Measurement of reduced, oxidized and total ascorbate content in plants.
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 alpha-alpha'-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 alpha-alpha'-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.
Gillespie
Kelly M
KM
Program in Physiological and Molecular Plant Biology and Institute for Genomic Biology, University of Illinois Urbana-Champaign, 147 ERML, 1201 W. Gregory Drive, Urbana, Illinois 61801, USA.
Ainsworth
Elizabeth A
EA
D000975:Antioxidants / Q000378:metabolism; D001205:Ascorbic Acid / Q000378:metabolism*; D003124:Colorimetry / Q000379:methods*; D003683:Dehydroascorbic Acid / Q000378:metabolism; D010084:Oxidation-Reduction; D010944:Plants / Q000378:metabolism*
D000975:Antioxidants; D001205:Ascorbic Acid; D003683:Dehydroascorbic Acid