An Aluminum-Activated Citrate Transporter in Barley
Jun Furukawa1, Naoki Yamaji1, Hua Wang1, Namiki Mitani1, Yoshiko Murata2, Kazuhiro Sato1,
Maki Katsuhara1, Kazuyoshi Takeda1and Jian Feng Ma1, ?
1Research Institute for Bioresources, Okayama University, Chuo, Kurashiki, Okayama, 710-0046 Japan
2Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, 618-8503 Japan
Soluble ionic aluminum (Al) inhibits root growth and
reduces crop production on acid soils. Al-resistant cultivars of
barley (Hordeum vulgare L.) detoxify Al by secreting citrate
from the roots, but the responsible gene has not been identified
yet. Here, we identified a gene (HvAACT1) responsible for
the Al-activated citrate secretion by fine mapping combined
with microarray analysis, using an Al-resistant cultivar,
Murasakimochi, and an Al-sensitive cultivar, Morex. This
gene belongs to the multidrug and toxic compound extrusion
(MATE) family and was constitutively expressed mainly in
the roots of the Al-resistant barley cultivar. Heterologous
expression of HvAACT1 in Xenopus oocytes showed efflux
electrode voltage clamp analysis also showed transport
activity of citrate in the HvAACT1-expressing oocytes in
the presence of Al. Overexpression of this gene in tobacco
enhanced citrate secretion and Al resistance compared
with the wild-type plants. Transiently expressed green
fluorescent protein-tagged HvAACT1 was localized at the
plasma membrane of the onion epidermal cells, and immu-
nostaining showed that HvAACT1 was localized in the
epidermal cells of the barley root tips. A good correlation
was found between the expression of HvAACT1 and citrate
secretion in 10 barley cultivars differing in Al resistance.
Taken together, our results demonstrate that HvAACT1
is an Al-activated citrate transporter responsible for Al
resistance in barley.
14C-labeled citrate, but not for malate. Two-
Keywords: Aluminum — Barley — Citrate transporter —
MATE — Resistance — Root.
CaMV, cauliflower mosaic virus; EST, expressed sequence tag;
GFP, green fluorescent protein; MATE, multidrug and toxic
compound extrusion; ORF, open reading frame; QTL, quantita-
tive trait locus; SNP, single nucleotide polymorphism.
The nucleotide sequence data reported in this paper have been
deposited in the DDBJ/EMBL/GenBank nucleotide sequence
databases with the accession number AB302223 (cDNA) and
AB331641 (genomic DNA).
Aluminum (Al) is the most abundant metal in the
earth’s crust. Under acidic conditions, Al is solublized to its
ionic form, which shows toxicity to plants (Foy 1988).
Al rapidly inhibits root elongation and subsequently the
uptake of water and nutrients, resulting in significant
reduction of crop production on acid soils, which comprise
30–40% of the world’s arable soils (von Uexku ¨ ll and Mutert
1995). However, some plant species have developed
mechanisms to cope with Al toxicity both internally and
externally (Ryan et al. 2001, Ma et al. 2001, Rengel 2004,
Kochian et al. 2005). The most documented mechanism of
Al resistance is the secretion of organic acid anions from the
roots (Ma 2000, Ma et al. 2001, Ryan et al. 2001, Kochian
et al. 2005). Since the first report on Al-induced malate
secretion in wheat (Kitagawa et al. 1986), a wide range of
plant species has been reported to secrete organic acid
anions in response to Al, including monocots and dicots
such as wheat, maize, rye and soybean. Physiological
studies have been carried out extensively to understand
the nature of Al-induced secretion of organic acid anions
(Ma et al. 2001, Ryan et al. 2001, Kochian et al. 2005).
Plants differ in the species of organic acid anions secreted,
temporal secretion patterns, temperature sensitivity and
dosage responses to Al (Ma 2000). Up to now, citrate,
oxalate and/or malate have been identified as the organic
acid anions secreted by roots in response to Al. In some
plant species, two organic acid anions are secreted in
response to Al. These anions are able to form a complex
with Al, thereby detoxifying Al externally. Two patterns of
organic acid anion release can be identified on the basis of
the timing of secretion (Ma 2000). In Pattern I-plants,
secretion occurs almost immediately following the addition
of Al, suggesting that Al activates a pre-existing anion
channel in the plasma membrane and that the induction of
genes is not required. In contrast, in Pattern II-plants,
organic acid anion secretion is delayed for several hours
?Corresponding author: E-mail, email@example.com; Fax, þ81-86-434-1209.
