EPR evidence for generation of hydroxyl radical triggered byN-acetylchitooligosaccharide elicitor and a protein phosphatase inhibitor in suspension-cultured rice cells

Abstract

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-oxideN-tert-butyl nitrone (4-POBN) and ethanol. OH generation was detected as the α-hydroxyethyl adduct of 4-POBN after elicitation. Superoxide dismutase, catalase or diethylenetriamine pentaacetic acid, a metal chelator, inhibited $${}^ \cdot O$$ generation, proposing the following reaction sequence: generation of $${}^ \cdot O_{2^ - } $$ in response to the oligosaccharide elicitor, followed by dismutation to H2O2, then generation of $${}^ \cdot OH$$ by the reaction of H2O2 with Fe2+ that is generated by the reduction of Fe3+ by $${}^ \cdot O_{2^ - } $$ . 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.