## 5 Conclusions  
Our study analyzed the changes in gleditsioside content and pod length of _G. sinensis_ from GS1 to GS6. A total of 163,319 genes were analyzed by high-throughput sequencing of pods at the six developmental stages. In differential gene expression analysis, 162 and 703 unigenes were annotated to the triterpene and sesquiterpene biosynthesis pathway (STTB) and terpene backbone synthesis pathway (TBB), respectively. In addition, CTB, ZTB, NGB, and ten interactive pathways were elucidated, along with their interrelationships. Among these synthesis pathways, we analyzed key DEGs encoding enzymes including ENIN, cytochrome P450 (CYP93E1), and UDP-glucosyltransferase, and key genes such as CL5845.Contig1 (HMGCR), CL8823.Contig2 (LUP4), and CL11248.Contig4 (CYP93E1). Additionally, we identified several TFs associated with gleditsioside synthesis, including NAC, EIN3, ERF, AGL, SEP1, and two ARFs (CL4767.Contig2 and Unigene28867). Through WGCNA analysis, two highly expressed genes, CL5845.Contig1 (HMGCR) and CL8823.Contig2 (LUP4), exhibited notably downward trends in gleditsioside synthesis. Figure 10: Regulatory network of gleditsioside synthesis in _G. sinensis_. Squares represent genes, and circles represent miRNA. Furthermore, CL5845.Contig1 (HMGCR), CL8823.Contig2 (LUP4), and CL11248.Contig4 significantly enhanced gleditsioside biosynthesis through transient expression validation in _G. sinensis_ pods. Additionally, this investigation revealed candidate key genes and pathways in gleditsioside biosynthesis and provided new insights into the understanding of the basic mechanism of saponin biosynthesis, which should be of great value and have broad application prospects in multiple fields, such as theoretical research, agricultural production, and drug development. The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/f16030393/s1](https://www.mdpi.com/article/10.3390/f16030393/s1)]([https://www.mdpi.com/article/10.3390/f16030393/s1](https://www.mdpi.com/article/10.3390/f16030393/s1)), Figure S1: Full wavelength scanning of EA in _G. sinensis_. Figure S2: Statistical analysis of GS pods. Figure S3: Differentially expressed unigene statistics in comparable groups of _G.sinensis_ pods. Figure S4: Gene dendrogram obtained by hierarchical clustering with themodule color. Table S1: Quality of clean reads in 12 samples of _Gleditsia sinensis_ pods after mRNA sequencing. Table S2: Differential expressed enzyme genes related in saponin biosynthesis of _Gleditsia sinensis_ pods from GO enrichment. Table S3: Enzymes during gleditsioside biosynthesis in _Gleditsia sinensis_ pods. Table S4: Differentially expressed unigenes with their corresponding enzymes during gleditsioside biosynthesis of _Gleditsia sinensis_ pods. Table S5: Enzymes to link TBB with other pathways of terpenoid and ramification. Table S6: Differential expressed unigenes of enzymes linking TBB with other parallel pathways in _Gleditsia sinensis_ pods. Table S7: Classification of TFs in GO enrichment from _Gleditsia sinensis_. Table S8: Primers used for RT-qPCR. [PERSON] and [PERSON] designed the experiment and edited the manuscript. [PERSON], [PERSON] and [PERSON] performed most of the experiments and analyzed the data. [PERSON], [PERSON] and [PERSON] wrote the manuscript; [PERSON], [PERSON], [PERSON], [PERSON] and [PERSON] revised the manuscript. All authors have read and agreed to the published version of the manuscript. This work was supported by Forestry New Varieties and Patent Protection Application Funds of the National Forestry and Grassland Administration (KJZXXP202308), the Science and Technology Innovation Team of Henan Academy of Agricultural Sciences (2023 TD28), Basic Research Funds of Henan Academy of Forestry (2022 JB01008), and the National Natural Science Foundation of China (32071504). No ethics approval or consent was required for this manuscript. _G. sinensis_ pods were collected at Beijing Forestry University.