Journal Article

Peroxynitrite Generation and Tyrosine Nitration in Defense Responses in Tobacco BY-2 Cells

Syuhei Saito, Ayako Yamamoto-Katou, Hirofumi Yoshioka, Noriyuki Doke and Kazuhito Kawakita

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 47, issue 6, pages 689-697
Published in print June 2006 | ISSN: 0032-0781
Published online June 2006 | e-ISSN: 1471-9053 | DOI: https://dx.doi.org/10.1093/pcp/pcj038
Peroxynitrite Generation and Tyrosine Nitration in Defense Responses in Tobacco BY-2 Cells

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  • Biochemistry
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  • Plant Sciences and Forestry

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Peroxynitrite (ONOO) is a compound formed by reaction of superoxide (O2 ) with nitric oxide (NO) and is expected to possess characteristics of both O2 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.

Keywords: Defense responses; INF1 elicitin; Nitrotyrosine; Peroxynitrite; Tobacco BY-2 cells; Urate; APF, aminophenyl fluorescein; DAF-2 DA, diaminofluorescein-2 diacetate; HR, hypersensitive responses; NO, nitric oxide; ROS, reactive oxygen species; SIN-1, 3-(4-morpholinyl) sydnonimine hydrochloride; SOD, superoxide dismutase

Journal Article.  6391 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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