Magnesium enhances aluminum-induced citrate secretion in rice bean roots (Vigna umbellata) by restoring plasma membrane H+-ATPase activity

We demonstrated that magnesium (Mg) can alleviate aluminum (Al) toxicity in rice bean [Vigna umbellata (Thunb.) Ohwi & Ohashi] more effectively than is expected from a non-specific cation response. Micromolar concentrations of Mg alleviated the inhibition of root growth by Al but not by lanthanum, and neither strontium nor barium at the micromolar level alleviates Al toxicity. Aluminum also induced citrate efflux from rice bean roots, and this response was stimulated by inclusion of 10 microM Mg in the treatment solution. The increase in the Al-induced citrate efflux by Mg paralleled the improvement in root growth, suggesting that the ameliorative effect of Mg might be related to greater citrate efflux. Vanadate (an effective H+-ATPase inhibitor) decreased the Al-induced citrate efflux, while addition of Mg partly restored the efflux. Mg addition also increased the activity of Al-reduced plasma membrane H+-ATPase, as well as helping to maintain the Mg and calcium contents in root apices. We propose that the addition of Mg to the toxic Al treatment helps maintain the tissue Mg content and the activity of the plasma membrane H+-ATPase. These changes enhanced the Al-dependent efflux of citrate which provided extra protection from Al stress.     

Brain and cerebrospinal fluid uptake of zidovudine (AZT) in rats after intravenous injection

Uptake kinetics of zidovudine into cerebrospinal fluid (CSF) and brain tissue were determined in adult Sprague Dawley male rats after single intravenous injection of 6.7 mg/kg (25 mumol/kg). The drug kinetics in plasma followed biexponential disposition with an initial distribution half-life of approximately 11 minutes and an elimination half-life of 40 minutes. Over the plasma concentration range of 0.2 to 10 micrograms/ml, the cerebrospinal fluid to plasma ratio averaged 14.8 +/- 1.9% whereas the mean brain tissue to plasma ratio was 8.2 +/- 1.2% (uncorrected) or 2.3 +/- 1.8% (corrected) for the brain vascular space contribution. Simultaneous nonlinear regression analysis of brain, CSF and plasma concentration data indicate that the overall rate constant for efflux of drug from brain is approximately 75-fold higher and from CSF is 8-fold higher than the respective rate constants for influx. Thus, the ratio of the efflux to influx appears to be the predominant factor in determining the net accumulation of drug into CSF and brain parenchymal tissue.     

Inhibition of Ca2+ uptake in freshwater carp, Cyprinus carpio, during short-term exposure to aluminum.

In carp exposed to pH 5.2 in fresh water, the Ca2+ influx from the water is reduced by 31% when compared to fish in water of neutral pH. At pH 5.2, the Ca2+ influx but not Na+ uptake is decreased by aluminum (Al). Al reduces Ca2+ influx dose-dependently: a maximum 55% reduction was observed after 1-2 h exposure to 200 micrograms.1(-1) (7.4 microM) Al. Branchial Ca2+ efflux is less sensitive to Al and affected only by exposure for more than 1 h to high Al concentrations. Na+ influx is not affected by concentrations Al up to 400 micrograms.1(-1). Na+ efflux, similarly to Ca2+ efflux, increased when fish were exposed for more than 1 h to 400 micrograms.1(-1) Al.     

Salicylic acid-induced aluminum tolerance by modulation of citrate efflux from roots of<Emphasis Type="Italic"> Cassia tora</Emphasis> L.

Aluminum-induced exudation of organic acids from roots has been proposed as a mechanism for Al tolerance in plants. To better understand the regulatory process leading to efflux of organic acids, the possible involvement of salicylic acid (SA) in regulating Al-induced citrate release in Cassia tora L. was identified. The response of citrate efflux to exogenous SA was concentration-dependent. Application of SA at 5 microM in solution containing 20 microM Al increased citrate efflux to levels 1.76-fold higher than in controls (20 microM Al alone). However, inhibition of citrate release was observed when SA concentrations increased to more than 20 microM. Increased citrate efflux due to the SA treatment was associated with decreased inhibition of root growth and Al content in root tips, suggesting that exogenous SA could confer Al tolerance by increasing citrate efflux. We also examined citrate synthase activities (EC 4.1.3.7) and citrate concentrations in root tips exposed to Al and/or SA. However, both citrate synthase activities and citrate accumulation remained unaffected. These results indicate that SA-promotion of Al-induced citrate efflux is not correlated with increase in citrate production. Total endogenous SA concentrations were measured in root tips and the SA concentrations were significantly enhanced by Al at levels of 10-50 microM.     

