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. H₂O₂ acts as a hub for critical information flow in plants. Despite such key roles in fundamental cellular processes, reliable determination of H₂O₂ levels in plant tissues is hard to achieve. We optimized an Amplex Red-based quantitation method for H₂O₂ estimation 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 H₂O₂ in three model plants, Pinus nigra (Austrian pine), Oryza sativa (rice), and Arabidopsis thaliana. Overall, assay sensitivity was in the nmolg^-1 FW range. Commonly used additives for H₂O₂ extraction such as activated charcoal, ammonium sulfate, perchloric acid, polyvinylpolypyrrolidone, and trichloroacetic acid either degraded H₂O₂ directly 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.

Keywords: Amplex Red; Analytical method; Fluorescence; Hydrogen peroxide; ROS.