title: Quantification of hydrogen peroxide in plant tissues using Amplex Red.
abstract: Reactive oxygen species (ROS) are by-products of photosynthesis and respiration in plant tissues. Abiotic and biotic stressors also induce the production and temporary accumulation of ROS in plants, including hydrogen peroxide (H2O2), whereby they can act as secondary messengers/chemical mediators in plant defense signaling and lead to programmed cell death. H2O2acts as a hub for critical information flow in plants. Despite such key roles in fundamental cellular processes, reliable determination of H2O2levels in plant tissues is hard to achieve. We optimized an Amplex Red-based quantitation method for H2O2estimation from plant tissue lysate. The standard limit of detection and quantitation was determined as 6 and 18picomol respectively. In this study we also quantified constitutive and/or induced levels of H2O2in three model plants, Pinus nigra (Austrian pine), Oryza sativa (rice), and Arabidopsis thaliana. Overall, assay sensitivity was in the nmolg-1FW range. Commonly used additives for H2O2extraction such as activated charcoal, ammonium sulfate, perchloric acid, polyvinylpolypyrrolidone, and trichloroacetic acid either degraded H2O2directly or interfered with the Amplex Red assay. Finally, We measured stability of Amplex Red working solution over one month of storage at -80°C and found it to be significantly stable over time. With appropriate modifications, this optimized method should be applicable to any plant tissue.
authors.affiliation: Department of Plant Pathology, The Ohio State University, 2021 Coffey Road, 201 Kottman Hall, Columbus, OH 43210, USA. Electronic address: schakraborty@ccsu.edu.
authors.forename: Sourav
authors.initials: S
authors.lastname: Chakraborty
authors.affiliation: Department of Plant Pathology, The Ohio State University, 2021 Coffey Road, 201 Kottman Hall, Columbus, OH 43210, USA.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Hill
authors.affiliation: Department of Plant Pathology, The Ohio State University, 2021 Coffey Road, 201 Kottman Hall, Columbus, OH 43210, USA.
authors.forename: Gautam
authors.initials: G
authors.lastname: Shirsekar
authors.affiliation: Department of Horticulture and Crop Science, The Ohio State University, 2021 Coffey Road, Columbus, OH 43210, USA.
authors.forename: Ahmed J
authors.initials: AJ
authors.lastname: Afzal
authors.affiliation: Department of Plant Pathology, The Ohio State University, 2021 Coffey Road, 201 Kottman Hall, Columbus, OH 43210, USA.
authors.forename: Guo-Liang
authors.initials: GL
authors.lastname: Wang
authors.affiliation: Department of Horticulture and Crop Science, The Ohio State University, 2021 Coffey Road, Columbus, OH 43210, USA.
authors.forename: David
authors.initials: D
authors.lastname: Mackey
authors.affiliation: Department of Plant Pathology, The Ohio State University, 2021 Coffey Road, 201 Kottman Hall, Columbus, OH 43210, USA.
authors.forename: Pierluigi
authors.initials: P
authors.lastname: Bonello
mesh_terms: D017360:Arabidopsis / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000302:isolation & purification*; D012275:Oryza / Q000737:chemistry / Q000378:metabolism; D010788:Photosynthesis / Q000235:genetics*; D028223:Pinus / Q000737:chemistry / Q000378:metabolism; D010936:Plant Extracts / Q000737:chemistry* / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000302:isolation & purification*; D015398:Signal Transduction
chemical_list: D010936:Plant Extracts; D017382:Reactive Oxygen Species; D006861:Hydrogen Peroxide
keywords: Amplex Red; Analytical method; Fluorescence; Hydrogen peroxide; ROS