Use of Dimethyl Sulfoxide to Detect Hydroxyl Radical during Bacteria-Induced Hypersensitive Reaction

Excess active oxygen is generated during the hypersensitive reaction (HR), an incompatible reaction of plants to bacterial pathogens. During HR, lipid peroxidation correlates chronologically with production of the oxygen species, superoxide (O(2) (.-)). However, O(2) (.-) may not be the active oxygen species that initiates lipid peroxidation. Evidence from other systems suggest that O(2) (.-) is converted to the hydroxyl radical (HO(.)) before lipid peroxidation is initiated. Until recently, HO(.) could not be detected directly in vivo. This study utilizes a newly reported method to directly detect and quantify the formation of HO(.)in vivo. Dimethyl sulfoxide (DMSO), used as a molecular probe, is oxidized by HO(.), forming the stable compound methanesulfinic acid. The methanesulfinic acid can be easily extracted from plant tissues and measured with a colorimetric assay. This study demonstrates significant increases in HO(.) concentration after simultaneous infiltration of cucumber (Cucumis sativa L.) plants with paraquat and DMSO. The concentration of HO(.) did not increase significantly when cucumber plants were infiltrated simultaneously with the HR-inducing bacteria, Pseudomonas syringae pv. pisi, and with DMSO. Lipid peroxidation, however, could be measured at times when HO(.) was not detectable. It appears that HO(.) is not generated during bacteria-induced HR; therefore, HO(.) is not responsible for the initiation of lipid peroxidation.