Fluorescence microscopy as a tool for visualization of metal-induced oxidative stress in plants

Various fluorescence reagents are often used for the detection of oxidative stress, but more extensive comparison in the same study is rather rare. We tested five ROS-related and two non-ROS-related reagents in the roots of Zea mays germinated and growing in 1 or 100 µM cadmium-enriched solution. Namely, “general” ROS (CellROX Deep Red reagent and 2′,7′-dichlorodihydrofluorescein diacetate), hydrogen peroxide (Amplex UltraRed), superoxide radical (dihydroethidium), hydroxyl radical/peroxynitrite (aminophenyl fluorescein), nitric oxide (1,2-diaminoanthraquinone), and glutathione/-SH (monochlorobimane) indicators were tested. Both Cd doses stimulated signal of various ROS often extensively compared to control and mainly HO differed between 1 and 100 µM Cd. Signal of ROS was clearly visible in exodermis and vascular tissue through which cadmium is transported via transpiration stream. CellROX Deep Red reagent gives bright and clear signal on the root cross sections. The standard spectrophotometry (detection of HO and superoxide) did not reveal differences between control and 1 µM Cd, indicating that fluorescence microscopy is more sensitive to detect low change in oxidative balance. Nitric oxide and glutathione/-SH signal was less different between control and Cd treatments, indicating their contribution to metal tolerance. Cd accumulation and translocation and the use of reagents under the excess of other metals are also discussed.