## 4 Discussion  
Many genes involved in terpene metabolism and biosynthesis were identified, cloned, and are well characterized. In this study, we identified 67 genes related to enzymes involved in flavor synthesis, analyzed their physicochemical properties, and performed subcellular localization predictions. Among these 67 genes, nearly all encode hydrophilic proteins, except for CaAACT-1-2, CaHMGR-1-2-3, CaMVK-1, and CaDXS-1, which are hydrophobic. However, the influence of pH within distinct cellular microenvironments on the synthesis, stability, and function of these proteins remains to be further investigated. Figure 8: Relationship between volatile compound content and gene expression levels in _C. annuum_. Compounds with \(p\) < 0.05 are highlighted in picture. Chromosome localization shows that most genes are distributed on Chr8, with only _CaFPPS-2_ located on Chr10. Tandem and fragment duplication analysis revealed multiple gene clusters in the duplicated regions, indicating that some terpenoid synthesis genes may be generated through gene duplication events. Multiple copies of these genes may buffer mutation effects [22], thereby enhancing adaptability under adverse environmental conditions. Tandem genes provide genetic redundancy, maintaining functional stability in the face of gene-specific mutations or enabling novel functions through mutational changes [23]. Additionally, environmental stress and adaptive evolution are likely to increase the probability of tandem duplication within populations. Identifying these duplication events provides valuable insights into evolutionary relationships among genes and aids in predicting their functions. We further hypothesize the presence of metabolic cluster genes among them. These metabolic cluster genes, which are linked closely in clusters, synergistically interact to regulate multiple secondary metabolic pathways [24], acting as co-regulators within biological systems [25]. Gene clusters for various compounds, including terpenoids, have already been identified in species such as _Zea mays_ L. [26], _Avena sativa_ L. [27], and _Oryza sativa_ L. [28]. The existence of such clusters may elucidate genome evolution and offer insights into the expansion and functional diversification of terpenoid metabolic pathways, providing clues about species' evolutionary history. Studying the transcriptional regulatory mechanisms of these clusters, including the ways co-regulated genes achieve synergistic expression via shared promoters, enhancers, and other regulatory elements, holds significant potential for advancing our understanding of metabolic pathway regulation. The phylogenetic tree revealed complex evolutionary relationships among terpenoid metabolism genes in peppers and those in other species. A total of 67 enzyme-encoding genes exhibited high sequence similarity to terpenoid pathway-related genes in the model plants _Arabidopsis thaliana_ L., _Solanum Lycopersicum_ L., and _Oryza sativa_ L., which clustered within the same branches. We hypothesize that these genes may perform functions analogous to those in other species, providing a basis for future functional verification experiments. An analysis of the gene aggregation patterns and branch length patterns suggests that these genes experienced gene duplication, functional divergence, and other evolutionary events. The overexpression of AACT, HMGR in _Arabidopsis_ confirmed an increase in the content of triterpen compounds [29]. In tomato, [PERSON] showed [30] that the GGPPS enzyme is involved in the increase in diterpeneoid content in leaves as well as roots during stress. The _KS_ gene encodes a protein with ent-kaurene synthase activity, which catalyzes the second step in the gibberell biosynthesis pathway in the cyclisation of GGPP to ent-kaurene. This gene has been shown to be involved in the _Oryza_ gibberell anabolic pathway [31] but has been poorly studied in flavor.