Plant Cell Physiol. 48(8): 1081–1091 (2007)
doi:10.1093/pcp/pcm091, available FREE online at www.pcp.oxfordjournals.org
? The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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after the exposure to Al, suggesting that gene induction
is required. Some inducible proteins could be involved in
organic acid metabolism or in the transport of organic acid
Physiological studies have shown that the secretion of
organic acid anions is mediated through anion channels
or transporters. Two studies with maize revealed that Al
activates Cl?efflux and the citrate-permeable anion channel
(Kollmeier et al. 2001, Pin ˜ eros and Kochian 2001).
These studies also indicated that at least a subset of the
Al-activated channels requires extracellular Al3þto main-
tain channel activity and that the activation machinery
is localized to the plasma membrane. Recently, a gene,
ALMT1 (Al-activated malate transporter 1), which is
responsible for malate release, has been identified in
wheat by a subtraction approach between near-isogenic
lines of wheat ET8 and ES8 (Sasaki et al. 2004). The
protein encoded by this gene is localized to the plasma
membrane (Yamaguchi et al. 2005), which is predicted
to have between six and eight putative transmembrane
regions. Heterologous expression of this gene in Xenopus
oocytes showed transport activity for malate, but not for
citrate. Homologs of wheat ALMT1 have been cloned from
Arabidopsis, rape and rye, although the expression patterns
of these genes differ among these plant species (Hoekenga
et al. 2006, Ligaba et al. 2006, Fontecha et al. 2007).
Barley (Hordeum vulgare L.) is one of the most
Al-sensitive species among small grain cereals; however,
there is a wide variation in Al resistance among cultivars.
A physiological study showed that the Al-resistant cultivars
of barley rapidly secrete citrate from the roots in response to
Al and that there is a good correlation between Al resistance
and the amount of citrate secretion among different cultivars
(Zhao et al. 2003). Previously, we identified a major
quantitative trait locus (QTL) for Al-induced secretion of
citrate in barley, and we also showed that the QTL is
controlled by a single dominant gene, flanked by micro-
satellite markers Bmac310 and Bmag353 on the long arm of
chromosome 4H (Ma et al. 2004). The locus for Al-induced
secretion of citrate was also mapped to the same region
as that for Al resistance (Alp) (Minella and Sorrells 1997,
Tang et al. 2000, Raman et al. 2002, Ma et al. 2004), indi-
cating that Al resistance in barley is mainly controlled by the
secretion of citrate. However, the responsible gene has not
been identified yet. In the present study, we cloned a gene
responsible for Al-induced secretion of citrate by using a
combination of positional cloning and microarray analysis.
Cloning of the candidate gene
Al-induced secretion of citrate, we used an F4 mapping
mappingof the generesponsible for
population from heterozygous plants for the QTL on
chromosome 4H, derived from a cross between an
Al-resistant cultivar, Murasakimochi, and an Al-sensitive
cultivar, Morex (Ma et al. 2004). Murasakimochi secreted
a large amount of citrate from the roots in response to Al,
but Morex did not (Ma et al. 2004). We developed new
markers between Bmac310 and Bmag353 based on the
expressed sequence tag (EST) information of the genetic
map from Haruna Nijo/H602 (Sato et al. 2004) and on the
synteny of rice (Supplementary Table S1). By genotyping
with a total of 793 F4lines, we delimited the gene to a region
equivalent to approximately 140kb of the rice genome
containing 21 annotated gene models (Fig. 1A). We also
performed a microarray analysis with Barley 1 GeneChip
(Affymetrix Co.) to identify up- or down-regulated tran-
scripts between Murasakimochi and Morex with and
without Al treatment. Based on the EST information of
the genetic map from Haruna Nijo/H602, there are
25 mapped genes on the chip between the markers
Bmac310 and Bmag353 (Table 1). Comparison of the
expression of these 25 genes between the two cultivars
showed that only one gene was up-regulated by420-fold in
Murasakimochi, irrespective of Al treatment (Table 1). This
transcript encodes a member of the multidrug and toxic
compound extrusion (MATE) family (Barley1 probe name:
Contig9960_at). Combined with fine mapping data, this
gene may encode an aluminum-activated citrate transporter
(referred to as HvAACT1 later). The homolog of this gene
exists on chromosome 3 of rice, which corresponds to
HvAACT1 on barley (Fig. 1A). We then cloned the
full-length cDNA of HvAACT1 from the roots of both
HvAACT1 was 1,668bp long, and the deduced polypep-
tide was 555 amino acids (Supplementary Fig. S1).
