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. 2014 Oct 16;166(4):2091–2099. doi: 10.1104/pp.114.250860

Block of ATP-Binding Cassette B19 Ion Channel Activity by 5-Nitro-2-(3-Phenylpropylamino)-Benzoic Acid Impairs Polar Auxin Transport and Root Gravitropism1,[OPEN]

Misuk Cho 1,2,2, Elizabeth M Henry 1,2,3, Daniel R Lewis 1,2, Guosheng Wu 1,2, Gloria K Muday 1,2, Edgar P Spalding 1,2,*
PMCID: PMC4256873  PMID: 25324509

Studies of an auxin transport protein identify an inhibitor of polar auxin transport and auxin-mediated gravitropism.

Abstract

Polar transport of the hormone auxin through tissues and organs depends on membrane proteins, including some B-subgroup members of the ATP-binding cassette (ABC) transporter family. The messenger RNA level of at least one B-subgroup ABCB gene in Arabidopsis (Arabidopsis thaliana), ABCB19, increases upon treatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), possibly to compensate for an inhibitory effect of the drug on ABCB19 activity. Consistent with this hypothesis, NPPB blocked ion channel activity associated with ABCB19 expressed in human embryonic kidney cells as measured by patch-clamp electrophysiology. NPPB inhibited polar auxin transport through Arabidopsis seedling roots similarly to abcb19 mutations. NPPB also inhibited shootward auxin transport, which depends on the related ABCB4 protein. NPPB substantially decreased ABCB4 and ABCB19 protein levels when cycloheximide concomitantly inhibited new protein synthesis, indicating that blockage by NPPB enhances the degradation of ABCB transporters. Impairing the principal auxin transport streams in roots with NPPB caused aberrant patterns of auxin signaling reporters in root apices. Formation of the auxin-signaling gradient across the tips of gravity-stimulated roots, and its developmental consequence (gravitropism), were inhibited by micromolar concentrations of NPPB that did not affect growth rate. These results identify ion channel activity of ABCB19 that is blocked by NPPB, a compound that can now be considered an inhibitor of polar auxin transport with a defined molecular target.

The directed flow of auxin from cell to cell, through tissues and organs, from sites of synthesis to sites of action underlies the coordination of many processes during plant growth and development. Arabidopsis (Arabidopsis thaliana) PIN-FORMED (PIN) genes were the first found to be necessary for the phenomenon known as polar auxin transport (Okada et al., 1991; Chen et al., 1998; Gälweiler et al., 1998). Asymmetric localization of PIN proteins to the downstream ends of each cell in auxin-transporting tissues was correctly suggested to be a molecular component of the efflux mechanisms (Gälweiler et al., 1998) originally hypothesized as necessary for a directionally biased, or polar movement of auxin through tissues (Rubery and Sheldrake, 1974; Raven, 1975; Goldsmith, 1977; Goldsmith et al., 1981). Other members of the eight-gene PIN family in Arabidopsis were subsequently shown to affect auxin distribution in various tissues and stages of development (Křeček et al., 2009).

Shortly after the breakthrough work on PIN1, members of the B subfamily of ATP-binding cassette (ABCB) transporters were discovered to be equally necessary for the phenomenon of polar auxin transport. They were originally called P-GLYCOPROTEIN1 (Dudler and Hertig, 1992; Sidler et al., 1998) and MULTIDRUG RESISTANCE1 (Noh et al., 2001) and ultimately renamed AtABCB1 and AtABCB19, respectively (Verrier et al., 2008). The connection between ABCB transporters and auxin transport was first made through the analysis of Arabidopsis knockout mutants. Polar auxin flow through abcb19 mutant stems is impaired by approximately 80% compared with the wild type and further reduced in abcb1 abcb19 double mutants (Noh et al., 2001). Resultant effects on development include abnormal hypocotyl tropisms (Noh et al., 2003) and the photomorphogenic control of hypocotyl elongation (Wu et al., 2010). Import of indole-3-acetic acid (IAA) to cotyledons through the petiole is reduced by 50% in abcb19 mutants, and this is correlated with an equivalent reduction in cotyledon blade expansion (Lewis et al., 2009). In roots, loss of ABCB19 greatly impairs auxin flow toward the tip without any detectable effect on shootward flow (Lewis et al., 2007). Surprisingly, the only defect detected in abcb19 primary roots associated with this major disruption of auxin transport is greater meandering of the tip during elongation down a vertical agar surface; gravitropism is unaffected (Lewis et al., 2007). Outgrowth of lateral roots, although not their initiation, depends significantly on ABCB19-mediated tipward auxin transport (Wu et al., 2007). The emergence of adventitious roots at the base of hypocotyls from which roots have been excised from Arabidopsis seedlings depends strongly on ABCB19-mediated auxin accumulation at the sites of primordium initiation (Sukumar et al., 2013).

