Rapid paper

An Aluminum-Activated Citrate Transporter in Barley

Jun Furukawa1, Naoki Yamaji

1, Hua Wang

1, Namiki Mitani

1, Yoshiko Murata

2, Kazuhiro Sato

1,

Maki Katsuhara1, Kazuyoshi Takeda

1and Jian Feng Ma

1, �

1 Research Institute for Bioresources, Okayama University, Chuo, Kurashiki, Okayama, 710-0046 Japan2 Suntory 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

activity for 14C-labeled citrate, but not for malate. Two-

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.

Keywords: Aluminum — Barley — Citrate transporter —

MATE — Resistance — Root.

Abbreviations: BAC, bacterial artificial chromosome;CaMV, cauliflower mosaic virus; EST, expressed sequence tag;GFP, green fluorescent protein; MATE, multidrug and toxiccompound extrusion; ORF, open reading frame; QTL, quantita-tive trait locus; SNP, single nucleotide polymorphism.

The nucleotide sequence data reported in this paper have beendeposited in the DDBJ/EMBL/GenBank nucleotide sequencedatabases with the accession number AB302223 (cDNA) andAB331641 (genomic DNA).

Introduction

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 Uexkull 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, maj@rib.okayama-u.ac.jp; Fax, þ81-86-434-1209.

Edito

r-in-Chief’s

choice

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.All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

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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

anions.

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, Pineros and Kochian 2001).

These studies also indicated that at least a subset of the

Al-activated channels requires extracellular Al3þ to 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.

Results

Cloning of the candidate gene

For fine mapping of the gene responsible for

Al-induced secretion of citrate, we used an F4 mapping

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 F4 lines, we delimited the gene to a region

equivalent to approximately 140 kb 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

Murasakimochi and Morex. The coding region of

HvAACT1 was 1,668 bp 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 85% identity in rice

(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

the phenotype (citrate secretion) (data not shown),

confirming that this gene is involved in citrate secretion.

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Fig. 1 Cloning of HvAACT1 from barley. (A) Fine mapping of HvAACT1. The candidate gene (HvAACT1) was mapped on the long armof chromosome 4H between markers HvP1 and K06496. The number of recombinants between the molecular markers is indicatedbelow 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.

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Citrate transport activity of HvAACT1 in Xenopus laevis

oocytes

To determine whether HvAACT1 has transport

activity for citrate, we expressed HvAACT1 from

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

Probe Annotation Fold-change

Mu(þ)/Mo(þ) Mu(�)/Mo(�) Mu(þ)/Mu(�) Mo(þ)/Mo(�)

Contig5872_at Toc34-1 protein 1.1� 1.2 1.5� 1.1 0.8� 1.1 1.1� 1.3

Contig5692_s_at Actin depolymerizing

factor 5

1.1� 1.3 0.9� 1.0 0.8� 1.1 0.7� 1.2

Contig3097_at Allene oxide synthase 0.6� 1.2 0.6� 1.1 1.1� 1.1 1.3� 1.1

Contig12365_at Unknown protein 1.2� 1.4 1.1� 1.2 0.8� 1.1 0.6� 1.2

Contig12508_s_at Putative cytochrome P450 2.2� 1.3 0.9� 1.3 1.0� 2.9 0.3� 2.6

Contig3376_at Putative trehalose-

6-phosphate

synthase

1.3� 1.1 1.0� 1.2 1.2� 1.2 0.8� 1.2

Contig1646_at Glutamine synthetase

root isozyme

2.7� 1.5 1.4� 1.1 1.0� 1.1 0.4� 1.2

Contig23128_at Steroid 22-a-hydroxylase(DWF4)