We sequenced the bacterial artificial chrmosome (BAC)
clone of Haruna Nijo that contains HvAACT1. The gene
consisted of 13 exons and 12 introns (Fig. 1B). It is
predicted to encode a membrane protein which contains
seven putative transmembrane domains (Supplementary
Fig. S2). BLAST search showed that there is one close
homolog (Os03g0216700) with
(Fig. 1C). In Arabidopsis MATE family members, FRDL
showed the highest homology to HvAACT1 with 59%
identity and 86% similarity.
HvAACT1 in Murasakimochi and Morex only differed
in two nucleotides and one amino acid in their open reading
frames (ORFs; Supplementary Fig. S1). We developed a
cleaved amplified polymorphic sequence (CAPS) marker
to genotype the haplotypes of Murasakimochi and Morex.
In an F4 segregating line with 100 individuals, the
segregation of the genotype was consistent with that of
confirming that this gene is involved in citrate secretion.
The coding regionof
85% identity in rice
1082 Al-activated transporter of citrate
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of chromosome 4H between markers HvP1 and K06496. The number of recombinants between the molecular markers is indicated
below the high resolution map. Corresponding genes on rice chromosome 3 are also shown at the bottom. (B) HvAACT1 gene structure.
Thirteen exons are boxed. (C) Phylogenetic relationship of HvAACT1 proteins in other plant species.
Cloning of HvAACT1 from barley. (A) Fine mapping of HvAACT1. The candidate gene (HvAACT1) was mapped on the long arm
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Citrate transport activity of HvAACT1 in Xenopus laevis
To determine whether HvAACT1 has transport
activity forcitrate, weexpressed
Murasakimochi in Xenopus laevis oocytes. The two-
electrode voltage clamp analysis showed that Al activated
an inward current (consistent with the anion efflux) only
in oocytes injected with both HvAACT1 cRNA and citrate
(Fig. 2A). The Al-activated currents in oocytes injected
with HvAACT1 cRNA and citrate were 2-fold greater than
in the control oocytes injected with water and citrate. The
Al-activated currents were also observed in oocytes injected
with HvAACT1 cRNA from Morex (data not shown).
We also investigated the substrate specificity for HvAACT1
Table 1 Changes in expression of genes between the markers Bmac310 and Bmag353 in two cultivars of barley
Allene oxide synthase
Putative cytochrome P450
Putative mitotic checkpoint
Putative Zn-finger protein
Putative MATE family
Contig1646_at 2.7?1.51.4?1.1 1.0?1.10.4?1.2
Contig9662_at 1.3?1.7 1.0?1.21.7?1.11.5?1.7
Tubulin a-2 chain
Glycosyl hydrolase family 31
RING zinc finger protein-like
Microarray analysis was performed with total RNA extracted from the root apices from Murasakimochi (Mu) and Morex (Mo) exposed
to Al (þ) or not (?) for 6h. Data are means?SD.
1084Al-activated transporter of citrate
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and found that HvAACT1 had transport activity for citrate,
but not for malate (Fig. 2B). Furthermore, to measure
the efflux of citrate directly, we injected14C-labeled citrate
into oocytes with or without HvAACT1 expression.
Oocytes expressing HvAACT1 showed enhanced efflux
activity for citrate compared with oocytes not expressing
HvAACT1 (Fig. 2C). Oocytes expressing HvAACT1 did
not show efflux activity for malate (Fig. 2D).
Overexpression of HvAACT1 in tobacco
We overexpressed HvAACT1 in tobacco under the
control of a cauliflower mosaic virus (CaMV) 35S RNA
promoter. All T1 plants were checked for HvAACT1
insertion in the genome and for HvAACT1 expression
showed significantly higher citrate secretion in the presence
of Al, compared with the control plants not carrying
HvAACT1 (P50.01) (Fig. 3B). The citrate secretion was
very low in both lines in the absence of Al. The relative
root elongation of HvAACT1-overexpressing tobacco was
70% after 24h Al exposure, whereas that of the control
was 31% (P50.05) (Fig. 3C), indicating that the Al
resistance was also enhanced in the transgenic plants.
