The identification of aluminium-resistance genes provides opportunities for enhancing crop production on acid soils

Abstract

Acid soils restrict plant production around the world. One of the major limitations to plant growth on acid soils is the prevalence of soluble aluminium (Al³⁺) ions which can inhibit root growth at micromolar concentrations. Species that show a natural resistance to Al³⁺ toxicity perform better on acid soils. Our understanding of the physiology of Al³⁺ resistance in important crop plants has increased greatly over the past 20 years, largely due to the application of genetics and molecular biology. Fourteen genes from seven different species are known to contribute to Al³⁺ tolerance and resistance and several additional candidates have been identified. Some of these genes account for genotypic variation within species and others do not. One mechanism of resistance which has now been identified in a range of species relies on the efflux of organic anions such as malate and citrate from roots. The genes controlling this trait are members of the ALMT and MATE families which encode membrane proteins that facilitate organic anion efflux across the plasma membrane. Identification of these and other resistance genes provides opportunities for enhancing the Al³⁺ resistance of plants by marker-assisted breeding and through biotechnology. Most attempts to enhance Al³⁺ resistance in plants with genetic engineering have targeted genes that are induced by Al³⁺ stress or that are likely to increase organic anion efflux. In the latter case, studies have either enhanced organic anion synthesis or increased organic anion transport across the plasma membrane. Recent developments in this area are summarized and the structure–function of the TaALMT1 protein from wheat is discussed.

Acid soils, aluminium, citrate, malate, resistance, roots, tolerance

Introduction

Aluminium (Al³⁺) toxicity limits plant production on acidic soils. The primary symptom of toxicity is inhibition of root growth but Al³⁺ can disrupt many other functions including root hair elongation, nutrient uptake (especially Ca and K), induce oxidative stress, disrupt the cytoskeleton and apoplastic processes, and affect intracellular transport (Kochian, 1995; Matsumoto, 2000; Sivaguru et al., 2000; Yamamoto et al., 2002; Kochian et al., 2004; Horst et al., 2010). Acid soils now encompass ∼35% of arable land so Al³⁺ toxicity is a major selection pressure for adaptation. Many species have evolved mechanisms to improve their survival on acid soils. Even before these mechanisms were fully understood they were divided into those that were likely to exclude Al³⁺ from the root (exclusion or resistance mechanisms) and those that would enable plants to accommodate Al³⁺ safely once it enters the symplast (tolerance mechanisms). In this review, tolerance mechanisms are distinguished from resistance mechanisms even though these terms are often used interchangeably in the literature. Exclusion mechanisms were predicted to depend on transport systems that export Al³⁺ from the symplast or on the exudation of ligands which bind Al³⁺ and limit its uptake into the cytosol. Tolerance mechanisms include those that chelate the aluminium entering the cytosol to form harmless complexes or that safely store it in subcellular compartments. We now know that these two mechanisms exist and that both can operate in parallel. Many thorough reviews provide a detailed discussion of these areas (Taylor, 1991; Kochian et al., 2004; Hiradate et al., 2007; Ma, 2007; Poschenrieder et al., 2008). This review will summarize recent developments and offer opinions on current research fronts.

Identification of Al³⁺ resistance genes

Genetic studies over the last 30 years have established that Al³⁺ resistance in many cereal species is a multigenic trait (Aniol and Gustafson, 1984; Luo and Dvorak, 1996; Reide and Anderson, 1996; Papernik et al., 2001; Garvin and Carver, 2003; Magalhaes et al., 2004; Ryan et al., 2009; Shi et al., 2009). Nevertheless one or two genetic loci can account for most of the phenotypic variation in some populations (Luo and Dvorak, 1996; Reide and Anderson, 1996; Garvin and Carver, 2003; Ma et al., 2004; Raman et al., 2005; Wang et al., 2007). As explained below, a thorough understanding of both the genetics and physiology of resistance was pivotal for finally identifying the first Al³⁺ resistance genes. Not surprisingly, these first genes accounted for much of the genotypic variation in specific plant species. Genes later identified from mutational analyses are also required for resistance but do not appear to explain genotypic variation within the species (Table 1).

Table 1.

Summary of genes involved in Al³⁺ resistance or tolerance

Genes are organized into different groups: (i) demonstrated Al³⁺-resistance genes that explain some genotypic variation, (ii) genes demonstrated to be involved in Al³⁺ resistance based on mutational analysis but to date do not explain genotypic variation, and (iii) likely Al³⁺ resistance genes based on segregation analysis, homology with other known genes, and/or functional analysis.

