Aluminum-activated root malate and citrate exudation play an important role in plant Al tolerance. This paper characterizes AtMATE, a homolog of the recently discovered sorghum and barley Al-tolerance genes, shown here to encode an Al-activated citrate transporter in Arabidopsis. Together with the previously characterized Al-activated malate transporter, AtALMT1, this discovery allowed us to examine the relationship in the same species between members of the two gene families for which Al-tolerance genes have been identified. AtMATE is expressed primarily in roots and is induced by Al. An AtMATE T-DNA knockdown line exhibited very low AtMATE expression and Al-activated root citrate exudation was abolished. The AtALMT1 AtMATE double mutant lacked both Al-activated root malate and citrate exudation and showed greater Al sensitivity than the AtALMT1 mutant. Therefore, although AtALMT1 is a major contributor to Arabidopsis Al tolerance, AtMATE also makes a significant but smaller contribution. The expression patterns of AtALMT1 and AtMATE and the profiles of Al-activated root citrate and malate exudation are not affected by the presence or absence of the other gene. These results suggest that AtALMT1-mediated malate exudation and AtMATE-mediated citrate exudation evolved independently to confer Al tolerance in Arabidopsis. However, a link between regulation of expression of the two transporters in response to Al was identified through work on STOP1, a transcription factor that was previously shown to be necessary for AtALMT1 expression. Here we show that STOP1 is also required for AtMATE expression and Al-activated citrate exudation.
Aluminum (Al) is the third most abundant element in the Earth’s crust. At low pH values (pH <5.5), the toxic species of aluminum, Al3+, is solubilized from aluminosilicate clay minerals into soil solutions and is toxic to crop plants (Kochian et al., 2004). Al toxicity mainly targets the root apex, resulting in inhibited root growth and function. As a result, Al toxicity leads to severe impairment in the acquisition of water and nutrients from the soil, which results in a significant reduction in crop yields on acid soils (Delhaize et al., 2007; Kochian et al., 2004; Ma et al., 2001; Matsumoto, 2000; Ryan et al., 2001). As up to 50% of the world’s potentially arable soils are acidic, with a significant proportion of these acid soils found in the tropics and sub-tropics in developing countries where food security is most tenuous, Al stress represents one of the most important constraints for agricultural production worldwide (Kochian et al., 2004).
As a strong correlation between root OA exudation and differential Al tolerance is found for many Al-tolerant crop species, it is not surprising that the first two plant Al-tolerance genes isolated were those encoding an Al-activated malate transporter in wheat (Sasaki et al., 2004) and an Al-activated citrate transporter in sorghum and barley (Furukawa et al., 2007; Magalhaes et al., 2007). TaALMT1 (Al-activated malate transporter) from wheat (Triticum aestivum), the first Al-tolerance gene identified in plants, encodes an Al-activated malate transporter that is expressed specifically and constitutively in the root apices of Al-tolerant wheat lines (Sasaki et al., 2004). Expression of the TaALMT1 transgene facilitated Al-activated malate efflux and increased Al tolerance in both tobacco suspension cells (Sasaki et al., 2004) and transgenic barley plants (Delhaize et al., 2004).
In sorghum (Sorghum bicolor), Al-activated citrate exudation from root apices of an Al-tolerant sorghum line is controlled at the AltSB locus, which explains more than 80% of the phenotypic variation in Al tolerance in the mapping populations studied (Magalhaes et al., 2004, 2007). SbMATE, the gene that underlies the AltSB locus, encodes a plasma membrane-localized citrate transporter that belongs to the multi-drug and toxic compound extrusion (MATE) family (Hvorup et al., 2003; Magalhaes et al., 2007). SbMATE is expressed primarily in the root apices of Al-tolerant lines and is induced by Al, ultimately being responsible for the observed Al-activated root citrate exudation. Furthermore, over-expression of SbMATE in transgenic Arabidopsis plants conferred a significant increase in Al tolerance and root citrate exudation (Magalhaes et al., 2007).
