Molecular Soil Biology 2025, Vol.16, No.5, 230-240 http://bioscipublisher.com/index.php/msb 233 enhanced in the mitochondria of root nodules, which improves the efficiency of ATP production. In addition, nitrogenase complexes (nifD, nifH, nifK) and regulatory proteins, as well as glutamine synthase and urease, are more abundant in highly efficient nitrogen-fixing rhizoma, promoting ammonia assimilation and transport (Oehrle et al., 2008; Cooper et al., 2017). Figure 1 General workflow of bioinformatics analysis in mass spectrometry-based proteomics. (a) MA-plot from protein differential abundance analysis. X-axis is the log2 transformed fold change and Y-axis is the average protein abundance from replicates. (b) Distribution of protein abundance data before and after normalization. (c) Heatmap for protein abundance with clustering; (d) Protein set enrichment analysis, Y-axis in the above plot shows the ranked list metric, and in the bottom plot shows the running enrichment score. X-axis is the ranked position in protein list. (e) Machine learning-based sample clustering. (f) Illustration of a network inferred from proteomics data. (g) Dimensionality reduction of proteomics expression profile (Adopted from Chen et al., 2020) 4.2 Stress-responsive proteins influencing nodule function (oxidative stress, drought, salinity) Under conditions of phosphorus deficiency, drought or salt stress, multiple stress-resistant proteins are upregulated in root nodules. Antioxidant enzymes, such as peroxidase and superoxide dismutase, enhance activity and eliminate excessive ROS, thereby protecting the activity of nitrogenase (Muneer et al., 2012; Yang et al., 2022; Yao et al., 2022). Some small molecule heat-shock proteins, such as GmHSP17.1, can regulate ROS levels, help cell wall growth, and ensure the normal development of root nodules and nitrogen fixation efficiency. During drought, proteins related to carbon metabolism, protein synthesis and amino acid metabolism undergo significant changes, thereby locally affecting nitrogen fixation activity (Gil-Quintana et al., 2013). 4.3 Post-translational modifications and their impact on protein activity Post-translational modifications of proteins (such as phosphorylation, acetylation, nitration and acylation) play an important role in the function of root nodule proteins. Apical proteomics (TDP) has discovered a variety of modifications, such as myoylation, palmitoylation and thiocyanation, which can affect the stability, localization and activity of proteins. Some modifications are closely related to nitrogen-fixing associated proteins, such as hemoglobin and nitrogenase (Matamoros and Becana, 2021; Zhou et al., 2022; Balparda et al., 2023) (Figure 2). In addition, red oxidation modifications (such as S-thionylation, S-nitroylation, etc.) can regulate the activity of stress resistance proteins and signaling proteins, helping root nodules adapt to environmental stress. 4.4 Crosstalk between proteomics and transcriptomics/metabolomics The combination of multiple omics shows that the proteome is closely linked with the transcriptome and metabolome, jointly regulating the nitrogen fixation efficiency. The combined analysis of proteomics and metabolomics revealed that exogenous nitrogen inhibits malic acid synthesis while increasing the synthesis of signaling molecules such as spermidine, nitric oxide, and asparagine, ultimately leading to a decrease in nitrogenase activity (Lyu et al., 2022; Xu et al., 2024). Combined proteomic and transcriptomic studies have also
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