The ABCB19 protein is present predominantly in the central cylinder and cortex of the root, consistent with its role in rootward auxin transport (Lewis et al., 2007; Mravec et al., 2008), whereas the closely related ABCB4 is restricted to the lateral root cap and epidermis (Cho et al., 2007), where it functions in shootward auxin transport (Lewis et al., 2007). Loss of ABCB4 function alters the timing and spatial pattern of gravitropic curvature development, apparently because the gravity-induced auxin gradient across the root is less rapidly dissipated by normal shootward (basipetal) transport of the hormone through the elongation zone (Lewis et al., 2007). Root hairs are significantly longer in abcb4 mutants, a phenotype attributed to auxin accumulation due to impaired efflux (Cho et al., 2007). ABCB4 is reported to conduct auxin influx or efflux, depending on the prevailing external auxin concentration (Kubeš et al., 2012).

Noh et al. (2001) originally isolated ABCB19 in a molecular screen for genes encoding an ion channel activity in Arabidopsis cells shown by patch-clamp electrophysiology to be blocked by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB). The rationale for the screen was that a plant challenged with a channel blocker would overexpress the gene encoding the blocked activity. A hypothesis emerging from the Noh et al. (2001) study is that ABCB19 encodes such an ion channel, which is required for polar auxin transport. If true, NPPB would be established as a blocker of polar auxin transport.

Pharmacological inhibitors, used for decades in auxin transport research, have some advantages over mutations. Mutations can create complicating pleiotropic effects by inhibiting the process throughout development, while inhibitors can be used to impose an effect at a specific time. 1-Naphthylphthalamic acid (NPA) is the most commonly used inhibitor of polar auxin transport (Katekar and Geissler, 1980), but others are being discovered (Rojas-Pierce et al., 2007; Kim et al., 2010; Tsuda et al., 2011). Inhibitors are especially useful when their targets are well defined, which would be the case if NPPB blocked ABCB19 and induced its expression as hypothesized. The experiments reported here were designed to test this hypothesis with electrophysiological measurements of ABCB19 transport activity, radiotracer measurements of polar auxin transport in roots, levels of fluorescently tagged ABCB19 proteins, auxin reporter expression patterns, and machine-vision measurements of a root growth response that depends on auxin redistribution.

RESULTS

ABCB19 Displays Ion Channel Activity When Expressed in Human Embryonic Kidney Cells

To test the hypothesis that ABCB19 functions as an ion channel, cultured HEK cells were transfected with a control vector containing GFP cDNA as a marker or a vector containing the marker and the ABCB19 cDNA in separate open reading frames. Cells displaying GFP fluorescence were subjected to patch-clamp recording in the whole-cell mode. Figure 1, A and B, shows ionic currents recorded across the plasma membrane of HEK cells at a series of applied membrane voltages ranging from −140 to 100 mV in 20-mV steps. Cells expressing ABCB19 displayed substantially greater currents. Figure 1C shows the steady-state current-voltage relationship for the control and ABCB19-expressing cells. Both inward and outward currents were enhanced by the presence of ABCB19, but the experiments were not designed to determine which ions in the auxin-free solutions were being transported. Although these experiments indicate that ABCB19 possesses ion transport activity, it is always a concern that the appearance of new currents in a heterologous system is due to a secondary effect of a foreign protein on an endogenous transporter. A rigorous test of this caveat is to determine the effect of rendering the protein nonfunctional by a point mutation. An abcb19 allele identified by the Targeting Induced Local Lesions in Genomes process (Till et al., 2006) resulted in an Asp-to-Asn change at position 1,173, near the second ATP-binding domain. This allele, abcb19-6, is phenotypically very similar to a well-studied knockout allele, abcb19-3 (Fig. 1D). Therefore, the abcb19-6 mutation was used to control for potential secondary effects of a large, foreign membrane protein on HEK cell channels. Currents recorded from HEK cells expressing the ABCB19D1173N cDNA did not differ from control cell currents (Fig. 1E), indicating that the mutation abolished function. Additional evidence that currents associated with ABCB19 expression reflect bona fide ABCB19 ion transport activity is found in the effect of coexpressing the immunophilin-like protein TWISTED DWARF1, which has been shown to bind to ABCB19. Figure 1F shows that TWD1 suppressed ABCB19 ion current activity, possibly indicating that its presumed chaperoning function includes maintaining a low level of ABCB19 function during maturation and trafficking of the transporter. Regardless of how relevant the observed suppression is to the natural function of TWD1, it is consistent with the channel activity being an intrinsic function of ABCB19 rather than a secondary effect on HEK cell functions.

