Genome-Wide Identification of 109 NAC Genes and Dynamic Expression Profiles Under Cold Stress in Madhuca longifolia

<i>Madhuca longifolia</i> (<i>M. longifolia</i>), a tropical tree valued for its medicinal, nutritional, and industrial applications, exhibits severe sensitivity to low-temperature stress in subtropical regions, particularly during seedling establishment. To address this challenge, this study systematically identified 109 NAC genes in <i>M. longifolia</i> and characterized their functional roles in cold adaptation via multi-omics analyses. All NAC proteins were hydrophilic. Key members (e.g., <i>MlNAC026</i>, <i>MlNAC077</i>, <i>MlNAC076</i>) were localized in the nucleus. Phylogenetic analysis grouped them with <i>ANAC072</i> (RD26), a homolog involved in leaf senescence and ABA-regulated cold stress responses. The NAC family expanded primarily through segmental duplication. And low Ka/Ks ratios (<1) indicated purifying selection. Promoter analysis highlighted the prevalence of dehydration-responsive DRE and LTR cis-acting elements. Transcriptomic profiling under cold stress identified five continuous differentially expressed genes (<i>MlNAC026</i>, <i>MlNAC040</i>, <i>MlNAC059</i>, <i>MlNAC077</i>, and <i>MlNAC078</i>) linked to regulatory functions. Homology modeling predicted 3D structures of cold-responsive NAC proteins, and STRING network analysis indicated independent regulatory mechanisms due to the absence of prominent interaction nodes. These findings advance our understanding of NAC-mediated cold tolerance and offer genetic targets to enhance <i>M. longifolia</i> resilience in subtropical climates.