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Journal of Plant Physiology 171 (2014) 429–437

Contents lists available at ScienceDirect

Journal of Plant Physiology

journa l h om epage: www.elsev ier .com/ locate / jp lph

hysiology

haracterization of transmembrane auxin transport in Arabidopsisuspension-cultured cells

aniela Seifertováa, Petr Skupaa, Jan Rychtárb, Martina Lankováa, Markéta Parezováa,etre I. Dobreva, Klára Hoyerováa, Jan Petráseka, Eva Zazímalováa,∗

Institute of Experimental Botany ASCR, Rozvojová 263, 165 02 Prague 6, Czech RepublicDepartment of Mathematics and Statistics, The University of North Carolina at Greensboro, 130 Petty Building, NC 27403, USA

r t i c l e i n f o

rticle history:eceived 1 June 2013eceived in revised form4 September 2013ccepted 28 September 2013

eywords:uxin influxuxin effluxuxin metabolic profilingrabidopsis thaliana cell suspension (LE)

s u m m a r y

Polar auxin transport is a crucial process for control and coordination of plant development. Studies ofauxin transport through plant tissues and organs showed that auxin is transported by a combination ofphloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissuesare 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 havebeen established for auxin transport assays. However, there are very few data available on the specificityand kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-culturedcells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the nativeauxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyaceticacids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells

ell culture phenotype (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly totobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies ofauxin 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 experimentalsystem for measurements of kinetics of auxin carriers on the cellular level that is complementary totobacco cells.

© 2013 Elsevier GmbH. All rights reserved.

ntroduction

The plant hormone auxin is one of the most important regulatorsf plant growth and development. In addition to local biosynthesisnd metabolic changes, its directional transport generates auxin

oncentration gradients needed for the transduction of develop-ental cues during both embryogenesis and postembryonic devel-

pment of plants, including reactions to external environmental

Abbreviations: BY-2, Nicotiana tabacum L., cv. Bright Yellow 2 cell line;HPAA, 3-chloro-4-hydroxyphenylacetic acid; 2,4-D, 2,4-dichlorophenoxyaceticcid; IAA, indole-3-acetic acid; LE, Arabidopsis thaliana, ecotype Landsberg erectaell line; NAA, naphthalene-1-acetic acid; 1-NOA, 1-naphthoxyacetic acid; 2-NOA, 2-aphthoxyacetic acid; NPA, 1-naphthylphthalamic acid; PBA, 2-(l-pyrenoyl)benzoiccid; PM, plasma membrane; TIBA, 2,3,5-triiodobenzoic acid; VBI-0, Nicotianaabacum 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.czP. Skupa), j rychta@uncg.edu (J. Rychtár), lankova@ueb.cas.cz (M. Lanková),arezova@ueb.cas.cz (M. Parezová), dobrev@ueb.cas.cz (P.I. Dobrev),oyerova@ueb.cas.cz (K. Hoyerová), petrasek@ueb.cas.cz (J. Petrásek),azimalova@ueb.cas.cz (E. Zazímalová).

176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.jplph.2013.09.026

stimuli. In general, auxin is transported to longer distances in thephloem, but it is also subject to cell-to-cell transport, where passivediffusion is combined with the activity of plasma membrane (PM)-localized carriers. The polarity of auxin transport across the PM hasbeen 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 ofauxin molecules. Undissociated auxin molecules in the more acidicextracellular environment enter cells by diffusion. In the more alka-line intracellular environment, dissociated auxin molecules havingvery low membrane permeability are trapped and are exported outof the cell almost entirely by active auxin efflux via auxin carriers.Generally, several groups of transporters are currently known toexhibit auxin influx or efflux activities (recent reviews by Peer et al.,2011; Petrásek 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; Petrásek 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 asto regulatory mechanisms involved in their action, auxin transport

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30 D. Seifertová et al. / Journal of P

as been studied in plant tissues/organs by the measurement ofovement of radioactively-labeled auxin (Lewis and Muday, 2009)

oth in roots (basipetal and acropetal) and shoots (basipetal) of Ara-idopsis plants (Garbers et al., 1996; Geisler et al., 2003; Murphyt al., 2000; Noh et al., 2001; Rashotte et al., 2003) or other plantystems (reviewed in Morris, 2000; Morris et al., 2004). However,hese approaches on the tissue/organ level cannot be used to deter-

ine kinetic parameters of auxin transport across membrane ando distinguish between cellular auxin influx and efflux. Therefore,implified models of yeasts and cell cultures derived from plants,nimals, and even humans are frequently utilized (Delbarre et al.,996; Geisler et al., 2005; Hrycyna et al., 1998; Luschnig et al., 1998;oh et al., 2001; Petrásek et al., 2006; Yang et al., 2006; Yang andurphy, 2009; and references therein).Cell lines represent the major experimental system that can be

sed for both qualitative and quantitative studies of various pro-eins’ activity at the cellular level in vivo. In fact, studies of theransport of radiolabeled indole-3-acetic acid (IAA) using crownall suspension culture of Parthenocissus tricuspidata resulted inhe chemiosmotic polar diffusion model of polar auxin transportRubery and Sheldrake, 1974). Suspension-cultured soybean rootells were used for intimate studies of IAA transport by Loper andpanswick (1991), and the authors described IAA uptake via pas-ive diffusion and saturable influx carrier and active efflux. Rapidetabolism of IAA molecules (about 80% after 15 min uptake) was

hown as well.Nowadays, the best characterized models are homogeneous,

ighly friable populations of tobacco suspension-cultured cells,here the active auxin influx and efflux parameters were deter-ined quantitatively for native auxin IAA and for its synthetic

nalogs (Delbarre et al., 1996; Petrásek et al., 2002, 2003; Petráseknd Zazímalová, 2006). The proportions of the active auxin influxnd efflux and diffusion rates for IAA, naphthalene-1-acetic acidnd 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively)ere determined in suspension-cultured cells of Nicotiana tabacum

