Analysis of Stress-Induced Generation of Reactive Oxygen Species in Phalaenopsis × hybridium Blume Protocorms Using a Redox-Sensitive Dihydroethidium Fluorescent Probe

Methods were developed for analyzing and studying the most important stress responses of protocorm epidermal cells of Phalaenopsis × hybridium Blume involving the synthesis of reactive oxygen species (ROS), in particular, the superoxide radical anion $$\left({O}_{2}^{\bullet -}\right)$$ . Epifluorescence microscopy was adapted for this purpose in combination with a dihydroethidium fluorescent probe and a Nikon FITC B-2E/C narrow-band fluorescent filter cube. This approach made it possible to avoid artifacts arising from the intense red fluorescence generated when ethidium (a product of the nonspecific oxidation of dihydroethidium) binds to DNA and to record the green signal of 2-hydroxyethidium (2-OH-E+) specific for the reaction with $${O}_{2}^{\bullet -}$$ . Using the developed methods, ROS generation in Orchid protocorms subjected to mechanical damage as well as osmotic and salt stress was analyzed for the first time. The abovementioned factors were shown to induce $${O}_{2}^{\bullet -}$$ generation in the protocorm epidermis identified by enhanced 2-OH-E+ fluorescence. The addition of enzymatic and low-molecular-weight antioxidants such as superoxide dismutase (SOD), catalase, dimethyl sulfoxide (DMSO), and thiourea suppressed the stress-induced ROS synthesis in protocorms. The greatest decrease in the fluorescence was seen in the presence of SOD and thiourea, indicating specificity of the reaction of dihydroethidium with $${O}_{2}^{\bullet -}$$ . These approaches can be applied to the analysis of the early stages of the oxidative imbalance arising in the cells of Orchidaceae and other higher plants due to the effect of abiotic stress factors.