Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 2008 Sep;148(1):529–535. doi: 10.1104/pp.108.122044

The Binding of Auxin to the Arabidopsis Auxin Influx Transporter AUX11,[OA]

David J Carrier 1, Norliza Tendot Abu Bakar 1, Ranjan Swarup 1, Richard Callaghan 1, Richard M Napier 1, Malcolm J Bennett 1, Ian D Kerr 1,*
PMCID: PMC2528085  PMID: 18614710

Abstract

The cellular import of the hormone auxin is a fundamental requirement for the generation of auxin gradients that control a multitude of plant developmental processes. The AUX/LAX family of auxin importers, exemplified by AUX1 from Arabidopsis (Arabidopsis thaliana), has been shown to mediate auxin import when expressed heterologously. The quantitative nature of the interaction between AUX1 and its transport substrate indole-3-acetic acid (IAA) is incompletely understood, and we sought to address this in the present investigation. We expressed AUX1 to high levels in a baculovirus expression system and prepared membrane fragments from baculovirus-infected insect cells. These membranes proved suitable for determination of the binding of IAA to AUX1 and enabled us to determine a Kd of 2.6 μm, comparable with estimates for the Km for IAA transport. The efficacy of a number of auxin analogues and auxin transport inhibitors to displace IAA binding from AUX1 has also been determined and can be rationalized in terms of their physiological effects. Determination of the parameters describing the initial interaction between a plant transporter and its hormone ligand provides novel quantitative data for modeling auxin fluxes.

A plethora of plant developmental processes is controlled by auxins, including tropic responses to light and gravity, tissue differentiation, development, and senescence (Delker et al., 2008). The most naturally abundant auxin is indole-3-acetic acid (IAA; Estelle, 1996; Perrot-Rechenmann and Napier, 2005), gradients of which are dependent on the directional import and export of the hormone into and out of cells. The chemiosmotic hypothesis of auxin transport (Rubery and Sheldrake, 1974; Raven, 1975; Goldsmith, 1977) states that the weak organic acid IAA (pKa of 4.8) is taken up by cells by a combination of carrier-mediated uptake (presumably of the IAA− anion) and diffusion of the undissociated lipophilic acid (IAAH). Within the neutral cytoplasm, auxin will exist almost entirely as IAA−, effectively becoming trapped and requiring an IAA− transporter to efflux it from the cell. The coordinated actions of influx and efflux carriers of IAA are necessary to drive polar auxin transport and to generate the hormone gradients that regulate cellular events. Despite their importance, there is still relatively little biochemical characterization of these transport proteins (Estelle, 1996; Friml, 2003; Blakeslee et al., 2005; Kerr and Bennett, 2007).

In recent years the proteins responsible for auxin influx (AUX/LAX) and efflux (PINs and multidrug resistance-type ATP-binding cassette transporters [ABCBs]) have been identified in genetic screens (Palme and Galweiler, 1999; Noh et al., 2001). AUX1, from Arabidopsis (Arabidopsis thaliana; Bennett et al., 1996), represents the paradigm for auxin influx carrier proteins (Kerr and Bennett, 2007), which are represented in all plant species analyzed to date. The protein is believed to act as a proton-auxin symporter and shares a high degree of sequence homology to the amino acid auxin permease family of transporters (Saier, 2000) and indeed may be part of the general amino acid polyamine cation transport family (Jack et al., 2000).

Recent characterization of AUX/LAX (and PIN) family members has shifted from in planta assays to in vitro assays following expression of the proteins in heterologous systems (Petrasek et al., 2006; Yang et al., 2006; Kerr and Bennett, 2007). The most notable achievement of this research has been the demonstration that AUX1 is a high-affinity IAA importer when expressed heterologously in Xenopus oocytes (Yang et al., 2006). Importantly, this research was able to demonstrate that AUX1 is likely to function as a transporter of the anionic form of IAA (IAA−), which coexists with uncharged IAA (IAAH) at the mildly acidic pH (approximately 5.2–5.5) of the apoplast (Yang et al., 2006). The Km for IAA uptake into Xenopus oocytes was determined to be 800 nm, compared to previous estimates obtained in studies of crown gall suspension cells of 1 to 5 μm (Rubery and Sheldrake, 1974).

