High resolution imaging of temporal and spatial changes of subcellular ascorbate, glutathione and H₂O₂ distribution during Botrytis cinerea infection in Arabidopsis.

Abstract

In order to study the mechanisms behind the infection process of the necrotrophic fungus Botrytis cinerea, the subcellular distribution of hydrogen peroxide (H₂O₂) was monitored over a time frame of 96 h post inoculation (hpi) in Arabidopsis thaliana Col-0 leaves at the inoculation site (IS) and the area around the IS which was defined as area adjacent to the inoculation site (AIS). H₂O₂ accumulation was correlated with changes in the compartment-specific distribution of ascorbate and glutathione and chloroplast fine structure. This study revealed that the severe breakdown of the antioxidative system, indicated by a drop in ascorbate and glutathione contents at the IS at later stages of infection correlated with an accumulation of H₂O₂ in chloroplasts, mitochondria, cell walls, nuclei and the cytosol which resulted in the development of chlorosis and cell death, eventually visible as tissue necrosis. A steady increase of glutathione contents in most cell compartments within infected tissues (up to 600% in chloroplasts at 96 hpi) correlated with an accumulation of H₂O₂ in chloroplasts, mitochondria and cell walls at the AIS indicating that high glutathione levels could not prevent the accumulation of reactive oxygen species (ROS) which resulted in chlorosis. Summing up, this study reveals the intracellular sequence of events during Botrytis cinerea infection and shows that the breakdown of the antioxidative system correlated with the accumulation of H₂O₂ in the host cells. This resulted in the degeneration of the leaf indicated by severe changes in the number and ultrastructure of chloroplasts (e.g. decrease of chloroplast number, decrease of starch and thylakoid contents, increase of plastoglobuli size), chlorosis and necrosis of the leaves.