Mechanisms of Aluminum Tolerance in Wheat : An Investigation of Genotypic Differences in Rhizosphere pH, K, and H Transport, and Root-Cell Membrane Potentials

Control of rhizosphere pH and exclusion of Al by the plasma membrane have been hypothesized as possible mechanisms for Al tolerance. To test primarily the rhizosphere pH hypothesis, wheat cultivars (Triticum aestivum L. `Atlas 66' and `Scout'), which differ in Al tolerance, were grown in either complete nutrient solution, or 0.6 millimolar CaSO₄, with and without Al at pH 4.50. A microelectrode system was used to simultaneously measure rhizosphere pH, K⁺, and H⁺ fluxes, and membrane potentials (E_m) along the root at various distances from the root apex. In complete nutrient solution, the rhizosphere pH associated with mature root cells (measured 10-40 millimeters from the root apex) of Al-tolerant `Atlas 66' was slightly higher than that of the bulk solution, whereas roots of Al-sensitive `Scout' caused a very small decrease in the rhizosphere pH. In CaSO₄ solution, no significant differences in rhizosphere pH were found between wheat cultivars, while differential Al tolerance was still observed, indicating that the rhizosphere pH associated with mature root tissue is not directly involved in the mechanism(s) of differential Al tolerance. In Al-tolerant `Atlas 66', growth in a CaSO<sub>4</sub> solution with 5 micromolar Al (pH 4.50) had little effect on net K<sup>+</sup> influx, H<sup>+</sup> efflux, and root-cell membrane potential measured in cells of mature root tissue (from 10-40 mm back from apex). However, in Al-sensitive `Scout', Al treatment caused a dramatic inhibition of K⁺ influx and both a moderate reduction of H⁺ efflux and depolarization of the membrane potential. These results demonstrate that increased Al tolerance in wheat is associated with the increased ability of the tolerant plant to maintain normal ion fluxes and membrane potentials across the plasmalemma of root cells in the presence of Al.     

Aluminium and calcium transport interactions in intact roots and root plasmalemma vesicles from aluminium-sensitive and tolerant wheat cultivars

Recent research from our laboratory indicates that aluminium (Al) and calcium (Ca) transport interactions may play an important role in the mechanisms of Al phytotoxicity. In this study, we investigated the effects of Al on Ca²⁺ transport in intact roots of winter wheat (Triticum aestivum L.) cultivars (Al-tolerant Atlas 66 and Al-sensitive Scout 66). We used both a vibrating Ca²⁺-microelectrode technique and ⁴⁵Ca²⁺ to monitor Ca²⁺ influx in intact roots. Root apical Ca²⁺ uptake was immediately inhibited, when roots were exposed to Al levels that ultimately decreased root growth in Al-sensitive Scout 66. The Al-tolerant cultivar was able to resist this Al inhibition of Ca²⁺ uptake, and to resist Al inhibition of ⁴⁵Ca²⁺ translocation from roots to shoots. We also studied Ca²⁺ transport in right-side out plasmalemma vesicles isolated from roots of Al-sensitive and tolerant wheat cultivars. Calcium influx into the vesicles was mediated by a voltage-gated Ca²⁺ channel. Aluminium blocks the Ca²⁺ channel equally well in the plasmalemma vesicles isolated from Al-sensitive and Al-tolerant wheat roots. The results indicate that the differential response observed in intact roots is not due to differences in Ca²⁺ channels. The Al-tolerant wheat cultivar may have an ability to reduce Al³⁺ activity in the rhizosphere, thus reducing the Al-inhibition of Ca²⁺ influx.     

