The Arabidopsis concentration-dependent influx/efflux transporter ABCB4 regulates cellular auxin levels in the root epidermis

Authors


(fax +1 765 494 0391; e-mail peerw@purdue.edu (W.A.P.) for Arabidopsis, S. pombe and HeLa cell materials, fax +420 225106446; e-mail hoyerova@ueb.cas.cz (K.H.) for BY-2 materials)

Summary

Arabidopsis ATP-binding cassette B4 (ABCB4) is a root-localised auxin efflux transporter with reported auxin uptake activity in low auxin concentrations. Results reported here demonstrate that ABCB4 is a substrate-activated regulator of cellular auxin levels. The contribution of ABCB4 to shootward auxin movement at the root apex increases with auxin concentration, but in root hair elongation assays ABCB4-mediated uptake is evident at low concentrations as well. Uptake kinetics of ABCB4 heterologously expressed in Schizosaccharomyces pombe differed from the saturation kinetics of AUX1 as uptake converted to efflux at threshold indole-3-acetic acid (IAA) concentrations. The concentration dependence of ABCB4 appears to be a direct effect on transporter activity, as ABCB4 expression and ABCB4 plasma membrane (PM) localisation at the root apex are relatively insensitive to changes in auxin concentration. However, PM localization of ABCB4 decreases with 1-naphthylphthalamic acid (NPA) treatment. Unlike other plant ABCBs studied to date, and consistent with decreased detergent solubility, ABCB4pro:ABCB4-GFP is partially internalised in all cell types by 0.05% DMSO, but not 0.1% ethanol. In trichoblasts, ABCB4pro:ABCB4-GFP PM signals are reduced by >200 nm IAA and 2,4-dichlorophenoxyacetic acid (2,4-D). In heterologous systems and in planta, ABCB4 transports benzoic acid with weak affinity, but not the oxidative catabolism products 2-oxindole-3-acetic-acid and 2-oxindole-3-acetyl-β-d-glucose. ABCB4 mediates uptake, but not efflux, of the synthetic auxin 2,4-D in cells lacking AUX1 activity. Results presented here suggest that 2,4-D is a non-competitive inhibitor of IAA transport by ABCB4 and indicate that ABCB4 is a target of 2,4-D herbicidal activity.

Introduction

The ATP-binding cassette class B (ABCB) transporters are found in all eukaryotic phyla and have diversified to mobilise a wide range of substrates. Three Arabidopsis members of the family, ABCB1, -4 and -19, transport the phytohormone auxin and function primarily in auxin export and exclusion from the plasma membrane (PM) (reviewed in Titapiwatanakun and Murphy, 2009). ABCB1 and -19 constitute an independent auxin transport system that functions primarily in the loading and maintenance of long-distance rootward auxin transport streams that contribute to vegetative development and tropic responses, but not early development or organogenesis (reviewed in Zažímalováet al., 2010).

ABCB4/PGP4 is a root-specific ABCB transporter that functions in shootward epidermal transport of auxin from the root apex, conditional regulation of primary and lateral root elongation, and regulation of auxin movement into root hair cells (Santelia et al., 2005; Terasaka et al., 2005; Cho et al., 2007; Lewis et al., 2007). In root hair elongation, ABCB4 appears to regulate export of auxin from trichoblasts, as abcb4 root hairs are longer than wild-type ones, and overexpression of ABCB4 under the control of a root hair-specific promoter results in shorter root hairs similar to those resulting from overexpression of PIN efflux transporters (Cho et al., 2007).

The increased rates of root hair elongation observed in abcb4 are difficult to reconcile with reports of decreased rates (30–50%) of shootward auxin transport in abcb4 roots (Santelia et al., 2005; Terasaka et al., 2005; Lewis et al., 2007), as those results suggest that less auxin is available to elongating trichoblasts in abcb4 than in the wild type. However, other evidence suggests that the abcb4 root epidermis contains sufficient auxin to stimulate enhanced root hair elongation when ABCB4 auxin efflux activity is absent in those cells. Roots of abcb4 exhibit a slight increase rather than the decrease in gravitropic bending rates associated with decreased shootward auxin flows (Lewis et al., 2007). Further, although abcb4 root apices have higher free indole-3-acetic acid (IAA) levels than the wild type (Santelia et al., 2005; Terasaka et al., 2005), IAA levels in the abcb4 differentiation zone and mature lower root are similar to wild-type levels (Terasaka et al., 2005; Peer and Murphy, 2007). As increases in ABCB4 expression after auxin treatment are not evident during the time course of shootward auxin transport experiments in roots (Terasaka et al., 2005), ABCB4 appears to be regulated by post-transcriptional processes.

Previous findings support that ABCB4 efflux activity is directly activated by auxin levels. There are several lines of evidence that support this hypothesis: (i) Free IAA levels are significantly increased in the abcb4 root apex where auxin levels are normally the highest, while little or no difference is observed in tissues where auxin levels are normally lower (Terasaka et al., 2005). (ii) Lateral and primary root elongation phenotypes in abcb4 are highly dependent on growth conditions and generally correlate with conditions that increase endogenous auxin levels (Terasaka et al., 2005; Santelia et al., 2005; Lewis et al., 2007). (iii) Altered DR5revpro:GFP signals in abcb4 are only observed in graviresponding roots where auxin levels are increased in the elongation zone (Lewis et al., 2007). (iv) Reductions in auxin transport in abcb4 roots reported in radioisotope tracer and ion selective electrode assays appear to increase proportionately to the amount of auxin applied (Santelia et al., 2005; Terasaka et al., 2005; Lewis et al., 2007; Peer and Murphy, 2007).

Experiments in heterologous systems also support direct induction of ABCB4 export by threshold concentrations of IAA. In mammalian and yeast cells, ABCB4 expression results in increased net influx of IAA at low concentrations (10–60 nm) over short periods of time, but produces net efflux when intracellular IAA levels increase (Terasaka et al., 2005; Yang and Murphy, 2009). Overexpression of ABCB4 in tobacco BY-2 cells was reported to enhance 1-naphthalene acetic acid (NAA) efflux (Cho et al., 2007). However, NAA is taken up more readily than IAA by plant cells as it bypasses carrier-mediated uptake mechanisms via passive diffusion (Petrášek et al., 2006), and BY-2 cells are routinely cultured in micromolar concentrations of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D). As such, sub-threshold intracellular auxin levels may be difficult to achieve in this system.