Understanding the transport of IAA by AUX1 requires quantitative characterization of a multi-step process involving ligand recognition at the extracellular face, protein conformational changes, and ligand release into the cytoplasm. In the current article, we have investigated the initial step of auxin transport, namely the interaction of AUX1 with IAA. Determination of AUX1-IAA interactions has been achieved using a radio-ligand binding assay, having expressed the protein to high levels in a baculovirus-infected insect cell system. We have determined that AUX1 is able to bind IAA and that the affinity (Kd) of this binding is comparable with the Km obtained for transport (Rubery and Sheldrake, 1974; Yang et al., 2006). A range of auxin analogues has also been investigated for their relative affinity for the AUX1 transporter, and these data can be reconciled with in vivo effects of these compounds.

RESULTS

AUX1 Expression in Insect Cells

To investigate the binding of IAA and related auxins and auxin-like compounds to the auxin influx transporter AUX1, we expressed an epitope-tagged version of AUX1 in Spodoptera frugiperda (Sf9) insect cells. The N-terminal tagging of AUX1 with the haemagluttinin (HA) tag (amino acid sequence YPYDVPDY) does not affect the localization or function of the protein, and, indeed, an HA-AUX1 transgene is able to rescue the agravitropic phenotype of an aux1-22 phenotype in plants (Swarup et al., 2001). The presence of the epitope tag enables easy identification of the protein following heterologous expression. Optimum expression conditions were determined (Fig. 1), and, routinely, cells were harvested 72 h postinfection. Comparable expression data in several different prokaryotic and eukaryotic expression systems indicated that the baculovirus expression system was most suited to production of large quantities of HA-AUX1-containing membranes (D.J. Carrier and I.D. Kerr, unpublished data).

Figure 1.

Figure 1.

Open in a new tab

High-level expression of HA-tagged AUX1 in insect cell membranes. Sf9 cells were infected with recombinant HA-AUX1-expressing baculovirus at the indicated multiplicity of infection (MOI) and harvested at the indicated hours postinfection (hpi). Cell lysates (10 μg) were resolved on 10% SDS-PAGE gels, electroblotted, and recombinant AUX1 protein (approximately 40–45 kD) identified by western blotting with anti-HA monoclonal antibodies.

IAA Binds Specifically to AUX1 with Low Micromolar Affinity

Initial attempts to establish a robust binding assay to measure the AUX1-IAA interaction tested the separation of free IAA from bound IAA by rapid filtration through a vacuum manifold. Despite employing numerous different filters (and combining these with protein precipitation methods), we were unable to show linear dependence of nonspecific binding (NSB) on protein load (Fig. 2). Consequently, we tested centrifugation and determined that a brief (5 min) centrifugation (20,000g) of Sf9 membranes was sufficient to pellet in excess of 70% of the protein (data not shown) and give a linear dependence of NSB on both sample size and IAA concentration, affording confidence in the assay's ability to measure specific AUX1-IAA interactions (Fig. 3B).

Figure 2.

Figure 2.

Open in a new tab

Filtration binding assays fail to display linear NSB of IAA to HA-AUX1-containing membranes. [3H]IAA was incubated with increasing quantities of HA-AUX1 membranes for 90 min at room temperature and bound ligand separated from free ligand by rapid vacuum filtration. Filters were washed and nonspecifically bound radioactivity determined as described in “Materials and Methods.”

Figure 3.

Figure 3.

Open in a new tab

Saturation binding of IAA to HA-AUX1. A, The affinity of IAA binding to HA-AUX1 was determined by merging several independent data sets, each of which represents a homologous displacement assay using increasing concentrations of unlabeled IAA to extend the saturation isotherm, as described in “Results.” Fitting of a standard binding isotherm to the data reported a Kd of 2.6 μm. B, The linearity of NSB of [3H]IAA to HA-AUX1 membranes with respect to the amount of protein in the assay (left) and the amount of ligand (right) is displayed.