Aluminum effects on calcium fluxes at the root apex of aluminum-tolerant and aluminum-sensitive wheat cultivars

The role of Ca²⁺ transport in the mechanism of Al toxicity was investigated, using a Ca²⁺-selective microelectrode system to study Al effects on root apical Ca²⁺ fluxes in two wheat (Triticum aestivum L.) cultivars: Al-tolerant Atlas 66 and Al-sensitive Scout 66. Intact 3-day-old low-salt-grown (100 micromolar CaCl₂, pH 4.5) wheat seedlings were used, and it was found that both cultivars maintained similar rates of net Ca²⁺ uptake in the absence of Al. Addition of Al concentrations that were toxic to Scout (5-20 micromolar AlCl₃) immediately and dramatically inhibited Ca²⁺ uptake in Scout, whereas Ca²⁺ transport in Atlas was relatively unaffected. The Al-induced inhibition of Ca²⁺ uptake in Scout 66 was rapidly reversed following removal of Al from the solution bathing the roots. Similar studies with morphologically intact root cell wall preparations indicated that the Al effects did not involve Al-Ca interactions in the cell wall. These results suggest that Al inhibits Ca²⁺ influx across the root plasmalemma, possibly via blockage of calcium channels. The differential effect of Al on Ca²⁺ transport in Al-sensitive Scout and Al-tolerant Atlas suggests that Al blockage of Ca²⁺ channels could play a role in the cellular mechanism of Al toxicity in higher plants.     

Aluminum Effects on Calcium (45Ca2+) Translocation in Aluminum-Tolerant and Aluminum-Sensitive Wheat (Triticum aestivum L.) Cultivars (Differential Responses of the Root Apex versus Mature Root Regions)

The influence of Al exposure on long-distance Ca2+ translocation from specific root zones (root apex or mature root) to the shoot was studied in intact seedlings of winter wheat (Triticum aestivum L.) cultivars (Al-tolerant Atlas 66 and Al-sensitive Scout 66). Seedlings were grown in 100 [mu]M CaCl2 solution (pH 4.5) for 3 d. Subsequently, a divided chamber technique using 45Ca2+-labeled solutions (100 [mu]M CaCl2 with or without 5 or 20 [mu]M AlCl3, pH 4.5) was used to study Ca2+ translocation from either the terminal 5 to 10 mm of the root or a 10-mm region of intact root approximately 50 mm behind the root apex. The Al concentrations used, which were toxic to Scout 66, caused a significant inhibition of Ca2+ translocation from the apical region of Scout 66 roots. The same Al exposures had a much smaller effect on root apical Ca2+ translocation in Atlas 66. When a 10-mm region of the mature root was exposed to 45Ca2+, smaller genotypic differences in the Al effects effects on Ca2+ translocation were observed, because the degree of Al-induced inhibition of Ca2+ translocation was less than that at the root apex. Exposure of the root apex to Al inhibited root elongation by 70 to 99% in Scout 66 but had a lesser effect (less than 40% inhibition) in Atlas 66. When a mature root region was exposed to Al, root elongation was not significantly affected in either cultivar. These results demonstrate that genotypic differences in Al-induced inhibition of Ca2+ translocation and root growth are localized primarily in the root apex. The pattern of Ca2+ translocation within the intact root was mainly basipetal, with most of the absorbed Ca2+ translocated toward the shoot. A small amount of acropetal Ca2+ translocation from the mature root regions to the apex was also observed, which accounted for less than 5% of the total Ca2+ translocation within the entire root. Because Ca2+ translocation toward the root apex is limited, most of the Ca2+ needed for normal cellular function in the apex must be absorbed from the external solution. Thus, continuous Al disruption of Ca2+ absorption into cells of the root apex could alter Ca2+ nutrition and homeostasis in these cells and could play a pivotal role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars.     

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.     

Organic acid exudation as an aluminum-tolerance mechanism in maize (Zea mays L.)