. cv. Xanthi XHFD8 (Delbarre et al., 1996). Based on the deter-ination of accumulation kinetics, metabolic degradation and

ompetition assays, it was shown that the accumulation of IAAomprises passive diffusion and the activity of both auxin influxnd efflux carriers. In contrast, synthetic auxin NAA was trans-orted into cells preferentially by passive diffusion and out ofhe cell by active efflux, while 2,4-D accumulation inside cellsesulted primarily from the active auxin influx. Based on thesendings, Delbarre et al. (1996) suggested a simple methodol-gy for the measurements of active auxin influx and efflux bysing the accumulation assays of radioactively labeled 2,4-D andAA, respectively. Active transport of auxin across PM has beenharacterized further using inhibitors of auxin influx, such as 1-aphthoxyacetic acid (1-NOA), 2-naphthoxyacetic acid (2-NOA)nd 3-chloro-4-hydroxyphenylacetic acid (CHPAA) (Imhoff et al.,000; Parry et al., 2001) and auxin efflux, 1-naphthylphthalamiccid (NPA) and 2-(l-pyrenoyl)benzoic acid (PBA) (Keitt and Baker,966; Delbarre et al., 1996; Petrásek et al., 2003). The applica-ion of inhibitors of auxin influx in the heterologous system ofenopus laevis oocytes (Yang et al., 2006; Swarup et al., 2008)nd in tobacco BY-2 cells (Lanková et al., 2010) revealed thathe amount of auxin taken up actively into the cells by spe-ific influx carriers is significant. Basic characteristics of auxinfflux in other tobacco cell lines (Nicotiana tabacum L., cv.BI-0; Petrásek et al., 2002, and BY-2; Petrásek et al., 2003;anková et al., 2010) were similar to tobacco Xanthi XHFD8ells, although in VBI-0 cells there was higher proportion of the

ctive efflux of 2,4-D (Paciorek et al., 2005). This activity waslso enhanced for 2,4-D after the inducible overexpression of PIN-ype auxin efflux carriers (namely PIN7) in BY-2 cells (Petrásekt al., 2006), suggesting differential affinity and/or capacity of

hysiology 171 (2014) 429–437

auxin carriers to various auxins in various experimental mod-els.

Recently, due to its genetic ‘accessibility,’ Arabidopsis repre-sents the main model for studies of auxin action and its transportin planta. In spite of this, there is still a significant lack ofknowledge of detailed auxin transport characteristics at the level ofcultured Arabidopsis cells. Arabidopsis cell suspensions derived fromecotypes 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 ecotypeLandsberg erecta cell line has already been used for IAA transportassays (Geisler et al., 2005), more information on the specificityand dynamics of IAA, NAA and 2,4-D cellular transport is stillneeded.

This report provides basic kinetic and specificity parametersof carrier-mediated auxin uptake (influx) and efflux in Arabidop-sis ecotype Landsberg erecta suspension-cultured cells (LE line),together with data about metabolism of exogenously added aux-ins, and compares these characteristics with the already establishedmodel 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). NPAand 2-naphthoxyacetic acid (2-NOA) were dissolved in ethanol toyield stock solutions 10 mM. NPA for the results presented in Fig-ure S4 was prepared in 0.1 mM, 1 mM, 10 mM, 100 mM ethanolicstock solutions. Stock solutions of non-labeled auxins were pre-pared in concentration 5 �M, 1 mM, 10 mM, 30 mM and 300 mMdissolved in ethanol. HPLC-grade methanol and acetonitrile wereobtained from Merck KGaA (Darmstadt, Germany). Formic acid andammonium hydroxide (both of p.a. grade) were from Lachemaa.s. (Neratovice, Czech Republic). Oasis MCX columns (150 mg/6cc) were obtained from Waters (Milford, MA, USA). The follow-ing radiolabeled auxins were used for accumulation and metabolicassays: [3H]naphthalene-1-acetic acid (NAA), [3H]indole-3-aceticacid (IAA), [3H]2,4-dichlorophenoxyacetic acid (2,4-D) (specificradioactivity 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 Lands-berg erecta (May and Leaver, 1993) LE cells were cultured in thedarkness at 24 ◦C (LE) and 27 ◦C (BY-2) on the orbital incubator(Sanyo Gallenkamp PLC, IOI400.XX2.C; 130 rpm and 150 rpm, LEand 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 KH2PO4, pH 5.8) and subculturedweekly (1 mL suspension to 30 mL fresh media for both LE and BY-2). Stock calli were maintained on the same media solidified with0.6% (w/v) agar and subcultured monthly.

