Research ArticleResearch Article

PGP4, an ATP Binding Cassette P-Glycoprotein, Catalyzes Auxin Transport in Arabidopsis thaliana Roots

Kazuyoshi Terasaka, Joshua J. Blakeslee, Boosaree Titapiwatanakun, Wendy A. Peer, Anindita Bandyopadhyay, Srinivas N. Makam, Ok Ran Lee, Elizabeth L. Richards, Angus S. Murphy, Fumihiko Sato, Kazufumi Yazaki

Published November 2005. DOI: https://doi.org/10.1105/tpc.105.035816

Abstract

Members of the ABC (for ATP binding cassette) superfamily of integral membrane transporters function in cellular detoxification, cell-to-cell signaling, and channel regulation. More recently, members of the multidrug resistance P-glycoprotein (MDR/PGP) subfamily of ABC transporters have been shown to function in the transport of the phytohormone auxin in both monocots and dicots. Here, we report that the Arabidopsis thaliana MDR/PGP PGP4 functions in the basipetal redirection of auxin from the root tip. Reporter gene studies showed that PGP4 was strongly expressed in root cap and epidermal cells. PGP4 exhibits apolar plasma membrane localization in the root cap and polar localization in tissues above. Root gravitropic bending and elongation as well as lateral root formation were reduced in pgp4 mutants compared with the wild type. pgp4 exhibited reduced basipetal auxin transport in roots and a small decrease in shoot-to-root transport consistent with a partial loss of the redirective auxin sink in the root. Seedlings overexpressing PGP4 exhibited increased shoot-to-root auxin transport. Heterologous expression of PGP4 in mammalian cells resulted in 1-N-naphthylthalamic acid–reversible net uptake of [3H]indole-3-acetic acid. These results indicate that PGP4 functions primarily in the uptake of redirected or newly synthesized auxin in epidermal root cells.

INTRODUCTION

Members of the ATP binding cassette (ABC) transporter superfamily have been identified in all prokaryotic and eukaryotic organisms (Henikoff et al., 1997). Although ABC transporters are often associated with the export of cytotoxic compounds (Gottesman and Pastan, 1993), they also function in mating hormone transport (Ketchum et al., 2001), ATP/ADP sensing (Bryan and Aguilar-Bryan, 1999), channel regulation (Anderson et al., 1991), and iron homeostasis (Kushnir et al., 2001). ABC transporters have also been shown to mediate the cellular uptake of urea (Beckers et al., 2004), iron (Brown et al., 2002; Danese et al., 2004), small peptides (Lamarque et al., 2004), choline (Dupont et al., 2004), and bilirubin (Ieiri et al., 2004).

In contrast with other organisms, a broad proliferation of ABC transporters has occurred in plants (Sanchez-Fernandez et al., 2001; Martinoia et al., 2002; Jasinski et al., 2003; Garcia et al., 2004). Members of the multidrug resistance–associated protein (MRP) subfamily involved in the vacuolar sequestration of toxins or xenobiotics are the best characterized (Martinoia et al., 1993; Rea et al., 1998; Rea, 1999; Theodoulou, 2000). MRP1, MRP2, and MRP3 transport glutathione conjugates of endogenous and artificial substrates into vacuoles (Lu et al., 1997, 1998; Tommasini et al., 1998), and MRP4 and MRP5 have been implicated in the complex regulation of stomatal aperture and guard cell ion flux (Gaedeke et al., 2001; Klein et al., 2003, 2004). Members of the pleiotropic drug resistance protein (PDR) subfamily, such as PDR1 from Nicotiana plumbaginifolia (Jasinski et al., 2001; Stukkens et al., 2005) and TURION2 from Spirodela polyrrhiza (van den Brule et al., 2002), export antifungal diterpene defense compounds to the leaf surface, and a half-size ABC protein, ECERIFERUM5, is responsible for wax export to the plant cuticle (Pighin et al., 2004).

Although first identified in an unsuccessful attempt to enhance herbicide resistance (Sidler et al., 1998), the majority of plant multidrug resistance P-glycoproteins (MDR/PGPs; hereafter referred to as PGPs) characterized to date have been implicated in the transport of the phytohormone auxin: PGP1 and PGP19 were identified as the principal Arabidopsis thaliana plasma membrane (PM) proteins binding the auxin efflux inhibitor 1-N-naphthylthalamic acid (NPA) with high affinity (Murphy and Taiz, 1999a, 1999b; Noh et al., 2001; Murphy et al., 2002). Loss of PGP19 and PGP1 function results in reduced auxin transport and growth defects of varying severity in Arabidopsis (pgp1, pgp19/mdr1), maize (Zea mays; brachytic2), and sorghum (Sorghum bicolor; dwarf3) (Noh et al., 2001; Geisler et al., 2003, 2005; Multani et al., 2003). Auxin transport reduction and dwarf phenotypes are more exaggerated in Arabidopsis pgp1 pgp19 double mutants, suggesting overlapping function (Geisler et al., 2003, 2005). Recently, PGP1 and PGP19 have been shown to mediate the ATP-dependent cellular efflux of natural and synthetic auxins as well as oxidative auxin breakdown products in Arabidopsis protoplasts and whole plants (Geisler et al., 2003, 2005). Furthermore, heterologous expression of PGP1 in yeast and mammalian cells resulted in increased auxin efflux (Geisler et al., 2005). Although all of the evidence reported to date indicates a role for PGPs in auxin efflux, structural differences observed among the 21 expressed members of the PGP family of Arabidopsis (Jasinski et al., 2003; E.L. Richards and A.S. Murphy, unpublished data) suggest that energy-dependent PGP mediation of active auxin uptake is also possible.

Previously, we identified a PM PGP transporter, MDR1, in the model isoquinoline alkaloid producer, Coptis japonica, and demonstrated that the protein mediated ATP-dependent berberine uptake (Yazaki et al., 2001; Sakai et al., 2002; Shitan et al., 2003; Yazaki, 2005). A survey of the Arabidopsis genome for a homolog of CjMDR1 indicated that a predicted protein of 1286 amino acids, AtPGP4 (At2g47000; hereafter referred to as PGP4), was the most similar Arabidopsis homolog (71% amino acid identity, 68% nucleotide identity). Because Arabidopsis does not produce isoquinoline alkaloids, we considered the possibility that PGP4 might be involved in auxin transport, as PGP4 shares sequence similarity with both PGP1 and PGP19 (60 and 61%, respectively). However, the predicted PGP4 amino acid sequence also contains a unique coiled-coil protein interactive domain at its N terminus and exhibits substantial sequence divergence from PGP1 and PGP19 in the loop region adjoining the first conserved nucleotide binding domain. To clarify the physiological function of PGP4 in Arabidopsis and determine whether PGP4 might function as an uptake transporter, we have analyzed PGP4 expression and characterized PGP4 protein function.

RESULTS

Expression of PGP4

RNA gel blot analysis with a PGP4-specific 0.7-kb probe showed that PGP4 expression is tissue-specific. PGP4 was strongly expressed in roots and weakly expressed in stems and leaves, but it was not detected in flowers (Figure 1A). PGP4 expression (determined by quantitative real-time PCR) in both primary and lateral root tips remains high throughout development (data not shown), although a transient decrease in seedling expression has been reported between 4 and 7 d after germination (Santelia et al., 2005).

Figure 1.
Figure 1.

Expression Profile of PGP4 in Arabidopsis.

