## 3 Discussion  
The phylogenetic analysis relative to the _A. thaliana_ TPS gene family members (Figure 1) showed that 39 ApTPSs of _A. paniculata_ were similar to those of _A. thaliana_ (40 AtTPSs), while the number ofiterpene synthase enzymes (members of the TPS-c and TPS-e/f subfamilies) in _A. paniculata_ was much higher than that in _A. thaliana_. A total of ten diterpene synthases were screened from the _A. paniculata_ genome, including four class II diterpene synthases (CPSs) and six class I diterpene synthases. By identifying the biological function of ApTPSs, we found that three of the class II TPSs (ApTPS1, ApTPS2 and ApTPS4) could convert GGPP to _ent_-CPP. _ent_-CPP is the direct precursor of andrographolide (the activity of ApTPS1 and ApTPS2 was analogous to that of ApCPS1 and ApCPS2) [19; 20; 25]. ApCPS4 is newly discovered here as an _ent_-copalyl diphosphate synthase that differs from ApCPS1, ApCPS2 and ApCPS3 [19; 20; 25]. Three of four class II diterpene synthases are _ent_-CPP synthases in the genome of _A. paniculata_; this provides a genetic explanation for the specific accumulation of _ent_-type labdane-related diterpenoids as the main chemical constituents in _A. paniculata_, which are the main specialized metabolites in _A. paniculata_ (only the content of andrographolide was greater than 1%). ApTPS5 is only an _ent_-kaurene synthase whose sequence is the same as that of ApKSL2 [19; 20; 25], and its biological function is different from that of ApKSL2, which has multiple biological functions. Thus, some unknown factors may still exist causing the results to differ, and this needs further study. The main chemical constituents of _A. paniculata_ are labdane-type diterpenoids and flavonoids, while other components in the plant are rare. Sesquiterpenes and monoterenes are important in plants but have not been reported in _A. paniculata_. We hypothesize that the content of other compounds may be present at low levels, as we failed to detect them in the original plant. Terpenoids are one of the largest groups of natural products in plants. However, only diterpene lactones of terpenoids have been reported in _A. paniculata_[31; 32]. The present study provides the first comprehensive annotation of the enzymatic functions of members of the ApTPS gene family within the A. paniculata genome. By identifying the biological function of terpene synthases of _A. paniculata_ to indicate the presence of other classes of terpenoids, we can study the chemical diversity in greater depth, which can also help us more effectively study the pharmacological activity of _A. paniculata_. In this study, we found seven multifunction or single-function sesquiterpene synthases (Figure 4 and Figure S1). These findings explain the diversity of the chemical composition by identifying the biological function of ApTPSs and provide evidence for the existence of \(\beta\)-bisabolene, cyclopropazulene, 4,7-methanoazulene and other sesquiterpenes, which could serve as a reference for future pharmacological research on _A. paniculata_. All monoterene syntheses in the type-b subfamily contain a conserved motif (RR(x)\({}_{8}\)W) downstream of their N-terminal transporters [33; 34]. It is thought that the RR(x)\({}_{8}\)W motif is required for the conversion of GPP to cyclic monoterpenes [13; 35; 36], but only two of ten ApTPSs of the TPS-b subfamily contain the complete structure of RR(x)\({}_{8}\)W (Figure 2). The absence or substitution of amino acids within the conserved domain may cause the monoterenoid synthase to lose its biofunction, which might be the reason that no accumulation of monoterenoid compounds was seen in _A. paniculata_. As a result of site-directed mutagenesis, ApTPS16 exhibited a nonpolar amino acid (Val) at position 533 compared to the wild-type (WT) ApTPS16. The mutation of this residue to a polar amino acid (Tyr) led to a diversification in product formation, resulting in the production of multiple products.