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Plant and Cell Physiology Advance Access originally published online on March 23, 2006
Plant and Cell Physiology 2006 47(6):689-697; doi:10.1093/pcp/pcj038
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Peroxynitrite Generation and Tyrosine Nitration in Defense Responses in Tobacco BY-2 Cells

Syuhei Saito1, Ayako Yamamoto-Katou1,2, Hirofumi Yoshioka1, Noriyuki Doke1 and Kazuhito Kawakita1,*

1 Plant Pathology Laboratory, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601 Japan
2 Department of Molecular Genetics, National Institute of Agrobiological Resources, Tsukuba, Ibaraki, 305-8602 Japan

* Corresponding author: E-mail, kkawakit{at}agr.nagoya-u.ac.jp; Fax, +81-52-789-5525.

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.

(Received December 12, 2005; Accepted March 16, 2006)
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