pgp4, an atp binding cassette p-glycoprotein, catalyzes ...pgp4, an atp binding cassette...
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PGP4, an ATP Binding Cassette P-Glycoprotein, CatalyzesAuxin Transport in Arabidopsis thaliana Roots W
Kazuyoshi Terasaka,a,1 Joshua J. Blakeslee,b,1 Boosaree Titapiwatanakun,b,1 Wendy A. Peer,b
Anindita Bandyopadhyay,b Srinivas N. Makam,b Ok Ran Lee,b Elizabeth L. Richards,b
Angus S. Murphy,b,2 Fumihiko Sato,a and Kazufumi Yazakic
a Laboratory of Molecular and Cellular Biology of Totipotency, Division of Integrated Life Science, Graduate School
of Biostudies, Kyoto University, Kitashirakawa, Kyoto 606-8502, Japanb Department of Horticulture, Purdue University, West Lafayette, Indiana 47907-2010c Laboratory of Plant Gene Expression, Research Institute for Sustainable Humanosphere, Kyoto University,
Gokasho Uji 611-0011, Japan
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.
Rootgravitropicbendingandelongationaswell as lateral root formationwere reduced inpgp4mutantscomparedwith thewild
type.pgp4exhibited reducedbasipetal auxin transport in roots andasmall decrease in shoot-to-root transport consistentwith
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 super-
family have been identified in all prokaryotic and eukaryotic or-
ganisms (Henikoff et al., 1997). AlthoughABC transporters are often
associated with the export of cytotoxic compounds (Gottesman
and Pastan, 1993), they also function in mating hormone trans-
port (Ketchumet al., 2001), ATP/ADP sensing (Bryan andAguilar-
Bryan, 1999), channel regulation (Anderson et al., 1991), and iron
homeostasis (Kushnir et al., 2001). ABC transporters have also
been shown tomediate 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 arti-
ficial 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 resistanceprotein (PDR) subfamily, suchasPDR1
from Nicotiana plumbaginifolia (Jasinski et al., 2001; Stukkens
et al., 2005) andTURION2 fromSpirodela polyrrhiza (vandenBrule
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 re-
ferred to as PGPs) characterized to date have been implicated in
the transport of the phytohormone auxin: PGP1 and PGP19were
identified as the principal Arabidopsis thaliana plasma mem-
brane (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
1These authors contributed equally to this work.2 To whom correspondence should be addressed. E-mail [email protected]; fax 765-494-0391.The authors responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) are: Angus S. Murphy([email protected]) and Kazufumi Yazaki ([email protected]).WOnline version contains Web-only data.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.105.035816.
The Plant Cell, Vol. 17, 2922–2939, November 2005, www.plantcell.orgª 2005 American Society of Plant Biologists
Figure 1. Expression Profile of PGP4 in Arabidopsis.
(A) Organ-specific expression of PGP4. Total RNA (5 mg) 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 PGP4mRNA
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 mM
1-naphthaleneacetic acid (NAA), 10 mM IAA, 10 mM 2,4-D, 10 mM 6-benzyladenine (BA), 10 mM kinetin (kin.), 10 mM abscisic acid (ABA), 10 mM gibberellic
acid (GA3), 10 mM brassinolide (BL), 100 mM methyl jasmonate (JA), 100 mM salicylic acid (SA), and 10 mM stigmasterol (St) and at 48C (cold)/low light
and dark (dark). Total RNA (8 mg) prepared fromwhole seedlings was probed with a 32P-labeled PGP4 fragment (top gel). Loading controls are shown by
b-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.
PGP4 Catalyzes Auxin Transport in Roots 2923
(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 syn-
thetic 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 in-
dicates a role for PGPs in auxin efflux, structural differences
observed among the 21 expressedmembers of the PGP family of
Arabidopsis (Jasinski et al., 2003; E.L. Richards andA.S.Murphy,
unpublished data) suggest that energy-dependent PGP media-
tion 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 ber-
berine 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 in-
teractive 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 trans-
porter, we have analyzed PGP4 expression and characterized
PGP4 protein function.
RESULTS
Expression of PGP4
RNA gel blot analysis with aPGP4-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).
In 14-d-old seedlings, the effects of hormones, signaling
molecules, and abiotic factors on PGP4 expression were ana-
lyzed: PGP4 expression was upregulated by treatment with
natural and synthetic auxins and the cytokinin kinetin, whereas
abscisic acid and cold treatments downregulated PGP4 expres-
sion (Figure 1B). A time course of PGP4 expression in 5-d seed-
lings 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 (P¼ 0.002) (Table 1). The abundances of PGP4
transcripts in Figure 1 were confirmed via radioimaging, semi-
quantitative 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).
Wild-type plants were transformed with ProPGP4:GUS, con-
sisting of a 2.2-kb sequence upstream of the PGP4 start site
fused to a b-glucuronidase (GUS) reporter gene, and the T2 or
T3 generations were histochemically stained with 5-bromo-4-
chloro-3-indolyl-b-glucuronic acid (X-Gluc) at different develop-
mental 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,
PGP4was 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).