ABCB4 may also transport compounds other than IAA in Arabidopsis roots. ABCB4 exhibits sequence homology with the alkaloid uptake transporter CjMDR1/ABCB1 from Coptis japonica (Shitan et al., 2003) and is associated with a clade that is phylogenetically distinct from the ABCB1 and -19 auxin transporters (Knöller et al., 2010). ABCB4 transport appears to be biochemically distinct from ABCB1 and -19 as well, since expression of ABCB4, but not ABCB1 or -19, in Schizosaccharomyces pombe resulted in export of benzoic acid at levels even greater than IAA (Yang and Murphy, 2009). Further, metabolomic analyses of root exudates from abcb4 detected decreases in a root exudate compound (Mr 186) that does not correspond to IAA or known IAA catabolites (Badri et al., 2008). Other ABCBs outside the ABCB1/19 clade may also be mixed-affinity transporters, as the malate/citrate transporter ABCB14 has recently been implicated in the modulation of auxin transport (Kaneda et al., 2011).

ABCB models based on high-resolution crystal structures from other species (Dawson and Locher, 2006; Aller et al., 2009; Yang and Murphy, 2009) predict that ABCB4 has a canonical ABCB protein organisation consisting of two transmembrane and two nucleotide-binding domains (TMDs, NBDs) in the ‘twisted’ TMD–NBD structure associated with eukaryotic ABC exporters (Hollenstein et al., 2007; Kos and Ford, 2009; Rees et al., 2009). Substrate docking simulations identified an additional high-probability TMD-binding site for auxin molecules in ABCB4 that is not found in the ABCB1/19 auxin efflux transporters (Yang and Murphy, 2009). Without IAA bound to this site, the model predicted transfer of IAA into the PM cytoplasmic leaflet and subsequent anionic trapping in the cytosol, whereas IAA binding would enhance the efflux activity of the protein. In support of this mechanism, 2,4-D, which was predicted to occlude this site, was experimentally shown to non-competitively inhibit ABCB4 auxin efflux (Yang and Murphy, 2009).

Here we re-examine ABCB4-mediated auxin transport in Arabidopsis roots, BY-2 cells, and S. pombe and provide evidence that ABCB4 functions as a homeostatic switch on the plasma membrane for regulation of cellular auxin levels. We show that substrate regulation of ABCB4 is initially a direct effect on the protein that precedes subsequent alterations in membrane localisation. We show that ABCB4 is activated by the primary auxin catabolite oxindole-3-acetic acid (oxIAA), but does not export this compound. Finally, we confirm that ABCB4 enhances uptake of 2,4-D, but not its efflux, and that this activity is likely to contribute to the herbicidal effects of 2,4-D.

Results

ABCB4 expression

Primary root and root hair elongation phenotypes of abcb4 mutants vary with growth conditions (Santelia et al., 2005; Terasaka et al., 2005; Cho et al., 2007). Quantitative real-time PCR (qRT-PCR) and microarray data indicate that ABCB4 expression is regulated by light and, to a much lesser extent, sucrose (Fig. S1 in Supporting Information). Etiolated seedlings shifted to the light showed a more than two-fold reduction in ABCB4 expression, while transfer to darkness or dim light increased expression (Fig. S1). However, no change in localisation or intensity of ABCB4pro:ABCB4-GFP signals was detected in the root tip up to the elongation zone with different light treatments (see below). Variation of mineral nutrients had no effect on abcb4 phenotypes or ABCB4pro:ABCB4-GFP signals, although <2 μm Fe inhibited root hair extension in both wild-type and abcb4. Exogenous treatment with IAA only slightly increased ABCB4 expression, although 2,4-D treatment increased ABCB4 expression about three-fold after 5 h (Terasaka et al., 2005; Fig. S1A). Based on these results, the experiments reported here utilised seedlings grown in 100 μE m−1 sec−1light on 1/4 MS salts, 0.5% sucrose and transferred to dim light unless noted.

IAA stimulation of ABCB4-mediated shootward auxin transport in the root apex

A combination of activities contribute to auxin transport at the root tip, and auxin transport mutant phenotypes are consistent with transporter distribution and function (reviewed in Petrášek and Friml, 2009; Zažímalováet al., 2010). Loss of AUX1 auxin influx carrier function abolishes root gravitropic growth and reduces root hair formation (Bennett et al., 1996; Yamamoto and Yamamoto, 1998). Loss of PIN2 and, to a lesser extent, PIN3, inhibits gravitropic bending (Vieten et al., 2005; Abas et al., 2006), loss of ABCB1 and -19 results in highly variable root gravitropic growth (Noh et al., 2003), while loss of ABCB4 results in variable elongation growth (Terasaka et al., 2005), altered root hair elongation (Santelia et al., 2005; Cho et al., 2007) and slightly increased root gravitropism (Lewis et al., 2007).

In the nanoscale experimental system used in our lab to monitor shootward auxin transport from the root apex, a 10-nL droplet of 1 μm3H-IAA (10 fmol) is placed on either the root cap/columella or over the quiescent centre (QC) of the root (Peer and Murphy, 2007). For the experiments described here, the 1 μm3H-IAA was diluted with an equal part of 0.1% ethanol or 10 μm (100 fmol) unlabelled IAA and administered in a 20-nl droplet overlaying the columella and QC to assess the contribution of AUX1, PIN2, ABCB1, ABCB4 and ABCB19 to shootward flows from the root apex (Fig. 1a). Assays were conducted in a gas exchange chamber to eliminate the effects of ethylene accumulation (columella placement and ethylene controls are shown in Fig. S2A,B).

Figure 1.

 ABCB4-mediated auxin transport is activated by indole-3-acetic-acid (IAA).
(a) Shootward 3H-IAA [20 nl (25 fmol) placed on root cap/S3 columella] transport from the quiescent centre in 5-day-old Arabidopsis roots: ‘a’3H-IAA; ‘b’+10× cold IAA; anova, Dunnet’s post-hoc, P < 0.05.
(b) ABCB4pro:ABCB4-GFP signals in control and 3 h after 200 nl 10 nmol IAA placed on root cap/S3 columella. Bar: 15 μm.
(c) Application of increasing 3H-IAA concentrations result in greater reductions in shootward transport in abcb4 compared to Col-0 and pin2.
(d) Root tip (0.5 mm) net 3H-IAA accumulation assays decreased in abcb4 and increased in ABCB4 overexpressor (ABCB4-OX) at 15 min. The pattern is reversed at 30 min. a,b,cP < 0.05.
(e) Free IAA levels in differentiation/maturation zone of root (2-mm segment starting 1.5 mm from the apex). Error bars, SD (n = 3 pools of 10 seedlings for assays, except D, n = 50 seedlings).

