Common bean is generally less adapted to acid soil environments and improving Al resistance of common bean to reduce the dependence of small-scale farmers on lime and nutrient inputs is a major challenge (Rao, 2001). However, efforts to develop adapted genotypes indicate that there are genotypic differences in Al resistance in the bean germplasm (Rao, 2001; Rangel et al., 2005; Manrique et al., 2006). Comparing two contrasting bean cultivars ‘Quimbaya’ (Al-resistant) and ‘VAX 1’ (Al-sensitive), Rangel et al. (2010) found that Al resistance in common bean is attributed to the release of citrate by the root apex. Organic acid anions such as citrate, malate and oxalate detoxify Al through forming a non-phytotoxic organic acid–Al complex. Ma et al. (2001) described two patterns of organic acid secretion: pattern I plants release organic anions immediately after the onset of Al treatment while in pattern II plants, organic anion release starts after a lag phase of several hours. This suggests that in pattern I the organic anion release mechanism is constitutively expressed, whereas in pattern II plants the induction of the resistance mechanism involves gene expression, and new protein synthesis. Common bean proved to be a typical pattern II plant (Rangel et al., 2007, 2010). The delay in citrate exudation was not due to the limitation of internal citrate reserve but to the absence of citrate permeases. The role of organic anion permeases in Al resistance was recently reviewed by Delhaize et al. (2007). Aluminium resistance genes of several plant species have been identified and found to encode membrane proteins which mediate the exudation of organic acid anions from the root. These proteins belong to two families, ALMT (Al-activated malate transporter) and MATE (multidrug and toxic compound extrusion). The ALMT facilitates malate efflux in plant species that depend on malate exudation as the Al resistance mechanism (Sasaki et al., 2004; Hoekenga et al., 2006; Ligaba et al., 2006). On the other hand, the MATE proteins are citrate transporters which play a decisive role in Al-induced citrate exudation (Furukawa et al., 2007; Magalhaes et al., 2007; Ryan et al., 2009).

Differential gene expression in Al-resistant genotype ‘Quimbaya’, in response to Al stress was assessed by using SSH. Through forward and reverse subtraction of cDNA samples (4 h Al treatment vs. control) 127 Al-induced differentially expressed transcripts, 61 up-regulated and 66 down-regulated genes were obtained (see Tables S1 and S2 in Supplementary data, available online). These genes were grouped into several functional categories as shown in Fig. 2. Al treatment affected the transcription of genes involved in a wide range of functions. Transcripts which were up-regulated due to Al stress include genes involved in stress response, plant defence, signal transduction and translation. In contrast, genes involved in primary metabolism, transport processes, cytoskeleton and cell wall organization were largely down-regulated. This indicates that Al toxicity induces manifold changes in the plant root, ranging from perception of stress signals, gene transcription and translation to downstream physiological processes. The up-regulated stress-responsive genes include those encoding peroxidases, heat shock proteins and dehydrins. Peroxidases may contribute to the plant Al resistance by detoxifying reactive oxygen species which are produced as a result of oxidative stress induced by Al. Down-regulated genes such as auxin influx-carrier and cytoskeleton-associated proteins confirm the physiological characterization of the mechanism of Al toxicity. However, this study was restricted to the genes which could be involved in citrate exudation and metabolism which was the focus of the physiological studies (Fig. 1). These include genes encoding isocitrate dehydrogenase, an anion-selective channel, a mitochondrial ATP synthase and cytochrome P450 monooxygenase. Their possible contribution to Al resistance is briefly summarized in Table 2.

In addition candidate genes were selected based on the physiological mechanisms of Al resistance in bean, which is a sustained release of citrate from the root tips (Fig. 1). The expression of two groups of genes was closely investigated using qRT-PCR. The first group included genes encoding the enzymes involved in citrate metabolism, while the second group consisted of genes encoding ion transporters. Aluminium treatment did not significantly alter the expression of genes coding for enzymes which play a role in citrate synthesis and degradation (Fig. 3). Besides, there were few or no genotypic differences in Al-induced changes in gene expression. However, constitutively lower expression of genes coding for phosphoenolpyruvate carboxylase (PEPC) and isocitrate dehydrogenase (ICDH) was observed in ‘VAX 1’ than in ‘Quimbaya’. The expression pattern did not change when the plants were treated for 8 or 24 h (data not shown).

