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A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

A Rapid and Specific Microplate Assay for the Determination of Intra- and Extracellular Ascorbate in Cultured Cells

Published on: April 11, 2014

A critical comparison of two high-throughput ascorbate analyses methods for plant samples.

Yoshiaki Ueda¹, Linbo Wu, Michael Frei

¹Institute of Crop Science and Resource Conservation (INRES) - Plant Nutrition, University of Bonn, Karlrobert-Kreiten Strasse 13, 53115 Bonn, Germany.

Plant Physiology and Biochemistry : PPB

|July 10, 2013

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Quantifying plant ascorbate (AsA) is crucial. The ascorbate oxidase (AO) assay offers higher sensitivity for low AsA levels and avoids interference from pigments and iron, unlike the dipyridyl (DPD) assay which provides greater precision.

Area of Science:

Plant Science
Biochemistry
Analytical Chemistry

Background:

Ascorbate (AsA) is a vital plant metabolite involved in stress responses and development.
Accurate quantification of AsA is essential for various plant science applications, including high-throughput screening.

Purpose of the Study:

To compare the suitability of two microplate-based assays, ascorbate oxidase (AO) and dipyridyl (DPD), for quantifying plant AsA.
To evaluate assay performance under challenging conditions, such as low AsA concentrations, presence of interfering pigments, and high iron content.

Main Methods:

Comparison of an ascorbate oxidase (AO)-based assay and a dipyridyl (DPD)-based assay.
Evaluation of assay performance with low AsA concentrations (apoplastic extracts).
Assessment of interference from plant pigments and high iron concentrations.

Keywords:

2,2′-dipyridyl AO Apoplast AsA Ascorbate DHA DPD DTT High-throughput analysis IWF Iron toxicity MPA N-ethylmaleimide NEM Oryza sativa Pigments ROS TCA ascorbate ascorbate oxidase dehydroascorbate dithiothreitol intercellular washing fluid metaphosphoric acid reactive oxygen species trichloroacetic acid

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Main Results:

The DPD method demonstrated higher measurement precision, indicated by superior recovery rates of internal AsA standards.
The AO method exhibited greater sensitivity for detecting low AsA concentrations, particularly in rice apoplastic extracts.
The AO method was unaffected by plant pigments and high iron concentrations, whereas the DPD assay was compromised by these factors.

Conclusions:

Both AO and DPD assays have distinct advantages for quantifying plant ascorbate.
The choice between AO and DPD methods depends on the specific requirements of the application, considering factors like AsA concentration and potential interfering substances.