Reductions in 3H-IAA transport were observed in aux1, pin2, abcb1 and abcb4, but not abcb19 in the assays shown in Fig. 1a. Although the decrease in 3H-IAA transport in aux1 and abcb19 was less than expected (P > 0.05; Fig. 1a), aux1 and abcb19 exhibited a greater decrease in shootward 3H-IAA transport compared with the wild type when the droplet was limited to the columella region or when ethylene was added to the system (P < 0.05; Fig. S2A,B). An ABCB4 overexpression line with higher ABCB4 expression levels in the root (Terasaka et al., 2005; Cho et al., 2007) exhibited shootward 3H-IAA transport that was no different from wild type (P > 0.05).

Shootward 3H-IAA transport was reduced in the wild type when an equal part of 0.1% ethanol in the 20-nl droplet was replaced with 10 μm cold IAA (P < 0.05; Fig. 1a), suggesting that the cold IAA competed with transport of the radiotracer or suppressed its transport in some other manner. By far the greatest competitive reduction in auxin transport was seen in the abcb4 loss-of-function mutant, implying that increased auxin application inhibited ABCB4 transport less than AUX1, PIN2 and the other ABCBs. A lack of competitive inhibition of shootward auxin transport observed in an ABCB4 overexpressor (ABCB4OX) (P < 0.05; Fig. 1a) suggests that ABCB4 is non-transcriptionally activated by increased auxin levels. In support of this interpretation, ABCB4pro:ABCB4-GFP signals decreased very slightly after a 1× nanoscale IAA application (Fig. 1b), and no further signal change was observed with increasing IAA application.

Transport assays were repeated in the wild type, pin2 and abcb4 utilising increasing amounts of 3H-IAA in a 30-nl droplet to maintain ethanol concentrations at 0.1% (Fig. 1c). Reductions in auxin transport observed in pin2 were consistent across all 3H-IAA amounts applied. However, significant reductions in 3H-IAA transport compared to the wild type (P < 0.05) were not observed in abcb4 until more than 25 fmol was applied and only reached the 30–50% reductions previously reported at 50 fmol and above (R2 = 0.96, 0.95, 0.85 for Col-0, pin2, abcb4, respectively; Fig. 1c).

Short-term (1 h) root tip incubation with radiolabelled compounds has previously been used to assess the role of the ABCG36/37 transporters in uptake of 3H-IAA, indole-3-butyric acid (IBA) and benzoic acid (BeA) (Růžička et al., 2010). This method was adapted to measure uptake of 3H-IAA for 15 or 30 min with the addition of 100 nm of cold IAA to the incubation mixture in the wild type and abcb4. After 15 min, net accumulation of 3H-IAA was lower than wild type in abcb4 root tips and higher in ABCB4 overexpression lines (P < 0.05; Fig. 1d). However, after 30 min, this trend was reversed with slightly increased net accumulation observed in abcb4 and decreased net accumulation in the ABCB4 overexpression line (P < 0.05; Fig. 1d). These results support a model where ABCB4 mediates uptake of low concentrations of IAA, but exports IAA as intracellular levels increase.

ABCB4 regulation of root hair length

It is difficult to reconcile decreased shootward auxin delivery to the root differentiation/maturation zone with evidence that ABCB4-mediated auxin efflux activity inversely correlates with root hair length (Cho et al., 2007). However, free IAA levels in a 2-mm segment containing the differentiation/maturation zone where root hair elongation is particularly pronounced in abcb4 (starting 1.5 mm above the root apex) were equal to or even slightly greater than those in equivalent segments from wild-type roots (Fig. 1e). This suggests that auxin supply to trichoblasts in abcb4 is sufficient for root hair elongation and that ABCB4 limits movement of auxin out of the elongation zone as previously proposed (Terasaka et al., 2005).

Consistent with ABCB4 regulation of auxin levels in root hairs, the addition of 10 nm IAA to the medium resulted in increased root hair elongation in both the wild type and abcb4, although root hairs were still significantly longer in abcb4 (P < 0.05, Fig. 2a). As reported previously (Santelia et al., 2005), treatment with a higher concentration (50 nm) of IAA completely eliminated the growth difference between wild-type and abcb4, and higher IAA concentrations (up to 1 μm) inhibited root hair elongation in both lines (Fig. 2a). Attempts to correlate growth with direct or indirect auxin quantification in root hairs were unsuccessful, as free IAA levels in root hairs collected by vacuum could not be reliably normalised and DR5revpro:GFP signals could not be detected in wild-type or abcb4 trichoblasts. Further analyses of root hair-specific growth responses utilising expression of ABCB4 driven by the Expansin A7 (PE7) promoter (Cho and Cosgrove, 2002; Cho et al., 2007) were not pursued, as, in our hands, weaker PE7:GUS and GFP fusion signals were present in atrichoblasts.

Figure 2.

 ABCB4 localisation in Arabidopsis root hairs (5-day-old Arabidopsis seedlings).
(a) Treatment with increasing indole-3-acetic-acid (IAA) concentrations inhibits root hair elongation. Mean lengths and SD from the first 10 root hairs measured starting 1.5 mm from the root apex (10 seedling pools, n = 3). *P < 0.05.
(b) Root hairs in a region 1.5 mm above the abcb4 root apex are consistently longer than in the wild type. Bar = 100 μm.
(c) Visualisation of ABCB4pro:ABCB4-GFP shows a signal at the plasma membrane in atrichoblast (a) and trichoblast (t) cells in light-grown seedlings acclimated to dim light.
(d) ABCB4-GFP signals in roots hairs after 5 h of 20 nm IAA treatment in light-grown seedlings acclimated to dim light.
(e) ABCB4-GFP signals in root hairs in light-grown seedlings. Bar = 50 μm (c–e).
(f–k) ABCB4pro:ABCB4-GFP localisation in epidermal cells is sensitive to auxins, 1-naphthylphthalamic acid (NPA), and DMSO: (f) Ethanol (EtOH), 0.1%, 5 h; (g) IAA, 1 μm, 5 h; (h) 2,4-dichlorophenoxyacetic acid (2,4-D), 1 μm, 5 h; (i) DMSO, 0.05%, 5 h; (j) NPA, 10 μm, 5 h; (k) In twd1-3. Bar = 10 μm.