The cell suspension of A. thaliana missense mutant aux1-7 (NASCN3074; Pickett et al., 1990) was established from a mixture ofcotyledons, hypocotyls and leaves of 1-week-old seedling plants

(based on the protocol by Blackhall, 1993). These were cut andplaced on callus induction medium (3.2 g L−1 Gamborg’s B5 Basalmedium, 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

D. Seifertová et al. / Journal of Plant Physiology 171 (2014) 429–437 431

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 cellsgrow 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. LEand 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).L ars =

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E cells are more spherical while BY-2 cells are more elongated (cylindrical). Scale b

ere transferred onto solid MS medium (3% sucrose, 4.3 g L−1

urashige and Skoog salts, 100 mg L−1 inositol, 1 mg L−1 thiamin,.2 mg L−1 2,4-D, and 200 mg L−1 KH2PO4, pH 5.8, 0.6%, w/v agar)nd subcultured monthly. The cell suspension was derived fromalli and cultured in the darkness at 24 ◦C on an orbital incubatorSanyo Gallenkamp PLC, IOI400.XX2.C; 130 rpm) in liquid medium3% sucrose, 4.3 g L−1 Murashige and Skoog salts, 100 mg L−1 ino-itol, 1 mg L−1 thiamin, 0.5 mg L−1 2,4-D, 0.2 mg L−1 kinetin and00 mg L−1 KH2PO4, pH 5.8) and subcultured weekly (16 mL sus-ension to 100 mL fresh medium).

icroscopy and image analysis

A Nikon Eclipse E600 microscope equipped with appropriatelter sets and Nomarski DIC optics was used. DIC images were cap-ured with a digital camera (DVC 1310C, USA). Lucia image analysisoftware (Laboratory Imaging, Prague, Czech Republic) was used forhe measurement of cell length and diameter (n = 700 and 500 for LEnd BY-2, respectively). From these values, the cell surface was cal-ulated using an approximation of the cell shape as a cylinder andsing the dimensions of the average cell (LE: 2782.29 �m2; BY-2:297.56 �m2). The cells were counted in a Fuchs-Rosenthal haemo-ytometer and cell density was expressed as the number of cells perilliliter of cell suspension. For the results presented in Fig. 1C, cellsere counted in 10 aliquots for each suspension culture. The dilu-

ion of the suspension for counting was used appropriately so thathe final number of counted cells was between 500 to 4000 cells for

E; and 300 to 900 cells for BY-2 in each aliquot during the entire-day growth cycle. For accumulation assays, cells were counted

n at least 8 aliquots (typically, cell suspensions were diluted: LE-times, BY-2.5-times).

100 �m.

Auxin accumulation assays

Accumulation assays were performed as described in Petráseket al. (2003, 2006). Briefly, the final density of the cell sus-pension was adjusted to about 1.5 × 106 cells mL−1 for LE and6 × 105 cells mL−1 for BY-2. The cultivation medium was removedusing filtration through nylon cloth (20 �m mesh), and cells werere-suspended in the uptake buffer (20 mM MES, 10 mM sucrose,0.5 mM CaSO4, pH adjusted to 5.7 with KOH) and equilibratedfor 45 min on the orbital shaker (LE, 24 ◦C; BY-2, 27 ◦C). Then,cells were collected by filtration, re-suspended in the fresh uptakebuffer, incubated for 1.5 h under the same conditions and celldensity was counted (see above). For all experiments, the finalconcentration of radiolabeled auxin was 2 nM. Radiolabeled aux-ins were added directly into the cell suspension in time-courseexperiments. In short-term experiments (modified from Delbarreet al., 1996), radiolabeled auxins were mixed with non-labeledauxins in uptake buffer prior to the experiment, and the equili-brated cells were added at the beginning of the experiment. Aftera timed uptake period (depending on experiment), 0.5 mL aliquotsof suspension were withdrawn and accumulation of label in thecells was terminated by rapid filtration under reduced pressure on22-mm-diameter cellulose filters. The cell cakes and filters weretransferred to scintillation vials, extracted in ethanol for 30 min,and radioactivity was determined by liquid scintillation counting(Packard Tri-Carb 2900TR scintillation counter, Packard InstrumentCo., Meriden, CT, USA). Counts were corrected for surface radioac-tivity by subtracting counts obtained for aliquots of cells collected

immediately after the addition of radiolabeled auxins in courseexperiments. 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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tion of radiolabeled auxins was studied in LE cells and comparedwith BY-2 cells in the same experimental setup. In both LE and BY-2cells, the accumulation kinetics for [3H]2,4-D showed a steep initialincrease followed by the saturation steady state, while the kinetics

32 D. Seifertová et al. / Journal of P

as determined by automatic external standardization, and countsere corrected for quenching automatically. NPA, 2-NOA andon-labeled auxins were added from ethanolic stock solutions toield the desired final concentration. The accumulation values forarious auxins were expressed with SEs (n = 4; 2 for short timexperiments), and treatments with inhibitors or competition assaysere expressed as the proportion to control variant considered as

00%. Treatments with inhibitors or non-labeled auxins were per-ormed either immediately after addition of radiolabeled auxins,n-flight, or as pretreatment in time-points specified below for aarticular experiment.

