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Singlet Oxygen and Superoxide Anion Radical Detection by EPR Spin Trap** in Thylakoid Preparations

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ROS Signaling in Plants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2798))

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Abstract

Reactive oxygen species (ROS) are produced by energy transfer and electron transport in plant chloroplast thylakoids at non-toxic levels under normal growth conditions, but at threatening levels under adverse or fluctuating environmental conditions. Among chloroplast ROS, singlet oxygen and superoxide anion radical, respectively, produced by photosystem II (PSII) and PSI, are known to be the major ROS under several stress conditions. Both are very unlikely to diffuse out of chloroplasts, but they are instead capable of triggering ROS-mediated chloroplast operational retrograde signalling to activate defence gene expression in concert with hormones and other molecular compounds. Therefore, their detection, identification and localization in vivo or in biological preparations is a priority for a deeper understanding of their role in (concurrent) regulation of plant growth and defence responses. Here, we present two EPR spin traps, abbreviated as TEMPD-HCl and DEPMPO, to detect and identify ROS in complex systems, such as isolated thylakoids, together with some hints and cautions to perform reliable spin trap** experiments.

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Acknowledgements

We thank Dr. Anja Krieger-Liszkay for her careful and critical reading. The research was funded by MCIN/AEI/10.13039/501100011033 (Project n° PID2019-107154RB-100) and the regional government of Castilla y León (Project n° CSI260P20). The Project “CLU-2019–05—IRNASA/CSIC Unit of Excellence” funded by the Junta de Castilla y León and co-financed by the European Union (ERDF, “Europe Drives Our Growth”) and the CSIC Interdisciplinary Thematic Platform (PTI) Optimization of Agricultural and Forestry Systems (PTI-AGROFOR) are also acknowledged.

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Authors and Affiliations

  1. Departamento de Estrés Abiótico, Instituto de Recursos Naturales y Agrobiología de Salamanca (IRNASA-CSIC), Salamanca, Spain

    Ouardia Bendou, Nara Bueno-Ramos, Emilio L. Marcos-Barbero, Rosa Morcuende & Juan B. Arellano

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  1. Ouardia Bendou
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  2. Nara Bueno-Ramos
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  3. Emilio L. Marcos-Barbero
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  4. Rosa Morcuende
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  5. Juan B. Arellano
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Correspondence to Juan B. Arellano .

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Editors and Affiliations

  1. Spanish National Research Council, Estación Experimental del Zaidín, Granada, Granada, Spain

    Francisco J. Corpas

  2. Spanish National Research Council, Estación Experimental del Zaidín, Granada, Spain

    José M. Palma

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Bendou, O., Bueno-Ramos, N., Marcos-Barbero, E.L., Morcuende, R., Arellano, J.B. (2024). Singlet Oxygen and Superoxide Anion Radical Detection by EPR Spin Trap** in Thylakoid Preparations. In: Corpas, F.J., Palma, J.M. (eds) ROS Signaling in Plants . Methods in Molecular Biology, vol 2798. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3826-2_2

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  • DOI: https://doi.org/10.1007/978-1-0716-3826-2_2

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