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Arsenic (As) contamination and accumulation in rice is a serious concern causing severe oxidative damage. Melatonin acts as a protective agent in plant defence against multiple abiotic stresses. The mechanism of antioxidant function of melatonin during As stress in rice genotypes is less studied.• In this study, hydroponically-grown As-susceptible (Khitish) and As-tolerant (Muktashri) rice cultivars, subjected to 150 µM arsenate stress, were supplemented with exogenously applied melatonin (20 µM) to examine the plant defence mechanism.• Melatonin (Mel) increased root and shoot length, fresh and dry weight, chlorophyll a and b content and activated reducing power and free radical scavenging capacity in both rice cultivars. The role of Mel in the sensitive variety appeared to be more prominent with respect to reduced water saturation deficit by reducing endogenous As and H 2 O 2 accumulation, and enhancing overall antioxidant capacity by imposing reduced requirement of catalase for ROS detoxification, and restoring As-inhibited activity of glutathione-S-transferase, glutathione peroxidase and dehydroascorbate reductase. In contrast, melatonin treatment in the tolerant cultivar required reduced involvement of ascorbate peroxidase to deal with As toxicity, and complemented the stress-mediated inhibition of guaiacol peroxidase activity. Isozyme profiling also established extensive varietal differences with regard to induction of new isoform(s) by Mel during As treatment.• This study provides clear insights into mechanistic details of the regulation of antioxidative enzymes by melatonin in contrasting rice genotypes, which may prove helpful in generating As tolerance in susceptible rice varieties grown in marginalized soils, thereby improving crop yield and productivity.

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