hort-term competition experiments and their evaluation

Short-term competition experiments were performed accord-ng to Delbarre et al. (1996) and Imhoff et al. (2000). Data werebtained from at least two independent experiments, each in dupli-ate. [3H]NAA or [3H]2,4-D (2 nM) were displaced by increasing theoncentration of non-labeled auxins, and the value IC50 (the con-entration of competitor needed to reduce the tracer uptake by0%) was determined using non-linear square analysis accordingo the Michaelis–Menten model:

= Vm

IC50 + x+ nsr

represents accumulation of radiolabeled auxin retained in theells after incubation with competitor, Vm represents the maximalransport capacity of the carrier, x is competitor concentration andsr represents non-saturable component.

To confirm that the observed difference between IC50 meansas not caused by accidental bias or measurement imprecision,

he following procedure was performed. In a computer simulation,very measured value was perturbed randomly by as much as 5%,nd the Michaelis–Menten model was fitted again. 103 simulatedata sets were generated for a given curve, and the IC50 valuesf the perturbed data were compared. If at least 95% of compar-sons of simulated data sets corresponded to results for measuredata, it was concluded that the difference between IC50 values wasignificant.

uxin metabolic profiling

48 h after inoculation, cells were adjusted to the same densitiess for auxin accumulation assays, incubated with 15 nM [3H]NAA or3H]IAA or [3H]2,4-D under standard cultivation conditions for 1, 2nd 20 min, collected and frozen in liquid nitrogen (200 mg of fresheight per sample). Extraction and purification of auxin metabo-

ites was performed as described in Dobrev and Kamínek (2002).he radioactive metabolites of [3H]NAA or [3H]IAA or [3H]2,4-

were separated on HPLC using column Luna C18(2), 150 × 4.6, �m column (Phenomenex, Torrance, CA, USA), mobile phase A:0 mM CH3COONH4, pH 4.0, and mobile phase B: CH3CN/CH3OH,/1, v/v. The flow rate was 0.6 mL/min−1 with a linear gradientf 30–50% B for 10 min, 50–100% B for 1 min, 100% B for 2 min,00–30% B for 1 min. The column eluate was monitored by aamona 2000 flow-through radioactivity detector (Raytest GmbH,traubenhardt, Germany) after online mixing with three volumes1.8 mL min−1) of liquid scintillation cocktail (Flo-Scint III, Perkinlmer Life and Analytical Sciences, Shelton, CT, USA). Integratedrea of chromatogram peaks was normalized based on the equaliza-ion of total accumulated radiolabel. Metabolic profiles have been

ecalculated to the total sum of radiolabel, to express the relativeontributions of labeled auxins and aggregation of their metabo-ites at 2 and 20 min of accumulation. The identity of NAA, IAA and,4-D peaks was verified by comparison with standard.

hysiology 171 (2014) 429–437

Results

Growth characteristics and phenotype of Arabidopsis suspensioncultures

Preconditions for the usage of cell suspensions for auxin trans-port assays are their good friability and sufficient growth rate, anda stable phenotype (Petrásek and Zazímalová, 2006). Therefore, LEcell culture that is typically cultured in medium supplemented withboth auxin (NAA, 2.7 �M) and cytokinin (kinetin, 0.232 �M) (Mayand Leaver, 1993; Fuerst et al., 1996; Riou-Khamlichi et al., 2000)was cultured in the same medium and continuous darkness astobacco BY-2 cells (see section “Materials and methods”), i.e. using2,4-D (0.9 �M) as auxin supply and without addition of cytokinin.Under these conditions, 2-day-old LE cell culture formed only smallclusters of 15–20 spherical cells (Fig. 1A) and multiplied 23 timesduring a 7-day subculture period (which is similar to the multiplica-tion rate of tobacco BY-2 cells, Fig. 1C). The spherical character of LEcells was further documented by measurement of cell lengths anddiameters in a representative sample of 700 cells (Fig. 1D). For com-parison, 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 cellline satisfied the basic preconditions for the auxin transport assays,including friability, sufficient growth rate as well as phenotype sta-bility.

Kinetics of auxin accumulation in A. thaliana suspension-culturedcells

To characterize the mode of transport of the native auxin IAA andtwo synthetic auxins NAA and 2,4-D across the PM, the accumula-

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

D. Seifertová et al. / Journal of Plant Physiology 171 (2014) 429–437 433

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, competitionassays showing the effect of cold 2,4-D and NAA (both 2 nM, 1 �M, 2 �M), respectively, on the accumulation of [3H]2,4-D at time-point 10 min. Non-labeled auxins wereadded immediately after addition of [3H]2,4-D (2 nM). Right panels, the effect of 2-NOA (10 �M) on the accumulation of [3H]2,4-D at time-point 10 min. 2-NOA was addedi AA, [3

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mmediately after the addition of [3H]2,4-D. (C and D) The accumulation of [3H]Nhe auxin efflux inhibitor 1-naphthylphthalamic acid (NPA, 10 �M), added in-flighon-treated (control) cells at the time-point 20 min represent 100%. Error bars = SE

f [3H]IAA had a much slower initial increase and did not reach aully saturated steady state (Fig. 2A and B). In contrast, accumula-ion of [3H]NAA in LE cells reached a steady state quickly (Fig. 2A),hile in BY-2 cells (Fig. 2B), the steady state was not reached within

0 min and these cells can accumulate much higher amounts of theadiolabel. This kinetics pattern indirectly suggests that [3H]NAAs not metabolized in LE cells within the given time frame, andhe uptake and efflux of this synthetic auxin are balanced quicklyhere. To exclude the possibility that slightly higher cultivationemperature used for BY-2 cells influences the kinetics of measureduxins, the same time course experiments were done in LE cells alsoultured at higher temperature, optimal for BY-2 cells (27 ◦C). Noifference in the accumulation kinetics of the three tested auxinsas observed (Figure S1).

