## 3 Discussion
### Metabolome and Transcriptome Analysis of the Metabolite Biosynthesis Pathway
In this study, fourteen terpene synthase genes, twenty-three cytochrome P450 oxidase genes, eight methyltransferase genes, and six aminotransferase genes were found in the downstream pathway that may be involved in dendrobine biosynthesis. The typical plant terpene synthases (TPSs) are a medium-sized gene family with two functions. Class I terpene synthases usually catalyze the cyclization of the lysis of the allylic diphosphate ester bond in acyclic isoprenyl diphosphate precursors [51]. Class II terpene synthases generally catalyze protonation-initiated bicyclization of the diterpene precursor (_E,E_,_E_)-geranylgeranyl diphosphate (GGPP) to produce _ent_-kaurene [52]. The TPS-e/f and TPS-c subfamilies belong to the class I and class II TPSs, respectively. The typical product of the TPS-e/f subfamily is _ent_-atiSTEM-16-ol, and the typical products of the TPS-c subfamily are _ent_-copalyl diphosphate, _ent_-kaurene, and _ent_-isopimara-8, 15-diene [51]. In addition, TPSs can be divided into the TPS-a, TPS-b, TPS-d, TPS-g, and TPS-h subfamilies. The major product of the TPS-h subfamily is terpenenedienyl-PP, whereas the major product of TPS-a/b/g/d is levopimaradiene [51]. According to the evolutionary analysis, the TPS-d subfamily can be subdivided into TPS-d1, TPS-d2, and TPS-d3. These TPS subfamilies are the basis for the formation of colorful terpenoids. Sesquiiterpene synthase (_PtTPS5_) has been reported to be a terpene synthase that can catalyze FPP to produce two products, (IS,S5,7R,10R)-guaia-4(15)-en-11-ol and (IS,7R,10R)-guaia-4-en-11-ol as the major products, and hedycayrol as a minor product [53]. In this study, we found a sesquiterpene synthase (_SQS2_) that catalyzes the formation of FPP into different compounds, for example, germacrene D, (_E_)-\(\beta\)-farnesene, \(\alpha\)-humulene, \(\beta\)-caryophyllene, 4-hydroxy-germacradiene, nerolidol, germacrene B, germacrene C, and bicyclo-germacrene [31]. _TPS21_ also affects dendrobine biosynthesis pathways, so reducing or knocking out this gene may be more beneficial for dendrobine biosynthesis [17]. Combined with the compounds isolated from _D. nobile_, we can further elaborate on the biosynthetic pathway of dendrobine. We speculate the possible cycle-reaction of dendrobine intermediate compounds (dendronobilin G): firstly, under the catalysis of terpene synthases, FPP cyclizes intramolecularly to form dendronobilin G. The intermediate compound of dendrobine is formed through a series of reactions such as molecular rearrangement, oxidation, and cyclization. Cytochrome P450 (_CYP450_) enzymes play significant roles in this pathway. In the dendrobine biosynthesis pathway, we hypothesized that dendronobilin G forms a type I skeleton under the action of CYP450 oxidase. The type I skeleton structures formed by intramolecular rearrangement and cyclization are dendronobilin A and dendronobilin M. These intermediate compounds are further oxidized to form type II skeleton structures. The type II skeleton structures are subsequently hydrolyzed, ring-opened, and oxidized to form type III and IV compound skeleton structures. The picrooxane-type sesquiterpene skeleton (type V and VI) is formed by intramolecular esterification of the type IV skeleton structure [16]. At least nine picrooxane-type sesquiterpene compounds have been identified. Picrooxane-type sesquiterpene compounds (type VI) may be cyclized to form dendronobilin C, which is catalyzed by aminotransferase to form the skeleton VII compound, which is further methylated to form dendrobine [16]. In addition, the picrooxane-type sesquiterpene compound is directly aminated to nobiline, which is cyclically decarboxylated to dendrobine. The analysis of these methyltransferases has played a role in promoting the biosynthesis of dendrobine. This indicates the direction for us to construct the synthesis pathway of dendrobine and provides a reference for the synthesis of dendrobine (Figure 12).