Aluminium triggers malate-independent potassium release via ion channels from the root apex in wheat

The regulatory mechanisms for the aluminium (Al)-induced efflux of K(+) and malate from the root apex of Al-resistant wheat ( Triticum aestivum L. cv. Atlas) were characterized. Treatment with 20 mM tetraethylammonium (TEA) chloride, a K(+)-channel inhibitor, blocked the Al-induced K(+) efflux by 65%, but blocked the Al-induced malate efflux only slightly. Lanthanum (La(3+)) or ytterbium (Yb(3+)) strongly inhibited the K(+) efflux, but slightly increased malate efflux. These lanthanides applied together with Al did not affect the Al-induced malate efflux, but reduced the Al-induced K(+) efflux by 57% for La(3+) and by 35% for Yb(3+). By contrast, pretreatment with 50 microM niflumic acid, an anion-channel inhibitor, strongly suppressed the Al-induced malate efflux, but did not affect the Al-induced K(+) efflux. The efflux of K(+) uncoupled with that of malate resulted in the alkalization of intracellular pH in the root apex, suggesting that the release of K(+) coupled with malate plays an important role in stabilizing intracellular pH. Copper (Cu(2+)) induced the release of K(+) via a TEA-insensitive pathway without the release of malate in both Al-resistant and Al-sensitive (cultivar Scout) wheat. Simultaneous application of Al and Cu(2+) to the root apices resulted in TEA-sensitive K(+) efflux in Atlas but not in Scout, suggesting that Al competes with Cu(2+) for K(+) efflux. Taken together, these results suggest that Al-induced K(+) efflux is mediated by both TEA- and lanthanide-sensitive K(+) channels, although this induction is not a prerequisite for the induction of the release of malate.     

Effect of K-252a and abscisic acid on the efflux of citrate from soybean roots

The Al-induced release of organic acid has been suggested as an important mechanism for Al resistance in plants. In this study, the effect of K-252a and abscisic acid (ABA) on the efflux of citrate was investigated in soybean (Glycine max L.) roots. Al initiated citrate efflux from the root apices 30 min after the addition of Al. The Al-triggered efflux of citrate was sensitive to metabolic inhibitors and anion channel inhibitors. Pretreatment or treatment with K-252a, an inhibitor of protein kinase, severely inhibited the Al-induced efflux of citrate accompanying an increase in Al accumulation and intensified Al-induced root growth inhibition. Al-treatment increased the endogenous level of abscisic acid (ABA) in soybean roots in a dose- and time-dependent manner, while K-252a failed to inhibit the Al-induced increase in endogenous ABA. Exogenous application of ABA increased the activity of citrate synthase (EC 4.1.3.7) by 26.2%, and decreased Al accumulation by 32.3%, respectively. ABA-induced increases in citrate efflux and root elongation were suppressed by K-252a, while ABA could not reverse the K-252a effects. Taken together, these results suggest that ABA is probably involved in the early response, after which K-252a-sensitive protein kinases play a key step in regulating the activity of an anion channel, through which citrate is released from the apical cells of soybean roots.     

Al-induced efflux of organic acid anions is poorly associated with internal organic acid metabolism in triticale roots

The secretion of organic acid anions from roots has been identified as a mechanism of resistance to Al. However, the process leading to the secretion of organic acid anions is poorly understood. The effect of Al on organic acid metabolism was investigated in two lines of triticale (xTriticosecale Wittmark) differing in Al-induced secretion of malate and citrate and in Al resistance. The site of Al-induced secretion of citrate and malate from a resistant line was localized to the root apices (terminal 5 mm). The levels of citrate (root apices and mature root segments) and malate (mature segments only) in roots increased during exposure to Al, but similar changes were observed in both triticale genotypes. The in vitro activities of four enzymes involved in malate and citrate metabolism (citrate synthase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and NADP-isocitrate dehydrogenase) were similar for sensitive and resistant lines in both root apices and mature root segments. The response of these enzymes to pH did not differ between tolerant and sensitive lines or in the presence and absence of Al. Moreover, cytoplasmic and vacuolar pH were not affected by exposure to Al in either line. Together, these results indicate that the Al-dependent efflux of organic acid anions from the roots of triticale is not regulated by their internal levels in the roots or by the capacity of the root cells to synthesize malate and citrate.     

