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.     

Probing the role of calmodulin in Al toxicity in maize

The role of calmodulin on Al toxicity was studied in two maize (Zea mays L.) inbred lines, Cat 100-6 (Al-tolerant) and S 1587-17 (Al-sensitive). Increasing levels of Al induced the release of malate at similar rate by roots of both genotypes, while the exudation of citrate, a stronger Al-binding compound, was 3.5 times higher in Cat 100-6 seedlings exposed to 16.2x10(-6) Al(3+) activity. The calmodulin inhibitor trifluoperazine significantly reduced the root growth in both genotypes, mimicking the main effect of Al. However, when Cat 100-6 and S 1587-17 seedlings were challenged with Al in conjunction with trifluoperazine, no further reduction in root growth or any other effect of Al toxicity was observed. The rate of Al-induced citrate exudation by both genotypes was not affected by treatment with trifluoperazine or calmidazolium, another calmodulin inhibitor. The Al(3+) interaction with cytoplasmic CaM was estimated using models for the binding of Al(3+) and Mg(2+) with CaM and physiological concentrations of citrate, CaM, InsP(3), ATP, ADP, Al(3+) and Mg(2+). In this simulation, Al(3+) associated with citrate and InsP(3), but not with CaM. We conclude that calmodulin is not relevant to the physiological processes leading to the Al tolerance in maize, nor is it a primary target for Al toxicity.     

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.     

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.     

Citrate transporters play an important role in regulating aluminum-induced citrate secretion in Glycine max

To further understand the process of Al-induced citrate secretion from soybean roots, the effect of protein synthesis inhibitor, anion channel blockers, and citrate carrier inhibitors on Al-induced citrate exudation was investigated in Al-resistant soybean cultivar PI 416937. Citrate exudation from roots increased with the increase of Al concentration from 10 to 50 μM and initiated after 4 h of Al exposure. Protein synthesis inhibitor, cycloheximide (CHM; 25 μM) completely inhibited Al-induced citrate secretion during 12-h exposure, suggesting that novel protein synthesis was necessary in Al-induced citrate efflux. Also both anion channel blocker anthracene-9-carboxylic acid (A-9-C) and citrate carrier inhibitor mersalyl acid (Mersalyl) significantly reduced citrate secretion, suggesting that both anion channels in plasma membrane and citrate carriers in mitochondria membrane were the rate limiting factors of Al dependent citrate release. However, Al-induced citrate secretion was insensitive to anion channel blockers phenylglyoxal (PG), 4,4′-diisothiocyanostibene-2,2′-disulfonat (DIDS) and citrate carrier inhibitor pyridoxal 5′-P (PP).     

Differential Al resistance and citrate secretion in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

While barley ( Hordeum vulgare L.) is the most sensitive species to Al toxicity among small-grain crops, variation in Al resistance between cultivars does exist. We examined the mechanism responsible for differential Al resistance in 21 barley varieties. Citrate was secreted from the roots in response to Al stress. A positive correlation between citrate secretion and Al resistance [(root elongation with Al)/(root elongation without Al)] and a negative correlation between citrate secretion and Al content of root apices, were obtained, suggesting that citrate secretion from the root apices plays an important role in excluding Al and thereby detoxifying Al. The Al-induced secretion of citrate was characterized using an Al-resistant variety (Sigurdkorn) and an Al-sensitive variety (Kearney). In Sigurdkorn, Al-induced secretion of citrate occurred within 20 min, and the secretion did not increase with increasing external Al concentration. The Al-induced citrate secretion ceased at low temperature (6 degrees C) and was inhibited by anion-channel inhibitors. Internal citrate content of root apices was increased by Al exposure in Sigurdkorn, but was not affected in Kearney. The activity of citrate synthase was unaffected by Al in both Al-resistant and Al-sensitive varieties. The secretion rate of organic acid anions from barley was the lowest among wheat, rye and triticale.     

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.     