Figure 1.

Figure 1.

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Ion channel activity of ABCB19 expressed in human embryonic kidney (HEK) cells. A and B, Ionic currents across the whole cell membrane of a HEK cell transfected with a vector carrying only the GFP marker complementary DNA (cDNA; A) or carrying GFP and ABCB19 cDNA (B). Membrane potential was successively clamped at −140 to 100 mV in 20-mV increments. C, Whole-cell current (I) versus voltage (V) curves show the average membrane current associated with ABCB19 expression (n = 10) compared with the control (n = 10). D, The abcb19-6 allele encodes Asn at position 1,173 instead of Asp (ABCB19D1173N), but with respect to cotyledon epinasty and hypocotyl length in 5-d-old seedlings, this single-site mutant is phenotypically similar to the abcb19-3 knockout allele. WT, Wild type. E, When expressed in HEK cells, ABCB19D1173N does not produce new ionic currents (n = 6) compared with the control (n = 5). F, Coexpression of the TWD1 and ABCB19 proteins (n = 7) suppressed the ABCB19 ionic currents (n = 10) to the level of the controls (n = 10). Each I-V curve is the mean ± se of the indicated number of independent experiments each performed on a different cell.

NPPB But Not NPA Inhibits ABCB19 Channel Activity

The electrophysiological assay thus established was used to test the hypothesis that NPPB is an inhibitor of ABCB19 function. Figure 2A shows that 20 µm NPPB, a concentration that maximally blocks an Arabidopsis anion channel (Cho and Spalding, 1996; Noh and Spalding, 1998), completely blocked the ABCB19-associated ionic currents, confirming the hypothesis. The most widely used inhibitor of polar auxin transport in plants is NPA. Figure 2B shows that 20 µm NPA did not affect ABCB19 ion channel activity. The experiment in Figure 2B was designed to address an additional important point: whether transport of the auxin anion (IAA−) by ABCB19 could be detected. In these experiments, a 300-fold gradient in IAA was imposed across the plasma membrane by including 30 mm of the sodium salt of IAA in the pipette and 0.1 mm in the bath solution. If the ABCB19 channel was significantly permeable to IAA− relative to other ions present in the electrolytes, the reversal voltage of the current-voltage (I-V) curve (the membrane potential at which no current flows) would have a positive value. Figure 2B shows that the reversal voltage was near zero, not different from values obtained in the absence of auxin (Figs. 1, C, E, and F, and 2A). Thus, no evidence of IAA− transport by ABCB19 was obtained with this experiment.

Figure 2.

Figure 2.

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Effects of NPPB and NPA on the ion channel activity of ABCB19. A, The channel blocker NPPB completely blocked ABCB19-dependent ionic currents (n = 14 or 15). B, The polar auxin transport inhibitor NPA did not affect ABCB19-dependent ionic currents (n = 7–10). Both inhibitors were present at 20 µm. Each I-V curve is the mean ± se of the indicated number of independent experiments each performed on a different cell.

