Pangenome-Wide Identification, Evolutionary Analysis of Maize ZmPLD Gene Family, and Functional Validation of ZmPLD15 in Cold Stress Tolerance

<i>Phospholipase D (PLD)</i> genes play key roles in plant abiotic stress responses, but the systematic identification of the maize (<i>Zea mays</i>) PLD family and its cold tolerance mechanism remain unclear. Using 26 maize genomes (pangenome), we identified 21 ZmPLD members via Hidden Markov Model (HMM) search (Pfam domain PF00614), including five private genes-avoiding gene omission from single reference genomes. Phylogenetic analysis showed ZmPLD conservation with Arabidopsis and rice PLDs; Ka/Ks analysis revealed most <i>ZmPLDs</i> under purifying selection, while three genes (including <i>ZmPLD15</i>) had positive selection signals, suggesting roles in maize adaptive domestication. For <i>ZmPLD15</i>, five shared structural variations (SVs) were found in its promoter; some contained ERF/bHLH binding sites, and SVs in Region1/5 significantly regulated <i>ZmPLD15</i> expression. Protein structure prediction and molecular docking showed conserved ZmPLD15 structure and substrate (1,2-diacyl-sn-glycero-3-phosphocholine) binding energy across germplasms. Transgenic maize (B73 background) overexpressing ZmPLD15 was generated. Cold stress (8-10 °C, 6 h) and recovery (24 h) on three-leaf seedlings showed transgenic plants had better leaf cell integrity than wild type (WT). Transgenic plants retained 45.8% net photosynthetic rate (Pn), 47.9% stomatal conductance (Gs), and 55.8% transpiration rate (Tr) versus 7.6%, 21.3%, 13.8% in WT; intercellular CO₂ concentration (Ci) was maintained properly. This confirms ZmPLD15 enhances maize cold tolerance by protecting photosynthetic systems, providing a framework for <i>ZmPLD</i> research and a key gene for cold-tolerant maize breeding.