Species	Gene	Protein function	Evidence	Reference
Al3+ resistance genes that explains genotypic variation
Wheat	TaALMT1	Malate transport	Segregation, function	Sasaki et al., 2004
Arabidopsis	AtALMT1	Malate transport	Homology, function, mutational	Hoekenga et al., 2006
Sorghum	SbMATE1	Citrate transport	Segregation, function	Magalhaes et al., 2007
Barley	HvAACT1	Citrate transport	Segregation, function	Furukawa et al., 2007
Rye	ScALMT gene cluster	Malate transport	Segregation, homology	Collins et al., 2008
Maize	ZmMATE1	Citrate transport	Segregation, function	Maron et al., 2010
Al3+ resistance genes that do not explain genotypic variation
Arabidopsis	AtMATE	Citrate transport—efflux	Mutational	Liu et al., 2009
Arabidopsis	AtSTOP1	C2H2-type Zn finger transcription factor	Mutational	Iuchi et al., 2007
Rice	OsSTAR1 and OsSTAR2	UDP-glucose transport	Mutational	Huang et al., 2009
Rice	ART1	C2H2-type Zn finger transcription factor	Mutational	Yamaji et al., 2009
Arabidopsis	ALS3	Partial ABC protein—function unclear	Mutational	Larsen et al., 2005
Arabidopsis	ALS1	Partial ABC protein—function unclear	Mutational	Larsen et al., 2007
Arabidopsis	AtSTAR1	Partial ABC protein—function unclear	Mutational	Huang et al., 2010
Likely Al3+ resistance genes
Wheat	TaMATE1	Citrate transport—efflux	Segregation, homology (no mutational or functional data)	Ryan et al., 2009
Brassica napus	BnALMT1
	BnALMT2	Malate transport—efflux	Homology, function (no mutational or segregation data)	Ligaba et al., 2006
Rye	ScMATE2	Citrate transport—efflux	Homology, biology (no functional or segregation data)	Yokosho et al., 2010

Al³⁺-resistance genes that contribute to genotypic variation

In the 1990s the genetics and physiology of Al³⁺ resistance in wheat (Triticum aestivum L.) was among the best characterized of any species due to previous studies with segregating populations and near-isogenic lines (Delhaize et al., 1993,a, b; Basu et al., 1994; Ryan et al., 1995). Those studies demonstrated that resistance in a wide range of genotypes relied on the Al³⁺-activated efflux of malate from root apices, a trait largely controlled by a single genetic locus (Delhaize et al., 1993,b; Ryan et al., 1995). The model developed suggested that malate anions chelate and reduce the toxicity of the Al³⁺ cations in the apoplasm around the sensitive root apices. Sasaki et al. (2004) later identified a cDNA that was more highly expressed in the root apices of Al³⁺-resistant plants than sensitive plants of a pair of near-isogenic lines. The gene named ALMT (aluminium-activated malate transporter) belonged to a previously uncharacterized gene family and was the first Al³⁺ resistance gene identified in any plant species (Sasaki et al., 2004; Delhaize et al., 2007; Meyer et al., 2010). The higher expression of TaALMT1 in most Al³⁺-resistant genotypes of wheat is associated with tandemly-repeated elements in the promoter (Sasaki et al., 2006; Raman et al., 2008). Promoter analysis demonstrated that promoters containing these multiple repeats drive higher expression than promoters without repeats (Ryan et al., 2010). Heterologous expression of TaALMT1 in tobacco-suspension cells (Nicotiana tabaccum L.), barley (Hordeum vulgare L.) and wheat conferred Al³⁺-activated malate efflux and enhanced their resistance to Al³⁺ stress (Delhaize et al., 2004; Sasaki et al., 2004; Pereira et al., 2010). It is demonstrated here that TaALMT1 functions equally well in whole-plants of a dicotyledonous species. Transgenic Arabidopsis plants expressing TaALMT1 show the same Al³⁺-activated malate efflux and enhanced resistance to Al³⁺ toxicity observed in the transgenic cereals (Fig. 1). TaALMT1 is likely to be a useful tool in the future for engineering greater resistance into a wide range of crop and pasture plants.

Fig. 1.

Expression of the wheat gene TaALMT1 enhances the Al³⁺ resistance of Arabidopsis. (A) Al³⁺ resistance of wild-type Arabidopsis (Col) (white bars) and T2 homozygous transgenic lines (lines 1 and 3) expressing TaALMT1 (grey bars). Relative root growth was calculated by comparing the length of the longest root on plants grown on control plates (no Al³⁺) and on plates containing AlCl₃. Plants were grown for 14 d on agar plates containing a minimal nutrient solution with different concentrations of AlCl_3. Relative root growth was calculated as ((length+Al³⁺)×100)/(length–Al³⁺). Data show mean and SE (n=20–25 plants). (B) Malate efflux from wild-type control plants (white bars) and T₂ homozygous transgenic lines 1 and 3 (grey bars). Plants were first grown over a simple nutrient solution for 2 weeks. The solution was then replaced with 0.5 mM CaCl₂ (pH 4.9), containing 100 μM AlCl₃. Malate released from the roots over 24 h was measured by enzyme assay. Data show the mean and SE (n=3) where one replicate represented approximately 100 seedlings. TaALMT1 cDNA was ligated in the pPLEX502 binary vector behind the CaMV 35S promoter and transformed into Arabidopsis (Col) as described previously (Ryan et al., 2007).

Kinraide et al. (2005) modelled the diffusion of malate away from the root cells and concluded that the effectiveness of malate efflux as a resistance mechanism is unlikely to rely solely on the chelation of Al³⁺ in the rhizosphere. Their theoretical models indicated that the concentration of malate at the root surface would be too low to reduce Al³⁺ stress. Instead, they proposed that the epidermal cells near the apex are resilient to Al³⁺ stress and that malate may be more important for reducing Al³⁺ concentrations in and around the developing cortical cells of root apices.

