Peroxynitrite generation and tyrosine nitration in defense responses in tobacco BY-2 cells [Nicotiana tabacum]
2006
Saito, S.(Nagoya Univ. (Japan)) | Yamamoto Katou, A. | Yoshioka, H. | Doke, N. | Kawakita, K.
Peroxynitrite (ONOOsup(-)) is a compound formed by reaction of superoxide (O2sup(-)) with nitric oxide (NO) and is expected to possess characteristics of both O2sup(-) reactivity and NO mobility in order to function as a signal molecule. Although there are several reports that describe the role of ONOOsup(-) in defense responses in plants, it has been very difficult to detect ONOOsup(-) in bioimaging due to its short half-life or paucity of methods for ONOOsup(-)-specific detection among reactive oxygen species or free radicals. Aminophenyl fluorescein (APF), a recently developed novel fluorophore for direct detection of ONOOsup(-) in bioimaging, was used for intracellular ONOOsup(-) detection. ONOOsup(-) generation in tobacco BY-2 cells treated with INF1, the major elicitin secreted by the late blight pathogen Phytophthora infestans, occurred within 1 h and reached a maximum level at 6-12 h after INF1 treatment. Urate, a ONOOsup(-) scavenger, abolished INF1-induced ONOOsup(-) generation. It is well known that ONOOsup(-) reacts with tyrosine residues in proteins to form nitrotyrosine in a nitration reaction as an ONOOsup(-)-specific reaction. Western blot analysis using anti-nitrotyrosine antibodies recognized nitrotyrosine-containing proteins in 20 and 50 kDa bands in BY-2 protein extract containing SIN-1 [3-(4-morpholinyl) sydnonimine hydrochloride; an ONOOsup(-) donor]. These bands were also recognized in INF1treated BY-2 cells and were found to be slightly suppressed by urate. Our study is the first to report ONOOsup(-) detection and tyrosine nitration in defense responses in plants.
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