Molecular Soil Biology 2025, Vol.16, No.5, 230-240 http://bioscipublisher.com/index.php/msb 230 Research Article Open Access Proteomic Analysis of Soybean Nodules: Insights into Efficient Nitrogen Fixation Xiaoxi Zhou, Tianxia Guo Institute of Life Sciences, Jiyang College, Zhejiang A&F University, Zhuji, 311800, Zhejiang, China Corresponding email: tianxia.guo@jicat.org Molecular Soil Biology, 2025, Vol.16, No.5 doi: 10.5376/msb.2024.15.0021 Received: 19 Jul., 2025 Accepted: 26 Aug., 2025 Published: 12 Sep., 2025 Copyright © 2025 Zhou and Guo, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Zhou X.X., and Guo T.X., 2025, Proteomic analysis of soybean nodules: insights into efficient nitrogen fixation, Molecular Soil Biology, 16(5): 230-240 (doi: 10.5376/msb.2024.15.0021) Abstract The protein research on soybean root nodules has provided effective assistance for humans to understand the molecular mechanism of efficient nitrogen fixation. This study mainly summarizes the progress made in the field of soybean rhizoma protein research in recent years, with a focus on several aspects: energy metabolism, signal transduction, antioxidant defense, and nutrient transport, etc. By comparing different proteomes, research has found that the levels of related proteins in highly efficient nitrogen-fixing root nodules significantly increase during processes such as energy supply, stress resistance and defense, and signal regulation. This is directly related to the enhanced activity of nitrogenase and the improved assimilation capacity of ammonia. Further research has found that when nutrients such as phosphorus and nitrogen are insufficient, the protein expression and nitrogen fixation efficiency in root nodules will also be affected. Some proteins (such as GmHSP17.1, GmSPX8 and GmPAP12) play an important regulatory role in nitrogen fixation under adverse conditions. There are still some undeniable limitations in current proteomics research: insufficient coverage, limited dynamic range, and inadequate spatial resolution, etc. However, with the development of new technologies, these limitations are expected to be broken through in the future. The combination of single-cell and spatial proteomics, multi-omics, and artificial intelligence modeling may all lead to deeper research and development in this field. The aim of this study is to promote the improvement of soybean nitrogen fixation capacity at the molecular level in the future by summarizing these advancements. Keywords Soybean root nodules; Proteomics; Biological nitrogen fixation; Energy metabolism; Molecular breeding 1 Introduction Soybeans are one of the most important leguminous crops in the world. It is not only an important source of protein for humans and animals, but also plays a significant role in the sustainable development of agriculture. Soybeans have a special symbiotic nitrogen fixation capacity. It can cooperate with rhizobia to convert nitrogen in the air into nitrogen that plants can utilize, thereby reducing the use of chemical fertilizers. This can reduce environmental pollution, improve soil quality and increase yield (Chen et al., 2018; Wang et al., 2020; Yang et al., 2021). The nitrogen fixation process of soybeans occurs in the root nodules at the roots. This process is very complex, involving signal communication between plants and microorganisms, as well as efficient energy and substance transport, and multi-level metabolic regulation (Yang et al., 2022; Sun et al., 2023). The formation and function of root nodules rely on the cooperation of many proteins, which are related to energy metabolism, signal transduction, antioxidant defense and nutrient transport, etc. (Oehrle et al., 2008). During the nitrogen fixation process, significant metabolic changes occur in root tumor cells, such as the preferential allocation of carbon sources, the regulation of mitochondrial energy, and the efficient utilization of nutrients such as phosphorus and iron (Chen et al., 2018; Brear et al., 2020). Proteomics is an important tool for studying biological functions and molecular mechanisms. It can help systematically analyze the development process of root nodules, nitrogen fixation efficiency, and the response mechanism to environmental stress (such as phosphorus and nitrogen deficiency) (Oehrle et al., 2008; Lyu et al., 2022; Yao et al., 2022). Through quantitative and spatially resolved proteomics analysis, researchers have
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