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Plant and Cell Physiology Advance Access published online on March 23, 2006
Plant and Cell Physiology, doi:10.1093/pcp/pcj038
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Plant and Cell Physiology 2006 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received December 12, 2005
Accepted March 16, 2006

Regular Paper

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

Syuhei Saito 1, Ayako Yamamoto-Katou 2, Hirofumi Yoshioka 1, Noriyuki Doke 1, and Kazuhito Kawakita 1 *

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

* To whom correspondence should be addressed.
Kazuhito Kawakita, E-mail: kkawakit{at}agr.nagoya-u.ac.jp


  Abstract

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 halflife 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 kDa and 50 kDa bands in SIN-1 (ONOO- donor) added BY-2 protein extract. 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.
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