(A) Organ-specific expression of PGP4. Total RNA (5 μg) prepared from each Arabidopsis organ was probed with 32P-labeled PGP4 fragment (0.7 kb) (top gel). The amount of total RNA applied to each lane is shown by 18S rRNA (bottom gel). RL, rosette leaf; CL, cauline leaf; S, stem; R, root; F, flower. Abundances of PGP4 transcripts were quantified and confirmed via in silico analysis of microarray expression data and semiquantitative real-time PCR analysis. Relative expression values were determined using the radioimaging analyzer BAS 1800 (Fuji Film Co.). The expression level of the PGP4 mRNA was normalized by the 18S rRNA values. The experiment was repeated twice with similar results.

(B) Response of PGP4 expression to various treatments. Cont., untreated control. Fourteen-day seedlings were treated for 24 h with 10 μM 1-naphthaleneacetic acid (NAA), 10 μM IAA, 10 μM 2,4-D, 10 μM 6-benzyladenine (BA), 10 μM kinetin (kin.), 10 μM abscisic acid (ABA), 10 μM gibberellic acid (GA3), 10 μM brassinolide (BL), 100 μM methyl jasmonate (JA), 100 μM salicylic acid (SA), and 10 μM stigmasterol (St) and at 4°C (cold)/low light and dark (dark). Total RNA (8 μg) prepared from whole seedlings was probed with a 32P-labeled PGP4 fragment (top gel). Loading controls are shown by β-actin (bottom gel). Relative expression values were determined using the radioimaging analyzer BAS 1800. The expression level of the PGP4 mRNA was normalized by the actin values. The experiment was repeated twice with similar results.

In 14-d-old seedlings, the effects of hormones, signaling molecules, and abiotic factors on PGP4 expression were analyzed: PGP4 expression was upregulated by treatment with natural and synthetic auxins and the cytokinin kinetin, whereas abscisic acid and cold treatments downregulated PGP4 expression (Figure 1B). A time course of PGP4 expression in 5-d seedlings in response to indole-3-acetic acid (IAA), kinetin, and abscisic acid was analyzed by quantitative real-time PCR to further refine PGP4 responsiveness to these stimuli (Table 1). Like PGP1 and PGP19 (Noh et al., 2001; Geisler et al., 2005), PGP4 expression increased after exogenous IAA treatment (P < 0.001) (Table 1); however, the expression did not increase until 8 h after treatment, indicating that PGP4 is a late auxin response gene. Consistent with a correlation between late auxin response and stress gene induction by stress and wounding (Smith et al., 2003), abscisic acid treatment resulted in a 4-h oscillation pattern (peak to peak) of PGP4 expression (P < 0.001), whereas only a small transient increase in expression was seen at 4 to 6 h after kinetin treatment (P = 0.002) (Table 1). The abundances of PGP4 transcripts in Figure 1 were confirmed via radioimaging, semiquantitative RT-PCR (see Supplemental Figure 1 online), and comparisons with published microarray data (http://bbc.botany.utoronto.ca/affydb/cgi-bin/affy_db_exprss_browser_in.cgi?p; http://www.arexdb.org/index.jsp; http://signal.salk.edu/cgi-bin/tdnaexpress).

View this table:
Table 1.

Time Course of PGP4 Expression in Response to Various Hormones via Quantitative Real-Time PCR Analysis

Wild-type plants were transformed with ProPGP4:GUS, consisting of a 2.2-kb sequence upstream of the PGP4 start site fused to a β-glucuronidase (GUS) reporter gene, and the T2 or T3 generations were histochemically stained with 5-bromo-4-chloro-3-indolyl-β-glucuronic acid (X-Gluc) at different developmental stages. Consistent with the RNA gel blot analysis, PGP4 was strongly expressed in the roots of seedlings and mature plants (Figures 2A and 2E). In the mature portion of the root, PGP4 was expressed in the epidermis and cortex (Figures 2F and 2G). In the root elongation zone, GUS staining was restricted to the epidermis (Figures 2H and 2I), and in the root tip, GUS signal was observed only in the root cap, S3 columella, and epidermal cells (Figures 2J and 2K). No staining was evident in aerial tissues, although extended X-Gluc treatment resulted in weak staining at hydathodes and silique junctions (Figures 2B to 2D).

Figure 2.
Figure 2.

GUS Staining of ProPGP4:GUS Transformants.

(A) GUS activity is seen throughout the root in a 5-d ProPGP4:GUS seedling.

(B) GUS activity in a ProPGP4:GUS rosette leaf.

(C) GUS activity in a ProPGP4:GUS silique.

(D) GUS activity in the hydathodes of a ProPGP4:GUS rosette leaf (magnification of [B]).

(E) GUS activity is seen primarily in the roots of a 14-d ProPGP4:GUS plant.

(F) GUS activity is seen in the epidermis and the cortex in a radial section of the mature root region in a ProPGP4:GUS plant. Cx, cortex; Ed, endodermis; Ep, epidermis.

(G) GUS activity in a longitudinal section of the mature root region in a ProPGP4:GUS plant.

(H) GUS activity in the epidermis of a radial section of the root elongation zone in a ProPGP4:GUS plant.

(I) Magnification of (H).

(J) GUS activity in the root cap and the epidermis in a radial section of a ProPGP4:GUS root tip. Rc, root cap.

(K) GUS activity in the root cap, the S3 columella cells, and the epidermis in a longitudinal section of a ProPGP4:GUS root tip.

Bars = 1 mm in (A) to (D), 2.5 cm in (E), and 50 μm in (F) to (K).

Localization of PGP4

Previously, an unidentified high molecular mass protein band was observed after SDS-PAGE of NPA-affinity purification of PGP1 and PGP19 from microsomal fractions (Murphy et al., 2002; Geisler et al., 2003). Amino acid sequencing of that band indicated that it contained a mixture of PGP1, PGP19, and PGP4 (Table 2). Sucrose density gradient fractionation of microsomal membranes suggested that PGP4 is found in multiple membrane fractions (Figure 3A). However, the PGP4 peak fraction coincided with that of the PM H+-ATPase W1D (Figure 3A) to a greater extent than with the endoplasmic reticulum lumenal marker binding protein (BiP) (Haas, 1994) and the H+-pyrophosphatase (H+-PPase) multiple-compartment marker ARABIDOPSIS VACUOLAR PYROPHOSPHATASE1 (AVP1) (Li et al., 2005). Furthermore, when wild-type microsomal membranes were separated via aqueous two-phase partitioning, PGP4 was found predominantly in the PM-enriched upper phase, along with the PM H+-ATPase W1D (Figure 3B). This distribution is consistent with a recent report of PGP4, H+-ATPase, and BiP retention in detergent-resistant microdomains (Borner et al., 2005). PGPs have also been localized in detergent-resistant microdomains involved in vesicular cycling between endomembrane compartments and the PM in mammals (Kipp and Arias, 2002; Brown and London, 1998a, 1998b).

View this table:
Table 2.

PGPs Identified in NPA Binding Fractions

Figure 3.
Figure 3.

Subcellular Localization of PGP4.

(A) Membrane fractionation and immunodetection of PGP4. Fractionation of total microsomes from Arabidopsis seedlings was done on a noncontinuous sucrose gradient consisting of 20, 30, 40, and 50% (w/v) sucrose. Membrane fractions were collected from the interfaces between different sucrose concentrations and analyzed via SDS-PAGE and protein gel blotting. Blots were probed with antisera to PGP4, PM H+-ATPase (W1D), multicompartment H+-PPase (AVP1), and endoplasmic reticulum BiP (BiP).