In this study, the role of root organic acid synthesis and exudation in the mechanism of aluminum tolerance was examined in Al-tolerant (South American 3) and Al-sensitive (Tuxpeño and South American 5) maize genotypes. In a growth solution containing 6 M Al³⁺, Tuxpeño and South American 5 were found to be two- and threefold more sensitive to Al than South American 3. Root organic acid content and organic acid exudation from the entire root system into the bulk solution were investigated via high-performance liquid chromatographic analysis while exudates collected separately from the root apex or a mature root region (using a dividedroot-chamber technique) were analyzed with a more-sensitive ion chromatography system. In both the Al-tolerant and Al-sensitive lines, Al treatment significantly increased the total root content of organic acids, which was likely the result of Al stress and not the cause of the observed differential Al tolerance. In the absence of Al, small amounts of citrate were exuded into the solution bathing the roots. Aluminum exposure triggered a stimulation of citrate release in the Al-tolerant but not in the Al-sensitive genotypes; this response was localized to the root apex of the Al-tolerant genotype. Additionally, Al exposure triggered the release of phosphate from the root apex of the Al-tolerant genotype. The same solution Al³⁺ activity that elicited the maximum difference in Al sensitivity between Al-tolerant and Al-sensitive genotypes also triggered maximal citrate release from the root apex of the Al-tolerant line. The significance of citrate as a potential detoxifier for aluminum is discussed. It is concluded that organic acid release by the root apex could be an important aspect of Al tolerance in maize.Abbreviations SA3 South American 3, an Al-tolerant maize cultivar - SA5 South American 5, an Al-sensitive maize cultivar The authors would like to express their appreciation to Drs. John Thompson, Ross Welch and Mr. Stephen Schaefer for their training and guidance in the use of the chromatography systems. This work was supported by a Swiss National Science Foundation Fellowship to Didier Pellet, and U.S. Department of Agriculture/National Research Initiative Competitive Grant 93-37100-8874 to Leon Kochian. We would also like to thank Drs. S. Pandey and E. Ceballos from the CIMMYT Regional office at CIAT Cali, Colombia for providing seed for the maize varieties and inbred line.     

The effect of aluminium on polypeptide pattern of cell wall proteins isolated from the roots of Al-sensitive and Al-resistant barley cultivars

Accumulation of some proteins isolated from the cell wall of roots of the Al-sensitive (Alfor) and the Al-resistant (Bavaria) barley cultivars were followed during treatment with different Al³⁺ concentrations, pH changes of the root medium, and several heavy metals (Cu²⁺, Cd²⁺, Co²⁺). SDS-PAGE analysis revealed an Al-induced accumulation of polypeptides with molecular mass of 14, and 16 kDa and a group of polypeptides around 27 kDa. The accumulation pattern of Al-induced polypeptides was very similar in both cultivars but in the Al-resistant Bavaria it was induced at lower Al concentration and earlier than it was in the Al-sensitive cultivar Alfor. Changes in pH values of root medium (pH 3.5–6.5) did not show any effect on the accumulation of Al-induced cell wall polypeptides either in Al-sensitive or in Al-tolerant barley cultivar. Heavy metals (Cu, Cd, and Co) at concentration of 10 μM resulted in similar accumulation of individual polypeptides as we found after Al treatment. In comparison to Al, quantitative differences in polypeptides accumulation induced by Cu, Cd and Co were less expressed that of Al treatment. More pronounced accumulation and earlier induction of individual cell wall polypeptides in roots of Al-resistant barley cultivar than in Al-sensitive, might indicate some possible role of these polypeptides in plant resistance to Al stress.     

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.     

Inhibition of growth and development of root border cells in wheat by Al

The production and development of border cells vary with genotype, and they are released in wheat at an earlier stage of root development than other species studied so far. No significant difference was observed in the maximum number of border cells between Al-tolerant (Atlas 66) and Al-sensitive (Scout 66) cultivars in the absence of Al treatment. Al seriously inhibited the production and release of border cells, resulting in clumping of border cells in Scout 66, but less clustering in Atlas 66. The number of border cells released from roots treated with Al is significantly less than that from roots grown without Al treatment. Al treatment induced the death of detached border cells in vitro and they were killed by a 20-h treatment with 25 µ m Al. No significant difference in survival percentage of detached border cells was observed between Atlas 66 and Scout 66, regardless of the presence or absence of Al. The removal of border cells from root tips of both Atlas 66 and Scout 66 enhanced the Al-induced inhibition of root elongation concomitant with increased Al accumulation in the root. These results suggest that border cells adhered to the root tips play a potential role in the protection of root from Al injury in wheat.     

