Generation of hydrogen peroxide is not required for harpin-induced apoptotic cell death in tobacco BY-2 cell suspension culture

To characterize molecular and biochemical mechanisms of hypersensitive reaction (HR) in plants, a tobacco suspension culture of BY-2 was treated with the proteinaceous HR elicitor harpin from several pathovars of Pseudomonas syringae. Tobacco BY-2 cells are sensitive to harpins derived from non-pathogenic pathovars of P. syringae, such as pvs. pisi, tomato and glycinea. These three harpins induce apoptotic cell death accompanied by DNA fragmentation in BY-2. Because the cell death was also accompanied by rapid generation of hydrogen peroxide (H₂O₂), one of the active oxygen species, we investigated the role of H₂O₂ in harpin-induced cell death. Although treatment with diphenylene iodium chloride (DPI) reduced, and catalase completely abolished, the harpin-induced generation of H₂O₂, the frequency of cell death was not affected at all. Treatment with superoxide dismutase (SOD) enhanced the generation of H₂O₂, but the cell death was unaffected. These results indicate that harpin-induced apoptotic cell death does not require oxidative burst.