A solid understanding of the genetics and physiology of resistance in sorghum (Sorghum bicolor) and barley was also pivotal in identifying the first members of a second family of resistance genes. Aluminium resistance in each of these species is controlled by a major genetic locus that segregates with the Al³⁺-dependent efflux of citrate from roots (Magalhaes et al., 2004, 2007; Furukawa et al., 2007; Wang et al., 2007). Fine mapping of these loci identified the SbMATE gene in sorghum and the HvAACT1 gene in barley, both of which belong to the multidrug and toxic compound exudation (MATE) family (Table I). The MATE family of transporter proteins is a large and diverse group present in prokaryotic and eukaryotic cells. Many appear to function as secondary carriers (mostly with protons) to remove small organic compounds from the cytosol (Omote et al., 2006; Magalhaes, 2010). Heterologous expression of SbMATE in Arabidopsis plants and HvAACT1 in Xenopus oocytes and tobacco plants established that they encode transport proteins which facilitate the Al³⁺-activated efflux of citrate (Furukawa et al., 2007; Magalhaes et al., 2007). Additional resistance genes were isolated based on their homology to these ALMT1 and MATE genes and by exploiting the available information on the physiology and genetics of resistance in other species. This approach has helped identify candidate resistance genes in Arabidopsis (Hoekenga et al., 2006), Brassica napus (Ligaba et al., 2006), rye (Secale cereale (Collins et al., 2008), wheat (Ryan et al., 2009), sorghum (Eticha et al., 2010), and maize (Maron et al., 2010). A role for some of these candidates in Al³⁺ resistance was confirmed using mutational analysis in Arabidopsis (Hoekenga et al., 2006) and by segregation or functional analysis in rye and maize (Collins et al., 2008; Maron et al., 2010).

Resistance genes that do not explain genotypic variation

Subsequently, a different set of Al³⁺ resistance genes was identified using an approach that requires no prior knowledge or assumptions regarding the genetics or mechanisms involved. In this approach, seed are mutagenized by chemical treatments, radiation or by the random insertion of a DNA fragment (T-DNA) into the genome. M₂ seedlings are screened and those that grow similarly to wild-type plants under control conditions, but are hypersensitive to Al³⁺ stress or acidity, may carry mutations in genes necessary for Al³⁺ resistance. The genes are finally identified by mapping or by obtaining sequence flanking the T-DNA and, in both cases, are verified by complementation of the mutants. These genes need not show allelic variation which means they are not necessarily responsible for natural genotypic variation within a species. At least six genes have been identified in rice and Arabidopsis by mutational analysis as discussed below.

As model species, rice and Arabidopsis are attractive systems for mutational analysis. Rice is an important crop worldwide with a high basal level of resistance compared with other small-grained cereals (Famoso et al., 2010). The striking similarities between the resistance genes isolated from rice and Arabidopsis using a mutational approach is intriguing given that rice is considerably more resistant (Table I). In contrast to other cereals, Al³⁺ resistance in rice is not explained by organic anion efflux. Indeed, the Al³⁺-activated efflux of citrate from rice roots is considerably lower than the efflux measured in rye where it does represent a major mechanism of resistance (Li et al., 2000; Collins et al., 2008).

A recent breakthrough in uncovering the molecular basis of Al³⁺ resistance in rice was achieved when the gene underlying an Al³⁺-sensitive mutant, star1, was identified (Huang et al., 2009). The wild-type gene named OsSTAR1 (sensitive to Al rhizotoxicity) encodes the nucleotide binding domain of a bacterial-type ABC-transporter. The OsSTAR1 protein interacts with OsSTAR2 which possesses a transmembrane domain from this same ABC-transporter family. Both OsSTAR1 and OsSTAR2 are predominantly expressed in roots and expression of both is specifically induced by Al³⁺ treatment. The OsSTAR1:OsSTAR2 complex localizes to vesicular membranes and transports UDP-glucose, but it is not clear how this function confers resistance. The OsSTAR proteins may release UDP-glucose to the apoplasm by exocytosis and provide protection by modifying the cell walls. It is also plausible that the OsSTAR proteins confer Al³⁺ ‘tolerance’ rather than ‘resistance’ by performing other functions in the cytosol. Neither of the OsSTAR genes underlies QTLs for Al³⁺ resistance in rice, but they are possibly responsible for its high basal level of resistance (Huang et al., 2009). Subsequently, Yamaji et al. (2009) identified a zinc-finger transcription factor (ART1) that regulates the Al³⁺-induced expression of both OsSTAR1 and OsSTAR2 along with over 30 other genes. Some of these other genes might also contribute to Al³⁺ resistance because they map to previously identified QTLs for Al³⁺ resistance on the rice genome. The observation that the star1 mutant is as Al³⁺ sensitive as the art1 mutant suggests that the OsSTAR genes control a major mechanism of Al³⁺ resistance in rice.

AtALS1 and AtALS3 are Al³⁺-resistance genes isolated by mutational analysis of Arabidopsis. They also encode ‘half’ ABC transporters with AtALS1 having significant sequence similarity to OsSTAR2 (Larsen et al., 2005, 2007). Nevertheless Larsen (2009) speculated that the AtALS proteins transport aluminium, possibly in a chelated form, between cells (AtALS3) or to the vacuole (AtALS1). A zinc finger transcription factor, AtSTOP1, was initially identified from a pH-sensitive Arabidopsis mutant. AtSTOP1 is related to OsART1 from rice but regulates a different range of genes including the known resistance genes AtALMT1, AtMATE1, and ALS3 (Sawaki et al., 2009) and several genes associated with proton tolerance (Iuchi et al., 2007). More recently, the closest homologue to OsSTAR1 in Arabidopsis, named AtSTAR1, was shown to contribute to Al³⁺ tolerance as well because plants carrying a knockout mutation in AtSTAR1 were hypersensitive to Al³⁺ stress (Huang et al., 2010). Furthermore, some evidence suggests AtSTAR1 may be interacting with AtALS3 (Huang et al., 2010).

