Post-Doc Chemical Signalling, INRES, University of Bonn
Metabolic changes in chloroplasts and mitochondria trigger retrograde signals to feedback information to nuclei. One proposed signal for this are reactive oxygen species (ROS), high concentrations of which may cause oxidation of protein thiol switches and also glutathione (GSH) to form glutathione disulfide (GSSG). An imbalance of the GSH/GSSG pool is considered an important marker for stress responses. Genetically encoded biosensors Grx1-roGFP2 and roGFP2-Orp1 enable in vivo visualization of the GSH redox potential and H2O2 production, respectively. At the subcellular level, however, microscope-based approaches are limited by sample throughput and our ability to do long-term dynamic recordings. To overcome these constraints, we implemented a plate reader-based system that enables monitoring the GSH/GSSG dynamics and H2O2 production in a high-throughput manner in intact samples exposed to different types of stress over time spans of several hours. The use of compartment-targeted versions of Grx1-roGFP2 and roGFP2-Orp1 together with specific inhibitors of photosynthetic electron transport and the non-selective herbicide methyl viologen (MV), reveal a more sensitive GSH pool in plastids compared to the cytosol upon MeV treatments. Similarly, MeV induces an autonomous oxidation in the mitochondrial matrix that is dependent on electron flux through the mitochondrial electron transport chain. In addition, we show that under illumination with photosynthetically active light the initial oxidation triggered by MeV in chloroplasts propagates into the cytosol and mitochondria where it results in oxidation surpassing the mitochondria-autonomous oxidation. Based on this so far inaccessible spatiotemporal resolution, we discuss the subcellular compartment-specific oxidative responses in the context of retrograde signaling and general stress acclimation in plants.
Coauthors: Andreas Meyer – Chemical Signalling, INRES, University of Bonn