ZmbHLH30 Enhances Cold Tolerance During Maize Germination

Low temperature is a major abiotic stress that affects maize across its entire growth cycle, with the germination stage being particularly sensitive. To investigate the genetic basis of early-stage cold tolerance, we used quantitative trait locus mapping and identified <i>ZmbHLH30</i> as a candidate gene regulating maize responses to low temperature. The ZmbHLH30 protein is localized in the cytoplasm of maize protoplasts, and <i>ZmbHLH30</i> promoter drives <i>β</i>-glucuronidase (GUS) expression in <i>Arabidopsis thaliana</i> leaves. The promoter region of <i>ZmbHLH30</i> contains multiple environmental stress-responsive elements, including motifs associated with cold and auxin responses. Overexpression of <i>ZmbHLH30</i> significantly enhanced cold tolerance at the germination, bud, and seedling stages, with the strongest effect observed during germination, where the cold-tolerance <i>D</i>-value increased by 0.366 relative to the control. In contrast, CRISPR/Cas9 knockout lines showed a 0.399 decrease in <i>D</i>-value. Under cold stress, <i>ZmbHLH30</i> expression was markedly induced in overexpression lines but suppressed in knockout lines. Integrated transcriptomic and metabolomic analyses further identified <i>ZmbHLH30</i> as a key regulator of cold tolerance in maize.