Other genes were isolated by a strategy that identified mutations able to suppress the hypersensitivity of Arabidopsis als3-1 mutants (Gabrielson et al., 2006). als3-1 seeds were further mutagenized and M₂ seedlings screened for mutations that recovered Al tolerance. A description of AtATR (Ataxia telangiectasia-mutated and RAD3-related) and other genes identified in this way can be found elsewhere (Gabrielson et al., 2006; Larsen, 2009). Not all of these genes can be considered Al³⁺-resistance genes in the strict sense. For instance, a mutation in AtATR improved root growth on Al³⁺ toxic media by altering fundamental processes of the cell cycle that detect DNA damage and these may carry other costs for the plant in the absence of Al. Interestingly, those studies led Larsen and colleagues to radically re-interpret the mechanism of Al³⁺ toxicity. Instead of root growth being inhibited by a series of lesions in the symplasm and apoplasm that disrupt cell function, they proposed that growth is actually inhibited by the plant's own responses to the stress rather than by the stress itself (Larsen, 2009).

Transgenic approaches for increasing Al resistance: Why bother?

By 2050 world population will have increased by two billion people. Crop production will need to increase by 50% or more to meet the added demand for food, fibre, and animal feed. Most of these gains will have to be met by increasing yields and cropping intensity, but additional land, perhaps previously considered unsuitable for farming, will also need to be cultivated. Acid soils are widespread in sub-Sarahan Africa, Asia, and other regions likely to see the largest increases in population. While the application of lime (calcium carbonate) is the most efficient way of ameliorating soil acidity and improving its suitability for agriculture, it is not practical or common in developing countries that rely on small subsistence farms for food production. A combination of Al³⁺-resistant germplasm with the application of lime or some other ameliorant, where possible, is a common management strategy. Some crops perform adequately on acid soils and others can be improved with conventional breeding strategies. However, for crops that possess insufficient natural variation to breed for Al³⁺ resistance, other solutions are required. Demands on our production systems from population growth and climate change require all options to be considered, including transgenic strategies (Hoisington, 2002; Bhalla, 2006).

Once the role of organic anion efflux in Al resistance was understood, many researchers tried genetically engineering this trait into model species. The two main strategies were to increase organic anion synthesis with the hope that this leads to greater efflux, and to increase organic anion transport across the plasma membrane. Some argued that the transport step was likely to be the rate-limiting step for efflux because the capacity to synthesize citrate and malate is probably in excess in wild-type cells or is tightly regulated (Ratledge, 2000; Ryan et al., 2001). Furthermore, the large electrochemical gradient across the plasma membrane favours anion release from cells indicating that malate and citrate efflux should occur spontaneously once a pathway is provided. Nevertheless, early attempts to increase resistance targeted organic anion synthesis because the genes encoding the transport proteins were not available at the time.

The first influential study of this type was from de la Fuente et al. (1997) who over-expressed a bacterial citrate synthase gene in tobacco. They reported greater citrate efflux and enhanced Al³⁺ resistance in several transgenic lines. Perhaps expressing a bacterial gene in plants helped the enzyme avoid normal regulatory mechanisms. In any case this publication triggered a flurry of activity as groups tried to repeat this study and perform similar experiments using other genes involved in anion synthesis (e.g. citrate synthase, malate dehydrogenase, isocitrate hydrogenase). A number of reports claimed success (Table 2) while others were unable to repeat the original study of de la Fuente and colleagues (Delhaize et al., 2001). However, even when successful, the gains in resistance were relatively modest. Rarely is relative root growth more than 3-fold greater than the controls (Table 2).

Table 2.

Studies that have enhanced Al³⁺ resistance by genetic engineeringShown are studies that have enhanced the Al³⁺ resistance of whole plants by genetic engineering. Listed are the transgenes, their species of origin, and the species that were transformed with the transgenes. Also included are estimates for the fold-increases in relative root growth compared with control plants (wild-type plants or null segregants) as well as the proposed mechanism for the phenotype. Relative root growth refers to net root growth in the presence of Al³⁺ divided by net root growth in the absence of Al³⁺.

