## 3 Results
### Gene Expression in the GSL-TGG Pathway  
From 1 d to 5 d, the contents of GRA and GER and their breakdown products changed greatly. Thus, we conducted RNA-seq to reveal the changes in the GSL metabolism pathway in this period. The RNA-seq analysis showed 3519 up-regulated and 613 down-regulated genes in the 3d_vs_1d comparison, 333 up-regulated and 183 down-regulated genes in the 5d_vs_3d comparison, and 4070 up-regulated and 1778 down-regulated genes in the 5d_vs_1d comparison (Figure S1). The KEGG pathway enrichment analysis  
Figure 2: Changes in ITCs and nitriles derived from GER and GRA during broccoli sprout development. (**A**) The concentrations of erucin, erucin nitrile, sulforaphane, and sulforaphane nitrile; (**B**) the ratio of erucin/erucin nitrile and sulforaphane/sulforaphane nitrile. Values are expressed as mean \(\pm\) SD (n = 6).  
revealed that many genes were enriched in \"metabolic pathways\" and \"biosynthesis of secondary metabolism\" (Figure S2). In general, the DEGs in the 5d_vs_3d comparison were notably fewer compared to 3d_vs_1d and 5d_vs_1d, indicating a relatively stable development from 3 d to 5 d. The DEGs related to GSL metabolism were selected and analyzed (Table S2). A total of 43 genes (25 _Arabidopsis_ homologous genes) related to GSL biosynthesis were identified. These genes, specifically involved in aliphatic GSL biosynthesis, such as _MYB28_, _CYP83A1_, and _MAMs_[1], had low expression levels at the beginning of seed germination but were slightly induced at 3 d and 5 d (Figure 3). Nevertheless, most genes, including _SURI_, _SOT16_, _SOT18_, _GPP1_, _UGT74B1_, and _GSTs_[1], involved in the core structure biosynthesis of both aliphatic GSL and indole GSL showed high expression from 1 d to 5 d (Figure 3). Similarly, transcription factors such as _MYB51_, _MYB122_, _MYB34_, _WRKY33_, and _MYC2_, as well as critical biosynthetic genes such as _CYP79B2_ and _CYP83B1_ involved in the indole GSL biosynthesis pathway [29], were up-regulated in broccoli sprouting (Table S2).  
Figure 3: transcriptomic analysis of TGG-dependent metabolic pathways of GRA and GER. (**A**) The GRA and GER metabolic pathways. (**B**) Analysis of gene expression in the GRA and GER metabolic pathways. Values represent the means of three replicates. The expression levels were visualized using TBtools. The heatmap scale ranges from \(-4\) to \(+12\) on a log2 fold change.  
In comparison to biosynthesis and regulation, the genes involved in the hydrolysis of GRA and GER were up-regulated at the beginning of seed germination (Figure 3). Eight TGG1 homologous genes and five TGG2 homologous genes were identified. The current investigation revealed the high expression of TGGs at 3 d and 5 d, particularly _TGG1_ homologous genes. Also, two homologous genes (_BolC4t28607H_ and _BolC4t22177H_) of _BGLU28_ were observed with a high expression from 1 d to 5 d. In addition, the RNA-seq analysis showed that two _ESP2_ homologous genes (_BolC7t3254H_ and _BolC7t43256H_) exhibited higher expression levels and gradually increased from 1 d to 5 d (Figure 3), which was confirmed using qRT-PCR (Figure 4A). Our results also showed that all four _EMS1_ homologous genes were highly expressed at 3 d and 5 d. In this study, apart from one _BolNSP1_ (_BolC5t33748H_) and two _BolNSP5_ (_BolC7t44414H_ and _BolC2t11690H_) genes expressed at 1 d, all the _BolNPS_ genes identified in the samples using RNA-seq were highly expressed at 3 d and 5 d, which was verified using qRT-PCR (Figure 4B).  
Figure 4: The relative expression levels of _BolESPs_ (**A**) and _BolNSPs_ (**B**) in broccoli sprouts. Values are expressed as mean \(\pm\) SD (n = 3).