Auxin binding to AUX1 was not fully saturable within the constraints of the specific activity of the commercially available ligand (supplied at 40 μm in ethanol). Thus, to determine a Kd for IAA binding to AUX1, we effectively carried out a homologous displacement assay (see e.g. Martin et al., 2001), whereby a fixed concentration of [3H]IAA was employed together with increasing concentrations of unlabeled IAA. Correction of the measured binding for this isotope dilution enabled the quantification of the AUX1-IAA interaction at concentrations up to 8 μm IAA. A composite saturation binding isotherm of data obtained from multiple independent experiments, each with triplicate determinations of specific binding, is displayed in Figure 3. Nonlinear regression of the data fitted to a single site saturation isotherm (see “Materials and Methods”) yielded a Kd of 2.6 (±0.30) μm and a maximal binding of 11,800 (±675) fmol IAA/mg membrane protein. Under identical conditions, we determined that specific binding of IAA to Sf9 cell membranes lacking AUX1 was <1% of HA-AUX1 membranes (data not shown), ruling out anion trapping as an explanation for our observations.

To determine a Kd for the AUX1-IAA interaction by kinetic means (i.e. Kd = koff/kon, where koff and kon are the measured rates of dissociation and association, respectively), we investigated the association rate for IAA at 21°C and 2°C (Fig. 4, A and B). We estimate from these data that the half-life for auxin binding to AUX1 is <10 s, and thus the process is essentially complete within the 5 min required to pellet the membranes and separate bound from free ligand, even at 2°C. This rapid association precludes determination of Kd from kon and koff values. By contrast, the dissociation of IAA from HA-AUX1-expressing membranes was slow, even at 21°C, with more than 90% of the specific binding being retained 60 min after resuspension of the membranes in IAA-free buffer, pH 6.0 (data not shown).

Figure 4.

Figure 4.

Open in a new tab

Time dependence of auxin binding. AUX1 membranes were incubated at 21°C (A, black triangles) or 2°C (B, white squares) with 500 nm [3H]IAA for the indicated time intervals. Specific binding was determined as described in “Materials and Methods” and is represented as a percentage of the maximal specific binding observed. Data are fitted to a single-phase exponential isotherm, and the t1/2 values are 0.9 s at 21°C and 2.0 s at 2°C, although, as discussed in the text, these are approximations.

AUX1 Interacts Primarily with IAA between pH 5.0 and 6.0

The pH of IAA-AUX1 interaction was determined over the range 4.5 to 7.5. At each pH, membranes were resuspended in a citrate-phosphate buffer of the desired pH prior to incubation with ligand. The binding showed a clear pH dependence with maximal specific binding occurring between pH 5 and 6 (Fig. 5, solid line, black squares). The reduction in specific binding at pH > 7.0 is discussed below. Within the apoplastic space (pH 5.2), and assuming a pKa of 4.8 for IAA, this is consistent with an interaction with primarily the anionic (IAA−) rather than the protonated (IAAH) form of the hormone (Fig. 5, broken line). This is also consistent with structure-activity analyses of IAA and related compounds that proposed a key stereo-chemical role for the acetic acid group in determination of auxin specificity (Katekar, 1979).

Figure 5.

Figure 5.

Open in a new tab

IAA displays a pH-dependent binding to HA-AUX1-containing membranes. Binding assays were performed as described with a fixed concentration of [3H]IAA (500 nm), with membranes prepared by centrifugation and resuspension in citrate-phosphate buffers at the indicated pH. After 90 min at room temperature, bound IAA was separated from free IAA and the percentage maximal specific binding (left-hand axis) is plotted as a function of pH. The percentage of anionic IAA (right-hand axis) at each pH is displayed as a dotted line.

