# Unveiling Monoterpene Biosynthesis in _Taiwania cryptorneioides_ via Functional Characterization
## 2 Results and Discussion  
### TcTPS Isolation  
To identify any potential candidates for monoterpene synthase, we searched previously established _T. cryptomerioide_ transcriptome libraries, which include needles, cones, and wood. After conducting BLAST annotation against the NCBI nonredundant (NR) database and removing duplicates, three contigs (IDs 29358, 5958, and 2226) from the cone library and two contigs (IDs 17110 and 10575) from the needle library were identified as candidates (Table 1). Although monoterpene synthase was also observed in the wood transcriptome library, the lengths were shorter than 500 bp and identical to the contigs from needles and cones. This result might be caused by the low abundance of monoterpene synthases in wood, which corresponded to the lower concentration of monoterpene in the metabolite profile of _T. cryptomerioides_ wood extracts and essential oils in previous studies. Thus, we decided to use contigs from needles and cones for further experiments and renamed them _TcTPS13_ (_TF_29358_), _TcTPS14_ (_TL_17110_), _TcTPS15_ (_TF_5958_), _TcTPS16_ (_TF_2226_), and _TcTPS17_ (_TL_10575_).  
These transcript lengths ranged from 600 to 628 amino acids (Table 1), and the length distribution was close to that of known conifer sesquiterpenes and monoterpene synthases, compared with diterpene synthases. Sequence alignment with selective terpene synthase  
\begin{table}
\begin{tabular}{c c c c} \hline \hline \multirow{2}{*}{**Sequence ID**} & \multirow{2}{*}{**Gene Name**} & **Coding Region** & **Best Blast Hit 1**  \\ \hline TF\_29358 & _TcTPS13_ & 628 & \(\alpha\)-Pinene synthase [_Chamaczyparis formosensis_] 2 \\ TL\_17110 & _TcTPS14_ & 607 & Terpinolene synthase [_Chamaczyparis obtusa_] 3 \\ TF\_5958 & _TcTPS15_ & 610 & \(\alpha\)-Pinene synthase [_Chamaczyparis formosensis_] 2 \\ TF\_2226 & _TcTPS16_ & 617 & \(\alpha\)-Pinene synthase [_Chamaczyparis formosensis_] 2 \\ TL\_10575 & _TcTPS17_ & 593 & Terpene synthase [_Triumaria cryptomerioides_] 4 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Summary of _T. cryptomerioides_ monoterpene synthases identified in this study.  
showed a conserved structure of the \(\beta\)-\(\gamma\) domain (Figure S1), which suggests their possible function as monoterpene or sesquiterpene synthases.  
Transcript annotation against the NCBI database showed that _Tc_TPS13, _Tc_TPS15, and _Tc_TPS16 have similar sequences to the \(\alpha\)-pinene synthase from _Chamanecyparis formosensis_. _Tc_TPS14 shares its highest similarity with the terpionelene synthase from _Chamanecyparis obtusa_, and _Tc_TPS17 has the best result against the previously identified monoterpene synthase from _T. cryptomerioides_. According to these results, the monoterpene syntheses sequences from Cupressaceae are closely related, and they might form a phylogenetic cluster, which was also observed in sesquiterpene and diterpene synthases.  
To further investigate the genome structure of the identified TPSs, we attempted to amplify their genome sequences, and successfully obtained the sequences of _Tc_TPS14, _Tc_TPS15, and _Tc_TPS16. Compared with the ancestral form of terpene synthase (Figure 1, _Abies grandis_ abietadiene synthase [26]), the _Tc_TPS14-16 has fewer members of exons due to possible intron loss events, but the structures share the same composition as _Abies grandis_ monoterpene synthase (limonene synthase) [27]. The previously published intron XII loss was not observed in this study [19], thus it is likely an independent evolutionary event that occurred after the differentiation of sesquiterpene and monoterpene synthases.