Isolation and characterization of a rice mutant hypersensitive to Al

Rice (Oryza sativa L.) is a highly Al-resistant species among small grain crops, but the mechanism responsible for the high Al resistance has not been elucidated. In this study, rice mutants sensitive to Al were isolated from M(3) lines derived from an Al-resistant cultivar, Koshihikari, irradiated with gamma-rays. Relative root elongation was used as a parameter for evaluating Al resistance. After initial screening plus two rounds of confirmatory testing, a mutant (als1) was isolated from a total of 560 lines. This mutant showed a phenotype similar to the wild-type plant in the absence of Al. However, in the presence of 10 microM Al, root elongation was inhibited 70% in the mutant, but only 8% in the wild-type plant. The mutant also showed poorer root growth in acid soil. The Al content of root apices (0-1 cm) was much lower in the wild-type plant. The sensitivity to other metals including Cd and La did not differ between the mutant and the wild-type plants. A small amount of citrate was secreted from the roots of the mutant in response to Al stress, but there was no difference from that secreted by the wild-type plant. Genetic analysis of F(2) populations between als1 and wild-type plants showed that the Al-resistant seedlings and Al-sensitive seedlings segregated at a 3 : 1 ratio, indicating that the high sensitivity to Al in als1 is controlled by a single recessive gene. The gene was mapped to the long arm of chromosome 6, flanked by InDel markers MaOs0619 and MaOs0615.     

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.     

Aluminum resistance of cowpea as affected by phosphorus-deficiency stress

Plants growing in acid soils suffer both phosphorus (P) deficiency and aluminum (Al) toxicity stresses. Selection of genotypes for adaptation to either P deficiency or Al toxicity has sometimes been unsuccessful because these two soil factors often interact. Two experiments were conducted to evaluate eight cowpea genotypes for Al resistance and to study the combined effect of P deficiency and Al toxicity stress on growth, P uptake, and organic acid anion exudation of two genotypes of contrasting Al resistance selected from the first experiment. Relative root inhibition by 30 μM Al ranged from 14% to 60% and differed significantly among the genotypes. Al significantly induced callose formation, particularly in Al-sensitive genotypes. P accumulation was significantly reduced (28% and 95%) by Al application for both the Al-resistant and the Al-sensitive genotypes. Al supply significantly enhanced malate release of root apices of both genotypes. However, the exudation rate was significantly higher in the Al-resistant genotype. P deprivation induced an enhanced malate exudation in the presence of Al only in the Al-resistant genotype IT89KD-391. Citrate exudation rate of the root apices was lower than malate exudation by a factor of about 10, and was primarily enhanced by P deficiency in both genotypes. Al treatment further enhanced citrate exudation in P-sufficient, but not in P-deficient plants. The level of citrate exudation was consistently higher in the Al-resistant genotype IT89KD-391 particularly in presence of Al.It is concluded that the Al-resistant genotype is better adapted to acid Al-toxic and P-deficient soils than the Al-sensitive genotype since both malate and citrate exudation were more enhanced by combined Al and P-deficiency stresses.     

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.     

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.     

Secretion of citrate from roots in response to aluminum and low phosphorus stresses in Stylosanthes

Excess aluminum (Al) ions and phosphorus (P) deficiency are the key factors that limit plant growth in acid soils. Secretion of organic acids (OA) from roots has been proposed as an Al-resistance mechanism. Nonetheless, the correlation between Al resistance and this mechanism has not been tested beyond a very small number of Al-resistant and Al-sensitive genotypes. To elucidate the mechanisms responsible for plant adaptability to acid soils, we studied the secretion of OA from roots of Stylosanthes in response to high-Al and low-P stresses using six different genotypes. Relative root inhibition by 50?µM Al ranged from 25–71% and differed significantly among six Stylosanthes genotypes. Al treatment induced the secretion of citrate from the roots of Stylosanthes seedling in a dose- and time-dependent manner. Moreover, the secretion rate was significantly higher in the Al-resistant genotype. On the other hand, inhibition of Al-induced citrate secretion by phenylisothiocyanate or 9-anthracenecarboxylic acid resulted in an increase in Al content in Stylosanthes root apices. P deficiency also induced citrate secretion from Stylosanthes seedling roots. Furthermore, citrate secretion was much more robust with exposure to both excess-Al and P-deficiency stresses than under either stress alone. Unlike Al-induced citrate secretion, which was rapid, low-P-induced secretion was a slow process, with significant increases in secretion only becoming evident after 6 d of treatment with free phosphate. The lag between treatment with Al and citrate secretion was approximately 4 h. These results suggest that the secretion of citrate is a mechanism for resistance to both excess-Al and low-P stresses in Stylosanthes.     