Auxin Transport Inhibitors Displace IAA Binding from AUX1

The displacement of IAA from AUX1 was investigated with a series of auxin analogues and auxin transport inhibitors. All compounds tested were able to completely displace (>95%) the binding of IAA to AUX1 (Fig. 6). The specificity of AUX1-competitor interactions was substantiated by the failure of a structurally unrelated weak organic acid (benzoic acid) to displace binding in the same concentration range (Fig. 6). By determining full dose-response curves for the displacement of IAA binding to AUX1, we were able to determine IC50 values (i.e. the concentration required to reduce the specific binding of IAA to AUX1 by 50%) for five selected compounds (Fig. 6; Table I). IC50 values represent a measure of the relative affinity of each compound for the IAA binding site on AUX1. The synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), which requires a carrier for uptake into cells (Delbarre et al., 1996), inhibits IAA binding to AUX1 at pH 6.4 (at which value essentially all of the IAA and 2,4-D exist in the anionic form) with an IC50 of 40 μm. The freely membrane-permeable auxin 1-naphthylacetic acid (1-NAA; Delbarre et al., 1996) also inhibits binding of IAA to AUX1 with a comparable IC50 (Fig. 6). The IC50s of the auxin influx inhibitors 1-naphthoxyacetic acid (Parry et al., 2001) and 3-chloro-4-hydroxyphenoxyacetic acid (Parry et al., 2001) are not significantly different from those of 1-NAA and 2,4-D (P > 0.1), suggesting they have comparable affinities for AUX1. In contrast, the auxin influx inhibitor 2-NAA (Parry et al., 2001) has an IC50 of 3.6 μm, suggesting an approximate 10-fold more effective displacement of IAA in our assay conditions.

Figure 6.

Figure 6.

Open in a new tab

IAA binding is displaced by competing auxinic compounds. IAA binding to HA-AUX1-containing membranes was allowed to come to equilibrium in the presence of increasing concentrations of the indicated competing ligands. After 90 min, the specific binding of IAA was determined and plotted as a function of the competing ligand concentration and fitted to the general dose-response curve to determine an IC50 for each compound.

Table I.

Displacement of IAA by related auxin transport inhibitors

*, Denotes IC50 (μm) for 2-NAA is significantly different (P < 0.01) from other values (ANOVA).

2,4-D 1-Naphthoxyacetic Acid 1-NAA 2-NAA 3-Chloro-4-hydroxyphenoxyacetic Acid
IC50 (sd) 39.7 (14.6) 53.6 (22.0) 70.8 (21.3) 3.6* (0.6) 32.4 (11.1)
Open in a new tab

DISCUSSION

The transport of auxin into plant cells occurs down both a proton and auxin concentration gradient (Lomax et al., 1995). Members of the amino acid auxin permease family of membrane transporters (Saier, 2000) are proposed to be the primary auxin influx carriers, in addition to which there is evidence of auxin import facilitated by the ABC transporter AtABCB4 (previously known as AtPgp4; Santelia et al., 2005; Terasaka et al., 2005; Cho et al., 2007; Verrier et al., 2008). Members of the AUX/LAX family of membrane transporters, conserved in all higher plant species, are believed to act as IAA-H+ symporters. In the current investigation, we provide direct determination of the affinity of an auxin importer for its cognate ligand, although a preliminary estimate for the Kd of naphthylphthalamic acid binding to AtABCB1 (an auxin efflux pump previously known as AtMDR1; Verrier et al., 2008) expressed in yeast membranes has been obtained (Noh et al., 2001).

We believe that we are directly measuring IAA binding for three primary reasons. First, we see essentially no specific interaction of IAA with Sf9 membranes isolated from non-AUX1-expressing cells. This suggests that anion trapping is not occurring in our experiments. Consistent with this, our membrane preparations are formed in a low-ionic-strength buffer precluding the formation of vesicles with a sizeable internal volume, which we have confirmed by electron microscopy (data not shown). Finally, we show that IAA displacement is specific to auxin analogues, because benzoic acid is not able to displace IAA binding. Our current data suggest that the initial event in the translocation pathway, namely, the interaction of auxin with its ligand, occurs with a measured Kd of 2.6 μm (Fig. 4), which compares well with the estimated auxin concentration in root tips of 1 μm (Ljung et al., 2001; Ljung et al., 2005). Similarly, our data are comparable to the Km for IAA transport measured in EYFP-AUX1-expressing Xenopus oocytes (0.8 μm; Yang et al., 2006) and with earlier estimates for the Km for auxin transport in plant suspension cell culture experiments (1–5 μm; Rubery and Sheldrake, 1974).

