## 4 Discussion
### miRNAs Involved in terpenoid Synthesis of Gleditsia sinensis Pods  
Brassinosteroids (BR) synthesized from squalene and then cyclized to become cycloartenol are a kind of terpene with biological activity. They were also identified as an endogenous steroid hormone that promotes plant growth and development [59]. For example, BR was reported to regulate the stabilization of microtubules to regulate plant pavement cells and leaf growth [60]. Thus far, only _miR848_, which modulates the quality of seeds by cleaving _OsCYP51G3_, has been proved to regulate brassinosteroid biosynthesis in rice [61]. On the other hand, _miR172_ could control the BR sensitivity of plants by regulating _BAKI_ in BR signaling [62]. In this investigation, the target of _miR838-3p_, which was enriched in the BR pathway, encodes the enzyme of PHYB activation tagged suppressor 1 (_CYP34A1_, _BAS1_). In addition, _miR838-3p_ was only expressed in May and September, and the expression in September was more than that in May. This implied that _miR838-3p_ might suppress the oxidoreductase of brassinosteroids to promote the production and regulate the biosynthesis of brassinosteroids in different developmental stages of _G. sinensis_ pods (Figure 6).  
Figure 6: Differential expression of miRNAs in the synthesis of terpenoids of _Gleditsia sinensis_. Note: The upward arrow indicates upregulation of miRNAs; the downward arrow indicates downregulation of miRNAs, and the dotted arrow represents miRNAs indirectly involved in the synthesis of related terpenoids by inhibiting the expression of target genes.  
Thiamine, called vitamin B1, is a vital cofactor for plant development and growth. In wheat, thiamine thiazole synthase was enriched, underlying the vernalization response [63]. In oil palm seedlings, thiamine biosynthesis was enhanced by endophytic colonization [64]. In soybean, thiamine was increased as ROS signaling after injection of _Rhizoctonia solani_[65]. It was demonstrated that _miR829_ could resist _Exserohilum turcicum_ in maize [66]. In this study, _miR829-3p.1_ was highly expressed in May but dropped to zero in July and was then upregulated in September. Additionally, the targets of _miR829-3p.1_ were predicted to take part in thiamine biosynthesis (Tables S5 and S6).  
In addition, the IAB pathway related to the development of saponin was enriched. IAB, which is derived from tryptophan with secologanin, is an important secondary metabolite [67]. In _Cathamuthus roseus_, large numbers of monoterene indole alkaloids (MIAs) have been identified to possess pharmacological activities, such as anticancer activity. In this study, four target genes of _mir2093-5p (CL1216.Contig13, CL1216.Contig6, CL1216.Contig7_, and _CL1216.Contig8_) were predicted to encode the enzyme of polyurethane-aldehyde esterase (PNAE) in the IAB process (Figure 6). However, _miR2093-5p_ was largely expressed in July and September, implying that indole alkaloids might be abundantly produced at an early stage. It was then significantly inhibited by _miR2093-5p_, possibly to reduce the consumption of tryptophan and provide a substrate for the synthesis of other essential protein components in plants. On the other hand, the accumulation of saponins may be regulated by feedback. The pathway of IAB shared the terpenoid backbone biosynthesis with other terpenoids, such as triterpenoids. Hence, this miRNA mechanism could balance the contents of the production of various terpenoids and play different roles at each stage. Additionally, the indole zeatin biosynthesis (ZTB) pathway was also enriched. The enriched targets of _miR838-3p_ were _cl1552.contig1_, _cl1552.contig2_, and _cl1552.contig6_ encoding the BAS1 enzyme in the BRB pathway and belonging to the P450 family (Figure 6). The enriched target genes of _miR4414b_ were _cl9352.Contig1_, _cl9352.Contig7_, and _cl9352.Contig8_ encoding IPT. These miRNAs should negatively regulate these terpenoid pathways by regulating the catalytic enzyme genes.