Proteomic, Transcriptomic, Biochemical and Physiological Investigations Shed Light on Responses to Low Temperature Stress in Two Contrasting Soybean Varieties

ABSTRACT Cold stress is the major abiotic factor limiting crop productivity. However, compared to model plant Arabidopsis Thaliana , crucial genes and underlying molecular mechanisms involved in soybean cold stress remain underexplored. Here, we investigate two national soybean cultivars, HH43 and HX3, bred from the northeast and southwest regions of China, which exhibit significant differences in cold tolerance. Morphological and biochemical examinations show that, compared to HH43, HX3 exhibits delayed wilting, reduced oxidative damage, and elevated antioxidant enzyme activities under low temperature (4°C). Transcriptomic analyses show distinct patterns between HH43 and HX3, in which HH43 displays a rapid gene response, while HX3 exhibits a gradual increase. Particularly, we identify MEblue and MEgreen modules related to cold stress and construct their GENIE3 networks. Key TFs such as ATAF1 and its potential targets were identified, which likely contribute to the cold tolerance differences between HH43 and HX3. Moreover, proteomic analyses reveal a broader and more sustained protein upregulation associated with maintaining metabolic activity and cellular homeostasis in cold‐tolerant HX3, while limited proteomic response was observed in cold‐sensitive HH43 and degradation of certain early responsive proteins. These findings provide substantial resources for further functional research and breeding cold‐tolerant soybean cultivars.