The fluorescent protein sensor roGFP2-Orp1 monitors in vivo H O and thiol redox integration and elucidates intracellular H O dynamics during elicitor-induced oxidative burst in Arabidopsis.

Hydrogen peroxide (H O ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H O 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 any meaningful H O sensing in living plants. We established H O monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2-Orp1 using confocal microscopy and multiwell fluorimetry. We confirmed sensor oxidation by H O , show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H O , pharmacologically-induced H O release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H O dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants. We provided a well defined toolkit for H O monitoring in planta and showed that intracellular H O measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H O dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.