Synergistic responses of physiological, transcriptomic, and metabolomic levels in soybean (Glycine max (Linn.) Merr) under combined salt-alkali stress

Salt and alkali stresses frequently co-occur in natural environments, substantially inhibiting soybean ( Glycine max (Linn.) Merr) growth and yield. However, the regulatory networks underlying soybean adaptation to combined salt-alkali stress remain largely unclear. This study aimed to elucidate these mechanisms by integrating phenotypic, physiological, transcriptomic, and metabolomic analyses. Four soybean varieties with differing salt and alkali tolerance— Hedou NO. 6 , Heihe 35 , Zhonghuang 929 , and Zhonghuang 911 —were screened based on comprehensive evaluations conducted at the Agricultural Research Institute. Phenotypic and agronomic traits confirmed their tolerance/sensibility at the seedling stage. The physiological results of the four soybean varieties under control, salt, alkali, and combined salt-alkali stress conditions indicated that combined salt-alkali stress induced unique physiological and biochemical responses, including increased reactive oxygen species and altered photosynthetic parameters. Targeted metabolomic and transcriptomic analyses of the tolerant varieties revealed that key regulatory pathways related to flavonoids, starch and sucrose, α-linolenic acid metabolism, and amino acid biosynthesis were identified as involved in this response, with significant upregulation of relevant genes and metabolites. Notably, key metabolites such as L-homocysteine (C00155), catechin (C06562) and kaempferol (C05903) were upregulated 2–64 fold under combined salt-alkali stress compared with the control. Additionally, key genes such as HIDH (K13258) and AAT (K14454) were upregulated by 2–29-fold under combined salt-alkali stress compared with the control group, indicating that they may be central to key regulatory pathways. These findings advance the understanding of soybean responses to multiple abiotic stresses and provide a theoretical foundation for breeding varieties with enhanced tolerance to salt, alkali, and combined salt-alkali stress. Regulatory mechanisms underlying soybean responses to combined salt-alkali stress. Upward and downward arrows represent positive and negative responses, respectively, and are displayed to the right of the corresponding genes, metabolites, and physiological parameters. The four pathways in the lower left corner are shown in different colors. The CK, S, A and SA indicated control, salt, alkali and combined stress；POD (peroxidase), CAT (catalase), SOD (superoxide dismutase), Phi2 (net photosynthetic rate), PhiNPQ (non-photochemical quenching coefficient), qP (photochemical quenching coefficient), ETR (photosynthetic electron transport efficiency), and Fv/Fm (maximum photochemical quantum efficiency of photosystem II). • Revealed the molecular mechanisms of soybean responses to combined salt-alkali stress. • Identified the key pathways involved in soybean responses to combined salt-alkali stress. • Screened specific DEGs and DAMs responding to combined salt-alkali stress within the key pathways. • Protein-protein interaction (PPI) analysis determined core DEGs in the key pathways. • WGCNA identified key genes associated with soybean responses to combined salt-alkali stress.