Spatial distribution of apoplastic antioxidative constituents in maize root.

Apoplastic antioxidative constituents (enzymes, primary and secondary metabolites, ROS) from different root zones of hydroponically grown maize (Zea mays L.) were investigated using a noninvasive isolation procedure: filter strip method. Filter strips were placed at specific positions on the root surface: apical zone (tip) and basal zone (base) to absorb apoplastic fluid. Three major classes of low-weight metabolites (organic acids, sugars, and phenolics) have been identified by HPLC-ECD. The longitudinal distribution of sugars and organic acids had the same pattern: higher concentration in the tip than the base, while it was vice versa for phenolics. The specific activities of guaiacol peroxidase, superoxide dismutase, and ascorbate peroxidase were higher in the apoplastic fluid from the root base than the tip, and their different isoforms were separated by isoelectric focusing. Electron paramagnetic resonance (EPR) spectroscopy coupled with the spin-trapping method using DEPMPO showed a persistent generation of hydroxyl radical in the root tip. In vivo EPR imaging of the whole maize root with membrane-permeable and impermeable aminoxyl spin-probes, enabling real-time detection of ROS formation within and outside the membranes, demonstrated ROS accumulation on the root surface, while endodermis and central cylinder were ROS free. For the first time in plant research, 2D EPR images enabled the direct demonstration of site-specific free radical production along the root. Highly sensitive analytical techniques combined with the filter strips, as a non-invasive tool, have increased our knowledge of metabolic processes occurring in the apoplast and their spatial-temporal changes in small regions of the intact root tissue.