Cutting off the dual-stress ROS tap: NADPH oxidase inhibition rescues maize roots.

Maize roots suffer severe growth inhibition under combined drought and cold, yet the underlying trigger of this damage remains elusive. To address this, seeds with 5-6 mm radicles were placed on moistened filter paper for stress treatments with or without 1 mM imidazole (IMZ) treatment, and we quantified NADPH oxidase (NOX) activity, reactive oxygen species (ROS), antioxidant enzyme activities, redox status, lipid peroxidation, stress-responsive gene expression, root viability, and root growth. Combined stress synergistically upregulated ZmRbohA and ZmRbohB, boosting NOX activity by 540% and triggering O2-· and H2O2 bursts that overwhelmed antioxidant defenses despite upregulated superoxide dismutase, catalase, and ascorbate peroxidase activities, reduced AsA/DHA ratios by 96%, and repressed ZmEXPA by 79%. IMZ treatment suppressed NOX hyperactivation by 52%, mitigated ROS accumulation, modulated antioxidant enzyme activities, preserved redox homeostasis and root viability, reduced lipid peroxidation by 56%, partially derepressed ZmEXPA, and increased root length and fresh weight to ∼309% and 304% of the combined-stress level, approaching single-stress levels. These findings identify NOX hyperactivation as a critical bottleneck in compound stress-induced growth collapse, establishing "ROS source control" as a mechanistic advance. Hence, IMZ holds potential as a seed-coating agent for spring-sown maize in high-latitude regions.