Molecular Soil Biology 2025, Vol.16, No.5, 230-240 http://bioscipublisher.com/index.php/msb 237 in the nitrogen fixation process. For instance, the proteome of the mitochondria in root nodules has undergone reprogramming, which makes the supply of carbon sources such as malic acid more abundant and simultaneously enhances ATP synthesis, directly supporting the high-energy-consuming reactions of nitrogenase. The rich variety of membrane proteins, transport proteins and signal proteins indicates that the exchange of substances and signals at the symbiotic interface is very complex. Proteomics also reveals that environmental conditions such as phosphorus stress and nitrogen supply can significantly affect the expression of root nodule proteins and nitrogen fixation efficiency. Comparative studies have found that in root nodules with high nitrogen fixation efficiency, the levels of proteins related to energy metabolism, stress resistance and defense, and signal regulation are significantly increased, which is directly related to stronger nitrogenase activity and ammonia assimilation capacity. Small heat shock proteins such as GmHSP17.1/17.9, phosphatase GmPAP12, and phosphorus transporter GmPT7 have been verified, providing specific targets for molecular breeding and bioengineering. These results not only deepen the understanding of the molecular mechanism of nitrogen fixation, but also provide theoretical basis and tools for the screening and cultivation of highly efficient nitrogen-fixing soybeans. Proteomics will continue to drive research on nitrogen fixation mechanisms in the future and also provide new ideas for sustainable agriculture. With the integration of proteomics with transcriptomics, metabolomics, and epigenomics, as well as the development of single-cell and spatial proteomics, we will be able to more clearly observe the cellular differences and dynamic changes in root tumor development, nitrogen fixation regulation, and environmental adaptation. The application of proteomic markers and functional proteins is expected to accelerate the breeding of highly efficient nitrogen-fixing soybean varieties, reduce the use of chemical fertilizers, increase yield and quality, and promote the development of green and low-carbon agriculture. Meanwhile, proteomics can also provide a molecular basis for root nodule bioengineering and climate-smart agriculture, helping to address global nitrogen management and environmental challenges. Acknowledgments We would like to thank our research team members for their dedicated work and collaborative spirit, which were instrumental to the success of this project. Conflict of Interest Disclosure The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References Arifuzzaman M., Mamidi S., Sanz-Saez A., Zakeri H., Scaboo A., and Fritschi F., 2023, Identification of loci associated with water use efficiency and symbiotic nitrogen fixation in soybean, Frontiers in Plant Science, 14: 1271849. https://doi.org/10.3389/fpls.2023.1271849 Balparda M., Bouzid M., Del Pilar Martinez M., Zheng K., Schwarzländer M., and Maurino V., 2023, Regulation of plant carbon assimilation metabolism by post-translational modifications, The Plant Journal: for Cell and Molecular Biology, 114(5): 1059-1079. https://doi.org/10.1111/tpj.16240 Baysoy A., Bai Z., Satija R., and Fan R., 2023, The technological landscape and applications of single-cell multi-omics, Nature Reviews. Molecular Cell Biology, 1-19. https://doi.org/10.1038/s41580-023-00615-w Brandão A., Brandão A., De Sousa Barbosa H., De Sousa Barbosa H., Arruda M., and Arruda M., 2010, Image analysis of two-dimensional gel electrophoresis for comparative proteomics of transgenic and non-transgenic soybean seeds, Journal of Proteomics, 73(8): 1433-1440. https://doi.org/10.1016/j.jprot.2010.01.009 Brear E., Bedon F., Gavrin A., Kryvoruchko I., Torres-Jerez I., Udvardi M., Day D., and Smith P., 2020, GmVTL1a is an iron transporter on the symbiosome membrane of soybean with an important role in nitrogen fixation, The New Phytologist, 228(2): 667-681. https://doi.org/10.1111/nph.16734 Brewis I., and Brennan P., 2010, Proteomics technologies for the global identification and quantification of proteins, Advances in Protein Chemistry and Structural Biology, 80: 1-44. https://doi.org/10.1016/B978-0-12-381264-3.00001-1
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