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The fluorescent protein sensor ro GFP 2‐Orp1 monitors in vivo H 2 O 2 and thiol redox integration and elucidates intracellular H 2 O 2 dynamics during elicitor‐induced oxidative burst in Arabidopsis
Author(s) -
Nietzel Thomas,
Elsässer Marlene,
Ruberti Cristina,
Steinbeck Janina,
Ugalde José Manuel,
Fuchs Philippe,
Wagner Stephan,
Ostermann Lara,
Moseler Anna,
Lemke Philipp,
Fricker Mark D.,
MüllerSchüssele Stefanie J.,
Moerschbacher Bruno M.,
Costa Alex,
Meyer Andreas J.,
Schwarzländer Markus
Publication year - 2019
Publication title -
new phytologist
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.742
H-Index - 244
eISSN - 1469-8137
pISSN - 0028-646X
DOI - 10.1111/nph.15550
Subject(s) - intracellular , cytosol , green fluorescent protein , microbiology and biotechnology , arabidopsis , context (archaeology) , glutathione , yellow fluorescent protein , biochemistry , biophysics , chemistry , biology , redox , mutant , gene , enzyme , paleontology , organic chemistry
Summary Hydrogen peroxide (H 2 O 2 ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H 2 O 2 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 2 O 2 sensing in living plants. We established H 2 O 2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein ro GFP 2‐Orp1 using confocal microscopy and multiwell fluorimetry. We confirmed sensor oxidation by H 2 O 2 , 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 2 O 2 , pharmacologically‐induced H 2 O 2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H 2 O 2 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 2 O 2 monitoring in planta and showed that intracellular H 2 O 2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H 2 O 2 dynamics and redox regulation, including intracellular NADPH oxidase‐mediated ROS signalling.