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Live Monitoring of ROS-Induced Cytosolic Redox Changes with roGFP2-Based Sensors in Plants

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Reactive Oxygen Species in Plants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2526))

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Abstract

Plant cells produce reactive oxygen species (ROS) as by-products of oxygen metabolism and for signal transduction. Depending on their concentration and their site of production, ROS can cause oxidative damage within the cell and must be effectively scavenged. Detoxification of the most stable ROS, hydrogen peroxide (H2O2), via the glutathione-ascorbate pathway may transiently alter the glutathione redox potential (EGSH). Changes in EGSH can thus be considered as an indicator of the oxidative load in the cell. Genetically encoded probes based on roGFP2 enable extended opportunities for in vivo monitoring of H2O2 and EGSH dynamics. Here, we provide detailed protocols for live monitoring of both parameters in the cytosol with the probes Grx1-roGFP2 for EGSH and roGFP2-Orp1 for H2O2, respectively. The protocols have been adapted for live cell imaging with high lateral resolution on a confocal microscope and for multi-parallel measurements in whole organs or intact seedlings in a fluorescence microplate reader. Elicitor-induced ROS generation is used for illustration of the opportunities for dynamic ROS measurements that can be transferred to other research questions and model systems.

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Acknowledgments

Support by the Deutsche Forschungsgemeinschaft through the Research Training Group GRK2064 (to AJM, MS and SJM-S) and through the priority program SPP1710 (to AJM and MS) is gratefully acknowledged. We thank our former lab members Thomas Nietzel, Stephan Wagner, and Philippe Fuchs for their seminal work in establishing protocols and analysis routines for live redox imaging and plate reader assays.

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Authors and Affiliations

  1. Institute of Crop Science and Resource Conservation (INRES), Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany

    José Manuel Ugalde, Lara Fecker & Andreas J. Meyer

  2. Institute of Plant Biology and Biotechnology, Westfälische Wilhelms-Universität Münster, Münster, Germany

    Markus Schwarzländer

  3. Department of Biology, Molecular Botany, Technische Universität Kaiserslautern, Kaiserslautern, Germany

    Stefanie J. Müller-Schüssele

Authors
  1. José Manuel Ugalde
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  2. Lara Fecker
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  3. Markus Schwarzländer
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  4. Stefanie J. Müller-Schüssele
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  5. Andreas J. Meyer
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Corresponding author

Correspondence to Andreas J. Meyer .

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Editors and Affiliations

  1. VIB Center for Plant Systems Biology, Ghent University, Zwijnaarde, Belgium

    Amna Mhamdi

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Ugalde, J.M., Fecker, L., Schwarzländer, M., Müller-Schüssele, S.J., Meyer, A.J. (2022). Live Monitoring of ROS-Induced Cytosolic Redox Changes with roGFP2-Based Sensors in Plants. In: Mhamdi, A. (eds) Reactive Oxygen Species in Plants. Methods in Molecular Biology, vol 2526. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2469-2_5

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  • DOI: https://doi.org/10.1007/978-1-0716-2469-2_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2468-5

  • Online ISBN: 978-1-0716-2469-2

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