Cytotoxic thio-malate is transported by both an aluminum-responsive malate efflux pathway in wheat and the MAE1 malate permease in Schizosaccharomyces pombe

Aluminum (Al) tolerance in wheat (Triticum aestivum L.) is mainly achieved by malate efflux, which is regulated by the expression of the recently identified gene, presumably encoding an Al-activated malate efflux transporter (ALMT1). However, the transport mechanism is not fully understood, partly as a result of the rapid turnover of its substrate. We developed a tool to study malate transport in wheat by screening biological compounds using the well-characterized Schizosaccharomyces pombe malate transporter (SpMAE1). Expression of SpMAE1 in both S. pombe and Saccharomyces cerevisiae, which has no SpMAE1 homologue, caused hypersensitivity to thio-malic acid. This hypersensitivity was prominent at pH 3.5, but not pH 4.5, and was accompanied by an increase in thiol content, indicating that SpMAE1 mediates the uptake of thio-malic acid at a specific low pH. In wheat, root apices were able to accumulate thio-malic acid without growth reduction at pH values above 4.2. Pretreatment of root apices with thio-malic acid followed by Al treatment induced thio-malate efflux. Al-induced thio-malate efflux was much higher in Al-resistant cultivars/genotypes than in Al-sensitive ones, and was accompanied by a decrease in thiol-content. Thio-malate efflux in the Al-resistant cultivar was slightly activated by lanthanum or ytterbium ion. Thio-malic acid did not alleviate the Al-induced inhibition of root elongation in wheat. Taken together, our results suggest that thio-malate acts as an analogue for malate in malate transport systems in wheat and yeast, and that it may be a useful tool for the analysis of malate transport involved in Al-tolerance and of other organic ion transport processes.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Possible Involvement of Al-Induced Electrical Signals in Al Tolerance in Wheat

The relationship between Al-induced depolarization of root-cell transmembrane electrical potentials (Em) and Al tolerance in wheat (Triticum aestivum L.) was investigated. Al exposure induced depolarizations of Em in the Al-tolerant wheat cultivars Atlas and ET3, but not in the Al-sensitive wheat cultivars Scout and ES3. The depolarizations of Em occured in root cap cells and as far back as 10 mm from the root tip. The depolarization was specific to Al3+; no depolarization was observed when roots were exposed to the rhizotoxic trivalent cation La3+. The Al-induced depolarization occurred in the presence of anion-channel antagonists that blocked the release of malate, indicating that the depolarization is not due to the electrogenic efflux of malate2-. K+-induced depolarizations in the root cap were of the same magnitude as Al-induced depolarizations, but did not trigger malate release, indicating that Al-induced depolarization of root cap cell membrane potentials is probably linked to, but is not sufficient to trigger, malate release.     

The role of phosphorus in aluminium-induced citrate and malate exudation from rape (Brassica napus)

Exudation of organic anions is believed to be a common tolerance mechanism for both aluminium toxicity and phosphorus deficiency. Nevertheless, which of these stresses that actually elicit the exudation of organic anions from rape ( Brassica napus L) remains unknown, and the combined effects of Al toxicity and P deficiency on rape have not been reported before. Therefore, in the current study, Brassica napus var. Natane nourin plants grown with or without 0.25 m M P were exposed to 0 or 50 µ M AlCl₃ and several parameters related to the exudation of organic anions from the roots were investigated. Eight days of P deficiency resulted in a significant growth reduction, but P deficiency alone did not induce exudation of organic anions. In contrast, Al strongly induced organic acid exudation, while simultaneously inhibiting root growth. Increased in-vitro activity of citrate synthase (CS, EC 4.1.3.7), malate dehydrogenase (MDH, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), together with reduced root respiration, indicated that the Al-induced accumulation and subsequent exudation of citrate and malate were associated with both increased biosynthesis and reduced metabolism of citric and malic acid. Phosphorus-sufficient plants showed more pronounced aluminium-induced accumulation and exudation of organic anions than P-deficient plants. A divided root chamber experiment showed the necessity of direct contact between Al and roots to elicit exudation of organic anions. Prolonged exposure (10 days) to Al resulted in a decrease in the net exudation of citrate and malate, and the rate of decrease was much more rapid in P-deficient plants than in P-sufficient plants. It is concluded that P nutrition affects the level of Al-induced synthesis and exudation of organic anions. However, the mechanism needs further investigation.     