0.7� 2.5 0.6� 1.6 1.2� 1.4 2.1� 3.9

Contig7497_at Putative mitotic checkpoint

protein

1.6� 1.4 1.2� 1.1 0.9� 1.1 0.7� 1.3

Contig22801_at Putative Zn-finger protein 1.3� 1.1 2.6� 2.7 1.0� 1.1 2.7� 3.3

Contig9960_at Putative MATE family

protein

36.1� 1.2 20.4� 1.3 1.1� 1.1 0.6� 1.1

Contig9662_at Hexose transporter-like

protein

1.3� 1.7 1.0� 1.2 1.7� 1.1 1.5� 1.7

Contig20435_at None 0.2� 3.7 1.7� 4.6 0.8� 4.5 3.7� 2.4

HVSMEf0003

E10r2_s_at

Fructose 1,6-bisphosphatase 0.5� 1.4 0.6� 1.2 1.0� 1.1 1.1� 1.3

Contig24154_at None 3.7� 2.3 0.5� 2.3 2.7� 2.1 0.6� 4.4

Contig930_at Tubulin a-2 chain 0.6� 2.8 1.1� 1.1 1.4� 1.1 3.2� 2.5

Contig11793_at Unknown protein 1.2� 1.1 0.9� 1.1 1.3� 1.2 1.0� 1.2

baak20j05_s_at Unknown protein 1.3� 1.5 0.9� 1.0 0.9� 1.0 0.6� 1.4

HVSMEb0007

M04r2_s_at

None 1.3� 1.4 1.0� 1.1 1.1� 1.0 0.7� 1.5

Contig10831_at Unknown protein 1.0� 1.1 0.9� 1.1 1.2� 1.1 1.0� 1.2

Contig9432_at Glycosyl hydrolase family 31 1.0� 1.2 1.1� 1.1 0.8� 1.1 0.8� 1.2

Contig13208_at Unknown protein 1.5� 1.4 1.0� 1.1 1.0� 1.1 0.7� 1.3

Contig24332_at RING zinc finger protein-like 1.0� 1.2 1.1� 1.2 1.1� 1.3 1.3� 1.3

Contig8063_at Putative CuZn-superoxide

dismutase

0.9� 1.9 1.2� 1.3 1.2� 1.2 2.0� 1.6

Contig14019_at Putative GDP-mannose

pyrophosphorylase A

1.1� 1.3 0.9� 1.2 1.1� 1.2 0.8� 1.2

Microarray analysis was performed with total RNA extracted from the root apices from Murasakimochi (Mu) and Morex (Mo) exposedto Al (þ) or not (�) for 6 h. Data are means� SD.

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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 injected 14C-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

by PCR and reverse transcription–PCR (RT–PCR),

respectively, using gene-specific primers (Fig. 3A).

Transgenic tobacco plants overexpressing HvAACT1

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 24 h 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

−120 −100 −80 −60 −40 −20 00

−100

−200

−300

−400

−500

Current (nA

)

Holding potential (mV)

water + citrate − AI HvAACT1 + citrate − AIHvAACT1 + citrate + AIwater + citrate + AI

140

120

100

80

60

40

20

0

Rel

ativ

e cu

rren

t (%

)

Citrate Malate Citrate Malate

Citrate MalateHvAACT1

HvAACT1

HvAACT1

HvAACT1

Water

Water

Water

Water

5.0

4.0

3.0

2.0

1.0

0.0

Effl

ux (

% r

adio

activ

ity)

5.0

4.0

3.0

2.0

1.0

0.014C

-citr

ate

efflu

x (%

rad

ioac

tivity

)

0 10 20 30 40 50 60 70

Time (min)

A

B

C

D

Fig. 2 Heterologous expression of HvAACT1. (A) Mean current–voltage curves from oocytes expressing HvAACT1 and water-injectedoocytes. Sodium citrate was injected before measurement and the electrical potential (mV) was clamped from �100mV to the potentialwhich indicated current of 0A in 10mV steps in the presence or absence of Al. Data are means� SD (n¼ 3–6). (B) Substrate specificity ofHvAACT1. Citrate or malate was injected into oocytes with or without HvAACT1 expression, and the inward current produced by Al wasmeasured at �100mV. Relative values are shown. Data are means� SD (n¼ 3–6). (C) Efflux activity of citrate due to HvAACT1. Oocyteswith or without HvAACT1 expression were injected with 14C-labeled citrate and the release of 14C-labeled citrate from the oocyteswas determined at various times. Data are means� SD (n¼ 4). (D) Efflux activity of citrate and malate due to HvAACT1. Oocytes withor without HvAACT1 expression were injected with 14C-labeled citrate or malate and the release of 14C-labeled citrate or malate fromthe oocytes was determined 1 h later. Data are means� SD (n¼ 4).

