Superoxide production by plant homologues of the gp91(phox) NADPH oxidase.Modulation of activity by calcium and by tobacco mosaic virus infection

Genes encoding homologs of the gp91(phox) subunit of the plasma membrane NA DPH oxidase complex have been identified in plants and are hypothesized to be a source of reactive oxygen species during defense responses. However, t he direct involvement of the gene products in superoxide (O-2(-)) productio n has yet to be shown. A novel activity gel assay based on protein fraction ation in native or sodium dodecyl sulfate (SDS)-denaturing polyacrylamide g els was developed. In native polyacrylamide gel electrophoresis, one or two major O-2(-)-producing formazan bands were detected in tomato (Lycopersicu m esculentum Mill. cv Moneymaker) and tobacco (Nicotiana tabacum var. Samsu n, NN) plasma membranes, respectively. Denaturing fractionation of tomato a nd tobacco plasma membrane in SDS-polyacrylamide gel electrophoresis, follo wed by regeneration of the in-gel activity, revealed NADPH-dependent O-2(-) -producing formazan bands of 106-, 103-, and 80- to 75-kD molecular masses. The SDS and native activity bands were dependent on NADPH and completely i nhibited by diphenylene iodonium or CuZn- O-2(-) dismutase, indicating that the formazan precipitates were due to reduction by O-2(-) radicals catalyz ed by an NADPH-dependent flavin containing enzyme. The source of the plasma membrane activity bands was confirmed by their cross-reaction with antibod y prepared from the C terminus of the tomato gp91(phox) homolog. Membrane e xtracts as well as the in-gel NADPH oxidase activities were stimulated in t he presence of Ca2+. In addition, the relative activity of the gp91(phox) h omolog was enhanced in the plasma membrane of tobacco mosaic virus-infected leaves. Thus, in contrast to the mammalian gp(91phox), the plant homolog c an produce O-2(-) in the absence of additional cytosolic components and is stimulated directly by Ca2+.