CISLAGHI ANA PAULA
Congresos y reuniones científicas
Título:
USE OF A FLUOROMETER-BASED METHOD FOR REAL-TIME MONITORING OF CYTOSOL REDOX STATUS IN Arabidopsis thaliana
Autor/es:
CISLAGHI ANA PAULA; ALVAREZ MARIA ELENA
Lugar:
virtual
Reunión:
Congreso; IV JOINT MEETING OF THE BIOLOGY SOCIETIES OF ARGENTINA; 2020
Institución organizadora:
SOCIEDADES DE BIOLOGÍA DE LA REPÚBLICA ARGENTINA
Resumen:
Oxidative stress is a conserved defense mechanism that plants activate under biotic and abiotic stresses. Several assays enable the analysis ofoxidative changes into plant tissues, including the use of proteins that function as redox sensors. roGFP-GRX, is a ratiometric probe that allowsobtaining redox information of the glutathione redox couple (GSH:GSSG) throughout their excitation at different wavelengths. The GFPfluorescence depends on the oxidation of its Cys residues and its fusion to GRX (glutaredoxin) accelerates the equilibrium between Cys oxidationand glutathione redox state. Confocal fluorescence imaging allows monitoring redox changes on roGFP-GRX localized at different subcellularcompartments. However, the heterogeneity of responses activated by different cells limits real-time monitoring of redox changes in leaf tissues. Forthis reason, we set up a fluorometric assay to study cytoplasmic redox changes in roGFP-GRX in Arabidopsis leaves exposed to biotic stress andwere able to monitor a large number of samples. Several oxidant- and reducing-agent concentrations were tested to determine the full dynamic rangebetween the oxidized and reduced protein configurations. A high H2O2 concentration was required to achieve full oxidation of the probe, possiblydue to catalase, peroxidase, and superoxide dismutase activity. By contrast, full reduction requires fewer reducer levels. The elicitor peptide flg22was used to trigger defense responses in leaf tissues 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 G6PD provides NADPH to cytosol, its activity could modify other NADPH-dependent enzymes, including the membrane NADPH oxidase actingas a main source of apoplastic ROS. Therefore, the accumulation of apoplastic ROS in response to flg22 would be associated with changes in thecytoplasmic redox homeostasis regulated by NADPH/NADP and GSH/GSSG.