Opposing roles for superoxide and nitric oxide in the NaCl stress-induced upregulation of antioxidant enzyme activity in cotton callus tissue

The roles of superoxide and NO in the NaCl-induced upregulation on antioxidant enzyme activity were investigated in NaCl-tolerant cotton calli. Both NaCl and paraquat treatments resulted in significant increases in superoxide production. The activities of ascorbate peroxidase (APX), catalase, glutathione reductase (GR), and peroxidase also increased significantly within 2 h after applying the stress. Pre-treatment with the superoxide scavenger, N-acetyl l-cysteine (NAC), completely removed the superoxide and inhibited the upregulation of antioxidant enzyme activity in the tissue treated with either NaCl or paraquat. NaCl stress also resulted in a significant increase in the NO level. Experiments were also carried out to measure antioxidant enzyme activity in cotton calli exposed to NO, the NO producer sodium nitroprusside (SNP), and the NO scavenger 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) under different salt stress conditions. The direct addition of NO gas produced no change in the activities of catalase and GR and caused a significant decrease in APX activity when compared to the controls. When the calli was treated with SNP in the absence of NaCl stress, APX and GR activities decreased significantly and catalase activity was only slightly higher than the control. Treatment with SNP in the presence of NaCl stress resulted in a significant decrease in APX activity, and GR and APX activities were not significantly different from those observed in the NaCl treatment alone. In the presence of PTIO, the activities of all three enzymes increased in the presence or absence of NaCl stress. These results suggest that reactive oxygen species (ROS) such as superoxide radicals may serve as signal transduction molecules to switch “on” the early NaCl-induced up-regulation of antioxidant enzyme activity, while NO may play a role in switching “off” the response after other mechanisms in the cascade of events responsible for NaCl tolerance have been activated.