(B) Two-phase separation of PM and immunodetection of PGP4. Microsomal membranes (M) from Arabidopsis seedlings were fractionated by the aqueous two-phase portioning method, by which PM was enriched in the upper phase (U) and other intracellular membranes remained in the lower phase (L). Proteins from each fraction (5 μg per lane) were blotted and probed with antisera to PGP4, PM H+-ATPase, multicompartment H+-PPase, and endoplasmic reticulum BiP.

(C) DIC image of a 5-d seedling root. Top bracket, mature root ([D], [E], [J], and [K]); middle bracket, root transition zone ([F], [G], and [L] to [O]); bottom bracket, root tip ([H] and [I]). Bar = 100 μm.

(D) Confocal image of immunohistochemical localization of PGP4 in a wild-type mature root region. Apical (bottom) PGP4 signal is observed.

(E) DIC overlay of (D).

(F) Confocal image of immunohistochemical localization of PGP4 in a wild-type root transition zone. Basal (top) PGP4 signal is restricted to three cell stories in the root transition zone.

(G) DIC overlay of (F).

(H) Confocal image of immunohistochemical localization of PGP4 in a wild-type root tip. PGP4 signal is seen in the root cap, S3 columella cells, and epidermis.

(I) DIC overlay of (H).

(J) Confocal image of immunohistochemical localization of PGP4 in pgp4-1 in a mature root region. Only nonspecific epidermal signal is observed.

(K) DIC overlay of (J).

(L) Confocal image of immunohistochemical localization of PGP4 in pgp4-1 in the root transition zone. Only nonspecific epidermal signal is observed.

(M) DIC overlay of (L).

(N) Confocal image of immunohistochemical localization of PGP4 in PGP4OX in the root transition zone. Basal and lateral PGP4 signal is restricted to three cell stories in the root transition zone.

(O) DIC overlay of (N).

Both PM and endomembrane localization of PGP4 was apparent in immunofluorescence localizations of the protein (Figures 3D to 3I) using antisera generated against the unique N-terminal domain of PGP4 (see mutant characterization below). Apical (bottom) PM localization of PGP4 was seen in mature root epidermal cells from the proximal elongation zone to two to three cell stories below the root–shoot junction (Figures 3D and 3E); although PGP4 is expressed in mature root cortical tissues, cortical PGP4 signals were very weak, attributable perhaps to posttranscriptional regulation, poor confocal laser penetration, or limited antibody penetration in this tissue. These tissues coincided exactly with tissues where the PGP1 efflux transporter was previously shown to be predominantly basally localized (Geisler et al., 2005). However, in a short region (three cell stories) of the root transition (distal elongation) zone, basal (top) PM localization of PGP4 was observed (Figures 3F and 3G). Interestingly, PGP1 was shown previously to exhibit increased apical PM localization in this region (Geisler et al., 2005). Similar to PGP1 (Geisler et al., 2005), apolar PM and some apparent endomembrane localization of PGP4 was observed at the root apex. Apolar PGP4 localization was restricted to S3 columella and adjacent root cap cells (Figures 3H and 3I), where PGP1 localization is not seen (Geisler et al., 2005) and where ALTERED RESPONSE TO AUXIN AND GRAVITY1 (AUX1) also exhibits apolar localization (Swarup et al., 2001, 2004). Figure 3C shows a differential interference contrast (DIC) image of a root for reference.

Phenotypic Characterization of pgp4 Mutants

Five T-DNA insertional lines in the PGP4 gene were obtained from the SALK collection (Alonso et al., 2003), and three homozygous mutant lines were selected after PCR analysis of the insertion sites: pgp4-1 (SALK_063720), pgp4-3 (SALK_067653), and pgp4-4 (SALK_088311) (Figure 4A). In all three lines, no PCR product was obtained with primers designed for either the full-length cDNA or sequences flanking the insertion site. In pgp4-1 seedlings, PGP4 mRNA was not detected via RNA gel blot analysis using a gene-specific probe (Figure 4B). Furthermore, PGP4 protein was not detected when pgp4-1 membrane fractions were immunoblotted with PGP4-specific antisera (Figure 4B). The PGP4 antisera cross-reacted with a single band of the expected apparent molecular mass (140 kD) on SDS gels prepared from wild-type Arabidopsis seedling membranes. The antisera also did not cross-react with the closest homolog, PGP21 (∼129 kD), which is expressed in seedlings at levels similar to those of PGP4 (Figure 4B; see Supplemental Figure 1 online). No signal was detected when the PGP4 antisera was used in immunofluorescence localizations of pgp4-1 mutants at the dilution used with the wild type (Figures 3J to 3M).

Figure 4.
Figure 4.

Genomic Structure of PGP4 and Phenotypes of pgp4.

(A) Genomic structure of PGP4 T-DNA insertion sites of pgp4-1 (SALK_063720), pgp4-3 (SALK_067653), and pgp4-4 (SALK_088311). Rectangles represent exons, and lines represent untranslated regions, including the introns of the PGP4 gene. T-DNA, represented by triangles, is not drawn to scale. The black box in the 6th exon indicates the first nucleotide binding fold (NBF1), and those in 10th and 11th exons indicate the second nucleotide binding fold (NBF2).

(B) RNA and protein gel blot analyses of pgp4-1. Ten micrograms of total RNA and 30 μg of microsomal protein from either wild-type or mutant seedlings were analyzed. Neither PGP4 mRNA nor protein was detected in pgp4-1, indicating that this is a null allele.

(C) Root phenotype of pgp4-1and PGP4 overexpressor 7-d seedlings, and complementation of pgp4-1. Bar = 1 cm.

(D) pgp4-1 seedlings have shorter roots. Asterisk indicates statistically significant difference in root length between the wild type and the pgp4-1 mutant (P < 0.01, n = 16). Experiments were done in triplicate.

(E) pgp4-1 seedlings have fewer lateral roots. Asterisk indicates statistically significant differences in lateral root number between the wild type and pgp4-1 mutant (P < 0.01, n = 16). Experiments were done in triplicate.

(F) Gravitropic phenotypes in pgp4-3 and pgp4-4. The angle of curvature is reduced in both pgp4 alleles compared with the wild type. Twenty seedlings were used per replicate (n = 3). Asterisks indicate significant difference from the wild type by Student's t test (P < 0.05).

The root lengths of 10-d pgp4-1 seedlings were 30% shorter than those of the wild type, and the number of lateral roots was also significantly reduced (∼61% of wild type; P < 0.01) (Figures 4C to 4E). Similar results were observed for pgp4-3 and pgp4-4 (see Supplemental Table 1 online). These root phenotypes were reproducible when seedlings were grown at moderate light levels (100 to 120 μmol·m−2·s−1) using sucrose concentrations of 0.5 to 1% (see Methods). The conditions under which these phenotypes are most visible in pgp4 (Figures 4C and 4D; see Supplemental Figure 2 online) are similar to those under which pgp1 growth phenotypes are most evident (Geisler et al., 2005). However, when pgp4 mutants were grown under high light or on sucrose concentrations >1.5%, the root lengths and number of lateral roots observed were greater than or equal to those of the wild type (data not shown). Root phenotypes in pgp4-1 could be complemented by transformation with Pro35S:PGP4 (P > 0.05) (Figure 4D; see Supplemental Figure 2 online).

pgp4-3 and pgp4-4 seedlings exhibited reductions in the rate of root gravitropic bending similar to the observed decreases in linear root growth (P < 0.05) (Figure 4F); pgp4-1 root gravitropic responses were not statistically different from those of the wild type, perhaps because of the large standard deviations observed (P > 0.05). Furthermore, no changes in ProPGP4:GUS expression patterns were evident in roots up to 6 h after gravistimulation (data not shown). Because of this, observed differences in root gravitropism were assumed to be the result of reduced linear root growth in pgp4 mutants, not an intrinsic defect in the gravitropic response. An alternative explanation could be that the slight gravitropism phenotype observed in pgp4-3 and pgp4-4 may be a dominant negative effect.

