Involvements of H2O2 and metallothionein in NO-mediated tomato tolerance to copper toxicity

Both H2O2 and NO are involved in multiple physiological responses in plants. Metallothionein (MT) can bind heavy metal ions and reduce metal toxicity. Copper toxicity has become a major problem with increasing agricultural and environmental pollution. Here, we investigated the possible roles of ROS, NO and metallothionein in tomato plant responses to copper toxicity. We found that Cu2+ stress caused the rapid release of H2O2 and chlorotic leaves, and it stunted root growth and development. Cu treatment also caused an increase in NOS enzyme activity and NO release in roots and leaves. Application of the NO donor SNP efficiently alleviated the copper toxicity effect, as shown by increases in chlorophyll content and the biomass of fresh/dry leaves. SNP treatment also induced the transcription and increased activities of anti-oxidant enzymes, including catalase, peroxidase, superoxide dismutase and ascorbate peroxidase, and led to reduced H2O2 accumulation in the leaves. Special inhibitors or scavengers of NO synthesis diminished the ameliorating effect of NO on copper toxicity. NO application induced MT transcription and accumulation in leaves. Furthermore, the antisense-MT transgenic tomato was more sensitive to copper stress, and this effect could not be efficiently reversed by NO treatment. From these data, we propose that NO induces tomato tolerance to copper toxicity through antioxidant enzyme activity and metallothionein accumulation, and that metallothionein acts downstream of NO signaling.