Interaction between Aluminum Toxicity and Calcium Uptake at the Root Apex in Near-Isogenic Lines of Wheat (Triticum aestivum L.) Differing in Aluminum Tolerance

Aluminum (Al) is toxic to plants at pH < 5.0 and can begin to inhibit root growth within 3 h in solution experiments. The mechanism by which this occurs is unclear. Disruption of calcium (Ca) uptake by Al has long been considered a possible cause of toxicity, and recent work with wheat (Triticum aestivum L. Thell) has demonstrated that Ca uptake at the root apex in an Al-sensitive cultivar (Scout 66) was inhibited more than in a tolerant cultivar (Atlas 66) (J.W. Huang, J.E. Shaff, D.L. Grunes, L.V. Kochian [1992] Plant Physiol 98: 230-237). We investigated this interaction further in wheat by measuring root growth and Ca uptake in three separate pairs of near-isogenic lines within which plants exhibit differential sensitivity to Al. The vibrating calcium-selective microelectrode technique was used to estimate net Ca uptake at the root apex of 6-d-old seedlings. Following the addition of 20 or 50 [mu]M AlCl3, exchange of Ca for Al in the root apoplasm caused a net Ca efflux from the root for up to 10 min. After 40 min of exposure to 50 [mu]M Al, cell wall exchange had ceased, and Ca uptake in the Al-sensitive plants of the near-isogenic lines was inhibited, whereas in the tolerant plants it was either unaffected or stimulated. This provides a general correlation between the inhibition of growth by Al and the reduction in Ca influx and adds some support to the hypothesis that a Ca/Al interaction may be involved in the primary mechanism of Al toxicity in roots. In some treatments, however, Al was able to inhibit root growth significantly without affecting net Ca influx. This suggests that the correlation between inhibition of Ca uptake and the reduction in root growth may not be a mechanistic association. The inhibition of Ca uptake by Al is discussed, and we speculate about possible mechanisms of tolerance.     

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex

The effects of aluminum on the concentration-dependent kinetics of Ca²⁺ uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 M CaCl₂ solution (pH 4.5) for 3 d. Subsequently, net Ca²⁺ fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca²⁺-selective microelectrode. The kinetics of Ca²⁺ uptake into cells of the root apex, for external Ca²⁺ concentrations from 20 to 300 M, were found to be quite similar for both cultivars in the absence of external Al; Ca²⁺ transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 M total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca²⁺ absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca²⁺ uptake over the entire Ca²⁺ concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca²⁺ uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 M) caused the K _m for Ca²⁺ uptake to increase with each rise in Al concentration, from approx. 100 M in the absence of Al to approx. 300 M in the presence of 10 M Al, while having no effect on the V _max. The same Al exposures had little effect on the kinetics of Ca²⁺ uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca²⁺ transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca²⁺-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.We would like to thank Dr. Lionel F. Jaffe, Director of the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA, for making his calcium-selective vibrating-mi-croelectrode system available for a portion of this work. The research presented here was supported in part by USDA/NRI Competitive Grant number 91-37100-6630 to Leon Kochian. Contribution from the USDA-ARS, U.S. Plant, Soil and Nutrition Laboratory, Cornell University, Ithaca, N.Y. This research was part of the program of the Center for Root-Soil Research, Cornell University, Ithaca, N.Y. Department of Soil, Crop and Atmosphere Science, paper No. 1741.     

