Plant and Cell Physiology Advance Access originally published online on July 18, 2007
Plant and Cell Physiology 2007 48(8):1081-1091; doi:10.1093/pcp/pcm091
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Rapid Paper |
An Aluminum-Activated Citrate Transporter in Barley
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
*Corresponding author: E-mail, maj{at}rib.okayama-u.ac.jp; Fax, +81-86-434-1209.
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Abstract |
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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 immunostaining 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 toxic compound extrusion; ORF, open reading frame; QTL, quantitative trait locus; SNP, single nucleotide polymorphism
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsSupplementary materialAcknowledgmentsReferences |
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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 Uexküll 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 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, Piñeros and Kochian 2001). These studies also indicated that at least a subset of the Al-activated channels requires extracellular Al3+ to maintain 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 microsatellite 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), indicating 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.
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Results |
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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 transcripts 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 by >20-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 polypeptide 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.
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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.
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 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).
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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 (P < 0.01) (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% (P < 0.05) (Fig. 3C), indicating that the Al resistance was also enhanced in the transgenic plants.
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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 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 10–20 mm from the root tip than in the 0–10 mm region (Fig. 4B). Murasakimochi constitutively showed a higher expression level than Morex in both regions, irrespective of Al treatment (Fig. 4B).
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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 (r = 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 (r = 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).
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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.
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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.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsSupplementary materialAcknowledgmentsReferences |
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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 overexpression of this gene in tobacco resulted in enhanced 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 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 (Piñeros 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 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 transporters 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). Furthermore, 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.
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Materials and Methods |
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Fine mapping of the candidate gene
A barley F4 segregating 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 recombination 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 F4 plants were used for construction of a fine map.
Microarray analysis
Four-day-old seedlings were transferred to a 1.0 mM CaCl2 solution (pH 5.0, aerated) containing 0 or 5 µM Al for 6 h at 23°C. Root apices (0–10 mm from root tip, 40 root tips/sample) were harvested and stored at –80°C 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
The full-length HvAACT1 cDNA derived from Murasakimochi was amplified by PCR using high-fidelity KOD plus DNA polymerase (Toyobo, Tokyo, Japan). Gene-specific primers 5'-TGCAGGATCCAAGCATCCGCTGTGTATGGAG-3' and 5-'TGCAGGATCCTCACTTCCGGAGGAAAACCC-3' were used to create BamHI sites on both ends and then inserted into the BglII site in the oocyte expression vector pXßG-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 experiment, 50 nl of water containing 50 ng of cRNA was injected into each X. laevis oocyte. The cRNA-injected oocytes were incubated in Modified Barth's Saline (MBS) solution at 18°C. After a 24 h incubation, 50 nl of 25 mM sodium citrate or 25 mM sodium malate were injected into the oocytes and then incubated for 1–3 h. Before measurement, the oocytes were exposed to a modified MBS solution containing 100 µM 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 –100 mV to the potential which indicated 0 A current, in 10 mV steps.
Efflux transport activity of citrate
Oocytes with or without HvAACT1 expression as described above were injected with 50 nl of 2.4 mM 14C-labeled citrate or malate (Amersham, 2.3 nCi/oocyte) (4–5 oocytes/replicate). The oocytes were washed for 5 min in modified MBS buffer (pH 5.0) and then transferred into 500 µl of fresh buffer at 18°C. For the time-course experiment, 500 µl 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 0.1 N HNO3. The radioactivity of the buffer and homogenized oocytes was measured with a liquid scintillation counter (Aloka LIQUID SCINTILLATION SYSTEM).
Overexpression of HvAACT1 in tobacco
The full-length HvAACT1 cDNA derived from Murasakimochi was amplified by PCR using KOD plus DNA polymerase with the gene-specific primers 5'-AAGCATCCGCT GTGTATGGAG-3'and 5'-TCACTTCCGGAGGAAAACCC-3' 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 transformed as described previously (Yamaji and Kyo 2006). Transformed calluses were selected by hygromycin resistance, and from them regenerated plants were obtained. Transgenic lines carrying HvAACT1 were selected from T1 lines by PCR using the primers described above. The 3-week-old T1 plants carrying HvAACT1 or not were transferred to 1.2 liter plastic pots (three plants per pot) containing 1/10 Hoagland solution. After 2 weeks, the plants were exposed to 0 or 30 µM Al in 1.0 mM CaCl2 solution (pH 5.0) for 6 h. 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 30 µM Al at pH 4.5 for 24 h. The root elongation was measured with a ruler.
Tissue-dependent expression of HvAACT1
Four-day old barley seedlings exposed to a 1.0 mM CaCl2 solution (pH 5.0, aerated) containing 0 or 5 µM Al for 6 h 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 5'-GTTCGCCAAGAACGATCACA-3' and 5'-AGAGACCAAGCACCACCGTC-3' Expression data were normalized with the expression level of Actin, and the data for the root tips of Murasakimochi were compared with those of Morex (0 µM Al) by the 
Ct method. The primers used for Actin were 5'-GACTCTGGTGATGGTGTCAGC-3' and 5'-GGCTGGAAGAGGACCTCAGG-3'. The expression level at different root segments (0–10 and 10–20 mm) was also examined with three replicates.
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 1.0 mM CaCl2 solution containing 0 or 5 µM Al for 24 h. Root exudates of each cultivar exposed to 10 µM Al were collected for 6 h 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, 5'-AAGCATCCGCTGTGTATGGAG-3', and reverse primer, 5'-TCACTTCCGGAGGAAAACCC-3', 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 12 µm cryosections of Murasakimochi or Morex roots as described elsewhere (Jackson 1991).
For immunostaining, the synthetic peptide C-HGPEEKAAEDLPAA (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 25 nmol ml–1 for 1 h at room temperature before staining.
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 5'-TGCACTCGAGAAGCATCCGCTGTGTATGGAG-3' and 5'-TGCAGGATCCCTTCCGGAGGAAAACCCATG-3' were used to create XhoI and BamHI sites on both ends. After BamHI treatment, the 3' end 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).
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Supplementary material |
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Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oxfordjournals.org.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsSupplementary materialAcknowledgmentsReferences |
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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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Footnotes |
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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).
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References |
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(Received June 26, 2007; Accepted July 9, 2007)
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