ABCB4 expression in trichoblast-forming epidermal cells is initially low, increases as the root hair elongates, and then decreases after elongation ceases (Fig. S3). Under the growth conditions used in the assays described here (3-day light-grown seedlings acclimated to 20 μmol m−2 sec−1 light for 24 h before and during assays), elongated root hairs were readily observed in abcb4 in a region starting 1.5 mm above the root apex (Fig. 2b). Examination of an ABCB4pro:ABCB4-GFP line (Cho et al., 2007) confirmed that, unlike AUX1, which is not expressed in trichoblasts (Jones et al., 2009), PM-localised ABCB4 is observed in all epidermal cells (Fig. 2c). The PM-localised ABCB4-GFP signal intensified slightly as root hairs began to form and extended with the root hair during all subsequent stages. Attempts to discern whether intracellular signals observed in root hairs were a result of ABCB4-GFP secretion using cycloheximide were unsuccessful, as cycloheximide completely inhibited growth of the root hair tip. However, fluorescence recovery after photobleaching (FRAP) analysis detected rapid restoration of signal to the PM (not shown), suggesting that these signals represented anterograde trafficking.

Like ABCB19, ABCB4 has been shown to exhibit stable membrane localisation and resistance to brefeldin A (Cho et al., 2007; Wu et al., 2007; Titapiwatanakun et al., 2009). In the proximal elongation and maturation zones, PM ABCB4-GFP signals decreased slightly after treatment with 20–100 nm IAA or 2,4-D (Fig. 2d; 2,4-D not shown) and decreased substantially after 5 h of treatment with 1 μm IAA or 2,4-D (Fig. 2g,h). Auxin treatment also produced an increased intracellular signal not associated with endomembrane compartments and not seen in the control (Fig. 2c,d). Presumably this signal represents free GFP, as post transcriptional inactivation of ectopically overexpressed ABCB4 and proteolytic truncation of the ABCB4 C-terminus have been reported previously (Geisler et al., 2003; Terasaka et al., 2005; Titapiwatanakun et al., 2009).

ABCB4-GFP seedlings grown in continuous elevated light (100 μmol m−2 sec−1) exhibited very weak signals in trichoblasts, but signals in the atrichoblast PM were greatly increased (Fig. 2e). These results suggest that studies of root hair elongation in response to low concentrations of auxins in dim light are likely to primarily reflect effects on ABCB4 transport activity, not protein localisation/abundance. However, they also underscore the sensitivity of these assays to light conditions and auxin levels.

Solvent control experiments also revealed a sensitivity of ABCB4 membrane localisation to low concentrations of dimethyl sulphoxide (DMSO) in epidermal cells. Substantial internalisation of both N- and C-terminal GFP fusions of ABCB4 were observed after treatment with 0.05% DMSO (Fig. 2f,i). Presumably this is a result of dissociation of ABCB4 from hydrophobic membrane domains (Borner et al., 2005; Titapiwatanakun et al., 2009) as a result of introduction of water molecules at points of lipid–protein interaction (Notman et al., 2006). As a result, all assays of ABCB4 activity in Arabidopsis, BY-2 cells and S. pombe described herein were conducted with low auxin concentrations and compounds solubilised in ethanol.

Plasma membrane localisation of ABCB4 is sensitive to 1-naphthylphthalamic acid (NPA) and high auxin concentrations

A standard efflux inhibitor used in analyses of auxin transport is NPA (Rubery, 1990; Muday, 2001). Functional secretion of ABCB19 and ABCB4 out of the endoplasmic reticulum requires the immunophilin-like chaperone FKBP42/TWD1, and ABCB secretion is inhibited by overnight treatment with NPA which interrupts TWD1-ABCB interactions (Geisler et al., 2003; Bouchard et al., 2006; Rojas-Pierce et al., 2007; Wu et al., 2007, 2010). Treatment of ABCB4-GFP transformants with 10 μm NPA (5 h) resulted in ABCB4-GFP localisation to intracellular inclusions similar to those seen in the twd1-3 background (Fig. 2j,k). These results suggest that it would be difficult to differentiate between direct and indirect effects of NPA on ABCB4-mediated efflux, so NPA was not utilised in any of the Arabidopsis growth and transport studies described herein.

2-Oxindole-3-acetic acid is not an ABCB4 transport substrate

Previously, ABCB4 was shown to transport BeA when expressed in S. pombe (Yang and Murphy, 2009). In root tip net uptake assays, 3H-BeA uptake in wild-type, abcb4 and ABCB4OX was similar at 15 min, but was increased in abcb4 and decreased in ABCB4OX after 30 min (Fig. 3a). This suggested that ABCB4 may mobilise other organic acid substrates or conjugates in addition to IAA in planta. In Arabidopsis, oxIAA and 2-oxindole-3-acetyl-β-glucose (oxIAA-Gluc) are the initial oxidative products generated when IAA is present at excessive levels (Ernstsen et al., 1987; Ostin et al., 1998), and thus are possible substrates for a transporter that regulates physiologically relevant cellular auxin levels. Liquid chromatography (LC)-MS analysis of extracts indicated that increased levels of oxIAA and oxIAA-Gluc were generated in the root when shoot-derived auxin streams were increased by application of increasing amounts of 3H-IAA to the shoot tip in wild-type seedlings (Fig. 3b) and that oxIAA is not polarly transported (Fig. 3c).

Figure 3.

 ABCB4 transports benzoic acid (BeA), but not oxindole-3-acetic acid (oxIAA) out of the root.
(a) Root tip uptake assays indicate that ABCB4 mediates 3H-BeA efflux.
(b) Application of increasing amounts of 3H-indole-3-acetic-acid (IAA) to the shoot apex results in increased oxindole-3-acetic acid (oxIAA) and oxIAA-Gluc accumulation in the root (50 seedling pools, n = 3, error bars, SD).
(c) 3H-oxIAA purified from extracts (see Experimental Procedures) is not transported polarly in Arabidopsis seedlings.
(d) The ox-IAA and oxIAA-Gluc levels are not increased in abcb4. oxIAA-Gluc levels are normalized against a 13C-IAA standard so are relative values.
(e) Oxindole-3-acetic acid does not compete with shootward transport of 3H-IAA from the root apex and activates ABCB4-mediated 3H-IAA transport in planta (10 seedling pools, n = 3). Error bars, SD. *,a,bP < 0.05.

However, levels of oxIAA and oxIAA-Gluc in roots of abcb4 were not significantly higher compared with the wild type (P > 0.05; Fig. 3d). As would be expected, levels of oxIAA and oxIAA-Gluc were slightly higher in abcb1 and lower in abcb19 due to changes in auxin movement in these mutants (Fig. 3d). Competition of IAA transport with oxIAA was also analysed in assays of shootward transport of 3H-IAA from the root apex. No competitive inhibition was detected (Fig. 3e), and oxIAA appeared to enhance 3H-IAA transport as was previously described for ABCB1 (Geisler et al., 2005). A small amount of competitive inhibition of 3H-IAA uptake was observed in yeast expressing ABCB4 (Fig. S4A; P > 0.05), but no inhibition of efflux was observed (Fig. S4B).