Our pH profile for IAA binding to AUX1 affords further confidence that the function of AUX1 is largely preserved in our heterologous system. The pH optimum for specific binding was observed between pH 5.0 and 6.0, where IAA would be expected to be 60% to 95% in the dissociated state. The reduction in observed specific binding at pH greater than 7.0 is consistent with a reduced interaction with IAA following transport of the hormone to the neutral cytoplasm. It is also tempting to speculate that one or more ionizable residues within AUX1, with pKas close to neutrality, may be key in the transport process, as seen for conserved His residues in the related human proton-coupled amino acid transporter (Metzner et al., 2008).

The role of auxin in plant development has led to the development of numerous synthetic auxin analogues as specific inhibitors of the influx and efflux pathways. In the current study, two synthetic auxins and three auxin influx inhibitors (Imhoff et al., 2000; Parry et al., 2001) were analyzed for their ability to displace IAA binding from AUX1. All were able to completely displace the bound IAA, and the IC50 values for this displacement can be compared with their relative abilities to inhibit radiolabeled 2,4-D uptake into tobacco (Nicotiana tabacum) suspension cells (Delbarre et al., 1996; Imhoff et al., 2000). Notably, our study identifies 2-NAA as having the highest relative affinity of the auxin influx inhibitors analyzed, which parallels the conclusions from suspension cell studies (Delbarre et al., 1996; Imhoff et al., 2000), membrane vesicle experiments (Jacobs and Hertel, 1978; Hertel et al., 1983), and tissue transport data (Sussman and Goldsmith, 1981). There is an approximate 10-fold difference in our measured IC50 values (low micromolar) and the values responsible for inhibition of 2,4-D uptake (usually 10-fold higher; Delbarre et al., 1996; Imhoff et al., 2000). Because IC50 values are strictly assay dependent with variation due to, for example, protein concentration and temperature, direct comparisons of the data are precluded.

The aux1 agravitropic phenotype in plants is to some extent mirrored by an alternative mutation, axr4. Further analysis of this mutation has led to the determination that the AUX1 protein requires an endoplasmic reticulum-localized accessory protein, AXR4, for correct targeting to the plasma membrane (Dharmasiri et al., 2006; Hobbie, 2006). In the current study and in heterologous system transport studies, it has now been determined that the overall Km for auxin transport and the Kd for auxin binding are similar to estimates for transport Km in plant cells (Yang et al., 2006). Because AXR4 homologues are apparently absent in insects (Adams et al., 2000) and amphibians, this reinforces the suggestion that although AUX1 requires AXR4 for targeting in plants, it is not a fundamental requirement for correct function.

The elucidation of the Kd for IAA binding to AUX1, together with the recent determination of the Km for transport (Yang et al., 2006) and the generation of a set of allelic AUX1 isoforms (Swarup et al., 2004) are all important steps toward a full biochemical understanding of the transport cycle of AUX1, which has important implications for our understanding of the generation and maintenance of physiologically relevant auxin gradients (Kramer and Bennett, 2006) and for mathematical modeling of such processes.

MATERIALS AND METHODS

All reagents were of the highest grade and were obtained from Sigma or Fisher. Molecular biology reagents were from New England Biolabs, Invitrogen, or Fermentas. [3H]IAA, specific activity 880 to 980 GBq/mmol, was obtained from GE Healthcare.

Expression of HA-Tagged AUX1 in Sf9 Insect Cells

Spodoptera frugiperda (Sf9) cells were grown as orbital cultures at 27°C to 28°C in InsectXpress medium (Lonza) supplemented with 10% fetal calf serum and 50 units/mL penicillin and streptomycin. AUX1 was expressed as an N-terminally HA-tagged construct, HA-AUX1, in Sf9 cells following viral infection. Recombinant baculoviral DNA (bacmid DNA) was generated using Bac-2-Bac technology (Invitrogen) following the manufacturer's instructions. Bacmid DNA was screened by PCR to ensure integration of the HA-AUX1 cDNA, and recombinant virus was then produced by Cellfectin-mediated transfection of Sf9 cell monolayers. Viral titres were determined and amplified as previously described (King and Possee, 1992). HA-AUX1 expression was routinely induced by infecting Sf9 cells in suspension culture (2.0 × 106/mL) with a multiplicity of infection of 1.0 for 72 h.

