Al-Induced, 51-Kilodalton,Membrane-Bound Proteins Are Associated with Resistance to Al in a Segregating Population of Wheat

Incorporation of 35S into protein is reduced by exposure to Al in wheat (Triticum aestivum), but the effects are genotype-specific. Exposure to 10 to 75 [mu]M Al had little effect on 35S incorporation into total protein, nuclear and mitochondrial protein, microsomal protein, and cytosolic protein in the Al-resistant cultivar PT741. In contrast, 10 [mu]M Al reduced incorporation by 21 to 38% in the Al-sensitive cultivar Katepwa, with effects becoming more pronounced (31-62%) as concentrations of Al increased. We previously reported that a pair of 51-kD membrane-bound proteins accumulated in root tips of PT741 under conditions of Al stress. We now report that the 51-kD band is labeled with 35S after 24 h of exposure to 75 [mu]M Al. The specific induction of the 51-kD band in PT741 suggested a potential role of one or both of these proteins in mediating resistance to Al. Therefore, we analyzed their expression in single plants from an F2 population arising from a cross between the PT741 and Katepwa cultivars. Accumulation of 1,3-[beta]-glucans (callose) in root tips after 24 h of exposure to 100 [mu]M Al indicated that this population segregated for Al resistance in about a 3:1 ratio. A close correlation between resistance to Al (low callose content of root tips) and accumulation of the 51-kD band was observed, indicating that at least one of these proteins cosegregates with the Al-resistance phenotype. As a first step in identifying a possible function, we have demonstrated that the 51-kD band is most clearly associated with the tonoplast. Whereas Al has been reported to stimulate the activity of the tonoplast H+-ATPase and H+-PPase, antibodies raised against these proteins did not cross-react with the 51-kD band. Efforts are now under way to purify this protein from tonoplast-enriched fractions.     

Differential Exudation of Polypeptides by Roots of Aluminum-Resistant and Aluminum-Sensitive Cultivars of Triticum aestivum L. in Response to Aluminum Stress

Cultivars of Triticum aestivum differing in resistance to Al were grown under aseptic conditions in the presence and absence of Al and polypeptides present in root exudates were collected, concentrated, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon exposure to 100 and 200 [mu]M Al, root elongation in Al-sensitive cultivars was reduced by 30 and 65%, respectively, whereas root elongation in resistant cultivars was reduced by only 15 and 30%. Accumulation of polypeptides in the growth medium increased with time for 96 to 120 h, with little additional accumulation thereafter. This pattern of exudation was virtually unaffected by exposure to 100 [mu]M Al in the Al-resistant cultivars Atlas 66 and Maringa, whereas total accumulation was reduced in sensitive cultivars. Changes in exudation were consistent with alterations in root elongation. Al-induced or Al-enhanced polypeptide bands were detected in Atlas 66 and Maringa after 72 h of exposure to Al. Increased accumulation of 12-, 22-, and 33-kD bands was observed at 75 [mu]M Al in Atlas 66 and 12-, 23-, and 43.5-kD bands started to appear at 50 [mu]M Al in Maringa. In the Al-sensitive cultivars Roblin and Katepwa, no significant effect on polypeptide profiles was observed at values up to 100 [mu]M Al. When root exudates were separated by ultrafiltration and the Al content was measured in both high molecular mass (HMM; >10 kD) and ultrafiltrate (<10 kD) fractions, approximately 2 times more Al was detected in HMM fractions from Al-resistant cultivars than from Al-sensitive cultivars. Dialysis of HMM fractions against water did not release this bound Al;digestion with protease released between 62 and 73% of total Al, with twice as much released from exudates of Al-resistant than of Al-sensitive cultivars. When plants were grown in the presence of 0 to 200 [mu]M Al, saturation of the Al-binding capacity of HMM exudates occurred at 50 [mu]M Al in Al-sensitive cultivars. Saturation was not achieved in resistant cultivars. Differences in exudation of total polypeptides in response to Al stress, enhanced accumulation of specific polypeptides, and the greater association of Al with HMM fractions from Al-resistant cultivars suggest that root exudate polypeptides may play a role in plant response to Al.     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

Aluminum resistance in wheat (Triticum aestivum) is associated with rapid, Al-induced changes in activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in root apices

