Phase-Dependent Redox and Antioxidant Response During Early Heat Stress in Hordeum vulgare L. Seedlings

Rising global temperatures and increasingly frequent heat waves threaten cereal crop productivity, but the temporal coordination of early redox, oxidative, and heat-protective reactions in barley remains insufficiently characterized. In this study, we investigated these responses in Hordeum vulgare L. seedlings exposed to short-term heat stress (STHS; 40 °C for 15 min, 30 min, 1 h, and 6 h). Spectrophotometrically measured hydrogen peroxide (H2O2) decreased from 15 min onward, whereas superoxide (O2–) remained near control levels during the first 30 min and declined at 1 and 6 h, indicating the absence of a sustained oxidative burst. In contrast, nitroblue tetrazolium (NBT) staining revealed a transient increase in O2–-related staining intensity and a sustained response in the apical leaf region, while 3,3′-diaminobenzidine (DAB) staining showed no detectable H2O2 accumulation. Lipid peroxidation increased significantly, peaking at 1 h, despite a decrease in overall reactive oxygen species (ROS) levels. The initial increase in ascorbate and proline levels was accompanied by various enzymatic reactions, including delayed regulation of antioxidant enzymes and increased aldehyde oxidase activity during prolonged exposure. Hsp70 accumulated rapidly and extensively, while Hsp90 exhibited a moderate increase. The obtained results support three partially overlapping stages of early heat acclimation in barley, progressing from an early redox and heat-shock response through transient oxidative membrane damage and spatial ROS heterogeneity to a later acclimation phase.