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was 26-fold higher in the Al-resistant cultivar

(Murasakimochi) than in the Al-sensitive cultivar

(Morex), and the expression level was not induced by Al

in either cultivar (Fig. 4A). These results are consistent with

those obtained by microarray analysis (Table 1).

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).

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 (r¼ 0.93) between the

expression level of HvAACT1 mRNA in the roots and

the amount of Al-induced citrate secretion (Fig. 4C) as

well as (r¼ 0.89) Al resistance (relative root elongation)

(Fig. 4D). We compared the ORF of HvAACT1 in all these

cultivars and found four single nucleotide polymorphisms

(SNPs) between cultivars (Supplementary Fig. S1).

However, these SNPs cannot explain the differences in

HvAACT1 expression.

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

membrane.

Discussion

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 T0 line was

examined, analysis of several T1 lines showed that over-

expression of this gene in tobacco resulted in enhanced

1200

1000

800

600

400

200

0

140120100

806040200

Citr

ate

secr

etio

n(n

mol

g−1

roo

t dry

wt.

6 h−1

)

−HvAACT1

HvAACT1

−HvAACT1+HvAACT1

+HvAACT1

−AI +AI

−AI +AI

Rel

ativ

e ro

ot e

long

atio

n(%

of c

ontr

ol)

Over-expressed lines Wild type

A

B

C

Fig. 3 Overexpression of HvAACT1 in tobacco. (A) Expressionof HvAACT1 in the selected overexpressing T1 lines carryingHvAACT1 and in the wild type. (B) Citrate secretion fromtransgenic tobacco overexpressing HvAACT1. The plants with orwithout HvAACT1 expression were exposed to 0 or 30mM Alfor 6 h. Data are means� SD (n¼ 3–5). (C) Al resistance in tobaccocarrying HvAACT1. Plants were exposed to 0 or 30mM Al andtheir root length was measured before and after the treatment.Relative root elongation is shown. Data are means� SD (n¼ 3–10).

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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

barley.

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

Root Shoot

Mo MoMu Mu

−AI +AI −AI +AI −AI +AI −AI +AI

Actin

HvAACT1

120

100

80

60

40

20

0

AI−AI+

Mo Mu Mo Mu

Root tip Basal root tip

450400350300250200150100500

Citr

ate

secr

eted

(nm

ol g

−1 r

oot f

resh

wt.

6h−1

)

r = 0.93, P<0.01

r = 0.89, P<0.01

0 20 40 60

Relative HvAACT1 expression level(Normalized by Morex)

Relative HvAACT1 expression level(Normalized by Morex)

200

150

100

50

0

Rel

ativ

e H

vAA

CT

1 ro

ot e

xpre

ssio

n le

vel

(nor

mal

ized

by

Mor

ex, 0

mM

AI)

Rel

ativ

e ro

ot e

long

atio

n(%

of c

ontr

ol)

0 10 20 30 40 50 60

A C

B D

Fig. 4 Expression of HvAACT1. (A) Expression of HvAACT1 in different tissues of two barley cultivars with (þAl) or without (�Al) Altreatment. Mu, Murasakimochi; Mo, Morex. (B) Expression of HvAACT1 in different root segments of two barley cultivars with (þAl)or without (�Al) Al treatment for 6 h. Data are means� SD (n¼ 3). The relative value of Morex is shown. (C) Correlation betweenexpression of HvAACT1 in the roots and Al-induced secretion of citrate in 10 barley cultivars. The root exudates were collected for 6 hin the presence of 10 mM Al. Data are means� SD (n¼ 3). (D) Correlation between HvAACT1 expression and the relative rootelongation in 10 barley cultivars. The roots were exposed to a solution with or without 5 mM Al for 24 h. Data for root elongation aremeans� SD (n¼ 10).