Table 1. Time Course of PGP4 Expression in Response to Various Hormones via Quantitative Real-Time PCR Analysis
Time (h)
Hormone 0 2 4 6 8 12
IAA 1.0 6 0.8 3.0 6 0.4 3.3 6 0.2 2.3 6 0.7 22 6 2.0a 44 6 2.5a
Abscisic acid 1.0 6 0.8 7.6 6 2.2a 2.0 6 0.5 8.4 6 0.7a 2.5 6 0.7 5.0 6 0.5a
Kinetin 1.0 6 0.8 0.9 6 0.6 4.3 6 1.5a 5.1 6 1.6a 1.9 6 0.3 3.0 6 1.1
PGP4 expression was measured relative to time 0 with b-tubulin as the endogenous correction factor. Values shown are means 6 SD.a Significantly different from time 0 as determined by analysis of variance, followed by Dunnett’s posthoc analysis (P < 0.05).
2924 The Plant Cell
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.
PGP4 Catalyzes Auxin Transport in Roots 2925
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 amixture of PGP1, PGP19, and PGP4
(Table 2). Sucrose density gradient fractionation of microsomal
membranes suggested that PGP4 is found inmultiple membrane
fractions (Figure 3A). However, the PGP4peak fraction coincided
with that of the PM Hþ-ATPase W1D (Figure 3A) to a greater
extent than with the endoplasmic reticulum lumenal marker bind-
ing protein (BiP) (Haas, 1994) and the Hþ-pyrophosphatase (Hþ-
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 Hþ-ATPase W1D
(Figure 3B). This distribution is consistent with a recent report of
PGP4,Hþ-ATPase, andBiP retention in detergent-resistantmicro-
domains (Borner et al., 2005). PGPs have also been localized
in detergent-resistantmicrodomains involved in vesicular cycling
between endomembrane compartments and the PM in mam-
mals (Kipp and Arias, 2002; Brown and London, 1998a, 1998b).
Both PM and endomembrane localization of PGP4 was appar-
ent 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 antibodypenetration 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 PGP4was 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 pgp4Mutants
Five T-DNA insertional lines in the PGP4 genewere 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
seedlingmembranes. The antisera also did not cross-react with the
closest homolog, PGP21 (;129 kD), which is expressed in seed-
lings at levels similar to those ofPGP4 (Figure 4B; seeSupplemental
Table 2. PGPs Identified in NPA Binding Fractions
Band Size
125 to 140 kD Protein Sequence Data
PGP4 EEEEEVKa–GYTGGQVLNIIIAVLTG–
VLLLDEATSALDAESERb
PGP10 PSVSFLK–LFSFADFYDXVLc–LLEPSE–
VVQQALDRc
PGP19 FDYLLMFVGSLGAIVHGSS–GFAGDTAK–
DGATESEVIDAA
Microsomal proteins were isolated and subjected to NPA-affinity chro-
matography, as described by Murphy et al. (2002). Select protein bands
from SDS-PAGE were isolated and sequenced. Unique sequence data
were used to identify proteins present in each band. –, break in amino
acid sequence; X, indeterminate amino acid.a Sequence in PGP4 and also in four other unrelated sequences.b Sequence also found in PGP17.c Sequence also found in PGP2.
Figure 2. (continued).
(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 ¼ 1 mm in (A) to (D), 2.5 cm in (E), and 50 mm in (F) to (K).
2926 The Plant Cell
Figure 3. Subcellular Localization of PGP4.
(A) Membrane fractionation and immunodetection of PGP4. Fractionation of total microsomes from Arabidopsis seedlings was done on a non-
continuous sucrose gradient consisting of 20, 30, 40, and 50% (w/v) sucrose. Membrane fractions were collected from the interfaces between different
PGP4 Catalyzes Auxin Transport in Roots 2927
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).
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 atmoderate light levels
(100 to 120mmol�m�2�s�1) using sucrose concentrations of 0.5 to
1% (see Methods). The conditions under which these pheno-
types are most visible in pgp4 (Figures 4C and 4D; see Supple-
mental 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 ob-
served (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 seed-
lings and adult plants. The lack of a visible phenotype of pgp4-1
mutants in aerial tissues may also result from functional re-
dundancy in these tissues because other PGPs implicated in
auxin transport (such as PGP19, PGP6, and PGP10) are ex-
pressed 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.
PGP4Overexpression
PGP4 was also overexpressed under the control of a 35S pro-
moter (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 mul-
tiple 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 posttran-
scriptionally 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.
Figure 3. (continued).
sucrose concentrations and analyzed via SDS-PAGE and protein gel blotting. Blots were probed with antisera to PGP4, PM Hþ-ATPase (W1D),
multicompartment Hþ-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 mg per lane) were blotted and probed with antisera to PGP4, PM Hþ-ATPase, multicompartment Hþ-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 ¼ 100 mm.
(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).
2928 The Plant Cell
Figure 4. Genomic Structure of PGP4 and Phenotypes of pgp4.
PGP4 Catalyzes Auxin Transport in Roots 2929
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 thesameepidermal andcorticalcells asPGP4 (Chenetal., 1998;
Luschnig et al., 1998; Muller 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 con-
centrations 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 trans-
port 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, aggre-
gations 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).
