SUBCELLULAR-LOCALIZATION OF H2O2 IN PLANTS - H2O2 ACCUMULATION IN PAPILLAE AND HYPERSENSITIVE RESPONSE DURING THE BARLEY-POWDERY MILDEW INTERACTION

Active oxygen species (AOS) are believed to have important roles in pl ants in general and in plant-pathogen interactions in particular. They are believed to be involved in signal transduction, cell wall reinfor cement, hypersensitive response (HR) and phytoalexin production, and t o have direct antimicrobial effects. Since current methods are inadequ ate for localizing AOS in intact plant tissue, most studies have been conducted using cell suspension culture/elicitors systems. 3,3-diamino benzidine (DAB) polymerizes instantly and locally as soon as it comes into contact with H2O2 in the presence of peroxidase, and it was found that, by allowing the leaf to take up this substrate, in-vivo and in- situ detection of H2O2 can be made at subcellular levels. This method was successfully used to detect H2O2 in developing papillae and surrou nding haloes (cell wall appositions) and whole cells of barley leaves interacting with the powdery mildew fungus. Thus, H2O2 can be detected in the epidermal cell wall subjacent to the primary germ tube from 6 h after inoculation, and subjacent to the appressorium from 15 h. The earliest time point for observation of H2O2 in relation to epidermal c ells undergoing HR is 15 h after inoculation, first appearing in the z ones of attachment to the mesophyll cells underneath, and eventually i n the entire epidermal cell. Furthermore, it was observed that protein s in papillae and HR cells are cross-linked, a process believed to be fuelled by H2O2. This cross-linking reinforces the apposition, presuma bly assisting the arrest of the pathogen.