Legume Genomics and Genetics 2026, Vol.17, No.1, 14-31 http://cropscipublisher.com/index.php/lgg 21 morphogenesis (Ayra et al., 2025). MicroRNA-centered modules add additional layers of control: in common bean and soybean, the miR156–SPL and miR172–AP2 circuits form an interconnected cascade, where SPL transcription factors and MADS-domain proteins directly activate MIR172c transcription, and miR172c in turn modulates AP2-like repressors of nodulation. These miRNA–TF feed-forward loops fine-tune nodule number and function and are responsive to nitrogen supply and developmental stage (De Lima et al., 2022). Integrative genomic and transcriptomic analyses across legumes suggest that many nodulation-related TF families (NAC, GRAS, NF-Y, MADS, SRS, WRKY) have undergone lineage-specific expansions, with paralogs showing nodule-enriched and condition-specific expression patterns, consistent with subfunctionalization and specialization within the broader SNF regulatory landscape (Qiao et al., 2016). 5.2 Epigenetic regulatory mechanisms Epigenetic regulation is emerging as a key dimension shaping transcriptional programs during nodulation and nitrogen fixation in major legume crops. Studies in peanut provide one of the first high-resolution views of three-dimensional chromatin organization in roots and nodules, revealing that approximately 2% of the genome switches from a repressive B compartment to an active A compartment in nodules, accompanied by extensive remodeling of topologically associated domains and increased long-range cis-interactions at symbiosis-related loci. Integration of Hi-C with ATAC-seq and histone modification profiling has identified dynamic local open chromatin regions and histone mark changes around nodulation genes, including a chromatin loop mediated by the repressive H3K27me3 mark that appears to regulate NIN expression, and another loop that highlights the nodule-expressed gene AhMsrA as a positive regulator of nodulation. DNA methylation also contributes to fine-tuning symbiotic gene activity: in Medicago truncatula, the DNA demethylase DEMETER is essential for proper nodule development, regulating expression of genes involved in nodule differentiation, while nodule-specific cysteine-rich (NCR) peptide genes show extensive hypomethylation correlated with high expression and active histone marks (Wang et al., 2025). In common bean and soybean, DNA methylation, histone modifications, and small RNAs together form a multilayered regulatory system that influences both nodule initiation and lifespan. Adenine methylation patterns in rhizobial genomes change during symbiosis, and rhizobial tRNA-derived small RNA fragments are transferred into plant cells, where they exploit the host RNA interference machinery to cleave specific mRNA targets and thereby increase nodule number. On the plant side, histone lysine methyltransferases have been implicated in modulating nodule number and bacterial colonization, while numerous nodulation-associated microRNAs (including, but not limited to, miR156 and miR172) regulate transcription factors and transporters important for nodule development and nitrogen metabolism (De Lima et al., 2022). Integrative chromatin-accessibility and expression studies in soybean diversity panels have shown that expression quantitative trait loci for many SNF-related genes are enriched within open chromatin regions in nodules, providing population-level evidence that variation in cis-regulatory elements and local chromatin state underlies natural diversity in SNF performance (Li et al., 2025). Together, these findings indicate that epigenetic remodeling of chromatin architecture, DNA methylation landscapes, histone marks, and small RNA profiles is tightly intertwined with transcription factor networks in controlling where, when, and how strongly nitrogen fixation genes are expressed in legume crops. 5.3 Functional gene validation techniques The rapid growth in candidate nitrogen fixation-related genes identified by comparative genomics and expression profiling has spurred extensive use of functional validation approaches in legumes. Classical forward genetics, often using fast-neutron or chemical mutagenesis, remains a powerful tool: in Medicago truncatula, fast-neutron mutagenesis combined with whole-genome sequencing enabled the identification of a symbiotic nitrogen fixation-defective mutant harboring a large deletion on chromosome 3, providing direct functional links between lost genomic segments and SNF phenotypes (Shen et al., 2023). Transcriptomic analysis of chromosome segment substitution lines in peanut with severely impaired nitrogen fixation similarly uncovered differential expression of key symbiotic regulators (including NIN, EFD and SNF-related transporters) and pinpointed FEN1-encoding a homocitrate synthase essential for nitrogenase activity-as a strong candidate gene underlying the defective
RkJQdWJsaXNoZXIy MjQ4ODYzNA==