The kinetics pattern of the three auxins suggests that LEuspension-cultured cells can be a good model for auxin transporttudies.

he carrier-mediated auxin influx

The specificity of auxin uptake carriers was tested by auxin com-etition assays using radiolabeled 2,4-D as it is a well-establishedubstrate for the auxin influx carriers (Delbarre et al., 1996). Non-abeled auxins 2,4-D and NAA were added immediately after theddition of [3H]2,4-D, and the data from 10 min after the onsetf the accumulation assay (i.e. after the addition of radiolabeleduxin) are shown in Fig. 3A. The application of cold (non-labeled),4-D induced a dose-dependent decrease in the accumulation of

3H]2,4-D (Fig. 3A, left panel), and the same behavior was observedhen cold 2,4-D was added in flight (Figure S2A). NAA reduced

he accumulation with lower efficiency (Fig. 3A, middle panel). Foromparison, in BY-2 cells, the competition with non-labeled 2,4-D

H]IAA and [3H]2,4-D is shown at the time-point 20 min. Cells were treated withe time-point 14 min. The values of accumulation of particular labeled auxin in the).

and NAA did not have an effect on [3H]2,4-D accumulation (Fig. 3B,left and middle panel, and Figure S2B).

To test the sensitivity of LE cells to the established specificinhibitor of the active auxin influx, 2-NOA (Lanková et al., 2010 andreferences therein) was applied at the beginning of the accumu-lation assay. Under such conditions, [3H]2,4-D accumulation wasdramatically reduced, and at 10 min after the onset of the accumu-lation assay, it reached only about 13% (Fig. 3A, right panel) of thecontrol.

These results show a high level of the active uptake of 2,4-D in LEcells, 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 unambigu-ous because of the interference with necessary previous ‘loading’of the cells with the experimental compound and its possiblemetabolism. Therefore, activity of auxin efflux carriers was testedusing the widely used auxin efflux inhibitor NPA, even if it isnot clear how specific NPA is towards various types of auxinefflux carriers. This approach has been used previously for tobaccocells (Delbarre et al., 1996; Petrásek et al., 2006), where NPA wasreported to block the saturable efflux of NAA efficiently. At 20 min(i.e. 6 min after in-flight addition of NPA), the accumulation of allthree of the tested auxins increased (Fig. 3C and D; Figure S3A–C).In LE cells, the most noticeable increase was observed for [3H]NAA(more than 4.5-times). For [3H]2,4-D and [3H]IAA, the increase wasnot more than 2-times (Fig. 3C). Interestingly, in BY-2 cells, the

increase of accumulation after NPA treatment was less than 2-timesfor all of the auxins tested (Fig. 3D). Moreover, the relationshipbetween [3H]NAA accumulation and NPA concentration suggestedthat LE cells tolerate concentrations of NPA that are already toxic

434 D. Seifertová et al. / Journal of Plant P

Table 1Short-time experiments on auxin influx and efflux.

A: Influx–incubation time: 30 s

IC50 values (�M) IC50 values (�M) Division quotient[3H]2,4-D + 2,4-D [3H]2,4-D + NAA

LE 1 ± 0.01 8.5 ± 0.01 8.5BY-2 1.18 ± 0.01 7.03 ± 0.01 5.96

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

IC50 values (�M) IC50 values (�M) Division quotient[3H]NAA + NAA [3H]NAA + 2,4-D

LE 6.82 ± 0.01 719 ± 1 105.42BY-2 1.36 ± 0.01 60 ± 0.5 44.12

Data show IC50 values derived from ‘displacement’ curves for auxin influx (A) andauxin efflux (B) for both LE and BY-2 cells. Statistical evaluation confirmed that dif-ferences between all IC50 values shown are significant. Division quotient representstrc

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he logarithmic difference between the two presented values in each row. Its valueeflects the difference between affinities towards NAA and 2,4-D of each type ofarriers in each plant material.

or BY-2 cells (Figure S4, cf. Petrásek et al., 2003). The competitionssay between [3H]IAA, i.e. the radiolabeled form of native auxinnd thus the natural substrate for efflux carriers, and non-labeledAA showed that in both LE and BY-2 cells, cold NAA affected accu-ulation of [3H]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 setf efflux carriers which have high sensitivity towards NPA, and thatE cells are more resistant to high concentrations of this inhibitor.

hort-term auxin competition assays

The accumulation and/or competition assays performed for longer time period reflect the complex behavior of cells andhey involve various auxin-related processes. Thus, more precisenformation about the relative affinity of auxin influx and effluxrocesses towards various auxins can be obtained in short-termxperiments, as this experimental setup minimizes the impact ofrocesses other than auxin transport occurring in cells (in particularegradation and/or metabolic changes of auxins).