Efflux of cyclic adenosine monophosphate from cells: mechanisms and physiological implications

Although cyclic nucleotides are hydrophilic compounds, extracellular cAMP (cAMPo) rapidly accumulates during the activation of adenylate cyclase. This review considers the kinetic characteristics of cAMP transport through the plasma membrane and its physiological implications. The influx and efflux of cAMP occur via different carriers. At physiological concentrations of cAMPo, the influx of cAMP does not significantly contribute to regulation of the intracellular content of the cyclic nucleotide, but it is responsible for the accumulation of cAMPi in experiments at [cAMP]o approximately 1 mM. In contrast, the high rate of cAMP efflux is mainly responsible for normalization of [cAMP]i during long-term activation of adenylate cyclase. The possible involvement of ATP-binding cassette proteins (ABC proteins) in the efflux of cAMP from the cell is considered. In procaryotes cAMPo is a signal molecule during the generation of cell colonies, acting on special receptors that interact with GTP-binding proteins. Such receptors have not been found in vertebrates, and in most cases the signal functions of cAMPo are mediated by its degradation by extracellular enzymes with subsequent activation of adenosine receptors.     

Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.     

Glutamate release and neuronal damage in ischemia.

Neuronal injury caused by ischemia after occlusion of cerebral arteries is believed to be mediated by excessive activation of glutamate receptors. In the ischemic brain, extracellular glutamate is elevated rapidly after the onset of ischemia and declines following reperfusion. The mechanisms of the elevation of extracellular glutamate include enhanced efflux of glutamate and the reduction of glutamate uptake. The early efflux of glutamate occurring immediately after the onset of ischemia is mediated by a calcium-dependent process through activation of voltage-dependent calcium channels. The calcium-independent efflux at later stages is thought to be mediated primarily by glutamate transporters operating in the reverse mode owing to the imbalance of sodium ions across plasma membranes. Although high levels of glutamate in the extracellular space are well established to appear rapidly after the onset of ischemia, a direct linkage between the enhanced release of glutamate and the neuronal injury has not been fully established. In cultured neurons, ischemia induces efflux of glutamate into the extracellular space, but subsequent neuronal loss is not solely caused by the high glutamate concentration. In addition, cultured neurons can be rescued by NMDA antagonists added to the medium after exposure to glutamate receptor agonists. Two mechanisms can be proposed for neuroprotection by late NMDA receptor blockade, i.e., blocking of presynaptic release of glutamate after excessive activation of glutamate receptors, and blocking of postsynaptic sensitization of NMDA receptors.     

Differential aluminum tolerance in soybean: An evaluation of the role of organic acids

The role of organic acids in aluminum (Al) tolerance has been the object of intensive research. In the present work, we evaluated the roles of organic acid exudation and concentrations at the root tip on Al tolerance of soybean. Exposing soybean seedlings to Al³⁺ activities up to 4.7 μ M in solution led to different degrees of restriction of primary root elongation. Al tolerance among genotypes was associated with citrate accumulation and excretion into the external media. Citrate and malate efflux increased in all genotypes during the first 6 h of Al exposure, but only citrate efflux in Al-tolerant genotypes was sustained for an extended period. Tolerance to Al was correlated with the concentration of citrate in root tips of 8 genotypes with a range of Al sensitivities (r²=0.75). The fluorescent stain lumogallion indicated that more Al accumulated in root tips of the Al-sensitive genotype Young than the Al-tolerant genotype PI 416937, suggesting that the sustained release of citrate from roots of the tolerant genotype was involved in Al exclusion. The initial stimulation of citrate and malate excretion and accumulation in the tip of all genotypes suggested the involvement of additional tolerance mechanisms. The experiments included an examination of Al effects on lateral root elongation. Extension of lateral roots was more sensitive to Al than that of tap roots, and lateral root tips accumulated more Al and had lower levels of citrate.     