In Arabidopsis, Al stress induces a high level of root malate exudation and a lower level of root citrate exudation (Magalhaes et al., 2007). The Al-activated root malate exudation is facilitated by AtALMT1, a functional homolog of TaALMT1 (Hoekenga et al., 2006; Magalhaes, 2006). Expression of AtALMT1 requires STOP1, a putative zinc finger transcription factor involved in low pH resistance and Al tolerance in Arabidopsis (Iuchi et al., 2007). In the absence of STOP1, Al-induced AtALMT1 expression and Al-activated root malate exudation were completely suppressed in Arabidopsis, indicating that STOP1 is essential for AtALMT1 gene function (Iuchi et al., 2007).
In the current study, we have utilized an integrated genetic and physiological analysis to identify a member of the Arabidopsis MATE family, AtMATE, as the functional homolog of SbMATE. We then demonstrate that the Al-activated citrate exudation facilitated by AtMATE functions independently from and along withAtALMT1 to confer full expression of Arabidopsis Al tolerance.
AtMATE shares highest sequence identity with SbMATE
In Arabidopsis, the MATE family contains at least 56 members, which can be further classified into several clusters based on sequence similarity (Li et al., 2002; Rogers and Guerinot, 2002). AtMATE, which was originally named AtFRDL as it is the closest homolog of AtFRD3 in the AtFRD3 clade (Rogers and Guerinot, 2002), is the Arabidopsis MATE family member with the highest sequence similarity to SbMATE in sorghum (Figure 1a). The AtFRD3 clade has four members, including AtFRD3 (At3g08040), AtMATE (At1g51340), At2g38330 and At4g38380. Figure 1(a) shows a phylogenetic tree in which SbMATE, AtMATE and AtFRD3 are more closely related to each other than to the other two members of this clade, At2g38330 and At4g38380.
If HvMATE, the functional homolog of SbMATE in barley (Furukawa et al., 2007), and Os01g69010, the rice MATE with the closest sequence similarity to SbMATE, are also included in the phylogenetic tree, the monocot homologs are found to be closely clustered together, and AtMATE is more closely related to its monocot counterparts than to AtFRD3 (Figure 1b). In addition, AtMATE and Os01g69010 are closely clustered with the known citrate transporters (i.e. SbMATE, HvMATE and AtFRD3), suggesting that they might also function as citrate transporters. It should be noted that Os01g69010 is located in a region of the rice genome that is syntenic to the region in sorghum in which SbMATE is found. Furthermore, several rice Al-tolerance QTLs have been identified in this region by studies from several laboratories (see discussion in Magalhaes et al., 2004; Nguyen et al., 2001, 2002; Wu et al., 2000).
AtMATE expression in roots is induced by Al treatment
To monitor gene expression patterns for the members of the AtFRD3 clade, we performed quantitative real-time RT-PCR analysis. Under standard (−Al) growth conditions, AtMATE transcripts in the wild-type (WT) background were primarily detected in roots, but not in shoots (Figure 2). The level of the AtMATE transcript in roots was significantly increased in the wild-type after 1 day of Al treatment and remained at the same level of expression after 3 or 6 days of Al treatment, suggesting that Al stress induced de novo AtMATE transcription (Figure 2). Although Al treatment also increased AtMATE transcript levels in shoots of the wild-type plants, the corresponding levels were much lower in shoots than those in roots (Figure 2). The patterns of AtALMT1 gene expression in the wild-type background were similar to those of AtMATE (Figure 2), consistent with results previously reported by Hoekenga et al. (2006).
To demonstrate the biological function of AtMATE, we obtained an AtMATE T-DNA insertion line (SALK_081671) from the Arabidopsis Biological Resource Center (ABRC). We found that the T-DNA insertion, 370 bp upstream from the start codon of AtMATE, caused a significant reduction in the level of AtMATE transcript in both root and shoot tissues, and the induction of AtMATE expression by Al stress seen in wild-type plants was no longer present in any tissues of this T-DNA insertion line (Figure 2). These results suggest that the T-DNA insertion in the promoter region disrupts the cis-element essential for Al-induced/enhanced AtMATE gene expression, but that the cis-element required for basal AtMATE expression is not disrupted by the T-DNA insertion. Thus, the T-DNA insertion line represents an AtMATE knockdown line (AtMATE-KD). The pattern of AtALMT1 expression in the AtMATE-KD line was comparable to that of the wild-type (Figure 2). In the AtALMT1 T-DNA insertion knockout line (AtALMT1-KO) (SALK_009629), a T-DNA insertion in the AtALMT1 coding region caused loss of detectable AtALMT1 transcripts (Figure 2). However, the pattern of AtMATE gene expression in the AtALMT1-KO background was not affected by the loss of AtALMT1 expression (Figure 2). These results indicate that expression of AtMATE and AtALMT1 is independent of the presence of the other gene.
