Accumulation of Microsomal Polypeptides in Barley Roots during Aluminium Stress

Accumulation of two peripheral membrane polypeptides (20 and 28 kDa) in roots of Al-sensitive (cv. Alfor) and Al-resistant (cv. Bavaria) barley cultivars were analysed during Al stress. Both cultivars were subjected to Al concentration ranging from 0 to 150 µM for 24, 48, 72 and 96 h. Accumulation of both polypeptides was determined 24 h after exposure of plants to Al and content of both polypeptides showed only small depedence upon Al concentration and duration of Al treatment. Although, based on root growth test, Bavaria showed significantly greater resistance to Al than Alfor, analysis of 20 and 28 kDa polypeptide pattern has not revealed significant difference between the two cultivars. However, accumulation of 20 and 28 kDa polypeptides in Alfor was selectively induced by Al treatment because different pH of the root media (pH 3.5 to 6.5) or application of other metals (Cu, Co, or Cd) failed to induce these two bands. On the other hand, accumulation of these polypeptides in Bavaria was induced not only by Al, but also by Cd and in a lesser extent by Co treatment.     

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.     

A possible role of sphingolipids in the aluminium resistance of yeast and maize

The plasma membrane is most likely the major target for sensing of aluminium (Al), leading to inhibition of plant root-growth. As a result of high external Al, alterations in plasma membrane composition may be expected in order to maintain its properties. As sphingolipids are characteristic components of this membrane, their involvement in membrane adjustment to increased Al concentrations was investigated. Heterologous expression of a stereounselective long-chain base (LCB) (8E/Z)-desaturase from Arabidopsis thaliana, Brassica napus and Helianthus annuus in Saccharomyces cerevisiae improved the Al resistance of the transgenic yeast cells. This encouraged us to investigate whether Al affects the LCB composition, and whether genetic engineering of the LCB profile modifies the Al resistance of the Al-sensitive plant species maize (Zea mays, L.). Constitutive expression of the LCB (8E/Z)-desaturase from Arabidopsis thaliana in maize roots led to an 8- to 10-fold increase in (8E)-4-hydroxysphing-8-enine in total roots. Less marked but similar changes were observed in 3 mm root apices. Al treatment of the Al-sensitive maize cv Lixis resulted in a significant increase in the proportion of (8Z)-LCB and in the content of total LCBs in root tips, which was not observed in the Al-resistant cv ATP-Y. When root tips of transgenic plants were exposed to Al, only minor changes of both (8Z)- and (8E)-unsaturated LCBs as well as of the total LCB were observed. Al treatment of the wild type parental line H99 decreased the (8Z)-unsaturated LCBs and the total LCB content. Based on Al-induced callose production, a marker for Al sensitivity, the parental line H99 was as Al-resistant as cv ATP-Y, whereas the transgenic line became as sensitive as cv Lixis. Taken together, these data suggest that, in particular, the loss of the ability to down-regulate the proportion of (8Z)-unsaturated LCBs may be related to increased Al sensitivity.     

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.     

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.     

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.     

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.     

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 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.     

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-induced deposition of (1,3)-β-glucans (callose) inTriticum aestivum L.

Aluminum (Al)-induced damage to leaves and roots of two Al-resistant (cv. Atlas 66, experimental line PT741) and two Al-sensitive (cv. Scout 66, cv. Katepwa) lines ofTriticum aestivum L. was estimated using the deposition of (1, 3)--glucans (callose) as a marker for injury. Two-day-old seedlings were grown for forty hours in nutrient solutions with or without added Al, and callose deposition was quantified by spectrofluorometry (0–1000 µM Al) and localized by fluorescence microscopy (0 and 400 µM Al). Results suggested that Al caused little damage to leaves. No callose was observed in leaves with up to 400 µM Al treatment. In contrast, root callose concentration increased with Al treatment, especially in the Al-sensitive lines. At 400 µM Al, root callose concentration of Al-sensitive Scout 66 was nearly four-fold that of Al-resistant Atlas 66. After Al treatment, large callose deposits were observed in the root cap, epidermis and outer cortex of root tips of Scout 66, but not Atlas 66. The identity of callose was confirmed by a reduced fluorescence in Al-treated roots: firstly, after adding an inhibitor of callose synthesis (2-deoxy-D-glucose) to the nutrient solution, and secondly, after incubating root sections with the callosedegrading enzyme -D-glucoside glucohydrolase [EC 3.2.1.21]. Root callose deposition may be a good marker for Al-induced injury due to its early detection by spectrofluorometry and its close association with stress perception.Abbreviations DDG 2-deoxy-D-glucose - PAS periodic acid - Schiffs reagent - PE pachyman equivalents     

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.     

