Analysis of Stress-Induced Generation of Reactive Oxygen Species in Phalaenopsis ×hybridum Blume Protocorms Using the Redox-Dependent Fluorescent Dihydroethidium Probe

Methods for analyzing and studying the most important stress responses of epidermal cells of the protocorms of Phalaenopsis ×hybridum Blume – the synthesis of reactive oxygen species (ROS), in particular, the superoxide anion radical (О₂^●–) – were developed. Epifluorescence microscopy was adapted for this purpose in combination with the fluorescent probe dihydroethidium and a narrow-band fluorescent filter cube Nikon FITC B-2E/C. This approach made it possible to avoid artifacts arising from intense fluorescence in the red region of the spectrum upon binding of ethidium (a product of nonspecific oxidation of dihydroethidium) to DNA, and to successfully record the signal of 2-hydroxyethidium (2-OH-E⁺) in the green region of the spectrum, specific for the reaction with О₂^●–. 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. It was shown that the above factors induced О₂^●–generation in the protocorm epidermis (registered as an increase in 2-OH-E⁺ fluorescence). The addition of enzymatic and low-molecular antioxidants, such as superoxide dismutase (SOD), catalase, dimethyl sulfoxide (DMSO), and thiourea, reduced stress-induced ROS synthesis in protocorms. The greatest decrease in fluorescence was observed in the presence of SOD and thiourea, indicating the specificity of the reaction of dihydroethidium with О₂^●–. The developed approaches can be applied to the analysis of the early stages of oxidative imbalance developing in the cells of Orchidaceae and other higher plants under the influence of abiotic stress factors.

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