Journal of Plant Physiology 171 (2014) 429–437

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Physiology

Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells a a ˇ ˚ Daniela Seifertová a , Petr Skupa , Jan Rychtáˇr b , Martina Lanková , Markéta Paˇrezová a , a a a a,∗ Petre I. Dobrev , Klára Hoyerová , Jan Petráˇsek , Eva Zaˇzímalová a b

Institute of Experimental Botany ASCR, Rozvojová 263, 165 02 Prague 6, Czech Republic 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 Available online 14 February 2014 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 cellular 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 proportion 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 developmental cues during both embryogenesis and postembryonic development 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, 2naphthoxyacetic 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: [email protected] (D. Seifertová), [email protected] ˇ ˚ (P. Skupa), j [email protected] (J. Rychtáˇr), [email protected] (M. Lanková), [email protected] (M. Paˇrezová), [email protected] (P.I. Dobrev), [email protected] (K. Hoyerová), [email protected] (J. Petráˇsek), [email protected] (E. Zaˇzímalová). 0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.09.026

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 permeability 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 alkaline 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; Petráˇsek et al., 2011). Recent progress in understanding mechanisms of auxin transport in planta comes mainly from studies in Arabidopsis thaliana plants (Benjamins and Scheres, 2008; Petráˇsek and Friml, 2009; Leyser, 2011; Löfke et al., 2013). In addition to the molecular biological characterization of auxin influx and efflux carriers, as well as to regulatory mechanisms involved in their action, auxin transport

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has been studied in plant tissues/organs by the measurement of movement of radioactively-labeled auxin (Lewis and Muday, 2009) both in roots (basipetal and acropetal) and shoots (basipetal) of Arabidopsis plants (Garbers et al., 1996; Geisler et al., 2003; Murphy et al., 2000; Noh et al., 2001; Rashotte et al., 2003) or other plant systems (reviewed in Morris, 2000; Morris et al., 2004). However, these approaches on the tissue/organ level cannot be used to determine kinetic parameters of auxin transport across membrane and to distinguish between cellular auxin influx and efflux. Therefore, simplified models of yeasts and cell cultures derived from plants, animals, and even humans are frequently utilized (Delbarre et al., 1996; Geisler et al., 2005; Hrycyna et al., 1998; Luschnig et al., 1998; Noh et al., 2001; Petráˇsek et al., 2006; Yang et al., 2006; Yang and Murphy, 2009; and references therein). Cell lines represent the major experimental system that can be used for both qualitative and quantitative studies of various proteins’ activity at the cellular level in vivo. In fact, studies of the transport of radiolabeled indole-3-acetic acid (IAA) using crown gall suspension culture of Parthenocissus tricuspidata resulted in the chemiosmotic polar diffusion model of polar auxin transport (Rubery and Sheldrake, 1974). Suspension-cultured soybean root cells were used for intimate studies of IAA transport by Loper and Spanswick (1991), and the authors described IAA uptake via passive diffusion and saturable influx carrier and active efflux. Rapid metabolism of IAA molecules (about 80% after 15 min uptake) was shown as well. Nowadays, the best characterized models are homogeneous, highly friable populations of tobacco suspension-cultured cells, where the active auxin influx and efflux parameters were determined quantitatively for native auxin IAA and for its synthetic analogs (Delbarre et al., 1996; Petráˇsek et al., 2002, 2003; Petráˇsek and Zaˇzímalová, 2006). The proportions of the active auxin influx and efflux and diffusion rates for IAA, naphthalene-1-acetic acid and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) were determined in suspension-cultured cells of Nicotiana tabacum L. cv. Xanthi XHFD8 (Delbarre et al., 1996). Based on the determination of accumulation kinetics, metabolic degradation and competition assays, it was shown that the accumulation of IAA comprises passive diffusion and the activity of both auxin influx and efflux carriers. In contrast, synthetic auxin NAA was transported into cells preferentially by passive diffusion and out of the cell by active efflux, while 2,4-D accumulation inside cells resulted primarily from the active auxin influx. Based on these findings, Delbarre et al. (1996) suggested a simple methodology for the measurements of active auxin influx and efflux by using the accumulation assays of radioactively labeled 2,4-D and NAA, respectively. Active transport of auxin across PM has been characterized further using inhibitors of auxin influx, such as 1naphthoxyacetic acid (1-NOA), 2-naphthoxyacetic acid (2-NOA) and 3-chloro-4-hydroxyphenylacetic acid (CHPAA) (Imhoff et al., 2000; Parry et al., 2001) and auxin efflux, 1-naphthylphthalamic acid (NPA) and 2-(l-pyrenoyl)benzoic acid (PBA) (Keitt and Baker, 1966; Delbarre et al., 1996; Petráˇsek et al., 2003). The application of inhibitors of auxin influx in the heterologous system of Xenopus laevis oocytes (Yang et al., 2006; Swarup et al., 2008) ˇ and in tobacco BY-2 cells (Lanková et al., 2010) revealed that the amount of auxin taken up actively into the cells by specific influx carriers is significant. Basic characteristics of auxin efflux in other tobacco cell lines (Nicotiana tabacum L., cv. VBI-0; Petráˇsek et al., 2002, and BY-2; Petráˇsek et al., 2003; ˇ Lanková et al., 2010) were similar to tobacco Xanthi XHFD8 cells, although in VBI-0 cells there was higher proportion of the active efflux of 2,4-D (Paciorek et al., 2005). This activity was also enhanced for 2,4-D after the inducible overexpression of PINtype auxin efflux carriers (namely PIN7) in BY-2 cells (Petráˇsek et al., 2006), suggesting differential affinity and/or capacity of

