Multi-Omics Integration Profiles Coordinated Regulatory Layers in Soybean Response to Salt Stress

Soybean production is severely threatened by soil salinization. Plants adapt to salt stress through multi-layered molecular responses, but whether these layers act cooperatively remains unclear. This study integrated six layers of data—H3K27me3 ChIP-seq, m6A DART-seq, RNA-seq, proteomics, phosphoproteomics, and metabolomics—to explore the regulatory architecture of the soybean salt stress response. DART-seq and RNA-seq were generated in this study; the ChIP-seq, proteomic, phosphoproteomic, and metabolomic datasets were reanalysed from public repositories. Through the intersection of three layers—H3K27me3 reduction, DART-seq detection, and RNA upregulation—245 multi-omics candidate genes were obtained, of which 66 were independently supported by public RNA-seq data. The protein abundance of these candidate genes showed modest changes under salt stress, whereas phosphosite changes were significantly but modestly directionally consistent (53.6% of multi-site proteins remained bidirectional; two-sided permutation p < 0.0001). Among 346 upstream transcription factors, 28 showed large changes in inferred activity. Metabolite-level enrichment was observed for starch and sucrose metabolism (FDR = 0.043), with key metabolite directions biologically consistent with gene-level trends; joint gene–metabolite enrichment was not statistically supported (intersection–union test maximum p = 0.795). This study provides an exploratory multi-omics resource of multi-layer regulation in the soybean salt stress response, and offers a candidate gene network and testable regulatory hypotheses for salt-tolerant breeding.