Antisense Sense

A B

C D

Mu Mu

Mo Mo 100 µm

Fig. 5 Expression of HvAACT1 transcripts in barley roots.Cryosections of root tips (5mm) from Murasakimochi (A, B) orMorex roots (C, D) were hybridized with antisense (A, C) and sense(B, D) probes labeled with digoxigenin. Scale bar¼ 100 mm.

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function of this gene has not been characterized

(Uhde-Stone et al. 2005). Lupin secretes citrate from the

roots in response to phosphorus deficiency, suggesting that

MATE is also involved in the phosphorus deficiency-induced

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

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 5 cm from the

apex (Pineros 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 future.

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

Mo

A B C

Mu Mu + peptide

Fig. 6 Localization of the HvACCT1 protein in barley roots. Immunostaining was performed using anti-HvAACT1 polyclonal antibodyat the root tip (3mm) of Morex (A) and Murasakimochi (B). The specificity of the antibody was tested by pre-incubating the antibodywith the epitope peptide (C). Scale bar¼ 100mm.

A B

C D

HvAACT1-GEP GEP

Fig. 7 Subcellular localization of HvAACT1. A gene fusionbetween HvAACT1 and GFP (A, C) or GFP protein alone (B, D)was introduced into onion epidermal cells. A and B, GFP-derivedfluorescence; C and D, fluorescence superimposed over the trans-mission image. Scale bar¼ 100mm.

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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

resistance.

Materials and Methods

Fine mapping of the candidate gene

A barley F4 segregating population from the heterozygousplants for the QTL of Al resistance and citrate secretion, which wasderived from a cross between Al-resistant (Murasakimochi)and Al-sensitive (Morex) cultivars, was used for fine mapping ofthe gene. A total of 793 individuals were grown hydroponicallyas described (Ma et al. 2004), and the leaves were sampled forDNA extraction. The samples were genotyped first with twomarkers: Bmac310 and Bmag353. Individuals with the recombina-tion were chosen for further genotyping with the developedmarkers. Markers K00500, K02565, K02338, K03066, K04725and K06496 between Bmac 310 and Bmag353 were developed

according to the corresponding EST sequence from the barley ESTdatabase (Supplementary Table S1). Marker HvP1 was developedbased on the sequence of a rice BAC clone OSJNBa0090D11 onrice chromosome 3 of Oryza sativa ssp. japonica ‘Nipponbare’. ThemRNA sequence of a pyridoxal phosphate-dependent enzymefamily protein gene (Os03g0215800) located on the BAC washomologous with an EST (bags5e04) from the barley database.The Al-induced secretion of citrate was also examined in therecombinants as described previously (Ma et al. 2004). Allrecombinant F4 plants were used for construction of a fine map.

Microarray analysis

Four-day-old seedlings were transferred to a 1.0mM CaCl2solution (pH 5.0, aerated) containing 0 or 5mM Al for 6 h at 238C.Root apices (0–10mm from root tip, 40 root tips/sample) wereharvested and stored at �808C until RNA extraction. Microarrayanalysis was performed with a Barley 1 GeneChip according to themanufacturer’s protocol (Affymetrix). Two replicates were madefor each sample. Gene expression was examined in Murasakimochiand Morex with and without Al treatment.

Screening and sequence of BAC clones

The BAC clones containing the candidate gene werescreened with a pair of primers: TGGAGGAAGCATAGTATCand CACCTGGAGGTATGAA from a BAC library of HarunaNijo (Saisho et al. 2007). The selected BAC clone was sequenced.