auxin carriers to various auxins in various experimental models. Recently, due to its genetic ‘accessibility,’ Arabidopsis represents the main model for studies of auxin action and its transport in planta. In spite of this, there is still a significant lack of knowledge of detailed auxin transport characteristics at the level of cultured Arabidopsis cells. Arabidopsis cell suspensions derived from ecotypes Landsberg erecta (May and Leaver, 1993) and Columbia (Axelos et al., 1992) are available. Similar to BY-2 tobacco cells, they can be transformed (Mathur et al., 1998) and synchronized (Menges and Murray, 2002). Even though the A. thaliana ecotype Landsberg erecta cell line has already been used for IAA transport assays (Geisler et al., 2005), more information on the specificity and dynamics of IAA, NAA and 2,4-D cellular transport is still needed. This report provides basic kinetic and specificity parameters of carrier-mediated auxin uptake (influx) and efflux in Arabidopsis ecotype Landsberg erecta suspension-cultured cells (LE line), together with data about metabolism of exogenously added auxins, and compares these characteristics with the already established model of tobacco cells. Materials and methods Chemicals All chemicals were obtained from Sigma–Aldrich (St. Louis, MO, USA) unless otherwise noted. 1-Naphthylphthalamic acid (NPA) was obtained from OlChemIm (Olomouc, Czech Republic). NPA and 2-naphthoxyacetic acid (2-NOA) were dissolved in ethanol to yield stock solutions 10 mM. NPA for the results presented in Figure S4 was prepared in 0.1 mM, 1 mM, 10 mM, 100 mM ethanolic stock solutions. Stock solutions of non-labeled auxins were prepared in concentration 5 ␮M, 1 mM, 10 mM, 30 mM and 300 mM dissolved in ethanol. HPLC-grade methanol and acetonitrile were obtained from Merck KGaA (Darmstadt, Germany). Formic acid and ammonium hydroxide (both of p.a. grade) were from Lachema a.s. (Neratovice, Czech Republic). Oasis MCX columns (150 mg/6 cc) were obtained from Waters (Milford, MA, USA). The following radiolabeled auxins were used for accumulation and metabolic assays: [3 H]naphthalene-1-acetic acid (NAA), [3 H]indole-3-acetic acid (IAA), [3 H]2,4-dichlorophenoxyacetic acid (2,4-D) (specific radioactivity 20 Ci/mmol each, American Radiolabeled Chemicals, ARC, Inc., St. Louis, MO, USA). Plant material Tobacco BY-2 cells Nicotiana tabacum L. cv. Bright Yellow 2 (Nagata et al., 1992), and Arabidopsis thaliana, ecotype Landsberg erecta (May and Leaver, 1993) LE cells were cultured in the darkness at 24 ◦ C (LE) and 27 ◦ C (BY-2) on the orbital incubator (Sanyo Gallenkamp PLC, IOI400.XX2.C; 130 rpm and 150 rpm, LE and BY-2 cells, respectively) in liquid medium (3% sucrose, 4.3 g L−1 Murashige and Skoog salts, 100 mg L−1 inositol, 1 mg L−1 thiamin, 0.2 mg L−1 2,4-D, and 200 mg L−1 KH2 PO4 , pH 5.8) and subcultured weekly (1 mL suspension to 30 mL fresh media for both LE and BY2). Stock calli were maintained on the same media solidified with 0.6% (w/v) agar and subcultured monthly. The cell suspension of A. thaliana missense mutant aux1-7 (NASC N3074; Pickett et al., 1990) was established from a mixture of cotyledons, hypocotyls and leaves of 1-week-old seedling plants (based on the protocol by Blackhall, 1993). These were cut and placed on callus induction medium (3.2 g L−1 Gamborg’s B5 Basal medium, 2% glucose, 0.5 g L−1 MES, 0.05 mg L−1 kinetin, 0.5 mg L−1 2,4-D, agar 0.6%, w/v, pH 5.7). After 4 weeks, newly formed calli

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Fig. 1. Phenotype of 2-day-old Arabidopsis thaliana, ecotype Landsberg erecta (LE, panel A) and Nicotiana tabacum, cv. Bright Yellow 2 (BY-2, panel B) cell cultures. LE cells grow in small spherical clusters, BY-2 form cell chains. Growth curve in panel (C) shows the multiplication rate of LE and BY-2 cell cultures during 7-day subculture interval. LE and BY-2 multiply 23-times and 32-times, respectively, in one subculture interval. Starting density (day 0) and final density (day 7): LE – 240 938; 5 648 438, BY-2 – 111 781; 3 541 000 cells per mL. Error bars = SEs (n = 10). (D) Distribution of cell lengths and cell diameters in 2-day-old cell populations (n = 700 or 500 for LE and BY-2, respectively). LE cells are more spherical while BY-2 cells are more elongated (cylindrical). Scale bars = 100 ␮m.

