Molecular Soil Biology 2025, Vol.16, No.5, 230-240 http://bioscipublisher.com/index.php/msb 232 process consumes a very large amount of energy. For every 1 mole of nitrogen gas fixed, at least 16 moles of ATP and a large amount of reducing power are required (Wang et al., 2020). To ensure the activity of nitrogenase, root nodules provide support through efficient carbohydrate supply (such as sucrose and malic acid) and mitochondrial energy metabolism (Yang et al., 2021; Ke et al., 2022; Lyu et al., 2022). External nutritional conditions such as nitrogen and phosphorus can also affect the activity of nitrogenase and the lifespan of root nodules by regulating carbon flow, energy metabolism and some signaling molecules (such as nitric oxide and isoflavones) (Chen et al., 2018). 3 Advances in Proteomic Technologies Applied to Soybean Nodules 3.1 Historical overview: from 2D-gel electrophoresis to modern LC–MS/MS In the early stage, the protein research of soybean root nodules mainly used two-dimensional gel electrophoresis (2D-GE). This method can separate and quantify proteins, but it has low resolution, limited sensitivity and is easily interfered by high-abundance proteins (Brandao et al., 2010). Later, as mass spectrometry technology continued to advance, liquid chromatography-tandem mass spectrometry (LC-MS /MS) gradually replaced 2D-GE. It greatly increases the quantity and scope of protein identification, especially suitable for complex samples such as root nodules (Brewis and Brennan, 2010; Komatsu et al., 2017; Min et al., 2019; Zhou et al., 2022). Recently, proteomics based on independent data acquisition (DIA) has enabled us to study subcellular parts such as mitochondria of root nodules more clearly. 3.2 Quantitative proteomics (label-free, iTRAQ, TMT) There are an increasing number of quantitative proteomics methods, including label-free and isotope-labeled ones (such as iTRAQ, TMT). Label-free methods are suitable for large-scale samples and can quickly observe the changes in protein abundance. iTRAQ and TMT can quantitatively analyze multiple samples simultaneously, significantly enhancing the efficiency and accuracy of comparative studies. These methods have been widely applied in the studies of different developmental stages, adverse conditions and subcellular components of soybean root nodules (Komatsu et al., 2017; Min et al., 2020; Moradi et al., 2021). 3.3 Integration with subcellular proteomics (membrane, mitochondrial, peribacteroid) Subcellular proteomics helps reveal the distribution and function of nitrogen-fixing related proteins by isolating parts such as membranes, mitochondria and bacterioid-like membranes. For instance, the proteome of root tumor mitochondria shows that the energy metabolism pattern ADAPTS to the high energy consumption of nitrogen fixation (Wang and Komatsu, 2023). The proteomes of mycorrhizal membranes and Spaces reveal key processes of amino acid metabolism, nutrient transport and signal transduction. Meanwhile, the quantitative analysis of membrane proteins, mitochondrial proteins and peroxisome proteins also enables us to better understand the response of root tumors under environmental stress (Komatsu et al., 2017). 3.4 Bioinformatics pipelines for protein identification, annotation, and network analysis Proteomics is inseparable from bioinformatics. Commonly used software includes MaxQuant, ProteinPilot, Proteome Discoverer, OpenMS and Peaks Studio, etc. These tools can convert raw mass spectrometry data into protein quantitative information and analyze post-translational modifications and subcellular localization (Komatsu et al., 2017; Chen et al., 2020; Moradi et al., 2021) (Figure 1). Network analysis and machine learning methods can assist researchers in inferring protein interactions, signaling pathways and regulatory networks, and gain a more comprehensive understanding of the complex processes in root nodules (Hartman et al., 2023). 4 Proteomic Insights into Nitrogen Fixation Efficiency 4.1 Key proteins involved in carbon metabolism, ATP supply, and nitrogenase regulation The nitrogen fixation process of soybean root nodules requires a large amount of energy, so there must be sufficient carbon sources and ATP. Through proteomics research, both Ke et al. (2022) discovered that root tumor cells adjust carbon metabolism and prioritize the use of phosphoenolpyruvate (PEP) for the synthesis of malic acid. After entering the mitochondria, malic acid becomes the main source of NADH and ATP, directly providing energy for the nitrogenase reaction. Proteins related to the respiratory chain (such as complexes I and IV) are
RkJQdWJsaXNoZXIy MjQ4ODYzNA==