A comparative study on the aluminium- and copper-induced organic acid exudation from wheat roots

It is well established that aluminium (Al) and some heavy metals can elicit organic acid exudation from a range of species. In the present research we found that copper (Cu) can also induce organic acid exudation from the roots of wheat, rye, triticale, maize and soybean. Using intact wheat plants, we made a comparative study of Al- and Cu- induced organic acid exudation. In 5-day-old wheat seedlings, severe Cu stress (40 µ M CuCl₂) mainly induced the exudation of malate and citrate, and Al-tolerant genotypes could release significantly greater amounts of malate than Al-sensitive genotypes. The time course of the exudation of malate and citrate from the roots of 5-day-old seedlings of wheat (cv. Atlas) in 200 µ M AlCl₃ was similar to that in 40 µ M CuCl₂. In older wheat plants (15-day-old), moderate Cu stress (12 µ M CuCl₂) induced the exudation of large amounts of citrate and addition of Al or La sharply reduced Cu-induced citrate exudation, while Cu or La did not affect Al-induced malate efflux. When half of the root system of Atlas wheat was immersed in Al- or Cu-containing solution and the remaining half in Al- or Cu-free solution, organic acids were only exuded into the solution containing Al or Cu. This suggests that no long distance signal transport is involved in organic acid exudation induced by Al or Cu, and that direct contact of Al or Cu with plant roots is a prerequisite for the induction of organic acid exudation. The anion-channel inhibitor niflumic acid (NIF) significantly stimulated the exudation of both citrate and malate from 5-day-old wheat seedlings under severe Al or Cu stress. Our results suggest that Cu-induced organic acid efflux may be a common response, which may play a role in alleviating Cu toxicity in plants.     

Multiple Aluminum-Resistance Mechanisms in Wheat (Roles of Root Apical Phosphate and Malate Exudation)

Although it is well known that aluminum (Al) resistance in wheat (Triticum aestivum) is multigenic, physiological evidence for multiple mechanisms of Al resistance has not yet been documented. The role of root apical phosphate and malate exudation in Al resistance was investigated in two wheat cultivars (Al-resistant Atlas and Al-sensitive Scout) and two near-isogenic lines (Al-resistant ET3 and Al-sensitive ES3). In Atlas Al resistance is multigenic, whereas in ET3 resistance is conditioned by the single Alt1 locus. Based on root- growth experiments, Atlas was found to be 3-fold more resistant in 20 [mu]M Al than ET3. Root-exudation experiments were conducted under sterile conditions; a large malate efflux localized to the root apex was observed only in Atlas and in ET3 and only in the presence of Al (5 and 20 [mu]M). Furthermore, the more Al-resistant Atlas exhibited a constitutive phosphate release localized to the root apex. As predicted from the formation constants for the Al-malate and Al-phosphate complexes, the addition of either ligand to the root bathing solution alleviated Al inhibition of root growth in Al-sensitive Scout. These results provide physiological evidence that Al resistance in Atlas is conditioned by at least two genes. In addition to the alt locus that controls Al-induced malate release from the root apex, other genetic loci appear to control constitutive phosphate release from the apex. We suggest that both exudation processes act in concert to enhance Al exclusion and Al resistance in Atlas.     

