LOCALIZED CHANGES IN PEROXIDASE-ACTIVITY ACCOMPANY HYDROGEN-PEROXIDE GENERATION DURING THE DEVELOPMENT OF A NONHOST HYPERSENSITIVE REACTIONIN LETTUCE

Peroxidase activity was characterized in lettuce (Lactuca sativa L.) l eaf tissue. Changes in the activity and distribution of the enzyme wer e examined during the development of a nonhost hypersensitive reaction (HR) induced by Pseudomonas syringae (P. s.) pv phaseolicola and in r esponse to an hrp mutant of the bacterium. Assays of activity in tissu e extracts revealed pH optima of 4.5, 6.0, 5.5 to 6.0, and 6.0 to 6.5 for the substrates tetramethylbenzidine, guaiacol, caffeic acid, and c hlorogenic acid, respectively. Inoculation with water or with wild-typ e or hrp mutant strains of P. s. pv phaseolicola caused an initial dec line in total peroxidase activity; subsequent increases depended on th e hydrogen donor used in the assay. Guaiacol peroxidase recovered more rapidly in tissues undergoing the HR, whereas changes in tetramethylb enzidine peroxidase were generally similar in the two interactions. In contrast, increases in chlorogenic acid peroxidase were significantly higher in tissues inoculated with the hrp mutant. During the HR, incr eased levels of Mn2+/2,4-dichlorophenol-stimulated NADH and NADPH oxid ase activities, characteristic of certain peroxidases, were found in i ntercellular fluids and closely matched the accumulation of H2O2 in th e apoplast. Histochemical analysis of peroxidase distribution by elect ron microscopy revealed a striking, highly localized increase in activ ity within the endomembrane system and cell wall at the sites of bacte rial attachment. However, no clear differences in peroxidase location were observed in tissue challenged by the wild-type strain or the hrp mutant. Our results highlight the significance of the subcellular cont rol of oxidative reactions leading to the generation of reactive oxyge n species, cell wall alterations, and the HR.