Aluminum Resistance in the Arabidopsis Mutant alr-104 Is Caused by an Aluminum-Induced Increase in Rhizosphere pH

A mechanism that confers increased Al resistance in the Arabidopsis thaliana mutant alr-104 was investigated. A modified vibrating microelectrode system was used to measure H⁺ fluxes generated along the surface of small Arabidopsis roots. In the absence of Al, no differences in root H⁺ fluxes between wild type and alr-104 were detected. However, Al exposure induced a 2-fold increase in net H⁺ influx in alr-104 localized to the root tip. The increased flux raised the root surface pH of alr-104 by 0.15 unit. A root growth assay was used to assess the Al resistance of alr-104 and wild type in a strongly pH-buffered nutrient solution. Increasing the nutrient solution pH from 4.4 to 4.5 significantly increased Al resistance in wild type, which is consistent with the idea that the increased net H⁺ influx can account for greater Al resistance in alr-104. Differences in Al resistance between wild type and alr-104 disappeared when roots were grown in pH-buffered medium, suggesting that Al resistance in alr-104 is mediated only by pH changes in the rhizosphere. This mutant provides the first evidence, to our knowledge, for an Al-resistance mechanism based on an Al-induced increase in root surface pH.     

边缘细胞对荞麦根尖铝毒的防护效应和对细胞壁多糖的影响

以2个荞麦(Fygopyrum esculentum Moench)基因型‘江西荞麦’(耐性)和‘内蒙荞麦’(敏感)为材料,采用悬空培养(保持边缘细胞附着于根尖和去除根尖边缘细胞),研究边缘细胞对根尖铝毒的防护效应以及对细胞壁多糖组分的影响。结果表明,铝毒抑制荞麦根系伸长,导致根尖Al积累。去除边缘细胞的根伸长抑制率和根尖Al含量高于保留边缘细胞的根。去除边缘细胞使江西荞麦和内蒙荞麦根尖的酸性磷酸酶(APA)活性显著升高,前者在铝毒下增幅更大。同时,铝毒胁迫下去除边缘细胞的根尖果胶甲酯酶(PME)活性和细胞壁果胶、半纤维素1、半纤维素2含量显著高于保留边缘细胞的酶活性和细胞壁多糖含量。表明边缘细胞对荞麦根尖的防护效应,与其阻止Al的吸收,降低根尖细胞壁多糖含量及提高酸性磷酸酶活性有关,以此缓解Al对根伸长的抑制。     

Aluminum-induced citric acid secretion is not the sole mechanism of Al-resistance in maize

Aluminum-induced citric acid (CA) root secretion is a widely accepted mechanism to explain Al-resistance in maize. Nonetheless, several aspects of this mechanism remain controversial. In this study, we used paclobutrazol (PBZ), a plant growth retardant, to gain new insights into the relationship between Δ⁵-sterol composition, membrane permeability, (PM) H⁺-ATPase activity and CA secretion in an Al-sensitive (UFVM-100) and Al-resistant (UFVM-200) maize genotypes challenged with Al. The Al-sensitive genotype displayed greater concentrations of Al in the root tips and greater inhibition of root elongation (RE), which was accompanied by greater electrolyte leakage and greater reduction in the Δ⁵-sterols content after Al treatment. CA secretion by roots increased in both genotypes after Al treatment but to a greater extent in the Al-resistant genotype. The (PM) H⁺-ATPase activity was down-regulated in the sensitive cultivar and up-regulated in its resistant counterpart upon Al treatment. A significant correlation between (PM) H⁺-ATPase activity and CA secretion was observed, but only in the Al-resistant genotype. Upon adding PBZ to the Al-treated plants, differences in the RE and Δ⁵-sterol composition between the maize genotypes were fully abolished, whereas genotypic differences in CA secretion and (PM) H⁺-ATPase activity were reduced but not completely eliminated. Taken together, this information suggests the existence of other processes or mechanisms operating in the Al resistance in these two maize genotypes.     

