## 3 Results  
### Sequence, Phylogenetic, and Motif Analysis of Six Pm TPSs  
To characterize the terpene synthase genes involved in the _M. alternatus_ feeding induced defense in _P. massoniana_, six genes, Pm TPS (+)-\(\alpha\)-pinene, Pm TPS (-)-\(\alpha\)-pinene, Pm TPS (-)-\(\beta\)-pinene, Pm TPS limonene, Pm TPS solienene, and Pm TPS longifolene, which were differentially expressed after _M. alternatus_ inoculation, were identified in the _P. massoniana_ transcriptome [38]. The BLAST alignment showed hits with known TPS genes based on the presence of conserved sequence characteristics shared by all TPSs. The six predicted proteins encoded by the above Pm TPSs ranged between 580-635 amino acids in size, with predicted pIs of 5.71, 5.53, 5.74, 5.44, 5.6, and 5.24, respectively. All Pm mono-TPSs had signal peptide sequences at the N-terminus, ranging from 36-48 aa in length, and localized to chloroplasts (Table S2). In contrast, Pm sesqui-TPSs did not contain signal peptide sequences (Table S2). Hydrophobic/hydrophilic analyses showed that all six Pm TPSs were hydrophilic (Figure S1). As shown in Figure 2a, the mono-TPSs _Pm TPS (+)-\(\alpha\)-pinene, Pm TPS (-)-\(\alpha\)-pinene, Pm TPS (-)-\(\beta\)-pinene, Pm TPS limonene_ and the sesqui-TPS _Pm TPS solienene_ contain the arginine-tryptophan motif RRX\({}_{8}\)W, which is conserved among most mono-TPSs and some sesqui-TPSs, near the N-terminus. The RRX\({}_{8}\)W motif was missing in _Pm TPS longifolene_. The highly conserved aspartate-rich motif DDXXD and RXR motif were present in these six _Pm TPSs_. However, another conserved motif, DTE, was not found in _P. massoniana_. This is crucial for chelating divalent cations, typically Mg\({}^{2+}\), in the carboxyl-terminal (C-terminal) domain [50]. Phylogenetic analysis revealed that four monoterpene and two sesquiterpene synthases of _P. massoniana_ were clustered into two subfamilies (TPS-d1 and TPS-d2) with monotperone and sesquiterpene synthases from other pine plants, except for _P. Abies_ TPS \(E\) (_E_-\(\alpha\)-farnesene), _P. engelmannii \(\times\) P. glauca_ TPS \(E\) (_E_-\(\alpha\)-farnesene synthase/_E_-\(\beta\)-ocimene), and _P. teada_ TPS (\(\alpha\)-farnesene), which formed into one clade as monoterpene synthases, in line with previous studies [25; 29; 51; 52] (Figure 2b). Among them, Pm TPS (+)-\(\alpha\)-pinene grouped with \(\alpha\)-pinene of _P. sesiya_. Furthermore, these were grouped with TPS \(\alpha\)-pinene of _P. contorta_ and _P. banksiana_. The Pm TPS limonene was grouped with _P. sitchensis_ TPS (-)-limonene and _P. abies_ TPS (-)-limonene. The Pm TPS (-)-\(\beta\)-pinene clustered with TPS (-)-\(\beta\)-pinene and TPS \(\alpha\)-terpineol from several _Pinus_ spp. Two sesqui-TPSs, Pm TPS longifolene and _P. sylvestris_ TPS longifolene, were grouped, whereas Pm TPS selinene was grouped with _P. sylvestris_ TPS carophyllene/humulene (cary/humu), and slightly distant from the _A. grandies_\(\delta\)-selinene synthase, annotated as monofunctional but heterologous. The motif types and permutations of _P. massoniana_ (Figure 2c) and its homologous TPSs (Table S3) were analyzed using the online software MEME (Version 5.5.5). The number and species of the 20 predicted motifs ranged from 11 to 50 aa in length. All Pm mono-TPSs contained 20 motifs, except for Pm TPS (-)-\(\beta\)-pinene. Thirteen motifs (motifs 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 14, and 17) were found in all Pm TPSs. The conserved domains DDXXD, RRX\({}_{8}\)W, and RXR were found in motifs 1, 11, and 3, respectively. Motifs 15, 18, 19, and 20 were found only in the Pm mono-TPSs but not in the Pm sesqui-TPSs. Most of the Pm mono-TPSs contained all 20 motif structures in the order 18-11-12-20-7-13-8-19-9-5-17-3-15-1-6-4-2-14-10-16. Motifs 1, 5, 7, 8, 11, and 12 appeared in all sequences. None of the Pm sesqui-TPSs contained motifs 18 or 20. The motifs of TPS D1 and TPS D2 had their characteristics.