Gene function	Transgenic strategy	Phenotype (RRG)	Proposed mechanism	Reference
Organic anion metabolism
Citrate synthase	Pseudomonas aeruginosa gene expressed	2-fold	Enhanced organic acid efflux	de la Fuente et al., 1997
	in tobacco
Malate dehydrogenase	Nodule-enhanced gene expressed in alfalfa	2.0-fold	Enhanced organic acid efflux	Tesfaye et al., 2001
Citrate synthase	Mitochondrial cDNA from carrot was	1.3-fold	Enhanced citrate efflux	Koyama et al., 2000
	expressed in Arabidopsis
Citrate synthase	Mitochondrial cDNA from Arabidopsis	2.0-fold	Enhanced citrate efflux	Anoop et al., 2003
	expressed in Brassica napus
Citrate synthase	Pseudomonas aeruginosa gene	2.5-fold		Barone et al., 2008
	expressed in alfalfa
Pyruvate phosphate dikinase	Mesembryanthemum crystallinum gene	1.2-fold	Enhanced malate and citrate efflux	Trejo-Tellez et al., 2010
	expressed in tobacco
Malate dehydrogenase	Arabidopsis and E.coli genes	2.4-fold	Increase in malate efflux	Wang et al., 2010
	expressed in tobacco
Stress responsive
Glutathione S-transferase	Tobacco gene expressed in Arabidopsis	1.5-fold	Enhanced oxidative stress tolerance	Ezaki et al., 2000
Peroxidase	Tobacco gene expressed in Arabidopsis	1.5-fold	Protection from oxidative stress	Ezaki et al., 2000
GDP-dissociation inhibitor	Tobacco gene expressed in Arabidopsis	1.5-fold	Enhanced stress tolerance	Ezaki et al., 2000
Blue copper protein	Arabidopsis gene expressed in Arabidopsis	1.5-fold	Protection from oxidative stress	Ezaki et al., 2000
Dehydroascorbate reductase	Arabidopsis gene expressed in tobacco	1.5-fold	Higher ascorbic acid protects from	Yin et al., 2010
			oxidative stress
Manganese superoxide dismutase	Wheat gene expressed in Brassica napus	2.5-fold	Protection from oxidative stress	Basu et al., 2001
Transport
Al3+-activated malate transporter (TaALMT1)	Wheat gene expressed in barley	20-fold	Enhanced malate efflux	Delhaize et al., 2004
Multidrug and toxic compound efflux gene (MATE) called Frd3	Arabidopsis gene expressed in Arabidopsis	3.0-fold	Enhanced citrate efflux	Durrett et al., 2007
Al3+-activated malate transporter (TaALMT1)	Wheat gene expressed in wheat	8-fold	Enhanced malate efflux	Pereira et al., 2010
Al3+-activated malate transporter (TaALMT1)	Wheat gene expressed in Arabidopsis	4-fold	Enhanced malate efflux	Fig. 1
Multidrug and toxic compound efflux gene (HvAACT1)	Barley gene expressed in tobacco plants	2.3-fold	Enhanced citrate efflux	Furukawa et al., 2007
Al3+-activated malate transporter (AtALMT1)	Arabidopsis gene expressed in Arabidopsis	3-fold	Enhanced malate efflux	Ryan PR, Dong B, DelhaizeE Hoekenga OAKochian LV (unpublished results)
H+-pyrophosphatase AVP1	Over-expression of endogenous genes in	1.8-fold	Enhanced organic acid efflux	Yang et al., 2007
	Arabidopsis, tomato, and rice
Multidrug and toxic compound efflux gene (SbMATE)	Sorghum gene expressed in the Arabidopsis Atalmt1 mutant	2.5-fold	Enhanced citrate efflux	Magalhaes et al., 2007
Al3+-activated malate transporter family (HvALMT1)	Barley gene expressed in barley.	3.4-fold	Enhanced malate efflux	Gruber, 2009
Multidrug and toxic compound efflux gene (ZmMATE1)	Maize gene expressed in Arabidopsis	3-fold	Enhanced citrate efflux	Maron et al., 2010
Other
Cell wall associated receptor kinase	Arabidopsis WAK1 gene expressed in Arabidopsis	3-fold	Enhances stress responsiveness	Sivaguru et al., 2003
Auxilin-like protein	Over-expression of endogenous Arabidopsis gene	3-fold	Reduction in endocytosis	Ezaki et al., 2007
Δ8 sphingolipid desaturase	Stylosanthes gene expressed in Arabidopsis	2-fold	Changes to membrane lipid composition	Ryan et al., 2007
Ced-2 (Bcl2 homologue)	Caenorhabditis elegans gene expressed in tobacco	2-fold	Reduced Al3+-induced programmed cell death	Wang et al., 2009

Other studies enhanced Al³⁺ resistance in plants by over-expressing genes whose expression is induced by Al³⁺ treatment, particularly those associated with oxidative stress (Ezaki et al., 2000, 2005). For instance, increasing the expression of glutathione S-transferase, peroxidase, GDP-dissociation inhibitor, and a blue copper protein in Arabidopsis increased relative root growth by 1.5–2.5-fold compared with the controls. These and other studies (Basu et al., 2001) demonstrate that boosting a plant's normal defences to oxidative stress can also enhance Al³⁺ tolerance. Once again, the benefits were small, with most improving relative root growth by less than 2-fold.

Modest gains in the tolerance of transgenic plants were also obtained using an approach that assumed no prior knowledge of genetics or physiology. Candidate genes were first identified by screening plant cDNA libraries in yeast cells grown on agar plates containing sufficient Al³⁺ to suppress the growth of wild-type cells (Delhaize et al., 1999; Ryan et al., 2007). cDNAs that allowed yeast cells to grow were isolated and then systematically expressed in plants. A Δ8 sphingolipid desaturase isolated in this way slightly increased the Al³⁺ resistance of Arabidopsis by modifying the composition of cell lipids (Ryan et al., 2007).

By far the largest increases in Al³⁺ resistance have been achieved by over-expressing organic anion transport proteins (Table 2). These studies targeted the known Al³⁺ resistance genes discussed above (TaALMT1, AtALMT1, SbMATE) or related genes that performed different functions (e.g. Frd3 from Arabidopsis and HvALMT1 from barley). The most striking examples to date have resulted from over-expressing TaALMT1 in barley, wheat, and Arabidopsis. These plants showed greater relative root growth by 20, 8, and 4-fold, respectively (Table 2). Several important conclusions can be drawn from these studies. The first is that the transport of organic anions across the plasma membrane is a more important bottleneck to efflux than their synthesis. These studies also explain why Al³⁺-resistance relies on malate efflux in some species and citrate efflux in others. This difference is determined primarily by the substrate specificity of the transport protein in the membrane, and not by the metabolism of the cells (Ryan and Delhaize, 2010). For example, Al³⁺ resistance in wild-type barley relies on citrate efflux from roots (mediated by HvAACT) yet transgenic plants expressing the wheat TaALMT1 gene also release malate anions indicating that barley is capable of synthesizing and releasing malate provided a transport pathway is present (Delhaize et al., 2004).

