DOI: 10.1007/s10535-016-0634-2 BIOLOGIA PLANTARUM 60 (4): 695-705, 2016 695 Tracing root permeability: comparison of tracer methods E. PECKOVÁ, E. TYLOVÁ, and A. SOUKUP* Department of Experimental Plant Biology, Faculty of Natural Sciences, Charles University in Prague, CZ-12844 Prague, Czech Republic Abstract Root epidermis and apoplastic barriers (endodermis and exodermis) are the critical root structures involved in setting up plant-soil interface by regulating free apoplastic movement of solutes within root tissues. Probing root apoplast permeability with “apoplastic tracers” presents one of scarce tools available for detection of “apoplastic leakage” sites and evaluation of their role in overall root uptake of water, nutrients, or pollutants. Although the tracers are used for many decades, there is still not an ideal apoplastic tracer and flawless procedure with straightforward interpretation. In this article, we present our experience with the most frequently used tracers representing various types of chemicals with different characteristics. We examine their behaviour, characteristics, and limitations. Here, we show that results gained with an apoplastic tracer assay technique are reliable but depend on many parameters – chemical properties of a selected tracer, plant species, cell wall properties, exposure time, or sample processing. Additional key words: apoplast, berberine, endodermis, exodermis, ferrous ions, PAS reaction, propidium iodide, PTS. Introduction Root permeability is one of the key features determining root-soil communication, resources acquisition, or resistance to pollutants with implication to plant stress tolerance or food quality. Passive non-selective transport via apoplast is restricted by apoplastic barriers. Among them, endodermis is the essential barrier of vascular plant roots. Its function is related to cell wall modifications and tight membrane adhesion that prevents apoplastic transport across the endodermal layer (Kroemer 1903, De Lavison 1910, Esau 1953, Enstone et al. 2003, Geldner 2013). A similar barrier (exodermis) may occur in the outer cortex (Enstone et al. 2003). Exodermis is not an obligatory structure and its formation is under a strong environmental influence (Perumalla et al. 1990, Peterson and Perumalla 1990). Apoplastic barriers play a crucial role in water and nutrient uptake and are involved in stress ecophysiology (North and Nobel 1995, Armstrong et al. 2000, Soukup et al. 2007, Redjala et al. 2011). They should not be perceived as strictly impermeable boundaries (Hose et al. 2001, Ranathunge et al. 2005b), and their properties are modified along the root, in different root types, or in response to stress factors (Moon et al. 1984, Degenhardt and Gimmler 2000, Colmer 2003, Meyer et al. 2009, Krishnamurthy et al. 2014, Shiono et al. 2014). Apoplast permeability modulates root uptake characteristics substantially, but there is a limited set of methodological tools to evaluate its extent and spatial variation. Quantitative measurements of root transport parameters present the first set of available methods, e.g., a root pressure probe technique (Peterson et al. 1993, Zimmermann and Steudle 1998, Ranathunge et al. 2003, 2005a,b, Bramley et al. 2007, Knipfer and Fricke 2010) or a vacuum perfusion technique (Knipfer and Fricke 2010). These methods allow quantifying root pressure (Wegner 2014) and water and solute flows in intact root systems or excised root segments. In addition, techniques following radial O 2 loss with a root-sleeving O 2 electrode or a methylene blue indicator dye detecting O 2 escaped from the root were successfully used to monitor O 2 permeability of barriers (Armstrong and Armstrong 2001, Soukup et al. 2007, Shiono et al. 2011). All these methods provide quantitative data for modelling of transport flows at whole plant/root levels, but their spatial resolution is rather limited. In contrast, apoplastic tracer assays allow analysis of spatial variability in root apoplast permeability. Roots are exposed to compounds with a limited penetration cross  Submitted 22 October 2015, last revision 1 February 2016, accepted 31 March 2016. Abbreviations: PAS - periodic acid – Schiff’s reagent; PTS - trisodium 3-hydroxy-5,8,10-pyrenetrisulfonate. Acknowledgements: We gratefully acknowledge financial support by project LO1417. The first two authors contributed equally to this work. * Corresponding author; e-mail: asoukup@natur.cuni.cz