## 3 Results
### Transcription Factors Related to Gleditsioside Synthesis  
Transcription factors (TFs) are vital in regulating secondary metabolism and coordinating transcriptional responses to corresponding hormones. In various plants, such as _P. ginseng_, _P. notoginseng_, and _Panax quinquefolius_, several transcription factors have been shown to regulate saponin production [21; 34]. TF families contain many members, such as bHLH, MYB, and WRKY, and play essential roles in regulating triterpenoid and interpene biosynthesis in diverse plant species. Specifically, in _P. notoginseng_, the basic helix-loop-helix (bHLH)-type transcription factor PhbHLH1 exerts a positive regulatory effect on the biosynthesis of triterpene saponins [35; 36; 37; 38]. In addition, TFs like EIN3, AGBH, ARF, and MADS have also been reported to play critical regulatory roles in plant fruit growth and development [39; 40; 20; 41]. By applying more stringent screening criteria [FPKM \(\geq\) 1 and FDR \(\leq\) 0.05], a total of 22 TFs were obtained (Table S7). Some EIN3 and AGBH genes exhibited high expression levels in the initial stage, while others showed elevated expression, specifically in the final stage. Similarly, transcription factors such as GRAS, AGL, and SCL28 were highly expressed during the early period but demonstrated a subsequent decline. In contrast, ARF (CL4767.Contig2, Unigene28867), ERF (CL2043.Contig2), and bHLH exhibited predominant high expression levels during the later stage (Figure 5A,B). These transcription factors likely play a crucial role in the synthesis of saponins, with specific functional validation still required for further elucidation. Figure 5: Differentially expressed unigenes in _G. sinensis_ spots. (**A**) Differentially expressed enzyme genes related to gleditsioside biosynthesis from GO enrichment. (**B**) Highly differentially expressed unigenes coding transcription factors. ENIN: term of enzyme inhibitor activity (GO: 0004857), AGBH: term of hydrolase activity acting on glycosyl bonds (GO: 0016798). From left to right in the heatmap, these values are the logarithmic values of GS2, GS3, GS4, GS5, and GS6 relative to GS1. The red triangles represent the key differential genes in the gleditsioside synthesis pathway. Identification of a WGCNA Module Related to Gleditsioside Biosynthesis During Pod Development of G. sinensis  
To explore the correlation between phenotype and gene expression, a weighted gene co-expression network analysis (WGCNA) was conducted on the variables associated with the six developmental stages of _G. sinensis_. We categorized 8000 DEGs into 21 hierarchically clustered modules (Figure S4). Within the 21 modules, the turquoise module contained the highest number of genes (n = 2240). Specifically, the MEgrey module demonstrated a positive correlation in gleditsioside content with a correlation coefficient of 0.76 (\(p\) = 0.08) (Figure 6A). Consequently, this module was selected as a hub module for this study. Subsequently, GO enrichment and KEGG pathway enrichment analyses were performed to further characterize the functional differences and relationships of the MEgrey module. KEGG enrichment analysis revealed that the grey module was enriched in the flavonoid biosynthesis, terpene skeleton synthesis, sesquiterpenoid, and other pathways. The biosynthetic pathway of triterpenoid saponins consists of both terpene skeleton biosynthesis and triterpenoid synthesis pathways (Figure 6B). GO enrichment results revealed  
Figure 6: Construction of the gene co-expression network. (**A**) Heat map of correlation between modules and traits. (**B**) Hub genes were associated with gleditsioside. The circles represent nodes in the network. The size of the circles and their colors indicate the degree of the nodes. (**C**) GO enrichment analysis results of gleditsioside. (**D**) KEGG enrichment analysis results of gleditsioside. Solid lines represent relationships between nodes, with the thickness and color of the lines indicating the weight of these relationships.