Tissue-dependent expression of HvAACT1 in barley
HvAACT1 mRNA was expressed in both the roots and
shoots (Fig. 4A), but the level was higher in the roots than
in the shoots. The amount of HvAACT1 mRNA transcript
Holding potential (mV)
water + citrate − AI
water + citrate + AI
HvAACT1 + citrate − AI
HvAACT1 + citrate + AI
Relative current (%)
Efflux (% radioactivity)
14C-citrate efflux (% radioactivity)
010 20 30
40 5060 70
oocytes. Sodium citrate was injected before measurement and the electrical potential (mV) was clamped from ?100mV to the potential
which indicated current of 0A in 10mV steps in the presence or absence of Al. Data are means?SD (n¼3–6). (B) Substrate specificity of
HvAACT1. Citrate or malate was injected into oocytes with or without HvAACT1 expression, and the inward current produced by Al was
measured at ?100mV. Relative values are shown. Data are means?SD (n¼3–6). (C) Efflux activity of citrate due to HvAACT1. Oocytes
with or without HvAACT1 expression were injected with14C-labeled citrate and the release of14C-labeled citrate from the oocytes
was determined at various times. Data are means?SD (n¼4). (D) Efflux activity of citrate and malate due to HvAACT1. Oocytes with
or without HvAACT1 expression were injected with14C-labeled citrate or malate and the release of14C-labeled citrate or malate from
the oocytes was determined 1h later. Data are means?SD (n¼4).
Heterologous expression of HvAACT1. (A) Mean current–voltage curves from oocytes expressing HvAACT1 and water-injected
Al-activated transporter of citrate 1085
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(Morex), and the expression level was not induced by Al
in either cultivar (Fig. 4A). These results are consistent with
Furthermore, the expression level was higher in the root
segments 10–20mm from the root tip than in the 0–10mm
region (Fig. 4B). Murasakimochi constitutively showed
a higher expression level than Morex in both regions,
irrespective of Al treatment (Fig. 4B).
analysis (Table 1).
Correlation between HvAACT1 expression and Al-induced
citrate secretion and Al resistance in barley
Analysis of 10 barley cultivars differing in Al resistance
revealed a good positive correlation (r¼0.93) between the
expression level of HvAACT1 mRNA in the roots and
the amount of Al-induced citrate secretion (Fig. 4C) as
well as (r¼0.89) Al resistance (relative root elongation)
(Fig. 4D). We compared the ORF of HvAACT1 in all these
cultivars and found four single nucleotide polymorphisms
However, these SNPs cannot explain the differences in
Localization of HvAACT1
In situ hybridization analysis showed that HvAACT1
mRNA was expressed in the epidermal cells of the root tips
(Fig. 5A, C). Furthermore, the expression level of mRNA
was higher in Murasakimochi than in Morex, which is
consistent with the expression level of HvAACT1 in these
cultivars (Fig. 4A, B).
We also examined the localization of HvAACT1
protein by means of rabbit anti-HvAACT1 polyclonal
antibody staining. The peptide used for preparing the
antibody was designed specifically for HvAACT1, based
on the database of all MATE sequences. Consistent with
the in situ hybridization result, HvAACT1 protein was
also localized in the epidermal cells of the root tips
(Fig. 6A, B), and a higher signal intensity was observed in
Murasakimochi. To examine the specificity of the antibody
used, the antibody was pre-incubated with the peptide
epitope before staining. As a result, a strong signal in the
epidermal cells disappeared (Fig. 6C), suggesting that
the antibody has a high specificity for HvACCT1.
We further investigated the subcellular localization of
HvAACT1 by introducing green fluorescent protein (GFP)
alone or GFP-fused HvAACT1 (HvAACT1–GFP) into
onion epidermal cells under the control of a CaMV 35S
RNA promoter. The GFP signal was observed only at the
plasma membrane of the cells expressing HvAACT1–GFP
(Fig. 7A, C), whereas the signal was observed in the nuclei
and cytoplasm when GFP was expressed alone (Fig. 7B, D).
This indicates that HvAACT1 is localized at the plasma
Barley has a large genome size (12 times that of rice)
and the complete genome sequence is still not available.
Therefore, it is often difficult to clone a gene based on the
information of a QTL identified in barley. In the present
study, a combination of fine mapping and microarray
analysis led us to clone a gene (HvAACT1) which is
responsible for Al-induced secretion of citrate (Fig. 1,
Table 1). Heterologous expression of HvAACT1 in the
oocytes showed efflux transport activity for citrate (Fig. 2).
Furthermore, although only one independent T0line was
examined, analysis of several T1lines showed that over-
expression of this gene in tobacco resulted in enhanced
(nmol g−1 root dry wt. 6 h−1)
Relative root elongation
(% of control)
Over-expressed linesWild type
of HvAACT1 in the selected overexpressing T1 lines carrying
HvAACT1 and in the wild type. (B) Citrate secretion from
transgenic tobacco overexpressing HvAACT1. The plants with or
without HvAACT1 expression were exposed to 0 or 30mM Al
for 6h. Data are means?SD (n¼3–5). (C) Al resistance in tobacco
carrying HvAACT1. Plants were exposed to 0 or 30mM Al and
their root length was measured before and after the treatment.
