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Measurement of reduced, oxidized and total ascorbate content in plants

Abstract

Ascorbate is one of the major antioxidant metabolites in plant tissues. This protocol describes a microplate-adapted colorimetric ascorbate assay, in which ferric ion is reduced by ascorbate to the ferrous ion. The ferrous ion reacts with α-α′-bipyridl to form a complex with characteristic absorbance at 525 nm. With the chemical reduction of any dehydroascorbate (DHA) in a sample, total ascorbate can be assayed using the α-α′-bipyridl method, and DHA can be estimated by subtracting the reduced portion from the total ascorbate pool. The assay is performed in microcentrifuge tubes and assessed in a 96-well plate reader. Reduced ascorbate, DHA and total ascorbate of at least 64 experimental samples can be analyzed easily in 1 d.

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Figure 1: Example of an ascorbate standard curve.

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Acknowledgements

This research was supported by the Office of Science (BER), U.S. Department of Energy, Grant no. DE-FG02-04ER63849.

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Correspondence to Elizabeth A Ainsworth.

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Gillespie, K., Ainsworth, E. Measurement of reduced, oxidized and total ascorbate content in plants. Nat Protoc 2, 871–874 (2007). https://doi.org/10.1038/nprot.2007.101

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