The fluorescent protein sensor roGFP2‐Orp1 monitors in vivo H2O2 and thiol redox integration and elucidates intracellular H2O2 dynamics during elicitor‐induced oxidative burst in Arabidopsis

Hydrogen peroxide (H2O2) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H2O2 notoriously hard to detect dynamically, specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour in vivo have hampered meaningful H2O2 sensing in living plants. We establish H2O2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2‐Orp1 using confocal microscopy and multiwell fluorimetry. We confirm sensor oxidation by H2O2, show insensitivity to physiological pH changes, and demonstrate that glutathione dominates sensor reduction in vivo. We show the responsiveness of the sensor to exogenous H2O2, pharmacologically‐induced H2O2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H2O2 dynamics in response to elicitor exposure reveals late and prolonged impact of the oxidative burst in the cytosol, which is modified in redox mutants. We provide a well‐defined toolkit for H2O2 monitoring in planta and show that intracellular H2O2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H2O2 dynamics and redox regulation, including intracellular NADPH‐oxidase‐mediated ROS signalling.