In the AtFRD3 clade, AtFRD3 was shown to play a role in iron nutrition and is mainly expressed in the stelar tissues of Arabidopsis roots (Green and Rogers, 2004). Our quantitative real-time RT-PCR results indicate that AtFRD3 expression was not induced by Al treatment (Figure 2). Of the other two members in the AtFRD3 clade, At2g38330 was primarily expressed in shoots, while At4g38380 was constitutively expressed in all tissues, and expression of these genes was not increased by Al exposure (Figure 2).
Thus, within the AtFRD3 clade, AtMATE is the only member whose gene expression is induced by Al and the expression is localized primarily to the root, which is the site where Al tolerance occurs. Furthermore, AtMATE shares highest sequence similarity with SbMATE (Figure 1). Based on these lines of evidence, AtMATE was considered the best candidate for the functional homolog of SbMATE within the AtFRD3 clade.
AtMATE is responsible for Al-activated root citrate exudation
In the Arabidopsis thaliana Columbia ecotype (Col-0), Al induces a high level of malate exudation and a lower level of citrate exudation from roots (Figure 3a,b). The Al-activated malate exudation has already been shown to be facilitated by AtALMT1 (Hoekenga et al., 2006), while the Al-activated root citrate exudation is consistent with the Al-induced AtMATE gene expression pattern shown in Figure 2.
To examine the role of AtMATE in Al-activated OA exudation, we tested root OA exudation profiles in the AtMATE-KD line. As seen in Figure 3(b), the level of Al-activated citrate exudation was greatly reduced in the roots of the AtMATE-KD line compared with that in the roots of the wild-type (Figure 3b). These results suggest that AtMATE encodes the Al-activated citrate transporter, and no other genes in the Arabidopsis thaliana Columbia ecotype (Col-0) are redundant withAtMATE.
The AtMATE AtALMT1 double mutant is highly hypersensitive to Al stress
T-DNA insertion lines were also obtained from the ABRC for other members of the AtFRD3 clade. Homozygous T-DNA insertion lines were evaluated for Al tolerance expressed as percentage root growth in the presence of Al divided by control root growth (% relative net root growth, %RNRG). At 1.5 μm Al3+ activity, only the AtMATE-KD line displayed a small decrease (approximately 10%) in Al tolerance compared with the wild-type (Figure 4). The T-DNA knockout lines AtFRD3-KO (SALK_122235), At2g38330-KO (SALK_048133) and At4g38380-KO (SALK_023501) did not display any reduction in Al tolerance compared with wild-type plants (data not shown).
AtALMT1 is clearly the major determinant of Al tolerance in the Columbia ecotype, as knocking out this gene caused an approximately 60% reduction in Al tolerance with respect to the wild-type at 1.5 μm Al3+ activity (Figure 4) (Hoekenga et al., 2006). The lack of a strong phenotype for AtMATE-KD in response to Al stress could be due to the presence of an intact AtALMT1 locus in this background, based on the observation that strong Al-induced AtALMT transcription (Figure 2) and Al-activated malate exudation (Figure 3a) were still present in the AtMATE-KD background. Thus, the effect of the abolished root citrate exudation on Al tolerance in the AtMATE-KD line might be more clearly seen in a genetic background lacking the AtALMT1 transporter.
