In Arabidopsis thaliana, one of the very early immune-related responses induced after elicitor perception is the oxidative burst, i.e. reactive oxygen species (ROS) generation including superoxide anion and hydrogen peroxide (H2O2). ROS production plays different roles in a wide range of biotic and abiotic stress responses, including the closure of stomata and the regulation of cell expansion. In particular, elicitor-induced H2O2 is produced mainly by the membrane localized NAD(P)H oxidases RESPIRATORY BURST OXIDASE HOMOLOGUE D and F. In this protocol, we describe a simple and reproducible luminol/peroxidase-based assay to detect and evaluate immunity-related accumulation of H2O2 produced in Arabidopsis leaf discs treated with immunity elicitors, such as oligogalacturonides (OGs), flagellin (flg22) or the elongation factor-thermo-unstable (EF-Tu - elf18). This method is based on the detection of the luminescence released by excited-luminol molecules generated after the horseradish peroxidase (HRP)-catalyzed oxidation of luminol molecules in the presence of H2O2. Levels as well as duration of the luminescence are proportional to the amount of H2O2 produced by elicited leaf discs.

Luminol-based Assay for Detection of Immunity Elicitor-induced Hydrogen Peroxide Production in Arabidopsis thaliana Leaves / Bisceglia, Nora Gigli; Gravino, Matteo; Savatin, DANIEL VALENTIN. - In: BIO-PROTOCOL. - ISSN 2331-8325. - ELETTRONICO. - 5:24(2015), pp. 1-7. [10.21769/BioProtoc.1685]

Luminol-based Assay for Detection of Immunity Elicitor-induced Hydrogen Peroxide Production in Arabidopsis thaliana Leaves

GRAVINO, MATTEO;SAVATIN, DANIEL VALENTIN
2015

Abstract

In Arabidopsis thaliana, one of the very early immune-related responses induced after elicitor perception is the oxidative burst, i.e. reactive oxygen species (ROS) generation including superoxide anion and hydrogen peroxide (H2O2). ROS production plays different roles in a wide range of biotic and abiotic stress responses, including the closure of stomata and the regulation of cell expansion. In particular, elicitor-induced H2O2 is produced mainly by the membrane localized NAD(P)H oxidases RESPIRATORY BURST OXIDASE HOMOLOGUE D and F. In this protocol, we describe a simple and reproducible luminol/peroxidase-based assay to detect and evaluate immunity-related accumulation of H2O2 produced in Arabidopsis leaf discs treated with immunity elicitors, such as oligogalacturonides (OGs), flagellin (flg22) or the elongation factor-thermo-unstable (EF-Tu - elf18). This method is based on the detection of the luminescence released by excited-luminol molecules generated after the horseradish peroxidase (HRP)-catalyzed oxidation of luminol molecules in the presence of H2O2. Levels as well as duration of the luminescence are proportional to the amount of H2O2 produced by elicited leaf discs.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11573/873832
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