We have investigated the effect of aluminum (Al) on the activity of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) isolated from 5-mm root apices of 4-day-old wheat ( Triticum aestivum ) cultivars differing in resistance to Al. Rapid increases in G6PDH and 6PGDH activities were observed in Al-resistant cultivars (PT741 and Atlas 66) during the first 10 h of treatment with 100 μ M Al, while no change in the activity of either enzyme was observed in Al-sensitive cultivars (Katepwa and Neepawa) during a 24-h exposure to Al. The Al-induced increases in enzyme activities observed in the Al-resistant PT741 appear to reflect an induction of protein synthesis since the increases were completely abolished by 1 m M cycloheximide. No differences in G6PDH and 6PGDH activities were observed between the Al-sensitive and the Al-resistant genotypes when Al was supplied in vitro. Under these conditions, an increase in Al concentration from 0 to 1.4 m M caused a gradual decrease in activity of both enzymes, irrespective of the Al-resistance of whole seedlings. Aluminum-sensitive and aluminum-resistant cultivars also differed in the rate and extent of accumulation of slowly-exchanging Al in 5-mm root apices. During the first 6 h of Al treatment, Al accumulation was only 10% more rapid in Katepwa than in PT741. After 24-h exposure, accumulation in the Al-sensitive Katepwa, was two-fold higher. A decline in Al accumulation in a slowly-exchanging compartment as well as a decrease in activities of G6PDH and 6PGDH were found in the Al-resistant PT741, when seedlings were transferred to Al-free treatment solutions after 16-h exposure to 100 μ M Al. These results suggest that rapid induction of G6PDH and 6PGDH in the Al-resistant line PT741 by Al may play a role in the mechanism of Al resistance, possibly by regulation of the pentose phosphate pathway.     

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.     

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.     

Aluminum Tolerance in Wheat (Triticum aestivum L.) (II. Aluminum-Stimulated Excretion of Malic Acid from Root Apices)

We investigated the role of organic acids in conferring Al tolerance in near-isogenic wheat (Triticum aestivum L.) lines differing in Al tolerance at the Al tolerance locus (Alt1). Addition of Al to nutrient solutions stimulated excretion of malic and succinic acids from roots of wheat seedlings, and Al-tolerant genotypes excreted 5- to 10-fold more malic acid than Al-sensitive genotypes. Malic acid excretion was detectable after 15 min of exposure to 200 [mu]M Al, and the amount excreted increased linearly over 24 h. The amount of malic acid excreted was dependent on the external Al concentration, and excretion was stimulated by as little as 10 [mu]M Al. Malic acid added to nutrient solutions was able to protect Al-sensitive seedlings from normally phytotoxic Al concentrations. Root apices (terminal 3-5 mm of root) were the primary source of the malic acid excreted. Root apices of Al-tolerant and Al-sensitive seedlings contained similar amounts of malic acid before and after a 2-h exposure to 200 [mu]M Al. During this treatment, Al-tolerant seedlings excreted about four times the total amount of malic acid initially present within root apices, indicating that continual synthesis of malic acid was occurring. Malic acid excretion was specifically stimulated by Al, and neither La, Fe, nor the absence of Pi was able to elicit this response. There was a consistent correlation of Al tolerance with high rates of malic acid excretion stimulated by Al in a population of seedlings segregating for Al tolerance. These data are consistent with the hypothesis that the Alt1 locus in wheat encodes an Al tolerance mechanism based on Al-stimulated excretion of malic acid.     

Growth and cell wall properties of two wheat cultivars differing in their sensitivity to aluminum stress

The present study was conducted to investigate the cell wall properties in two wheat (Triticum aestivum L.) cultivars differing in their sensitivity to Al stress. Seedlings of Al-resistant, Inia66 and Al-sensitive, Kalyansona cultivars were grown in complete nutrient solutions for 4 days and then subjected to treatment solutions containing Al (0, 50 microM) in a 0.5 mM CaCl(2) solution at pH 4.5 for 24 h. Root elongation was inhibited greatly by the Al treatment in the Al-sensitive cultivar compared to the Al-resistant cultivar. The Al-resistant cultivar accumulated less amount of Al in the root apex than in the Al-sensitive cultivar. The contents of pectin and hemicellulose in roots were increased with Al stress, and this increase was more conspicuous in the Al-sensitive cultivar. The molecular mass of hemicellulosic polysaccharides was increased by the Al treatment in the Al-sensitive cultivar. The increase in the content of hemicellulose was attributed to increase in the contents of glucose, arabinose and xylose in neutral sugars. Aluminum treatment increased the contents of ferulic acid and p-coumaric acid especially in the Al-sensitive cultivar by increasing the activity of phenylalanine ammonia lyase (PAL, EC 4.3.1.5). Aluminum treatment markedly decreased the beta-glucanase activity in the Al-sensitive cultivar, but did not exert any effect in the Al-resistant cultivar. These results suggest that the modulation of the activity of beta-glucanase with Al stress may be involved in part in the alteration of the molecular mass of hemicellulosic polysaccharides in the Al-sensitive cultivar. The increase in the molecular mass of hemicellulosic polysaccharides and ferulic acid synthesis in the Al-sensitive cultivar with Al stress may induce the mechanical rigidity of the cell wall and inhibit the elongation of wheat roots.     

