# Unveiling Monoterpene Biosynthesis in _Taiwania cryptorneioides_ via Functional Characterization
###### Abstract  
_Taiwania cryptorneioides_ is a monotypic species, and its terpenoid-rich property has been reported in recent years. To uncover monoterpene biosynthesis in _T. cryptomerioides_, this study used transcriptome mining to identify candidates with tentative monoterpene synthase activity. Along with the phylogenetic analysis and in vitro assay, two gernaul synthases (_Tc_TPS13 and _Tc_TPS14), a linalool synthase (_Tc_TPS15), and a \(\beta\)-pinene synthase (_Tc_TPS16), were functionally characterized. Via the comparison of catalytic residues, the Cys/Ser at region 1 might be crucial in determining the formation of \(\alpha\)-pinene or \(\beta\)-pinene. In addition, the Cupressaceae monoterpene synthases were phylogenetically clustered together; they are unique and different from those of published conifer species. In summary, this study aimed to uncover the ambiguous monoterpenoid network in _T. cryptomerioide_, which would expand the landscape of monoterpene biosynthesis in Cupressaceae species.  
Taiwania cryptorneioides; monoterpene synthases; gas chromatography-mass spectrometry (GC-MS) +
Footnote †: journal: _plants_  
0000-0002-3199-2240-3199][PERSON]. [PERSON]  
0000-0002-3199-240-3199][PERSON]  
0000-0002-3199-240-3199-24  
the exact product of monoterpene synthases based on sequence similarity. Nevertheless, the product specificity of monoterpene syntheses is determined by catalytic residues around the substrate, and phylogenetically related homologs likely share similar plasticity residues [17; 21; 22]. Thus, it is a challenge to predict the function of monoterpene synthases among non-model species such as _T. cryptomerioides_, and it requires a complete biochemical characterization of monoterpene syntheses as a template for further research. To address this issue, high-throughput transcriptome sequencing is a reliable tool to discover potential participants in the network of _T. cryptomerioides_ monoterpene biosynthesis.  
In previous studies, few Cupressaceae-associated clusters were identified in sesquiterpene and diterpene synthases via phylogenetic analysis [23; 24; 25], and Cupressaceae monoterrene synthases might hypothetically be close in phylogenetic relationships. Compared with diterpene synthases, monoterpene and sesquiterpene synthases evolved much later after losing \(\gamma\)-domains, and the remaining \(\beta\)-\(\alpha\) domain exhibited highly conserved organization. However, the diverse catalytic functions of sesqui- and monoterpene synthases suggest that the plasticity residues in these enzymes have been the major cause of divergent evolution, and few are known in gymnosperm species, especially Cupressaceae.  
In this study, we identified monoterpene synthases from _T. cryptomerioides_ needle and cone transcriptome library and aimed to provide complete information on monoterpene biosynthesis in _T. cryptomeoides_ by conducting phylogenetic analysis, an in vitro enzymatic assay, and catalytic residue comparison. The new discovery of a Cupressaceae-specific cluster of monoterpene synthases revealed the phylogenetically and biochemically divergent relationship of conifer monoterpene synthases and unveiled the hidden terpene biosynthetic network, which is crucial to plant defense and ecological communication.