Use of a fluorometer-based method for real time monitoring of cytosol redox status in Arabidopsis thaliana method for real time monitoring of cytosol redox status in Arabidopsis thaliana

CISLAGHI A; ALVAREZ ME

Virtual

Congreso; IV Reunión Conjunta de Sociedades de Biología de la República Argentina; 2020

Sociedades de Biología de la República Argentina

Oxidative stress is a conserved defense mechanism that plants actives under biotic and abiotic stresses. Several assays enable to analyzeoxidative changes into plant tissues, including the use of proteins that function as redox sensors. roGFP-GRX, is a ratiometric probe that allows obtaining redox information of the glutathione redox couple (GSH:GSSG) throughout their excitation at different wavelengths. The GFP fluorescence depends on the oxidation of its Cys residues and its fusion to GRX (glutarredoxin) accelerates the equilibrium between Cys oxidation and glutathione redox state. Confocal fluorescence imaging allows monitoring redox changes on roGFP-GRX localized at different subcellular compartments. However, the heterogeneity of responses activated by different cells limits real-time monitoring of redox changes in leaf tissues. For this reason, we set up a fluorometric assay to study citoplasmic redox changes in roGFP-GRX in Arabidopsis leaves exposed to biotic stress, and were able to monitor a large number of samples. Several oxidant and reducing agent concentrations were testedto determine the full dynamic range between the oxidized and reduced protein configurations. A high H2O2 concentration was required to achieve full oxidation of the probe, possibly due to catalase, peroxidase and superoxide dismutase activity. By contrast, full reduction require fewer reducer levels. The elicitor peptide flg22 was used to trigger defense responses in leaf tisues, and was found to alter the redox state of the sensor in a concentration-dependent manner. Furthermore, incubation with flg22 and a glucose-6-phosphate dehydrogenase (G6PD) inhibitor produced a lower oxidation of the sensor than flg22. As G6PDH provides NADPH to cytosol, its activity could modify other NADPH-dependent enzymes, including the membrane NADPH oxidase acting as a main source of apoplastic ROS. Therefore, the accumulation of apoplastic ROS in response to flg22 would be associated to changes in the cytoplasmic redox homeostasis regulated by NADPH/NADP and GSH/GSSG.