NPPB Inhibits Polar Auxin Transport

The channel-blocking effect of NPPB created an important opportunity to test the relationship between ABCB19 electrogenic activity and polar auxin transport. If the channel activity is causally related to the phenomenon of polar auxin transport, NPPB should block the latter as it does the former. A direct test based on the movement of radioactivity provided as [3H]IAA gave unequivocal results. Figure 3A shows that 20 µm NPPB inhibited rootward, or acropetal, auxin flow almost as severely as an abcb19 null mutation, or approximately 60%, although a statistically significant difference remained between the two (P = 0.05). NPPB did not affect the amount of transport persisting in the mutant, the non-ABCB19 component. NPPB also inhibited transport in the opposite direction (shootward), which depends on ABCB4 (Lewis et al., 2007) and possibly different ABCB family members, by approximately 80% (Fig. 3B). The inhibition of polar auxin transport by NPPB demonstrated here is as strong as the effect of NPA demonstrated in numerous other studies. Thus, NPPB and NPA inhibit polar auxin transport by different mechanisms, and ion channel activity is associated with the mechanism by which ABCB19 controls directional flow of the hormone from cell to cell through tissues.

Figure 3.

Figure 3.

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NPPB blocks polar auxin transport in roots. A, NPPB blocks the entire ABCB19-dependent component of rootward or acropetal auxin transport measured by a radioactive IAA tracer method in intact seedlings. B, NPPB strongly inhibits shootward or basipetal auxin transport through roots, which depends on ABCB transporters other than ABCB19. Six seedlings were used in each of three independent trials for each treatment. Values shown are means ± se. The mutant allele used was abcb19-3.

NPPB Promotes the Turnover of ABCB19:GFP Fusion Proteins

Stably transformed plants expressing fully functional GFP-tagged ABCB4 and ABCB19 proteins in their respective transfer DNA insertion mutant backgrounds (Lewis et al., 2007) were used to determine the effect of NPPB on ABCB protein accumulation. Examining the root meristem/elongation zone area, in the epidermis where ABCB4 is expressed and in the cortex for ABCB19, showed that the protein levels were not higher after NPPB treatment (Fig. 4), contrary to what could be expected given the positive effect of NPPB on ABCB19 mRNA level (Noh et al., 2001), which was hypothesized to compensate for blocked activity. The hypothesis was modified to include the degradation of NPPB-blocked ABCB proteins and tested by cotreating roots with NPPB and cycloheximide (CHX) to inhibit protein synthesis. Figure 4 shows that cotreatment reduced ABCB4 and ABCB19 protein levels to a greater extent than either single treatment. A reasonable explanation is that NPPB-inhibited ABCB proteins are degraded. When new protein synthesis is concomitantly inhibited, this NPPB-dependent degradation is detectable.

Figure 4.

Figure 4.

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NPPB reduces the levels of ABCB4 and ABCB19 proteins when new protein synthesis is inhibited by CHX. A, Confocal microscopy images of Arabidopsis root apices expressing GFP-tagged ABCB4 or ABCB19 acquired after a brief treatment with NPPB, CHX, or both inhibitors together. B, Quantification of the GFP fluorescence signal in images relative to the average signal intensity of controls, which received only the solvent used for the inhibitors. The values plotted are means ± se of between 17 and 22 independent measurements. NPPB + CHX reduced ABCB protein levels significantly more than CHX alone: **P = 0.01 and *P = 0.05

NPPB Impairs Gravity-Stimulated Auxin Redistribution

Inhibition of at least two principal auxin transport streams by NPPB blockage of ABCB transporters would be expected to have a significant impact on auxin distribution and growth responses such as gravitropism that depend on auxin redistribution (Spalding, 2013). This expectation was tested by pretreating wild-type seedlings expressing the ProDR5:GFP or ProDR5:GUS auxin reporter with NPPB for 2 h or with only the solvent as a control followed by 90° reorientation to initiate the gravitropic response. In control seedlings, GFP signal indicative of auxin levels was clearly high along the lower flank of the root, compared with the upper flank, 5 h after reorientation (Fig. 5A). In seedlings pretreated with 10 μm NPPB, asymmetry in the auxin reporter signal between the upper and lower flanks of the root was less apparent. In roots pretreated with 20 μm NPPB, no auxin reporter asymmetry was detectable. Instead, a diffuse spread of the reporter signal was observed equally on the upper and lower flanks of the root. The ProDR5:GUS auxin reporter, which requires histochemical staining to observe, produced a similar result (Fig. 5B). These data demonstrate that short-term treatment with NPPB disrupts the auxin distribution mechanism, preventing the creation of lateral asymmetry induced by gravity.

Figure 5.

Figure 5.