Electrophysiological studies in Xenopus laevis oocytes

The full-length HvAACT1 cDNA derived fromMurasakimochi was amplified by PCR using high-fidelity KODplus DNA polymerase (Toyobo, Tokyo, Japan). Gene-specificprimers 50-TGCAGGATCCAAGCATCCGCTGTGTATGGAG-30 and 5-0TGCAGGATCCTCACTTCCGGAGGAAAACCC-30 were used to create BamHI sites on both ends andthen inserted into the BglII site in the oocyte expression vectorpXbG-ev1. The plasmid was linearized with BamHI, and cRNAwas transcribed in vitro with T3 RNA polymerase (mMESSAGEmMACHINE kit; Ambion, Austin, TX, USA). For each experi-ment, 50 nl of water containing 50 ng of cRNA was injected intoeach X. laevis oocyte. The cRNA-injected oocytes were incubatedin Modified Barth’s Saline (MBS) solution at 188C. After a 24 hincubation, 50 nl of 25mM sodium citrate or 25mM sodiummalate were injected into the oocytes and then incubated for 1–3 h.Before measurement, the oocytes were exposed to a modified MBSsolution containing 100 mM Al at pH 4.5 according to Sasaki et al.(2004). The net current across the oocyte membrane was measuredusing the two-electrode voltage clamp system with the amplifier(MEZ-7200 and CEZ-1200, Nihon Kohden, Tokyo, Japan) atdifferent membrane voltages. The electrical potential differenceacross the membrane was clamped from �100mV to the potentialwhich indicated 0A current, in 10mV steps.

Efflux transport activity of citrate

Oocytes with or without HvAACT1 expression as describedabove were injected with 50 nl of 2.4mM 14C-labeled citrateor 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 500 ml of fresh buffer at 188C.For the time-course experiment, 500 ml of buffer was carefullysampled, and replaced with fresh buffer at the time pointsindicated. At the end of the experiments, the oocytes werehomogenized with 0.1N HNO3. The radioactivity of the buffer

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and homogenized oocytes was measured with a liquid scintillationcounter (Aloka LIQUID SCINTILLATION SYSTEM).

Overexpression of HvAACT1 in tobacco

The full-length HvAACT1 cDNA derived fromMurasakimochi was amplified by PCR using KOD plus DNApolymerase with the gene-specific primers 50-AAGCATCCGCTGTGTATGGAG-30and 50-TCACTTCCGGAGGAAAACCC-30

and then cloned into pTA2 vector (Toyobo, Tokyo, Japan)according to the manufacturer’s protocol. After XhoI andBamHI treatment, HvAACT1 cDNA with XhoI and BamHI siteson both ends was ligated into an upstream SalI and a downstreamBamHI restriction site in pPZP2Ha3(�) Agrobacterium-mediatedtransformation vector (Fuse et al. 2001). The vectors weretransferred to Agrobacterium tumefaciens (strain EHA101) byelectroporation. Tobacco (Nicotiana tabacum) plants were trans-formed as described previously (Yamaji and Kyo 2006).Transformed calluses were selected by hygromycin resistance,and from them regenerated plants were obtained. Transgenic linescarrying HvAACT1 were selected from T1 lines by PCR using theprimers described above. The 3-week-old T1 plants carryingHvAACT1 or not were transferred to 1.2 liter plastic pots (threeplants per pot) containing 1/10 Hoagland solution. After 2 weeks,the plants were exposed to 0 or 30mMAl in 1.0mM CaCl2 solution(pH 5.0) for 6 h. The citrate in the root exudates was measuredaccording to Delhaize et al. (1993). For evaluation of Al resistance,the transformed tobacco plants (5 weeks old) were exposed to the1/10 Hoagland solution containing 0 or 30 mM Al at pH 4.5 for24 h. The root elongation was measured with a ruler.

