Quantification of hydrogen peroxide in plant tissues using Amplex Red

Sourav Chakraborty, Amy L. Hill, Gautam Shirsekar, Ahmed 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 (H 2 O 2 ), whereby they can act as secondary messengers/chemical mediators in plant defense signaling and lead to programmed cell death. H 2 O 2 acts as a hub for critical information flow in plants. Despite such key roles in fundamental cellular processes, reliable determination of H 2 O 2 levels in plant tissues is hard to achieve. We optimized an Amplex Red-based quantitation method for H 2 O 2 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 H 2 O 2 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 H 2 O 2 extraction such as activated charcoal, ammonium sulfate, perchloric acid, polyvinylpolypyrrolidone, and trichloroacetic acid either degraded H 2 O 2 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

Fingerprint

Hydrogen Peroxide
Tissue
Assays
Reactive Oxygen Species
Trichloroacetic Acid
Photosynthesis
Charcoal
Ammonium Sulfate
Cell death
Byproducts
Pinus
Arabidopsis
Limit of Detection
Respiration
Cell Death

Keywords

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

ASJC Scopus subject areas

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

Cite this

Chakraborty, S., Hill, A. L., Shirsekar, G., Afzal, A., 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

Quantification of hydrogen peroxide in plant tissues using Amplex Red. / Chakraborty, Sourav; Hill, Amy L.; Shirsekar, Gautam; Afzal, Ahmed; Wang, Guo Liang; Mackey, David; Bonello, Pierluigi.

In: Methods, Vol. 109, 15.10.2016, p. 105-113.

Research output: Contribution to journalArticle

Chakraborty, S, Hill, AL, Shirsekar, G, Afzal, A, Wang, GL, Mackey, D & Bonello, P 2016, 'Quantification of hydrogen peroxide in plant tissues using Amplex Red', Methods, vol. 109, pp. 105-113. https://doi.org/10.1016/j.ymeth.2016.07.016
Chakraborty S, Hill AL, Shirsekar G, Afzal A, Wang GL, Mackey D et al. Quantification of hydrogen peroxide in plant tissues using Amplex Red. Methods. 2016 Oct 15;109:105-113. https://doi.org/10.1016/j.ymeth.2016.07.016
Chakraborty, Sourav ; Hill, Amy L. ; Shirsekar, Gautam ; Afzal, Ahmed ; Wang, Guo Liang ; Mackey, David ; Bonello, Pierluigi. / Quantification of hydrogen peroxide in plant tissues using Amplex Red. In: Methods. 2016 ; Vol. 109. pp. 105-113.
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