The light/sucrose dependence of pgp4 seedling growth phe-
notypes suggested that differences in basipetal auxin transport
observed between pgp4 and thewild typewould decrease under
light conditions of >120 mmol�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 chemios-
motically 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 pHwould 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 locali-
zation of PGP4 seen in root transition/distal elongation zone cells
has either a negative or a negligible impact on basipetal trans-
port. 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 PGPHsMDR1 (Ueda et al., 1997), this basal
level of PGP4 ATPase activity required both Mg2þ and vanadate
(Figure 6B).
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
Figure 4. (continued).
(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 mg 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 ¼ 1 cm.
(D) pgp4-1 seedlings have shorter roots. Asterisk indicates statistically significant difference in root length between the wild type and the pgp4-1mutant
(P < 0.01, n ¼ 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 ¼ 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 ¼ 3). Asterisks indicate significant difference from the wild type by Student’s t test (P < 0.05).
2930 The Plant Cell
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 ¼ 2 groups of 10 seedlings each. Asterisks indicate significant difference from the wild type by Student’s t test (P # 0.05).
PGP4 Catalyzes Auxin Transport in Roots 2931
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 comparedwith 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).
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, cyclo-
propyl 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 mM) resulted in a 35% 6 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 oxida-
tive auxin breakdown products in addition to IAA (Geisler et al.,
2005). When cells expressing PGP4 were incubated with a mix-
ture 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 rhoda-
mine 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 local-
ization 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 chemios-
motic 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 IAAmust 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 trans-
port 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
Figure 5. (continued).
(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 ¼ 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 ¼ 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 ¼ 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 ¼ 100 mm for (E) and (F).
(F) pgp4-1 seedlings accumulate aggregations of quercetin in the corresponding region of the root elongation zone.
2932 The Plant Cell
(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 gravisti-
mulation (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).
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 mg (lanes 1 to 3) or 30 mg (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 mM 8-azido-[a-32P]ATP in the presence or absence of 3 mM MgSO4 (Mg2þ) and 0.2 mM orthovanadate (Vi) for 10 min at 378C.
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 mM
naphthaleneacetic acid (NAA), 50 mM IAA, 50 mM 2,4-D, 50 mM 6-benzyladenine (BA), 50 mM kinetin (kin.), 50 mM abscisic acid (ABA), 50 mM gibberellic
acid (GA3), 25 mM brassinolide (BL), 50 mM vanillic acid (Va), 50 mM syringic acid (Sa), 50 mM indole-3-propionic acid (IPA), 50 mM NPA, or 50 mM
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.
PGP4 Catalyzes Auxin Transport in Roots 2933
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 un-
derstand 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 localiza-
tion 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 interac-
tions 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 mmol�m�2�s�1 light in a 16-h-light/8-h-
dark cycle at 218C. 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 distilledwater;
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-
strengthMurashige 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.
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 ¼ 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 mM NPA, 200 nM quercetin (Quer.), 1 mM cyclosporin A (CsA), or 5 mM
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 þ inhibitors was compared with empty vector control þ inhibitors.
(B) Immunolocalization of hemagglutinin-tagged PGP4 expressed in HeLa cells. Hemagglutinin-tagged PGP4 was detected with an anti-hemagglutinin
antibody. Bar ¼ 10 mm.
2934 The Plant Cell
To analyze the regulation of PGP4 transcripts by various growth
regulators, seeds were sown onto nylon mesh (20-mm 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 59 untranslated region and the 58-bp 39
untranslated region (forward primer, 59-CGTCTAGAGCTTAGGATCGT-
GAGGTATCTG-39; reverse primer, 59-GCTGAGCTCATATAACGGGG-
CATAATTGAC-39). The underlined positions are nonnative sequences
representing XbaI and SacI restriction sites, respectively. The PCR
product was subcloned into pBluescript II SK� (Stratagene) for sequenc-
ing. The full-length cDNA was transferred into pENTR1A vector (Invitro-
gen) using SalI andNotI 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 ArabidopsisColumbia 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 trans-
fers onto Hybond-Nþ membranes (Amersham Bioscience), and hybrid-
izationwith the 0.7-kbAtPGP4 fragment (positionsþ1989 toþ2711) were
performed using standard procedures. The last stringent wash was
performed with 0.23 SSC (13 SSC is 0.15 M NaCl and 0.015 M sodium
citrate) and 0.1% SDS at 608C.
Promoter:GUS Fusion and GUS Expression Analysis
To create thePGP4 promoter:reporter construct (ProPGP4:GUS), twoPCR
primers flanking the regions from þ15 to �2196 were made (forward
primer, 59-GCTAAGCTTGGTAAAGGATTTGGGTCTATTCG-39; reverse
primer, 59-CTGTCTAGAGCTCTCTGAAGCCATTAGAGTTT-39). The un-
derlined positions are nonnative sequences representingHindIII and XbaI
restriction sites, respectively. PCRwas 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 1mMX-Gluc), and incubated at 378C 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
themanufacturer’s protocol. Tissue sections (10 mm thick) weremounted
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 phenyl-
methylsulfonyl 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 phenyl-
methylsulfonyl fluoride) and centrifuged at 100,000g for 3 h on a non-
continuous 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 parti-
tioning in an aqueous polymer two-phase system, as described pre-
viously (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 mg/mL pyronine Y, and 2% [w/v] SDS) for 10 min at 508C, subjected to
SDS-PAGE (7%gel), and then transferred to an Immobilon polyvinylidene
difluoridemembrane (Millipore). Themembranewas treatedwithBlocking
One (Nacalai Tesque) for blocking, then incubated with primary anti-
bodies 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 Hþ-ATPase, multicompart-
ment Hþ-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 localiza-
tions 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 micros-
copy. 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 pgp4Mutants
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 (59-CAGAGGAAACAGCCGCC-39), PGP4-
int-Rv (59-GCTACTTGGCTTGCTTCCCCGTAC-39), pgp4-3 forward
(59-CGAAAAGCCCAATAACGATTT-39), pgp4-3 reverse (59-CCTGAAG-
TACCCGAGCTGCAA-39), pgp4-4 forward (59-TCTGCAACAATGCAATC-
ACCG-39), and pgp4-4 reverse (59-CAGCGCTGATGCAAAGGTAAAC-39).