To test this hypothesis, a double AtALMT1 AtMATE T-DNA knockout/knockdown line (Double-KO/KD) was generated via a cross between AtALMT1-KO (SALK_009629) and AtMATE-KD. Like plants from the individual single mutant lines, the Double-KO/KD plants grew normally in standard (−Al) nutrient solution, pH 4.2 (Figure 4a). At a low level (0.5 μm) of Al3+ activity, Al tolerance was similar for the wild-type (Col-0), AtMATE-KD, AtALMT1-KO and Double-KO/KD lines (data not shown). At higher Al levels (1.0 and 1.5 μm Al3+ activity), the lack of AtMATE expression did decrease Al tolerance, which is most readily seen when the Double-KO/KD mutant is compared with AtALMT1-KO (Figure 4). At 1.0 and 1.5 μm Al3+ activities, the Al tolerances based on %RNRG in the Double-KO/KD line were 75 and 50% of those of the AtALMT1-KO line, respectively (Figure 4b). As the only difference between AtALMT1-KO and Double-KO/KD lies in the knockdown mutation of AtMATE in the Double-KO/KD line, the additional Al sensitivity displayed in Double-KO/KD can only be explained by the lack of Al-induced AtMATE expression and the concomitant loss of Al-activated root citrate exudation (Figure 3). Therefore, our results indicate that the Al-activated root citrate exudation facilitated by AtMATE makes a moderate contribution (approximately 30%) to the overall Arabidopsis Al tolerance.
AtMATE and AtALMT1 function independently in conferring Al tolerance
Both AtALMT1 and AtMATE are mainly expressed in root tissues, and expression of both genes is induced by Al stress (Figure 2). In the AtALMT1-KO background, no detectable AtALMT1 transcripts were found in either the root or shoot tissues (Figure 2), concomitant with abolishment of the Al-activated malate exudation from roots (Figure 3a) and the severe reduction in Arabidopsis Al tolerance (Figure 4). Despite these dramatic physiological and developmental changes under Al stress, the pattern of AtMATE gene expression (Figure 2) and the magnitude of Al-activated root citrate exudation (Figure 3b) in the AtALMT1-KO line remained unaltered compared to those of the wild-type.
In AtMATE-KD, we observed a sharp reduction in the AtMATE transcript levels in roots, as well as a complete loss of Al inducibility of AtMATE gene expression (Figure 2), which is concurrent with loss of Al-activated citrate exudation in this line (Figure 3b). However, the patterns of AtALMT1 gene expression and Al-activated root malate exudation in the AtMATE-KD line remained largely the same as those of the wild-type (Figures 2 and 3a).
These results indicate that induction of AtMATE and AtALMT1 gene expression by Al stress is independent of each other, as are the processes that lead to AtMATE-mediated citrate exudation and AtALMT1-mediated malate exudation. Moreover, it appears that these processes are mainly responsive to Al stress, but not to other physiological cues, based on the data here and those presented by Kobayashi et al. (2007).
Transcriptional regulation of AtMATE and AtALMT1 gene expression by STOP1
To determine whether the function of AtMATE is also controlled by STOP1, as is the case for AtALMT1 (Iuchi et al., 2007), we examined AtMATE gene expression and Al-activated root OA exudation profiles in the STOP1 T-DNA knockout line STOP1-KO (SALK_114108), which has been shown to be hypersensitive to low pH stress (Iuchi et al., 2007). We observed that Al-induced AtMATE gene expression and associated Al-activated root citrate exudation were strongly suppressed in the STOP1-KO background. This is similar to what was seen previously with regard to suppression of both Al-induced AtALMT1 gene expression and Al-activated root malate exudation in the same STOP1-KO background (Figure 5a,b). Our results indicate that the Arabidopsis STOP1 gene is essential for Al-induced expression of both AtMATE and AtALMT1.
In the wild-type background, low pH alone triggers a small increase in the levels of AtALMT1 and AtMATE transcripts (Figure 5a). For instance, when 6-day-old wild-type seedlings were transferred from the high-pH growth medium (pH 7.0) to the low-pH growth medium (pH 4.2) for 24 h, the levels of AtALMT1 and AtMATE transcripts in roots increased by 3.8- and 2-fold, respectively (Figure 5a). However, the increases in the levels of AtAMLT1 and AtMATE transcripts were much more profound in the presence of Al3+ in the low-pH medium. For example, 6 h after the wild-type seedlings were transferred from high-pH growth medium to low-pH growth medium supplemented with 1.5 μm Al3+ activity, the levels of AtALMT1 and AtMATE expression in roots increased by 13- and 9-fold, respectively (Figure 5a). These results indicate that, although gene expression for both AtALMT1 and AtMATE is responsive to low-pH stress, their increased expression is much greater in response to Al stress.
