Detection of peroxynitrite accumulation in Arabidopsis thaliana during the hypersensitive defense response

Nitric oxide (NO) is synthesized in plants in response to stress, and its role in signaling is well-documented. In contrast, very little is known about the physiological role of its derivate peroxynitrite (ONOO−), which forms when NO reacts with O2- and induces protein modification by tyrosine nitration. Infection with an avirulent pathogen triggers the simultaneous production of NO and reactive oxygen species, as well as an increase in tyrosine nitration, so peroxynitrite could be physiologically relevant during this process. To gain insight into the role of peroxynitrite in plants, we measured its accumulation during the hypersensitive response in Arabidopsis thaliana using the specific peroxynitrite-sensitive fluorescent dye HKGreen-2 in a leaf disc assay. The avirulent pathogen Pseudomonas syringae pv. tomato, carrying the AvrB gene (Pst AvrB), induced a strong increase in fluorescence 3–4 h post-infiltration (hpi) which peaked 7–8 hpi. The increase in HKGreen-2 fluorescence was inhibited by co-injecting the peroxynitrite-scavenger urate together with the pathogen, and was almost completely eliminated by co-infiltrating urate with HKGreen-2, confirming that HKGreen-2 fluorescence in planta is induced specifically by peroxynitrite. This establishes a link between peroxynitrite synthesis and tyrosine nitration, and we therefore propose that peroxynitrite transduces the NO signal by modifying protein functions.