Expression of ABCB4 in BY-2 cells produces phenotypes consistent with auxin efflux

Overexpression of ABCB4 under the control of a 35S promoter has previously been shown to enhance 3H-NAA efflux in BY-2 cells (Cho et al., 2007). Expression of Arabidopsis ABCB4-GFP in BY-2 resulted in abundance and localisation similar to that reported for 35S-driven expression in BY-2 (Cho et al., 2007) as well as a partial inhibition of cell division compared with untransformed cells (measured by cell density during a 7-day subculture period). This inhibition was similar to that produced by PIN1pro:PIN1-GFP expression (Fig. 4a), but was less severe than in auxin-starved BY-2 cells or in cells expressing Arabidopsis PIN4, PIN6, PIN7 and ABCB19 (Petrášek et al., 2006; Mravec et al., 2008). Further, cell elongation observed with expression of Arabidopsis ABCB19 or PIN1 (Mravec et al., 2008) was not observed in 3-day ABCB4-GFP cells (Fig. 4b), perhaps due to decreased transverse PM localisation of ABCB4-GFP (Fig. 4c and Cho et al., 2007) compared to PIN1-GFP (Fig. 4c). This decrease in transverse localisation may be a consequence of the higher PM stability of ABCB4 compared with PIN1 as indicated by higher accumulation of ABCB4-GFP fluorescence compared with PIN1-GFP in PM during FRAP (Fig. 4d). The PM abundance and localisation of ABCB4-GFP also exhibited decreased sensitivity to IAA, NPA and DMSO in BY-2 cells compared with Arabidopsis (Figs 2g,i,j and 4c), presumably due to differences in FKBP content, membrane lipid composition, and cultivation of BY-2 cells in 2,4-D.

Figure 4.

 ABCB4 overexpression in BY-2 cells shows auxin related phenotypes. (a) Decreased cell multiplication/density during 7-day sub-cultivation interval in PIN1-GFP and ABCB4-GFP cell lines. Error bars, SE (n = 8). (b) Cell size distribution in control and ABCB4-GFP cells scored 3 days post-inoculation. (c) ABCB4-GFP and PIN1-GFP fluorescence from single confocal scans and differential interference contrast, DMSO solvent control, 2-day cell cultures. Bars = 40μm. (d) Fluorescence recovery after photobleaching dynamics in ABCB4-GFP and PIN1-GFP expressing cells.

Auxin transport in BY-2 cells expressing ABCB4

As reported previously (Petrášek et al., 2002; Petrášek et al., 2006), PIN1 expression in BY-2 resulted in increased 3H-NAA efflux, and NPA treatment abolished background ABCB activity and reduced PIN1-mediated efflux (Fig. 5a). BY-2 cells expressing ABCB4pro:ABCB4-GFP exhibited little or no significant change in 3H-NAA (2 nm) uptake or efflux at 15 min, but did exhibit increased net uptake after NPA treatment (Fig. 5a), suggesting that ABCB4 also has NPA-sensitive efflux activity. The weak uptake activity could not be further analysed due to the residual lipophilic uptake of 3H-NAA observed in BY-2 cells, even after effective inhibition of AUX1/LAX transporters by 2-naphthoxyacetic acid (2-NOA) (Laňkováet al., 2010).

Figure 5.

 Indole-3-acetic-acid (IAA) transport by ABCB4 in BY-2 cells.
(a) Accumulation of 3H-1-naphthalene acetic acid (NAA) (2 nm) in control, PIN1-GFP and ABCB4-GFP BY-2 cell lines increased after addition of 1-naphthylphthalamic acid (NPA) (10 μm). Error bars, SE (n = 3). EtOH, ethanol.
(b) ABCB4-GFP expression reduces 3H-benzoic acid (BeA) retention.
(c) Cold BeA (10μm) does not compete with 3H-NAA transport in control or ABCB4-GFP cells.
(d, e) 10 μm IAA, but not oxindole-3-acetic acid (oxIAA), competes with 3H-IAA net retention in control and ABCB4-GFP cells. Error bars, SE (n = 3). Comparisons were made with respect to net efflux, not absolute values.

BY-2 cells expressing ABCB4 showed only a slight decrease in net 3H-BeA retention (Fig. 5b), but cold BeA (10 μm) did not compete with 3H-NAA or 3H-IAA transport (Fig. 5c, for IAA, data not shown). We selected 3H-IAA as a more informative substrate for oxIAA competition assays, and cold oxIAA did not compete with 3H-IAA in control or ABCB4 cells (Fig. 5d,e). However, some caution is required in comparing BY-2 competition assays with those in Arabidopsis and S. pombe because IAA catabolism is more rapid in BY-2 (Delbarre et al., 1994), presumably a consequence of BY-2 cells being cultured in 2,4-D as well as the required use of selective auxin uptake and efflux inhibitors for differentiation of heterologous and endogenous auxin transport activities in this system (Petrášek et al., 2006; Titapiwatanakun et al., 2009; Laňkováet al., 2010).

ABCB4 uptake kinetics

All of the Arabidopsis auxin transporters examined to date are at least partially functional when expressed in the yeast S. pombe (Yang and Murphy, 2009; Růžička et al., 2010; Tsuda et al., 2011). If ABCB4 functions as an uptake transporter at low (<50 nm) IAA concentrations, saturation kinetics should be evident with expression of ABCB4 in this system. For comparison, the kinetics for AUX1 were determined, and AUX1 exhibited uptake saturation at >2 μm3H-IAA with an apparent Km of approximately 340 nm based on initial rates of uptake (Fig. 6a,b). This observed Km was lower than that reported for AUX1 in Xenopus oocytes (Yang et al., 2006), and it is likely due to cell size differences and the presence of a cell wall in S. pombe. In comparison, ABCB4 exhibited an apparent Km of approximately 470 nm based on initial rates (Fig. 6c). In short-term assays, ABCB4 showed influx activity at lower IAA concentrations and ABCB4 transport activity switched to efflux at concentrations >250 nm or with exposure to IAA for a longer period of time (Fig. 6d, vector control in Fig. S4C). This transport profile was also observed when ABCB4 was expressed in HeLa cells (Fig. 6e). This uptake effect may be specific to ABCB4 and related ABCB4 isoforms (Knöller and Murphy, 2011), as no IAA uptake could be observed in the ABCB19 auxin efflux transporter under any conditions. Western blot analysis of membrane fractions confirmed that ABCB4 and ABCB19 proteins were expressed (Fig. S4D).

Figure 6.