PGP4 Catalyzes Auxin Transport in Roots 2935
PCR was performed using these primers in combination with LBb1
(59-GCGTGGACCGCTTGCTGCAACT-39) or LBa1 (59-TGGTTCACGTAG-
TGGGCCATCG-39), 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 mg/mL kanamycin, 7 mg/mL gentami-
cin, 10 mg/mL tetracycline, 100 mg/mL X-Gluc, and 40 mg/mL isopro-
pylthio-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 48C. Sf9 cells were infected at a cell density of 0.63 106 cells/mL
in 25-cm2 tissue culture flasks. Cells were collected and disrupted via
Dounce homogenization to prepare membranes that contain the re-
combinant 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 mM 8-azido-[a-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 mL for 10 min at 378C (Senior et al.,
1995). The reactions were stopped by the addition of 400 mL of ice-cold
TGMbuffer (40mMTris-Cl, pH 7.5, 0.1mMEGTA, and 1mMMgSO4), and
the membrane proteins were sedimented by centrifugation (15,000g,
10 min, 48C) to separate unbound ATP. Pellets were washed with fresh
ice-cold TGMbuffer, then irradiated with UV light at 254 nm (5.5mW/cm2)
in 8 mL 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-mL
microdroplet of 1 mM 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 collectedwhole after 20min
and washed three times in 100 mM 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 59, 59-TCCCCCGGGGCATGGCTTCAGA-
GAGCGGC-39; and PGP4 39, 59-CGCCCCGGGAGCGTAATCTGGTACG-
TCGTAAGAAGCCGCGGTTAGAT-39. Assays for the accumulation of
radiolabeled substrates were performed according to the method de-
scribed by Geisler et al. (2005). For inhibitor studies, cells were incubated
with radiolabeled IAA in the presence of 10 mM NPA, 200 nM quercetin,
1 mM cyclosporin A, or 5 mM verapamil.
Flavonoid Localization in pgp4
Seedlings were grown on 0.253 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 epifluor-
escence 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 908, 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 MSmedium, pH 5.8, under
16 h of light and then treated with 1 mM 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 (59-AAATGCGGATATGATCGCTG-39), PGP4
reverse (59-TGAACGACGTTTCCGTCAAT-39), b-tubulin forward (59-TGG-
GAACTCTGCTCATATCT-39), and b-tubulin reverse (59-GAAAGGAAT-
GAGGTTCACTG-39). The primer efficiencies forPGP4 and b-tubulinwere
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 acces-
sion numbers: PGP4 (At2g47000), PIN1 (At1g73590), PIN2 (At5g57090),
b-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.
2936 The Plant Cell
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 (Universite Catholique de Louvain) for providing anti-Hþ-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-Hþ-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.).
Received July 6, 2005; revised September 22, 2005; accepted
September 27, 2005; published October 21, 2005.
REFERENCES
Alonso, J.M., et al. (2003). Genome-wide insertional mutagenesis of
Arabidopsis thaliana. Science 301, 653–657.
Anderson, M.P., Gregory, R.J., Thompson, S., Souza, D.W., Paul, S.,
Mulligan, R.C., Smith, A.E., and Welsh, M.J. (1991). Demonstration
that CFTR is a chloride channel by alteration of its anion selectivity.
Science 253, 202–205.
Beckers, G., Bendt, A.K., Kramer, R., and Burkovski, A. (2004).
Molecular identification of the urea uptake system and transcriptional
analysis of urea transporter- and urease-encoding genes in Coryne-
bacterium glutamicum. J. Bacteriol. 186, 7645–7652.
Bennett, M.J., Marchant, A., Green, H.G., May, S.T., Ward, S.P.,
Millner, P.A., Walker, A.R., Schulz, B., and Feldmann, K.A. (1996).
Arabidopsis AUX1 gene: A permease-like regulator of root gravitrop-
ism. Science 273, 948–950.
Blakeslee, J.J., Peer, W.A., and Murphy, A.S. (2005a). Auxin transport.
Curr. Opin. Plant Biol. 8, 494–500.
Blakeslee, J.J., Peer, W.A., and Murphy, A.S. (September 22, 2005b).
MDR/PGP auxin transport proteins and endocytotic cycling. In Plant
Cell Monographs, Vol. 1, J. Samaj, F. Baluska, and D. Menzel, eds
(Berlin: Springer-Verlag), doi/10.1007/7089_010.
Borner, G.H., Sherrier, D.J.,Weimar, T., Michaelson, L.V., Hawkins, N.D.,
MacAskill, A., Napier, J.A., Beale, M.H., Lilley, K.S., and Dupree, P.