The isolation of genes from mutational studies provides another avenue for engineering Al³⁺ resistance. To date, these genes have been used successfully to complement their respective mutants, but in at least two instances (AtALS3 from Arabidopsis and OsART1 from rice), their overexpression did not enhance Al³⁺ resistance in transgenic plants (Larsen et al., 2007; Yamaji et al., 2009). Nevertheless, other genes, such as those encoding the OsSTAR proteins, need to be tested to determine whether they confer high levels of resistance if expressed in more Al³⁺-sensitive species.

Focus topic: structure–function of TaALMT

It is now clear that many Al³⁺-tolerance and resistance mechanisms operate in plants and these are controlled by numerous genes. Our understanding remains rudimentary in almost all aspects of their physiology and molecular biology. For instance, much of the plant research has been carried out on young seedlings, often grown in nutrient solution, so the behaviour of these mechanisms in older plants and in different parts of a mature root system is unknown. It is also possible that Al³⁺ resistance genes contribute to other stress pathways, nutrition acquisition, or even pathogen responses (Pineros et al., 2008b; Rudrappa et al., 2008; Delhaize et al., 2009). This section reviews recent studies on the structure of TaALMT1 from wheat which are beginning to reveal details of its function.

Does TaALMT1 function as an anion channel?

Early physiological studies suggested that malate efflux from wheat roots was mediated by anion channels. The electrochemical gradient for malate across the plasma membrane is consistent with the involvement of a channel and known antagonists of anion channels (niflumate and anthracene-9-carboxylate) reduced the Al³⁺-activated malate efflux (Ryan et al., 1995). A series of electrophysiological studies on protoplasts isolated from wheat roots (Ryan et al., 1997; Zhang et al., 2001), transgenic Xenopus oocytes (Sasaki et al., 2004; Pineros et al., 2008,a) and tobacco suspension cells (Zhang et al., 2008) found that TaALMT1 facilitated Al³⁺-activated inward and outward currents. The inward currents were generated by organic and inorganic anion efflux that were inhibited by niflumate.

On the basis of those experiments it was initially concluded (see later) that TaALMT1 functions as an inwardly rectifying, ligand-gated anion channel (Ryan et al., 1997; Zhang et al., 2001). What does this mean? Ligand-gated refers to the requirement for Al³⁺ to activate channel function and inwardly rectifying means it passes more inward current than outward current across the voltage range. Inwardly rectifying channels would typically conduct more current as the electrical potential difference across the membrane becomes more negative (hyperpolarized). The loss of malate anions from the cell is accompanied by the release of K⁺ which appears to satisfy electroneutrality (Ryan et al., 1995). The model proposed that malate efflux, via TaALMT1, depolarizes the plasma membrane and triggers K⁺ efflux via a voltage-sensitive, outwardly rectifying K⁺ channel. However, uncertainties with this model persisted because malate-dependent, single-channel traces, diagnostic of channel involvement, were not detected in those early studies, and this cast doubt on whether TaALMT1 functioned as an anion channel or as some other type of transporter. Secondly, since Al³⁺ depolarizes the apical cell membranes of Al³⁺-resistant wheat (Wherrett et al., 2005) it was perplexing how the rapid and sustained efflux of malate could occur via a channel that was supposedly more active in hyperpolarized membranes.

Stronger evidence that TaALMT1 functions as an anion channel was later collected on protoplasts prepared from wild-type wheat roots and tobacco-suspension cells expressing TaALMT1 (Zhang et al., 2008). Transient channel activity was detected in out-side out patches pulled from whole cells already activated by Al³⁺ and single-channel events were also recorded in the whole-cell configuration, especially during voltage ramps and as currents deactivated. Zhang et al. (2008) calculated that TaALMT1 was ∼18-fold more permeable to malate than Cl^– which was similar to estimates by Pineros et al. (2008,a) working with Xenopus oocytes at the same time. Perhaps more importantly, Zhang et al. (2008) revised our understanding of TaALMT1 function by establishing that it is not an inwardly-rectifying channel as first thought. They established that the Al³⁺-activated currents were fully active at depolarized membrane potentials and partially deactivated at more negative potentials. This deactivation was ‘tuned’ so that the channel acts like a constant current device. As a result TaALMT1 facilitates an Al³⁺-activated inward current that is relatively insensitive to membrane potential. This has the effect of providing a relatively constant efflux of malate over a range of physiological membrane potentials. The previous reports of ‘inward rectification’ (Ryan et al., 1997; Zhang et al., 2001) were artefacts of the concentration gradients of the permeating anions across the plasma membrane and did not result from voltage-dependent changes in gating behaviour. This is an important observation because it helps explain the question raised above of how TaALMT1 sustains malate efflux from depolarized cells. Sustained efflux of malate and K⁺ was now feasible because both occurred via separate channels active in depolarized membranes.