Coding sequences of the plant citrate transporters in the MATE family are highly conserved, and differ from those of other family members
To date, only a few citrate transporters have been identified and characterized in plants. These include AtFRD3 and AtMATE from the dicot species Arabidopsis (Rogers and Guerinot, 2002; this study), and SbMATE and HvMATE from the monocot species sorghum (Magalhaes et al., 2007) and barley (Furukawa et al., 2007), respectively. A common attribute of these citrate transporters is that they all belong to the MATE family of membrane transporters.
To examine the evolutionary relationship among the MATE citrate transporters from various plant species, we performed a sequence alignment for these plant citrate transporters. Our results indicate that the known and putative citrate transporters in plants display a high sequence similarity throughout their entire lengths (Figure 6a).
These citrate transporters also share a common predicted topology that is absent from other members of the MATE family, namely a predicted large intracellular loop (approximately 100 amino acids in length) between the 2nd and 3rd transmembrane domains, and the amino acid sequences of the first half of the loop are highly conserved (Figure 6a,b). The remainder of the predicted intracellular loops for these citrate transporters are short (20–30 amino acids long) (Figure 6b). Topological surveys based on the HMMTOP program (http://www.enzim.hu/hmmtop) indicated that members of the Arabidopsis MATE family other than AtMATE and AtFRD3 lack any large intracellular loops in their entire sequences (Figure 6c). It is intriguing to speculate that the large cytoplasmic loop unique to the citrate transporters AtFRD3 and AtMATE may play a role in citrate binding and transmembrane transport, and this possibility will be the focus of future research.
In this report, we have demonstrated that AtMATE is the functional homolog of the sorghum Al-tolerance gene, SbMATE, and the Al-activated citrate exudation facilitated by AtMATE makes a moderate contribution to overall Al tolerance in Arabidopsis. Our conclusions are based on the following evidence: (1) AtMATE is the closest sequence homolog of SbMATE in Arabidopsis (Figure 1); (2) AtMATE is mainly expressed in roots, where Al toxicity and tolerance take place (Figure 2); (3) AtMATE expression is induced by Al stress (Figure 2); (4) in the AtMATE-KD background, in which AtMATE gene expression is sharply reduced by a T-DNA insertion in the 5′ promoter region, Al-activated root citrate exudation is strongly suppressed (Figure 3b); and (5) the AtALMT AtMATE double knockout/knockdown mutant is measurably more Al-sensitive than the single AtALMT1-KO mutant; AtMATE appears to contribute approximately 30% of the total Arabidopsis Al tolerance (Figure 4).
In general, members of the MATE family share low sequence similarity (Li et al., 2002; Rogers and Guerinot, 2002). However, the coding sequences of the known citrate transporters from both monocots (SbMATE, HvMATE) and dicots (AtFRD3, AtMATE) are highly conserved at the amino acid level (Figure 6), and also share a unique predicted topological structure (i.e. a large cytoplasmic loop in the N-terminal portion of the protein) that distinguishes them from the rest of the members of the MATE family (Figure 6). Thus, it appears that the common ancestor of these citrate transporters evolved before the divergence of monocots and dicots, and that selection pressures have been imposed to keep their coding sequences conserved after this divergence.
In contrast to the coding sequences, the 5′ regulatory sequences of these citrate transporter genes are highly divergent. For instance, although SbMATE and AtMATE display a similar gene expression pattern in response to Al stress (Figure 2 and Magalhaes et al., 2007), their promoter regions share little sequence similarity. In sorghum, there is a miniature inverted transposable element (MITE)-containing region in the promoter of SbMATE and this region is repeated a number of times, with the number of repeats showing some correlation with the level of SbMATE expression and Al tolerance in various sorghum lines (Magalhaes et al., 2007). However, no MITEs or other transposons and no repeat regions are present in the promoter of the AtMATE gene. These results suggested that the regulatory regions of these genes have undergone significant independent evolution. However, we cannot rule out the possibility that some uncharacterized regulatory elements in the promoter regions are highly conserved among these Al-inducible citrate transport genes. Further promoter characterizations will be necessary to clarify this.