 ABCB4-mediated 3H-indole-3-acetic-acid (IAA) uptake in Schizosaccharomyces pombe and HeLa cells.
(a) Uptake of 3H-IAA by AUX1 (8-min assay).
(b) Uptske of 3H-IAA by AUX1 saturates at 2 μm (8-min assay). DPM, disintegrations per minute.
(c) Uptake of 3H-IAA by ABCB4 exhibits an apparent Km of 470 nm (4-min. assay).
(d) Efflux of 3H-IAA by ABCB4 and ABCB19. Uptake of 3H-IAA by ABCB4 saturates at >250 nm and reverses to efflux after 5 min. Error bars = SD, n = 4, two independent experiments. Values are subtracted from vector controls (see Fig. S2C for vector).
(e) Retention of 3H-IAA in HeLa cells expressing ABCB4 or ABCB19 at increasing concentrations of IAA; substrates were co-incubated for 40 min prior to assay; SC, solvent control.

Uptake of 2,4-D by ABCB4 inhibits efflux activity

2,4-D is often used as a substitute for IAA in growth assays as it is highly stable (Delbarre et al., 1996) and poorly distributed through the plant by auxin polar transport mechanisms (Pitts et al., 1998). This synthetic auxin is taken up via AUX1/LAX permeases (Bennett et al., 1996; Yang et al., 2006; Yang and Murphy, 2009), it was shown to be an uptake substrate for ABCB4 expressed in S. pombe, and to non-competitively inhibit ABCB4 3H-IAA efflux activity (Yang and Murphy, 2009). 2,4-D is a weak efflux substrate for all ABCB and PIN proteins expressed in non-plant heterologous systems, but is a preferred efflux substrate for the ABCG37/PDR9 pleiotropic substrate transporter (Titapiwatanakun et al., 2009; Růžička et al., 2010).

Analysis of the effects of 2,4-D on root hair elongation confirmed that this compound is an ABCB4 uptake substrate in planta. Low concentrations of 2,4-D (10 nm) compensated for differences in abcb4 root hair elongation in a manner similar to what is seen with IAA, and higher 2,4-D concentrations inhibited root hair elongation equally in both abcb4 and the wild type (Fig. 7a). However, unlike IAA, an intermediate concentration of 2,4-D (50 nm) inhibited wild-type root hair elongation to a greater extent than in abcb4, suggesting a direct role of ABCB4 in 2,4-D uptake. This result cannot be attributed to enhanced AUX1 uptake in abcb4 root hairs, as AUX1 is not expressed in trichoblasts (Jones et al., 2009). 2,4-D has also been shown to inhibit actin polymerization (Rahman et al., 2007), and this may be why the effect is more pronounced in the root hairs.

Figure 7.

 ABCB4 mediates uptake but not efflux of 2,4-dichlorophenoxyacetic acid (2,4-D).
(a) Responses of root hairs to increasing concentrations of 2,4-D.
(b) Addition of 2-naphthoxyacetic acid (NOA) (10 μm) decreased accumulation of 3H-2,4-D in ABCB4-GFP BY-2 cells. EtOH, ethanol. Error bars, SE (n = 3).
(c) 2-NOA (10 μm, 30-min pre-treatment) initially blocked accumulation of 3H-2,4-D in ABCB4-GFP BY-2 cells but accumulation gradually increased after 15 min. Error bars, SE (n = 3).
(d) Accumulation of 3H-2,4-D in ABCB4-GFP cell lines pre-treated with 1-naphthylphthalamic acid (NPA) (30 min, 10 μm) was the highest. When NPA was applied (time 13 min) to cells pre-treated with 2-NOA (30 min, 10 μm) 2,4-D accumulation increased compared with 2-NOA only. Error bars, SE (n = 3).
(e) In binding assays of ABCB4 or -19 expressed in HeLa cells, 3H-2,4-D binds ABCB4 with greater affinity than ABCB19 and is not competed away by indole-3-acetic-acid (IAA). Error bars, SD (n = 3).*P < 0.05.
(f) Modelling of binding of IAA and 2,4-D.

2,4-D is also the preferred substrate for native 2-NOA-sensitive auxin influx assays in BY-2, and ABCB4 is resistant to 2-NOA inhibition (Laňkováet al., 2010). As expected, 2-NOA (10 μm) decreased the net uptake of 3H-2,4-D (2 nm in control and PIN1-expressing cells, but had a lesser effect on the accumulation of 2,4-D in ABCB4-overexpressing cells (Fig. 7b). However, 2,4-D uptake was enhanced in cells expressing ABCB4 after background auxin uptake was blocked with 2-NOA (Fig. 7c), suggesting a non-competitive interaction of 2,4-D with the ABCB4-based auxin efflux activity. This notion was further supported by the fact that pre-treatment of cells expressing ABCB4 with cold 2,4-D, but not cold IAA, resulted in an enhanced net uptake of 3H-2,4-D (Figs S5 and S6). An enhancement of auxin influx in BY-2 cells expressing ABCB4 was also observed following addition of NPA to cells pre-treated with 2-NOA. This treatment resulted in an increase in accumulation of 2,4-D, consistent with continued import of 2,4-D by ABCB4 in combination with inhibition of the weak PIN and ABCB (including ABCB4) efflux activity (Fig. 7d).

One explanation for the lack of 3H-NAA efflux seen in BY-2 cells expressing ABCB4 might be that the cells are routinely maintained on medium containing the synthetic auxin 2,4-D (Nagata et al., 1992). Although routinely washed out with uptake buffer before auxin transport assays are conducted (Cho et al., 2007; Fig. S7), 2,4-D from the BY-2 cell culture medium bound to ABCB4 may be difficult to completely eliminate, especially as 2,4-D is poorly metabolized in tobacco cells compared with IAA and NAA (Delbarre et al., 1994, 1996). No condition, time point, expression level (35S or native promoter) or regimen could be identified where net 3H-NAA efflux greater than that shown in Fig. 5d was observed in BY-2 cells expressing ABCB4.

Assays of binding of 3H-2,4-D to ABCB4 and ABCB19 expressed in mammalian HeLa cells suggest that the affinity of ABCB4 for 2,4-D is higher than that of ABCB19 (Fig. 7e). Further, the increased 3H-2,4-D-specific binding observed in cells expressing ABCB4 was not displaced with excess IAA. Modelling of the docking of 2,4-D and IAA to ABCB4 is consistent with allosteric rather than competitive regulation by 2,4-D, and binding of 2,4-D to ABCB4 predicts a binding configuration that would enhance efflux (Fig. 7f).

Discussion

The experimental evidence presented here indicates that ABCB4 regulates cellular auxin levels in the Arabidopsis root epidermis by enhancing auxin uptake when cellular levels are low and catalysing efflux when internal levels rise. A role for ABCB4 in regulating auxin homeostasis at the plasma membrane in root epidermal cells is consistent with the need to balance auxin streams regulating programmed and tropic growth. This mechanism at the plasma membrane would be expected to complement auxin homeostasis at the endoplasmic reticulum membrane mediated by short PIN proteins (Mravec et al., 2009).

