Longitudinal distribution of apoplastic antioxidative components in maize root

Abstract

The apoplast is a liquid- and gas-filled extracellular continuum which includes cell wall polymer networks and the external surface of the plasma membrane. Apoplastic constituents, such as various organic molecules, enzymes and proteins play major roles in a wide range of physiological processes. In order to investigate apoplastic fluid, two isolation procedures were compared and critically evaluated: infiltration and/or centrifugation techniques to obtain Apoplastic Washing Fluid and filter paper strips which, based on our results, enable collection of experimental data from intact plants. Different components of the antioxidative system (enzymes, phenolics, sugars, organic acids) present in apoplastic fluid were analyzed using different techniques. Three classes of non-enzymatic compounds (organic acids, sugars and phenolics) have been identified and quantified by HPLC. Detection of hydroxyl radicals was performed by EPR methods using spin-trap DEPMPO, which is capable of forming different spin-adducts with hydroxyl and superoxide anion radicals. Spectrophotometrically estimated total protein concentrations, peroxidase and superoxide dismutase specific activities, as well as their different isoforms, were visually confirmed by isoelectric focusing. These high sensitivity techniques (HPLC-ECD, EPR), as well as electrophoresis, in combination with the filter strip method provided us with the tools necessary to study the components of the antioxidative system in the apoplast of developing plant organs and their spatial-temporal changes. Such an experimental setup provided a powerful non-invasive analytical tool for studying metabolic processes occurring in the apoplast and local changes in small regions of intact root tissue.