-curcumene, and \(\beta\)-sesquiphellandrene can be formed together from (6R,7S)-bisabolyl cation via enzymatic cyclization of FPP [40]. Interestingly, environmental stresses tested in this study did not increase transcript levels of _SrTPS4_ in Stevia roots, where it is highly expressed under normal conditions. However, its expression was induced in leaves upon phytohormone treatments and wounding stress (Figure 6A,C). Purified recombinant SrTPS5 produced mainly himachalol and \(\beta\)-himachalene with minor amounts of \(\alpha\)-longipinene, \(\alpha\)-himachalene, \(\gamma\)-himachalene, and 10s,11s-himachala-3(12),4-diene from FPP (Figure 4E,F). Until now, TPSs producing \(\alpha\)-longipinene have only been reported in gymnosperms as multifunctional TPSs [41; 42; 43]. PaTPS-Lon from Norway spruce produced \(\alpha\)-longipinene with other sesquiterpenoids, longfolene, \(\alpha\)-longicyclene, \(\beta\)-farresene, and longiborneol [41]. Similarly, in addition to \(\alpha\)-longipinene, PsTPS-Lonp from Sitka spruce (_Picea sistchenis_) was also able to form substantial amounts of longfolene, \(\gamma\)-himachalene, and \(\beta\)-farresene [43].