Consistent with the observed root-specific expression of PGP4, no obvious morphological differences between pgp4-1 and wild-type plants were observed in the aerial parts of seedlings and adult plants. The lack of a visible phenotype of pgp4-1 mutants in aerial tissues may also result from functional redundancy in these tissues because other PGPs implicated in auxin transport (such as PGP19, PGP6, and PGP10) are expressed in aerial tissues at much higher levels than PGP4 (Blakeslee et al., 2005b). This is in contrast with root tissues, in which PGP4 appears to be the most highly expressed PGP.

PGP4 Overexpression

PGP4 was also overexpressed under the control of a 35S promoter (Pro35S:PGP4; hereafter referred to as PGP4OX). Roots of PGP4OX lines were only slightly longer than wild-type roots (Figures 4C and 4D), and no changes were evident in shoot phenotypes. Similar growth phenotypes were observed in multiple independent PGP4OX lines. PGP4 expression in PGP4OX roots was twofold to fourfold that of the wild type (P < 0.05) but did not increase significantly in shoots (P > 0.05). As similar expression patterns were observed in all recovered PGP4OX lines, it appears that PGP4 transcript abundance is posttranscriptionally regulated in overexpressor lines.

Immunolocalization of PGP4 in PGP4OX was similar to that in the wild type, except that the signal was stronger in the root cap and elongation zone. No change in subcellular localization was seen in the root cap (data not shown), but PGP4 signals in three stories of epidermal cells in the elongation zone expanded slightly to lateral membranes as well (Figures 3N and 3O). This localization pattern was observed in all PGP4OX lines recovered, consistent with the posttranscriptional regulation of ectopic PGP4 expression.

Gain or loss of PGP4 expression does not alter the expression of PGP1 and PGP19, as expression of these genes (determined by quantitative real-time PCR) was not different from that of the wild type in pgp4-1 and PGP4OX (P > 0.05). Similarly, although the PIN-FORMED2 (PIN2) auxin efflux protein is basally localized in the same epidermal and cortical cells as PGP4 (Chen et al., 1998; Luschnig et al., 1998; Müller et al., 1998; Peer et al., 2004), as was the case with pgp1 mutants (Geisler et al., 2005), no alteration in PIN1 or PIN2 localization and abundance was apparent in pgp4-1 or PGP4OX (see Supplemental Figures 3 and 4 online).

Polar Auxin Transport in pgp4 and PGP4OX

Auxin transport and free IAA levels were measured in pgp4-1 seedlings as described previously (Geisler et al., 2003, 2005). A slight decrease of basipetal transport observed in pgp4-1 hypocotyls (P < 0.05) (Figure 5A) and increased free IAA concentrations in the lower part of the hypocotyls (P < 0.05) (Figure 5B) may be the result of the elimination of low levels of PGP4 expression in hypocotyls, but they are more likely the result of a diminished root tip auxin uptake sink. Consistent with this interpretation, reduced auxin uptake and basipetal auxin transport were observed at pgp4-1 root tips (P < 0.05) (Figure 5C), and free IAA levels were significantly increased in the first 1.5 mm of the root tip in pgp4-1 (P ≤ 0.01) (Figure 5D). Furthermore, aggregations of the flavonol quercetin have been shown to accumulate near the root tip when auxin concentrations increase (Peer et al., 2004). Similar aggregations were observed in pgp4-1 (Figures 5E and 5F; see Supplemental Figure 5 online).

Figure 5.
Figure 5.

Auxin Transport in pgp4-1 and PGP4OX.

(A) Basipetal hypocotyl and acropetal root transport in Columbia wild-type, pgp4-1, and PGP4OX 5-d seedlings. Data are presented as percentage of auxin transport, relative to Columbia wild type. Mean wild-type values were 4245.5 and 648 dpm for shoot basipetal and root acropetal transport, respectively. n = 2 groups of 10 seedlings each. Asterisks indicate significant difference from the wild type by Student's t test (P ≤ 0.05).

(B) Basipetal root auxin transport in a root segment 2 mm from the site of auxin application at the root apex. Data are presented as percentage of auxin transport, relative to Columbia wild type. The mean wild-type value was 6473.5 dpm. n = 2 groups of 10 seedlings each. Asterisk indicates significant difference from the wild type by Student's t test (P ≤ 0.05).

(C) Quantitations of free IAA in wild-type and pgp4-1 hypocotyl sections. Fifty seedlings were used per replicate (n = 3). Asterisk indicates significant difference from the wild type by Student's t test (P ≤ 0.05).

(D) Quantitations of free IAA in wild-type and pgp4-1 root sections. Fifty seedlings were used per replicate (n = 3). Asterisk indicates significant difference from the wild type by Student's t test (P ≤ 0.01).

(E) Wild-type seedlings do not accumulate quercetin in the root elongation zone. Bar = 100 μm for (E) and (F).

(F) pgp4-1 seedlings accumulate aggregations of quercetin in the corresponding region of the root elongation zone.

The light/sucrose dependence of pgp4 seedling growth phenotypes suggested that differences in basipetal auxin transport observed between pgp4 and the wild type would decrease under light conditions of >120 μmol·m−2·s−1. This proved to be the case (data not shown). Higher light conditions have been shown to increase both sucrose production and apoplastic acidification in roots (Le Bot and Kirkby, 1992; Stewart and Lieffers, 1994). Lower apoplastic pH was recently shown to increase chemiosmotically driven auxin efflux and uptake in Arabidopsis roots (Li et al., 2005). On the other hand, if PGP4 is a hydrophobic anion uptake transporter, decreased apoplastic pH would be expected to decrease PGP4-mediated transport.

Analysis of auxin transport in PGP4OX supports a primary function of PGP4 in establishing an auxin uptake sink in the root cap, as shoot basipetal and root acropetal auxin transport in PGP4OX seedlings both increased by ∼240% (P < 0.05) (Figure 5A), despite no increase in PGP4 expression in shoot tissues. The lack of increased root basipetal transport observed in PGP4OX (Figure 5C) suggests that the increased lateral localization of PGP4 seen in root transition/distal elongation zone cells has either a negative or a negligible impact on basipetal transport. However, root basipetal auxin transport was restored to wild-type levels after complementation of pgp4-1 with PGP4OX (see Supplemental Figure 2 online).

Vanadate-Induced Nucleotide Trapping of PGP4

As the nucleotide binding affinity and ATPase activity of ABC transporters greatly increases in the presence of preferred substrates when combined with vanadate (Urbatsch et al., 1995), vanadate-induced nucleotide trapping can be used to screen for ABC transporter substrates (Sakai et al., 2002; Terasaka et al., 2003). However, this method cannot be used with crude membranes from Arabidopsis because of the large number of ABC transporters present. PGP4 was expressed in Sf9 insect cells, which have low background vanadate-trapping activity and are used extensively to characterize the ATPase activity and substrate specificity of ABC transporters (Sarkadi et al., 1992; Rao, 1998; Cortes-Selva et al., 2005). Photoaffinity labeling of an ∼140 kD protein could be seen when recombinant PGP4 was expressed in Sf9 cells (Figures 6A and 6B). As was seen with the human PGP HsMDR1 (Ueda et al., 1997), this basal level of PGP4 ATPase activity required both Mg2+ and vanadate (Figure 6B).

