Nitrite is the driver, phytohormones are modulators while NO and H2O2 act as promoters of NO2-induced cell death

This study aimed to understand the molecular mechanisms of nitrogen dioxide (NO2)-induced toxicity and cell death in plants. Exposure of Arabidopsis to high concentrations of NO2 induced cell death in a dose-dependent manner. No leaf symptoms were visible after fumigation for 1 h with 10 parts per million (ppm) NO2. However, 20 ppm NO2 caused necrotic lesion formation and 30 ppm NO2 complete leaf collapse, which had already started during the 1 h fumigation period. NO2 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 NO2 fumigation. The onset of NO2-induced cell death correlated with NO and hydrogen peroxide (H2O2) signaling and a decrease in antioxidants. NO- and H2O2-accumulating mutants were more sensitive to NO2 than wildtype plants. Accordingly, experiments with specific scavengers confirmed that NO and H2O2 are essential promoters of NO2-induced cell death. Leaf injection of 100 mM nitrite caused an increase in S-nitrosylation, NO, H2O2, and cell death suggesting that nitrite functioned as a mediator of NO2-induced effects. A targeted screening of phytohormone mutants revealed a protective role of salicylic acid (SA) signaling in response to NO2. It was also shown that phytohormones were modulators rather than inducers of NO2-induced cell death. The established experimental set-up is a suitable system to investigate NO2 and cell death signaling in large-scale mutant screens.