## 4 Discussion  
In this study, we identified six TPS genes in _P. massoniana_, including four mono-TPS and two sesqui-TPS genes. We believe that these genes are involved in the response of _P. massoniana_ to _M. alternatus_ invasion. In vitro enzyme activity assay also verified that Pm mono-TPSs can catalyze substrate GPP to produce volatile terpenes. In our previous study [18; 38; 39], we proved that exogenous MeJA treating and _M. alternatus_ adult feeding could induce a resin-based defense of _P. massoniana_, including secondary resin duct formation, induced resin terpenoid accumulation, and gene transcriptional changes related to terpenoid synthesis in metabolic pathways (Figure 7). TPSs catalyze complex carbocation cascade reactions on the prenyl diphosphate substrate, resulting in cyclic or linear terpene backbones [53], which determines the formation and composition of oleoresin terpenes. Therefore, studying the relationship between TPS structure, action site and production is helpful to elucidate the mechanism behind the synthesis of resin with complex composition, as well as the understanding, development and application of terpene synthase in vitro terpene biosynthesis. The total length of Pm TPSs was similar to that reported in Pinaceae [54]. Each Pm mono-TPSs contained a peptide sequence of approximately 50 amino acid residues, similar to _SaTPS1_ identified from _Santalum album_[55]. Studies have shown that a lack of the N-terminal peptide FaNES1 leads to changes in subcellular localization, resulting in different products [56]. The _Pm sesqui-TPSs_ had no peptide sequence, suggesting that it may function in the cytoplasm, whereas the sesqui-TPSs from _Solanum haprochaites_ (santalene/bergamotene synthases) are located in chloroplasts, indicating that sesquiterpenes can also be synthesized in plastids [57]. Among the three characteristically conserved domains of TPS, RxR and DDxxD were found in all the sequences. The RRx8W domain was found not only in monoterpenes but also in _Pm TPS selinee_. Monoterpene synthases lacking the RRx8W motif are responsible for the production of acyclic mono-TPSs, a characteristic of the TPS-g subfamily [58]. Previous studies on the functional verification of menthol limonene synthase have shown that the tandem arginine sequence RR in RRx8W might catalyze the transformation of GPP to an intermediate product [59], suggesting that _Pm TPS selinee_ can bind the substrate GPP. Additionally, in _A. grandis_, RRx8W was also found  
Figure 7: _P. massoniana_ resin-based defense induced by _M. alternatus_ feeding/MeJA treatment. in the N-terminus of \(\delta\)-_selinene synthase_, and its heterologous expression could produce 52 sesquiterpenes [32]. The conserved domain NSE/DTE was not found in Pm TPSs nor the reported TPS sequences of _P. taeda, P. contorta, P. banksiana_, and _P. nigra_ subsp. _laricio_[22; 34; 60], suggesting that the conserved domain NSE/DTE may not be conserved in _Pinus_. In the phylogenetic tree, we found that the amino acid sequences of TPSs in congeners were more similar to those of TPSs with the same main products in species from different genera (Figure 2b). For example, the distance between Pm TPS seli (Pm TPS seliene) and _A. grandis_ TPS d-seli, both of which produce selinene, was greater than the distance between Pm TPS seli and _Pinus sylvestris_ TPS \(\mathrm{cary}/\mathrm{humu}\), which are congeners but have different main products. This feature is also found in other species. The similarity between Caryophyllene synthase from _Oryza sativa_, _Arabidopsis thaliana_, _Artemisia annuua_, and _Cucumis sativus_ is less than 40%. In contrast, Caryophyllene synthase from _Oryza sativa_ has 40%-42% similarity with the synthases that produce other sesquiterpenes from _Zea mays_[61]. No special motifs were found in the sesquiterpene sequences used to construct the phylogenetic tree. None of the Pm sesqui-TPSs contained motifs 18 or 20.