Aluminum-induced alterations in lipid composition of microsomal membranes from an aluminum-resistant and an aluminum-sensitive cultivar of Triticum aestivum

We have studied the effect of aluminum (Al) on the lipid composition of microsomal membranes isolated from 5-mm root tips of an Al-resistant (T 741) and an Al-sensitive (Katepwa) cultivar of Triticum aestivum L. Exposure of both genotypes to 10 and 50 μ M AeCl₃ for 1 day had no effect on lipid composition; however, decreases in phospholipids and increases in monogalactosyl diacylglycerols, free sterols, free fatty acids and triacylglycerols were observed with prolonged exposure (3 days) to 5O μ M AlCe₃. Several genotype-specific changes were also observed under these conditions. The content of digalactosyl diacylglycerols increased by 66.7% in Katepwa. but decreased slightly in PT 741. Thus, the ratio of rnonogalactosyl diacylglycerols to digalactosyl diacylglycerols increased by 46.2% in PT 741, but decreased by 21.3% in Katepwa. Genotype-specific differences were also observed in steryl lipids. Treatment with Al induced a 70.2% increase in sterylglucosides and a 23.3% increase in acylated sterylglucosides in Katepwa. In contrast, a 18.9% decrease in acylated sterylglucosides and no changes in sterylglucosides were observed in PT 741. Our limited understanding of the effect of membrane composition on membrane structure and function makes it difficult to predict how these changes relate to Al toxicity and resistance. While it is possible that many changes reflect the toxic effects of Al, we believe that changes observed only in the Al-resistant genotype could contribute to continuous growth in the face of Al stress.     

Aluminum Tolerance in Wheat (Triticum aestivum L.) (I. Uptake and Distribution of Aluminum in Root Apices)

We investigated the uptake and distribution of Al in root apices of near-isogenic wheat (Triticum aestivum L.) lines differing in Al tolerance at a single locus (Alt1: aluminum tolerance). Seedlings were grown in nutrient solution that contained 100 [mu]M Al, and the roots were subsequently stained with hematoxylin, a compound that binds Al in vitro to form a colored complex. Root apices of Al-sensitive genotypes stained after short exposures to Al (10 min and 1 h), whereas apices of Al-tolerant seedlings showed less intense staining after equivalent exposures. Differential staining preceded differences observed in either root elongation or total Al concentrations of root apices (terminal 2-3 mm of root). After 4 h of exposure to 100 [mu]M Al in nutrient solution, Al-sensitive genotypes accumulated more total Al in root apices than Al-tolerant genotypes, and the differences became more marked with time. Analysis of freeze-dried root apices by x-ray microanalysis showed that Al entered root apices of Al-sensitive plants and accumulated in the epidermal layer and in the cortical layer immediately below the epidermis. Long-term exposure of sensitive apices to Al (24 h) resulted in a distribution of Al coinciding with the absence of K. Quantitation of Al in the cortical layer showed that sensitive apices accumulated 5- to 10-fold more Al than tolerant apices exposed to Al solutions for equivalent times. These data are consistent with the hypothesis that Alt1 encodes a mechanism that excludes Al from root apices.     

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.     

