Visualizing ROS metabolism at subcellular level under fluctuating light conditions

Dr. Marino Expósito /Sainsbury Laboratory, Cambridge University, UK

Hydrogen peroxide (H₂O₂) and lipid hydroperoxides (LOOH) are initiators and transducer of inter- and intra-cellular signaling in response to diverse environmental, pathological and developmental cues. The accumulation of both H₂O₂ and LOOH is often coincident in tissues, but it is unknown if this happens in subcellular compartments. If it were to occur, then it would be a factor needing to be considered in determining peroxide signaling specificity. The fluorescent protein biosensor roGFP2-ORP1 provided a basis to address this issue. We show that recombinant roGFP2-ORP1 is not only oxidized by H₂O₂ as often reported, but also by the fatty acid peroxides of lecithin-derived liposomes.  We constructed a modified biosensor called roGFP2-synORP1, which has greatly diminished specificity towards LOOH and increased reactivity with lower H₂O₂ concentrations than roGFP2-ORP1. These two roGFP2-based biosensors, targeted to chloroplasts, cytosol and the nucleus, were quantitatively imaged in parallel in Nicotiana benthamiana abaxial epidermal cells experiencing high light- and herbicide-induced photo-oxidative stress. From differential patterns of oxidation of these probes, it was inferred that the chloroplasts accumulated both peroxide types. In contrast, LOOH and H₂O₂ accumulated exclusively in the cytosol and nucleus respectively, which would provide for spatially distinct peroxide signaling pathways.