A promoter-swap strategy between the AtALMT and AtMATE genes increased Arabidopsis aluminum resistance and improved carbon-use efficiency for aluminum resistance

The primary mechanism of Arabidopsis aluminum (Al) resistance is based on root Al exclusion, resulting from Al-activated root exudation of the Al(3+) -chelating organic acids, malate and citrate. Root malate exudation is the major contributor to Arabidopsis Al resistance, and is conferred by expression of AtALMT1, which encodes the root malate transporter. Root citrate exudation plays a smaller but still significant role in Arabidopsis Al resistance, and is conferred by expression of AtMATE, which encodes the root citrate transporter. In this study, we demonstrate that levels of Al-activated root organic acid exudation are closely correlated with expression of the organic acid transporter genes AtALMT1 and AtMATE. We also found that the AtALMT1 promoter confers a significantly higher level of gene expression than the AtMATE promoter. Analysis of AtALMT1 and AtMATE tissue- and cell-specific expression based on stable expression of promoter-reporter gene constructs showed that the two genes are expressed in complementary root regions: AtALMT1 is expressed in the root apices, while AtMATE is expressed in the mature portions of the roots. As citrate is a much more effective chelator of Al(3+) than malate, we used a promoter-swap strategy to test whether root tip-localized expression of the AtMATE coding region driven by the stronger AtALMT1 promoter (AtALMT1(P)::AtMATE) resulted in increased Arabidopsis Al resistance. Our results indicate that expression of AtALMT1(P)::AtMATE not only significantly increased Al resistance of the transgenic plants, but also enhanced carbon-use efficiency for Al resistance.     

Not all ALMT1-type transporters mediate aluminum-activated organic acid responses: the case of ZmALMT1 – an anion-selective transporter

The phytotoxic effects of aluminum (Al) on root systems of crop plants constitute a major agricultural problem in many areas of the world. Root exudation of Al-chelating molecules such as low-molecular-weight organic acids has been shown to be an important mechanism of plant Al tolerance/resistance. Differences observed in the physiology and electrophysiology of root function for two maize genotypes with contrasting Al tolerance revealed an association between rates of Al-activated root organic acid release and Al tolerance. Using these genotypes, we cloned ZmALMT1 , a maize gene homologous to the wheat ALMT1 and Arabidopsis AtALMT1 genes that have recently been described as encoding functional, Al-activated transporters that play a role in tolerance by mediating Al-activated organic acid exudation in roots. The ZmALMT1 cDNA encodes a 451 amino acid protein containing six transmembrane helices. Transient expression of a ZmALMT1 ::GFP chimera confirmed that the protein is targeted to the plant cell plasma membrane. We addressed whether ZmALMT1 might underlie the Al-resistance response (i.e. Al-activated citrate exudation) observed in the roots of the Al-tolerant genotype. The physiological, gene expression and functional data from this study confirm that ZmALMT1 is a plasma membrane transporter that is capable of mediating elective anion efflux and influx. However, gene expression data as well as biophysical transport characteristics obtained from Xenopus oocytes expressing ZmALMT1 indicate that this transporter is implicated in the selective transport of anions involved in mineral nutrition and ion homeostasis processes, rather than mediating a specific Al-activated citrate exudation response at the rhizosphere of maize roots.     

Not all ALMT1-type transporters mediate aluminum-activated organic acid responses: the case of ZmALMT1 - an anion-selective transporter.

The phytotoxic effects of aluminum (Al) on root systems of crop plants constitute a major agricultural problem in many areas of the world. Root exudation of Al-chelating molecules such as low-molecular-weight organic acids has been shown to be an important mechanism of plant Al tolerance/resistance. Differences observed in the physiology and electrophysiology of root function for two maize genotypes with contrasting Al tolerance revealed an association between rates of Al-activated root organic acid release and Al tolerance. Using these genotypes, we cloned ZmALMT1, a maize gene homologous to the wheat ALMT1 and Arabidopsis AtALMT1 genes that have recently been described as encoding functional, Al-activated transporters that play a role in tolerance by mediating Al-activated organic acid exudation in roots. The ZmALMT1 cDNA encodes a 451 amino acid protein containing six transmembrane helices. Transient expression of a ZmALMT1::GFP chimera confirmed that the protein is targeted to the plant cell plasma membrane. We addressed whether ZmALMT1 might underlie the Al-resistance response (i.e. Al-activated citrate exudation) observed in the roots of the Al-tolerant genotype. The physiological, gene expression and functional data from this study confirm that ZmALMT1 is a plasma membrane transporter that is capable of mediating elective anion efflux and influx. However, gene expression data as well as biophysical transport characteristics obtained from Xenopus oocytes expressing ZmALMT1 indicate that this transporter is implicated in the selective transport of anions involved in mineral nutrition and ion homeostasis processes, rather than mediating a specific Al-activated citrate exudation response at the rhizosphere of maize roots.     

