## 3 Discussion  
_U. rhynchophyllla_, an important medicinal plant resource in China, is rich in TIAs, which exhibit significant pharmacological activities, particularly in the treatment of hypertension and neurological disorders [29; 30; 31]. To investigate the biosynthetic mechanisms underlying TIA production in _U. rhynchophyllla_, genome sequencing of this species has recently been completed [25]. Elucidating the biosynthetic pathways and their regulatory mechanisms is essential for enhancing TIA accumulation and remains a key research focus [32; 33; 34]. Figure 5: Upregulation of _UrGES_ transcript levels and accumulation of terpenoid indole alkaloids (TIAs) in _U. rhynchophyllla_ in response to methyl jasmonate (MeJA). (**a**) HPLC chromatograms of standards and samples treated at different time points. The black line indicates mixed standards, including rhynchophylline, isorhorhophyllline, corynoxeine, and isocorynoxeine; the red line denotes the control not treated with MeJA, and the blue line denotes the leaf samples treated with MeJA for 3 h. (**b–e**) Correlation analysis between the _UrGES_ transcript levels and the concentrations of the four TIAs of _U. rhynchophyllla_ at different treatment times. Data indicate the mean \(\pm\) standard deviation from three repeats. The presence of distinct lowercase letters indicates statistical significance (\(p<0.05\)), and \(r\) stands for the Pearson correlation coefficient. In this study, a _GES_ gene was identified based on the genome sequencing results of _U. rhynchophylla_. Sequencing verification confirmed the successful cloning of a 1761 bp _UrGES_ gene encoding 586 amino acids. Bioinformatic characterization using the ExPASy website revealed that UrGES exhibits characteristics of a weakly acidic (pl 5.64) and unstable (instability index 51.14) protein. With a GRAVY value of \(-0.266\) and transmembrane region analysis showing no transmembrane domains, these results collectively suggested that UrGES is a hydrophilic protein. In plants, geraniol is known to be synthesized through two spatially separated pathways: the common pathway, which uses GPP produced by the MEP pathway as a substrate and is catalyzed by GES proteins in the plastid; and the atypical route, which involves cytoplasmic NUDX proteins converting GPP to geraniol [15; 16]. UrGES was predicted to localize to the chloroplast, along with the presence of a chloroplast transit peptide at its N-terminus, as determined using Plant-mPLoc and TargetP-2.0. These predictions aligned with the verified chloroplast targeting of GES proteins across various plant species, such as CoTPS4 (_Cananga odorata_) [20], CsTPS10 (_Camellia sinensis_) [35], PbTPS5, and PbTPS9 (_Phalanopsis bellina_) [36]. These findings suggest that the GES protein in _U. rhynchophylla_ likely functions in the plastid, using MEP-derived GPP as a substrate for monoterpene biosynthesis via the TPS-based pathway. During early land plant evolution, two duplications of the ancestral TPS led to the emergence of three major TPS clades, now classified as TPS-c, TPS-e/f, and the TPS-h/d/a/b/g subfamily [37]. Among these, the TPS-a, TPS-b, and TPS-g subfamilies, existing predominantly in angiosperms, play specialized roles in terpene biosynthesis [37; 38]. The TPS-a subfamily proteins are involved in the synthesis of sesquiterpenenes and terpen in monocotyledonous and dicotyledonous plants [39]. In contrast, the closely related TPS-b and TPS-g mainly take part in the synthesis of monoterpene, whereas TPS-b contains a RRXSW motif at N-terminus, which is a crucial domain for divalent metal ion binding and is absent in TPS-g [40]. Previous studies have shown that GES proteins from different plants have been classified into the TPS-b or TPS-g subgroup [35; 36; 41] (Figure 2). Amino acid sequence analysis revealed that UrGES possesses typical structural features of the TPS-g subfamily. Phylogenetic analysis further classified UrGES into a TPS-g subfamily that primarily consists of acyclic terpene synthases.