Integrated WGCNA and TO-GCN analysis reveals pathways and genes underlying salt tolerance in Sesbania cannabina

Salt stress seriously threatens the growth and development of plants. To investigate how Sesbania cannabina responds to salt stress, we constructed a temporal regulatory response network under salt stress by integrating the physiological and transcriptomic data from 'Zhongke Jing No.1' at five time points (0, 6, 12, 24, and 48 h, n = 3) after salt treatment. The results indicated that salt stress increased the relative electrical conductivity (REC) and malondialdehyde (MDA) content, induced the accumulation of osmotic regulators, and enhanced antioxidant enzyme activities. A total of 688,424,778 raw reads were generated by RNA-seq, and 21,422 DEGs were identified using |log2FC| ≥ 1 and adjusted p-value < 0.01. WGCNA and TO-GCN were constructed using β = 20 and PCC cutoff of 0.91, respectively, revealing three response stages: normal (0 h), early stage (6 h and 12 h), and late stage (24 h and 48 h). The MEturquoise module was associated with early osmotic and oxidative responses, while the MEbrown and MEblack modules were associated with late-stage antioxidant defense and secondary metabolism reprogramming. Early-stage genes were mainly involved in glycolysis/gluconeogenesis and plant hormone signal transduction, whereas late-stage genes were enriched in glutathione metabolism, phenylpropanoid biosynthesis, flavonoid biosynthesis, and isoflavonoid biosynthesis. Based on co-expression and network analyses, 9 putative candidate TFs, including ERF71, WRKY53, and bZIP9, were predicted to be associated with key salt-responsive pathway genes in the pathway subnetworks. These findings indicate that S. cannabina undergoes a transition from early carbon metabolism and hormone-mediated responses to late antioxidant defense and secondary metabolic reprogramming under salt stress.