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.     

Dependence of the Extent and Direction of Average Stomatal Response in Zea mays L. and Phaseolus vulgaris L. on the Frequency of Fluctuations in Environmental Stimuli

Three-day-old seedlings of an Al-sensitive (Neepawa) and an Al-resistant (PT741) cultivar of Triticum aestivum were subjected to Al concentrations ranging from 0 to 100 [mu]M for 72 h. At 25 [mu]M Al, growth of roots was inhibited by 57% in the Al-sensitive cultivar, whereas root growth in the Al-resistant cultivar was unaffected. A concentration of 100 [mu]M Al was required to inhibit root growth of the Al-resistant cultivar by 50% and resulted in almost total inhibition of root growth in the sensitive cultivar. Cytoplasmic and microsomal membrane fractions were isolated from root tips (first 5 mm) and the adjacent 2-cm region of roots of both cultivars. When root cytoplasmic proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, no changes in polypeptide patterns were observed in response to Al stress. Analysis of microsomal membrane proteins revealed a band with an apparent molecular mass of 51 kD, which showed significant accumulation in the resistant cultivar following Al exposure. Two-dimensional gel analysis revealed that this band comprises two polypeptides, each of which is induced by exposure to Al. The response of the 51-kD band to a variety of experimental conditions was characterized to determine whether its pattern of accumulation was consistent with a possible role in Al resistance. Accumulation was significantly greater in root tips when compared to the rest of the root. When seedlings were subjected to Al concentrations ranging from 0 to 150 [mu]M, the proteins were evident at 25 [mu]M and were fully accumulated at 100 [mu]M. Time-course studies from 0 to 96 h indicated that full accumulation of the 51-kD band occurred within 24 h of initiation of Al stress. With subsequent removal of stress, the polypeptides gradually disappeared and were no longer visible after 72 h. When protein synthesis was inhibited by cycloheximide, the 51-kD band disappeared even when seedlings were maintained in Al-containing media. Other metals, including Cu, Zn, and Mn, failed to induce this band, and Cd and Ni resulted in its partial accumulation. These results indicate that synthesis of the 51-kD microsomal membrane proteins is specifically induced and maintained during Al stress in the Al-resistant cultivar, PT741.     

Effect of aluminum on cell wall,plasma membrane,antioxidants and root elongation in triticale

Two triticale cultivars ZC 237 (Al-resistant) and ZC 1890 (Al-sensitive) were used to investigate the effects of 30 to 100 μM Al on antioxidative enzyme activity, lipid peroxidation and cell wall composition. In ZC 1890, the root elongation was significantly inhibited after 1-h exposure to 50 μM Al, the changes in hemicellulose fraction were clearly detected after 2-h Al exposure, while the peroxidase (POD) and superoxide dismutase (SOD) activities significantly increased after 6-h exposure, and the malondialdehyde (MDA) content after 12-h exposure. The similar patterns were also found in ZC 237. Treatment of ZC 1890 with 1 mM citrate for 30 min after 3-h exposure to Al resulted in significant decrease of Al bound to cell-wall and recovery of root elongation. These results suggested that Al affected cell wall before the damage of plasma membrane, but this was not the primary cause of root elongation inhibition.     

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.     

A 23-kDa, root exudate polypeptide co-segregates with aluminum resistance in Triticum aestivum