Another issue that arises from the sustained release of malate via TaALMT1 is why complexation of Al³⁺ in the immediate vicinity of the channel does not turn off the malate release. The reversibility of the Al³⁺ activation and the effect of external malate on channel activity after activation warrants further investigation. Some anion channels are trans-stimulated by permeating anions (Dietrich and Hedrich, 1998; Diatloff et al., 2004) which means efflux is stimulated by increasing the concentration of permeating anions on the external side. This response has been observed in TaALMT1 in response to external Cl^– (Pineros et al., 2008a) and in HvALMT1 in response to various organic anions (Gruber et al., 2010). This question raises the interesting possibility that malate itself and perhaps other anions that chelate Al³⁺, may also activate TaALMT1 in order to sustain the malate efflux.

Protein phosphorylation and activation by Al³⁺

One intriguing aspect of ALMT and MATE involvement in Al³⁺ resistance is the role of post-translational regulation and particularly the requirement for Al³⁺ to activate their function. ALMT1 proteins from wheat, rye, Arabidopsis, and Brassica napus, and the MATE proteins from sorghum and barley all require external Al³⁺ to trigger transport activity. This is clear from studies that have constitutively expressed these genes in a range of different cell types. In the absence of Al³⁺ the basal level of inward current (organic anion efflux) is consistently greater in transgenic cells compared with controls. However, the addition of Al³⁺ significantly increases transport activity in transgenic cells but not controls. These basal currents are similar to the Al³⁺-activated currents with regard to selectivity and sensitivity to antagonists, which suggests they reflect the normal function of TaALMT1 (Pineros et al., 2008,a; Zhang et al., 2008). How members of the ALMT and MATE protein families acquired such specific interactions with Al³⁺ is an interesting question which is discussed elsewhere (Ryan and Delhaize, 2010). The details of this activation by Al³⁺ remain a mystery, but recent studies investigating this issue are discussed below.

The secondary structure of TaALMT1 (459 amino acids) is predicted to consist of six transmembrane domains in the N-terminal half of the protein and a long C-terminal domain (∼240 residues) located extracellularly as shown in Fig. 2 (Motoda et al., 2007). It is tempting to propose that this extracellular domain interacts directly with external Al³⁺ to influence channel function and some support for this is presented later. However, well before TaALMT1 had even been identified, phosphorylation was predicted to be important because malate efflux from wheat roots was inhibited by the protein kinase inhibitor K252a (Osawa and Matsumoto, 2001). A similar inhibition was also reported for citrate release from soybean roots (Shen et al., 2004). It is now known that TaALMT1 is constitutively expressed in wheat (Sasaki et al., 2004) which suggests K252a may have been acting on the TaALMT1 protein rather than the pathways regulating expression. Ligaba et al. (2009) supported this conclusion by showing that protein kinase inhibitors (K252a and staurosporine) inhibit the Al³⁺-activated currents in Xenopus oocytes expressing TaALMT1. Interestingly, pretreatment with a protein kinase C activator, phorbol 12-myristate 13-acetate (PMA) slightly enhanced the TaALMT1-mediated currents. Ligaba et al. (2009) generated a series of mutations targeting possible phosphorylation sites on TaALMT1. Two mutations on the C-terminal domain altered protein function. One of these which substituted a threonine for an alanine at residue 323 (T323A) more than doubled the basal and Al³⁺-activated currents (Fig. 2). No clear explanation is available for this shift in conductance, but suggestions that the mutation altered protein structure are possible. Another mutation located on the C-terminal domain, S384A, significantly reduced both basal and Al³⁺-activated currents, with the modified proteins being insensitive to staurosporine and PMA treatments (Ligaba et al., 2009). While these results are consistent with phosphorylation being involved in TaALMT1 function, future work should confirm that the S384 residue is actually phosphorylated in wild-type alleles and that the S384A and T323A mutations do not affect the level of protein expression in oocytes. Furthermore, the S384A mutation may have resulted in a dysfunctional protein since both the basal and activated currents were abolished. Nevertheless, the model emerging from these studies indicates that phosphorylation is a prerequisite for TaALMT1 function and perhaps also for its activation by Al³⁺. It is easy to envision how the addition of a negatively- charged phosphate group on the protein might alter tertiary structure or form part of a binding site for Al³⁺ which then increases transport activity.

Fig. 2.

Diagram depicting the secondary structure of TaALMT1. TaALMT1 (459 amino acids) is predicted to possess six transmembrane regions with the N and C-terminal ends orientated extracellularly (Motoda et al., 2007). Residue D222 is located near the start of the hydrophilic C-terminal region (coloured red). Approximate positions of mutations to the C-terminal domain found to alter protein function are indicated by arrows. Two mutations affecting putative phosphorylation sites are coloured green (Ligaba et al., 2009) and three mutations targeting acidic residues are coloured blue (Furuichi et al., 2010).

Since the C-terminal domain is predicted to be extracellular (Motoda et al., 2007), a surprising corollary from this work is that TaALMT1 may be phosphorylated by a kinase acting in the apoplasm. No extracellular kinases that phosphorylate membrane proteins have been reported to date in plants although ecto-protein kinases that phosphorylate proteins on the cell surface have been characterized in animal cells. Indeed, in a recent review on carboxylate transport in plants, Meyer et al. (2010) considered it so unlikely that kinases act extracellularly that they reversed the orientation proposed by Motada et al. (2007). However, the direct experimental evidence supporting the topology predictions is likely to be more reliable than the physiology which could be influenced by other factors. Therefore, we conclude that either ecto-kinase activity does affect TaALMT1 function by processes that are currently unclear or the phosphorylation data require reconsideration.

