ZmEIL2 positively regulates drought tolerance in maize by modulating stomatal movement, hormonal homeostasis, and antioxidant defense

Drought stress severely constrains global maize productivity. Although ethylene (ETH)-responsive transcription factors play key roles in plant stress adaptation, the functional mechanism of ZmEIL2 in drought tolerance remains poorly understood. Here, transgenic maize lines with ZmEIL2 overexpressing (OE) and ZmEIL2 knockout (KO) were generated and subjected to progressive drought stress followed by rehydration. Phenotypic analysis indicated that ZmEIL2 OE lines exhibited improved plant survival and reduced leaf water loss, while KO lines exhibited displayed enhanced drought sensitivity. Physiological assessments demonstrated that ZmEIL2 preserves leaf relative water content by regulating stomatal movement and mitigates drought-induced membrane damage and lipid peroxidation, which correlated with increased proline (Pro) accumulation. Furthermore, ZmEIL2 modulated endogenous ETH and abscisic acid (ABA) levels under drought stress and enhanced the antioxidant defense system by increasing the activities of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX), thereby diminishing reactive oxygen species (ROS) accumulation, as evidenced by 3,3’-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining. We performed RNA sequencing (RNA-seq)on wild-type (WT), OE, and KO plants sampled at 0, 10, and 13 days of drought treatment and identified 120 core drought-responsive genes involved in stress perception, oxidoreductase activity, and metabolic reprogramming. Notably, 11 candidate genes, including ZmSOD16 , ZmPAL7 , ZmHSP101 , ZmHSP70 , ZmGA2ox3 , and ZmSMT1 , exhibited strictly opposite expression trends between OE and KO under normal conditions, suggesting their roles in antioxidant defense, protein protection, and hormone and carbohydrate metabolism. Collectively, these results demonstrate that ZmEIL2 as a positive regulator of drought tolerance in maize via a multi-layered regulatory mechanism encompassing stomatal regulation, hormonal balance, membrane integrity, osmotic adjustment, and ROS scavenging. This work provides valuable candidate genes for the breeding of drought-resilient maize varieties.