were transferred onto solid MS medium (3% sucrose, 4.3 g L−1 Murashige and Skoog salts, 100 mg L−1 inositol, 1 mg L−1 thiamin, 0.2 mg L−1 2,4-D, and 200 mg L−1 KH2 PO4 , pH 5.8, 0.6%, w/v agar) and subcultured monthly. The cell suspension was derived from calli and cultured in the darkness at 24 ◦ C on an orbital incubator (Sanyo Gallenkamp PLC, IOI400.XX2.C; 130 rpm) in liquid medium (3% sucrose, 4.3 g L−1 Murashige and Skoog salts, 100 mg L−1 inositol, 1 mg L−1 thiamin, 0.5 mg L−1 2,4-D, 0.2 mg L−1 kinetin and 200 mg L−1 KH2 PO4 , pH 5.8) and subcultured weekly (16 mL suspension to 100 mL fresh medium).

Microscopy and image analysis A Nikon Eclipse E600 microscope equipped with appropriate filter sets and Nomarski DIC optics was used. DIC images were captured with a digital camera (DVC 1310C, USA). Lucia image analysis software (Laboratory Imaging, Prague, Czech Republic) was used for the measurement of cell length and diameter (n = 700 and 500 for LE and BY-2, respectively). From these values, the cell surface was calculated using an approximation of the cell shape as a cylinder and using the dimensions of the average cell (LE: 2782.29 ␮m2 ; BY-2: 6297.56 ␮m2 ). The cells were counted in a Fuchs-Rosenthal haemocytometer and cell density was expressed as the number of cells per milliliter of cell suspension. For the results presented in Fig. 1C, cells were counted in 10 aliquots for each suspension culture. The dilution of the suspension for counting was used appropriately so that the final number of counted cells was between 500 to 4000 cells for LE; and 300 to 900 cells for BY-2 in each aliquot during the entire 7-day growth cycle. For accumulation assays, cells were counted in at least 8 aliquots (typically, cell suspensions were diluted: LE 3-times, BY-2.5-times).

Auxin accumulation assays Accumulation assays were performed as described in Petráˇsek et al. (2003, 2006). Briefly, the final density of the cell suspension was adjusted to about 1.5 × 106 cells mL−1 for LE and 6 × 105 cells mL−1 for BY-2. The cultivation medium was removed using filtration through nylon cloth (20 ␮m mesh), and cells were re-suspended in the uptake buffer (20 mM MES, 10 mM sucrose, 0.5 mM CaSO4 , pH adjusted to 5.7 with KOH) and equilibrated for 45 min on the orbital shaker (LE, 24 ◦ C; BY-2, 27 ◦ C). Then, cells were collected by filtration, re-suspended in the fresh uptake buffer, incubated for 1.5 h under the same conditions and cell density was counted (see above). For all experiments, the final concentration of radiolabeled auxin was 2 nM. Radiolabeled auxins were added directly into the cell suspension in time-course experiments. In short-term experiments (modified from Delbarre et al., 1996), radiolabeled auxins were mixed with non-labeled auxins in uptake buffer prior to the experiment, and the equilibrated cells were added at the beginning of the experiment. After a timed uptake period (depending on experiment), 0.5 mL aliquots of suspension were withdrawn and accumulation of label in the cells was terminated by rapid filtration under reduced pressure on 22-mm-diameter cellulose filters. The cell cakes and filters were transferred to scintillation vials, extracted in ethanol for 30 min, and radioactivity was determined by liquid scintillation counting (Packard Tri-Carb 2900TR scintillation counter, Packard Instrument Co., Meriden, CT, USA). Counts were corrected for surface radioactivity by subtracting counts obtained for aliquots of cells collected immediately after the addition of radiolabeled auxins in course experiments. Counts in short-term auxin competition experiments (30 s or 2 min, modified from Delbarre et al., 1996, see below) were not corrected for surface radioactivity. The counting efficiency

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was determined by automatic external standardization, and counts were corrected for quenching automatically. NPA, 2-NOA and non-labeled auxins were added from ethanolic stock solutions to yield the desired final concentration. The accumulation values for various auxins were expressed with SEs (n = 4; 2 for short time experiments), and treatments with inhibitors or competition assays were expressed as the proportion to control variant considered as 100%. Treatments with inhibitors or non-labeled auxins were performed either immediately after addition of radiolabeled auxins, in-flight, or as pretreatment in time-points specified below for a particular experiment. Short-term competition experiments and their evaluation Short-term competition experiments were performed according to Delbarre et al. (1996) and Imhoff et al. (2000). Data were obtained from at least two independent experiments, each in duplicate. [3 H]NAA or [3 H]2,4-D (2 nM) were displaced by increasing the concentration of non-labeled auxins, and the value IC50 (the concentration of competitor needed to reduce the tracer uptake by 50%) was determined using non-linear square analysis according to the Michaelis–Menten model: y=

Vm + nsr IC50 + x

y represents accumulation of radiolabeled auxin retained in the cells after incubation with competitor, Vm represents the maximal transport capacity of the carrier, x is competitor concentration and nsr represents non-saturable component. To confirm that the observed difference between IC50 means was not caused by accidental bias or measurement imprecision, the following procedure was performed. In a computer simulation, every measured value was perturbed randomly by as much as 5%, and the Michaelis–Menten model was fitted again. 103 simulated data sets were generated for a given curve, and the IC50 values of the perturbed data were compared. If at least 95% of comparisons of simulated data sets corresponded to results for measured data, it was concluded that the difference between IC50 values was significant.

