## 3 Discussion
Through comparative analysis, we found that the functional domains of EhFPS protein family members were highly conserved and contained DDXXD, GQXXD, and GKLNR domains that regulate enzyme activities. Previous studies have shown that plant FPPS have five conserved domains like those of other organisms, namely domains I (GKXXR), II (EXXXXXXXLXXDDXXDXXXXRRG), III (GQXXD), IV (KT), and VI (GXXKPQXXDDXXDXXXXXXXXGKKXXDXXXXK) [25]. Furthermore, it has been found that FPS contains two aspartate-rich regions, namely the DDXXD motifs in domains II and V. This motif plays a crucial role in the catalytic activity of FPS, which is responsible for binding magnesium ions (Mg\({}^{2+}\)), thereby stabilizing pyrophosphate groups and promoting substrate binding and reaction processes. The amino acid residues in this region participate in the formation of C-C bonds between IPP and allylic substrates and determine the chain length of the product. The aspartate and arginine residues in domain II are essential for FPPS function, and their substitution can lead to a decrease in the Vmax of IPP and geranyl pyrophosphate (GPP) while leaving Km largely unchanged. Conversely, mutation of the first aspartate residue in domain V significantly reduced FPS activity and increased the Km value for IPP, confirming the critical role of aspartate in enzymatic activity [26]. In our study, EhFPS8 lacked the DDXXD motif in the conserved domain II, suggesting a weakening of its core catalytic function. However, further experimental validation is required. Through physical and chemical property analyses, EhFPS proteins were found to be hydrophilic and have a high proportion of \(\alpha\)-helices. This is similar to the FPS proteins reported in rubber trees and other plants, the \(\alpha\)-helical structures of which are often closely related to their catalytic activity and substrate binding ability [27; 28]. Previous studieshave shown that prerequisite C5 units (DMAPP and IPP) are synthesized through both the plastitial methyl-D-erythitol phosphate (MEP) and cytosolic MVA pathway. However, the subsequent biosynthetic pathways for different terpenoid classes vary [29]. Specifically, sesquiterpenes and triterpenes are synthesized via the cytosolic MVA pathway, whereas monoterpenes, diterpenes, and tetraterpenes are synthesized through the plastitial MEP pathway [30]. For example, cytoplasmic localized _AtFPS1S_ can participate in the synthesis of sesquiterpenes, such as (+)-8-cadinene, and in inducible defense against aphid infestation in _Arabidopsis_[31]. In ginseng, _PcFPS_ localized in the cytosol is capable of regulating triterpenoids such as pachymic acid and eburicoic acid [32]. _AtFPS2_ encodes an FPS enzyme that localizes to the chloroplast. This enzyme is involved in the synthesis of monoterpenes and diterpenes, which are essential components of chlorophylls, carotenoids, and gibberellins. In this study, previous predictions using subcellular localization showed that _EhFPS1, EhFPS2, EhFPSS, EhFPS6, and EhFPS7_ were localized in the cytoplasm, whereas _EhFPS3, EhFPS4_, and _EhFPS8_ were localized in the chloroplasts. In addition, the subcellular localizations of _EhFPS1, EhFPS2, and _EhFPS7_ proteins in _N. benthamiana_ were confirmed (Figure 11). Subcellular localization prediction analysis indicated that, except for _EhFPS3, EhFPS4_, and _EhFPS8_, the remaining EhFPS proteins were primarily localized in the cytoplasm. This finding is consistent with their role in terpenoid biosynthesis, since most terpenoid synthases are active in the cytoplasm. In contrast, those localized in the chloroplast, such as _EhFPSS3, EhFPS4_, and _EhFPS8_, may play roles in the metabolic pathways associated with photosynthesis in plants. In this study, GO enrichment analysis revealed that _EhFPS_ genes were significantly enriched in biological processes related to isoprenoid metabolism and lipid biosynthesis, indicating that these genes play crucial roles in the secondary metabolism of _E. hirta_.