(2005). Analysis of detergent-resistant membranes in Arabidopsis. Evi-
dence for plasma membrane lipid rafts. Plant Physiol. 137, 104–116.
Brown, D., and London, E. (1998a). Functions of lipid rafts in biological
membranes. Annu. Rev. Cell Dev. Biol. 14, 111–136.
Brown, D., and London, E. (1998b). Structure and origin of ordered
lipid domains in biological membranes. J. Membr. Biol. 164, 103–114.
Brown, J., Gilliland, S., Ruiz-Albert, J., and Holden, D. (2002).
Characterization of pit, a Streptococcus pneumoniae iron uptake
ABC transporter. Infect. Immun. 70, 4389–4398.
Bryan, J., and Aguilar-Bryan, L. (1999). Sulfonylurea receptors: ABC
transporters that regulate ATP-sensitive Kþ channels. Biochim. Bio-
phys. Acta 1461, 285–303.
Chen, R.J., Hilson, P., Sedbrook, J., Rosen, E., Caspar, T., and
Masson, P.H. (1998). The Arabidopsis thaliana AGRAVITROPIC 1
gene encodes a component of the polar-auxin-transport efflux carrier.
Proc. Natl. Acad. Sci. USA 95, 15112–15117.
Clough, S.J., and Bent, A.F. (1998). Floral dip: A simplified method
for Agrobacterium-mediated transformation of Arabidopsis thaliana.
Plant J. 16, 735–743.
Cortes-Selva, F., Munoz-Martinez, F., Ilias, A., Jimenez, A., Varadi,
A., Gamarro, F., and Castanys, S. (2005). Functional expression of
a multidrug P-glycoprotein transporter of Leishmania. Biochem. Bio-
phys. Res. Commun. 329, 502–507.
Danese, I., Haine, V., Delrue, R.M., Tibor, A., Lestrate, P., Stevaux,
O., Mertens, P., Paquet, J.Y., Godfroid, J., De Bolle, X., and
Letesson, J.J. (2004). The ton system, an ABC transporter, and
a universally conserved GTPase are involved in iron utilization by
Brucella melitensis 16M. Infect. Immun. 72, 5783–5790.
De Michelis, M.I., Rasi-Caldogno, F., Pugliarello, M.C., and Olivari,
C. (1996). Fusicoccin binding to its plasma membrane receptor and
the activation of the plasmamembrane Hþ-ATPase. III. Is there a direct
interaction between the fusicoccin receptor and the plasma mem-
brane Hþ-ATPase? Plant Physiol. 110, 957–964.
Dupont, L., Garcia, I., Poggi, M.C., Alloing, G., Mandon, K., and Le
Rudulier, D. (2004). The Sinorhizobium meliloti ABC transporter cho is
highly specific for choline and expressed in bacteroids fromMedicago
sativa nodules. J. Bacteriol. 186, 5988–5996.
Elroy-Stein, O., and Moss, B. (1991). Gene expression using the
vaccinia virus/T7 RNA polymerase hybrid system. In Current Proto-
cols in Molecular Biology, F.M. Ausubel, R. Brent, R.E. Kinston, D.D.
Moore, J.A. Smith, J.C. Seidman, and K. Struhl, eds (New York: John
Wiley & Sons), pp. 16.19.11–16.19.19.
Gaedeke, N., Klein, M., Kolukisaoglu, U., Forestier, C., Muller, A.,
Ansorge, M., Becker, D., Mamnun, Y., Kuchler, K., Schulz, B.,
Mueller-Roeber, B., and Martinoia, E. (2001). The Arabidopsis
thaliana ABC transporter AtMRP5 controls root development and
stomata movement. EMBO J. 20, 1875–1887.
Garcia, O., Bouige, P., Forestier, C., and Dassa, E. (2004). Inventory
and comparative analysis of rice Arabidopsis ATP-binding cassette
(ABC) systems. J. Mol. Biol. 343, 249–265.
Geisler, M., et al. (2005). Cellular export of auxin catalyzed by the
Arabidopsis MDR/PGP transporter AtPGP1. Plant J. http://dx.
doi10.1111/j.1365–313X.2005.02519.x.
Geisler, M., et al. (2003). TWISTED DWARF1, a unique plasma
membrane-anchored immunophilin-like protein, interacts with Arabi-
dopsis multidrug resistance-like transporters AtPGP1 and AtPGP19.
Mol. Biol. Cell 14, 4238–4249.
Gottesman, M.M., and Pastan, I. (1993). Biochemistry of multidrug-
resistance mediated by the multidrug transporter. Annu. Rev. Bio-
chem. 62, 385–427.
Haas, I.G. (1994). BiP (GRP78), an essential HSP70 resident protein in
the endoplasmic reticulum. Experientia 50, 1012–1020.
Henikoff, S., Greene, E.A., Pietrokovski, S., Bork, P., Attwood, T.K.,
and Hood, L. (1997). Gene families: The taxonomy of protein paralogs
and chimeras. Science 278, 609–614.
Hou, G.C., Kramer, V.L., Wang, Y.S., Chen, R.J., Perbal, G., Gilroy,
S., and Blancaflor, E.B. (2004). The promotion of gravitropism in
Arabidopsis roots upon actin disruption is coupled with the extended
alkalinization of the columella cytoplasm and a persistent lateral auxin
gradient. Plant J. 39, 113–125.
