AS
Amir Sherman
  • Department of Fruit Trees Sciences, Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Israel
Research fields
  • Plant science
Luminol-based Assay for Detection of Immunity Elicitor-induced Hydrogen Peroxide Production in Arabidopsis thaliana Leaves
Authors:  Nora Gigli Bisceglia, Matteo Gravino and Daniel V. Savatin, date: 12/20/2015, view: 20671, Q&A: 1
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.
Camalexin Quantification in Arabidopsis thaliana Leaves Infected with Botrytis cinerea
Phytoalexins are heterogeneous low molecular mass secondary metabolites with antimicrobial activity produced at the infection site in response to pathogen invasion and represent an important part of the plant defense repertoire. Camalexin (3-Thiazol-2′-yl-indole) is a known phytoalexin first detected and isolated in Camelina sativa, from which it takes its name, infected with Alternaria brassicae (Browne et al., 1991). Production of camalexin is also induced in Arabidopsis thaliana leaves by a range of biotrophic and necrotrophic plant pathogens (bacteria, oomycetes, fungi and viruses) (Ahuja et al., 2012) as well as by abiotic stresses, such as UV and chemicals (e.g. acifluorfen, paraquat, chlorsulfuron and α-amino butyric acid) (Zhao et al., 1998; Tierens et al., 2002). Camalexin originates from tryptophan and CYP79B2 and CYP71B15 (PAD3) are P450 enzymes that catalyze important steps in its biosynthetic pathway (Glawischnig, 2007).

The detection and quantification of camalexin content is required to understand how it is produced upon various stress conditions. Here we describe an easy method for camalexin extraction from Arabidopsis leaves infected with the necrotrophic fungus Botrytis cinerea, and further determination of camalexin levels by liquid chromatography–mass spectrometry (LC-MS). The method is sensitive enough to trace amount of camalexin down to the low pico-gram (10 pg/mg FW) range.
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