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NPPB alters the distribution of auxin signaling in the apices of gravistimulated roots, as indicated by DR5 promoter-based reporters. A, Confocal microscope images of root apices expressing ProDR5:GFP treated with the indicated concentrations of NPPB or only the DMSO solvent for the control. The top row shows the GFP fluorescence channel, and the bottom row shows the GFP signal superimposed on the transmitted light image of the same root. Note how NPPB affects the auxin signal differential across the root, evident in the control. B, Bright-field microscope images of root apices expressing ProDR5:GUS taken after histochemical staining to show representative effects of NPPB on auxin signaling distribution. The displayed images are typical of at least 10 independent observations.

NPPB Impairs Gravitropism

It follows from the results in Figure 5 that NPPB should impair gravitropism, a differential growth response that results from the asymmetric auxin accumulation across the root. An automated image acquisition and analysis platform for measuring gravitropism with high spatiotemporal resolution (Durham Brooks et al., 2010) was employed to quantify the effects of NPPB on root tip angle and growth rate. Figure 6A shows that seedlings pretreated for 2 h with 5 µm NPPB responded slightly slower to a 90° reorientation than seedlings pretreated only with the equivalent amount of solvent. Growth rate was constant during the bending response and essentially unaffected by the NPPB treatment. Increasing the NPPB concentration to 10 μm greatly slowed the development of gravitropic bending while only slightly affecting root elongation rate (Fig. 6B). The control root tips had achieved an angle of 90° by 4 h, whereas seedlings treated with 10 μm NPPB had achieved a root tip angle of only 48° by this time. Impaired root gravitropism is an expected result of the reduced auxin differential in gravistimulated roots caused by NPPB blockage of ABCB channel function.

Figure 6.

Figure 6.

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NPPB slows root gravitropism more than growth rate. A, Average root tip angle versus time measured by automated image analysis at 2-min intervals over 8 h on agar plates containing 5 µm NPPB or the equivalent amount of DMSO solvent. At time 0, seedlings were rotated 90° to a horizontal position (0°) to initiate gravitropism. B, Same as in A, except that the NPPB concentration was 10 µm NPPB, requiring twice as much solvent for the control. C, Average elongation rate of the same roots used to produce the data in A. D, Average elongation rate of the same roots used to produce the data in B. se values at each time point, typically 8% to 12%, were omitted for clarity. The number of independent trials and results of Student’s t test of significant differences in average growth rate over the time course are shown.

DISCUSSION

Chemical inhibitors have long been used to learn about the physiological and developmental functions of their targets. Molecular-level understanding of phenomena in neuroscience such as synaptic transmission, for example, quickly followed from findings enabled by pharmacological agents such as ion channel blockers (Lodge, 2009). In the case of polar auxin transport, NPA has been the most commonly used inhibitor for decades (Katekar and Geissler, 1980). Its structure is shown in Figure 7. NPA is considered a reliably specific inhibitor of polar auxin transport when used in the micromolar concentration range (Lomax et al., 1995), but its molecular target(s) require clarification. Most studies that addressed the question of its targets conclude that NPA inhibits auxin transport mediated by both PIN and ABCB transporters to similar degrees (Bouchard et al., 2006; Petrásek et al., 2006; Blakeslee et al., 2007; Rojas-Pierce et al., 2007; Yang and Murphy, 2009), although at least one report concludes that ABCB transporters are the primary targets of NPA (Kim et al., 2010). The results presented in Figure 2 indicate that NPA does not affect the ion channel activity of ABCB19. If ABCB19 is a target of NPA, its endogenous channel activity is not the inhibited property. This conclusion is consistent with the suggestion that NPA exerts its effects on polar auxin transport at least in part by interfering with interactions between ABCB proteins and regulator proteins (Bailly et al., 2008; Kim et al., 2010), which may not be present in HEK cells.

Figure 7.

Figure 7.

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Chemical structures of four compounds that block polar auxin transport. BUM, 2-[4-(Diethylamino)-2-hydroxybenzoyl]benzoic acid; Bz-IAA, 5-benzyloxy-indole-3-acetic acid; Bz-NAA, 7-benzyloxy-naphthalene-1-acetic acid.

