## 2 Results
### Phylogenetic Analysis of ApTPSs  
Terpene synthases can be classified into three distinct classes according to their protein folds and different substrate catalytic mechanisms [21; 22; 23], which include ionization and induced carbocation formation (class I), protonation and induced carbocation formation (class II), or the use both of these mechanisms (bifunctional enzymes) [24]. All class I reprene synthases contain two conserved motifs, aspartate-rich regions (DDxXD) and (N,D)Dxx(S,T)xxxE, which utilize a trinuclear metal to trigger the ionization of the isoprenoid substrate prenyl diphosphate group and generate a carbocation intermediate to initiate catalysis [14]. Class II reprene synthases initiate the cyclization of GGPP through protonation, leading to the formation of a tertiary carbocation. This reactive intermediate subsequently undergoes a series of carbon-carbon bond-forming reactions, ultimately producing a bicyclic product. The general catalytic acid responsible for protonation is a central aspartate residue located within the conserved DxDD sequence motif [14; 24].  
By performing a phylogenetic analysis of ApTPSs, we found that ApTPS1, ApTPS2, ApTPS3 and ApTPS4 clustered with the characterized protein (AtCPS; AT4G02780) within the TPS-c subfamily belonging to class II diTPS (Figure 1), and contained the conserved motifs DxDD (Figure 2). The biological function of the two ApTPSs (ApTPS1 and ApTPS2) has been characterized; they convert GGPP to _ent_-CPP and were named ApCPS1 and ApCPS2 [19; 25], and ApTPS3 is similar to ApCPS3 [19; 20; 25] according to BLAST results, but the ApTPS4 enzymes of four class II TPS members have not been reported. And ApTPS5 to ApTPS35 were characterized as class I TPSs (Figure 1), which contained conserved DDxXD (or NDxXD) and clustered with the class I TPSs of _Arabidopsis thaliana_. Six of thirty-one class I TPSs clustered with the characterized AtKS genes within the TPS-e/f subfamily, which were characterized as encoding _ent_-kaurene synthase, and the members of this subfamily gene were also named class I diTPSs. Fourteen or ten of thirty-one class I TPSs clustered with the members of the TPS-a or TPS-b subfamilies of _A. thaliana_.  
Figure 1: Phylogenetic tree of _A. paniculate_ TPSs and those from other species. The tree was generated using the neighbour-joining method (bootstrap replications = 1000) and was condensed by a cut-off value of 70%. (These terpene synthase protein sequences of _A. thaliana_ were extracted from TAIR ([[http://www.arabidopsis.org](http://www.arabidopsis.org)]([http://www.arabidopsis.org](http://www.arabidopsis.org)), accessed on 14 April 2025, Table S1).  
Seven ApTPSs groups (e.g., group 1: TPS6, 7, 9, 10; group 2: TPS16, 17, 18, 19; group 3: TPS13, 14; group 4: TPS29, 30; group 5: TPS20, 24; group 6: TPS25, 26, 27, 28, 31; group 7: TPS34, 35) were found through the analysis results of scaffold classification, which also contained the similar intron-exon structure and the conserved motifs (Figure S1), and the same groups' \(\mathrm{ApTPSs}\) (except group 5) were clustered in the same branch. These results reveal that these \(\mathrm{ApTPSs}\) have a relatively close evolutionary relationship and similar catalytic activities.