Legume Genomics and Genetics 2026, Vol.17, No.1, 14-31 http://cropscipublisher.com/index.php/lgg 17 which generate a systemic inhibitory signal that limits further nodule formation (Ferguson et al., 2018; Chaulagain and Frugoli, 2021). More recent studies have revealed a complementary positive systemic pathway involving CEP peptides and the CRA2 receptor, as well as a miR2111/TML module, together forming a balanced regulatory system that allows legumes to adjust nodule numbers according to nitrogen availability and energy status (Li et al., 2022; Yanlin et al., 2024). Nutrient-dependent regulation adds another layer of control: nitrate and other mineral nutrients modulate the expression and activity of NIN, NIN-like proteins (NLPs), and multiple hormone pathways, thereby integrating soil nitrogen signals with symbiotic development and nodule senescence (Qiao et al., 2023; Yanlin et al., 2024). Comparative and functional genomic analyses across legumes indicate that key components of AON, nutrient signaling, and developmental control reside in gene families with species-specific expansions and allelic diversity, providing a genomic basis for natural variation in nodule number, size, and persistence in major legume crops (Tsyganov and Tsyganova, 2020; Li et al., 2022; Yanlin et al., 2024). 2.3 Molecular Mechanisms Related to Nitrogen Fixation and Metabolism Once nodules are established and rhizobia differentiate into bacteroids within plant-derived symbiosomes, effective nitrogen fixation depends on a highly integrated set of metabolic and regulatory mechanisms that couple bacterial nitrogenase activity to plant carbon supply and nitrogen assimilation. In the nodule infection and fixation zones, bacteroids express nif and fix gene clusters encoding the nitrogenase complex and associated functions for electron transfer and respiration, enabling the reduction of atmospheric N2 to ammonia under microaerobic conditions (Schulte et al., 2021; Lepetit and Brouquisse, 2023). Plant leghemoglobins, encoded by multigene families, buffer free oxygen to maintain a low-oxygen environment compatible with nitrogenase while supporting the high respiratory demand of bacteroids; their absence or disruption leads to impaired fixation and early nodule senescence (Chaulagain and Frugoli, 2021). Metabolic modelling and ^13C-based flux analysis indicate that bacteroids rely primarily on plant-supplied dicarboxylates such as malate as energy and carbon sources, and that oxygen limitation constrains the decarboxylating arm of the tricarboxylic acid cycle, favoring ammonia release over assimilation (Schulte et al., 2021). This metabolic configuration, shaped by both plant and bacterial genomes, promotes efficient export of reduced nitrogen to the host, a defining feature of rhizobium–legume symbioses. On the plant side, ammonia derived from bacteroids is rapidly assimilated through the glutamine synthetase/glutamate synthase (GS–GOGAT) pathway and further incorporated into amino acids or ureides, which are then transported to shoots (Schwember et al., 2019). Nodule metabolism is tightly coordinated with whole-plant nitrogen and carbon status, involving regulation of N and C metabolic enzymes, oxygen flux, redox balance, and responses to phosphorus and other nutrients (Lepetit and Brouquisse, 2023; Yanlin et al., 2024). Systemic changes in sucrose allocation, oxidative pentose phosphate pathway activity, and redox status appear to serve as integrators of plant N demand and nodule functioning, influencing both nitrogenase activity and nodule senescence (Schwember et al., 2019; Lepetit and Brouquisse, 2023). High-throughput transcriptomic and other “omics” approaches have identified extensive transcriptional reprogramming of nodule tissues in response to nitrate addition and abiotic stresses, including differential expression of nodulins, nodule-specific cysteine-rich peptides, transporters, and regulatory proteins (Schwember et al., 2019). MicroRNAs and other small RNAs are emerging as important regulators linking nitrogen fixation, stress responses, and developmental transitions, complementing transcription factor networks such as those centered on NAC proteins that govern N-induced nodule senescence (Schwember et al., 2019; Qiao et al., 2023). Comparative genomic analyses across legumes, combined with functional genomics, are beginning to reveal how gene family diversification, regulatory sequence evolution, and network rewiring in key metabolic and regulatory genes underlie species-specific strategies of nitrogen transport (amides vs. ureides), oxygen management, and stress resilience in nodules (Sharma et al., 2020). These insights provide a mechanistic and genomic foundation for designing legume cultivars and symbiotic combinations with enhanced nitrogen fixation efficiency and stability under variable field conditions. 3 Identification of Nitrogen Fixation-Related Genes in Major Legume Crops 3.1 Genes involved in nitrogen fixation signal recognition Comparative genetic analyses across model and crop legumes have identified a core set of plant genes required for
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