Relative root elongation is shown. Data are means?SD (n¼3–10).
Overexpression of HvAACT1 in tobacco. (A) Expression
1086Al-activated transporter of citrate
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Al-activated secretion of citrate and Al resistance (Fig. 3).
Taken together, all these results indicate that this gene
encodes an Al-activated efflux transporter of citrate in
Unexpectedly, the gene identified belongs to the
MATE family (Fig. 1C). MATEs are found in both
prokaryotes and eukaryotes (Omote et al. 2006), but there
is no apparent consensus sequence conserved in all MATE
proteins. MATE proteins are proposed to transport small,
organic compounds (Omote et al. 2006). In contrast to
MATE genes in the bacterial and animal kingdom, plants
contain more MATE-type transporters. For example, there
are 58 MATE orthologs in the genome of Arabidopsis
thaliana (Omote et al. 2006). However, the functions of
most genes are still unknown. Recently, AtFRD3 has been
reported to be involved in the xylem loading of citrate
(Durrett et al. 2007). In contrast to HvAACT1 (Figs. 5, 6),
AtFRD3 protein was localized to the pericycle and cells
internal to the pericycle cells in the roots of Arabidopsis
(Green and Rogers 2004). In white lupin, a MATE gene
was up-regulated by phosphorus deficiency, although the
Mo MoMu Mu
Root tip Basal root tip
(nmol g−1 root fresh wt. 6h−1)
r = 0.93, P<0.01
r = 0.89, P<0.01
0 2040 60
Relative HvAACT1 expression level
(Normalized by Morex)
Relative HvAACT1 expression level
(Normalized by Morex)
Relative HvAACT1 root expression level
(normalized by Morex, 0 mM AI)
Relative root elongation
(% of control)
0 10 203040 50 60
treatment. Mu, Murasakimochi; Mo, Morex. (B) Expression of HvAACT1 in different root segments of two barley cultivars with (þAl)
or without (?Al) Al treatment for 6h. Data are means?SD (n¼3). The relative value of Morex is shown. (C) Correlation between
expression of HvAACT1 in the roots and Al-induced secretion of citrate in 10 barley cultivars. The root exudates were collected for 6h
in the presence of 10mM Al. Data are means?SD (n¼3). (D) Correlation between HvAACT1 expression and the relative root
elongation in 10 barley cultivars. The roots were exposed to a solution with or without 5mM Al for 24h. Data for root elongation are
Expression of HvAACT1. (A) Expression of HvAACT1 in different tissues of two barley cultivars with (þAl) or without (?Al) Al
Cryosections of root tips (5mm) from Murasakimochi (A, B) or
Morex roots (C, D) were hybridized with antisense (A, C) and sense
(B, D) probes labeled with digoxigenin. Scale bar¼100mm.
Expression of HvAACT1 transcripts in barley roots.
Al-activated transporter of citrate 1087
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(Uhde-Stone et al. 2005). Lupin secretes citrate from the
roots in response to phosphorus deficiency, suggesting that
citrate secretion. These findings suggest that some MATE
proteins transport citrate, but their functions in the plants
differ in terms of localization, regulation, and so on.
The toxicity mechanisms of Al are complicated and the
exact mechanism by which Al initially causes the inhibition
of root elongation has not been understood (Kochian et al.
2005). However, it is clear that most events caused by Al
ofthisgene hasnot been characterized
basically result from the binding of Al to extracellular and
intracellular substances because of the high affinity of Al
for oxygen donor compounds. When the root elongation is
inhibited by Al, most of the Al is localized on the epidermis
and the outer cortex (Jones et al. 2006). HvAACT1 is
localized in the epidermal cells of root tips (Figs. 5, 6);
therefore, release of citrate from the epidermal cells through
HvAACT1 to the rhizosphere could protect the roots from
Al toxicity quickly. In addition, this localization pattern
gives the transporter the greatest likelihood of detecting
Al in the soils.
HvAACT1 was expressed not only in the root tips of
the Al-resistant cultivar, Murasakimochi, but also in the
mature regions of the roots (Fig. 4B). Root tips are the
target of Al toxicity, and physiological studies have shown
that the position of organic acid secretion is limited to the
root tips to protect the roots from Al toxicity in most plant
species (Ryan et al. 1993, Ryan et al. 1995, Zheng et al.