The release of organic acid anions is mediated by membrane-localized anion transporters, whose expressions were known to be regulated by the STOP1 transcription factor. In beans, short-term Al treatment (4 h) slightly increased the expression of STOP1 (Fig. 4) but the expression levels of MATE-a, MATE-b and ALS3 were greatly enhanced by Al treatment in both bean genotypes. Yet, Al did not affect the transcript abundance of MATE-c, ALMT1 and VDAC (voltage-dependent anion-selective channel; Fig. 4).

Upon extended Al treatment duration, the expression of MATE-a and MATE-b continued to increase in the Al-sensitive genotype ‘VAX 1’ but not in the Al-resistant genotype ‘Quimbaya’ (Fig. 5). In order to find out the exact timing of MATE-gene induction, root samples were collected at 1-h intervals during the first 4 h of Al treatment. Enhanced expression of the MATE-a gene was observed as early as 2 h Al treatment in ‘VAX 1’, but in ‘Quimbaya’ the expression was delayed until 3 h (Fig. 6). The expression levels continued to increase with time in both genotypes. Similar to Fig. 5, the expression level was much higher in ‘VAX 1’ than in ‘Quimbaya’. The earlier induction of MATE genes in this genotype is in agreement with the earlier induction of citrate exudation in ‘VAX 1’ (Fig. 1B).

Finally, the expression of a typically stress-induced gene coding the ACC-oxidase (1-aminocyclopropane-1-carboxylic acid oxidase) which was differentially expressed according to the SSH study (Table S1 in Supplementary data) was determined (Fig. 7). Early Al stress (4 h) resulted in a >10-fold increase in the expression of ACC-oxidase gene in both bean genotypes. In a subsequent analysis, enhanced gene expression could be found in both genotypes as early as 1 h after Al treatment (data not shown). But with Al treatment duration, it decreased to the original expression level in ‘Quimbaya’ while it continued to increase in ‘VAX 1’. This stress response is similar to the response in Al-induced inhibition of root growth (Fig. 1A) and thus might be used as an internal marker of Al stress.

Genes that may be related to the citrate exudation-mediated Al resistance of bean include those encoding a VDAC and an NADP-specific ICDH. Several studies indicate that the release of organic acid anions is mediated by anion channels located in the plasma membrane (Kochian et al., 2004; Delhaize et al., 2007; and references therein). The VDAC is a family of eukaryotic pore-forming proteins, originally discovered in the outer membrane of mitochondria where it allows free permeability of low molecular-weight solutes (Colombini, 1979). It is found to be not only expressed in the mitochondria, but also in the plasma membrane (Lawen et al., 2005) and in peroxisomes (Arai et al., 2008). Thus, VDAC may mediate organic anion exudation in bean. However, its differential expression in response to Al treatment could not be verified by qRT-PCR.

Similar to the current observation, cytochrome P450 monooxygenase expression was found to be up-regulated in Al-resistant near isogenic wheat lines under Al stress by Guo et al. (2007) and Houde and Diallo (2008) and was implicated to play a role in Al resistance. Cytochrome P450s may serve as monooxygenases in the biosynthethis of lignin, defence compounds, hormones, pigments, fatty acids and signalling molecules and in the detoxification catalysing numerous endogenous and exogenous toxic compounds encountered in the environment (Schuler and Werck-Reichhart, 2003). Thus it may contribute to sustained root growth under Al stress conditions. In addition, mitochondrial F1-ATPase (alpha-subunit) was also up-regulated in bean root tips upon Al treatment. F1-ATPase is involved in the mitochondrial oxidative phosphorylation by which ATP is produced through a proton gradient. The energy stored in ATP could fuel the metabolic processes involved in Al resistance. Hamilton et al. (2001) hypothesized that the induction of V-ATPase and the F1F0-ATPase plays a role in Al resistance of wheat. The subunits of these enzymes were newly synthesized upon Al treatment and the proteins accumulate in an Al dose-dependent manner (Basu et al., 1994). In addition, accumulation of V-ATPase and F1F0-ATPase subunits segregated with the Al-resistant phenotype (Taylor et al., 1997). This indeed suggests that the up-regulated ATPase may play a yet unspecified role in Al resistance.