We previously reported that treatment with aluminum (Al) leads to the accumulation of several polypeptides (12-, 23-, and 43.5-kDa) in root exudates of an Al-resistant cultivar of Triticum aestivum. In this report, we examine the segregation of the 23-kDa, Al-induced polypeptide and the Al-resistant phenotype in single F₂ plants arising from a cross between Al-resistant and Al-sensitive doubled-haploid (DH) lines. Single plants and plant populations were screened for sensitivity/resistance to Al using synthesis of 1,3-β-glucans (callose) as a sensitive marker for Al injury. Callose production in the Al-sensitive cv. Katepwa was approximately 3-fold higher than observed in the Al-resistant cv. Maringa, or a near-isogenic line derived from Katepwa and Maringa (Alikat), over a broad range of Al concentrations (0–100 μM). Similar results were observed with DH lines developed from cv. Katepwa, which produced two–four times more callose than DH lines developed from cv. Alikat. When single plants from F₁ and F₂ populations derived from a cross between DH Katepwa and DH Alikat were scored for Al-induced callose production after 4 days exposure to 100 μM Al, all F₁ plants were Al-resistant and F₂ plants segregated approximately 3:1 for Al-resistance/sensitivity. A backcross population derived from crossing Al-resistant F₁ with Al-sensitive Katepwa, segregated 1:1 for Al-resistance/sensitivity. Thus, the Al-resistant phenotype is inherited in a monogenic, dominant fashion in our DH lines. Enhanced accumulation of the Al-induced, 23-kDa polypeptide in root exudates was a trait which co-segregated with the Al-resistant phenotype in F₂ populations. This polypeptide was strongly labeled with S-methionine after 3 days of Al exposure and 6 h labeling. When root exudate polypeptides were separated by immobilized metal ion affinity chromatography, the 23-kDa polypeptide demonstrated significant Al-binding capacity. This polypeptide has been purified to near-homogeneity, providing an opportunity to isolate the gene(s) encoding this polypeptide.     

Effect of anion channel antagonists and La3+ on citrate release, Al content and Al resistance in maize roots

The correlation between organic acid anion release and Al content was examined in two maize (Zea mays L.) inbred lines, Cat 100-6 (Al-resistant) and S 1587-17 (Al-sensitive) treated with anion channel antagonists and La3+, a cation channel blocker. In the intact roots of Al-resistant maize, the Al-induced excretion of citrate was inhibited by the anion channel antagonists niflumic acid, anthracene-9-carboxylic and ethacrinic acid. Citrate release in excised root apices was reduced by 60% in the presence of 15 microM niflumic acid, while the Al content increased by 42%. Nevertheless, Cat 100-6 accumulated less Al than S 1587-17 when the rate of citrate release was similar in both lines, indicating that other mechanisms of Al-resistance are operating in Cat 100-6. The presence of 60 microM La3+ did not change the rate of citrate release, but the Al content in excised root apices of Al-resistant plants was reduced by 70%. These results suggest that the Al distributed uniformly in the roots does not contribute to citrate release and possibly the activity of anion channels is correlated with the free activities of extracellular Al3+ close to anion channels.     

Aluminum stress in the roots of naked barley

Phytotoxicity of aluminum (Al) is the major limiting factor for the crops grown in acid soils rapidly inhibiting root elongation. In this study, changes in root growth, total activity and isozyme patterns of antioxidant enzymes such as peroxidase, ascorbate peroxidase, catalase and glutathione reductase by Al stress were investigated in the roots of naked barley (Hordeum vulgare L. cv. Kwangwhalssalbori). As Al concentration increased up to 500 M, the rooting rate and root elongation substantially decreased. Growth results suggested that this cultivar is an Al-sensitive species. Total activities of antioxidant enzymes generally increased at lower Al concentrations and then gradually decreased at higher Al concentrations. They also increased when the exposure time to Al was extended up to 48 hr. Changes in the isozyme patterns of antioxidant enzymes were investigated byin situ enzyme activity staining on a non-denaturing PAGE gel. They generally coincided with the changes in the total activity in parallel. Changes in the total activity of antioxidant enzymes also coincided with the changes of the root growth. Since growth reduction in the roots by Al stress could be related with the changes in the activities of antioxidant enzymes, these results suggested that Al might cause the oxidative stress in the roots of this cultivar of naked barley.     

Changes in cell-wall properties of wheat (Triticum aestivum) roots during aluminum-induced growth inhibition

The effects of aluminum (Al) on root elongation, the mechanical extensibility of the cell wall, and the amount of cell-wall polysaccharides in the roots of Al-resistant (Atlas 66) and Al-sensitive (Scout 66) cultivars of wheat ( Triticum aestivum L.) were examined. Exposure to 10 μ M AlCl₃ for 6 h inhibited root elongation in Scout 66 but not in Atlas 66. It also decreased the mechanical extensibility of the cell wall in the roots of both cultivars, but prominently only in the roots of Scout 66. The amount of hemicellulose in the 10-mm region of root apex of Scout 66 was increased by the exposure to Al, especially in the apical regions. Al did not influence the neutral sugar composition of either pectin or hemicellulose in Scout 66 roots. However, Al increased the weight-average molecular mass of hemicellulosic polysaccharides and the amounts of wall-bound ferulic and diferulic acids in Scout 66 roots. These findings suggest that Al modifies the metabolism of cell-wall components and thus makes the cell wall thick and rigid, thereby inhibiting the growth of wheat roots.