In Arabidopsis, AtFRD3 and AtMATE encode two citrate transporters that are involved in different biological processes. AtFRD3 is responsible for loading citrate into xylem tissues, which helps facilitate iron transport to shoots (Durrett et al., 2007; Green and Rogers, 2004), while AtMATE is responsible for citrate exudation into the rhizosphere to protect roots from Al toxicity (Figures 2 and 3). In order to mediate the transport of OAs from root cells into the rhizosphere, the Al-activated citrate transporter must be localized to cells of the root epidermis and/or cortex. The AtFRD3 gene is expressed in the root stelar tissues, in which AtFRD3 is presumed to transport citrate into the xylem (Green and Rogers, 2004). As such, the AtFRD3-KO line did not show any change in Al tolerance compared with wild-type (data not shown), indicating that, although AtFRD3 is capable of transporting citrate, it is not involved in Al tolerance in Arabidopsis. However, ectopically over-expressing the AtFRD3 gene in Arabidopsis conferred enhanced Al tolerance in transgenic plants (Durrett et al., 2007). Therefore, it appears that the gene expression and tissue-specific localization patterns, rather than biochemical functions of the proteins, determine what biological functions the citrate transporters AtFRD3 and AtMATE perform.
Malate and citrate are the common OAs released from the root apices of many Al-tolerant crop plants under Al stress (Ryan et al., 2001). Recently, the Al-activated malate transporter TaALMT1 (Sasaki et al., 2004) and the Al-activated citrate transporters SbMATE (Magalhaes et al., 2007) and HvMATE (Furukawa et al., 2007) have been identified and characterized in wheat, sorghum and barley, respectively. However, the possible relationship between Al-activated malate and citrate release could not be directly studied because the OA transporters were identified from different plant species.
In this report, we identified and characterized AtMATE, a gene that encodes the Al-activated citrate transporter in Arabidopsis. Together with the previously identified Al-activated Arabidopsis malate transporter, AtALMT1, this discovery allowed us to study the regulation and interaction of the Al-activated citrate and malate transport systems that underlie the physiological mechanism for Al tolerance in the same plant species.
We have demonstrated that the patterns of AtMATE and AtALMT1 gene expression are not affected by each other, as do the physiological processes of Al-activated malate and citrate exudation from the Arabidopsis root (Figures 2 and 3). Thus, it is apparent that each transporter is not dependent on the existence of the other for normal function. Therefore, although convergent evolution appears to have occurred with regard to these genes that underlie the same trait, it appears that these two systems have evolved independently.
STOP1 encodes a putative zinc finger protein that is essential for Arabidopsis plants to withstand low-pH conditions (Iuchi et al., 2007). In the STOP1-KO mutant background, both Al-induced AtALMT1 gene expression and the associated Al-activated root malate exudation were strongly suppressed (Figure 5), and, as a result, the STOP1 mutant line showed a similar hypersensitive phenotype to Al stress as that seen in AtALMT1-KO (Iuchi et al., 2007). These results indicate that the putative transcription factor STOP1 is necessary for AtALMT1 gene expression and Al-activated root malate exudation (Iuchi et al., 2007). In this report, we have further demonstrated that a functional STOP1 gene is also required for expression of AtMATE and the associated Al-activated root citrate exudation (Figure 5). As the individual AtALMT1-KO and AtMATE-KD mutants and the double mutant of AtALMT1 and AtMATE (Double-KO/KD) showed no hypersensitivity to low-pH stress (Figure 4a, 0 μm Al3+), it is likely that another unknown cellular process whose gene expression and function are also controlled by STOP1 directly mediates low-pH tolerance in Arabidopsis. Therefore, STOP1 is likely to be a common transcription factor that controls at least three independent biological processes involved in Al tolerance and low-pH tolerance: the AtALMT1-mediated and Al-activated root malate exudation process, the AtMATE-mediated and Al-activated root citrate exudation process, and low-pH tolerance mediated by an unknown mechanism.
