ABSTRACT
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.
Subject(s)
Nicotiana/metabolism , Nicotiana/parasitology , Peroxynitrous Acid/metabolism , Tyrosine/metabolism , Algal Proteins , Aniline Compounds/analysis , Antibodies, Anti-Idiotypic/analysis , Antibodies, Anti-Idiotypic/immunology , Cells, Cultured , Cyclic GMP/analysis , Cyclic GMP/physiology , Fluoresceins/analysis , Fungal Proteins/metabolism , Fungal Proteins/pharmacology , Molsidomine/analogs & derivatives , Molsidomine/analysis , Nitric Oxide/analysis , Nitric Oxide/metabolism , Peroxynitrous Acid/analysis , Phytophthora/metabolism , Phytophthora/pathogenicity , Proteins , Reactive Oxygen Species/analysis , Superoxides/analysis , Superoxides/metabolism , Nicotiana/chemistry , Nicotiana/drug effects , Tyrosine/analogs & derivatives , Tyrosine/analysis , Tyrosine/immunology , Uric Acid/pharmacologyABSTRACT
Diseases activate the innate immune response which causes ancillary damage to the human body. Peroxynitrite (OONO-) or its carbon dioxide derivatives cause oxidation/nitration and hence mutation to various body polymers e.g. DNA, RNA, protein, lipids and sugars. The control of the ancillary damage can come from antioxidants which inhibit control the amount of peroxynitrite available for damage. In this paper we have developed three different levels of antioxidant screening: (i) Peroxynitrite or SIN-1 reaction with luminol to produce light, and the inhibition of light by substances therefore represents antioxidation. (ii) Nicking of plasmid DNA occurs via oxidants: and is prevented by antioxidants. (iii) Detection of plasmid luciferase activity post-oxidation and infection indicates either prevention or repair of damage: via antioxidants. We found green tea and a number of its polyphenolic constituents effective only at the first level of antioxidation, while extracts of various fruit help at all levels antioxidation. In the final analysis, a combination of green tea extracts and fruits is suggested to produce more complete antioxidant protection.
