Molecular Pathogens, 2025, Vol.16, No.5, 226-235 http://microbescipublisher.com/index.php/mp 232 Salt stress also interferes with the symbiotic nitrogen fixation process of soybean. The high-salt environment causes osmotic stress and ion toxicity to both soybeans and rhizobia: on the one hand, salt damage inhibits plant root growth, changes the structure and secretion composition of root hairs, and reduces the success rate of rhizobia infection and nodulation; on the other hand, excessive salt content dehydrates rhizobia cells, disrupts metabolism, and interferes with the function of its nitrogen-fixing-related enzymes (Wang et al., 2025). Heavy metal pollution poses severe challenges to symbiotic nitrogen fixation. Heavy metal ions such as cadmium (Cd), lead (Pb), and zinc (Zn) in the soil can poison rhizobia and damage plant root cells, hindering the normal nodulation process. These metal ions accumulate in root nodules, interfere with the structure and function of nitrogenase and induce oxidative stress, leading to premature root nodule aging and extremely low nitrogen fixation efficiency. Although some rhizobia strains have some heavy metal tolerance, overall heavy metal stress significantly reduces the contribution of biological nitrogen fixation (Miransari et al., 2013). 6.3 Interaction between nitrogen fertilizer application and symbiotic nitrogen fixation system The application of chemical nitrogen fertilizers and the symbiotic nitrogen fixation of leguminous crops wax and wane. When exogenous nitrogen levels in the soil are too high, soybeans reduce their reliance on symbiotic nitrogen fixation, preferentially absorb inorganic nitrogen, and inhibit new nodulation and nitrogenase activity through feedback mechanisms. This "nitrogen suppression" effect leads to a reduction in the number of soybean nodules, smaller nodules, a decrease in the expression of nitrogen fixation-related genes under nitrogen fertilization conditions, and the entire symbiotic nitrogen fixation process is significantly weakened (Wysokinski et al., 2024). The mechanism by which nitrogen fertilizer inhibits nitrogen fixation involves responses from both the host and rhizobia. On the one hand, high concentrations of nitrate nitrogen (NO₃⁻) induce the production of CLE signal peptide in plant roots, inhibiting the formation of new root nodules through self-regulatory pathways; on the other hand, too much ammonium nitrogen (NH₄⁺) can directly interfere with rhizobia nitrogenase synthesis - when exogenous nitrogen is sufficient, rhizobia nif gene expression is inhibited by nitrogen metabolites (Li et al., 2020). In addition, abundant nitrogen sources also cause plants to reduce carbon allocation to root nodules, resulting in a decrease in the nitrogen-fixing activity of existing root nodules. 7 Molecular Breeding and Gene Editing Improve Nitrogen Fixation Efficiency 7.1 Molecular markers and assisted breeding of nitrogen fixation-related genes The use of molecular marker-assisted breeding can accelerate the improvement of nitrogen fixation-related traits in soybeans. In recent years, researchers have identified some gene loci related to traits such as nodule number, nitrogenase activity or plant nitrogen accumulation through genome-wide association analysis (GWAS) and quantitative trait loci (QTL) mapping. Molecular markers developed based on these loci can be used to rapidly screen germplasm with high nitrogen fixation potential in breeding (Bopape and Hassen, 2013). By introducing excellent nitrogen-fixing traits into main cultivated varieties, it is expected to reduce the dependence of agricultural production on chemical fertilizer nitrogen. 7.2 Application of CRISPR/Cas system in soybean nitrogen fixation improvement Gene editing technologies (such as CRISPR/Cas) provide new tools for directly improving symbiotic nitrogen fixation in soybeans. CRISPR/Cas can be used to carry out targeted modification of key genes affecting nodulation and nitrogen fixation in the soybean genome. Researchers have tried to knock out certain genes in the self-inhibitory nodulation pathway to relieve the restriction on nodulation number; they have also tried to promote root nodule development and nitrogenase activity by editing the plant kinase signaling pathway. Recent cases have shown that knocking out a gibberellin receptor gene in soybean can significantly increase the number of root nodules and improve nitrogen fixation contribution and grain yield (Yao et al., 2023). This shows that gene editing can specifically enhance the nitrogen-fixing ability of soybeans without affecting other agronomic traits.
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