In Arabidopsis, malate is the dominant OA species in the root OA exudation profile (Figure 3). It is possible that the differences in the magnitudes of root malate and citrate release are caused by differential gene expression levels between AtMATE and AtALMT1 and/or post-transcriptional regulation. The tricarboxylate citrate3− anion is a better Al3+ chelator than the bicarboxylate malate2− ion (Ryan et al., 2001). So, if the larger Al-activated malate exudation in Arabidopsis is caused by a stronger AtALMT1 promoter, a promoter swap between AtMATE and AtALMT1 could lead to a higher level of AtMATE expression, and a concomitant increase in citrate exudation and Al tolerance. From a practical point of view, focusing on the regulatory regions of OA transporter genes that underlie an important agronomic trait such as Al tolerance, and breeding crops with enhanced expression of the most effective OA transporters might be an efficient approach for the generation of crop varieties with higher levels of Al tolerance.
Arabidopsis thaliana (Columbia ecotype, Col-0) was used for all of the control experiments. The T-DNA insertion knockout (KO) or knockdown (KD) mutants AtMATE-KD (SALK_081671), AtALMT1-KO (SALK_009629), AtFRD3-KO (SALK_122235), At2g38330-KO (SALK_048133), At4g38380-KO (SALK_023501) and STOP1-KO (SALK_114108) were obtained from the Arabidopsis Biological Resource Center (ABRC). The double AtALMT1-KO AtMATE-KD (Double-KO/KD) mutant was generated by crossing AtMATE-KD with AtALMT1-KO, followed by selection of plants with homozygous T-DNA insertions at both the AtMATE and AtALMT1 loci from the F2 population of this cross.
RNA isolation and quantitative real-time RT-PCR
Approximately 10 mg of surface-sterilized seeds from the wild-type (WT, Col-0 ecotype), AtALMT1-KO and AtMATE-KD lines were germinated individually in Magenta boxes containing sterile hydroponic growth solution (pH 4.2) inside a growth chamber with continuous light and a temperature of 23°C (Hoekenga et al., 2006). At day 6, seedlings were either harvested for the control experiment or transferred to new hydroponic growth solutions supplemented with 1.5 μm Al3+ activity for 1, 3 or 6 days of Al treatment. For harvesting, seedlings were rinsed with distilled water, wiped gently with paper towels, and root and shoot samples were separated with scissors and immediately frozen in liquid nitrogen.
Sample preparation for real-time RT-PCR experiments using the STOP1-KO mutant line was similar to the procedures mentioned above except for the following modifications: sterile WT and STOP1-KO seeds were germinated in modified hydroponic growth solution with neutral pH (pH 7.0) for 6 days before transferring the seedlings to the low-pH (pH 4.2) hydroponic growth solution (Hoekenga et al., 2006) for another day. The higher-pH growth solution was identical in composition to the standard pH 4.2 medium described by Hoekenga et al. (2006) except that homopipes was omitted from the solution and the pH was adjusted to 7.0. After 6 days of germination in high pH (7.0), seedlings of WT and the STOP1-KO line were transferred to standard, HOMOPIPES-buffered regular low-pH (pH 4.2) growth solutions supplemented with or without 1.5 μm Al3+ activity for an additional 1, 3, 6, 12 or 24 h.
Total RNA was extracted from individual samples using an RNeasy plant mini kit according to the manufacturer’s instructions (Qiagen, http://www.qiagen.com/). First-strand cDNA was synthesized in a reaction cocktail containing 5 μg of DNase I-treated total RNA, 20 μl of 5× reaction buffer, 2.5 μm oligo(dT)16, 1 mm of each dNTP, and 5 μl SuperScript III reverse transcriptase (Invitrogen, http://www.invitrogen.com/) in a total volume of 100 μl. The reaction was carried out at 37°C for 90 min, followed by heating at 72°C for 10 min. Subsequently, 2 μl of RNase H (Invitrogen) was added to each sample, and the samples were incubated at 37°C for 1.5 h and stored at −20°C.
