The fluorescent protein sensor ro<scp>GFP</scp>2‐Orp1 monitors <i>in vivo</i> H₂O₂ and thiol redox integration and elucidates intracellular H₂O₂ dynamics during elicitor‐induced oxidative burst in Arabidopsis

Abstract

Hydrogen peroxide (H₂ O₂ ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H₂ O₂ 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 H₂ O₂ sensing in living plants. We established H₂ O₂ 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 H₂ O₂ , show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H₂ O₂ , pharmacologically-induced H₂ O₂ release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H₂ O₂ 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 H₂ O₂ monitoring in planta and showed that intracellular H₂ O₂ measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H₂ O₂ dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.