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.     

Kinetics of Aluminum Uptake by Excised Roots of Aluminum-Tolerant and Aluminum-Sensitive Cultivars of Triticum aestivum L

Uptake of aluminum (Al) by excised roots of two Al-tolerant cultivars and two Al-sensitive cultivars of Triticum aestivum L. (wheat) was biphasic, with a rapid phase of uptake in the first 30 minutes followed by a linear phase of uptake up to 180 minutes. At the end of the uptake period, higher concentrations of Al were found in roots of the Al-sensitive cultivars (Neepawa and Scout-66) than in the Al-tolerant cultivars (Atlas-66 and PT-741), but differences were small. Experiments testing the effectiveness of several desorption agents demonstrated that citric acid was most effective in desorption of loosely bound Al (the putative apoplasmic compartment) followed by others in the order tartaric acid > EDTA > CaSO₄ = ScCl₃. In all cultivars, 30 minutes of desorption with citric acid depleted the rapidly exchanging, putative apoplasmic compartment, although some tightly bound Al remained in that compartment. The relationship between Al remaining after desorption and time in the uptake medium was nearly linear and no distinction was observed between Al-tolerant and Al-sensitive cultivars. However, uptake of Al by the Al-tolerant cultivars was increased by treatment with the protonophore 2,4-dinitrophenol (DNP), while uptake of Al by Al-sensitive cultivars was relatively unaffected. Such results suggest the possible involvement of an active exclusion mechanism in Al-tolerant cultivars of T. aestivum.     

Inhibition of Al-induced root elongation and enhancement of Al-induced peroxidase activity in Al-sensitive and Al-resistant barley cultivars are positively correlated

The quantitative changes in peroxidase activity and composition of anionic and cationic isoperoxidases were investigated in roots of two barley cultivars differing in Al resistance. Root growth of Al-resistant cv. Bavaria was in lesser extent reduced by Al treatment (23% after 24 h Al-treatment), whereas 40% reduction of the root growth was observed in Al-sensitive cv. Alfor. The strong root growth inhibition in Al-sensitive cv. Alfor correlated with a 6-fold enhancement of peroxidase activity by Al treatment. Al-induced enhancement of peroxidase activity was found also in roots of Al-resistant cv. Bavaria, but this increase was only half of the Al-sensitive cv. Alfor. Comparison of peroxidase isoenzyme composition of Al-treated and non-treated roots revealed that activity of at least five anionic and four cationic isoperoxidases was stimulated by Al treatment. Three of anionic isoperoxidases (aPOD2-4) were selectively induced only in the Al-sensitive cv. Alfor. A possible involvement of peroxidases in root-growth inhibition is discussed.     

Al Inhibits Both Shoot Development and Root Growth in als3, an Al-Sensitive Arabidopsis Mutant

In als3, an Al-sensitive Arabidopsis mutant, shoot development and root growth are sensitive to Al. Mutant als3 seedlings grown in an Al-containing medium exhibit severely inhibited leaf expansion and root growth. In the presence of Al, unexpanded leaves accumulate callose, an indicator of Al damage in roots. The possibility that the inhibition of shoot development in als3 is due to the hyperaccumulation of Al in this tissue was examined. However, it was found that the levels of Al that accumulated in shoots of als3 are not different from the wild type. The inhibition of shoot development in als3 is not a consequence of nonspecific damage to roots, because other metals (e.g. LaCl3 or CuSO4) that strongly inhibit root growth did not block shoot development in als3 seedlings. Al did not block leaf development in excised als3 shoots grown in an Al-containing medium, demonstrating that the Al-induced damage in als3 shoots was dependent on the presence of roots. This suggests that Al inhibition of als3 shoot development may be a delocalized response to Al-induced stresses in roots following Al exposure.     