Results Growth characteristics and phenotype of Arabidopsis suspension cultures Preconditions for the usage of cell suspensions for auxin transport assays are their good friability and sufficient growth rate, and a stable phenotype (Petráˇsek and Zaˇzímalová, 2006). Therefore, LE cell culture that is typically cultured in medium supplemented with both auxin (NAA, 2.7 ␮M) and cytokinin (kinetin, 0.232 ␮M) (May and Leaver, 1993; Fuerst et al., 1996; Riou-Khamlichi et al., 2000) was cultured in the same medium and continuous darkness as tobacco BY-2 cells (see section “Materials and methods”), i.e. using 2,4-D (0.9 ␮M) as auxin supply and without addition of cytokinin. Under these conditions, 2-day-old LE cell culture formed only small clusters of 15–20 spherical cells (Fig. 1A) and multiplied 23 times during a 7-day subculture period (which is similar to the multiplication rate of tobacco BY-2 cells, Fig. 1C). The spherical character of LE cells was further documented by measurement of cell lengths and diameters in a representative sample of 700 cells (Fig. 1D). For comparison, cells of the well-established BY-2 cell line are, on average, ca. 2.3-times bigger and more elongated (Fig. 1B and D). Altogether, under optimized cultivation conditions, the LE cell line satisfied the basic preconditions for the auxin transport assays, including friability, sufficient growth rate as well as phenotype stability. Kinetics of auxin accumulation in A. thaliana suspension-cultured cells To characterize the mode of transport of the native auxin IAA and two synthetic auxins NAA and 2,4-D across the PM, the accumulation of radiolabeled auxins was studied in LE cells and compared with BY-2 cells in the same experimental setup. In both LE and BY-2 cells, the accumulation kinetics for [3 H]2,4-D showed a steep initial increase followed by the saturation steady state, while the kinetics

Auxin metabolic profiling 48 h after inoculation, cells were adjusted to the same densities as for auxin accumulation assays, incubated with 15 nM [3 H]NAA or [3 H]IAA or [3 H]2,4-D under standard cultivation conditions for 1, 2 and 20 min, collected and frozen in liquid nitrogen (200 mg of fresh weight per sample). Extraction and purification of auxin metabolites was performed as described in Dobrev and Kamínek (2002). The radioactive metabolites of [3 H]NAA or [3 H]IAA or [3 H]2,4D were separated on HPLC using column Luna C18(2), 150 × 4.6, 3 ␮m column (Phenomenex, Torrance, CA, USA), mobile phase A: 40 mM CH3 COONH4 , pH 4.0, and mobile phase B: CH3 CN/CH3 OH, 1/1, v/v. The flow rate was 0.6 mL/min−1 with a linear gradient of 30–50% B for 10 min, 50–100% B for 1 min, 100% B for 2 min, 100–30% B for 1 min. The column eluate was monitored by a Ramona 2000 flow-through radioactivity detector (Raytest GmbH, Straubenhardt, Germany) after online mixing with three volumes (1.8 mL min−1 ) of liquid scintillation cocktail (Flo-Scint III, Perkin Elmer Life and Analytical Sciences, Shelton, CT, USA). Integrated area of chromatogram peaks was normalized based on the equalization of total accumulated radiolabel. Metabolic profiles have been recalculated to the total sum of radiolabel, to express the relative contributions of labeled auxins and aggregation of their metabolites at 2 and 20 min of accumulation. The identity of NAA, IAA and 2,4-D peaks was verified by comparison with standard.

Fig. 2. Representative auxin accumulation curves in LE (A) and BY-2 (B) cell cultures. Naphthalene-1-acetic acid (NAA, open symbols), indole-3-acetic acid (IAA, gray symbols), 2,4-dichlorophenoxyacetic acid (2,4-D, black symbols). Concentration of labeled auxins 2 nM. Error bars = SEs (n = 4).

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Fig. 3. Auxin influx (A and B) and efflux (C and D) characteristics in LE (A and C) and BY-2 (B and D) cell cultures, respectively. (A and B) Left and middle panels, competition assays showing the effect of cold 2,4-D and NAA (both 2 nM, 1 ␮M, 2 ␮M), respectively, on the accumulation of [3 H]2,4-D at time-point 10 min. Non-labeled auxins were added immediately after addition of [3 H]2,4-D (2 nM). Right panels, the effect of 2-NOA (10 ␮M) on the accumulation of [3 H]2,4-D at time-point 10 min. 2-NOA was added immediately after the addition of [3 H]2,4-D. (C and D) The accumulation of [3 H]NAA, [3 H]IAA and [3 H]2,4-D is shown at the time-point 20 min. Cells were treated with the auxin efflux inhibitor 1-naphthylphthalamic acid (NPA, 10 ␮M), added in-flight at the time-point 14 min. The values of accumulation of particular labeled auxin in the non-treated (control) cells at the time-point 20 min represent 100%. Error bars = SEs (n = 4).