To evaluate the affinity parameters of auxin influx carriers, firsthe net accumulation of radiolabeled 2,4-D (as a good ‘substrate’ foruxin uptake carriers; Delbarre et al., 1996) was measured 30 s afterhe addition of cells into the uptake buffer containing both 2 nM3H]2,4-D and non-labeled 2,4-D in concentrations 0; 0.1; 1; 5; 10;0; 100 and 500 �M (Figure S6A). The IC50 value (inflection pointt the logarithmic ‘displacement’ curve, showing the dependencef accumulated [3H]2,4-D on the concentration of non-labeled 2,4-) was determined and its significance was evaluated (see section

Materials and methods”). IC50 values for 2,4-D were 1.0 ± 0.01 �Mnd 1.18 ± 0.01 �M for LE and BY-2 cells, respectively (Table 1A).lthough the difference between these two values was significantnd the affinity towards 2,4-D was slightly higher in LE cells (i.e.ower concentration of 2,4-D is needed to displace the same pro-ortion of labeled 2,4-D in LE cells), auxin uptake carriers showedigh affinity towards 2,4-D in both of the cell lines tested.

To gain more information about auxin specificity of influxarriers in the two cell lines, the competition of [3H]2,4-D with non-abeled NAA was investigated (Figure S6B and F). The IC50 values forompetition between [3H]2,4-D and cold NAA were 8.5 ± 0.01 �Mnd 7.03 ± 0.01 �M (Table 1A) in LE and BY-2 cells, respectively,onfirming much lesser affinity of auxin uptake carriers towards

AA compared to 2,4-D (as ca. 7–8.5 higher concentration of NAAompared to cold 2,4-D was necessary to displace the same pro-ortion of labeled 2,4-D in BY-2 and LE cells, respectively), but alsoupporting the notion that NAA can also be taken up actively in both

hysiology 171 (2014) 429–437

experimental systems (as NAA is capable of competing with 2,4-Dfor saturable carriers). The division quotient values (i.e. the ‘dis-tance’ between inflection points on displacement curves, Table 1A)also point to lower relative affinity of auxin uptake carriers towardsNAA 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 usedas a basis for measurements. To allow auxins to penetrate into thecells and to reduce active transport by means of uptake carriers, thecells were pre-treated for 5 min with the inhibitor of auxin influx– 2-NOA (Imhoff et al., 2000; Lanková et al., 2010) and the loadingwith [3H]NAA (2 nM) was prolonged for 2 min. Non-labeled NAAwas used in concentrations 0; 0.1; 1; 5; 50; 100 �M (Figure S6Cand G). Under these conditions, IC50 values were 6.82 ± 0.01 �Mand 1.36 ± 0.01 �M for LE and BY-2 cells, respectively (Table 1B),suggesting that auxin efflux carriers show ca. 5-times lesser affin-ity towards NAA in LE cells compared to BY-2 cells (as ca. 5-timeshigher concentration of NAA was needed in LE cells to displace thesame proportion of labeled NAA as in BY-2 cells).

The competition of [3H]NAA with cold 2,4-D for efflux carrierswas 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, pretreat-ment with 2-NOA largely affected the predominantly active uptakeof 2,4-D, and so higher apparent concentrations of 2,4-D seem to beneeded for IC50 related to auxin efflux carriers. Even though IC50values for uptake of 2,4-D by auxin uptake carriers were very sim-ilar in both LE and BY-2 cells (see above and Table 1A), there wasa substantial difference between IC50 for 2,4-D and auxin effluxcarriers between both types of cells (719 ± 1 �M and 60 ± 0.5 �Mfor LE and BY-2, respectively; Table 1B). This suggests that the rela-tive affinity of auxin efflux carriers towards 2,4-D is much higher inBY-2 cells compared to LE cells (as ca. an order of magnitude lowerconcentration of 2,4-D is necessary to displace 50% of [3H]NAA inBY-2 compared to LE cells). This is consistent with the finding thata recognizable amount of 2,4-D can be transported from BY-2 cellsactively (Hosek et al., 2012).

Altogether, short-term measurements showed similar affinityof the auxin influx (uptake) carriers to 2,4-D in both LE and BY-2cells. However, the affinity of the auxin efflux carriers towards awell-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-2cells were also able to export distinct amounts of 2,4-D via saturableefflux 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 withincells. Therefore, HPLC-based profiling of [3H]NAA, [3H]IAA and[3H]2,4-D metabolism was performed both in LE and BY-2 cells.First, the [3H]NAA metabolic profile was studied, as there are majordifferences between LE and BY-2 cells in the time-course and shapeof the curves reflecting the amount of radiolabel in cells during theaccumulation of this compound. NAA metabolic profiles in LE cellsand BY-2 cells (Figure S7A–C) differed in both quantity and identityof particular metabolites. Already within 2 min, 41.3% of [3H]NAAwas converted into its metabolites in BY-2 cells, while in LE cellsthis proportion was only 28.5% (Fig. 4A). After 20 min, this differ-ence was even more obvious, with 80.9% of the original [3H]NAApresent 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 con-version of NAA is much slower in LE cells in comparison with BY-2cells. In contrast to synthetic auxin NAA, conversion of [3H]IAA intometabolites was faster in LE cells, as already after 1 min 41.2% and

