This study aimed to understand the molecular mechanisms of nitrogen dioxide (NO)-induced toxicity and cell death in plants. Exposure of Arabidopsis to high concentrations of NO induced cell death in a dose-dependent manner. No leaf symptoms were visible after fumigation for 1 h with 10 parts per million (ppm) NO However, 20 ppm NO caused necrotic lesion formation and 30 ppm NO complete leaf collapse, which had already started during the 1 h fumigation period. NO fumigation resulted in a massive accumulation of nitrite and in protein modifications by S-nitrosylation and tyrosine nitration. Nitric oxide (NO) at 30 ppm did not trigger leaf damage or any of the effects observed after NO fumigation. The onset of NO-induced cell death correlated with NO and hydrogen peroxide (HO) signaling and a decrease in antioxidants. NO- and HO-accumulating mutants were more sensitive to NO than wild-type plants. Accordingly, experiments with specific scavengers confirmed that NO and HO are essential promoters of NO-induced cell death. Leaf injection of 100 mM nitrite caused an increase in S-nitrosylation, NO, HO, and cell death suggesting that nitrite functioned as a mediator of NO-induced effects. A targeted screening of phytohormone mutants revealed a protective role of salicylic acid (SA) signaling in response to NO It was also shown that phytohormones were modulators rather than inducers of NO-induced cell death. The established experimental set-up is a suitable system to investigate NO and cell death signaling in large-scale mutant screens.
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