Legume Genomics and Genetics 2026, Vol.17, No.1, 14-31 http://cropscipublisher.com/index.php/lgg 20 Transcriptomic analyses across species indicate coordinated upregulation of genes involved in sucrose metabolism, the oxidative pentose phosphate pathway, and redox homeostasis in nodules, reflecting the tight coupling of carbon supply, oxygen flux, and nitrogenase regulation (Feng et al., 2021). Functional genomics has also identified a wide array of genes involved in ammonia assimilation, nitrogen transport, and nodule cell differentiation that collectively determine nitrogen fixation efficiency. Genes encoding glutamine synthetase, glutamate synthase, asparagine synthetase, ureide biosynthesis enzymes, and multiple transporter families (for ammonium, amino acids, ureides, and other metabolites) are highly expressed in nodules and display cell-type–specific expression patterns in soybean and L. japonicus (Feng et al., 2021; Wang et al., 2022). Single-nucleus and cell-type–resolved datasets in soybean mapped key steps of the ureide synthesis pathway to distinct nodule cell types, revealing spatial compartmentalization of nitrogen metabolism genes during fixation. In addition, large and variable families of NCR peptides in IRLC legumes such as pea and red clover play central roles in bacteroid differentiation, persistence, and, likely, nitrogen fixation efficiency; genomic surveys have identified hundreds of NCR genes clustered in legume genomes, with species-specific expansions and diverse expression patterns, suggesting rapid evolution of these effectors (Zorin et al., 2022). Transcriptomic comparison of red clover genotypes with contrasting biological nitrogen fixation efficiency identified nearly 500 differentially expressed genes, with significant enrichment of tandem- and dispersedly duplicated genes, underscoring the contribution of gene family expansion and regulatory divergence to fixation performance. Finally, regulatory genes, including NAC-type transcription factors controlling nitrate-induced nodule senescence, miRNAs modulating nodule metabolism, and numerous nodule-enriched transcription factors and transporters identified through high-throughput “omics” approaches, form higher-order networks that coordinate nitrogenase activity with plant nutritional and environmental cues (Schwember et al., 2019; Qiao et al., 2023). Together, these functional and regulatory gene sets illustrate how comparative genomics and transcriptomics across legumes are revealing both conserved and species-specific modules that can be targeted to enhance nitrogen fixation and nitrogen-use efficiency in major legume crops. 5 Transcriptional Regulation and Functional Validation of Nitrogen Fixation-Related Genes 5.1 Transcriptional regulatory networks Transcriptional regulation of symbiotic nitrogen fixation (SNF) in legumes is organized in hierarchical networks that integrate developmental, nutritional, and environmental signals. Central transcription factors such as NODULE INCEPTION (NIN) and NIN-LIKE PROTEINS act at early stages to couple Nod factor signaling with infection and nodule organogenesis, while downstream networks modulate nodule maturation, nitrogenase activity, and senescence (Qiao et al., 2023). Large-scale expression and co-expression analyses in soybean and other legumes have identified hundreds of transcription factors correlated with SNF traits, including components of the circadian clock (e.g., GmLHY1b), stress-responsive factors, and regulators of lipid and defense signaling, highlighting the polygenic and distributed nature of SNF control (Li et al., 2025). Weighted gene co-expression and independent component analyses have further delineated modules enriched in transcription factors that track variation in nodule number, nodule weight, and nitrogen fixation efficiency, many of which map to domestication sweeps, implying selection on regulatory circuitry during crop improvement (Qiao et al., 2023). In addition, transcriptional networks governing responses to fluctuating mineral nitrogen have been partially resolved, with NAC-type transcription factors (soybean SNAPs; Lotus LjNAC094) identified as master regulators of nitrate-induced nodule senescence, linking external nitrogen status to coordinated reprogramming of senescence-associated and metabolic genes in nodules (Haskett et al., 2025). Downstream branches of these networks often rely on interconnected transcription factor cascades and feedback loops involving both protein-coding genes and non-coding RNAs. In common bean, the SRS/STY family transcription factor PvSRS10 is transcriptionally activated by NF-Y complexes and by the MADS-box factor PvFUL-like, positioning it within the NIN–NF-Y cascade that conveys early symbiotic signals into developmental responses; protein interaction predictions suggest that other PvSRS members interface with auxin signaling components, providing a mechanistic link between transcriptional control and hormone-mediated nodule
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