Aluminum-induced oxidative stress in maize

The relation between Al-toxicity and oxidative stress was studied for two inbred lines of maize (Zea mays L.), Cat100-6 (Al-tolerant) and S1587-17 (Al-sensitive). Peroxidase (PX), catalase (CAT) and superoxide dismutase (SOD) activities were determined in root tips of both lines, exposed to different Al(3+) concentrations and times of exposure. No increases were observed in CAT activities in either line, although SOD and PX were found to be 1.7 and 2.0 times greater than initial levels, respectively, in sensitive maize treated with 36 microM of Al(3+) for 48 h. The results indicate that Al(3+) induces the dose- and time dependent formation of reactive oxygen species (ROS) and subsequent protein oxidation in S1587-17, although not in Cat100-6. After exposure to 36 microM of Al(3+) for 48 h, the formation of 20+/-2 nmol of carbonyls per mg of protein was observed in S1587-17. The onset of protein oxidation took place after the drop of the relative root growth observed in the sensitive line, indicating that oxidative stress is not the primary cause of root growth inhibition. The presence of Al(3+) did not induce lipid peroxidation in either lines, contrasting with the observations in other species. These results, in conjunction with the data presented in the literature, indicate that oxidative stress caused by Al may harm several components of the cell, depending on the plant species. Moreover, Al(3+) treatment and oxidative stress in the sensitive maize line induced cell death in root tip cells, an event revealed by the high chromatin fragmentation detected by TUNEL analysis.     

Immobilization of aluminum with phosphorus in roots is associated with high aluminum resistance in buckwheat

Oxalic acid secretion from roots is considered to be an important mechanism for aluminum (Al) resistance in buckwheat (Fygopyrum esculentum Moench). Nonetheless, only a single Al-resistant buckwheat cultivar was used to investigate the significance of oxalic acid in detoxifying Al. In this study, we investigated two buckwheat cultivars, Jiangxi (Al resistant) and Shanxi (Al sensitive), which showed significant variation in their resistance to Al stress. In the presence of 0 to 100 microM Al, the inhibition of root elongation was greater in Shanxi than that in Jiangxi, and the Al content of root apices (0-10 mm) was much lower in Jiangxi. However, the dependence of oxalic acid secretion on external Al concentration and the time course for secretion were similar in both cultivars. Furthermore, the variation in Al-induced oxalic acid efflux along the root was similar, showing a 10-fold greater efflux from the apical 0- to 5-mm region than from the 5- to 10-mm region. These results suggest that both Shanxi and Jiangxi possess an equal capacity for Al-dependent oxalic acid secretion. Another two potential Al resistance mechanisms, i.e. Al-induced alkalinization of rhizosphere pH and root inorganic phosphate release, were also not involved in their differential Al resistance. However, after longer treatments in Al (10 d), the concentrations of phosphorus and Al in the roots of the Al-resistant cultivar Jiangxi were significantly higher than those in Shanxi. Furthermore, more Al was localized in the cell walls of the resistant cultivar. All these results suggest that while Al-dependent oxalic acid secretion might contribute to the overall high resistance to Al stress of buckwheat, this response cannot explain the variation in tolerance between these two cultivars. We present evidence suggesting the greater Al resistance in buckwheat is further related to the immobilization and detoxification of Al by phosphorus in the root tissues.     

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.     

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.     

Cell-wall pectin and its degree of methylation in the maize root-apex: significance for genotypic differences in aluminium resistance

Cell-wall (CW) pectin content and its degree of methylation in root apices of selected maize cultivars were studied in relation to genotypic Al resistance. Maize cultivars differing in Al resistance were grown in nutrient solution treated with or without Al, and pectin content of the root tips was determined. Control plants did not differ in pectin content in the 5 mm root apex. Al treatment increased the pectin content of the root apex in all cultivars but more prominently in the Al-sensitive cultivars. Pectin and Al contents in 1 mm root sections decreased from the apex to the 3–4 mm zone. Pectin contents of the apical root sections were consistently higher although significantly different only in the 1–2 mm zone in the Al-sensitive cv Lixis. Al contents in most root sections were significantly higher in cv Lixis than in Al-resistant cv ATP-Y. Localization of pectins by immunofluorescence revealed that Al-sensitive cv. Lixis has a higher proportion of low-methylated pectin and thus a higher negativity of the cell wall than Al-resistant cv ATP-Y. This is in agreement with the higher Al content and Al sensitivity of cv Lixis. It is concluded that differences in CW pectin and its degree of methylation contribute to genotypic differences in Al resistance in maize in addition to the release of organic acid anions previously reported.     

