The function of salicylic acid (SA) in hypersensitive cell death was studied in a soybean (Glycine max)-Pseudomonas syringae pv glycinea system. The infection of cell cultures with bacteria leads to a hypersensitive reaction (HR), which is dependent on an appropriate avirulence gene and on low concentrations of SA. The requirement for SA is essential for a process shortly before the onset of the HR-caused cell death 5 to 6 h after infection with bacteria. SA

Soybean (Glycine max L. Merr.) Cell-suspension cultures inoculated with avirulent Pseudomonas syringae pv. glycinea bacteria generated a sustained oxidative burst 3±6 h after the infection. The H 2 O 2 production was not dependent on protein biosynthesis but, surprisingly, cycloheximide itself was a very strong inducer of the oxidative burst and of the alkalinization measured in the cell culture medium. Both responses were activated in a very similar manner by inhibitors of protein phosphatases, implicating a phosphorylation change evoked by cycloheximide as a trigger for the elicitation. The activation of the oxidative burst was totally blocked by the kinase inhibitor K252a. The alkalinization response preceded the oxidative burst. The generation of H 2 O 2 depleted the medium of H but the expected alkalinization of about one pH-unit did not occur. The H 2 O 2 production by the plasma membrane oxidase must therefore be charge-compensated, likely via H -channel activity.Abbreviations: CHX = cycloheximide; DPI = diphenylene iodonium chloride; HR = hypersensitive response; PP2A = protein phosphatase 2A; ROS = reactive oxygen species Correspondence to: R. Tenhaken;

Summary. Plant cells respond to a variety of external signals withthe production of reactive-oxygen species. The enzyme system generating these reactive-oxygen species is believed to be an NADPH oxidase located in the plasma membrane and sharing similarities with the NADPH oxidase from mammalian macrophages. Antibodies directed against individual subunits (p22 ply~ p47 ph~ p67 ph~ of the human NADPH oxidase cross-react with soybean proteins of a similar size and subceltular location. An extensive expression screening of a soybean cDNA-library with the anti-human NADPH oxidase antibodies gave a single class of cDNA-clones for each antibody. However, the sequence analysis of these clones clearly demonstrates that the different antibodies recognise proteins which are unrelated to the expected oxidase subunits. The anti-p22 p~~ antibody recognised a microsomal protein with no significant homology to any known protein in the database. One anti-p47 t#~~ antibody crossreacted with the UDP-glucose dehydrogenase and another antibody bound to the chaperon peptidyl protyl-cis-trans isomerase, both solubte cytosolic proteins, The anti-p67 ph~ antibody detected the soluble enzyme acetohydroxy acid ~vductoisomerase. Chromatography of soybean protein extracts on an ion-exchange column (MonoQ, FPLC) gave a perfect comigration of tbe enzyme activity with the antibody signal, thus confirming these unexpected results by independent biochemical experiments.