Filter strip as a method of choice for apoplastic fluid extraction from maize roots

Abstract

Apoplastic fluid was extracted from maize (Zea mays L.) roots using two procedures: collection from the surface of intact plant roots by filter paper strips (AF) or vacuum infiltration and/or centrifugation from excised root segments (AWF). The content of cytoplasmic marker (glucose-6-phosphate, G-6-P) and antioxidative components (enzymes, organic acids, phenolics, sugars, ROS) were compared in the extracts. The results obtained demonstrate that AF was completely free of G-6-P, as opposed to AWF where the cytoplasmic constituent was detected even at mildest centrifugation (200×g). Isoelectric focusing of POD and SOD shows the presence of cytoplasmic isoforms in AWF, and HPLC of sugars and phenolics a much more complex composition of AWF, due to cytoplasmic contamination. Organic acid composition differed in the two extracts, much higher concentrations of malic acid being registered in AF, while oxalic acid due to intracellular contamination being present only in AWF. EPR spectroscopy of DEPMPO spin trap in the extracts showed persistent generation of hydroxyl radical adduct in AF. The results obtained argue in favor of the filter strip method for the root apoplastic fluid extraction, avoiding the problems of cytoplasmic contamination and dilution and enabling concentration measurements in minute regions of the root.

Keywords: Apoplastic fluid; Filter paper strips; HPLC; Infiltration/centrifugation; Isoelectric focusing; Spin trapping electron paramagnetic resonance (EPR).