A patch-clamp study on the physiology of aluminum toxicity and aluminum tolerance in maize. Identification and characterization of Al(3+)-induced anion channels

The presence of Al(3+) in the rhizosphere induces citrate efflux from the root apex of the Al-tolerant maize (Zea mays) hybrid South American 3, consequently chelating and reducing the activity of toxic Al(3+) at the root surface. Because citrate is released from root apical cells as the deprotonated anion, we used the patch-clamp technique in protoplasts isolated from the terminal 5 mm of the root to study the plasma membrane ion transporters that could be involved in Al-tolerance and Al-toxicity responses. Acidification of the extracellular environment stimulated inward K(+) currents while inhibiting outward K(+) currents. Addition of extracellular Al(3+) inhibited the remaining K(+) outward currents, blocked the K(+) inward current, and caused the activation of an inward Cl(-) current (anion efflux). Studies with excised membrane patches revealed the existence of Al-dependent anion channels, which were highly selective for anions over cations. Our success in activating this channel with extracellular Al(3+) in membrane patches excised prior to any Al(3+) exposure indicates that the machinery required for Al(3+) activation of this channel, and consequently the whole root Al(3+) response, is localized to the root-cell plasma membrane. This Al(3+)-activated anion channel may also be permeable to organic acids, thus mediating the Al-tolerance response (i.e. Al-induced organic acid exudation) observed in intact maize root apices.     

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H⁺-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H⁺-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K⁺ for 40 min under various conditions. The rate of efflux of K⁺ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H⁺-ATPase of the plasma membrane, increased the efflux of K⁺. Al repressed this efflux at low pH, probably through an effect on K⁺ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K⁺ irrespective of the presence of vanadate. Ca²⁺ also had a repressive effect on the efflux of K⁺ at low pH. The effect of Ca²⁺, greater in Scout, might be related to the regulation of the net influx of H⁺, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H⁺, which in turn is counteracted by the efflux of K⁺ and H⁺. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H⁺ and the efflux of K⁺ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.     

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.     

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.     

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.     

Citrate transporters play a critical role in aluminium-stimulated citrate efflux in rice bean (Vigna umbellata) roots

BACKGROUND AND AIMS: Aluminium (Al) stimulates the efflux of citrate from apices of rice bean (Vigna umbellata) roots. This response is delayed at least 3 h when roots are exposed to 50 microm Al, indicating that some inducible processes leading to citrate efflux are involved. The physiological bases responsible for the delayed response were examined here. METHODS: The effects of several antagonists of anion channels and citrate carriers, and of the protein synthesis inhibitor, cycloheximide (CHM) on Al-stimulated citrate efflux and/or citrate content were examined by high-pressure liquid chromatography (HPLC) or an enzymatic method. KEY RESULTS: Both anion channel inhibitors and citrate carrier inhibitors can inhibit Al-stimulated citrate efflux, with anthracene-9-carboxylic acid (A-9-C, an anion channel inhibitor) and phenylisothiocyanate (PI, a citrate carrier inhibitor) the most effective inhibitors. A 6 h pulse of 50 microm Al induced a significant increase of citrate content in root apices and release of citrate. However, the increase in citrate content preceded the efflux. Furthermore, the release of citrate stimulated by the pulse treatment was inhibited by both A-9-C and PI, indicating the importance of the citrate carrier on the mitochondrial membrane and the anion channel on the plasma membrane for the Al-stimulated citrate efflux. CHM (20 microm) also significantly inhibited Al-stimulated citrate efflux, confirming that de novo protein synthesis is required for Al-stimulated citrate efflux. CONCLUSIONS: These results indicate that the activation of genes possibly encoding citrate transporters plays a critical role in Al-stimulated citrate efflux.     