Maize ZmALMT2 is a root anion transporter that mediates constitutive root malate efflux

Root efflux of organic acid anions underlies a major mechanism of plant aluminium (Al) tolerance on acid soils. This efflux is mediated by transporters of the Al-activated malate transporter (ALMT) or the multi-drug and toxin extrusion (MATE) families. ZmALMT2 was previously suggested to be involved in Al tolerance based on joint association-linkage mapping for maize Al tolerance. In the current study, we functionally characterized ZmALMT2 by heterologously expressing it in Xenopus laevis oocytes and transgenic Arabidopsis. In oocytes, ZmALMT2 mediated an Al-independent electrogenic transport product of organic and inorganic anion efflux. Ectopic overexpression of ZmALMT2 in an Al-hypersensitive Arabidopsis KO/KD line lacking the Al tolerance genes, AtALMT1 and AtMATE, resulted in Al-independent constitutive root malate efflux which partially restored the Al tolerance phenotype. The lack of correlation between ZmALMT2 expression and Al tolerance (e.g., expression not localized to the root tip, not up-regulated by Al, and higher in sensitive versus tolerance maize lines) also led us to question ZmALMT2's role in Al tolerance. The functional properties of the ZmALMT2 transporter presented here, along with the gene expression data, suggest that ZmALMT2 is not involved in maize Al tolerance but, rather, may play a role in mineral nutrient acquisition and transport.     

Overexpression of AtALMT1 in the Arabidopsis thaliana ecotype Columbia results in enhanced Al-activated malate excretion and beneficial bacterium recruitment

AtALMT1 (Arabidopsis thaliana ALuminum activated Malate Transporter 1) encodes an Arabidopsis thaliana malate transporter that has a pleiotropic role in Arabidopsis stress tolerance. Malate released through AtALMT1 protects the root tip from Al rhizotoxicity, and recruits beneficial rhizobacteria that induce plant immunity. To examine whether the overexpression of AtALMT1 can improve these traits, the gene, driven by the cauliflower mosaic virus 35S promoter, was introduced into the Arabidopsis ecotype Columbia. Overexpression of the gene enhanced both Al-activated malate excretion and the recruitment of beneficial bacteria Bacillus subtilis strain FB17. These findings suggest that overexpression of AtALMT1 can be used as an approach to enhance a plant's ability to release malate into the rhizosphere, which can enhance plant tolerance to some environmental stress factors.     

A multidrug and toxic compound extrusion (MATE) transporter modulates auxin levels in root to regulate root development and promotes aluminium tolerance

MATE (multidrug and toxic compound extrusion) transporters play multiple roles in plants including detoxification, secondary metabolite transport, aluminium (Al) tolerance, and disease resistance. Here we identify and characterize the role of the Arabidopsis MATE transporter DETOXIFICATION30. AtDTX30 regulates auxin homeostasis in Arabidopsis roots to modulate root development and Al-tolerance. DTX30 is primarily expressed in roots and localizes to the plasma membrane of root epidermal cells including root hairs. dtx30 mutants exhibit reduced elongation of the primary root, root hairs, and lateral roots. The mutant seedlings accumulate more auxin in their root tips indicating role of DTX30 in maintaining auxin homeostasis in the root. Al induces DTX30 expression and promotes its localization to the distal transition zone. dtx30 seedlings accumulate more Al in their roots but are hyposensitive to Al-mediated rhizotoxicity perhaps due to saturation in root growth inhibition. Increase in expression of ethylene and auxin biosynthesis genes in presence of Al is absent in dtx30. The mutants exude less citrate under Al conditions, which might be due to misregulation of AtSTOP1 and the citrate transporter AtMATE. In conclusion, DTX30 modulates auxin levels in root to regulate root development and in the presence of Al indirectly modulates citrate exudation to promote Al tolerance.     