This article establishes NPPB, the structure of which is shown in Figure 7, as an effective blocker of polar auxin transport (Fig. 3). Importantly, the results in Figure 2A give a mechanistic explanation of the macroscopic effect, namely that NPPB blocks an ion channel activity of ABCB19. Marketed commercially as an anion channel blocker and shown to act as such in Arabidopsis (Cho and Spalding, 1996; Noh and Spalding, 1998), NPPB may block ABCB-encoded anion channel activity required for polar auxin transport. The anion naturally transported by ABCB19 may be the auxin anion itself, because thermodynamic considerations favor channel-mediated efflux of IAA− being central to the polar auxin transport phenomenon (Goldsmith, 1977; Spalding, 2013), but the data in Figure 2B argue against this being the case. Imposition of a large IAA− concentration gradient across the membrane did not result in a measurable shift in the reversal voltage of the I-V curve. Although these experiments do not conclusively rule out IAA− permeation of the ABCB19 channel, they do not provide support for it.

If PIN channels are the principal release pathway for the auxin anion, an indirect but necessary effect of ABCB channel activity may explain the results. ABCB channels may be required to conduct the countercurrent necessary to satisfy Kirchoff’s first law, which states that the algebraic sum of all electric currents at a node must equal zero. This law pertains to ionic currents flowing across cellular membranes as well as wired circuits. As a rough mechanical analogy, consider how opening a second hole in a liquid-filled vessel facilitates outflow by allowing air to replace the vacating liquid. ABCB proteins may provide the holes that conduct an offsetting current required for PIN-mediated auxin efflux, unless blocked by NPPB.

If NPPB is a blocker of ABCB channel activity, its effect on ABCB19 mRNA levels (Noh et al., 2001) would be explained, based on the data in Figure 4, as a futile compensatory response to the chronic block and subsequent removal of ABCB protein due to continuously present NPPB. In this scenario, NPPB could be a useful tool for investigating the potential regulation of ABCB proteins by dynamic intracellular trafficking to and retrieval from the plasma membrane, which is known to be an important aspect of PIN regulation (Kleine-Vehn and Friml, 2008).

A chemical named gravicin, 3-(5-[3,4-dichlorophenyl]-2-furyl)acrylic acid, may act similarly to NPPB. Gravicin was discovered through a chemical screen for inhibitors of gravitropism (Surpin et al., 2005). ABCB19 is considered a target of gravicin because gravicin enhances auxin retention in cells expressing ABCB19, and a genetic screen for gravicin-resistant Arabidopsis seedlings isolated a point mutation in the ABCB19 gene that reduced gravicin binding (Rojas-Pierce et al., 2007). Another chemical that may act similarly to NPPB is 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid. It blocks polar auxin transport, binds to at least ABCB1, and interferes with ABCB1-TWD1 interactions (Kim et al., 2010). The structures of 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid and IAA derivatives that also block auxin transport at least in part by interfering with ABCB transport function (Tsuda et al., 2011) are shown in Figure 7.

The transport activity and pharmacological sensitivity of an ABCB transporter required for auxin transport has now been studied using the patch-clamp technique. The approach established NPPB as a blocker of ABCB19 activity, and the prediction that it would thereby impair polar auxin transport was borne out. If the same can be achieved with PIN proteins and other inhibitory compounds, pharmacological tools for dissecting auxin transport mechanisms and effects on development may be used with more certainty and precision.

