Peroxynitrite generation and tyrosine nitration in defense responses in tobacco BY-2 cells.
Peroxynitrite (ONOO(-)) is a compound formed by reaction of superoxide (O(2) (-)) with nitric oxide (NO) and is expected to possess characteristics of both O(2) (-) reactivity and NO mobility in order to function as a signal molecule. Although there are several reports that describe the role of ONOO(-) in defense responses in plants, it has been very difficult to detect ONOO(-) in bioimaging due to its short half-life or paucity of methods for ONOO(-)-specific detection among reactive oxygen species or free radicals. Aminophenyl fluorescein (APF), a recently developed novel fluorophore for direct detection of ONOO(-) in bioimaging, was used for intracellular ONOO(-) detection. ONOO(-) 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 ONOO(-) scavenger, abolished INF1-induced ONOO(-) generation. It is well known that ONOO(-) reacts with tyrosine residues in proteins to form nitrotyrosine in a nitration reaction as an ONOO(-)-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 ONOO(-) donor]. These bands were also recognized in INF1-treated BY-2 cells and were found to be slightly suppressed by urate. Our study is the first to report ONOO(-) detection and tyrosine nitration in defense responses in plants.
Saito
Syuhei
S
Plant Pathology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601 Japan.
Yamamoto-Katou
Ayako
A
Yoshioka
Hirofumi
H
Doke
Noriyuki
N
Kawakita
Kazuhito
K
D020418:Algal Proteins; D000814:Aniline Compounds / Q000032:analysis; D000888:Antibodies, Anti-Idiotypic / Q000032:analysis / Q000276:immunology; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000032:analysis / Q000502:physiology; D005452:Fluoresceins / Q000032:analysis; D005656:Fungal Proteins / Q000378:metabolism / Q000494:pharmacology; D008981:Molsidomine / Q000031:analogs & derivatives / Q000032:analysis; D009569:Nitric Oxide / Q000032:analysis / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000032:analysis / Q000378:metabolism*; D010838:Phytophthora / Q000378:metabolism / Q000472:pathogenicity; D011506:Proteins; D017382:Reactive Oxygen Species / Q000032:analysis; D013481:Superoxides / Q000032:analysis / Q000378:metabolism; D014026:Nicotiana / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism* / Q000469:parasitology*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D014527:Uric Acid / Q000494:pharmacology
D020418:Algal Proteins; D000814:Aniline Compounds; D000888:Antibodies, Anti-Idiotypic; D005452:Fluoresceins; D005656:Fungal Proteins; D011506:Proteins; D017382:Reactive Oxygen Species; C508569:aminophenyl fluorescein; C085101:elicitin, Phytophthora; D013481:Superoxides; D030421:Peroxynitrous Acid; D014527:Uric Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; C002385:linsidomine; D008981:Molsidomine; D006152:Cyclic GMP