A wheat gene encoding an aluminum-activated malate transporter

The major constraint to plant growth in acid soils is the presence of toxic aluminum (Al) cations, which inhibit root elongation. The enhanced Al tolerance exhibited by some cultivars of wheat is associated with the Al-dependent efflux of malate from root apices. Malate forms a stable complex with Al that is harmless to plants and, therefore, this efflux of malate forms the basis of a hypothesis to explain Al tolerance in wheat. Here, we report on the cloning of a wheat gene, ALMT1 (aluminum-activated malate transporter), that co-segregates with Al tolerance in F2 and F3 populations derived from crosses between near-isogenic wheat lines that differ in Al tolerance. The ALMT1 gene encodes a membrane protein, which is constitutively expressed in the root apices of the Al-tolerant line at greater levels than in the near-isogenic but Al-sensitive line. Heterologous expression of ALMT1 in Xenopus oocytes, rice and cultured tobacco cells conferred an Al-activated malate efflux. Additionally, ALMT1 increased the tolerance of tobacco cells to Al treatment. These findings demonstrate that ALMT1 encodes an Al-activated malate transporter that is capable of conferring Al tolerance to plant cells.     

The physiology and biophysics of an aluminum tolerance mechanism based on root citrate exudation in maize

Al-induced release of Al-chelating ligands (primarily organic acids) into the rhizosphere from the root apex has been identified as a major Al tolerance mechanism in a number of plant species. In the present study, we conducted physiological investigations to study the spatial and temporal characteristics of Al-activated root organic acid exudation, as well as changes in root organic acid content and Al accumulation, in an Al-tolerant maize (Zea mays) single cross (SLP 181/71 x Cateto Colombia 96/71). These investigations were integrated with biophysical studies using the patch-clamp technique to examine Al-activated anion channel activity in protoplasts isolated from different regions of the maize root. Exposure to Al nearly instantaneously activated a concentration-dependent citrate release, which saturated at rates close to 0.5 nmol citrate h(-1) root(-1), with the half-maximal rates of citrate release occurring at about 20 microM Al(3+) activity. Comparison of citrate exudation rates between decapped and capped roots indicated the root cap does not play a major role in perceiving the Al signal or in the exudation process. Spatial analysis indicated that the predominant citrate exudation is not confined to the root apex, but could be found as far as 5 cm beyond the root cap, involving cortex and stelar cells. Patch clamp recordings obtained in whole-cell and outside-out patches confirmed the presence of an Al-inducible plasma membrane anion channel in protoplasts isolated from stelar or cortical tissues. The unitary conductance of this channel was 23 to 55 pS. Our results suggest that this transporter mediates the Al-induced citrate release observed in the intact tissue. In addition to the rapid Al activation of citrate release, a slower, Al-inducible increase in root citrate content was also observed. These findings led us to speculate that in addition to the Al exclusion mechanism based on root citrate exudation, a second internal Al tolerance mechanism may be operating based on Al-inducible changes in organic acid synthesis and compartmentation. We discuss our findings in terms of recent genetic studies of Al tolerance in maize, which suggest that Al tolerance in maize is a complex trait.     

Two functionally distinct members of the MATE (multi‐drug and toxic compound extrusion) family of transporters potentially underlie two major aluminum tolerance QTLs in maize

Crop yields are significantly reduced by aluminum (Al) toxicity on acidic soils, which comprise up to 50% of the world’s arable land. Al‐activated release of ligands (such as organic acids) from the roots is a major Al tolerance mechanism in plants. In maize, Al‐activated root citrate exudation plays an important role in tolerance. However, maize Al tolerance is a complex trait involving multiple genes and physiological mechanisms. Recently, transporters from the MATE family have been shown to mediate Al‐activated citrate exudation in a number of plant species. Here we describe the cloning and characterization of two MATE family members in maize, ZmMATE1 and ZmMATE2, which co‐localize to major Al tolerance QTL. Both genes encode plasma membrane proteins that mediate significant anion efflux when expressed in Xenopus oocytes. ZmMATE1 expression is mostly concentrated in root tissues, is up‐regulated by Al and is significantly higher in Al‐tolerant maize genotypes. In contrast, ZmMATE2 expression is not specifically localized to any particular tissue and does not respond to Al. [¹⁴C]‐citrate efflux experiments in oocytes demonstrate that ZmMATE1 is a citrate transporter. In addition, ZmMATE1 expression confers a significant increase in Al tolerance in transgenic Arabidopsis. Our data suggests that ZmMATE1 is a functional homolog of the Al tolerance genes recently characterized in sorghum, barley and Arabidopsis, and is likely to underlie the largest maize Al tolerance QTL found on chromosome 6. However, ZmMATE2 most likely does not encode a citrate transporter, and could be involved in a novel Al tolerance mechanism.     