Influence of iron-saturation of plasma transferrin in iron distribution in the brain

Based on the evidence that iron distribution in the peripheral tissues is changed by iron-saturation of plasma transferrin, the influence of iron-saturation of plasma transferrin in iron delivery to the brain was examined. Mouse plasma was pre-incubated with ferric chloride in citrate buffer to saturate transferrin and then incubated with (59)FeCl(3). Peak retention time of (59)Fe was transferred from the retention time of transferrin to that of mercaptalbumin, suggesting that iron may bind to albumin in the plasma in the case of iron-saturation of transferrin. When mice were intravenously injected with ferric chloride in citrate buffer 10 min before intravenous injection of (59)FeCl(3), 59Fe concentration in the plasma was remarkably low. (59)Fe concentration in the liver of iron-loaded mice was four times higher than in control, while 59Fe concentration in the brain of iron-loaded mice was approximately 40% of that of control mice. Twenty-four hours after intravenous injection of (59)FeCl(3), brain autoradiograms also showed that (59)Fe concentrations in the brain of iron-loaded mice were approximately 40-50% of those of control mice in all brain regions tested except the choroid plexus, in which (59)Fe concentration was equal. These results suggest that the fraction of non-transferrin-bound iron is engulfed by the liver, resulting in the reduction of iron available for iron delivery to the brain in iron-loaded mice. Transferrin-bound iron may be responsible for the fraction of iron in circulation that enters the brain.     

Effects of Chemical Speciation on the Mineralization of Organic Compounds by Microorganisms

The mineralization of 1.0 to 100 ng each of four complexing compounds—oxalate, citrate, nitrilotriacetate (NTA), and EDTA—per ml was tested in media prepared in accordance with equilibrium calculations by a computer program so that the H, Ca, Mg, Fe, or Al complex (chemical species) was predominant. Sewage microorganisms mineralized calcium citrate more rapidly than iron, aluminum, or hydrogen citrate, and magnesium citrate was degraded slowest. Aluminum, hydrogen, and iron oxalates were mineralized more rapidly than calcium oxalate, and magnesium oxalate was decomposed slowest. Sewage microorganisms mineralized calcium NTA but not aluminum, magnesium, hydrogen, or iron NTA or any of the EDTA complexes. Pseudomonas sp. mineralized calcium and iron citrates but had no activity on hydrogen, aluminum, or magnesium citrate. Pseudomonas pseudoalcaligenes mineralized calcium, iron, hydrogen, and aluminum citrates but had little activity on magnesium citrate. Pseudomonas alcaligenes used calcium, iron, hydrogen, and aluminum oxalates readily, but it used magnesium oxalate at a slower rate. Listeria sp. destroyed calcium NTA but had no effect on hydrogen, iron, or magnesium NTA. Increasing the Ca concentration in the medium enhanced the breakdown of NTA by Listeria sp. The different activities of the bacterial isolates were not a result of the toxicity of the complexes or the lack of availability of a nutrient element. NTA mineralization was not enhanced by the addition of Ca to Beebe Lake water, but it was enhanced when Ca and an NTA-degrading inoculum were added to water from an oligotrophic lake. The data show that chemical speciation influences the mineralization of organic compounds by naturally occurring microbial communities and by individual bacterial populations.     

One Novel Mitochondrial Citrate Synthase from <Emphasis Type="Italic">Oryza sativa</Emphasis> L. can Enhance Aluminum Tolerance in Transgenic Tobacco

Rice exhibits the greatest aluminum (Al) tolerance compared with other cereals such as wheat, barley, maize, etc. A full-length gene, OsCS1, encoding citrate synthase, which is highly induced by aluminum toxicity in rice (Oryza sativa L.), was isolated. Sequence analysis and the sub-cellular localization of OsCS1 in yeast revealed that it is a mitochondrial citrate synthase. OsCS1 was induced by Al toxicity. Several independent transgenic tobacco lines expressing OsCS 1 exhibitted increased citrate efflux and extraordinary Al tolerance. Possible outlook for OsCS1 to be applied to enhance plant tolerance to Al toxicity was also discussed.     