MATERIALS AND METHODS

HEK Cell Culture, ABCB19 Expression, and Electrophysiology

HEK 293T cells were cultured in dishes on coverslips and transfected as described (Vincill et al., 2012). ABCB19 cDNA was amplified from cDNA template using primers 5′-ACAAGTCGACATGTCGGAAACTAACACAAC-3′ and 5′-ACTGCCCGGGTCAAATCCTATGTGTTTG-3′, then inserted into the SalI and XmaI sites (underlined sequences) of the pIRES-Enhanced Green Fluorescent Protein (EGFP) bicistronic vector used by Vincill et al. (2012) such that a single mRNA would separately code ABCB19 and EGFP. To produce the G-to-A mutation at +3,517 that generates the ABCB19D1173N allele, one-step reverse transcription-PCR was performed on mRNA isolated from abcb19-6 mutant seedlings (see below) to obtain a portion of the ABCB19 gene (+3,101 from ATG to +3,759) using primers 5′-CTCAGAATTCGAGCTGGACATAGCCAA-3′ and 5′-ACTGCCCGGGTCAAATCCTATGTGTTTG-3′. This region was subcloned into the EcoRI and XmaI sites. The +1 to 3,106 region of ABCB19 was amplified from wild-type ABCB19 cDNA using primers 5′-ACAAGTCGACATGTCGGAAACTAACACAAC-3′ and 5′-GCTCGAATTCTGAGGTTAAAGTCCCT-3′ and inserted into the XhoI and EcoRI sites to produce the mutant (D1173N) ABCB19 construct. For control experiments, the transfection vector did not contain the ABCB19 cDNA. For ABCB19 and TWD1 coexpression, Enhanced Yellow Fluorescent Protein (EYFP) was inserted in front of the ABCB19 cDNA in the pIRES-B19-EGFP bicistronic vector to create a translational fusion using primers 5′-ATCCGAGCTCACCATGGTGAGCAAG-3′ and 5′-AGTAGTCGACCTTGTACAGCTCGTCC-3′ (pIRES-EYFP-B19-EGFP). The TWD1 gene was cloned using primers 5′-TATGGCCACAACCATGGATGGATGAATCTCTGGAGCATC-3′ and 5′-TCGATCTAGATTAATCTGCTTTAACTCTG-3′ and replaced EGFP to create pIRES-EYFP-B19-TWD1. Results labeled ABCB19 in Figure 1F refer to the EYFP-ABCB19 fusion protein.

A coverslip with cells was placed in a recording chamber mounted on the fixed stage of an upright fluorescence microscope (Olympus BX51WI) mounted on an antivibration table equipped with a micromanipulator that controlled the head stage of the patch-clamp amplifier (Axopatch 200A; Molecular Devices; www.moleculardevices.com). A 40× dipping objective lens was used to view the cells in bright-field or fluorescence mode in the chamber, which was being continuously perfused with a bath solution containing 140 mm NaCl, 2 mm CaCl2, 2 mm MgCl2, 5 mm KCl, and 10 mm HEPES, adjusted to pH 6 with NaOH. The pipette was filled with 140 mm CsCl, 1 mm CaCl2, 2 mm MgCl2, 5 mm EGTA, 10 mm d-Glc, 10 mm HEPES, and 3 mm Mg-ATP, adjusted to pH 7.2 with CsOH. When 30 mm sodium IAA (Sigma-Aldrich) was added to the pipette solution, the concentration of CsCl was reduced to 110 mm. Cells displaying strong EGFP fluorescence, or EYFP fluorescence in the case of Figure 1F, were selected for whole-cell patch-clamp analysis using micropipettes pulled from borosilicate glass. Micropipette resistance was between 5 and 8 MΩ when filled. After achieving a GΩ seal, the patch was ruptured to obtain the whole-cell configuration. After the baseline current stabilized, a voltage clamp protocol was administered by pCLAMP 10.2 software (Molecular Devices). The measured membrane currents were low-pass filtered at 5 kHz and digitized at 10 kHz using a Digidata 1440A device (Molecular Devices). Data analysis was performed with Clampfit 10.2 (Molecular Devices) software.

Plant Materials and Growth Conditions

The Columbia-0 ecotype of Arabidopsis (Arabidopsis thaliana) was the wild type used in this study, and the genetic backgrounds of the mutant and transgenic lines employed were as follows: abcb19-3, a null phenotype transfer DNA insertion allele (Lewis et al., 2007); ProABCB19:GFP-ABCB19, an N-terminal GFP translational fusion expressed under the control of the ABCB19 promoter in the abcb19 mutant background, which it rescues (Lewis et al., 2007; Wu et al., 2007); and ProABCB4:ABCB4-GFP, a C-terminal GFP translational fusion expressed under the control of the ABCB4 promoter (Cho et al., 2007). The abcb19-6 allele, a D1173N substitution caused by a G-to-A mutation at position +3,517, was isolated using the Targeting Induced Local Lesions in Genomes method (Till et al., 2006). The mutant line was backcrossed to the Columbia-0 wild type four times to remove extraneous mutations, and a homozygous line, verified by DNA sequencing using the primer 5′-TGCTGGAGACACAGCTAAGGCTC-3′, was isolated.