Hrycyna, C.A., Ramachandra, M., Pastan, I., and Gottesman, M.M.
(1998). Functional expression of human P-glycoprotein from plasmids
using vaccinia virus-bacteriophage T7 RNA polymerase system.
Methods Enzymol. 292, 456–473.
Ieiri, I., Suzuki, H., Kimura, M., Takane, H., Nishizato, Y., Irie, S.,
Urae, A., Kawabata, K., Higuchi, S., Otsubo, K., and Sugiyama, Y.
(2004). Influence of common variants in the pharmacokinetic genes
(OATP-C, UGT1A1, and MRP2) on serum bilirubin levels in healthy
subjects. Hepatol. Res. 30, 91–95.
Ishikawa, H., and Evans, M.L. (1997). Novel software for analysis of
PGP4 Catalyzes Auxin Transport in Roots 2937
root gravitropism: Comparative response patterns of Arabidopsis
wild-type and axr1 seedlings. Plant Cell Environ. 20, 919–928.
Jasinski, M., Ducos, E., Martinoia, E., and Boutry, M. (2003). The ATP-
binding cassette transporters: Structure, function, and gene family com-
parison between rice and Arabidopsis. Plant Physiol. 131, 1169–1177.
Jasinski, M., Stukkens, Y., Degand, H., Purnelle, B., Marchand-
Brynaert, J., and Boutry, M. (2001). A plant plasma membrane ATP
binding cassette-type transporter is involved in antifungal terpenoid
secretion. Plant Cell 13, 1095–1107.
Ketchum, C.J., Schmidt, W.K., Rajendrakumar, G.V., Michaelis, S.,
and Maloney, P.C. (2001). The yeast a-factor transporter Ste6p,
a member of the ABC superfamily, couples ATP hydrolysis to
pheromone export. J. Biol. Chem. 276, 29007–29011.
Kipp, H., and Arias, I. (2002). Trafficking of canalicular ABC trans-
porters in hepatocytes. Annu. Rev. Physiol. 64, 595–608.
Klein, M., Geisler, M., Suh, S.J., Kolukisaoglu, H.U., Azevedo, L.,
Plaza, S., Curtis, M.D., Richter, A., Weder, B., Schulz, B., and
Martinoia, E. (2004). Disruption of AtMRP4, a guard cell plasma mem-
brane ABCC-type ABC transporter, leads to deregulation of stomatal
opening and increased drought susceptibility. Plant J. 39, 219–236.
Klein, M., Perfus-Barbeoch, L., Frelet, A., Gaedeke, N., Reinhardt,
D., Mueller-Roeber, B., Martinoia, E., and Forestier, C. (2003). The
plant multidrug resistance ABC transporter AtMRP5 is involved in
guard cell hormonal signaling and water use. Plant J. 33, 119–129.
Kushnir, S., Babiychuk, E., Storozhenko, S., Davey, M.W.,
Papenbrock, J., De Rycke, R., Engler, G., Stephan, U.W., Lange,
H., Kispal, G., Lill, R., and Van Montagu, M. (2001). A mutation of the
mitochondrial ABC transporter Sta1 leads to dwarfism and chlorosis
in the Arabidopsis mutant starik. Plant Cell 13, 89–100.
Lamarque, M., Charbonnel, P., Aubel, D., Piard, J.C., Atlan, D., and
Juillard, V. (2004). A multifunction ABC transporter (Opt) contributes
to diversity of peptide uptake specificity within the genus Lactococ-
cus. J. Bacteriol. 186, 6492–6500.
Le Bot, J., and Kirkby, E.A. (1992). Diurnal uptake of nitrate and
potassium during the vegetative growth of tomato plants. J. Plant
Nutr. 15, 247–264.
Li, J., et al. (2005). Arabidopsis Hþ-PPase AVP1 regulates auxin
mediated organ development. Science 310, 121–125.
Lu, Y.P., Li, Z.S., Drozdowicz, Y.M.,Hortensteiner, S.,Martinoia, E., and
Rea, P.A. (1998). AtMRP2, an Arabidopsis ATP binding cassette trans-
porter able to transport glutathione S-conjugates and chlorophyll catab-
olites: Functional comparisons with AtMRP1. Plant Cell 10, 267–282.
Lu, Y.P., Li, Z.S., and Rea, P.A. (1997). AtMRP1 gene of Arabidopsis
encodes a glutathione S-conjugate pump: Isolation and functional
definition of a plant ATP-binding cassette transporter gene. Proc.
Natl. Acad. Sci. USA 94, 8243–8248.
Luschnig, C., Gaxiola, R.A., Grisafi, P., and Fink, G.R. (1998). EIR1,
a root-specific protein involved in auxin transport, is required for
gravitropism in Arabidopsis thaliana. Genes Dev. 12, 2175–2187.
Marchant, A., Kargul, J., May, S.T., Muller, P., Delbarre, A., Perrot-
Rechenmann, C., and Bennett, M.J. (1999). AUX1 regulates root
gravitropism in Arabidopsis by facilitating auxin uptake within root
apical tissues. EMBO J. 18, 2066–2073.