Figure 6.
Figure 6.

Expression of Recombinant PGP4 in Sf9 Cells, and Vanadate Trapping.

(A) Immunoblot of PGP4 in Sf9 cells and Arabidopsis seedlings. Lane 1, membrane protein control from Sf9 cells; lane 2, 20,000g supernatant from cells expressing PGP4; lane 3, 20,000g pellet from cells expressing PGP4; lane 4, Arabidopsis seedling microsomal membrane fraction. For membrane proteins, 10 μg (lanes 1 to 3) or 30 μg (lane 4) was loaded per lane, respectively. CBB, Coomassie Brilliant Blue.

(B) Photoaffinity labeling of recombinant PGP4 expressed in Sf9 cells. Membrane proteins from recombinant AtPGP4 expressed in Sf9 cells were incubated with 10 μM 8-azido-[α-32P]ATP in the presence or absence of 3 mM MgSO4 (Mg2+) and 0.2 mM orthovanadate (Vi) for 10 min at 37°C. Proteins were photoaffinity-labeled with UV irradiation after removal of unbound ligands and analyzed by SDS-PAGE.

(C) Effect of putative substrates on the photoaffinity labeling of recombinant PGP4. Cont., control (no treatment). Seedlings were treated with 50 μM naphthaleneacetic acid (NAA), 50 μM IAA, 50 μM 2,4-D, 50 μM 6-benzyladenine (BA), 50 μM kinetin (kin.), 50 μM abscisic acid (ABA), 50 μM gibberellic acid (GA3), 25 μM brassinolide (BL), 50 μM vanillic acid (Va), 50 μM syringic acid (Sa), 50 μM indole-3-propionic acid (IPA), 50 μM NPA, or 50 μM tri-iodobenzoic acid. Relative expression values were determined using the radioimaging analyzer BAS 1800 (Fuji Film Co.). The experiment was done in duplicate. Representative results are shown.

Natural and synthetic growth regulators and their precursors were screened using this system as a first approximation to determine possible substrates of PGP4. IAA, the weak auxin indole-3-propionic acid, the competitive auxin transport inhibitor tri-iodobenzoic acid, and the PGP binding auxin efflux inhibitor NPA (Noh et al., 2001; Murphy et al., 2002; Geisler et al., 2003) all increased vanadate-trapping signals, whereas other growth regulators and artificial auxins (2,4-D and 1-naphthaleneacetic acid) did not (Figure 6C). In addition, two nonbioactive structural analogs of auxin, syringic acid and vanillic acid, also increased the vanadate-trapping signal (Figure 6C). The vanadate-trapping method could not be used to further resolve the auxin specificity of PGP4, as increased concentrations of IAA or indole-3-propionic acid did not increase PGP4 photoaffinity labeling (data not shown).

Cellular Transport Assays of Auxin by PGP4

PGP4 was expressed in the vaccinia virus–HeLa mammalian expression system commonly used to analyze PGP activity and recently used to demonstrate NPA-sensitive, PGP1-mediated net efflux of [3H]IAA and [3H]1-naphthaleneacetic acid (Elroy-Stein and Moss, 1991; Moss, 1991; Hrycyna et al., 1998; Geisler et al., 2005). PGP4 expression and protein localization on the PM were verified as described previously (Geisler et al., 2005) (Figure 7B). Unlike PGP1 (Geisler et al., 2005), expression of PGP4 resulted in a net increase in auxin retention compared with empty vector controls (Figure 7A), indicating that PGP4 may transport IAA into HeLa cells. A range of PGP4 expression levels were assayed to determine whether an increased abundance of PGP4 resulted in mistargeting of the protein: at all concentrations tested, net [3H]IAA retention was observed (data not shown). These IAA uptake data are consistent with a recent report of increased sensitivity to the toxic auxin analog 5-fluorol indole seen in JK93da yeast expressing Arabidopsis PGP4 and similar sensitivity to IAA seen in yap1-1 mutant yeast also expressing PGP4 (Santelia et al., 2005).

Figure 7.
Figure 7.

PGP4-Mediated Auxin Influx.

Net efflux is expressed as dpm/500,000 cells: the amount of auxin retained by cells transformed with empty vector minus the amount of auxin retained by cells transformed with the gene of interest. Reductions in retained auxin (efflux) are presented as positive values, and increases in retention (influx) are presented as negative values. Values shown are means with sum of the standard deviations (n = 3).

(A) PGP4 expressed in HeLa cells had increased influx of radiolabeled IAA compared with cells transformed with empty pTM1 vector. For inhibitor studies, cells were incubated with radiolabeled IAA in the presence of 10 μM NPA, 200 nM quercetin (Quer.), 1 μM cyclosporin A (CsA), or 5 μM verapamil (Ver.). NPA treatment reverses PGP4-mediated influx. For radiolabeled auxin degradation product assays, cells were loaded with 63 nM radiolabeled IAA and 63 nM nonlabeled IAA oxidative breakdown products (Deg.). Asterisks indicate significance as determined by Student's t test (P ≤ 0.006): PGP4 activity was compared with empty vector control; PGP4 activity + inhibitors was compared with empty vector control + inhibitors.

(B) Immunolocalization of hemagglutinin-tagged PGP4 expressed in HeLa cells. Hemagglutinin-tagged PGP4 was detected with an anti-hemagglutinin antibody. Bar = 10 μm.

The effects of auxin transport and ABC transport inhibitors on net auxin retention were examined. NPA treatment abolished IAA retention in HeLa cells expressing PGP4 (P ≤ 0.006) (Figure 7A), whereas treatment with the natural auxin transport inhibitor quercetin (Peer et al., 2004) and the ABC inhibitors verapamil and cyclosporin A significantly reduced IAA retention (P ≤ 0.006) (Figure 7A). Although auxin efflux inhibitors such as NPA, cyclopropyl propane dione, and quercetin have been shown to inhibit PGP-mediated auxin efflux both in planta and in membrane vesicles (Murphy et al., 2000; Geisler et al., 2005), in planta effects on PGP4-mediated uptake are difficult to determine in a background in which PGP1 and/or PGP19 are expressed. However, NPA treatment (5 μM) resulted in a 35% ± 12% reduction of IAA retention (P < 0.05) in pgp4-1 roots. Similar results have been reported by Santelia et al. (2005).

Both PGP1 and PGP19 have been shown to transport oxidative auxin breakdown products in addition to IAA (Geisler et al., 2005). When cells expressing PGP4 were incubated with a mixture of 50% radiolabeled auxin and 50% nonlabeled oxidative auxin breakdown products, the IAA retention activity was strongly inhibited (P ≤ 0.006) (Figure 7A), indicating that IAA metabolites are also likely PGP4 substrates. However, PGP4 does exhibit relative substrate specificity, as is the case with PGP1 and PGP19 expression (Geisler et al., 2005; J.J. Blakeslee and A.S. Murphy, unpublished data), PGP4 expressed in HeLa cells did not transport the mammalian PGP substrates rhodamine 123, daunomycin, or vinblastine (data not shown).