The best understood mechanism of Al resistance in plants is the release of organic acid anions such as citrate, malate and oxalate, which chelate Al and form non-toxic complexes (Ryan et al., 2001; Ma et al., 2001). Rangel et al. (2010) observed that Al-activated exudation of citrate plays a major role in Al resistance of common bean. Citrate exudation started after about 4 h of Al treatment despite the abundant citrate content in the root tissue (Fig. 1B and C). Moreover, the root growth of both Al-resistant and Al-sensitive bean genotypes was equally inhibited during this lag period, indicating that Al resistance in common bean is not constitutively expressed (Fig. 1A). This is in agreement with an earlier suggestion that Al resistance in bean is an inducible trait (Cumming et al., 1992). The lag phase between the beginning of Al treatment and the onset of citrate exudation shows that the induction process involves gene transcription and de novo synthesis of proteins which are necessary for citrate transport.

In the present study, the expression of genes encoding organic anion transporters was examined. Among the candidate genes tested, MATE-a (GenBank accession no. CV535133) and MATE-b (GenBank accession no. CV534527) were strongly expressed upon Al treatment in bean. Both ESTs (denoted as MATE-a and MATE-b) of common bean have high sequence similarity to previously characterized MATE genes of Lupinus albus (GenBank accession no. AY631874) and Glycine max (GenBank accession nos EU591739 and EU591741). Nucleotide sequences of MATE-a and MATE-b have no significant similarity and also they do not belong to the same contig assembly of ESTs in the TIGR database. Whether they are two different genes or just different sequence regions of the same gene will be clarified in the ongoing study through full-length cDNA sequencing.

The MATE proteins are a large family of membrane transport-proteins which have 58 members (paralogues) known just in the arabidopsis genome (Hvorup et al., 2003). The arabidopsis FRD3 gene which is important for iron transport in the xylem as ferric citrate is also a MATE protein (Durrett et al., 2007; Rogers et al., 2009). The role of a MATE protein for Al resistance was first observed in sorghum (Magalhaes et al., 2007) and barley (Furukawa et al., 2007) almost simultaneously, later in wheat (Ryan et al., 2009) and further characterized in arabidopsis (Liu et al., 2009). The MATE protein was described as an Al-activated citrate transporter which is responsible for Al resistance of both sorghum and barley (Furukawa et al., 2007; Magalhaes et al., 2007). In sorghum SbMATE was expressed only in the root tips of the Al-resistant genotype in an Al-inducible way. Similarly, barley HvMATE was constitutively expressed mainly in the root apices and correlated with Al-activated citrate exudation and Al resistance in a set of barley cultivars. In contrast, the MATE gene of bean is highly expressed in both resistant and sensitive genotypes used in the present study (Figs 4​4–6). This result corroborates the observation that citrate exudation was induced by Al in both Al-resistant and -sensitive genotypes (Fig. 1B). Regardless of the ample amount of citrate in the root tissue (Fig. 1B), exudation started only after about 4 h of Al treatment, the time lag which is required for activation of MATE-gene transcription, translation and formation of the functioning protein (Fig. 6). Similarly, Al enhanced the expression of a citrate transporter gene in soybean after 4 h (Z. M. Yang, Jilin University, China, pers. comm.). After the MATE protein is in place, citrate exudation progressed and resulted in a reduction in the citrate content of the root tissue (Fig. 1B and C). As a result of citrate exudation, both bean genotypes transiently recovered from the stress which equally affected both of them in the early hours of Al treatment (Fig. 1A). The remarkable difference between the two bean genotypes was observed in their capacity to replenish the tissue citrate reserve and to sustain citrate exudation in order to protect the growing root tip. The Al-resistant genotype ‘Quimbaya’ was able to restore the citrate pool in the root tissue and to continue to release citrate, whereas the Al-sensitive genotype ‘VAX 1’ was unable to restore the internal citrate pool and failed to further release citrate after the short recovery period (Fig. 1B and C). These observations underline that sustained synthesis of citrate as well as constant expression and activity of a citrate transporter are vital for Al resistance in common bean.

