Molecular Soil Biology 2025, Vol.16, No.5, 230-240 http://bioscipublisher.com/index.php/msb 235 5.3 Key findings: differential expression of energy metabolism, antioxidant defense, and signaling proteins In highly efficient root nodules, the levels of energy metabolism-related proteins (such as respiratory chain complex, malate metabolase, ATP synthase) are higher, which can enhance ATP supply and meet the high energy consumption requirements of nitrogenase. The abundance of antioxidant defense proteins (such as peroxidase and small heat shock protein GmHSP17.1/17.9) was also higher, helping to eliminate reactive oxygen species and protect the normal function of nitrogenase (Yang et al., 2021; 2022). Signal transduction and transport proteins (such as Nod factor synthase, SPX protein, phosphatase GmPAP12/4) were also significantly upregulated, promoting root tumor development and nitrogen fixation process (Cooper et al., 2017; Wang et al., 2020; Xing et al., 2022; Sun et al., 2024). 5.4 Functional interpretation: how proteomic shifts correlate with nitrogen fixation capacity The proteome of highly efficient root nodules has undergone reprogramming, manifested as carbon flow priority supply, enhanced energy metabolism, upregulation of stress resistance proteins, and optimized signal regulation. These changes jointly enhanced the activity of nitrogenase and the assimilation efficiency of ammonia (Wang et al., 2020; Xing et al., 2022). For instance, malic acid synthesis and upregulation of respiratory chain proteins directly increase ATP supply, while enhanced antioxidant proteins delay root tumor senescence and maintain nitrogen fixation activity (Cooper et al., 2017; Yang et al., 2021; 2022). The differential expression of signals and transporters helps root nodules respond better to nutritional and environmental changes, thereby enhancing the overall efficiency of the symbiotic nitrogen fixation system (Sun et al., 2024). 5.5 Lessons learned: strategies for improving symbiotic performance in breeding programs. The results of comparative proteomics indicate that to enhance nitrogen fixation efficiency, the key lies in strengthening energy metabolism, stress resistance and defense, as well as signal regulation capabilities. Future breeding can focus on screening or improving related genes, such as GmHSP17.1/17.9, GmPAP12/4, GmSPX8, GmPT7, etc. By combining molecular marker-assisted selection and transgenic technology simultaneously, it is expected to cultivate new soybean varieties with higher nitrogen fixation efficiency (Wang et al., 2020; Xing et al., 2022; Yang et al., 2022; Sun et al., 2024). 6 Applications and Translational Potential 6.1 Breeding for high-efficiency nitrogen fixation using proteomic markers Proteomics has identified many pathways and proteins related to nitrogen fixation, and these results provide candidate markers for molecular breeding. For instance, the abundance of root tumor membrane proteins, signal transduction proteins, amino acid metabolic proteins and nutrient transport proteins is closely related to nitrogen fixation efficiency and can be used as molecular markers for screening high-efficiency nitrogen-fixing soybeans (Cooper et al., 2017; Chen et al., 2018; Xing et al., 2022; Ni et al., 2024). In addition, by combining the miRNA regulatory network with the proteome, researchers have identified some key genes that regulate nitrogen fixation efficiency and are of great value for breeding highly efficient nitrogen fixation varieties (Arifuzzaman et al., 2023). High-throughput spectral phenotypic technology can also be used for large-scale nitrogen fixation trait screening under field conditions (Vollmann et al., 2022). 6.2 Integration into systems biology models for soybean-rhizobia symbiosis Proteomic data have been integrated into the metabolic networks and systems biology models of soybeans and rhizobia, which enables us to understand and predict nitrogen fixation mechanisms more intuitively. For instance, metabolic modeling combining proteomics and genomic annotation can simulate the changes in nitrogen fixation efficiency after different genes are knocked out, and can also quantify the nitrogen fixation capacity of different soybean varieties and rhizobia symbiotic systems (Contador et al., 2020; Sun et al., 2023; Liu et al., 2023). In addition, the combination of single-cell and spatial transcriptomes with proteomes has revealed the cellular differences and regulatory networks of root nodules in development and nitrogen fixation zones, providing a basis for more precise regulation of symbiosis.
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