Plant Science 223 (2014) 49–58 Contents lists available at ScienceDirect Plant Science j ourna l ho me pa ge: www.elsevier.com/locate/plantsci Filter strip as a method of choice for apoplastic fluid extraction from maize roots Jelena J. Dragiˇ si´ c Maksimovi ´ c a,∗ , Branka D. ˇ Zivanovi ´ c a , Vuk M. Maksimovi ´ c a , Miloˇ s D. Mojovi ´ c b , Miroslav T. Nikolic a , ˇ Zeljko B. Vuˇ cini ´ c a a Institute for Multidisciplinary Research, University of Belgrade, Kneza Viˇ seslava 1, 11030 Belgrade, Serbia b Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, 11000 Belgrade, Serbia a r t i c l e i n f o Article history: Received 10 December 2013 Received in revised form 24 February 2014 Accepted 5 March 2014 Available online 13 March 2014 Keywords: Apoplastic fluid Filter paper strips Infiltration/centrifugation Isoelectric focusing HPLC Spin trapping electron paramagnetic resonance (EPR) a b s t r a c t 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. © 2014 Elsevier Ireland Ltd. All rights reserved. 1. Introduction The apoplast is a complex plant compartment, delimited from the symplast by the plasma membrane. It consists of the rigid cell wall fibrillar polymer network, the external surface of plasma membrane, and the liquid- and gas-filled spaces within this net- work, which provide interactions between the environment and the plasma membrane enclosed cytoplasm. All of these three com- ponents of the apoplast are rich in various organic molecules, Abbreviations: AF, apoplastic fluid; AWF, apoplastic washing fluid; DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide; DEPMPO/OH, DEPMPO adduct with hydroxyl radical; EPR, spin trapping electron paramagnetic resonance; G-6-P, glucose-6-phosphate; IEF, isoelectric focusing; MDH, malate dehydroge- nase; • OH, hydroxyl radical; PPB, potassium phosphate buffer; POD, peroxidase (EC 1.11.1.7); SOD, superoxide dismutase (EC 1.15.1.1). ∗ Corresponding author. Tel.: +381 112078460; fax: +381 113055289. E-mail addresses: draxy@imsi.rs, draxy@imsi.bg.ac.rs (J.J. Dragiˇ si´ c Maksimovi ´ c), vunduk@imsi.bg.ac.rs (B.D. ˇ Zivanovi ´ c), maxivuk@imsi.bg.ac.rs (V.M. Maksimovi ´ c), milos@ffh.bg.ac.rs (M.D. Mojovi ´ c), mnikolic@imsi.bg.ac.rs (M.T. Nikolic), vucinic@imsi.bg.ac.rs ( ˇ Z.B. Vuˇ cini ´ c). enzymes and proteins attached, dissolved, or embedded in or to them. The apoplast plays a major role in a wide range of physiological processes, including transport of water, nutrients and metabolites [1], gas exchange, growth regulation, plant-pathogen interactions, and perception and transduction of environmental signals [2]. As a thermodynamically open system, in direct contact with the envi- ronment, the root apoplast is the first to encounter varying and sometime adverse environmental conditions that are determining the response of the whole plant [3,4]. For each of the three apoplastic constituents specific isolation procedures have been developed, allowing a detailed in depth anal- ysis of their composition, molecular organization, biochemistry and physiology. The application of perfusion and infiltration and/or centrifugation method initially developed by Söding and Klement [5,6], and further refined by numerous authors, is the most fre- quently used technique for the isolation of the apoplastic fluid. It is a simple, quick and inexpensive method that can yield suffi- cient quantities of the so called “Apoplastic Washing Fluid (AWF)”. Besides the unknown dilution capacity, the infiltration of buffers or distilled water into the plant tissue alters the ionic composition, http://dx.doi.org/10.1016/j.plantsci.2014.03.009 0168-9452/© 2014 Elsevier Ireland Ltd. All rights reserved.