Cells were harvested by centrifugation (500g, 5 min, 4°C) and resuspended in approximately 10 times the pellet volume in 10 mm Tris, pH 7.4, 250 mm Suc, 0.2 mm CaCl2 with protease inhibitors (Complete EDTA-free Protease Inhibitor; Roche) then passed twice through a pressure disruptor (Constant Systems) at 5 kpsi. Cellular debris was removed by centrifugation at 300g for 15 min at 4°C, and total cell microsomal membranes were then pelleted by centrifugation at 100,000g for 1 h at 4°C. The membrane pellet was resuspended by shearing 10 to 20 times through a 27.5-G needle, to a protein concentration of 10 to 20 mg/mL in citrate-phosphate buffer, pH 6.0, and aliquoted and frozen at −80°C.

Binding of [3H]IAA to HA-AUX1-Containing Cell Membranes

Saturation and competition radio-ligand binding assays were performed on HA-AUX1 membrane preparations. Membrane proteins (300 μg) were incubated with varying concentrations of [3H]IAA for 90 min at 20°C to 22°C in a total volume of 100 μL in citrate-phosphate buffer, pH 6. Unbound ligand was separated from bound ligand by centrifugation at 20,000g for 5 min, 4°C, and the supernatant removed. The supernatant accounted for over 95% of the radioactivity added, ruling out ligand depletion. The pellet was then washed twice in ice-cold citrate-phosphate buffer, with recentrifugation, and radioactivity associated with the membrane pellet was then determined by liquid scintillation counting. Parallel samples were treated with a large excess (100 μm) of unlabeled IAA to determine the contribution due to NSB. This NSB is due to interactions with plasticware, lipid membranes, and other insect cell proteins, and accounted for approximately 30% of the total binding observed. Competition assays were performed identically, except that [3H]IAA was maintained at a fixed concentration (500 nm) and membranes were co-incubated with increasing concentrations of analog/inhibitor (3 nm–1 mm in semi-log intervals).

Saturation binding data was fitted using nonlinear least squares regression of the equation Inline graphic, where B is the specific binding occurring at an IAA concentration, S. Statistical comparison of the fit of this equation compared to equations representing multiple binding sites was performed with an F-test. In subsequent competition binding experiments, data were fitted to the general dose-response equation of the form Inline graphic, where f is the fractional IAA binding observed in the presence of concentration, S, of a competing compound. The IC50 represents the concentration of competing ligand required to displace 50% of IAA specifically bound to AUX1-containing membranes. All data fitting was performed using GraphPad Prism 4.0, and each data point shown is the result of at least triplicate determinations within each experiment, and multiple, independent membrane preparations were employed.

1

This work was supported by the Biotechnology and Biological Sciences Research Council (grant no. BB/C514958/1 to I.D.K. and M.J.B.), by the Wellcome Trust (equipment grant no. 077212/Z/05/Z to I.D.K.), by the Biomedical Research Committee and the Schools of Biomedical Sciences and Biosciences (all University of Nottingham; to D.C.), and by the Malaysian Government (scholarship to N.A.B.).

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Ian D. Kerr (ian.kerr@nottingham.ac.uk).

[OA]

Open Access articles can be viewed online without a subscription.