A de novo synthesis citrate transporter, Vigna umbellata multidrug and toxic compound extrusion, implicates in Al-activated citrate efflux in rice bean (Vigna umbellata) root apex

Al-activated organic acid anion efflux from roots is an important Al resistance mechanism in plants. We have conducted homologous cloning and isolated Vigna umbellata multidrug and toxic compound extrusion (VuMATE), a gene encoding a de novo citrate transporter from rice bean. Al treatment up-regulated VuMATE expression in the root apex, but neither in the mature root region nor in the leaf. The degree of up-regulation of VuMATE was both partially Al concentration and time dependent, consistent with the delay in the onset of the Al-induced citrate efflux in rice bean roots. While La(3+) moderately induced VuMATE expression, Cd(2+) and Cu(2+) did not induce the expression. Electrophysiological analysis of Xenopus oocytes expressing VuMATE indicated this transporter can mediate significant anion efflux across the plasma membrane. [(14) C]citrate efflux experiments in oocytes demonstrated that VuMATE is a H(+) -dependent citrate transporter. In addition, expression of VuMATE in transgenic tomato resulted in increased Al resistance, which correlated with an enhanced citrate efflux. Taken together, these findings suggest that VuMATE is a functional homolog of the known citrate transporters in sorghum, barley, maize and Arabidopsis. The similarities and differences of all the known citrate transporters associated with Al stress in the MATE family are also discussed.     

The BnALMT1 and BnALMT2 genes from rape encode aluminum-activated malate transporters that enhance the aluminum resistance of plant cells

The release of organic anions from roots can protect plants from aluminum (Al) toxicity and help them overcome phosphorus (P) deficiency. Our previous findings showed that Al treatment induced malate and citrate efflux from rape (Brassica napus) roots, and that P deficiency did not induce the efflux. Since this response is similar to the malate efflux from wheat (Triticum aestivum) that is controlled by the TaALMT1 gene, we investigated whether homologs of TaALMT1 are present in rape and whether they are involved in the release of organic anions. We isolated two TaALMT1 homologs from rape designated BnALMT1 and BnALMT2 (B. napus Al-activated malate transporter). The expression of these genes was induced in roots, but not shoots, by Al treatment but P deficiency had no effect. Several other cations (lanthanum, ytterbium, and erbium) also increased BnALMT1 and BnALMT2 expression in the roots. The function of the BnALMT1 and BnALMT2 proteins was investigated by heterologous expression in cultured tobacco (Nicotiana tabacum) cells and in Xenopus laevis oocytes. Both transfection systems showed an enhanced capacity for malate efflux but not citrate efflux, when exposed to Al. Smaller malate fluxes were also activated by ytterbium and erbium treatment. Transgenic tobacco cells grew significantly better than control cells following an 18 h treatment with Al, indicating that the expression of BnALMT1 and BnALMT2 increased the resistance of these plant cells to Al stress. This report demonstrates that homologs of the TaALMT1 gene from wheat perform similar functions in other species.     

An aluminum-activated citrate transporter in barley

Soluble ionic aluminum (Al) inhibits root growth and reduces crop production on acid soils. Al-resistant cultivars of barley (Hordeum vulgare L.) detoxify Al by secreting citrate from the roots, but the responsible gene has not been identified yet. Here, we identified a gene (HvAACT1) responsible for the Al-activated citrate secretion by fine mapping combined with microarray analysis, using an Al-resistant cultivar, Murasakimochi, and an Al-sensitive cultivar, Morex. This gene belongs to the multidrug and toxic compound extrusion (MATE) family and was constitutively expressed mainly in the roots of the Al-resistant barley cultivar. Heterologous expression of HvAACT1 in Xenopus oocytes showed efflux activity for (14)C-labeled citrate, but not for malate. Two-electrode voltage clamp analysis also showed transport activity of citrate in the HvAACT1-expressing oocytes in the presence of Al. Overexpression of this gene in tobacco enhanced citrate secretion and Al resistance compared with the wild-type plants. Transiently expressed green fluorescent protein-tagged HvAACT1 was localized at the plasma membrane of the onion epidermal cells, and immunostaining showed that HvAACT1 was localized in the epidermal cells of the barley root tips. A good correlation was found between the expression of HvAACT1 and citrate secretion in 10 barley cultivars differing in Al resistance. Taken together, our results demonstrate that HvAACT1 is an Al-activated citrate transporter responsible for Al resistance in barley.