Martinoia, E., Grill, E., Tommasini, R., Kreuz, K., and Amrhein, N.
(1993). ATP-dependent glutathione S-conjugate export pump in the
vacuolar membrane of plants. Nature 364, 247–249.
Martinoia, E., Klein, M., Geisler, M., Bovet, L., Forestier, C.,
Kolukisaoglu, U., Muller-Rober, B., and Schulz, B. (2002). Multi-
functionality of plant ABC transporters—More than just detoxifiers.
Planta 214, 345–355.
Moss, B. (1991). Vaccinia virus—A tool for research and vaccine
development. Science 252, 1662–1667.
Multani, D.S., Briggs, S.P., Chamberlin, M.A., Blakeslee, J.J.,
Murphy, A.S., and Johal, G.S. (2003). Loss of an MDR transporter
in compact stalks of maize br2 and sorghum dw3 mutants. Science
302, 81–84.
Muller, A., Guan, C., Galweiler, L., Tanzler, P., Huijser, P., Marchant,
A., Parry, G., Bennett, M., Wisman, E., and Palme, K. (1998). AtPIN2
defines a locus of Arabidopsis for root gravitropism control. EMBO J.
17, 6903–6911.
Murphy, A., Peer, W.A., and Taiz, L. (2000). Regulation of auxin
transport by aminopeptidases and endogenous flavonoids. Planta
211, 315–324.
Murphy, A., and Taiz, L. (1995). A new vertical mesh transfer technique
for metal-tolerance studies in Arabidopsis—Ecotypic variation and
copper-sensitive mutants. Plant Physiol. 108, 29–38.
Murphy, A., and Taiz, L. (1999a). Naphthylphthalamic acid is enzymat-
ically hydrolyzed at the hypocotyl-root transition zone and other tis-
sues of Arabidopsis seedlings. Plant Physiol. Biochem. 37, 413–430.
Murphy, A., and Taiz, L. (1999b). Localization and characterization of
soluble and plasma membrane aminopeptidase activities in Arabi-
dopsis seedlings. Plant Physiol. Biochem. 37, 431–443.
Murphy, A.S., Hoogner, K.R., Peer, W.A., and Taiz, L. (2002).
Identification, purification, and molecular cloning of N-1-naphthylph-
thalmic acid-binding plasma membrane-associated aminopeptidases
from Arabidopsis. Plant Physiol. 128, 935–950.
Noh, B., Bandyopadhyay, A., Peer, W.A., Spalding, E.P., and
Murphy, A.S. (2003). Enhanced gravi- and phototropism in plant
mdr mutants mislocalizing the auxin efflux protein PIN1. Nature 423,
999–1002.
Noh, B., Murphy, A.S., and Spalding, E.P. (2001). Multidrug resis-
tance–like genes of Arabidopsis required for auxin transport and
auxin-mediated development. Plant Cell 13, 2441–2454.
Peer, W.A., Bandyopadhyay, A., Blakeslee, J.J., Makam, S.N., Chen,
R.J., Masson, P.H., and Murphy, A.S. (2004). Variation in expression
and protein localization of the PIN family of auxin efflux facilitator
proteins in flavonoid mutants with altered auxin transport in Arabi-
dopsis thaliana. Plant Cell 16, 1898–1911.
Peer, W.A., Brown, D.E., Tague, B.W., Muday, G.K., Taiz, L., and
Murphy, A.S. (2001). Flavonoid accumulation patterns of transparent
testa mutants of Arabidopsis. Plant Physiol. 126, 536–548.
Perez-Victoria, J.M., Chiquero, M.J., Conseil, G., Dayan, G., Di Pietro,
A., Barron, D., Castanys, S., and Gamarro, F. (1999). Correlation
between the affinity of flavonoids binding to the cytosolic site of
Leishmania tropica multidrug transporter and their efficiency to revert
parasite resistance to daunomycin. Biochemistry 38, 1736–1743.
Pighin, J.A., Zheng, H.Q., Balakshin, L.J., Goodman, I.P., Western,
T.L., Jetter, R., Kunst, L., and Samuels, A.L. (2004). Plant cuticular
lipid export requires an ABC transporter. Science 306, 702–704.
Rao, U. (1998). Drug binding and nucleotide hydrolyzability are essential
requirements in the vanadate-induced inhibition of the human
P-glycoprotein ATPase. Biochemistry 37, 14981–14988.
Rea, P.A. (1999). MRP subfamily ABC transporters from plants and
yeast. J. Exp. Bot. 50, 895–913.
Rea, P.A., Li, Z.S., Lu, Y.P., Drozdowicz, Y.M., and Martinoia, E.
(1998). From vacuolar GS-X pumps to multispecific ABC transporters.
Annu. Rev. Plant Physiol. 49, 727–760.
Sakai, K., Shitan, N., Sato, F., Ueda, K., and Yazaki, K. (2002).
Characterization of berberine transport into Coptis japonica cells and
the involvement of ABC protein. J. Exp. Bot. 53, 1879–1886.
Sanchez-Fernandez, R., Davies, T.G.E., Coleman, J.O.D., and Rea,
P.A. (2001). The Arabidopsis thaliana ABC protein superfamily,
a complete inventory. J. Biol. Chem. 276, 30231–30244.