DISCUSSION

Evidence is presented here that PGP4 is a mediator of auxin transport at the root apex and participates in basipetal auxin transport in root epidermal tissues. Support for this conclusion comes from analysis of PGP4 expression, PGP4 protein localization and function, pgp4 growth phenotypes, auxin transport, and auxin accumulation. These results also add to the growing body of evidence supporting a major role of PGPs in auxin transport (Noh et al., 2001, 2003; Geisler et al., 2003, 2005; Multani et al., 2003; Santelia et al., 2005). Analysis of PGP gene expression and pgp mutant growth phenotypes suggests that PGPs function in a tissue-specific manner similar to what is seen with the PIN proteins (Blakeslee et al., 2005a).

Until now, PGPs were thought to function exclusively in auxin efflux. Together with recently published evidence that PGP4 expression increases the sensitivity of yeast to auxins and toxic auxin analogs (Santelia et al., 2005), the results presented here suggest that PGP4 functions as an energy-dependent uptake transporter. Such a role for PGP4 is not unexpected in light of its homology with the uptake transporter CjMDR1 and the energetic requirements of transporting auxin anions against the chemiosmotic gradient. At the apoplastic pH of ∼5.5 seen in roots, only ∼16% of the IAA (pKa 4.75) present is protonated (neutral and lipophilic). As such, only a small portion of apoplastic IAA can be taken up by hydrophobic diffusion. Anionic IAA must be taken up by a process that directly uses ATP, as is the case with an ABC transporter such as PGP4, or by secondary active transport, as is seen with the putative proton symporter AUX1 (Marchant et al., 1999; Swarup et al., 2001). Although lipophilic uptake appears to be sufficient in most tissues, additional uptake mechanisms would be expected to be required at the root apex, where auxin must be retained and redirected rapidly into the basipetal transport stream.

However, based on the modest loss of root gravitropism seen in some pgp4 alleles (equivalent to that seen in agr1/pin2) compared with the severe agravitropism seen in aux1 (Bennett et al., 1996), the contribution of PGP4 to auxin uptake in these tissues appears to be less than that of AUX1. This makes sense, as a symporter such as AUX1 would be expected to be more sensitive to changes in pH associated with root gravistimulation (Hou et al., 2004). The amelioration of pgp4 growth phenotypes by increased light and sucrose also supports this relationship, as light and sucrose enhance ATP-driven proton extrusion (Le Bot and Kirkby, 1992; Stewart and Lieffers, 1994) and, thus, chemiosmotically driven transport in roots (Li et al., 2005).

The localization of PGP4 is also consistent with uptake function. Very little expression or protein abundance is seen in the columella initials, S1 or S2 cell layers. Expression in the S3 layer is consistent with an uptake function, especially because AUX1 has not been localized in these cells (Swarup et al., 2004), but exhibits a similar apolar localization in epidermal/lateral root cap cells. PGP4 localization in S3 and S3-derived cells in the lateral root cap is also consistent with a lack of expression of the PGP1 efflux transporter in those cells. PGP4 could also be expected to maintain minimal levels of auxin uptake under conditions of apoplastic alkalinization that would severely inhibit lipophilic or AUX1/LIKE AUX1 uptake.

The apical (bottom) localization of PGP4 in the epidermal cells of the proximal elongation zone and above is also consistent with both an uptake function and the observed pgp4 root growth phenotypes. What is more difficult to understand is the reversal of this orientation seen in three stories of epidermal cells within the root transition/distal elongation zone. However, it is interesting that PGP1 subcellular localization changes from apolar to basal in the root transition zone (Geisler et al., 2005). More significantly, these cells exhibit the first changes in elongation after gravistimulation (Ishikawa and Evans, 1997). As PGP4 expression does not appear to change in these tissues in response to gravistimulation, we hypothesize that PGP4 serves to increase auxin retention in these cells to enhance graviresponsiveness. Functional green fluorescent protein fusions of PGP4 now in progress will allow the analysis of possible dynamic protein relocalization during gravitropic responses.

Finally, these results suggest that PGP4-mediated auxin uptake requires other factors to enhance auxin specificity. PIN proteins are the most likely candidates for this role (Blakeslee et al., 2005a). Although PGP4 is localized in the same cells as PIN2, a direct interaction between these proteins is unlikely because they are at opposite ends of the cell. Furthermore, in the three cell stories of the transition zone where the two proteins colocalize, PIN2 is more weakly expressed. However, as it is likely that specific PIN–PGP pairings act to provide specificity and directionality to polar auxin transport (Blakeslee et al., 2005a), PGP4 interaction with one or more PIN proteins cannot be ruled out. Using recently developed cellular assays of auxin transport (Geisler et al., 2005), a functional analysis of interactions between PGP4 and members of the PIN protein family is now under way.

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana plants (ecotype Columbia) were grown on soil in growth chambers with 100 or 120 μmol·m−2·s−1 light in a 16-h-light/8-h-dark cycle at 21°C. For growth under sterile conditions, seeds were surface-sterilized (20-min incubation in 30% [v/v] sodium hypochlorite and 0.02% [v/v] Triton X-100 and rinsed four times in sterile distilled water; or 2-min incubation in 20% [v/v] sodium hypochlorite, rinsed in distilled water three times, with a final rinse of 95% ethanol) and sown on half-strength Murashige and Skoog (MS) salts supplemented with no sucrose, 0.5%, 1.0%, or 1.5% (w/v) sucrose, pH 5.8, and 0.8% (w/v) bactoagar or phytagar in Petri dishes.

To analyze the regulation of PGP4 transcripts by various growth regulators, seeds were sown onto nylon mesh (20-μm pore) over the MS medium and grown for 14 d under the same light cycle described above. Roots were subjected to various treatments by gentle transfer of the mesh to new medium. Treatments were stopped by immediate freezing of seedlings in liquid N2.

Cloning of AtPGP4 cDNA and Construction of the Plant Overexpression Construct

To isolate the cDNA of PGP4 (At2g47000) by RT-PCR, two primers were designed that allowed the amplification of the entire PGP4 open reading frame, adding to the 32-bp 5′ untranslated region and the 58-bp 3′ untranslated region (forward primer, 5′-CGTCTAGAGCTTAGGATCGTGAGGTATCTG-3′; reverse primer, 5′-GCTGAGCTCATATAACGGGGCATAATTGAC-3′). The underlined positions are nonnative sequences representing XbaI and SacI restriction sites, respectively. The PCR product was subcloned into pBluescript II SK− (Stratagene) for sequencing. The full-length cDNA was transferred into pENTR1A vector (Invitrogen) using SalI and NotI sites. This construct served as the entry vector to transfer the cDNA of PGP4 into the binary destination vector pGWB2 for constitutive expression via the Gateway system (Invitrogen) to create the Pro35S:PGP4 construct with the cauliflower mosaic virus 35S promoter. Agrobacterium tumefaciens GV3101 (pMP90) was transformed with the binary vector, which was introduced into Arabidopsis Columbia wild-type plants by floral dip (Clough and Bent, 1998). Kanamycin- and hygromycin-resistant plants in the T1 and T2 generations were examined by immmunoblot analysis.

RNA Isolation and RNA Gel Blot Analysis

Total Arabidopsis RNA was prepared from 200 mg of seedlings using the RNeasy plant mini kit (Qiagen). Agarose gel electrophoresis, RNA transfers onto Hybond-N+ membranes (Amersham Bioscience), and hybridization with the 0.7-kb AtPGP4 fragment (positions +1989 to +2711) were performed using standard procedures. The last stringent wash was performed with 0.2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate) and 0.1% SDS at 60°C.

