H2O2 localization in the green alga Micrasterias after salt and osmotic stress by TEM-coupled electron energy loss spectroscopy

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), are constantly generated as by-products of normal metabolic cellular pathways and can be overproduced in response to stress. In this study, we investigated ROS production and localization of H₂O₂ after salt (200 mM KCl) and osmotic (iso-osmotic sorbitol concentration) stress in the unicellular green alga Micrasterias. By means of the dye H₂DCFDA and confocal laser scanning microscopy, most ROS production could be detected in KCl-treated cells when compared to sorbitol-exposed cells and controls. For ultrastructural detection of H₂O₂, CeCl₃, which reacts with H₂O₂ and produces cerium perhydroxide deposits, has been used. Cerium was identified by transmission electron microscopy (TEM)-coupled electron energy loss spectroscopy (EELS) in organelles of KCl- and sorbitol-treated cells and in controls. Statistical measurements of the presence of the cerium M_4,5 edge were performed in mitochondria, chloroplasts, cell walls, and cytoplasmic sites of five individual cells after each treatment. The most pronounced increase in H₂O₂ production was found in chloroplasts of KCl- and sorbitol-treated cells. This shows that the chloroplast reveals the strongest response in H₂O₂ production after stress induction in Micrasterias. Significant elevation of H₂O₂ production also occurred in mitochondria and cytoplasm, whereas H₂O₂ levels remained unchanged or even slightly decreased in cell walls of treated cells. Additionally, TEM micrographs and EELS analyses provided indirect evidence for an increased H₂O₂ production at the plasma membrane of KCl-treated cells, indicating an involvement of the plasma membrane NADPH oxidase in H₂O₂ generation.