## 3 Results  
### Transcriptional Profiling  
To study the system-wide changes in the \(D\). _officiale_ leaves under salt stress, three time points (0, 4 and 12 h after treatment) were selected for analysis. We obtained 67.78 Gb of clean data in total after mRNA sequencing of nine samples with at least 6.14 Gb of clean data for each sample (Table S2). In each sample, more than 93.27% of bases had a score of Q30 or above, indicating that the sequencing results could be used for subsequent analysis. The clean data were mapped to the reference genome ([PERSON] et al., 2016), with the mapping ratio varying from 91.04% to 91.52% (Table S2). For the pairwise comparisons of 0 h vs. 4 h, 0 h vs. 12 h, and 4 h vs. 12 h, 2692 (1318 upregulated; 1374 downregulated), 2928 (1351 upregulated; 1577 downregulated), and 1102 (369 upregulated; 733 downregulated) DEGs, respectively, were identified (Figure 1A). Of these DEGs, 1586 were the same in both 0 h vs. 4 h and 0 h vs. 12 h; 527 were the same in both 0 h vs. 4 h and 4 h vs. 12 h; and 596 were the same in both 0 h vs. 12 h and 4 h vs. 12 h. Additionally, 230 DEGs were the same in 0 h vs. 4 h, 0 h vs. 12 h, and 4 h vs. 12 h (Figure 1B). KEGG analysis showed that several metabolic processes, including plant hormone signal transduction, alpha-linolenic acid metabolism, phenylpropanol biosynthesis, flavonoid biosynthesis, and flavone and flavanol biosynthesis were significantly enriched in DEGs under salt treatment (Figure 1C). Eight DEGs (three genes related to flavonoid synthesis and five genes related to JA synthesis) were selected for qRT-PCR analysis, and their expression patterns were consistent with the RNA-Seq results (Figure 2), indicating that the RNA-Seq data were reliable. Significant changes in KEGG pathways were identified using DEG co-expression analysis. To decipher the general trend in gene expression profiles, we subjected the 6722 DEGs to \(k\)-means clustering analysis. The 6722 DEGs were hierarchically clustered (Figure 3A) into six subclusters (Figure 3B). KEGG analysis was performed for DEGs belonging to each cluster with a \(q\) value < 0.05. According to subcluster 1, 3 and 6, salt stress induced the synthesis of secondary metabolites in \(D\). _officiale_ leaves. The \(\alpha\)-linoleic acid metabolic pathway is involved in the synthesis of JA. The expression levels of related genes in this pathway were significantly enriched after salt stress treatment, as shown in subcluster 1 (Figure 3B). Many genes involved in JA biosynthesis were upregulated under salt stress (determined by checking the detailed FPKM values of these DEGs, Figure 4). Under salt stress, genes involved in photosynthesis were significantly downregulated in subcluster 2 and 4, but genes related to glutathione metabolism were significantly upregulated in subcluster 6 (Figure 3B). Figure 1: Differentially expressed genes (DEGs) at different time points under salt treatments. (**A**) Number of DEGs. (**B**) Venn diagram of DEGs. (**C**) Top 20 enriched pathways for overlapping salt responsive genes of 0 h vs. 4 h, 0 h vs. 12 h, and 4 h vs. 12 h.  
Figure 2: qRT-PCR confirmation of expression profiles obtained by RNA-Seq transcriptome analysis at different times after salt treatment. Fold-change in transcript abundance obtained by both qRT-PCR and RNA-Seq are presented on the same graph for eight representative encoding genes. Column (**A**) shows the fold-change in the Fragments Per Kilobase of transcript sequence per Millions mapped reads (FPKM) value obtained by RNA-Seq, and column (**B**) shows the fold-change in the expression determined by qRT-PCR. DoANS, DoAOC, DoAOS, DoCHI, DoF3’5’H, DoLOX2S, DoMF, and DoOPR are anthocyanin synthase, allene oxide cyclase, allene oxide synthase, chalcone isomerase, flavonoid 3’,5’-hydroxylase, lipoxygenase, peroxisomal fatty acid beta-oxidation multifunctional protein, and 12-oxophyllodienoate reductase, respectively, in _D. officinal_. Data represent means and SDs.