Promoter:GUS Fusion and GUS Expression Analysis

To create the PGP4 promoter:reporter construct (ProPGP4:GUS), two PCR primers flanking the regions from +15 to −2196 were made (forward primer, 5′-GCTAAGCTTGGTAAAGGATTTGGGTCTATTCG-3′; reverse primer, 5′-CTGTCTAGAGCTCTCTGAAGCCATTAGAGTTT-3′). The underlined positions are nonnative sequences representing HindIII and XbaI restriction sites, respectively. PCR was performed using genomic DNA as a template and KOD-Plus polymerase (Toyobo). The PCR product was inserted upstream of the GUS coding sequence in the pBI101 binary vector. A. tumefaciens GV3101 (pMP90) was transformed with the binary vector and introduced into wild-type Arabidopsis as described above. Kanamycin-resistant plants in the T2 generation were histochemically stained to detect GUS activity. For GUS staining, tissues were prefixed in ice-cold 90% acetone for 20 min, rinsed with cold water, immersed with staining solution (50 mM sodium phosphate buffer, pH 7.0, 0.1% Triton X-100, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 1 mM X-Gluc), and incubated at 37°C for 2 to 5 h for roots and 20 h for rosette leaves and siliques. For higher integrity of the DIC imaging, destaining time was limited before fixation; therefore, very light staining does not indicate signal. The stained samples were fixed in 50% ethanol, 5% acetic acid, and 3.7% formaldehyde, dehydrated through an ethanol series, and embedded in Technovit 7100 (Heraeus Kulzer) according to the manufacturer's protocol. Tissue sections (10 μm thick) were mounted on slides and examined with a light microscope (Zeiss).

Peptide Antiserum against PGP4

A keyhole limpet hemocyanin conjugate of an oligopeptide of PGP4, at position 1 (n-MASESGLNGDPNILEEVSEC-c), was injected into rabbits according to a standard protocol (Kurabo Industries). After the fourth boost, the antiserum was recovered and used for immunoblot analysis and immunohistochemical analysis without further purification.

Density Gradient Fractionation of Membrane Vesicles

For sucrose gradient fractionation, the method described by van den Brule et al. (2002) was used with the following modifications. Arabidopsis seedlings (5 g) were homogenized in 3 volumes of 50 mM HEPES-KOH, pH 7.5, 5 mM EDTA, 2 mM DTT, 250 mM sucrose, and 1 mM phenylmethylsulfonyl fluoride using mortar and pestle. The homogenate was centrifuged for 15 min to remove the debris, and the microsomal membrane fraction was pelleted by ultracentrifugation at 100,000g for 30 min. The pellet was resuspended in 3 mL of gradient buffer (10 mM Tris-MES, pH 7.0, 1 mM DTT, 250 mM sucrose, and 1 mM phenylmethylsulfonyl fluoride) and centrifuged at 100,000g for 3 h on a noncontinuous sucrose gradient from 13 to 40% (w/v) in the same buffer using a Beckman SW41Ti rotor. Gradient fractions were collected from the interface between different sucrose concentrations.

PM fractions were separated from microsomal preparations by partitioning in an aqueous polymer two-phase system, as described previously (De Michelis et al., 1996). The upper (PM) phase as well as the lower phase were used for immunoblot analysis.

For immunoblotting, proteins were denatured in the denaturation buffer (10 mM Tris-HCl, pH 8.0, 40 mM DTT, 1 mM EDTA, 10% [w/v] sucrose, 10 μg/mL pyronine Y, and 2% [w/v] SDS) for 10 min at 50°C, subjected to SDS-PAGE (7% gel), and then transferred to an Immobilon polyvinylidene difluoride membrane (Millipore). The membrane was treated with Blocking One (Nacalai Tesque) for blocking, then incubated with primary antibodies and subsequently with the secondary horseradish peroxidase–conjugated anti-rabbit antibodies using standard procedures. The band was visualized by chemiluminescence (Perkin-Elmer). Antisera used for immunodetection were against PGP4, PM H+-ATPase, multicompartment H+-PPase AVP1, and endoplasmic reticulum BiP from Arabidopsis.

Immunohistochemical Localization of PGP4

Seedlings were grown on 1% (w/v) phytoagar plates, one-quarter MS basal salts, pH 4.85, and 1% sucrose. Immunohistochemical localizations and microscopy were as described previously (Geisler et al., 2005). The antibody dilutions were 1:800 and 1:1200 for anti-PGP4 antisera. Fluorescent staining was imaged by confocal laser-scanning microscopy. Indirectly visualized signals of the fluorescein isothiocyanate–conjugated antibody are indicated in green. A confocal laser-scanning microscope (Eclipse 800; Nikon) equipped with an argon laser (488 nm; Bio-Rad) was used for immunofluorescence imaging. A green HeNe laser (543 nm, 1.4 mW) and a red laser diode (638 nm, 5 mW) were used for autofluorescence detection.

Isolation of pgp4 Mutants

For identification of mutant lines carrying T-DNA insertions in the PGP4 gene, five T-DNA insertion lines available through the Arabidopsis stock centers at Ohio State University (ABRC) and the University of Nottingham (Nottingham Arabidopsis Stock Centre) were screened using the gene-specific primers PGP4-int-Fw (5′-CAGAGGAAACAGCCGCC-3′), PGP4-int-Rv (5′-GCTACTTGGCTTGCTTCCCCGTAC-3′), pgp4-3 forward (5′-CGAAAAGCCCAATAACGATTT-3′), pgp4-3 reverse (5′-CCTGAAGTACCCGAGCTGCAA-3′), pgp4-4 forward (5′-TCTGCAACAATGCAATCACCG-3′), and pgp4-4 reverse (5′-CAGCGCTGATGCAAAGGTAAAC-3′). PCR was performed using these primers in combination with LBb1 (5′-GCGTGGACCGCTTGCTGCAACT-3′) or LBa1 (5′-TGGTTCACGTAGTGGGCCATCG-3′), which anneals to the left border of the T-DNA insertion, according to the protocol published on the Internet (http://signal.salk.edu/tabout.html). We verified three homozygous T-DNA insertional lines: pgp4-1 (SALK_063720), pgp4-3 (SALK_067653), and pgp4-4 (SALK_088311).

Production of Recombinant Baculovirus

The recombinant donor plasmids were used to transform Escherichia coli–competent DH10Bac cells (Invitrogen), which contained the parent bacmid and a helper plasmid. Recombinant bacmids were selected on Luria-Bertani plates containing 50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline, 100 μg/mL X-Gluc, and 40 μg/mL isopropylthio-d-galactoside. Sf9 cells were transfected with the purified recombinant bacmids using Cellfectin reagent (Invitrogen). Three days later, supernatant was recovered and used to infect fresh Sf9 cells. Finally, amplified virus supernatants were prepared after two cycles of this infection procedure. Their titer was determined and the virus was stored at 4°C. Sf9 cells were infected at a cell density of 0.6 × 106 cells/mL in 25-cm2 tissue culture flasks. Cells were collected and disrupted via Dounce homogenization to prepare membranes that contain the recombinant PGP4.

