Generation of active oxygen in elicited cells of Arabidopsis thaliana is mediated by a NADPH oxidase-like enzyme

Abstract

Suspension-cultured cells of Arabidopsis thaliana generated active oxygen species (AOS) (measured by luminol-dependent chemiluminescence) following challenge with the bacterial protein elicitor harpin or the protein kinase activator phorbol 12-myristate 13-acetate. These responses were blocked by inhibitors of superoxide dismutase (SOD), NADPH oxidase and protein kinase. Harpin treatment also resulted in an increase in cell death, a response reduced by inhibitors of AOS generation or AOS scavengers. Extracellular SOD activity was found to be present in cell culture medium. Immunoblotting of Arabidopsis extracts revealed the presence of proteins immunologically related to the human neutrophil NADPH oxidase comples, and cell-free reconstitution assays showed that human neutrophil cytosol combined with Arabidopsis membranes could initiate superoxide generation. These data suggest that the enzyme catalysing the generation of superoxide in elicited Arabidopsis cells is similar to the mammalian NADPH oxidase and that a signalling cascade leading to AOS generation involves protein phosphorylation.