Strategies to isolate transporters that facilitate organic anion efflux from plant roots

The efflux of organic anions from roots plays an important role in plant nutrition. The release of simple carboxylic anions such as citrate, malate and oxalate have been implicated in mechanisms of aluminium (Al) tolerance and improved acquisition of soil phosphorus. These metabolites are likely to cross cell membranes as multivalent anions and recent evidence indicates that anion-permeable channels facilitate this flow in the Al-dependent efflux of malate and citrate from wheat and maize, respectively. However, the genes encoding these anion channels, or any other protein that facilitates the release of citrate, malate or oxalate have not been isolated. This is an obstacle for the application of biotechnology to combat Al toxicity and to improve P-acquisition efficiency in plants. We discuss several strategies aimed at isolating genes that facilitate organic anion release from plant roots.     

Roles of rootstocks and scions in aluminum-tolerance of <Emphasis Type="Italic">Citrus</Emphasis>

Four scion-rootstock combination [i.e., X/X and X/SP, ‘Xuegan’ (Citrus sinensis) grafted on ‘Xugan’ and ‘Sour pummelo’ (Citrus grandis), respectively, and SP/X and SP/SP, ‘Sour pummelo’ grafted on ‘Xuegan’ and ‘Sour pummelo’, respectively] plants were treated for 18 weeks with 0 (?Al) or 1.2 mM AlCl₃·6H₂O (+Al). Thereafter, leaf, stem and root concentrations of phosphorus and aluminum (Al), leaf and root levels of organic acids (OAs), Al-induced release of OA anions (i.e., malate and citrate), photosynthesis and chlorophyll a fluorescence (OJIP) transients were measured. Al-induced decrease of photosynthesis and damage of photosynthetic electron transport chain were less pronounced in X/X and X/SP leaves than in SP/SP and SP/X leaves, which might be related with the higher Al-induced root efflux of OA anions and leaf P concentration. C. sinensis rootstock alleviated the influences of Al-toxicity on leaf photosynthetic electron transport chain by enhancing Al-induced release of root OA anions, hence lessening Al-induced photosynthesis inhibition in SP/X plants, while the reverse was the case for C. grandis rootstock in X/SP plants. In conclusion, the tolerance of grafted Citrus plants to Al depends on the scion as well as rootstock genotype, and the scion-rootstock interaction.     

Nitric oxide exacerbates Al-induced inhibition of root elongation in rice bean by affecting cell wall and plasma membrane properties

Aluminum (Al) toxicity is one of the most widespread problems for crop production on acid soils, and nitric oxide (NO) is a key signaling molecule involved in the mediation of various biotic and abiotic stresses in plants. Here we found that exogenous application of the NO donor sodium nitroprusside (SNP) exacerbated the inhibition of Al-induced root growth in rice bean [Vigna umbellata (Thunb.) Ohwi & Ohashi ‘Jiangnan’, Fabaceae]. This was accompanied by an increased accumulation of Al in the root apex. However, Al treatments had no effect on endogenous NO concentrations in root apices. These results indicate that a change in NO concentration is not the cause of Al-induced root growth inhibition and the adverse effect of SNP on Al-induced root growth inhibition should result from increased Al accumulation. Al could significantly induce citrate efflux but SNP had no effects on citrate efflux either in the absence or presence of Al. On the other hand, SNP pretreatment significantly increased Al-induced malondialdehyde accumulation and Evans Blue staining, indicating an intensification of the disruption of plasma membrane integrity. Furthermore, SNP pretreatment also caused greater induction of pectin methylesterase activity by Al, which could be the cause of the increased Al accumulation. Taken together, it is concluded that NO exacerbates Al-induced root growth inhibition by affecting cell wall and plasma membrane properties.     

Probing the “malate hypothesis” of differential aluminum tolerance in wheat by using other rhizotoxic ions as proxies for Al

