Transcriptomic analysis reveals the physiological and molecular of wild soybean (Glycine soja Siebold & Zucc) leaves to saline-alkali stress

Saline-alkali stress represents a major abiotic threat to global agriculture, severely constraining the growth and productivity of crops. Although wild soybean ( Glycine soja ) constitutes a valuable reservoir of stress-tolerant alleles, gaps still exist in our understanding of the integrated physiological and molecular mechanisms governing its adaptation to composite saline-alkali stress. In this study, two wild soybean genotypes exhibiting contrasting tolerance to saline-alkali stress, designated F00142 (tolerant) and F0047 (sensitive), were subjected to a 16-day stress treatment. A comprehensive physiological analysis was conducted, followed by RNA sequencing of leaf tissues to profile the transcriptional landscape associated with stress responses. Weighted Gene Co-expression Network Analysis (WGCNA) was subsequently employed to integrate transcriptomic data with key physiological traits, thereby enabling the identification of candidate modules and hub genes potentially governing stress adaptation. Comprehensive physiological analysis revealed that F00142 exhibited superior resilience under saline-alkali stress compared to F0047. Specifically, F00142 maintained higher growth parameters, including plant height and leaf area, alongside elevated relative water content (RWC) and photosynthetic capacity ( P n and pigment content). Moreover, the tolerant genotype exhibited higher activities of antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD). Conversely, F00142 exhibited less oxidative damage, as evidenced by lower levels of reactive oxygen species (H 2 O 2 , O 2 ·⁻), malondialdehyde (MDA), and electrolyte leakage. It also exhibited stronger osmotic adjustment, as reflected by greater accumulation of proline, soluble sugars, and soluble proteins. Transcriptomic profiling identified 18,510 differentially expressed genes (DEGs) in the sensitive genotype and 15,859 in the tolerant genotype. KEGG enrichment analysis highlighted common stress-response pathways, such as plant hormone signaling, while also revealing key divergent strategies between the two genotypes. The tolerant genotype enriched pathways associated with antioxidant synthesis, including glutathione and flavonoid metabolism, barrier formation, including cutin and suberin biosynthesis, and amino acid metabolism. WGCNA was subsequently employed to integrate transcriptomic data with key physiological traits, thereby enabling the identification of candidate modules and hub genes. Notably, the MEblue module exhibited a strong positive correlation with oxidative damage markers, whereas the MEpink module showed a negative correlation with osmotic regulators and antioxidant enzymes. The MEturquoise and MEyellow modules were associated with stress resilience, exhibiting positive correlation with relative water content (RWC) and negative correlation with damage metrics. This study reveals that superior saline-alkali tolerance in wild soybean is conferred by a synergistic and multi-layered adaptive strategy. This adaptive strategy is characterized by three key features, including enhanced osmotic adjustment and antioxidant capacity, preferential activation of specific detoxification and cellular protection pathways, and a more targeted transcriptional response that may reflect a potentially energy-conserving strategy. The co-expression modules correlated with physiological traits, along with their hub genes identified by WGCNA, provide a systems-level framework for understanding saline-alkali tolerance. Collectively, these findings provide critical insights into the complex regulatory networks governing saline-alkali stress adaptation and establish a valuable genetic resource for the molecular breeding of stress-resilient soybeans.