Although the role of organic acid anion exudation for Al resistance and the importance of organic acid anion transporters are currently well defined, the significance of organic acid metabolism and accumulation in the root tissue are still not well understood (Ryan et al., 2001; Horst et al., 2007). In plant species, where organic acid anion release started directly after Al treatment, no correlations were observed between internal organic acid concentrations and efflux. For example, Al-sensitive and Al-resistant wheat genotypes did not differ in root concentrations of malate, although the Al-resistant genotypes released up to 10-fold more malate than the Al-sensitive genotypes (Delhaize et al., 1993). Similarly, contrasting maize genotypes did not differ in tissue citrate content and Al equally increased citrate accumulation in the root tissue of both genotypes but significant citrate exudation was only observed in the Al-resistant genotype (Pellet et al., 1995). In contrast, in soybean (pattern II plant) the Al-enhanced internal accumulation of citrate contributed to the enhanced citrate exudation (Silva et al., 2001). Reports on the role of enzymes involved in the organic acid metabolism for Al-induced organic acid anion efflux are also diverse. In wheat, Al-induced malate exudation occurred without significant changes to the activities of PEPC or malate dehydrogenase (NAD-MDH). Moreover, the activities of these enzymes were not significantly different between genotypes (Ryan et al., 1995). In contrast, an increased citrate synthase (CS) activity was reported in P. vulgaris (Mugai et al., 2000) and Cassia tara (Yang et al., 2004) after Al treatment. Similarly, Al treatment enhanced the gene expression as well as enzyme activity of mitochondrial CS in soybean (Z. M. Yang, Jilin University, China, pers. comm.). Furthermore, over-expression of enzymes involved in organic acid metabolism has been proven to be effective in enhancing exudation of organic acid anions leading to Al resistance in transgenic plants of arabidopsis (Koyama et al., 2000), alfalfa (Tesfaye et al., 2001) and canola (Anoop et al., 2003).

Rangel et al. (2010) studied the changes in activities of enzymes involved in citrate metabolism. Al treatment reduced the activity of ICDH leading to reduced internal citrate consumption and enhanced exudation. The citrate content in the root tissue is a function of citrate synthesis, exudation, degradation or consumption for other metabolic functions. Accordingly, continuous release of citrate while maintaining normal citrate concentration in root tissue requires enhanced synthesis and/or reduced degradation of citrate. Reduction in cytosolic NADP-isocitrate dehydrogenase activity resulted in citrate accumulation and subsequent release from mutant carrot cells which were able to grow on insoluble phosphate sources (Kihara et al., 2003). But according to Rangel et al. (2010), not only the reduction of NADP–ICDH but also maintaining the activities of CS and PEPC are important for sustained exudation of citrate in common bean. Failure of continuous citrate exudation in the Al-sensitive bean genotype ‘VAX 1’ was mainly attributed to the constitutively lower CS activity which was further inhibited by extended duration of Al treatment. In the current study, no significant change was observed in the expression of genes encoding enzymes involved in citrate metabolism (Fig. 3). Similarly, Kumari et al. (2008) who made a large-scale, transcriptomic analysis of root responses to Al, using a microarray representing about 93 % of the predicted genes in the genome of arabidopsis did not detect a significant increase in transcript abundance for any of the 52 genes of the TCA cycle present in the microarray, except for MDH. However this does not mean that there is no change in the activity of enzymes involved in the TCA cycle. Since Rangel et al. (2010) clearly demonstrate the changes in the enzyme activity of the above bean genotypes it can be concluded that the activities of these enzymes are regulated at the post-translational level.

The role of ATP-binding cassette transporter family proteins, ALS1 and ALS3 for Al resistance, was observed in arabidopsis (Larsen et al., 2005, 2007). Plant ATP-binding cassette transporters that have been functionally characterized so far were known to detoxify organic and inorganic compounds by sequestering in the vacuole (Schulz and Kolukisaoglu, 2006). Arabidopsis als1-1 and als3-1 mutants were hypersensitive to Al but the exact functions and substrates of ALS1 and ALS3 are not known. Whereas ALS1 is located in the tonoplast and the gene is expressed in root apices and the vascular system (Larsen et al., 2007), ALS3 is primarily located in the plasma membrane of leaf hydathode cells, the phloem and the root cortex (Larsen et al., 2005). The expression of ALS3 is induced by Al and was suggested to function in channelling accumulated Al away from Al-sensitive tissues in order to protect the growing root from Al toxicity. In agreement with Larsen et al. (2005) it was observed in the present study that Al treatment induced the expression of the ALS3 gene in both in Al-resistant and sensitive bean genotypes (Fig. 4). However, the suggested function of ALS3 could not be confirmed since the sensitive cultivar continued to accumulate Al in the root tissue (Rangel et al., 2010) regardless of ALS3 expression.