Involvement of ROS and calcium ions in developing heat resistance and inducing antioxidant system of wheat seedlings under melatonin’s effects

The stress-protective effect of melatonin (N-acetyl-5-methoxytryptamine) on plant cells is mediated by key signaling mediators, in particular calcium ions and reactive oxygen species (ROS). However, the links between changes in calcium and redox homeostasis and the formation of adaptive responses of cultivated cereals (including wheat) to the action of high temperatures have not yet been studied. In the present study, we investigated the possible involvement of ROS and calcium ions as signaling mediators in developing heat resistance in wheat (Triticum aestivum L.) seedlings and activating their antioxidant system. Treatment of 3-day-old etiolated seedlings with melatonin solutions at concentrations 0.01–10 µM increased their survival after exposure to 45 °C for 10 min. The most significant stress-protective effect was exerted by melatonin treatment at 1 µM concentration. Under the influence of melatonin, a transient enhancement of superoxide anion radical (O₂^•–) generation and an increase in hydrogen peroxide content were observed in roots, with a maximum at 1 h. Four hours after treatment with melatonin, the activity of catalase and guaiacol peroxidase increased in roots, while the activity of superoxide dismutase did not change significantly. After exposure to 45 °C, the activity of catalase and guaiacol peroxidase was higher in the roots of melatonin-treated wheat seedlings, and the indices of ROS generation, content of the lipid peroxidation product malonic dialdehyde, and cell membrane damage were lower than in control seedlings. Melatonin-induced changes in root ROS generation and antioxidant enzyme activities were eliminated by pretreatment with the hydrogen peroxide scavenger dimethylthiourea (DMTU), NADPH oxidase inhibitor imidazole, and calcium antagonists (the extracellular calcium chelator EGTA and phospholipase C inhibitor neomycin). Treatment with DMTU, imidazole, EGTA, and neomycin also abolished the melatonin-induced increase in survival of wheat seedlings after heat stress. The role of calcium ions and ROS, generated with the participation of NADPH oxidase, as signaling mediators in the melatonin-induced antioxidant system and heat stress resistance of wheat seedlings have been demonstrated.