Quantification of hydrogen peroxide in plant tissues using Amplex Red.

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 (HO), whereby they can act as secondary messengers/chemical mediators in plant defense signaling and lead to programmed cell death. HO acts as a hub for critical information flow in plants. Despite such key roles in fundamental cellular processes, reliable determination of HO levels in plant tissues is hard to achieve. We optimized an Amplex Red-based quantitation method for HO 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 HO in three model plants, Pinus nigra (Austrian pine), Oryza sativa (rice), and Arabidopsis thaliana. Overall, assay sensitivity was in the nmolg FW range. Commonly used additives for HO extraction such as activated charcoal, ammonium sulfate, perchloric acid, polyvinylpolypyrrolidone, and trichloroacetic acid either degraded HO 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.