Tissue-dependent expression of HvAACT1

Four-day old barley seedlings exposed to a 1.0mM CaCl2solution (pH 5.0, aerated) containing 0 or 5 mM Al for 6 h wereseparated into roots and shoots. The samples were ground in liquidnitrogen and RNA was immediately extracted with an RNeasyplant Mini Kit (Qiagen, Valencia, CA, USA). cDNA wassynthesized from the extracted RNA with a SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen), and the geneexpression level was quantified by quantitative RT–PCR withSYBR Green I reagent (SYBR Premix Ex Taq; TAKARASHUZO CO. LTD, Tokyo, Japan) on a Prism 7500 real-timePCR System (Applied Biosystems, Foster City, CA, USA)according to the manufacturer’s instruction. The primers usedfor HvAACT1 were 50-GTTCGCCAAGAACGATCACA-30 and50-AGAGACCAAGCACCACCGTC-30 Expression data werenormalized with the expression level of Actin, and the data forthe root tips of Murasakimochi were compared with those ofMorex (0mMAl) by the ��Ct method. The primers used for Actinwere 50-GACTCTGGTGATGGTGTCAGC-30 and 50-GGCTGGAAGAGGACCTCAGG-30. The expression level at different rootsegments (0–10 and 10–20mm) was also examined with threereplicates.

Correlation between HvAACT1 expression level and citrate secretion

Morex, Murasakimochi (CI5899), Haruna Nijo, ALP7,ALP21, ALP25, BC26, BC29, BC95 and Z504, which differed inAl resistance, were used (Zhao et al. 2003). Root length wasmeasured before and after the seedlings prepared as above wereexposed to a 1.0mM CaCl2 solution containing 0 or 5 mM Al for24 h. Root exudates of each cultivar exposed to 10 mM Al werecollected for 6 h and the HvAACT1 expression of the roots wasdetermined as described above.

In situ hybridization and immunostaining

The RNA probes were made by amplification of the ORFregion of HvAACT1 cDNA by PCR with the forward primer,50-AAGCATCCGCTGTGTATGGAG-30, and reverse primer,50-TCACTTCCGGAGGAAAACCC-30, and then cloned intopTA2 vector (Toyobo) according to the manufacturer’s protocol.After checking the direction of the inserted cDNA, the plasmidwas linearized with BamHI (sense strand) and HindIII (antisensestrand). In situ hybridization was done with 12 mm cryosectionsof Murasakimochi or Morex roots as described elsewhere(Jackson 1991).

For immunostaining, the synthetic peptide C-HGPEEKAAEDLPAA (positions 35–48 of HvAACT1) was used to immunizerabbits to obtain antibodies against HvAACT1. The roots of bothcultivars were used for immunostaining according to Ma et al.(2006). To check the specificity, the antibody (1 : 50 dilution) waspre-incubated with the epitope peptide used for preparation ofantibody at 25 nmol ml�1 for 1 h at room temperature beforestaining.

Subcellular localization of HvAACT1

For constructing a translational HvAACT1–GFP fusion,the full-length HvAACT1 ORF except for the stop codon derivedfrom Murasakimochi was amplified. Amplification was performedusing KOD plus DNA polymerase and the nucleotide sequencewas checked to confirm its identity. Gene-specific primers50-TGCACTCGAGAAGCATCCGCTGTGTATGGAG-30 and50-TGCAGGATCCCTTCCGGAGGAAAACCCATG-30 wereused to create XhoI and BamHI sites on both ends. After BamHItreatment, the 30 end was filled with T4 DNA polymerase(New England Biolabs, Ipswich, MA, USA) and then treatedwith XhoI restriction enzyme. The XhoI-blunt fragment ofHvAACT1 was inserted upstream of SalI and downstream ofa blunted NcoI restriction site between the 35S promoter and theGFP coding region in pBluescript vector. The fused gene wasintroduced into the onion epidermal cells as described by Murataet al. (2006).

Supplementary material

Supplementary material mentioned in the article is

available to online subscribers at the journal website

www.pcp.oxfordjournals.org.

Acknowledgments

This work was supported by the Program of Promotionof 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 OharaFoundation for Agricultural Science.

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(Received June 26, 2007; Accepted July 9, 2007)

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