LOCALIZATION OF HYDROGEN-PEROXIDE ACCUMULATION DURING THE HYPERSENSITIVE REACTION OF LETTUCE CELLS TO PSEUDOMONAS-SYRINGAE PV PHASEOLICOLA

The active oxygen species hydrogen peroxide (H2O2) was detected cytoch emically by its reaction with cerium chloride to produce electron-dens e deposits of cerium perhydroxides. In uninoculated lettuce leaves, H2 O2 was typically present within the secondary thickened walls of xylem vessels. inoculation with wild-type cells of Pseudomonas syringae pv phaseolicola caused a rapid hypersensitive reaction (HR) during which highly localized accumulation of H2O2 was found in plant cell walls ad jacent to attached bacteria. Quantitative analysis indicated a prolong ed burst of H2O2 occurring between 5 to 8 hr after inoculation in cell s undergoing the HR during this example of non-host resistance. Cell w all alterations and papilla deposition, which occurred in response to both the wild-type strain and a nonpathogenic hrpD mutant, were not as sociated with intense staining for H2O2, unless the responding cell wa s undergoing the HR. Catalase treatment to decompose H2O2 almost entir ely eliminated staining, but 3-amino-1,2,4-triazole (catalase inhibito r) did not affect the pattern of distribution of H2O2 detected. H2O2 p roduction was reduced more by the inhibition of plant peroxidases (wit h potassium cyanide and sodium azide) than by inhibition of neutrophil -like NADPH oxidase (with diphenylene iodonium chloride). Results sugg est that CeCl3 reacts with excess H2O2 that is not rapidly metabolized during cross-linking reactions occurring in cell walls; such an exces s of H2O2 in the early stages of the plant-bacterium interaction was o nly produced during the HR. The highly localized accumulation of H2O2 is consistent with its direct role as an antimicrobial agent and as th e cause of localized membrane damage at sites of bacterial attachment.