## 3 Results
### Gleditsioside Biosynthesis-Related Unigenes Were Differentially Expressed in G. sinensis  
The predominant pathways of gleditsioside biosynthesis encompass the mevalonate (MVA) pathway, the methylerythnitol phosphate (MEP/DOXP) pathway, and the triterpene cyclization process (TCP). The MVA and MEP/DOXP pathways are integral in forming the triterpene backbone [4]. The investigation identified 372 unigenes encoding 23 enzymes among the six developmental stages of the _G. sinensis_ pods. Only 99 unigenes associated with 17 enzymes showed differential expression (Tables S3 and S4). In the MVA and MEP/DOXP pathways, a notable trend of elevated expression levels of most enzymes and genes was observed during stages GS1 to GS4, with particular prominence noted in key enzymes such as HMGCS, HMGCR, MVK, and DXPS. Furthermore, specific contigs such as CL5845.Contig1 for HMGCR, CL1651.Contig1 for IDI, CL4174.Contig3 for DXPS, and both CL4067.Contig3 and CL4067.Contig4 for ISPG were identified as highly expressed DEGs in the MEP/DOXP pathways (Figure 3).  
During the triterpene cyclization process (TCP), various oxidosqualene cyclases (OSCs) catalyze the conversion of 2,3-oxidosqualene into either cycloartenol or other triterpenoids, forming diverse pentacyclic triterpenoid saponin skeletons [24; 33]. A total of 79 unigenes encoding four differentially expressed OSCs (named LUP1, LUP2, LUP4, and CAMS1) were annotated in this study. LUP1, LU2, and LUP4 were related to producing oleanolate- and lupane-type pentacyclic triterpenes. The structural diversity of triterpenoids is enhanced by P450-mediated hydroxylation and UGT-mediated glycosylation. Among these, 22 beta-amyrin 24-hydroxylases and eight UDP-glucosyltransferases (UGTs) were annotated. Additionally, P450s such as CYP93E1 exhibited differential expression corresponding to the oleanolane type. High expression levels of Unigene6088 (LUP4), Unigene29200 (CYP93E1), and CL11248.Contig4 (CYP93E1) were observed, with gradual up-regulation of these genes noted (Figure 3). Moreover, the high expression of Unigene37260 (UGT) indicated an initially increasing and then decreasing trend. The results suggest that Unigene6088, Unigene29200, CL11248.Contig4, CL8823.Contig2 and Unigene37260 potentially impact the synthesis of gleditsioside.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Pathway**} & \multicolumn{6}{c}{**Gene Numbers**} \\ \cline{2-7}  & **GS2 vs. GS1** & **GS3 vs. GS1** & **GS4 vs. GS1** & **GS5 vs. GS1** & **GS6 vs. GS1** \\ \hline Steroid biosynthesis & 90 ** & 100 ** & 94 ** & 82 ** & 106 ** \\ Synthesis and degradation of ketone bodies & 9 ** & 20 & 24 & 4 & 2 \\ Terpenoid backbone biosynthesis & 69 ** & 87 & 81 & 86 ** & 77 \\ Tropane, piperidine, and pyridine alkaloid & 21 ** & 31 ** & 30 ** & 25 * & 42 ** \\ biosynthesis & 22 ** & 32 & 37 * & 46 ** & 41 ** \\ Triyptophan metabolism & 26 ** & 44 * & 41 * & 39 * & 42 \\ biosynthesis & 72 ** & 78 ** & 64 * & 76 ** & 93 ** \\ \hline \hline \end{tabular}  
* represents significant difference (q-value \(\leq\) 0.05). ** represents extremely significant difference (q-value \(\leq\) 0.01); GS1: June, GS2: July, GS3: August, GS4: September, GS5: October, and GS6: November.  
\end{table}
Table 2: _Cont._Figure 3: Heatmap and schematic diagram for genes related to terpenoid biosynthesis in _G. sinensis_ pods. Red font: differentially expressed gene; IPP: isopentenyl pyrophosphate; DMAPP: dimethylallyl pyrophosphate; GPS: geranyl diphosphate; FDPS: farnesyl pyrophosphate. TCP: Triterpene cyclization process. MVA pathway: mevalonate pathway in the cytoplasm; MEP/DOXP pathway: methyleryth nitol phosphate pathway in the plastid. The heatmap from left to right shows the DEGs for log\({}_{2}\) ratios of GSZ/GS1, GS3/GS1, GS4/GS1, GS5/GS1, and GS6/GS1. The red boxes represent the key differential genes in the gleditsioside synthesis pathway.