A correlative study of hydrogen peroxide accumulation after mercury or copper treatment observed in root nodules of Medicago truncatula under light, confocal and electron microscopy.

Heavy metal stress affects both, nodulation and nitrogen fixation of legumes. Mercury triggers disturbances in cellular structure and metabolism but its influence on ROS generation is poorly understood. Copper is redox active metal which in opposition to mercury is an essential micronutrient for plants. Excess of copper is cytotoxic, as it participates in ROS generation via Fenton-type reaction. The present work describes changes in hydrogen peroxide (H₂O₂) accumulation in response to monthly stress caused by mercury (6 mg/L HgCl₂) or copper (60 mg/L CuCl₂) in root nodules. H₂O₂ accumulation viewed with a light microscopy was detected by the use of diaminobenzidine (DAB). 2',7'-Dichlorofluorescein diacetate (H2DCF-DA) was used as a probe for the intracellular localization of H₂O₂ with a confocal laser scanning system. H₂O₂ detection under transmission electron microscopy was performed by the use of cerium method. Histochemical localization and light and confocal microscopy investigations revealed that under Hg or Cu treatments distinct amount of H₂O₂ accumulated mainly in the interzone and nitrogen-fixing zone. Under normal conditions H₂O₂ accumulated predominantly in the interzone. Electron microscopy observations showed H₂O₂ accumulation under Hg or Cu- treatments around peribacteroid membranes of mature symbiosomes located within nitrogen-fixing zone. It should be underlined that under normal conditions H₂O₂ was not detected at the peribacteroid membranes. The main result of our observations is increased accumulation of H₂O₂ in response to mercury and copper treatments at the peribacteroidal membranes, to our knowledge shown for the first time. Therefore, our results revealed that an overproduction of H₂O₂ in response to copper or mercury-treatment may account for lowering of nitrogen fixation rates in heavy-metal affected root nodules.