## 3 Discussion  
In an attempt to identify genes of direct and putative indirect terpenoid defenses in _S. moellendorffii_ and to provide a better foundation for phylogenetic analysis of the MTPSLs in nonseed plants family, eight full-length _SmMTPSL_ genes were selected and functionally characterized from _S. moellendorffii_. The eight SmMTPSLs all contain two highly conserved motifs: 'DDxxD' and 'NSD/DITE' (Figure 1). Most MTPSLs, like typical plant TPSs, harbor a canonical aspartate-rich motif: 'DDxxD'. The aspartate-rich motif functions in binding a cluster of three Mg\({}^{2+}\) ions, which in term binds to the isoprenyl diphosphate substrates [24]. The binding leads to conformational changes of the active site, initiating the ionization and cyclization reaction. To the best of our knowledge, TPS function through divalent metal ion dependent generation of enzyme bound carbocation intermediates [25; 26; 27]. The phylogenetic analysis showed that all SmMTPSLs clustered with four MTPSLs from ferns and were similar to TPSs found in fungi and bacteria, which were consistent with the conclusion in the previous literature [12; 14]. According to the docking result of SmMTPSL19 with FPP or GPP, the amino acids D93, K238, R321, K57, 553, and S98 were easy to form hydrogen bond with the ligand, making the binding of ligand and receptor more stable (Figure 3). The remaining structure models and molecular docking also made clear that the other seven SmMTPSLs could successfully connect with the FPP or GPP. These models and molecular docking have painted a picture of some of the amino acids involved in the active sites of these SmMTPSLs and provided some level of confidence for modeling of SmMTPSLs, which will direct future experiments to analyze the involvement of these amino acids in catalysis. Through this, we hope to pinpoint amino acids involved in catalysis and provide a basis for future site directed mutagenesis of SmMTPSLs. As shown in our study, the eight SmMTPSLs were all determined to be functional, respectively, based on in vitro assays of the heterologously expressed proteins. All of them could use GPP or FPP as the substrate to display monoterpene synthase and sesquiterpene synthase activities (Figures 4 and 5). Like previously characterized MTPSLs from _S. moellendorffii_[12], we note that all functional SmMTPSLs were also multi-product enzymes possessing sesquiterpene synthase activity in the present study. With some of them producing two or three sesquiterpenes, SmMTPSL15 appeared capable of producing as many as six sesquiterpenes. In this context, it is noteworthy that many typical plant TPSs are multi-product enzymes [28; 29; 30]. Another interesting observation is that the product profiles of SmMTPSL5 and SmMTPSL33 share strong similarities and both produce farnesol and aromadendrene (Figure 5). However, SmMTPSL5 and SmMTPSL33 share only 38% identity at the protein sequence level. It is thus an interesting question whether such similarity in catalytic activity reflects common ancestry or convergent evolution. Terpenoids play significant ecological roles in the interactions between plants and stress conditions. As the critical gene in terpenoid biosynthesis, the expression level of _TPS_ genes in different plants under various stress conditions were widely reported [31; 32; 33; 34]. Both MeJA and ALA treatments regulated _SmMTPSL_ genes expression (Figure 6). The expression level of _SmMTPSL23_, -33, and -37 were enhanced after the MeJA treatment with the most notable enhancement occurred to _SmMTPSL33_ (13.5-fold) at 1.5 h, followed by _SmMTPSL37_ (7.9-fold) at 3 h. Besides, the most remarkably up-regulation was found for _SmMTPSL23_ (4-fold) at 6 h, followed by _SmMTPSL33_ (5.1-fold) at 3 h after the ALA treatment (Figure 6). The induction of _SmMTPSLs_ expression in _S. moellendorffii_ by MeJA or ALA supports that _TPS_ genes play a role in defense against abiotic stresses.