of [3 H]IAA had a much slower initial increase and did not reach a fully saturated steady state (Fig. 2A and B). In contrast, accumulation of [3 H]NAA in LE cells reached a steady state quickly (Fig. 2A), while in BY-2 cells (Fig. 2B), the steady state was not reached within 20 min and these cells can accumulate much higher amounts of the radiolabel. This kinetics pattern indirectly suggests that [3 H]NAA is not metabolized in LE cells within the given time frame, and the uptake and efflux of this synthetic auxin are balanced quickly there. To exclude the possibility that slightly higher cultivation temperature used for BY-2 cells influences the kinetics of measured auxins, the same time course experiments were done in LE cells also cultured at higher temperature, optimal for BY-2 cells (27 ◦ C). No difference in the accumulation kinetics of the three tested auxins was observed (Figure S1). The kinetics pattern of the three auxins suggests that LE suspension-cultured cells can be a good model for auxin transport studies. The carrier-mediated auxin influx The specificity of auxin uptake carriers was tested by auxin competition assays using radiolabeled 2,4-D as it is a well-established substrate for the auxin influx carriers (Delbarre et al., 1996). Nonlabeled auxins 2,4-D and NAA were added immediately after the addition of [3 H]2,4-D, and the data from 10 min after the onset of the accumulation assay (i.e. after the addition of radiolabeled auxin) are shown in Fig. 3A. The application of cold (non-labeled) 2,4-D induced a dose-dependent decrease in the accumulation of [3 H]2,4-D (Fig. 3A, left panel), and the same behavior was observed when cold 2,4-D was added in flight (Figure S2A). NAA reduced the accumulation with lower efficiency (Fig. 3A, middle panel). For comparison, in BY-2 cells, the competition with non-labeled 2,4-D

and NAA did not have an effect on [3 H]2,4-D accumulation (Fig. 3B, left and middle panel, and Figure S2B). To test the sensitivity of LE cells to the established specific ˇ inhibitor of the active auxin influx, 2-NOA (Lanková et al., 2010 and references therein) was applied at the beginning of the accumulation assay. Under such conditions, [3 H]2,4-D accumulation was dramatically reduced, and at 10 min after the onset of the accumulation assay, it reached only about 13% (Fig. 3A, right panel) of the control. These results show a high level of the active uptake of 2,4-D in LE cells, with high affinity of the auxin uptake carriers towards 2,4-D. The carrier-mediated auxin efflux Direct measurement of auxin efflux at the cell level is unambiguous because of the interference with necessary previous ‘loading’ of the cells with the experimental compound and its possible metabolism. Therefore, activity of auxin efflux carriers was tested using the widely used auxin efflux inhibitor NPA, even if it is not clear how specific NPA is towards various types of auxin efflux carriers. This approach has been used previously for tobacco cells (Delbarre et al., 1996; Petráˇsek et al., 2006), where NPA was reported to block the saturable efflux of NAA efficiently. At 20 min (i.e. 6 min after in-flight addition of NPA), the accumulation of all three of the tested auxins increased (Fig. 3C and D; Figure S3A–C). In LE cells, the most noticeable increase was observed for [3 H]NAA (more than 4.5-times). For [3 H]2,4-D and [3 H]IAA, the increase was not more than 2-times (Fig. 3C). Interestingly, in BY-2 cells, the increase of accumulation after NPA treatment was less than 2-times for all of the auxins tested (Fig. 3D). Moreover, the relationship between [3 H]NAA accumulation and NPA concentration suggested that LE cells tolerate concentrations of NPA that are already toxic

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Table 1 Short-time experiments on auxin influx and efflux. A: Influx–incubation time: 30 s

LE BY-2

IC50 values (␮M) [3 H]2,4-D + 2,4-D

IC50 values (␮M) [3 H]2,4-D + NAA

Division quotient

1 ± 0.01 1.18 ± 0.01

8.5 ± 0.01 7.03 ± 0.01

8.5 5.96

B: Efflux–incubation time: 2 min, pretreatment with 2-NOA (5 min)

LE BY-2

IC50 values (␮M) [3 H]NAA + NAA

IC50 values (␮M) [3 H]NAA + 2,4-D

Division quotient

6.82 ± 0.01 1.36 ± 0.01

719 ± 1 60 ± 0.5

105.42 44.12

Data show IC50 values derived from ‘displacement’ curves for auxin influx (A) and auxin efflux (B) for both LE and BY-2 cells. Statistical evaluation confirmed that differences between all IC50 values shown are significant. Division quotient represents the logarithmic difference between the two presented values in each row. Its value reflects the difference between affinities towards NAA and 2,4-D of each type of carriers in each plant material.

