Abstract
Madhuca longifolia (M. longifolia), 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 M. longifolia and characterized their functional roles in cold adaptation via multi-omics analyses. All NAC proteins were hydrophilic. Key members (e.g., MlNAC026, MlNAC077, MlNAC076) were localized in the nucleus. Phylogenetic analysis grouped them with ANAC072 (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 (MlNAC026, MlNAC040, MlNAC059, MlNAC077, and MlNAC078) 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 M. longifolia resilience in subtropical climates.