Reactive oxygen species (ROS) are integral component of cellular system. 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. H2O2 acts as a central hub for critical information flow in plants. Despite such key roles in fundamental 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 18 picomol respectively. We further 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 nmol g-1 FW range. Commonly used additives for H2O2 extraction 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 a variety of complex tissue matrices.

Hydrogen peroxide quantitation in complex matrices – A fluorimetric method using Amplex Red

253rd National Meeting (2017)

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