The low (nanomolar) auxin concentrations required to induce efflux activity in ABCB4 suggest that this protein functions primarily as an efflux transporter in the root apex and catalyses uptake only in cells distal to the meristematic region where auxin levels decrease (Petersson et al., 2009). However, as excess auxin levels destabilise or reduce the abundance of ABCB4 on membranes in cells in the elongation zone and above, it is unlikely that ABCB4 functions in the removal of toxic auxin concentrations in root epidermal cells. Therefore, it appears that excess IAA is oxidatively catabolised in these cells and the oxidative products are either internalised in the vacuole or exported. Results presented herein, as well as experiments documenting IAA induction of reactive oxygen species (Peer and Murphy, 2007), suggest that the threshold for oxidative IAA catabolism in non-apical tissues (100–500 nm) is well above the induction threshold for ABCB4 efflux.

A mechanistic basis for auxin uptake associated with ABCB4 may be derived from the characteristics of its association with the PM lipid bilayer. Protonated apoplastic IAA readily diffuses into the PM (Delbarre et al., 1994, 1996). ABCB4 is associated with sterol/sphingolipid-enriched detergent-resistant membranes (Borner et al., 2005) and exhibits greater hydrophobicity than ABCB1 and -19 (Titapiwatanakun et al., 2009). Further, the instability of ABCB4 in relatively low concentrations of the polar solvent DMSO suggests that the introduction of water molecules into the lipid bilayer disturbs hydrophobic interactions of the protein. Conformational changes in ABCB4 brought about by ATP hydrolysis may result in more perturbation of the outer membrane leaflet and consequent mobilisation of resident amphipathic molecules such as IAA, NAA and 2,4-D to promote cytosolic trapping of these molecules. The combination of more hydrophobic anchoring and additional IAA binding sites in ABCB4 differentiate this transporter from the ABCB1 or -19 efflux transporters.

Alternatively, ABCB4 transport of a substrate other than auxins or substrate-independent conformational change associated with the first of two ATP hydrolysis steps required for ABC transport function (Knöller and Murphy, 2011) may expose empty IAA binding sites to IAA within the plasma membrane and result in a net import activity when the ‘open’ conformation of the protein is restored (Aller et al., 2009). In this scenario, uptake would cease when increased IAA concentrations in the cytosol and inner leaflet result in IAA-binding sites being occupied in the ‘closed’ conformation of the protein (Dawson and Locher, 2006; Yang and Murphy, 2009) with the result that efflux activity would predominate. Such a model would explain the lack of saturable influx kinetics observed in ABCB4. Substrate activation and conformational change has been shown for other ABC transporters (Loo et al., 2003; Sauna et al., 2008), and apparent drug-induced reversal of mammalian ABCB1 and ABCG22 activity has recently been reported (Shi et al., 2011).

This report also underscores the point that radiotracer assays are highly dependent on the concentrations, amounts and types of auxin applied. The contribution of ABCB4 to physiologically relevant shootward auxin streams from the root apex appears to be relevant only as auxin levels increase, and ABCB4 may play a more important role in controlling auxin movement out of the elongation zone as previously proposed (Peer and Murphy, 2007). However, the localisation of ABCB4 in epidermal cells also suggests that this transporter could modulate the accumulation of microbially-produced auxins at the root surface. It is less likely that this is the function of ABCG36 and -37, as these proteins exhibit very low affinity for IAA (Růžička et al., 2010), the primary auxin produced by bacteria in the rhizosphere (Glick, 1995; Khakipour et al., 2008). It is also possible that ABCB4 modulates interactions with rhizobial communities by eliminating breakdown products or biosynthetic intermediates of indolic glucosinolate defence compounds in plants that produce these compounds.

Finally, ABCB4 appears to be a direct herbicidal target of 2,4-D. Binding of 2,4-D to ABCB4 results in increased accumulation of both 2,4-D and other auxins in root epidermal cells and is likely to amplify the herbicidal effects of the compound including swelling, separation of epidermal and cortical cell layers of the root, and decreased root surface area due to loss of root hairs (Johanson and Muzik, 1961; Calahan and Engel, 1965). As such, tissue-specific manipulation of ABCB4 expression may be a useful tool for decreasing damage to crop plants caused by off-site deposition 2,4-D in agriculture use.

Experimental procedures

Materials

The mutant alleles utilised were aux1-7 (Pickett et al., 1990), pin2/eir1-1 (Luschnig et al., 1998), abcb1/pgp1-1 (Geisler et al., 2005; abcb19/mdr19/pgp19-1 (Noh et al., 2001; Geisler et al., 2003) introgressed into Col-0 with three backcrosses, abcb4/pgp4-1 and Pro35S:PGP4 (Terasaka et al., 2005). Control, ABCB4-GFP and PIN1-GFP BY-2 cells (Nicotiana tabacum L., cv. Bright-Yellow-2) were as described (Nagata et al., 1992; Petrášek et al., 2002; Petrášek et al., 2006; Jelínková and Malínskáet al., 2010). Transgenic BY-2 cells and calli were maintained on media supplemented with 40 μg ml−1 hygromycin or 100 μg ml−1 kanamycin, and 300 μg ml−1 cefotaxim.

The IAA, 2,4-D, 2-NOA, DMSO, IAA-Gluc and IAA-Asp were from Sigma-Aldrich (http://www.sigmaaldrich.com/). 1-Naphthylphthalamic acid (NPA) was from OlChemIm Ltd (http://www.olchemim.cz/). The 3H-IAA (specific radioactivity 20 or 30 Ci mmol−1, 3H-BeA (26 or 20 Ci mmol−1), 3H-2,4-D (20 Ci mmol−1) and 3H-NAA (25 Ci mmol−1) were from American Radiolabeled Chemicals (http://www.arc-inc.com/). The oxIAA was from OlChemIm Ltd. We synthesised 3H-oxIAA and 3H-oxIAA-Gluc by incubating 1 g of 5-day Col-0 seedlings in 5 ml ½ MS medium (pH 5.5) containing 3 μM 3H-IAA for 3 h in low light with mild shaking. The seedlings were collected, frozen and ground in liquid nitrogen and extracted as previously described for IAA determinations (Kim et al., 2007), except that C8 rather than C18 solid phase extraction (SPE) was used. Fractions were separated as described (Ostin et al., 1998). Three primary peak fractions oxIAA-Gluc, oxIAA and IAA (in order of elution) were obtained, identities were confirmed by LC-MS analysis (LCT-Premier; Micromass, http://www.waters.com) and ester hydrolysis, and were used in transport assays.