D. Seifertová et al. / Journal of Plant P

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 (graycolumns) are presented for [3H]NAA (A), [3H]IAA (B) and [3H] 2,4-D (C). The amountof metabolites was examined at the time-points 1, 2 and 20 min after addition ofp(

aicriica

msb

D

tmlsp

articular radiolabeled auxin. Note the distinct amounts of non-metabolized NAAA) and IAA (B) at time 20 min in LE and BY-2 cells.

fter 20 min the majority (91.8%) of original radiolabeled IAA wasn the form of its metabolites (Fig. 4B, Figure S7D–F). Much sloweronversion of [3H]IAA occurred in BY-2 cells, where the metabolitesepresent 48.3% of original [3H]IAA after 20 min (Fig. 4B). Interest-ngly, the spectra of both [3H]NAA and [3H]IAA metabolites differedn LE and BY-2 cells (Figure S7A–F). There was almost no metaboliconversion when synthetic auxin [3H]2,4-D was applied to both LEnd BY-2 cells (Fig. 4C and Figure S7G–I).

These results show that both IAA and NAA are largelyetabolized in cells and that the way they are metabolized is

pecies-specific. In contrast, 2,4-D is metabolically very stable inoth LE and BY-2 cells.

iscussion

The use of simplified cell culture models for measurements ofhe cell-to-cell auxin transport in A. thaliana, the major experi-

ental plant model that is easily ‘genetically accessible,’ has beenimited by the fact that it has been difficult to derive stable andufficiently friable cell suspension lines having standard growtharameters from this species. However, the cell suspension derived

hysiology 171 (2014) 429–437 435

from stem explants of A. thaliana ecotype Landsberg erecta (Mayand Leaver, 1993) has been used for a one-shot comparative IAAtransport study (Geisler et al., 2005). As shown in this paper, afteroptimization of the cultivation protocol, this cell line can serveas valuable tool for tracking auxin influx and efflux activities atthe cellular level. If LE cells are repeatedly cultured in the samemedium as the well-established tobacco BY-2 cells, containing 2,4-D as the only phytohormone, they grow with a stable phenotypeand with a multiplication rate comparable to that of BY-2 cells.Under these conditions, the LE cell culture shows also sufficientcell friability to allow accurate microscopic determination of cellpopulation density and cell dimensions. Therefore, the amount ofradioactively labeled auxin (or another compound) accumulatedinside cells can be readily calculated in relation to parameters suchas cell surface, cell volume, cell number etc., so that it provides anidea of e.g. how many auxin molecules are present inside a cell at aparticular time point and/or physiological situation. Nevertheless,in comparison with tobacco cell lines BY-2 (Petrásek et al., 2003;Dhonukshe et al., 2005; Petrásek and Zazímalová, 2006) and VBI-0(Campanoni et al., 2003; Petrásek et al., 2002), LE cell suspensiondoes not form cell filaments (Menges and Murray, 2002) with clearaxiality that would allow analysis of morphoregulatory aspects ofauxin flow (Campanoni et al., 2003) in parallel to auxin accumula-tion measurements. Instead, LE cells grow radially, from one centerequally in all directions. In any case, LE cells can be proposed asan alternative experimental material for auxin transport assays atthe 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 infor-mation on transcriptome of auxin response is available (Paponovet 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 nec-essary to keep in mind that it was derived from X-ray mutagenizedLandsberg plants (Redei, 1962), so minor differences in auxin trans-port compared to the predominantly used A. thaliana lines cannotbe excluded (Jander et al., 2002; Ziolkowski et al., 2009).

Although LE cells have already been used to show the effect ofinhibitors on the IAA loading and efflux (Geisler et al., 2005), themore detailed auxin transport characteristics for IAA and both syn-thetic auxins NAA and 2,4-D as well as their comparison with theestablished models of tobacco cells (Delbarre et al., 1996; Petrásekand Zazímalová, 2006) have not yet been provided.

As shown here, the kinetics of IAA and 2,4-D accumulation incells and the absolute values expressed per the PM area are com-parable for both tobacco BY-2 cells and LE cells. However, it seemsthat for tracking the active auxin uptake into LE cells, the syntheticauxin analog 2,4-D is far better than native IAA, as IAA is metab-olized quite quickly here. The specific inhibitor of auxin influx2-NOA (Imhoff et al., 2000; Swarup et al., 2008; Yang et al., 2006;Lanková et al., 2010) blocked the influx of synthetic auxin 2,4-Dmore efficiently in LE cells than in BY-2 cells, suggesting a higherproportion of its active, 2-NOA-sensitive influx here. In agreementwith this, the affinity of auxin influx carriers towards 2,4-D wasslightly higher in LE cells. On the other hand, competition exper-iments performed in BY-2 cells showed that the accumulation of[3H]2,4-D was not influenced by the addition of cold 2,4-D in theconcentration range tested (2 nM–2 �M). However, a higher con-centration of cold 2,4-D (10 �M) decreased the accumulation of[3H]2,4-D in BY-2 cells as well (Simon et al., 2013). This, togetherwith the observation of the rapid increase of the 2,4-D accumu-lation curve, could be explained by the higher capacity of net

auxin uptake and by uptake carriers with lower affinity to 2,4-Din BY-2 cells. It could be speculated that BY-2 cells are using pref-erentially MDR/PGP/ABCB-based carriers, perhaps thanks to theirlong-lasting sub-culturing into the media supplemented with 2,4-D