Aluminium induces rapid changes in cytosolic pH and free calcium and potassium concentrations in root protoplasts of wheat (Triticum aestivum)

Addition of aluminium chloride (50 μM Al) caused different effects on the transmembrane electrical potential (PD) of root cells in Al-tolerant wheat (Triticum aestivum) cv. Kadett and Al-sensitive cv. WW 20299. As changes in PD of plant cells may depend on transient fluxes of protons, potassium and/or calcium through cell membranes, the effect of Al was investigated on the cytosolic concentrations of these ions in protoplasts isolated from root tips of the same cultivars. The tetra[acetoxymethyl] esters of the fluorescent dyes bis-carboxyethyl-carboxyfluorescein, BCECF, K⁺-binding benzofuran isophthalate, PBFI, and the stilbene chromophore Fura 2-AM were used to determine pH, K⁺ and Ca²⁺, respectively. Changes in fluorescence ratios, directly reflecting changes in [H⁺], [K⁺] and [Ca²⁺] in the cytosol, were determined by photometry fluorescence microscopy. Additions and removals of Al to and from both cultivars caused hyperpolarizations and depolarizations, respectively, but only in the sensitive cv. WW 20299 did the resting PD decrease gradually. Addition of Al to the protoplasts caused rapid changes in cytosolic pH, free [K⁺] and [Ca²⁺]. In both cultivars Al caused a transient oscillating increase in cytosolic [Ca²⁺] for 1 or 2 min and a rapid pH-dependent change in cytosolic [K⁺]. At pH 5 the presence of K⁺ in the medium diminished the Al-induced decrease in cytosolic [K⁺]. Aluminium (50 μM) induced a transient increase in cytosolic [H⁺] (pH decreased) in both cultivars, but the cytosolic pH returned to its initial value only in the Al-tolerant cv. Kadett. In the Alsensitive cv. WW 20299, repeated additions of Al caused a gradual decline in pH. Moreover, in the presence of 1 mM KCl, pH recovered completely in both cultivars. Since only the effect on pH differed in the two cultivars, the more toxic effect of Al on the cv. WW 20299 should be related to the change in pH.     

Zonal responses of sensitive vs. tolerant wheat roots during Al exposure and recovery

Aluminium (Al) irreversibly inhibits root growth in sensitive, but not in some tolerant genotypes. To better understand tolerance mechanisms, seedlings from tolerant ('Barbela 7/72' line) and sensitive ('Anahuac') Triticum aestivum L. genotypes were exposed to AlCl(3) 185 μM for: (a) 24 h followed by 48 h without Al (recovery); (b) 72 h of continuous exposure. Three root zones were analyzed (meristematic (MZ), elongation (EZ) and hairy (HZ)) for callose deposition, reserves (starch and lipids) accumulation, endodermis differentiation and tissue architecture. Putative Al-induced genotoxic or cytostatic/mytogenic effects were assessed by flow cytometry in root apices. Tolerant plants accumulated less Al, presented less root damage and a less generalized callose distribution than sensitive ones. Starch and lipid reserves remained constant in tolerant roots but drastically decreased in sensitive ones. Al induced different profiles of endodermis differentiation: differentiation was promoted in EZ and HZ, respectively, in sensitive and tolerant genotypes. No ploidy changes or clastogenicity were observed. However, differences in cell cycle blockage profiles were detected, being less severe in tolerant roots. After Al removal, only the 'Barbela 7/72' line reversed Al-induced effects to values closer to the control, mostly with respect to callose deposition and cell cycle progression. We demonstrate for the first time that: (a) cell cycle progression is differently regulated by Al-tolerant and Al-sensitive genotypes; (b) Al induces callose deposition >3 cm above root apex (in HZ); (c) callose deposition is a transient Al-induced effect in tolerant plants; and (d) in HZ, endodermis differentiation is also stimulated only in tolerant plants, probably functioning in tolerant genotypes as a protective mechanism in addition to callose.