1998). However, a study with an Al-resistant cultivar of
maize showed that citrate exudation was not confined to
the root apex, but could be found as far as 5cm from the
apex (Pin ˜ eros et al. 2002). Expression of HvAACT1 at the
mature region may also play a role in Al detoxification,
although the exact mechanism remains to be examined in
The expression of HvAACT1 was not induced by Al
exposure (Fig. 4A, B). This suggests that HvAACT1 is
constitutively expressed in the roots and that the secretion
of citrate is mediated through the activation of HvAACT1.
This result is in agreement with the rapid secretion of citrate
upon Al exposure (Zhao et al. 2003), confirming that
gene induction is not required in the Al-induced secretion
of citrate in barley.
Four SNPs were found in the ORF of HvAACT1 in
10 barley cultivars differing in Al resistance (Supplementary
Fig. S1), but these SNPs could not explain the differential
citrate secretion. In contrast, a good correlation was found
between the expression of HvAACT1 and the amount of
Mu Mu + peptide
at the root tip (3mm) of Morex (A) and Murasakimochi (B). The specificity of the antibody was tested by pre-incubating the antibody
with the epitope peptide (C). Scale bar¼100mm.
Localization of the HvACCT1 protein in barley roots. Immunostaining was performed using anti-HvAACT1 polyclonal antibody
between HvAACT1 and GFP (A, C) or GFP protein alone (B, D)
was introduced into onion epidermal cells. A and B, GFP-derived
fluorescence; C and D, fluorescence superimposed over the trans-
mission image. Scale bar¼100mm.
Subcellular localization of HvAACT1. A gene fusion
1088 Al-activated transporter of citrate
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citrate secretion in these cultivars (Fig. 4C, D). These
findings indicate that higher expression of HvAACT1 rather
than SNPs is required for greater release of citrate. In fact,
HvAACT1 from the Al-sensitive cultivar Morex also
showed transport activity for citrate in oocytes expressing
this gene (data not shown). In wheat, a recent study showed
that the expression of ALMT1 may be controlled by the
presence of the sequence repeats upstream of this gene in
69 wheat lines of non-Japanese origin (Sasaki et al. 2006).
It remains to be investigated whether the expression of
HvAACT1 is also controlled by the promoter regions.
HvAACT1 showed transport activity for citrate, but
not for malate (Fig. 2). On the other hand, ALMT1 is able
to transport malate, but not citrate (Sasaki et al. 2004).
These findings suggest that plant roots use different trans-
porters to release citrate or malate in response to Al.
In addition to barley, a number of plant species such as
soybean, Cassia tora, rye and triticale secrete citrate in
response to Al treatment (Ma 2000, Ma et al. 2001, Ryan
et al. 2001, Kochian et al. 2005). Identification of
HvAACT1 from barley in the present study will help
to clone genes related to citrate secretion in other plant
species, contributing to a better understanding of molecular
mechanisms of Al resistance.
Al toxicity limits the growth and productivity of barley
on acid soils and the expansion of barley as a crop into
many agricultural areas of the world (Alva et al. 1986).
Soil is limed in some areas to improve barley growth and
productivity on acid soils, but this practice is often not
economically feasible (Minella and Sorrells 1992). Further-
more, surface application of lime cannot alleviate toxic
subsoil Al, which presents a barrier to deep rooting and
the uptake of water and nutrients. A transgenic barley
overexpressing the malate transporter ALMT1 showed
increased Al resistance (Delhaize et al. 2004). Citrate has
6–8 times higher Al-chelating ability compared with malate.
As observed in transgenic tobacco (Fig. 3), overexpression
of HvAACT1 will enable us to develop more Al-resistant
barley and other important crops with enhanced Al
Materials and Methods
Fine mapping of the candidate gene
A barley F4segregating population from the heterozygous
plants for the QTL of Al resistance and citrate secretion, which was
derived from a cross between Al-resistant (Murasakimochi)
and Al-sensitive (Morex) cultivars, was used for fine mapping of
the gene. A total of 793 individuals were grown hydroponically
as described (Ma et al. 2004), and the leaves were sampled for
DNA extraction. The samples were genotyped first with two
markers: Bmac310 and Bmag353. Individuals with the recombina-
tion were chosen for further genotyping with the developed
markers. Markers K00500, K02565, K02338, K03066, K04725
and K06496 between Bmac 310 and Bmag353 were developed
according to the corresponding EST sequence from the barley EST
database (Supplementary Table S1). Marker HvP1 was developed
based on the sequence of a rice BAC clone OSJNBa0090D11 on
rice chromosome 3 of Oryza sativa ssp. japonica ‘Nipponbare’. The
mRNA sequence of a pyridoxal phosphate-dependent enzyme
family protein gene (Os03g0215800) located on the BAC was
homologous with an EST (bags5e04) from the barley database.