Photoaffinity Labeling of ABC Proteins with 8-Azido-ATP

Ten micrograms of membrane proteins from Sf9 cells was incubated in a buffer mixture composed of 10 μM 8-azido-[α-32P]ATP (specific activity, 12.5 Ci/mmol; Affinity Labeling Technologies), 2 mM ouabain, 0.1 mM EGTA, 3 mM MgSO4, 40 mM Tris-Cl, pH 7.5, and 0.2 mM orthovanadate in a total volume of 8 μL for 10 min at 37°C (Senior et al., 1995). The reactions were stopped by the addition of 400 μL of ice-cold TGM buffer (40 mM Tris-Cl, pH 7.5, 0.1 mM EGTA, and 1 mM MgSO4), and the membrane proteins were sedimented by centrifugation (15,000g, 10 min, 4°C) to separate unbound ATP. Pellets were washed with fresh ice-cold TGM buffer, then irradiated with UV light at 254 nm (5.5 mW/cm2) in 8 μL of TGM buffer for 5 min on ice. Samples were electrophoresed on a 7% SDS-polyacrylamide gel and autoradiographed. The radioactivity trapped by ABC protein was analyzed with the radioimaging analyzer BAS 1800 (Fuji Film).

Whole Plant Auxin Transport and Uptake Assays

Basipetal auxin transport assays in shoot tissues were performed as described previously (Geisler et al., 2003). For root transport assays, intact light-grown Arabidopsis seedlings were treated with a 0.1-μL microdroplet of 1 μM auxin at the root apical meristem using techniques described by Geisler et al. (2005), and root segments of 2 mm were collected at 2, 4, and 6 mm from the root tip.

Root auxin uptake assays were performed as described by Murphy et al. (2000) except that incubations used 2-mm root tip sections and [3H]IAA as in transport assays. Sections were collected whole after 20 min and washed three times in 100 μM cold IAA before scintillation counting.

HeLa Cell Assays

PGP4 was expressed in mammalian HeLa cells using a vaccinia virus cotransfection system as described previously (Geisler et al., 2005). Full-length PGP4 cDNA was subcloned into the multiple cloning site of the pTM1 vector (Hrycyna et al., 1998). For pTM1-PGP4, a PGP4 PCR fragment containing XmaI-XmaI restriction sites was generated using the following primers: PGP4 5′, 5′-TCCCCCGGGGCATGGCTTCAGAGAGCGGC-3′; and PGP4 3′, 5′-CGCCCCGGGAGCGTAATCTGGTACGTCGTAAGAAGCCGCGGTTAGAT-3′. Assays for the accumulation of radiolabeled substrates were performed according to the method described by Geisler et al. (2005). For inhibitor studies, cells were incubated with radiolabeled IAA in the presence of 10 μM NPA, 200 nM quercetin, 1 μM cyclosporin A, or 5 μM verapamil.

Flavonoid Localization in pgp4

Seedlings were grown on 0.25× MS, 1% phytoagar plates and vertical mesh transfer (Murphy and Taiz, 1995). Four-day seedlings were stained with diphenylboric acid 2-aminoethyl ester and imaged using epifluorescence microscopy as described previously (Murphy et al., 2000; Peer et al., 2001).

Auxin Quantitations

Quantitations of auxin in seedling tissues were performed as described previously (Geisler et al., 2005).

Root Gravitropism Assay

Seedlings were grown on half-strength MS medium with 1% (w/v) phytoagar, pH 4.85, for 5 d. Plates were kept in a vertical position. After reorienting the plates by 90°, the root tip position was marked every 3 h over a 15-h time period, and again at 27 and 30 h. The angles of curvature were measured with the Scion Image Beta 4.02 program (Scion), and the data were analyzed by Microsoft Excel.

Quantitative Real-Time PCR

Seedlings were grown for 5 d on half-strength MS medium, pH 5.8, under 16 h of light and then treated with 1 μM IAA, kinetin, or abscisic acid. Whole seedlings, shoots, and roots were harvested for RNA isolation at 0, 2, 4, 6, 8, and 12 h after treatment. RNA isolation and quantitative real-time PCR were performed as described previously (Peer et al., 2004) except that 25 nM fluorescein was added for normalization. The primers used were PGP4 forward (5′-AAATGCGGATATGATCGCTG-3′), PGP4 reverse (5′-TGAACGACGTTTCCGTCAAT-3′), β-tubulin forward (5′-TGGGAACTCTGCTCATATCT-3′), and β-tubulin reverse (5′-GAAAGGAATGAGGTTCACTG-3′). The primer efficiencies for PGP4 and β-tubulin were equal (1.95 and 1.96, respectively).

Accession Numbers

Sequence data for the genes and mutants used in this study can be found in the GenBank/EMBL data libraries under the following accession numbers: PGP4 (At2g47000), PIN1 (At1g73590), PIN2 (At5g57090), β-tubulin (At5g12250), pgp4-1 (SALK_063720), pgp4-3 (SALK_067653), and pgp4-4 (SALK_088311).

Supplemental Data

The following materials are available in the online version of this article.

  • Supplemental Table 1. Root Lengths of Wild-Type, pgp4-1, pgp4-3, and pgp4-4 7-d Seedlings.

  • Supplemental Figure 1. Regulation of PGP4 and PGP21 Gene Expressions by Chemical Stresses.

  • Supplemental Figure 2. Complementation of pgp4-1.

  • Supplemental Figure 3. PIN2 Localization in pgp4-1 and PGP4OX.

  • Supplemental Figure 4. PIN1 Localization in pgp4-1 and PGP4OX.

  • Supplemental Figure 5. Flavonol Accumulations in pgp4.

Acknowledgments

We thank Markus Geisler, Enrico Martinoia, Dan Lewis, and Edgar Spalding for discussions of the data and sharing their data with us before publication. We thank Malcolm Bennett for helpful discussions. We thank Jiri Friml and Klaus Palme for PIN1 antisera. We thank M. Boutry (Université Catholique de Louvain) for providing anti-H+-ATPase antisera, N. Koizumi (Nara Institute of Science and Technology) for providing anti-BiP antisera, and M.H. Sato (Kyoto University) and M. Maeshima (Nagoya University) for anti-H+-PPase antisera. We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. The pGWB vectors and A. tumefaciens GV3101 (pMP90) were generous gifts from T. Nakagawa (Shimane University) and T. Shikanai (Kyushu University), respectively. Funding for this project was provided by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (Grants 15031217 and 00L01605 to K.Y.) the Uehara Foundation (to K.Y.), and the National Science Foundation (to A.S.M.).

Footnotes

  • The authors 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.plantcell.org) are: Angus S. Murphy (murphy{at}purdue.edu) and Kazufumi Yazaki (yazaki{at}rish.kyoto-u.ac.jp).

  • Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.105.035816.

  • ↵ w⃞ Online version contains Web-only data.

  • ↵1 These authors contributed equally to this work.

  • Received July 6, 2005.
  • Revised September 22, 2005.
  • Accepted September 27, 2005.
  • Published October 21, 2005.

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PGP4, an ATP Binding Cassette P-Glycoprotein, Catalyzes Auxin Transport in Arabidopsis thaliana Roots
Kazuyoshi Terasaka, Joshua J. Blakeslee, Boosaree Titapiwatanakun, Wendy A. Peer, Anindita Bandyopadhyay, Srinivas N. Makam, Ok Ran Lee, Elizabeth L. Richards, Angus S. Murphy, Fumihiko Sato, Kazufumi Yazaki
The Plant Cell Nov 2005, 17 (11) 2922-2939; DOI: 10.1105/tpc.105.035816
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