Quantitative real-time RT-PCR was performed using an ABI 7500 real-time PCR system and SYBR Green kit (Applied Biosystems, http://www.appliedbiosystems.com/). Each real-time RT-PCR reaction contained 1 μg of first-strand cDNAs, 0.15 μm primers and 10 μl of 2× SYBR Green Master Mix in a final volume of 20 μl.
The optimal gene-specific RT-PCR primer sequences are 5′-GCATAGGACTTCCGTTTGTGGCA-3′ and 5′-CGAACACAAACGCTAAGGCA-3′ for AtMATE, 5′-CTCAGATTTTCAGATCCCAGTGGAC-3′ and 5′-TTCCCGATTCCGAGCTCATT -3′ for AtALMT1, 5′-CGAGTTGCATCTCTTCTTCCT-3′ and 5′-TGATAACGGTCTCTCGAACA-3′ for AtFRD3, 5′-ATTCTATGGTGATTGTCGGA-3′ and 5′-GATCCGCTTCATTCTGGCTT-3′ for At2g38330, 5′-GATCGCACTGGCTATTGTGT-3′ and 5′-GCAATCCCATGAACATGCTC-3′ for At4g38380, and 5′-GCTGACCGTATGAGCAAAGA-3′ and 5′-GATCCACATCTGTTGGAACG-3′ for the internal control AtACT gene.
The real-time PCR process comprised an initial denaturation at 95°C for 10 min, followed by 40 cycles of 94°C for 15 sec and 60°C for 1 min, and a final dissociation stage of 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. The data were analyzed using the ABI 7500 System SDS software (Applied Biosystems).
Detection of organic acid exudation from roots
Surface-sterilized seeds (approximately 2–3 mg) from each Arabidopsis line were germinated in Magenta boxes containing sterile hydroponic growth solution (pH 4.2) for 6 days, and then seedlings were transferred to 20 ml of filter-sterilized exudation solution (pH 4.2) with or without 1.5 μm Al3+ activity for 2 days (see Hoekenga et al., 2006; for details). For experiments involving the STOP1-KO mutant line, seeds for the STOP1-KO line and wild-type (WT, Col-0) were germinated in sterile modified hydroponic growth solution at pH 7.0 instead of pH 4.2 for 6 days. Then seedlings were transferred to regular hydroponic growth solution at pH 4.2 for another day. Seedlings of individual lines were then transferred to 20 ml of filter-sterilized exudation solution (pH 4.2) with or without a total Al concentration of 13.6 μm AlCl3 (Al3+ activity of 1.5 μm) in a sterile Petri dish for 1 day. The exudation solutions were collected and the numbers of plants were counted. Then the exudation solutions were passed through anionic and cationic chromatography columns to remove Al3+ and other inorganic anions that could interfere with the measurement of organic acid anions. Subsequently, 1 ml of treated samples was analyzed to determine the profiles of organic acids using the capillary electrophoresis system as described by Piñeros et al. (2002).
Growth conditions and root measurement
Root growth experiments were conducted in hydroponic solution as described by Hoekenga et al. (2006). In brief, surface-sterilized seeds were cold-treated (4°C) for 4 days before being sown onto plastic mesh floating on hydroponic solutions in Magenta boxes. The hydroponic solutions either contained no Al (control) or were supplemented with 4.8, 9.4 or 14 μm of AlCl3, which corresponds to 0.5, 1.0 or 1.5 μm Al3+ activity in the solutions, as calculated using Geochem-PC software (http://www.geochem.zip.com.au/geochem_modeling_software.htm). Root measurements were performed after 6 days of growth in hydroponic growth solutions.
This work was supported by United States Department of Agriculture-Agriculture Research Service (USDA-ARS) Competitive Grant number 2006-35301-16884 and Generation Challenge Grant number I69. The authors would like to thank Zhifang Cheng for technical assistance. Arabidopsis T-DNA insertion lines were obtained from the Arabidopsis Biological Resource Center (ABRC).