The Al-induced secretion of citrate was also examined in the
recombinants as described previously (Ma et al. 2004). All
recombinant F4plants were used for construction of a fine map.
Four-day-old seedlings were transferred to a 1.0mM CaCl2
solution (pH 5.0, aerated) containing 0 or 5mM Al for 6h at 238C.
Root apices (0–10mm from root tip, 40 root tips/sample) were
harvested and stored at ?808C until RNA extraction. Microarray
analysis was performed with a Barley 1 GeneChip according to the
manufacturer’s protocol (Affymetrix). Two replicates were made
for each sample. Gene expression was examined in Murasakimochi
and Morex with and without Al treatment.
Screening and sequence of BAC clones
The BAC clones containing the candidate gene were
screened with a pair of primers: TGGAGGAAGCATAGTATC
and CACCTGGAGGTATGAA from a BAC library of Haruna
Nijo (Saisho et al. 2007). The selected BAC clone was sequenced.
Electrophysiological studies in Xenopus laevis oocytes
Murasakimochi was amplified by PCR using high-fidelity KOD
plus DNA polymerase (Toyobo, Tokyo, Japan). Gene-specific
CC-30were used to create BamHI sites on both ends and
then inserted into the BglII site in the oocyte expression vector
pXbG-ev1. The plasmid was linearized with BamHI, and cRNA
was transcribed in vitro with T3 RNA polymerase (mMESSAGE
mMACHINE kit; Ambion, Austin, TX, USA). For each experi-
ment, 50nl of water containing 50ng of cRNA was injected into
each X. laevis oocyte. The cRNA-injected oocytes were incubated
in Modified Barth’s Saline (MBS) solution at 188C. After a 24h
incubation, 50nl of 25mM sodium citrate or 25mM sodium
malate were injected into the oocytes and then incubated for 1–3h.
Before measurement, the oocytes were exposed to a modified MBS
solution containing 100mM Al at pH 4.5 according to Sasaki et al.
(2004). The net current across the oocyte membrane was measured
using the two-electrode voltage clamp system with the amplifier
(MEZ-7200 and CEZ-1200, Nihon Kohden, Tokyo, Japan) at
different membrane voltages. The electrical potential difference
across the membrane was clamped from ?100mV to the potential
which indicated 0A current, in 10mV steps.
Efflux transport activity of citrate
Oocytes with or without HvAACT1 expression as described
above were injected with 50nl of 2.4mM
or malate (Amersham, 2.3 nCi/oocyte) (4–5 oocytes/replicate).
The oocytes were washed for 5min in modified MBS buffer
(pH 5.0) and then transferred into 500ml of fresh buffer at 188C.
For the time-course experiment, 500ml of buffer was carefully
sampled, and replaced with fresh buffer at the time points
indicated. At the end of the experiments, the oocytes were
homogenized with 0.1N HNO3. The radioactivity of the buffer
Al-activated transporter of citrate1089
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and homogenized oocytes was measured with a liquid scintillation
counter (Aloka LIQUID SCINTILLATION SYSTEM).
Overexpression of HvAACT1 in tobacco
Murasakimochi was amplified by PCR using KOD plus DNA
polymerase with the gene-specific primers 50-AAGCATCCGCT
and then cloned into pTA2 vector (Toyobo, Tokyo, Japan)
according to the manufacturer’s protocol. After XhoI and
BamHI treatment, HvAACT1 cDNA with XhoI and BamHI sites
on both ends was ligated into an upstream SalI and a downstream
BamHI restriction site in pPZP2Ha3(?) Agrobacterium-mediated
transformation vector (Fuse et al. 2001). The vectors were
transferred to Agrobacterium tumefaciens (strain EHA101) by
electroporation. Tobacco (Nicotiana tabacum) plants were trans-
Transformed calluses were selected by hygromycin resistance,
and from them regenerated plants were obtained. Transgenic lines
carrying HvAACT1 were selected from T1lines by PCR using the
primers described above. The 3-week-old T1 plants carrying
HvAACT1 or not were transferred to 1.2liter plastic pots (three
plants per pot) containing 1/10 Hoagland solution. After 2 weeks,
the plants were exposed to 0 or 30mM Al in 1.0mM CaCl2solution
(pH 5.0) for 6h. The citrate in the root exudates was measured
according to Delhaize et al. (1993). For evaluation of Al resistance,
the transformed tobacco plants (5 weeks old) were exposed to the
1/10 Hoagland solution containing 0 or 30mM Al at pH 4.5 for
24h. The root elongation was measured with a ruler.
