Enzymatic determination of ascorbic acid in leaf cell walls using acidic buffer during infiltration

Abstract

A modification of the procedure of extraction of cell wall solution for enzymatic determination of ascorbic acid and its reduction level in the apoplast of leaf cells is proposed. The modification consists in infiltration of leaves with citric acid/sodium phosphate buffer, pH 3, instead of customarily used neutral solutions. In acidic media autooxidation of ascorbic acid is effectively suppressed, so that infiltration could be performed at laboratory temperatures. Using polyacrylamide gel electrophoresis and infiltration solutions of pH down to 1.5 it is shown, that at pH 3 the extracted fluid is not contaminated with intracellular substances if appropriate vacuum and centrifugation forces are used. The modification is shown to be more effective for leaves of Phaseolus than for those of Spinacia. In cell walls of mature leaves of these species the concentration of ascorbic acid was found to be around 1 mM, with reduction level up to 0.90. The role of ascorbic acid in cell walls as ozone scavenger is discussed.