NO and H₂O₂ contribute to SO₂ toxicity via Ca²⁺ signaling in Vicia faba guard cells

NO and H₂O₂ have been implicated as important signals in biotic and abiotic stress responses of plants to the environment. Previously, we have shown that SO₂ exposure increased the levels of NO and H₂O₂ in plant cells. We hypothesize that, as signaling molecules, NO and H₂O₂ mediate SO₂-caused toxicity. In this paper, we show that SO₂ hydrates caused guard cell death in a concentration-dependent manner in the concentration range of 0.25 to 6 mmol L^-1, which was associated with elevation of intracellular NO, H₂O₂, and Ca²⁺ levels in Vicia faba guard cells. NO donor SNP enhanced SO₂ toxicity, while NO scavenger c-PTIO and NO synthesis inhibitors L-NAME and tungstate significantly prevented SO₂ toxicity. ROS scavenger ascorbic acid (AsA) and catalase (CAT), Ca²⁺ chelating agent EGTA, and Ca²⁺ channel inhibitor LaCl₃ also markedly blocked SO₂ toxicity. In addition, both c-PTIO and AsA could completely block SO₂-induced elevation of intracellular Ca²⁺ level. Moreover, c-PTIO efficiently blocked SO₂-induced H₂O₂ elevation, and AsA significantly blocked SO₂-induced NO elevation. These results indicate that extra NO and H₂O₂ are produced and accumulated in SO₂-treated guard cells, which further activate Ca²⁺ signaling to mediate SO₂ toxicity. Our findings suggest that both NO and H₂O₂ contribute to SO₂ toxicity via Ca²⁺ signaling.