for BY-2 cells (Figure S4, cf. Petráˇsek et al., 2003). The competition assay between [3 H]IAA, i.e. the radiolabeled form of native auxin and thus the natural substrate for efflux carriers, and non-labeled NAA showed that in both LE and BY-2 cells, cold NAA affected accumulation of [3 H]IAA with the same efficiency (Figure S5). Altogether, these experiments indicated that in LE cells, auxins (namely NAA) are transported out from cells preferentially by a set of efflux carriers which have high sensitivity towards NPA, and that LE cells are more resistant to high concentrations of this inhibitor. Short-term auxin competition assays The accumulation and/or competition assays performed for a longer time period reflect the complex behavior of cells and they involve various auxin-related processes. Thus, more precise information about the relative affinity of auxin influx and efflux processes towards various auxins can be obtained in short-term experiments, as this experimental setup minimizes the impact of processes other than auxin transport occurring in cells (in particular degradation and/or metabolic changes of auxins). To evaluate the affinity parameters of auxin influx carriers, first the net accumulation of radiolabeled 2,4-D (as a good ‘substrate’ for auxin uptake carriers; Delbarre et al., 1996) was measured 30 s after the addition of cells into the uptake buffer containing both 2 nM [3 H]2,4-D and non-labeled 2,4-D in concentrations 0; 0.1; 1; 5; 10; 50; 100 and 500 ␮M (Figure S6A). The IC50 value (inflection point at the logarithmic ‘displacement’ curve, showing the dependence of accumulated [3 H]2,4-D on the concentration of non-labeled 2,4D) was determined and its significance was evaluated (see section “Materials and methods”). IC50 values for 2,4-D were 1.0 ± 0.01 ␮M and 1.18 ± 0.01 ␮M for LE and BY-2 cells, respectively (Table 1A). Although the difference between these two values was significant and the affinity towards 2,4-D was slightly higher in LE cells (i.e. lower concentration of 2,4-D is needed to displace the same proportion of labeled 2,4-D in LE cells), auxin uptake carriers showed high affinity towards 2,4-D in both of the cell lines tested. To gain more information about auxin specificity of influx carriers in the two cell lines, the competition of [3 H]2,4-D with nonlabeled NAA was investigated (Figure S6B and F). The IC50 values for competition between [3 H]2,4-D and cold NAA were 8.5 ± 0.01 ␮M and 7.03 ± 0.01 ␮M (Table 1A) in LE and BY-2 cells, respectively, confirming much lesser affinity of auxin uptake carriers towards NAA compared to 2,4-D (as ca. 7–8.5 higher concentration of NAA compared to cold 2,4-D was necessary to displace the same proportion of labeled 2,4-D in BY-2 and LE cells, respectively), but also supporting the notion that NAA can also be taken up actively in both

experimental systems (as NAA is capable of competing with 2,4-D for saturable carriers). The division quotient values (i.e. the ‘distance’ between inflection points on displacement curves, Table 1A) also point to lower relative affinity of auxin uptake carriers towards NAA in LE cells compared to BY-2 cells. Similarly, the relative affinity of efflux carriers was investigated, and in this case the net accumulation of radiolabeled NAA (as a good ‘substrate’ for auxin efflux carriers; Delbarre et al., 1996) was used as a basis for measurements. To allow auxins to penetrate into the cells and to reduce active transport by means of uptake carriers, the cells were pre-treated for 5 min with the inhibitor of auxin influx ˇ – 2-NOA (Imhoff et al., 2000; Lanková et al., 2010) and the loading with [3 H]NAA (2 nM) was prolonged for 2 min. Non-labeled NAA was used in concentrations 0; 0.1; 1; 5; 50; 100 ␮M (Figure S6C and G). Under these conditions, IC50 values were 6.82 ± 0.01 ␮M and 1.36 ± 0.01 ␮M for LE and BY-2 cells, respectively (Table 1B), suggesting that auxin efflux carriers show ca. 5-times lesser affinity towards NAA in LE cells compared to BY-2 cells (as ca. 5-times higher concentration of NAA was needed in LE cells to displace the same proportion of labeled NAA as in BY-2 cells). The competition of [3 H]NAA with cold 2,4-D for efflux carriers was also investigated. 2,4-D was used in concentrations 0; 0.1; 5; 50; 100; 500; 1000 ␮M (Figure S6D and H). In this case, pretreatment with 2-NOA largely affected the predominantly active uptake of 2,4-D, and so higher apparent concentrations of 2,4-D seem to be needed for IC50 related to auxin efflux carriers. Even though IC50 values for uptake of 2,4-D by auxin uptake carriers were very similar in both LE and BY-2 cells (see above and Table 1A), there was a substantial difference between IC50 for 2,4-D and auxin efflux carriers between both types of cells (719 ± 1 ␮M and 60 ± 0.5 ␮M for LE and BY-2, respectively; Table 1B). This suggests that the relative affinity of auxin efflux carriers towards 2,4-D is much higher in BY-2 cells compared to LE cells (as ca. an order of magnitude lower concentration of 2,4-D is necessary to displace 50% of [3 H]NAA in BY-2 compared to LE cells). This is consistent with the finding that a recognizable amount of 2,4-D can be transported from BY-2 cells actively (Hoˇsek et al., 2012). Altogether, short-term measurements showed similar affinity of the auxin influx (uptake) carriers to 2,4-D in both LE and BY-2 cells. However, the affinity of the auxin efflux carriers towards a well-established ‘substrate’ for them in tobacco cells – i.e. NAA – was ca. 5-times lower in LE cells in comparison to BY-2 cells. BY-2 cells were also able to export distinct amounts of 2,4-D via saturable efflux carriers, and in higher quantity than LE cells. Metabolism of NAA, IAA and 2,4-D As radioactivity is the value measured in accumulation assays, the apparent kinetics of auxin accumulation for all tested auxins (Fig. 2) can be influenced by their metabolic conversions within cells. Therefore, HPLC-based profiling of [3 H]NAA, [3 H]IAA and [3 H]2,4-D metabolism was performed both in LE and BY-2 cells. First, the [3 H]NAA metabolic profile was studied, as there are major differences between LE and BY-2 cells in the time-course and shape of the curves reflecting the amount of radiolabel in cells during the accumulation of this compound. NAA metabolic profiles in LE cells and BY-2 cells (Figure S7A–C) differed in both quantity and identity of particular metabolites. Already within 2 min, 41.3% of [3 H]NAA was converted into its metabolites in BY-2 cells, while in LE cells this proportion was only 28.5% (Fig. 4A). After 20 min, this difference was even more obvious, with 80.9% of the original [3 H]NAA present in the form of metabolites in BY-2 cells and only ca. one-half (47.5%) in LE cells (Fig. 4A). This indicates that the metabolic conversion of NAA is much slower in LE cells in comparison with BY-2 cells. In contrast to synthetic auxin NAA, conversion of [3 H]IAA into metabolites was faster in LE cells, as already after 1 min 41.2% and