Tissue-dependent expression of HvAACT1
Four-day old barley seedlings exposed to a 1.0mM CaCl2
solution (pH 5.0, aerated) containing 0 or 5mM Al for 6h were
separated into roots and shoots. The samples were ground in liquid
nitrogen and RNA was immediately extracted with an RNeasy
plant Mini Kit (Qiagen, Valencia, CA, USA). cDNA was
synthesized from the extracted RNA with a SuperScript First-
Strand Synthesis System for RT-PCR (Invitrogen), and the gene
expression level was quantified by quantitative RT–PCR with
SYBR Green I reagent (SYBR Premix Ex Taq; TAKARA
SHUZO CO. LTD, Tokyo, Japan) on a Prism 7500 real-time
PCR System (Applied Biosystems, Foster City, CA, USA)
according to the manufacturer’s instruction. The primers used
for HvAACT1 were 50-GTTCGCCAAGAACGATCACA-30and
normalized with the expression level of Actin, and the data for
the root tips of Murasakimochi were compared with those of
Morex (0mM Al) by the ??Ct method. The primers used for Actin
were 50-GACTCTGGTGATGGTGTCAGC-30and 50-GGCTGG
AAGAGGACCTCAGG-30. The expression level at different root
segments (0–10 and 10–20mm) was also examined with three
Expression data were
Correlation between HvAACT1 expression level and citrate secretion
Morex, Murasakimochi (CI5899), Haruna Nijo, ALP7,
ALP21, ALP25, BC26, BC29, BC95 and Z504, which differed in
Al resistance, were used (Zhao et al. 2003). Root length was
measured before and after the seedlings prepared as above were
exposed to a 1.0mM CaCl2solution containing 0 or 5mM Al for
24h. Root exudates of each cultivar exposed to 10mM Al were
collected for 6h and the HvAACT1 expression of the roots was
determined as described above.
In situ hybridization and immunostaining
The RNA probes were made by amplification of the ORF
region of HvAACT1 cDNA by PCR with the forward primer,
50-AAGCATCCGCTGTGTATGGAG-30, and reverse primer,
50-TCACTTCCGGAGGAAAACCC-30, and then cloned into
pTA2 vector (Toyobo) according to the manufacturer’s protocol.
After checking the direction of the inserted cDNA, the plasmid
was linearized with BamHI (sense strand) and HindIII (antisense
strand). In situ hybridization was done with 12mm cryosections
of Murasakimochi or Morex roots as described elsewhere
For immunostaining, the synthetic peptide C-HGPEEKA
AEDLPAA (positions 35–48 of HvAACT1) was used to immunize
rabbits to obtain antibodies against HvAACT1. The roots of both
cultivars were used for immunostaining according to Ma et al.
(2006). To check the specificity, the antibody (1:50 dilution) was
pre-incubated with the epitope peptide used for preparation of
antibody at 25nmol ml?1for 1h at room temperature before
Subcellular localization of HvAACT1
For constructing a translational HvAACT1–GFP fusion,
the full-length HvAACT1 ORF except for the stop codon derived
from Murasakimochi was amplified. Amplification was performed
using KOD plus DNA polymerase and the nucleotide sequence
was checked to confirm its identity. Gene-specific primers
used to create XhoI and BamHI sites on both ends. After BamHI
treatment, the 30end was filled with T4 DNA polymerase
(New England Biolabs, Ipswich, MA, USA) and then treated
with XhoI restriction enzyme. The XhoI-blunt fragment of
HvAACT1 was inserted upstream of SalI and downstream of
a blunted NcoI restriction site between the 35S promoter and the
GFP coding region in pBluescript vector. The fused gene was
introduced into the onion epidermal cells as described by Murata
et al. (2006).
Supplementary material mentioned in the article is
available to online subscribers at the journal website
This work was supported by the Program of Promotion
of Basic Research Activities for Innovative Biosciences (BRAIN),
by a Grant-in-Aid for General Scientific Research (grant No.
18380052 to J.F.M.) from the Ministry of Education, Sports,
Culture, Science, and Technology of Japan, and by the Ohara
Foundation for Agricultural Science.
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(Received June 26, 2007; Accepted July 9, 2007)
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