Characterization of transmembrane auxin transport in Arabidopsis

Journal of Plant Physiology 171 (2014) 429–437
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Journal of Plant Physiology
journal homepage: www.elsevier.com/locate/jplph
Physiology
Characterization of transmembrane auxin transport in Arabidopsis
suspension-cultured cells
Daniela Seifertová a , Petr Skůpa a , Jan Rychtář b , Martina Laňková a , Markéta Pařezová a ,
Petre I. Dobrev a , Klára Hoyerová a , Jan Petrášek a , Eva Zažímalová a,∗
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
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]
(P. Skůpa), j [email protected] (J. Rychtář), [email protected] (M. Laňková),
[email protected] (M. Pařezová), [email protected] (P.I. Dobrev),
[email protected] (K. Hoyerová), [email protected] (J. Petrášek),
[email protected] (E. Zažímalová).
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; Petrášek 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; Petrášek 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; Petrášek 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; Petrášek et al., 2002, 2003; Petrášek
and Zažímalová, 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; Petrášek 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)
and in tobacco BY-2 cells (Laňková 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; Petrášek et al., 2002, and BY-2; Petrášek et al., 2003;
Laňková 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 (Petrášek
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
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 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 Petrášek
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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D. Seifertová et al. / Journal of Plant Physiology 171 (2014) 429–437
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 (Petrášek and Zažímalová, 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).
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, 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
inhibitor of the active auxin influx, 2-NOA (Laňková 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; Petrášek 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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D. Seifertová et al. / Journal of Plant Physiology 171 (2014) 429–437
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. Petrášek 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
– 2-NOA (Imhoff et al., 2000; Laňková 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 (Hošek 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
D. Seifertová et al. / Journal of Plant Physiology 171 (2014) 429–437
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
435
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 (Petrášek et al., 2003;
Dhonukshe et al., 2005; Petrášek and Zažímalová, 2006) and VBI-0
(Campanoni et al., 2003; Petrášek 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; Petrášek
and Zažímalová, 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;
Laňková 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
436
D. Seifertová et al. / Journal of Plant Physiology 171 (2014) 429–437
as the only auxin. This explanation is also in concert with the proposal by Yang and Murphy (2009) and Kubeš 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 (Petrášek 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 (Hošek 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; Petrášek et al., 2002; and BY-2, Cho et al., 2007; Lee and
Cho, 2006; Petrášek et al., 2003, 2006). However, in tobacco cell
s, metabolic changes of NAA are relatively quick and massive
(Delbarre et al., 1994; Hošek et al., 2012) and as shown for BY2 cells, NAA metabolites are not transported out of cells (Hošek
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.
Acknowledgements
This work was supported by the Grant Agency of the Czech
Republic, project P305/11/0797 (DS, PS, ML, PID, MP, KH, JP, EZ)
and Simons Foundation Grant #245400 (JR). Authors acknowledge
the service of Nottingham Arabidopsis Stock Centre (NASC).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.jplph.
2013.09.026.
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