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Fig. 4. Metabolic changes of auxins in 2-day-old LE (left) and BY-2 (right) cells. Remaining radiolabeled auxins (black columns) and their metabolites (gray columns) are presented for [3 H]NAA (A), [3 H]IAA (B) and [3 H] 2,4-D (C). The amount of metabolites was examined at the time-points 1, 2 and 20 min after addition of particular radiolabeled auxin. Note the distinct amounts of non-metabolized NAA (A) and IAA (B) at time 20 min in LE and BY-2 cells.

after 20 min the majority (91.8%) of original radiolabeled IAA was in the form of its metabolites (Fig. 4B, Figure S7D–F). Much slower conversion of [3 H]IAA occurred in BY-2 cells, where the metabolites represent 48.3% of original [3 H]IAA after 20 min (Fig. 4B). Interestingly, the spectra of both [3 H]NAA and [3 H]IAA metabolites differed in LE and BY-2 cells (Figure S7A–F). There was almost no metabolic conversion when synthetic auxin [3 H]2,4-D was applied to both LE and BY-2 cells (Fig. 4C and Figure S7G–I). These results show that both IAA and NAA are largely metabolized in cells and that the way they are metabolized is species-specific. In contrast, 2,4-D is metabolically very stable in both LE and BY-2 cells. Discussion The use of simplified cell culture models for measurements of the cell-to-cell auxin transport in A. thaliana, the major experimental plant model that is easily ‘genetically accessible,’ has been limited by the fact that it has been difficult to derive stable and sufficiently friable cell suspension lines having standard growth parameters from this species. However, the cell suspension derived

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from stem explants of A. thaliana ecotype Landsberg erecta (May and Leaver, 1993) has been used for a one-shot comparative IAA transport study (Geisler et al., 2005). As shown in this paper, after optimization of the cultivation protocol, this cell line can serve as valuable tool for tracking auxin influx and efflux activities at the cellular level. If LE cells are repeatedly cultured in the same medium as the well-established tobacco BY-2 cells, containing 2,4D as the only phytohormone, they grow with a stable phenotype and with a multiplication rate comparable to that of BY-2 cells. Under these conditions, the LE cell culture shows also sufficient cell friability to allow accurate microscopic determination of cell population density and cell dimensions. Therefore, the amount of radioactively labeled auxin (or another compound) accumulated inside cells can be readily calculated in relation to parameters such as cell surface, cell volume, cell number etc., so that it provides an idea of e.g. how many auxin molecules are present inside a cell at a particular time point and/or physiological situation. Nevertheless, in comparison with tobacco cell lines BY-2 (Petráˇsek et al., 2003; Dhonukshe et al., 2005; Petráˇsek and Zaˇzímalová, 2006) and VBI-0 (Campanoni et al., 2003; Petráˇsek et al., 2002), LE cell suspension does not form cell filaments (Menges and Murray, 2002) with clear axiality that would allow analysis of morphoregulatory aspects of auxin flow (Campanoni et al., 2003) in parallel to auxin accumulation measurements. Instead, LE cells grow radially, from one center equally in all directions. In any case, LE cells can be proposed as an alternative experimental material for auxin transport assays at the cellular level because the protocols for their synchronization (Menges and Murray, 2002), transformation and cryopreservation (Menges and Murray, 2006) are well established, and also the information on transcriptome of auxin response is available (Paponov et al., 2008). Also, transport of other plant hormones – cytokinins – has been described in the LE suspension culture (Cedzich et al., 2008). Nevertheless, to make use of this cell suspension, it is necessary to keep in mind that it was derived from X-ray mutagenized Landsberg plants (Redei, 1962), so minor differences in auxin transport compared to the predominantly used A. thaliana lines cannot be excluded (Jander et al., 2002; Ziolkowski et al., 2009). Although LE cells have already been used to show the effect of inhibitors on the IAA loading and efflux (Geisler et al., 2005), the more detailed auxin transport characteristics for IAA and both synthetic auxins NAA and 2,4-D as well as their comparison with the established models of tobacco cells (Delbarre et al., 1996; Petráˇsek and Zaˇzímalová, 2006) have not yet been provided. As shown here, the kinetics of IAA and 2,4-D accumulation in cells and the absolute values expressed per the PM area are comparable for both tobacco BY-2 cells and LE cells. However, it seems that for tracking the active auxin uptake into LE cells, the synthetic auxin analog 2,4-D is far better than native IAA, as IAA is metabolized quite quickly here. The specific inhibitor of auxin influx 2-NOA (Imhoff et al., 2000; Swarup et al., 2008; Yang et al., 2006; ˇ Lanková et al., 2010) blocked the influx of synthetic auxin 2,4-D more efficiently in LE cells than in BY-2 cells, suggesting a higher proportion of its active, 2-NOA-sensitive influx here. In agreement with this, the affinity of auxin influx carriers towards 2,4-D was slightly higher in LE cells. On the other hand, competition experiments performed in BY-2 cells showed that the accumulation of [3 H]2,4-D was not influenced by the addition of cold 2,4-D in the concentration range tested (2 nM–2 ␮M). However, a higher concentration of cold 2,4-D (10 ␮M) decreased the accumulation of [3 H]2,4-D in BY-2 cells as well (Simon et al., 2013). This, together with the observation of the rapid increase of the 2,4-D accumulation curve, could be explained by the higher capacity of net auxin uptake and by uptake carriers with lower affinity to 2,4-D in BY-2 cells. It could be speculated that BY-2 cells are using preferentially MDR/PGP/ABCB-based carriers, perhaps thanks to their long-lasting sub-culturing into the media supplemented with 2,4-D