Physiological basis of reduced Al tolerance in ditelosomic lines of Chinese Spring wheat

Aluminum tolerance was assessed in the moderately Al-tolerant wheat (Triticum aestivum L.) cultivar Chinese Spring and a set of ditelosomic lines derived from Chinese Spring. Three ditelosomic lines lacking chromosome arms 4DL, 5AS and 7AS, respectively, exhibited decreased Al tolerance relative to the euploid parent Chinese Spring based on reduced root growth in Al-containing solutions. The physiological basis of the reduced Al tolerance was investigated. Measurements by inductively coupled argon plasma mass spectroscopy of root apical Al accumulation demonstrated that two of these three lines had a decreased ability to exclude Al from the root apex, the site of Al phytotoxicity. As Al-induced malate exudation has been suggested to be an important physiological mechanism of Al tolerance in wheat, this parameter was quantified and malate exudation was shown to be smaller in all three deletion lines compared with Chinese Spring. These results suggest that the decreased Al tolerance in at least two of the three ditelosomic lines is due to the loss of different genes independently influencing a single Al-tolerance mechanism, rather than to the loss of genes encoding alternative Al-tolerance mechanisms. Received: 3 July 2000 / Accepted: 9 August 2000     

Identification and characterization of aluminum tolerance loci in Arabidopsis (Landsberg erecta x Columbia) by quantitative trait locus mapping. A physiologically simple but genetically complex trait

Aluminum (Al) toxicity, which is caused by the solubilization of Al3+ in acid soils resulting in inhibition of root growth and nutrient/water acquisition, is a serious limitation to crop production, because up to one-half of the world's potentially arable land is acidic. To date, however, no Al tolerance genes have yet been cloned. The physiological mechanisms of tolerance are somewhat better understood; the major documented mechanism involves the Al-activated release of Al-binding organic acids from the root tip, preventing uptake into the primary site of toxicity. In this study, a quantitative trait loci analysis of Al tolerance in Arabidopsis was conducted, which also correlated Al tolerance quantitative trait locus (QTL) with physiological mechanisms of tolerance. The analysis identified two major loci, which explain approximately 40% of the variance in Al tolerance observed among recombinant inbred lines derived from Landsberg erecta (sensitive) and Columbia (tolerant). We characterized the mechanism by which tolerance is achieved, and we found that the two QTL cosegregate with an Al-activated release of malate from Arabidopsis roots. Although only two of the QTL have been identified, malate release explains nearly all (95%) of the variation in Al tolerance in this population. Al tolerance in Landsberg erecta x Columbia is more complex genetically than physiologically, in that a number of genes underlie a single physiological mechanism involving root malate release. These findings have set the stage for the subsequent cloning of the genes responsible for the Al tolerance QTL, and a genomics-based cloning strategy and initial progress on this are also discussed.     

Aluminum resistance in maize cannot be solely explained by root organic acid exudation. A comparative physiological study

Root apical aluminum (Al) exclusion via Al-activated root citrate exudation is widely accepted as the main Al-resistance mechanism operating in maize (Zea mays) roots. Nonetheless, the correlation between Al resistance and this Al-exclusion mechanism has not been tested beyond a very small number of Al-resistant and Al-sensitive maize lines. In this study, we conducted a comparative study of the physiology of Al resistance using six different maize genotypes that capture the range of maize Al resistance and differ significantly in their genetic background (three Brazilian and three North American genotypes). In these maize lines, we were able to establish a clear correlation between root tip Al exclusion (based on root Al content) and Al resistance. Both Al-resistant genotypes and three of the four Al-sensitive lines exhibited a significant Al-activated citrate exudation, with no evidence for Al activation of root malate or phosphate release. There was a lack of correlation between differential Al resistance and root citrate exudation for the six maize genotypes; in fact, one of the Al-sensitive lines, Mo17, had the largest Al-activated citrate exudation of all of the maize lines. Our results indicate that although root organic acid release may play a role in maize Al resistance, it is clearly not the only or the main resistance mechanism operating in these maize roots. A number of other potential Al-resistance mechanisms were investigated, including release of other Al-chelating ligands, Al-induced alkalinization of rhizosphere pH, changes in internal levels of Al-chelating compounds in the root, and Al translocation to the shoot. However, we were unsuccessful in identifying additional Al-resistance mechanisms in maize. It is likely that a purely physiological approach may not be sufficient to identify these novel Al-resistance mechanisms in maize and this will require an interdisciplinary approach integrating genetic, molecular, and physiological investigations.