An Al-stimulated efflux of malate from the root apex has been proposed as the primary mechanism whereby some wheat (Triticum aestivum L.) genotypes demonstrate marked resistance to the rhixotoxic metal Al. Appealing in its simplicity, the model has not been unequivocally validated, and suffers from some significant discrepancies between estimated, steady-state concentrations of malate at the root surface and concentrations that are necessary to explain the resistance of the superior genotypes. Using two other rhizotoxic ions that are also chelated by malate, Cu(II) and La(III), we specifically probed whether the quantities of malate released by tolerant genotypes could effectively detoxify Al. Experiments with exogenous additions of malate to solution showed that ≥200 μM malate is required to account for the difference between Scout 66 (Al-sensitive) and Atlas 66 (Al-tolerant) wheats, and that this level of malate can also partially alleviate the toxicities of Cu and La. When simultaneously exposed to a mildly rhizotoxic level of Al (25 μM) to induce malate efflux, Atlas exhibited a pronounced reduction in sensitivity to Cu. When, La was used as the proxy ion, however, no such Al-induced tolerance to La was observed, a result that refutes the significance of malate efflux to Al tolerance. Additional experiments using Al, Cu, and La in combination suggested that a trivalent ion can alleviate Cu toxicity directly (i.e. via competition for apoplastic binding), providing an alternative explanation for the ability of Al to detoxify Cu in Atlas. Using a weight-of-evidence approach, we argue that malate efflux plays at most a minor role in the differential Al tolerance of wheat, and that a more integrative, multifaceted model of tolerance is needed. Received: 14 August 1997 / Accepted: 26 November 1997     

Apoplastic binding of aluminum is involved in silicon-induced amelioration of aluminum toxicity in maize

The alleviating effect of silicon (Si) supply on aluminum (Al) toxicity was suggested to be based on ex or in planta mechanisms. In our experiments with the Al-sensitive maize (Zea mays) cultivar Lixis, Si treatment but not Si pretreatment ameliorated Al-induced root injury as revealed by less root-growth inhibition and callose formation. Si treatment did not affect monomeric Al concentrations in the nutrient solution, suggesting an in planta effect of Si on Al resistance. A fractionated analysis of Si and Al in the 1-cm root apices revealed that more than 85% of the root-tip Al was bound in the cell wall. Al contents in the apoplastic sap, the symplastic sap, and the cell wall did not differ between -Si and +Si plants. Si did not affect the Al-induced exudation of organic acid anions and phenols from the root apices. However, Al treatment greatly enhanced Si accumulation in the cell wall fraction, reducing the mobility of apoplastic Al. From our data we conclude that Si treatment leads to the formation of hydroxyaluminumsilicates in the apoplast of the root apex, thus detoxifying Al.     

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.     

Recent progress in the research of external Al detoxification in higher plants: a minireview

Aluminum (Al) is highly toxic to plant growth. The toxicity is characterized by rapid inhibition of root elongation. However, some plant species and cultivars have evolved some mechanisms for detoxifying Al both internally and externally. In this review, the recent progress made in the research of external detoxification of Al is described. Accumulating evidence has shown that organic acids play an important role in the detoxification of Al. Some plant species and cultivars respond to Al by secreting citrate, malate or oxalate from the roots. Recently, the anion channel of malate and citrate in the plasma membrane has been characterized and a gene encoding the malate channel has been cloned. The metabolism of organic acids seems to be poorly correlated with the Al-induced secretion of organic acid anions. A number of QTLs (quantitative trait loci) for Al resistance have been identified in rice, Arabidopsis, and other species. Transgenic plants with enhanced resistance to Al have also been reported, but introduction of multiple genes may be required to gain high Al resistance in future.     

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.     

Functional characterization of plasma membrane-localized organic acid transporter (CsALMT1) involved in aluminum tolerance in <Emphasis Type="Italic">Camelina sativa</Emphasis> L.

