Journal of Plant Physiology 171 (2014) 429–437 Contents lists available at ScienceDirect Journal of Plant Physiology journal h om epage: www.elsevier.com/locate/jplph Physiology Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells Daniela Seifertová a , Petr Sk ˚ upa a , Jan Rychtᡠr b , Martina Laˇ nková a , Markéta Paˇ rezová a , Petre I. Dobrev a , Klára Hoyerová a , Jan Petrᡠsek a , Eva Zaˇ zímalová a,∗ a Institute of Experimental Botany ASCR, Rozvojová 263, 165 02 Prague 6, Czech Republic b Department of Mathematics and Statistics, The University of North Carolina at Greensboro, 130 Petty Building, NC 27403, USA a r t i c l e i n f o Article history: Received 1 June 2013 Received in revised form 24 September 2013 Accepted 28 September 2013 Keywords: Auxin influx Auxin efflux Auxin metabolic profiling Arabidopsis thaliana cell suspension (LE) Cell culture phenotype s u m m a r y Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cel- lular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small propor- tion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells. © 2013 Elsevier GmbH. All rights reserved. Introduction The plant hormone auxin is one of the most important regulators of plant growth and development. In addition to local biosynthesis and metabolic changes, its directional transport generates auxin concentration gradients needed for the transduction of develop- mental cues during both embryogenesis and postembryonic devel- opment of plants, including reactions to external environmental Abbreviations: BY-2, Nicotiana tabacum L., cv. Bright Yellow 2 cell line; CHPAA, 3-chloro-4-hydroxyphenylacetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; LE, Arabidopsis thaliana, ecotype Landsberg erecta cell line; NAA, naphthalene-1-acetic acid; 1-NOA, 1-naphthoxyacetic acid; 2-NOA, 2- naphthoxyacetic acid; NPA, 1-naphthylphthalamic acid; PBA, 2-(l-pyrenoyl)benzoic acid; PM, plasma membrane; TIBA, 2,3,5-triiodobenzoic acid; VBI-0, Nicotiana tabacum L., cv. Virginia Bright Italia cell line. ∗ Corresponding author. Tel.: +420 225 106 429; fax: +420 225 106 446. E-mail addresses: seifertova@ueb.cas.cz (D. Seifertová), skupa@ueb.cas.cz (P. Sk ˚ upa), j rychta@uncg.edu (J. Rychtᡠr), lankova@ueb.cas.cz (M. Laˇ nková), parezova@ueb.cas.cz (M. Paˇ rezová), dobrev@ueb.cas.cz (P.I. Dobrev), hoyerova@ueb.cas.cz (K. Hoyerová), petrasek@ueb.cas.cz (J. Petrᡠsek), zazimalova@ueb.cas.cz (E. Zaˇ zímalová). stimuli. In general, auxin is transported to longer distances in the phloem, but it is also subject to cell-to-cell transport, where passive diffusion is combined with the activity of plasma membrane (PM)- localized carriers. The polarity of auxin transport across the PM has been explained by the chemiosmotic polar diffusion model (Raven, 1975; Rubery and Sheldrake, 1974), based on the differential per- meability of the PM for dissociated and undissociated forms of auxin molecules. Undissociated auxin molecules in the more acidic extracellular environment enter cells by diffusion. In the more alka- line intracellular environment, dissociated auxin molecules having very low membrane permeability are trapped and are exported out of the cell almost entirely by active auxin efflux via auxin carriers. Generally, several groups of transporters are currently known to exhibit auxin influx or efflux activities (recent reviews by Peer et al., 2011; Petrᡠsek et al., 2011). Recent progress in understanding mechanisms of auxin trans- port in planta comes mainly from studies in Arabidopsis thaliana plants (Benjamins and Scheres, 2008; Petrᡠsek and Friml, 2009; Leyser, 2011; Löfke et al., 2013). In addition to the molecular bio- logical characterization of auxin influx and efflux carriers, as well as to regulatory mechanisms involved in their action, auxin transport 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.09.026