Al Partitioning Patterns and Root Growth as Related to Al Sensitivity and Al Tolerance in Wheat

Studies of Al partitioning and accumulation and of the effect of Al on the growth of intact wheat (Triticum aestivum L.) roots of cultivars that show differential Al sensitivity were conducted. The effects of various Al concentrations on root growth and Al accumulation in the tissue were followed for 24 h. At low external Al concentrations, Al accumulation in the root tips was low and root growth was either unaffected or stimulated. Calculations based on regression analysis of growth and Al accumulation in the root tips predicted that 50% root growth inhibition in the Al-tolerant cv Atlas 66 would be attained when the Al concentrations were 105 [mu]M in the nutrient solution and 376.7 [mu]g Al g-1 dry weight in the tissue. In contrast, in the Al-sensitive cv Tam 105, 50% root growth inhibition would be attained when the Al concentrations were 11 [mu]M in the nutrient solution and 546.2 [mu]g Al g-1 dry weight in the tissue. The data support the hypotheses that differential Al sensitivity correlates with differential Al accumulation in the growing root tissue, and that mechanisms of Al tolerance may be based on strategies to exclude Al from the root meristems.     

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.     

Effects of aluminium on root growth and apical root cells in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars. Reliability of screening tests to detect Al resistance at the seedling stage

The effects of Al₂(SO₄)₃·18H₂O on growth of root and apical root cells were studied in seedlings of rice cultivars differing in Al resistance including I Kong Pao and Aiwu (Al-sensitive) and IRAT 112 and IR6023-10-1-1 (Al-resistant). Inhibition of root growth was a typical effect of Al, and the extent of the inhibition depended on both cultivar and Al concentration. Al impaired the activity of the root meristem as indicated by reductions in its size, mitotic activity and the diameter of the meristematic cell nucleoli. Cell size in the elongation zone of the root was also reduced by Al. The reliability of the haematoxylin staining method to classify rice cultivars according to their Al-sensitivity failed to discriminate the Al-resistant IR6023-10-1-1 cultivar from the two sensitive cultivars. The results are discussed in relation to the Al resistance mechanisms operating in rice.     

Differential effects of aluminium on osmotic potential and sugar accumulation in the root cells of Al-resistant and Al-sensitive wheat

The changes in osmotic potential and the concentration of osmotic solutes in the cell sap of the root tips exposed to Al were examined in two cultivars of wheat ( Triticum aestivum ) differing in Al resistance. Root elongation was less influenced by an 8-h exposure to 20 μ M or 50 μ M Al in Al-resistant cv. Atlas 66 than in Al-sensitive cv. Scout 66. After Al treatment the osmotic potential of the root cells was decreased in Atlas 66 but increased in Scout 66 indicating that the Al treatment osmotically stimulated the driving force for water uptake in Atlas 66 but suppressed it in Scout 66. Al increased the concentration of soluble sugars, the major osmotic solute in the root cells in Atlas 66, but decreased it in Scout 66. Al at both low (5 μ M ) and high (50 μ M ) concentrations, also increased the concentration of soluble sugars in the Al-resistant genotype ET8 but a high Al concentration decreased it in Al-sensitive genotype ES8. Enzymatic analyses and thin-layer chromatography revealed that soluble sugars in the root cells of both Atlas 66 and Scout 66 mainly consisted of monosaccharides such as glucose, fructose and a small amount of sucrose. These results suggest that the accumulation of soluble sugars in Al-resistant wheat Atlas 66 keeps the osmotic potential in the root cells low and thus, enables the root cells to take up water and to elongate against the pressure produced by cell wall rigidification under Al stress.     

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.     

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 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.     

Molecular mapping of a gene responsible for Al-activated secretion of citrate in barley

Aluminium (Al) toxicity is an important limitation to barley (Hordeum vulgare L.) on acid soil. Al-resistant cultivars of barley detoxify Al externally by secreting citrate from the roots. To link the genetics and physiology of Al resistance in barley, genes controlling Al resistance and Al-activated secretion of citrate were mapped. An analysis of Al-induced root growth inhibition from 100 F2 seedlings derived from an Al-resistant cultivar (Murasakimochi) and an Al-sensitive cultivar (Morex) showed that a gene associated with Al resistance is localized on chromosome 4H, tightly linked to microsatellite marker Bmag353. Quantitative trait locus (QTL) analysis from 59 F4 seedlings derived from an F3 plant heterozygous at the region of Al resistance on chromosome 4H showed that a gene responsible for the Al-activated secretion of citrate was also tightly linked to microsatellite marker Bmag353. This QTL explained more than 50% of the phenotypic variation in citrate secretion in this population. These results indicate that the gene controlling Al resistance on barley chromosome 4H is identical to that for Al-activated secretion of citrate and that the secretion of citrate is one of the mechanisms of Al resistance in barley. The identification of the microsatellite marker associated with both Al resistance and citrate secretion provides a valuable tool for marker-assisted selection of Al-resistant lines.