Quantification of hydrogen peroxide in plant tissues using Amplex Red

Sourav Chakraborty, Amy L. Hill, Gautam Shirsekar, Ahmed J. Afzal, Guo Liang Wang, David Mackey, Pierluigi Bonello

Research output: Contribution to journalArticle

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 18 picomol 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 nmol g−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.

Original languageEnglish (US)
Pages (from-to)105-113
Number of pages9
JournalMethods
Volume109
DOIs
StatePublished - Oct 15 2016

Keywords

  • Amplex Red
  • Analytical method
  • Fluorescence
  • Hydrogen peroxide
  • ROS

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Chakraborty, S., Hill, A. L., Shirsekar, G., Afzal, A. J., Wang, G. L., Mackey, D., & Bonello, P. (2016). Quantification of hydrogen peroxide in plant tissues using Amplex Red. Methods, 109, 105-113. https://doi.org/10.1016/j.ymeth.2016.07.016