Seeds were sown on the surface of petri plates containing 0.8% phytoagar supplemented with one-half-strength Murashige and Skoog (MS) medium containing 2.15 g L−1 MS nutrient mix (Sigma-Aldrich), 1% (w/v) Suc, and 0.5 g L−1 MES, adjusted to pH 5.7 with KOH, or a simple medium consisting of 1 mm KCl, 1 mm CaCl2, 5 mm MES, and 1% (w/v) agar, adjusted to pH 5.7 with BisTris propane. Plates containing seeds were maintained at 4°C for at least 2 d. After this stratification treatment, plates were placed vertically at 23°C under a 16-h-light/8-h-dark photoperiod.

Observation of Reporter Genes and the Effects of Inhibitors

To observe and measure reporter gene signals and their spatial patterns, seedlings of wild-type, ProDR5:GUS, ProDR5:GFP, ProABCB19:GFP-ABCB19, and ProABCB4:ABCB4-GFP transgenic plants were grown on one-half-strength MS agar plates for 4 d. Inhibitors or only the amount of dimethyl sulfoxide (DMSO) used to dissolve them (0.5% maximum) were applied by transferring seedlings to fresh agar petri plates containing the treatments. For tests of the effects of 10 μm NPPB and/or 50 μm CHX on GFP-tagged ABCB4 and ABCB19 protein levels (Fig. 4), the seedlings remained on the treatment plates for 5 h prior to examination by confocal laser scanning microscope using a Zeiss LSM 510 set to excite GFP fluorescence with a 488-nm argon laser line and collect 505- to 530-nm emission. To quantify the GFP signal levels from the resulting digital images, the mean value of GFP fluorescence intensity within the entire field of view was determined with ImageJ software, essentially as described in a previous study of ABCB19 protein changes during photomorphogenesis (Wu et al., 2010). To test the effect of NPPB on the gravity-induced distribution of ProDR5:GFP reporter signal in Figure 4, seedlings were placed on treatment plates for 2 h prior to rotating them by 90° to initiate gravitropism. After 4 h, the ProDR5:GFP signal pattern was examined by confocal microscopy using the equipment and parameters described above. To determine the effects of NPPB on the ProDR5:GUS auxin reporter signal, roots were transferred to treatment plates as explained for 2 h, rotated to initiate gravitropism, and collected for histochemical staining 5 h later.

Polar Auxin Transport Measurements

IAA transport was measured as described (Lewis and Muday, 2009) using 100 nm [3H]IAA (American Radiolabeled Chemicals; 26 Ci mmol−1). For acropetal or rootward assays, a radioactive auxin-containing agar droplet was applied at the root-shoot junction, and after 18 h, the apical 5 mm of root tips (at least 7 mm from the site of application) was harvested and radioactivity was quantified by scintillation counting. For basipetal or shootward IAA transport assays, the agar droplet was applied to the root tip, and 5-mm root sections that were 2 mm from the site of application were assayed for radioactivity 5 h later.

Gravitropism and Growth Rate

Seedlings for these assays were grown on petri plates containing the simple media described above. Digital images were automatically collected every 2 min for 8 h using a bank of CCD cameras and infrared backlighting as described previously (Miller et al., 2007). The image files were automatically analyzed to calculate root tip angle time courses as described previously (Durham Brooks et al., 2010). Root growth rate time courses were calculated by differentiating the lengths of the extracted midlines with respect to time, then smoothing the curve with the method of Savitzky and Golay (1964) as implemented in the Origin software package (http://www.originlab.com/) using a window size of 20. To determine the effects of NPPB on the time course of gravitropism and growth rate, seedlings were transferred to plates containing the indicated amount of NPPB or only the same amount of DMSO solvent for control treatments 2 h before the agar plates were rotated to induce gravitropism.

The Arabidopsis Genome Initiative locus identifier for the gene product studied in this article is At3G28860 (ABCB19).

Acknowledgments

We thank Dr. Eric Vincill for assistance with the whole-cell patch-clamp analysis.

Glossary

IAA

indole-3-acetic acid

NPPB

5-nitro-2-(3-phenylpropylamino)-benzoic acid

NPA

1-naphthylphthalamic acid

HEK

human embryonic kidney

cDNA

complementary DNA

I-V

current-voltage

CHX

cycloheximide

MS

Murashige and Skoog

DMSO

dimethyl sulfoxide

Footnotes

1

This work was supported by the National Research Foundation of Korea (grant no. NRF–2009–352–C00130 to M.C.) and the National Science Foundation (grant nos. IOS–0921071 and IOS–1360751 to E.P.S.).

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