References

  1. Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, Scherer SE, Li PW, Hoskins RA, Galle RF, et al (2000) The genome sequence of Drosophila melanogaster. Science 287 2185–2195 [DOI] [PubMed] [Google Scholar]
  2. Bennett MJ, Marchant A, Green HG, May ST, Ward SP, Millner PA, Walker AR, Schulz B, Feldmann KA (1996) Arabidopsis AUX1 gene: a permease-like regulator of root gravitropism. Science 273 948–950 [DOI] [PubMed] [Google Scholar]
  3. Blakeslee JJ, Peer WA, Murphy AS (2005) Auxin transport. Curr Opin Plant Biol 8 494–500 [DOI] [PubMed] [Google Scholar]
  4. Cho M, Lee SH, Cho HT (2007) P-Glycoprotein4 displays auxin efflux transporter like action in Arabidopsis root hair cells and tobacco cells. Plant Cell 19 3930–3943 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Delbarre A, Muller P, Imhoff V, Guern J (1996) Comparison of mechanisms controlling uptake and accumulation of 2,4-dichlorophenoxyacetic acid, naphthalene-1-acetic acid, and indole-3-acetic acid in suspension-cultured cells. Planta 198 532–541 [DOI] [PubMed] [Google Scholar]
  6. Delker C, Raschke A, Quint M (2008) Auxin dynamics: the dazzling complexity of a small molecule's message. Planta 227 929–941 [DOI] [PubMed] [Google Scholar]
  7. Dharmasiri S, Swarup R, Mockaitis K, Dharmasiri N, Singh SK, Kowalchyk M, Marchant A, Mills S, Sandberg G, Bennett MJ, et al (2006) AXR4 is required for localization of the auxin influx facilitator AUX1. Science 312 1218–1220 [DOI] [PubMed] [Google Scholar]
  8. Estelle M (1996) Plant tropisms: the ins and outs of auxin. Curr Biol 6 1589–1591 [DOI] [PubMed] [Google Scholar]
  9. Friml J (2003) Auxin transport: shaping the plant. Curr Opin Plant Biol 6 7–12 [DOI] [PubMed] [Google Scholar]
  10. Goldsmith MHM (1977) The polar transport of auxin. Annu Rev Plant Physiol 28 439–478 [Google Scholar]
  11. Hertel R, Lomax TL, Briggs WR (1983) Auxin transport in membrane vesicles from Curcurbita pepo L. Planta 157 193–201 [DOI] [PubMed] [Google Scholar]
  12. Hobbie L (2006) Auxin and cell polarity: the emergence of AXR4. Trends Plant Sci 11 517–518 [DOI] [PubMed] [Google Scholar]
  13. Imhoff V, Muller P, Guern J, Delbarre A (2000) Inhibitors of the carrier-mediated influx of auxin in suspension-cultured tobacco cells. Planta 210 580–588 [DOI] [PubMed] [Google Scholar]
  14. Jack DL, Paulsen IT, Saier MH (2000) The amino acid/polyamine/organocation (APC) superfamily of transporters specific for amino acids, polyamines and organocations. Microbiology 146 1797–1814 [DOI] [PubMed] [Google Scholar]
  15. Jacobs M, Hertel R (1978) Auxin binding to subcellular fractions from Curcurbita hypocotyls: in vitro evidence for an auxin transport carrier. Planta 142 1–10 [DOI] [PubMed] [Google Scholar]
  16. Katekar GF (1979) Auxins: on the nature of the receptor site and molecular requriements for auxin activity. Phytochemistry 18 223–233 [Google Scholar]
  17. Kerr ID, Bennett MJ (2007) New insight into the biochemical mechanisms regulating auxin transport in plants. Biochem J 401 613–622 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. King L, Possee R (1992) The Baculovirus Expression System, A Laboratory Guide. Chapman, New York
  19. Kramer EM, Bennett MJ (2006) Auxin transport: a field in flux. Trends Plant Sci 11 382–386 [DOI] [PubMed] [Google Scholar]
  20. Ljung K, Bhalerao RP, Sandberg G (2001) Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth. Plant J 28 465–474 [DOI] [PubMed] [Google Scholar]
  21. Ljung K, Hull AK, Celenza J, Yamada M, Estelle M, Normanly J, Sandberg G (2005) Sites and regulation of auxin biosynthesis in Arabidopsis roots. Plant Cell 17 1090–1104 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lomax TL, Muday GK, Rubery PH (1995) Auxin transport. In PJ Davies, ed, Plant Hormones, Ed 2. Kluwer, Amsterdam, pp 509–530
