# Composition Diversity and Expression Specificity of the _Tps_ Gene Family among 24 _Ficus_ Species
## 3 Discussion
### Sequence Characteristics and Subfamily Categories of the FTPS Genes  
According to the amino acid motifs and catalytic mechanism, TPS enzymes can be divided into two classes: class I and II [21; 22; 23]. Class I TPSs contain two conserved motifs, DDX and (N,D)DX(S,T,G)XXX(E,D) (also named DTE), which facilitate ionization of an isoprenoid diphosphate moiety and generate a reactive carbocation intermediate. In contrast, Class II TPSs typically contain only a DXDD motif, which protonates substrates, catalyzes scaffold rearrangements without cleavage of the diphosphate ester bond, and produces initial carbocation intermediates [35]. In this study, at the protein level, the DXDD motif was only found in one sequence of FTPS25 (_fhir39312_). Phylogenetic analysis showed there was another paralog of the sequences in _F. hirta (fhir17484_). However, owing to the low expression levels of the two sequences (Table S3), we could not detect sufficient reads to assemble complete coding sequences, including the DXDD domain in the second sequence. Because the DXDD motif is the characteristic of Class II _TPS_ gene, the _FTPS_s identified in our transcriptomes should be dominated by Class I TPS enzymes [36]. This suggestion was also supported by the subsequent phylogenetic analysis. On the basis of genome-wide comparisons of multiple plants, the _TPS_ family has been split into seven subfamilies, i.e., _TPS-a_, _TPS-b_, _TPS-c_, _TPS-d_, _TPS-e_/_f_, _TPS-g_, and _TPS-h_[21; 22; 37]. Among the subfamilies, _TPS-d_ and _TPS-h_ are distributed only in Gymnosperms and _Selaginella moellena_, respectively [21]. Notably, in this study, we identified all of the other five _TPS_ subfamilies that can be distributed in angiosperms in the 24 _Ficus_ species, suggesting a diverse and complicated composition of terpen in the receptive stage of male syconia. In addition, compared with other plants such as _A. thaliana_[38], grapevine [24], and tomato [30], in which _TPS-a_ genes are the most abundant genes, here, we found that a majority of _FTPS_s were categorized into _TPS-b_ (46%) rather than the _TPS-a_ (38%) subfamily (Figure 1B). Previous studies have shown that the _TPS-b_ subfamily is mainly composed of angiosperm monoterpene synthases genes [39; 40], except a sesquiterpene, (_E_,_E_)-\(\alpha\)-farnesene synthase gene, found in apple [41], poplar [42], and soybean [43]. Here, we also identified an orthologous gene encoding (_E_,_E_)-\(\alpha\)-farnesene synthase (_FTPS_8) in 12 _Ficus_ species (Table S3). More interestingly, our study revealed that most _FTPS_s protein sequences in the _TPS-b_ subfamily (63%) lack the second R of the R(R)X8W motif, i.e., lacking the RR domain (Figure 2). Previous authors confirmed that the RR domain was functionally important to isomerize GPP to a cyclizable intermediate [44]. However, the RR domain may be absent in the monoterpene synthases producing only acyclic compounds, which do not require isomerization [45]. Therefore, our study suggests that a majority of the _Ficus TPS-b_ genes likely encode acyclic terpene synthases. This suggestion is supported by our current annotations that 70% of monoterpene were acyclic (Table S3). However, the result is inconsistent with previous findings that many of the enzymes in the plant _TPS-b_ group produce cyclic monoterrenes [21]. Therefore, the production of rich highly volatile acyclic monoterepenes may be a distinguishing characteristic for the _Ficus_ TPS-b enzymes. The _TPS-a_ subfamily, which included 14 _FTPS_ genes (Figure 1B), is the second largest _TPS_ subfamily in the _Ficus_ species. Similar to other plants, most _TPS_s in the _TPS-a_ clade were sesquiterpene synthase genes [21; 38]. A total of 13 out the 14 _FTPS_ genes identified are responsible for encoding sesquiterpen. Only _FTPS13_, which encodes a cadinene synthase, is an acyclic monoterpene. The third largest _TPS_ subfamily in the 24 _Ficus_ species is _TPS-g_. This subfamily includes 29 sequences (Figure 1B).