Evaluating the role of root citrate exudation as a mechanism of aluminium resistance in maize genotypes

Organic anion exudation by roots as a mechanism of aluminium (Al) resistance has been intensively studied lately. In the present study, we evaluated qualitative and quantitative aspects of root exudation of organic anions in maize genotypes of distinct sensitivity to Al in response to Al exposure. Maize seedlings were grown axenically in nutrient solution and root exudates were collected along the whole seminal root axis for a short period (4 h) using a divided-root-chamber technique. In root exudates collected from 10-mm long root apices, citrate accounted for 67% of the total organic anions found, followed by malate (29%), trans-aconitate (3%), fumarate (<1%), and cis-aconitate (1%). Rates of citrate exudation from root apices of two genotypes with differential resistance to Al were consistently higher in the Al resistant one, differing by a factor of 1.7 – 3.0 across a range of external Al concentrations. Furthermore, relative Al resistance of eight maize genotypes correlated significantly well with their citrate exudation rate measured at 40 M Al. Higher exudation rates were accompanied by a less inhibited root elongation. The exudation of citrate along the longitudinal axis of fully developed seminal roots showed a particular pattern: citrate was exuded mainly in the regions of root apices, either belonging to the main root or to the lateral roots in the most basal part of the main root. The involvement of citrate in a mechanism of Al resistance is evaluated in terms of protection of the root from the effects of excess Al on root elongation and on nutrient uptake along a root axis showing distinct sites of citrate exudation.     

Differential Al resistance and citrate secretion in the tap and basal roots of common bean seedlings

The common bean root system is composed of several types of root (e.g. tap, basal, and lateral roots), whose physiological functions may be of great difference. However, we do not know if the root system of common bean differs in organic acid secretion and thus aluminium (Al) resistance. In the present study, the tap and basal roots of three common bean genotypes (i.e. G19842, SQ12 and BAT881) from different origins were compared for their citrate secretion and Al resistance. Grown in a simple solution containing 30 µM Al³⁺ for 24 h, genotype G19842 maintained 75% relative tap root length [RTRL = (tap root length with Al)/(tap root length without Al)], 48% relative basal root length [RBRL = (basal root length with Al)/ (basal root length without Al)], genotype SQ12 maintained 62% RTRL and 57% RBRL, while BAT881 only maintained 31% RTRL and 19% RBRL, indicating differential sensitivity of bean genotypes and root types to Al stress. The amounts of Al‐induced citrate secretion by the tap/basal roots were 9.8/5.1, 8.2/5.9 and 5.4/4.1 nmol cm^?2 FR (fresh root) [12 h]^?1 for G19842, SQ12 and BAT881, respectively, indicating that both bean genotypes and root types differ in organic acid secretion. In G19842, the root surface area was 25% higher in tap root apex than that in basal root apex, and the amounts of citrate secretion per unit surface area and per root apex were 29 and 62% higher in tap root apex than those in basal root apex, respectively, suggesting that the higher citrate secretion in the tap root apex could be attributed to the larger surface area and the higher secretion activity. Stronger inhibition of Al‐induced citrate secretion in the basal than tap roots by anthracene‐9‐carboxylic acid, an inhibitor of anion channel and K‐252a, a broad range inhibitor of protein kinase may also imply the differences in the activities of anion channels and K‐252a‐sensitive protein kinases on the plasma membrane between the tap and basal roots, resulting in differential citrate secretion. We propose that the higher Al resistance in the tap root than in basal roots might be attributed to both greater number and higher activity of the anion channels in the former, thus allowing more citrate secretion in this root type.