  23. Martin C, Higgins CF, Callaghan R (2001) The vinblastine binding site adopts high- and low-affinity conformations during a transport cycle of P-glycoprotein. Biochemistry 40 15733–15742 [DOI] [PubMed] [Google Scholar]
  24. Metzner L, Natho K, Zebisch K, Dorn M, Bosse-Doenecke E, Ganapathy V, Brandsch M (2008) Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1. Biochim Biophys Acta 1778 1042–1050 [DOI] [PubMed] [Google Scholar]
  25. Noh B, Murphy AS, Spalding EP (2001) Multidrug resistance-like genes of Arabidopsis required for auxin transport and auxin-mediated development. Plant Cell 13 2441–2454 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Palme K, Galweiler L (1999) PIN-pointing the molecular basis of auxin transport. Curr Opin Plant Biol 2 375–381 [DOI] [PubMed] [Google Scholar]
  27. Parry G, Delbarre A, Marchant A, Swarup R, Napier R, Perrot-Rechenmann C, Bennett MJ (2001) Novel auxin transport inhibitors phenocopy the auxin influx carrier mutation aux1. Plant J 25 399–406 [DOI] [PubMed] [Google Scholar]
  28. Perrot-Rechenmann C, Napier RM (2005) Auxins. Vitam Horm 72 203–233 [DOI] [PubMed] [Google Scholar]
  29. Petrasek J, Mravec J, Bouchard R, Blakeslee JJ, Abas M, Seifertova D, Wisniewska J, Tadele Z, Kubes M, Covanova M, et al (2006) PIN proteins perform a rate-limiting function in cellular auxin efflux. Science 312 914–918 [DOI] [PubMed] [Google Scholar]
  30. Raven JA (1975) Transport of idoleacetic acid in plant cells in relation to pH and electrical potential gradients, and its significance for polar IAA transport. New Phytol 74 163–172 [Google Scholar]
  31. Rubery PH, Sheldrake AR (1974) Carrier-mediated auxin transport. Planta 118 101–121 [DOI] [PubMed] [Google Scholar]
  32. Saier MH Jr (2000) Families of transmembrane transporters selective for amino acids and their derivatives. Microbiology 146 1775–1795 [DOI] [PubMed] [Google Scholar]
  33. Santelia D, Vincenzetti V, Azzarello E, Bovet L, Fukao Y, Duchtig P, Mancuso S, Martinoia E, Geisler M (2005) MDR-like ABC transporter AtPGP4 is involved in auxin-mediated lateral root and root hair development. FEBS Lett 579 5399–5406 [DOI] [PubMed] [Google Scholar]
  34. Sussman MR, Goldsmith MHM (1981) Auxin uptake and action of N-1-naphtylphthalamic acid in corn coleoptiles. Planta 150 15–25 [DOI] [PubMed] [Google Scholar]
  35. Swarup R, Friml J, Marchant A, Ljung K, Sandberg G, Palme K, Bennett M (2001) Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex. Genes Dev 15 2648–2653 [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Swarup R, Kargul J, Marchant A, Zadik D, Rahman A, Mills R, Yemm A, May S, Williams L, Millner P, et al (2004) Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1. Plant Cell 16 3069–3083 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Terasaka K, Blakeslee JJ, Titapiwatanakun B, Peer WA, Bandyopadhyay A, Makam SN, Lee OR, Richards EL, Murphy AS, Sato F, et al (2005) PGP4, an ATP binding cassette P-glycoprotein, catalyzes auxin transport in Arabidopsis thaliana roots. Plant Cell 17 2922–2939 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Verrier PJ, Bird D, Burla B, Dassa E, Forestier C, Geisler M, Klein M, Kolukisaoglu U, Lee Y, Martinoia E, et al (2008) Plant ABC proteins: a unified nomenclature and updated inventory. Trends Plant Sci 13 151–159 [DOI] [PubMed] [Google Scholar]
  39. Yang Y, Hammes UZ, Taylor CG, Schachtman DP, Nielsen E (2006) High-affinity auxin transport by the AUX1 influx carrier protein. Curr Biol 16 1123–1127 [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES