EPR EVIDENCE FOR GENERATION OF HYDROXYL RADICAL TRIGGERED BY N-ACETYLCHITOOLIGOSACCHARIDE ELICITOR AND A PROTEIN PHOSPHATASE INHIBITOR IN SUSPENSION-CULTURED RICE CELLS

N-acetylchitooligosaccharides, fragments of the backbone of fungal cell wall, trigger rapid membrane responses such as transient depolarization, and elicit defense reactions including phytoalexin production in suspension-cultured rice cells. The generation of reactive oxygen species triggered by the oligosaccharide signal was analyzed with EPR spectroscopy using a spin trapping system, 4-pyridyl 1-oxide N-tert-butyl nitrone (4-POBN) and ethanol.(OH)-O-. generation was detected as the alpha-hydroxyethyl adduct of 4-POBN after elicitation. Superoxide dismutase, catalase or diethylenetriamine pentaacetic acid, a metal chelator, inhibited O-. generation, proposing the following reaction sequence: generation of O-.(2)- in response to the oligosaccharide elicitor, followed by dismutation to H2O2, then generation of (OH)-O-. by the reaction of H2O2 with Fe2+ that is generated by the reduction of Fe3+ by O-.(2)-. Generation of the same reactive oxygen species was also triggered by calyculin A, a protein phosphatase inhibitor, alone, suggesting the involvement of protein phosphorylation in its regulation during the oligosaccharide signal transduction.