Root auxin transport assays and determination of root hair length in Arabidopsis

Root transport and uptake assays were performed as described (Blakeslee et al., 2007; Peer and Murphy, 2007 and references therein; see http://www.hort.purdue.edu/hort/research/murphy/The%20more%20than%20complete%20hitchhiker%27s%20guide%20to%20auxin%20transport.pdf). Samples were incubated in chambers fitted with flowthrough gas controllers (ambient or ethylene) under continuous yellow light (40 μE m−2 sec−1). Root hair growth assays were performed using 5-day seedlings grown on ¼ MS (pH 5.5), 0.5% sucrose on 1.3% gellan gum. Root hair length was measured using a Zeiss Stemi 2000C microscope (http://www.zeiss.com/), Luminera Infinity2 camera (http://www.lumenera.com/), and Imagej software (NIH, http://rsbweb.nih.gov/ij/). Ten root hairs were measured in a 2-mm zone starting 1.5 mm above the apex. Only root hairs extending parallel to the surface were measured. Values are means of 10 root hairs per plant from 10 plants. Experiments were repeated three times.

Determinations of IAA, oxIAA, oxIAA-Gluc, and IBA in Arabidopsis tissues

Extraction, SPE preparation and analysis of prepared extracts were as described (Kim et al., 2007) using 13C-IAA as an internal standard, except that C8 SPE (Isolute, http://www.biotage.com) replaced C18 SPE, sample sizes were reduced to 220 μg, and un-derivatised samples were analysed directly by using either an QLCT Premier or Agilent HPLC-MSD/TOF (Agilent, http://www.agilent.com/). Determination of oxIAA, oxIAA-Gluc, IAA-Asp and IAA-Gluc generation in roots after application of 3H-IAA (30 nl, 10 μm, 50 seedling apices) was as described (Ostin et al., 1998). The oxIAA standard for LC-MS and HPLC was from OlChemIm Ltd.

Yeast and BY-2 auxin transport assays and HeLa transport and binding assays

Auxin transport assays in S. pombe were performed as described previously (Yang and Murphy, 2009) with the following modifications. Schizosaccharomyces pombe cells were grown to OD600 ∼ 2.0 in Edinburgh minimal medium (EMM) containing 15 μm thiamine. Then thiamine was removed by washing twice with EMM, and cells were transferred to fresh EMM and incubated for 19 h (final OD600 = 2–3) to induce the expression of proteins. Cells were spun at 6000 g for 30 sec. Pellets were washed once and resuspended in EMM (pH 4.5 for ABCB4/19, and pH 5.5 for AUX1) to OD600 = 2. Cells were kept at 4°C in all the following steps except where noted. The 3H-IAA (specific activity 20 Ci mmol−1; American Radiolabelled Chemicals) was added into 25-μl cells with final concentrations 0.1, 0.25, 0.5, 1 and 2 μm and incubated at 30°C for 5 min (ABCB4) or 8 min (AUX1). Cells were washed twice with EMM (pH 4.5) and resuspended in 250 ml EMM (pH 4.5). Five millilitres of EcoLite® scintillation cocktail was added and retained radioactivity was quantified by scintillation counting. The net transport of 3H-IAA was calculated by subtracting empty vector controls from ABCB4/19 and AUX1. All transport assays were performed with three or four repeats. BY-2 (control, PIN1-GFP, ABCB4-GFP) auxin accumulation assays were as described (Delbarre et al., 1996; Petrášek et al., 2006) with two or more repeats and 10 μm 2-NOA or NPA (in DMSO), 10 μm cold BeA, IAA, 2,4-D or oxIAA (in ethanol) added at 0 or 30 min (pretreatment) as indicated with the radiolabelled assay substrate at 2 nm. For transport assays in HeLa cells, 3H-IAA and unlabelled IAA substrates were co-incubated with the cells for 40 min prior to transport assays as described in Blakeslee et al. (2007). Substrate binding to ABCB4 expressed in HeLa cells was as described (Rojas-Pierce et al., 2007), using 60 nm3H-2,4-D as a substrate.

Molecular docking of ABCB4.  The sequence alignment of ABCBs (GenBank accession no. NP_182223) with Sav1866 (accession Q99T13) were performed in Multalin http://bioinfo.genotoul.fr/multalin/multalin.html (Corpet, 1988) and NCBI bl2seqhttp://blast.ncbi.nlm.nih.gov/Blast.cgi as described in Yang and Murphy (2009). Based on the sequence alignment and the crystal structure of Sav1866, the ABCB4 model was computed with Modeller structural modeling software http://salilab.org/modeller (Sali and Blundell, 1993; Eswar et al., 2000). Computational simulation of ligand binding to ABCB TMDs was performed using MEDock at http://medock.csbb.ntu.edu.tw (Chang et al., 2005).

Microscopy

Arabidopsis thaliana.  Images were collected using a Zeiss LSM 710 confocal laser scanning microscope with a 40× water immersion objective (numerical aperture 1.4, C-Apochromatic) and argon laser (Carl Zeiss) as in Christie et al. (2011).

BY-2 cells.  Cell densities were determined with eight or more aliquots per sample using a Fuchs–Rosenthal haemocytometer on a Zeiss Axiovert 40C. Cell lengths and diameters were observed with a Nikon Eclipse E600 (http://www.nikon.com/), DVC1310C camera (DVC Company, http://www.dvcco.com), and LUCIA image analysis software (Laboratory Imaging, http://www.lim.cz/). For each variant 150 cells were measured on five optical fields. Analysis of fluorescent protein fusions utilised a Zeiss LSM5 DUO (40× C-Apochromat water immersion, numerical aperture 1.2).

Fluorescence recovery after photobleaching (FRAP) analysis.  For bleaching, a 40 × 20 pixel rectangle centred on the transverse membrane was bleached or used as a control, then tracked for fluorescence recovery for 300 sec in 10-sec intervals. Values are means of 25 or more cells.

Acknowledgements

The work was supported by the Ministry of Education, Youth and Sports of the Czech Republic, LC06034 to MK, JP, KH and EZ, the Grant Agency of the Czech Republic, P305/11/0797 to MK, JP, KH and EZ, the Division of Energy Biosciences, US Department of Energy DE-FG02-06ER15804 to ASM. We thank Kateřina Malínská for help with confocal microscopy and Jana Žabová for technical assistance, Ambreen Ahmed for her assistance in setting up root hair measurement assays, and Joshua Blakeslee for helpful comments on the manuscript.

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