## 4 Discussion  
The primary objective of this study was to investigate the biosynthesis of terpen in _Z. candidia_ by identifying and characterizing the _TPS_ genes and their encoded enzymes. SPME-GC-MS analysis showed that 16 out of the 17 identified volatile compounds were terpenoids, with D-limonene, \(\beta\)-ocimene, and linalool being the most abundant monoterpenes. To date, more than 1700 VOCs from 90 different plant families have been categorized. These plant VOCs are divided into three main classes based on their biosynthetic origin: terpenoids, phenylpropanoids/benzenoids, and alcohols/aldehydes [36]. The dominance of terpenoid metabolic pathways in \(Z\). _candida_ flowers is indicated by the high proportion of terpenoids (16/17). Their potential significance in the ecological context of \(Z\). _candida_ and their potential value for use in the fragrance and flavor industry are indicated by this study. In flowering plants, terpenoids are widely present in the volatile metabolites of plants. For example, during the bloom period, over 20 volatile terpen are released from _Clematis florida_[37]. The wild species of the _Freesia_ genus displays a variety of terpenes [38]. Notably, the identification of four monoterpene derivatives in \(Z\). _candida_ suggests that monoterpene skeletons can be modified by other enzymes, including cytochrome P450 oxygenases (CYPs) and acyltransferases, and form a wider range of volatile compounds [39]. In addition to volatile compounds, biosynthetic pathways for nonvolatile specialized metabolites in plants should be further elucidated [40]. Through transcriptome sequencing, 17 unigenes were identified as potential TPSs. The _TPS_ gene family has been extensively studied in various plant species, with reported repertoires ranging from tens to hundreds of _TPS_ genes [18; 35; 41]. For the gene expression analysis of the transcriptome, ZcTPS01 and ZcTPS02 were selected for further investigation. Sequence alignment results showed the presence of several typical monoterpene synthase motifs. Notably, the RR\({}_{\text{XS}}\)W motif, located at the protein's N-terminus, is a hallmark of the N-terminal domain and has been demonstrated to undergo isomerization in the initial step from geranyl diphosphate to linalyl diphosphate [42; 43; 44]. The RR\({}_{\text{XS}}\)W motif is fully conserved and found in ZcTPS02, but it is absent in ZcTPS01; this finding may explain the unsuccessful characterization of the biochemical function of ZcTPS01. Phylogenetic analysis result showed that ZcTPS01 and ZcTPS02 belong to the TPS-b subfamily, which is distinct from the TPS-g group that includes (E)-\(\beta\)-ocimene synthase (encoded by Arabidopsis AtTPS14) and (E)-\(\beta\)-ocimene synthase (ama0a23) [45]. After examining the TPS-g subfamily of \(Z\). _candida_, only one TPS-g gene (TRINITY_DN1687_c0_g1. P1) is present in this clade. However, this gene seems to be a partial sequence with a remarkably low expression level. It is worth noting that (E)-\(\beta\)-ocimene synthases have emerged multiple times in separate lineages and have undergone convergent evolution at the molecular scale [46]. The enzymatic assay result showed that ZcTPS02 produces \(\beta\)-ocimene, which has been detected in the flowers of \(Z\). _candida_, suggesting that ZcTPS02 may produce this product in vivo. Despite numerous attempts, ZcTPS01 has not yet yielded any enzymatic products. Considering that information about terpene metabolism in \(Z\). _candida_ is limited, future studies may uncover enzymatic activity and enhance our understanding of the complex molecular networks involved in plant terpene biosynthesis. For example, ten monoterpenes were detected in this study, indicating a high rate of GPP formation in the flowers of \(Z\). _candida_. The molecular mechanism of GPP formation, specifically the action of geranyl diphosphate synthase (GPPS), requires further investigation.