4 lant P

app

m2tadmp

ihmbctstNlsD2eAoeettBo2

iD2Cs(2eswpsaccwa

burp

A(Naacl

Lanková M, Smith RS, Pesek B, Kubes M, Zazímalová E, Petrásek J, et al. Auxin influxinhibitors 1-NOA, 2-NOA, and CHPAA interfere with membrane dynamics intobacco cells. J Exp Bot 2010;61(13):3589–98.

36 D. Seifertová et al. / Journal of P

s the only auxin. This explanation is also in concert with the pro-osal by Yang and Murphy (2009) and Kubes et al. (2012) on theossible role of ABCB4 as a dual auxin influx and efflux transporter.

In LE cells, on the other hand, the kinetics of [3H]2,4-D accu-ulation with a gradual increase together with the ability of cold

,4-D and NAA to compete with the radiolabeled 2,4-D, could reflecthe activity of various types of auxin uptake carriers with variousffinity towards 2,4-D. This may correspond to the cultivation con-itions, as the LE suspension culture used here was maintained inedium supplemented with 2,4-D instead of NAA only for a short

eriod of time.With respect to auxin export from cells, in LE cells the activ-

ty of the NPA-sensitive efflux carriers transporting NAA was evenigher than in tobacco cells (i.e. the relative increase of NAA accu-ulation after NPA application was higher in LE cells). This could

e due to the higher capacity of relevant carriers and/or higher effi-iency of the NPA-based carriers’ regulation, etc. Based on the NPAreatments, synthetic auxin 2,4-D seemed to also be a good sub-trate for the auxin efflux carriers in LE cells. However, as shown inhe short-term experiments, 2,4-D was not able to compete withAA for the active efflux in LE cells. Therefore, it might be specu-

ated that at least two different sets of efflux carriers with differentpecificity are present in LE suspension cells. As originally noted byelbarre et al. (1996) for Xanthi tobacco cells, some degree of active,4-D efflux was also reported later for VBI-0 tobacco cells (Paciorekt al., 2005) and BY-2 cells overproducing the auxin efflux carriertPIN7 (Petrásek et al., 2006). Recently, careful testing in BY-2 cellsf the initial phases of the 2,4-D accumulation supported by math-matical modeling provided additional evidence for carrier-drivenfflux of 2,4-D (Hosek et al., 2012). Interestingly, NPA concentra-ion dependence assays showed that LE cells are relatively moreolerant to the higher concentrations of NPA in comparison withY-2 cells. This could partly justify the quite high concentrationsf this inhibitor used for some studies in planta (Geldner et al.,001).

Radiolabeled NAA has been considered the major tool for study-ng the active auxin efflux in tobacco cell lines (Xanthi XHFD8,elbarre et al., 1996; VBI-0, Campanoni et al., 2003; Paciorek et al.,005; Petrásek et al., 2002; and BY-2, Cho et al., 2007; Lee andho, 2006; Petrásek et al., 2003, 2006). However, in tobacco cell, metabolic changes of NAA are relatively quick and massiveDelbarre et al., 1994; Hosek et al., 2012) and as shown for BY-

cells, NAA metabolites are not transported out of cells (Hosekt al., 2012), thus complicating the interpretation of transport mea-urements using labeled NAA. One of the important findings hereas a much slower rate of NAA metabolic conversion in LE com-ared to BY-2 cells. Therefore, the observed differences in thehape of accumulation curves between LE and BY-2 cells can bettributed to the pattern of metabolism of radiolabeled NAA. Inontrast to NAA, IAA is massively metabolized in LE suspensionells. A similar rate of IAA metabolism (over 80% after 15 min)as observed previously in soybean root suspension cells (Loper

nd Spanswick, 1991).In addition to differences in metabolism of major auxins

etween tobacco and Arabidopsis, another advantage of makingse of Arabidopsis cells for this type of studies is possible prepa-ation of cell suspensions from mutant, transformed and crossedlants.

Altogether, based on: (1) the general ‘genetic accessibility’ ofrabidopsis, including possible use of mutants and transformants,2) the improved protocol for cultivation, (3) a lower rate ofAA metabolism, and (4) possible use of 2,4-D for tracking bothuxin influx and efflux, this study introduces LE cells as a useful,lternative tool to study auxin transport parameters on a single

ell level that is complementary to well-established tobacco cellines.

hysiology 171 (2014) 429–437

Acknowledgements

This work was supported by the Grant Agency of the CzechRepublic, project P305/11/0797 (DS, PS, ML, PID, MP, KH, JP, EZ)and Simons Foundation Grant #245400 (JR). Authors acknowledgethe service of Nottingham Arabidopsis Stock Centre (NASC).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.jplph.2013.09.026.

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