Another recent study generated different types of mutations to examine TaALMT1 function. A series of truncations were made to the C-terminal domain starting with the terminal 70 residues and working up to the entire C-terminal tail (Furuichi et al., 2010). All truncations abolished the basal currents (occurring in the absence of Al³⁺) as well as the Al³⁺-activated transport activity without affecting protein expression. Interestingly, full function was recovered by fusing the N-terminal region of TaALMT1 with the C-terminal region of AtALMT1 from Arabidopsis. These findings demonstrate that the C-terminal domain is required for all functions of TaALMT1 and highlight the close functional similarity between these homologues from wheat and Arabidopsis.

Furuichi et al. (2010) then mutated acidic residues on the C-terminal domain since these were considered most likely to interact with extracellular Al³⁺. Fifteen acidic residues were individually replaced with uncharged residues and the transport behaviour of the modified proteins were compared in Xenopus oocytes. Three mutations (E274Q, D275N, and E284Q) abolished the Al³⁺-activated transport activity without affecting the basal transport activity which remained the same as wild-type TaALMT1 (Fig. 2). The authors proposed that all three anionic residues are necessary for Al³⁺ to activate TaALMT1 function. They further argued that these mutations do not grossly disrupt the tertiary structure of the protein because the basal currents were unaffected.

The AtALMT1 gene from Arabidopsis requires Al³⁺ treatment both to induce gene expression and to activate protein function (Hoekenga et al., 2006; Kobayashi et al., 2007). This differs from TaALMT1 in wheat which is constitutively expressed and only requires Al³⁺ to activate function. Kobayashi et al. (2007) showed the induction of AtALMT1 expression by Al³⁺ is inhibited by staurosporine (kinase inhibitor) and calyculin A (phosphatase inhibitor) and K252a inhibited Al³⁺-dependent malate efflux without reducing gene expression. These results suggest that induction of AtALMT1 expression by Al³⁺ may involve reversible protein phosphorylation. Furthermore, either AtALMT1 itself or an upstream component needs to be phosphorylated for Al³⁺ to activate transport activity, which is similar to the conclusions drawn above for TaALMT1.

Insights into the structure–function relationships of AtALMT1 were also obtained from the Arabidopsis ecotype Warschau-1 which is hypersensitive to Al³⁺ stress. A single nucleotide substitution was responsible for the phenotype because it introduced a premature stop codon in the N-terminal half of the AtALMT1 coding region (Kobayashi et al., 2007). The resulting protein is a naturally occurring example of the truncation experiments described above for TaALMT1 but it is not known whether the truncated protein accumulates in the plant. The mutation in Warschau-1 generated a dominant negative phenotype in an F₂ population developed from Warschau-1 and Col-4 ecotypes and the authors speculated that the presence of truncated monomers disrupt the formation of functioning multimeric complex necessary for a mature transport protein. If correct, all F₁ plants from this cross should be sensitive of Al³⁺ but this requires confirmation.

Constraints to further progress

Much of our understanding of Al³⁺ resistance conferred by organic anions is derived from heterologous expression of ALMTs and MATEs in plant and animal cells. Further advances will rely on the systematic generation and characterization of mutations in these proteins. Mutations can be efficiently made in yeast and bacteria by standard techniques, but a suitable expression system remains an obstacle. ALMTs and MATEs have been successfully expressed in plants (wheat, barley, Arabidopsis, and rice) as well as tobacco suspension cells. While increasingly routine, these procedures take months to complete and it is difficult to carry scores of different lines through tissue culture.

Xenopus oocytes have proved themselves to be very useful for characterizing the electrophysiological behaviour of transport proteins and already many studies have used these cells to assess the effect that specific mutations have on ALMT transport activity. The procedure is rapid, only taking a few days and Xenopus oocytes will continue to be an important experimental system in the future. Nevertheless, there is always some concern whether the behaviour of proteins expressed in this animal system truly reflects their behaviour in planta. The ionic composition of the cytosol in oocytes and root-cells are not identical and so transport behaviour could differ. Furthermore, oocyte experiments require substantial infrastructure to raise the frogs and analyse protein function with electrophysiological techniques. Few laboratories can easily afford the expense to maintain this system.

Are there practical alternatives to Xenopus oocytes? Plant cells are preferred but single-celled systems or other animal cells offer some advantages. Insect cell lines (e.g. Spodoptera frugiperda and Trichoplusia ni) are available but these may present the same difficulties as oocytes. Attempts to express ALMTs in yeast and bacteria were unable to detect function, despite confirming protein expression in yeast (W. Chen and P. Ryan, unpublished; BD Gruber, personal communication; Sasaki T, personal communication). Unfortunately, this precludes the use of a number of powerful genetic and molecular techniques for analysing these proteins. One alternative to using stably-transformed plants is the hairy root systems which have been successfully established in several dicotyledonous species (Guillon et al., 2006). More rapid than whole-plant transformation, this technique provides a transgenic root system suitable for experiments ranging from whole roots to single cells. The choice of which system to use comes down to a balance between using plant cells to ensure a faithful phenotype and the time, effort and expense in generating the transgenic lines.

We are grateful to Dr Bei Dong for technical assistance in the transforming Arabidopsis with TaALMT1.