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as the only auxin. This explanation is also in concert with the proposal by Yang and Murphy (2009) and Kubeˇs et al. (2012) on the possible role of ABCB4 as a dual auxin influx and efflux transporter. In LE cells, on the other hand, the kinetics of [3 H]2,4-D accumulation with a gradual increase together with the ability of cold 2,4-D and NAA to compete with the radiolabeled 2,4-D, could reflect the activity of various types of auxin uptake carriers with various affinity towards 2,4-D. This may correspond to the cultivation conditions, as the LE suspension culture used here was maintained in medium supplemented with 2,4-D instead of NAA only for a short period of time. With respect to auxin export from cells, in LE cells the activity of the NPA-sensitive efflux carriers transporting NAA was even higher than in tobacco cells (i.e. the relative increase of NAA accumulation after NPA application was higher in LE cells). This could be due to the higher capacity of relevant carriers and/or higher efficiency of the NPA-based carriers’ regulation, etc. Based on the NPA treatments, synthetic auxin 2,4-D seemed to also be a good substrate for the auxin efflux carriers in LE cells. However, as shown in the short-term experiments, 2,4-D was not able to compete with NAA for the active efflux in LE cells. Therefore, it might be speculated that at least two different sets of efflux carriers with different specificity are present in LE suspension cells. As originally noted by Delbarre et al. (1996) for Xanthi tobacco cells, some degree of active 2,4-D efflux was also reported later for VBI-0 tobacco cells (Paciorek et al., 2005) and BY-2 cells overproducing the auxin efflux carrier AtPIN7 (Petráˇsek et al., 2006). Recently, careful testing in BY-2 cells of the initial phases of the 2,4-D accumulation supported by mathematical modeling provided additional evidence for carrier-driven efflux of 2,4-D (Hoˇsek et al., 2012). Interestingly, NPA concentration dependence assays showed that LE cells are relatively more tolerant to the higher concentrations of NPA in comparison with BY-2 cells. This could partly justify the quite high concentrations of this inhibitor used for some studies in planta (Geldner et al., 2001). Radiolabeled NAA has been considered the major tool for studying the active auxin efflux in tobacco cell lines (Xanthi XHFD8, Delbarre et al., 1996; VBI-0, Campanoni et al., 2003; Paciorek et al., 2005; Petráˇsek et al., 2002; and BY-2, Cho et al., 2007; Lee and Cho, 2006; Petráˇsek et al., 2003, 2006). However, in tobacco cell s, metabolic changes of NAA are relatively quick and massive (Delbarre et al., 1994; Hoˇsek et al., 2012) and as shown for BY2 cells, NAA metabolites are not transported out of cells (Hoˇsek et al., 2012), thus complicating the interpretation of transport measurements using labeled NAA. One of the important findings here was a much slower rate of NAA metabolic conversion in LE compared to BY-2 cells. Therefore, the observed differences in the shape of accumulation curves between LE and BY-2 cells can be attributed to the pattern of metabolism of radiolabeled NAA. In contrast to NAA, IAA is massively metabolized in LE suspension cells. A similar rate of IAA metabolism (over 80% after 15 min) was observed previously in soybean root suspension cells (Loper and Spanswick, 1991). In addition to differences in metabolism of major auxins between tobacco and Arabidopsis, another advantage of making use of Arabidopsis cells for this type of studies is possible preparation of cell suspensions from mutant, transformed and crossed plants. Altogether, based on: (1) the general ‘genetic accessibility’ of Arabidopsis, including possible use of mutants and transformants, (2) the improved protocol for cultivation, (3) a lower rate of NAA metabolism, and (4) possible use of 2,4-D for tracking both auxin influx and efflux, this study introduces LE cells as a useful, alternative tool to study auxin transport parameters on a single cell level that is complementary to well-established tobacco cell lines.

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