Aluminum (Al) toxicity is a major limiting factor that inhibits root elongation and decreases crop production in acidic soils. The symptoms of inhibited root growth include a reduced uptake of nutrients because the roots become stubby and brittle. The release of organic anions from roots can protect a plant from Al toxicity. The mechanism relies on the efflux of organic anions, such as malate or citrate, which protect roots by chelating the Al³⁺. In this study, homologs of TaALMT1, a Camelina gene that encodes an aluminum-activated malate transporter, were investigated. The expression of this gene was induced by Al in the root, but not in the shoots. Using green fluorescent protein (GFP) fusion constructs and Western-blot analysis, we observed that CsALMT1 was localized in the plasma membrane. Also, to determine the degree to which Al tolerance was affected by malate secretion in Camelina root, we generated CsALMT1 overexpressing plants. CsALMT1 overexpressing transgenic plants showed a higher root elongation rate than the wild-type plant. Damaged cell staining analysis by hematoxylin under 25 µM Al treatment for 2, 4, and 6 h showed a pattern of less damage in CsALMT1 transgenic plants than in wild-type plant, especially in the root elongation zone. Furthermore, the rate of increase of secretion of organic acid in overexpressed plants after Al treatment was higher than that in the wild-type plant. In addition, in the Al-specific dye morin staining on root protoplast under 50 µM Al treatment, less Al accumulation was observed in the CsALMT1 transgenic plants than in the wild-type plant. The Al contents in the roots of the transgenic plants were at a lower level than those in the wild-type plant. These results show that the overexpression of CsALMT1 improves Al tolerance by increasing the release of malate from the root to the soil and, thereby, detoxifies the Al³⁺.     

Aluminum Interactions with Voltage-Dependent Calcium Transport in Plasma Membrane Vesicles Isolated from Roots of Aluminum-Sensitive and -Resistant Wheat Cultivars

The role of Al interactions with root-cell plasma membrane (PM) Ca2+ channels in Al toxicity and resistance was studied. The experimental approach involved the imposition of a transmembrane electrical potential (via K+ diffusion) in right-side-out PM vesicles derived from roots of two wheat (Triticum aestivum L.) cultivars (Al-sensitive Scout 66 and Al-resistant Atlas 66). We previously used this technique to characterize a voltage-dependent Ca2+ channel in the wheat root PM (J.W. Huang, D.L. Grunes, L.V. Kochian [1994] Proc Natl Acad Sci USA 91: 3473-3477). We found that Al3+ effectively blocked this PM Ca2+ channel; however, Al3+ blocked this Ca2+ channel equally well in both the Al-sensitive and -resistant cultivars. It was found that the differential genotypic sensitivity of this Ca2+ transport system to Al in intact roots versus isolated PM vesicles was due to Al-induced malate exudation localized to the root apex in Al-resistant Atlas but not in Al-sensitive Scout. Because malate can effectively chelate Al3+ in the rhizosphere and exclude it from the root apex, the differential sensitivity of Ca2+ influx to Al in intact roots of Al-resistant versus Al-sensitive wheat cultivars is probably due to the maintenance of lower Al3+ activities in the root apical rhizosphere of the resistant cultivar.     

Aluminum activates a citrate-permeable anion channel in the aluminum-sensitive zone of the maize root apex. A comparison between an aluminum- sensitive and an aluminum-resistant cultivar

In search for the cellular and molecular basis for differences in aluminum (Al) resistance between maize (Zea mays) cultivars we applied the patch-clamp technique to protoplasts isolated from the apical root cortex of two maize cultivars differing in Al resistance. Measurements were performed on protoplasts from two apical root zones: The 1- to 2-mm zone (DTZ), described as most Al-sensitive, and the main elongation zone (3-5 mm), the site of Al-induced inhibition of cell elongation. Al stimulated citrate and malate efflux from intact root apices, revealing cultivar differences. In the elongation zone, anion channels were not observed in the absence and presence of Al. Preincubation of intact roots with 90 microM Al for 1 h induced a citrate- and malate-permeable, large conductance anion channel in 80% of the DTZ protoplasts from the resistant cultivar, but only 30% from the sensitive cultivar. When Al was applied to the protoplasts in the whole-cell configuration, anion currents were elicited within 10 min in the resistant cultivar only. La3+ was not able to replace or counteract with Al3+ in the activation of this channel. In the presence of the anion-channel blockers, niflumic acid and 4, 4'-dinitrostilbene-2, 2'disulfonic acid, anion currents as well as exudation rates were strongly inhibited. Application of cycloheximide did not affect the Al response, suggesting that the channel is activated through post-translational modifications. We propose that the Al-activated large anion channel described here contributes to enhanced genotypical Al resistance by facilitating the exudation of organic acid anions from the DTZ of the maize root apex.     

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.