Melatonin Enhances Heat Tolerance via Increasing Antioxidant Enzyme Activities and Osmotic Regulatory Substances by Upregulating zmeno1 Expression in Maize (Zea mays L.)

Heat stress severely affects the yield and quality of maize. Melatonin (N-acetyl-5-methoxy-tryptamin, MT) plays an important role in various types of stress resistance in plants, including heat tolerance. Enolase (ENO, 2-phospho-D-glycerate hydrolyase) contributes to plant growth, development, and stress response. As of now, the molecular mechanisms by which MT and ENO1 affect heat tolerance are unknown. In our research, we have revealed that heat stress (H) and heat stress + MT (MH) treatment upregulate <i>ZmENO1</i> expression levels by 15 and 20 times, respectively. <i>ZmENO1</i> overexpression and mutant maize lines were created by transgenic and genome editing. These results illustrate that heat stress has a significant impact on the growth of maize at the seedling stage. However, ZmENO1-OE lines showed a lower degree of susceptibility to heat stress, whereas the mutant exhibited the most severe effects. Under heat stress, exogenous application of MT improves heat resistance in maize. The ZmENO1-OE lines exhibited the best growth and highest survival rate, while the <i>zmeno1</i> mutants showed the least desirable results. Following treatment with H and MH, the level of MT in ZmENO1-OE lines exhibited the greatest increase and reached the maximum value, whereas the level of MT in the <i>zmeno1</i> mutant was the lowest. Heat stress decreased the maize's relative water content and fresh weight, although ZmENO1-OE lines had the highest and <i>zmeno1</i> mutants had the lowest. Heat stress led to an increase in the levels of MDA, hydrogen peroxide, and superoxide in all plants. Additionally, the ionic permeability and osmotic potential of the plants were significantly increased. However, the levels of MT were decreased in all plants, with the greatest decrease observed in the ZmENO1-OE lines. Interestingly, the <i>zmeno1</i> mutant plants had the highest expression levels of MT. Heat stress-induced upregulation of <i>ZmSOD</i>, <i>ZmPOD</i>, <i>ZmAPX</i>, <i>ZmCAT</i>, <i>ZmP5CS</i>, and <i>ZmProDH</i> in all plants. However, the ZmENO1-OE lines exhibited the greatest increase in expression levels, while the <i>zmeno1</i> mutants showed the lowest increase following MT spraying. The patterns of SOD, POD, APX, and CAT enzyme activity, as well as proline and soluble protein content, aligned with the variations in the expression levels of these genes. Our findings indicate that MT can upregulate the expression of the <i>ZmENO1 gene</i>. Upregulating the <i>ZmENO1 gene</i> resulted in elevated expression levels of <i>ZmSOD</i>, <i>ZmPOD</i>, <i>ZmAPX</i>, <i>ZmCAT</i>, <i>ZmP5CS,</i> and <i>ZmProDH</i>. This led to increased activity of antioxidant enzymes and higher levels of osmoregulatory substances. Consequently, it mitigated the cell membrane damage caused by heat stress and ultimately improved the heat resistance of maize. The results of this study provide genetic resources for molecular design breeding and lay a solid foundation for further exploring the molecular mechanism of MT regulation of heat stress tolerance in maize.