Detection of Peroxynitrite in Plants Exposed to Bacterial Infection

Peroxynitrite is a highly reactive derivative of nitric oxide (NO) which is gaining attention in the plant biology community because it may play a role in NO signaling during biotic stress. Peroxynitrite can react with many different biomolecules, but its ability to nitrate the tyrosine residues of proteins is particularly important because this may regulate defense signaling in response to pathogens. The analysis of peroxynitrite levels in the context of its proposed defense role requires an accurate and specific detection method. Here, we describe a photometric assay using the fluorescent dye Hong Kong Green 2 as a specific and quantitative probe for peroxynitrite in Arabidopsis thaliana plants challenged with an avirulent strain of Pseudomonas syringae pv. tomato. This protocol includes the preparation of plant samples, the assay procedure, the measurement of peroxynitrite-specific fluorescence, and data presentation.