GENERATION OF ACTIVE OXYGEN IN ARABIDOPSIS-THALIANA

Suspension cultures of Arabidopsis thaliana generated active oxygen sp ecies (AOS) on challenge with the bacterial protein elicitor harpin, t he protein kinase activator, phorbol 12-myristate 13-acetate (PMA), or the calcium ionophore, ionomycin. The release of AOS was inhibited by the protein kinase inhibitor, K-252a and by a NADPH oxidase inhibitor diphenylene iodonium (DPI). A substantial reduction in measured AOS w as also observed following the addition of a superoxide dismutase inhi bitor, diethyldithiocarbamate (DDC). Immunoblotting analysis revealed the presence of proteins immunologically related to those of the mamma lian phagocytic NADPH oxidase, and reconstitution experiments demonstr ated that a combination of human neutrophil cytosol and Arabidopsis me mbranes could generate superoxide. Current work is using RT-PCR techno logy to identify clones in an Arabidopsis suspension culture cDNA libr ary with potential homology to NADPH oxidase components.