Santelia, D., Vincenzetti, V., Azzarello, E., Bovet, L., Fukao, Y.,
Duchtig, P., Mancuso, S., Martinoia, E., and Geisler, M. (2005).
2938 The Plant Cell
MDR-like ABC transporter AtPGP4 is involved in auxin-mediated
lateral root and root hair development. FEBS Lett. 579, 5399–5406.
Sarkadi, B., Price, E.M., Boucher, R.C., Germann, U.A., and
Scarborough, G.A. (1992). Expression of the human multidrug
resistance cDNA in insect cells generates a high-activity drug-
stimulated membrane ATPase. J. Biol. Chem. 267, 4854–4858.
Senior, A.E., al-Shawi, M.K., Urbatsch, I.L. (1995). The catalytic cycle
of P-glycoprotein. FEBS Lett. 377, 285–289.
Shitan, N., Bazin, I., Dan, K., Obata, K., Kigawa, K., Ueda, K., Sato,
F., Forestier, C., and Yazaki, K. (2003). Involvement of CjMDR1,
a plant multidrug-resistance-type ATP-binding cassette protein, in
alkaloid transport in Coptis japonica. Proc. Natl. Acad. Sci. USA 100,
751–756.
Sidler, M., Hassa, P., Hasan, S., Ringli, C., and Dudler, R. (1998).
Involvement of an ABC transporter in a developmental pathway
regulating hypocotyl cell elongation in the light. Plant Cell 10, 1623–
1636.
Smith, A.P., Nourizadeh, S.D., Peer, W.A., Xu, J., Bandyopadhyay,
A., Murphy, A.S., and Goldsbrough, P.B. (2003). Arabidopsis
AtGSTF2 is regulated by ethylene and auxin, and encodes a
glutathione S-transferase that interacts with flavonoids. Plant J. 36,
433–442.
Stewart, J.D., and Lieffers, V.J. (1994). Diurnal cycles of rhizosphere
acidification by Pinus contorta seedlings. Plant Soil 162, 299–302.
Stukkens, Y., Bultreys, A., Grec, S., Trombik, T., Vanham, D., and
Boutry, M. (2005). NpPDR1, a pleiotropic drug resistance-type ATP-
binding cassette transporter from Nicotiana plumbaginifolia, plays
a major role in plant pathogen defense. Plant Physiol. 139, 341–352.
Swarup, R., Friml, J., Marchant, A., Ljung, K., Sandberg, G., Palme,
K., and Bennett, M. (2001). Localization of the auxin permease AUX1
suggests two functionally distinct hormone transport pathways oper-
ate in the Arabidopsis root apex. Genes Dev. 15, 2648–2653.
Swarup, R., et al. (2004). Structure-function analysis of the presumptive
Arabidopsis auxin permease AUX1. Plant Cell 16, 3069–3083.
Terasaka, K., Shitan, N., Sato, F., Maniwa, F., Ueda, K., and Yazaki, K.
(2003). Application of vanadate-induced nucleotide trapping to plant
cells for detection of ABC proteins. Plant Cell Physiol. 44, 198–200.
Theodoulou, F.L. (2000). Plant ABC transporters. Biochim. Biophys.
Acta 1465, 79–103.
Tommasini, R., Vogt, E., Fromenteau, M., Hortensteiner, S., Matile,
P., Amrhein, N., and Martinoia, E. (1998). An ABC-transporter of
Arabidopsis thaliana has both glutathione-conjugate and chlorophyll
catabolite transport activity. Plant J. 13, 773–780.
Ueda, K., Inagaki, N., and Seino, S. (1997). MgADP antagonism
to Mg2þ-independent ATP binding of the sulfonylurea receptor
SUR1. J. Biol. Chem. 272, 22983–22986.
Urbatsch, I.L., Sankaran, B., Weber, J., and Senior, A.E. (1995).
P-glycoprotein is stably inhibited by vanadate-induced trapping of
nucleotide at a single catalytic site. J. Biol. Chem. 270, 19383–19390.
van den Brule, S., Muller, A., Fleming, A.J., and Smart, C.C. (2002).
The ABC transporter SpTUR2 confers resistance to the antifungal
diterpene sclareol. Plant J. 30, 649–662.
Yazaki, K. (2005). Transporters of secondary metabolites. Curr. Opin.
Plant Biol. 8, 301–307.
Yazaki, K., Shitan, N., Takamatsu, H., Ueda, K., and Sato, F. (2001). A
novel Coptis japonica multidrug-resistant protein preferentially
expressed in the alkaloid-accumulating rhizome. J. Exp. Bot. 52,
877–879.
PGP4 Catalyzes Auxin Transport in Roots 2939
DOI 10.1105/tpc.105.035816; originally published online October 21, 2005; 2005;17;2922-2939Plant Cell
Sato and Kazufumi YazakiBandyopadhyay, Srinivas N. Makam, Ok Ran Lee, Elizabeth L. Richards, Angus S. Murphy, Fumihiko
Kazuyoshi Terasaka, Joshua J. Blakeslee, Boosaree Titapiwatanakun, Wendy A. Peer, Anindita Rootsthaliana
ArabidopsisPGP4, an ATP Binding Cassette P-Glycoprotein, Catalyzes Auxin Transport in
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Supplemental Data /content/suppl/2005/10/05/tpc.105.035816.DC1.html
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