## 4 Discussion  
Based on previous studies, the TPS family is typically organized into seven or eight subfamilies, including TPS-a, -b, -c, -d, -e/f or -e/-f, -g, and -h, distinguished by their sequence's heterogeneity and phylogenetic relationships [32]. The TPS-e/f subfamily, both classified as diterpene synthases, are prevalent in vascular plants, with TPS-f identified as a derivative of TPS-e and often grouped with it. TPS-c, a diterpene synthase, is distributed in both gymnosperms and angiosperms. The TPS-d subfamily, which is specific to gymnosperms, is responsible for the synthesis of a complex mixture of monoterpenes, sesquiterpennes, and diterpenenes. Conversely, TPS-h is exclusively detected in stone pines (_Pterocarpus indica_). Among these subfamilies, TPS-a primarily encodes for sesquiterpene synthase in monocotyledonous plants and diterpene synthase in dicotyledonous plants [33]. Predominantly found in angiosperms, TPS-b and TPS-g are responsible for monoterpene synthesis. Notably, TPS-b features the essential metal-binding domains C-terminal DDxND,  
Figure 7: Analysis of volatile metabolome results of GC-MS analysis between DcTPSb1-overexpressed tobacco and wild type. (**a**) Ring diagram of metabolite class composition. Each color represents a metabolite category, and the area of the color block indicates the proportion of that category; (**b**) Heat map of differential metabolite clustering. On the left side of the figure is the sample clustering line, and on the top is the sample clustering line. Class is the first level classification of substances. Different colors are the colors filled with different values obtained after standardizing the relative content. Red represents high content, and green represents low content; (**c**) Dynamic distribution of differences in levels of differential metabolites. Each point represents a substance, with green points representing the top ten substances ranked downwards and red points representing the top ten substances ranked upwards. NSE/DTE, and the N-terminal RRXSW motif, crucial for binding divalent metal ions such as Mg or Mn, all integral to monoterense synthesis. In contrast, TPS-g does not contain the RRXSW motif [34]. In this study, the DcTPSb1 from _D. chrysotoxum_, which consisted of 598 amino acids and possessed all three aforementioned motifs, was unequivocally classified within the TPS-b family. Moreover, experimental findings indicate its localization in the chloroplast, consistent with the majority of TPS-b family members. Phylogenetic analysis revealed that DcTPSb1 was clustered with alpha-terpineol synthase from _D. catenatum_ (PKU83075.1) and from _D. nobile_ (KAI0507613.1), indicating a closer genetic relation among species within _Dendrobium_ genus. This observation is consistent with the general principle that plants within the same genus are more closely related. However, discrepancies were noted in the examination of gene structure and amino acid alignment. Specifically, KAI0507613.1 from _D. nobile_ was found to lack the RRXSW domain, indicating that it does not belong to the TPS-b family, in contrast to the other four proteins utilized for sequence alignment, all of which contained the three requisite domains. This observation supports [PERSON]'s hypothesis that TPSs from closely related plant species tend to cluster together more prominently than enzymes with similar functions, posing challenges for predicting substrates/products based solely on sequence similarities [35]. The intricacies of studying plant affinities underscore the complexity of the process, wherein outcomes may differ based on the chosen sequences and methods. Variations in results may arise even when using different approaches on the same sequences. Therefore, a comprehensive strategy that integrates multiple methodologies is essential for achieving a more robust determination, underscoring the necessity for further exploration in this domain.