Expression of aluminum-induced genes in transgenic arabidopsis plants can ameliorate aluminum stress and/or oxidative stress

To examine the biological role of Al-stress-induced genes, nine genes derived from Arabidopsis, tobacco (Nicotiana tabacum L.), wheat (Triticum aestivum L.), and yeast (Saccharomyces cerevisiae) were expressed in Arabidopsis ecotype Landsberg. Lines containing eight of these genes were phenotypically normal and were tested in root elongation assays for their sensitivity to Al, Cd, Cu, Na, Zn, and to oxidative stresses. An Arabidopsis blue-copper-binding protein gene (AtBCB), a tobacco glutathione S-transferase gene (parB), a tobacco peroxidase gene (NtPox), and a tobacco GDP-dissociation inhibitor gene (NtGDI1) conferred a degree of resistance to Al. Two of these genes, AtBCB and parB, and a peroxidase gene from Arabidopsis (AtPox) also showed increased resistance to oxidative stress induced by diamide, while parB conferred resistance to Cu and Na. Al content of Al-treated root tips was reduced in the four Al-resistant plant lines compared with wild-type Ler-0, as judged by morin staining. All four Al-resistant lines also showed reduced staining of roots with 2',7'-dichloro fluorescein diacetate (H(2)DCFDA), an indicator of oxidative stress. We conclude that Al-induced genes can serve to protect against Al toxicity, and also provide genetic evidence for a link between Al stress and oxidative stress in plants.     

An aluminum-activated citrate transporter in barley

Soluble ionic aluminum (Al) inhibits root growth and reduces crop production on acid soils. Al-resistant cultivars of barley (Hordeum vulgare L.) detoxify Al by secreting citrate from the roots, but the responsible gene has not been identified yet. Here, we identified a gene (HvAACT1) responsible for the Al-activated citrate secretion by fine mapping combined with microarray analysis, using an Al-resistant cultivar, Murasakimochi, and an Al-sensitive cultivar, Morex. This gene belongs to the multidrug and toxic compound extrusion (MATE) family and was constitutively expressed mainly in the roots of the Al-resistant barley cultivar. Heterologous expression of HvAACT1 in Xenopus oocytes showed efflux activity for (14)C-labeled citrate, but not for malate. Two-electrode voltage clamp analysis also showed transport activity of citrate in the HvAACT1-expressing oocytes in the presence of Al. Overexpression of this gene in tobacco enhanced citrate secretion and Al resistance compared with the wild-type plants. Transiently expressed green fluorescent protein-tagged HvAACT1 was localized at the plasma membrane of the onion epidermal cells, and immunostaining showed that HvAACT1 was localized in the epidermal cells of the barley root tips. A good correlation was found between the expression of HvAACT1 and citrate secretion in 10 barley cultivars differing in Al resistance. Taken together, our results demonstrate that HvAACT1 is an Al-activated citrate transporter responsible for Al resistance in barley.     

Arabidopsis mutants with increased sensitivity to aluminum.

Al-sensitive (als) mutants of Arabidopsis were isolated and characterized with the aim of defining mechanisms of Al toxicity and resistance. Most als mutants selected on the basis of root growth sensitivity to Al were recessive, and together the mutants constituted eight complementation groups. Also, in most als mutants, Al sensitivity appeared to be specific for Al relative to La (another trivalent cation), except als2, which was more sensitive to La than wild type. The tendency of roots on mutant seedlings to accumulate Al was examined by staining with morin and hematoxylin, dyes used to indicate the presence of Al. A significant increase in morin staining was observed in als5, consistent with its increased sensitivity to Al. Unexpectedly, als7 and als4 showed less morin staining, suggesting that the roots on these mutants accumulate less Al than wild type seedlings after exposure to Al-containing solutions. Roots of wild-type seedlings produce callose in response to AlCl3 concentrations that inhibit root growth. Only als5 accumulated more callose than wild type in response to low levels (25 mu M) of AICI3 However, als4 and als7 did not accumulate callose at this AlCl3 concentration even though root growth was significantly inhibited. The lack of callose accumulation in als4 and als7 suggests that there is not an obligatory relationship between callose deposition and Al-induced inhibition of root growth.     

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.     

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

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

The sax1 dwarf mutant of Arabidopsis thaliana shows altered sensitivity of growth responses to abscisic acid, auxin, gibberellins and ethylene and is partially rescued by exogenous brassinosteroid

Genetic approaches using Arabidopsis thaliana aimed at the identification of mutations affecting events involved in auxin signalling have usually led to the isolation of auxin-resistant mutants. From a selection screen specifically developed to isolate auxin-hypersensitive mutants, one mutant line was selected for its increased sensitivity to auxin (x 2 to 3) for the root elongation response. The genetic analysis of sax1 (hypersensitive to abscisic acid and auxin) indicated that the mutant phenotype segregates as a single recessive Mendelian locus, mapping to the lower arm of chromosome 1. Sax1 seedlings grown in vitro showed a short curled primary root and small, round, dark-green cotyledons. In the greenhouse, adult sax1 plants were characterized by a dwarf phenotype, delayed development and reduced fertility. Further physiological characterization of sax1 seedlings revealed that the most striking trait was a large increase (x 40) in ABA-sensitivity of root elongation and, to a lesser extent, of ABA-induced stomatal closure; in other respects, hypocotyl elongation was resistant to gibberellins and ethylene. These alterations in hormone sensitivity in sax1 plants co-segregated with the dwarf phenotype suggesting that processes involved in cell elongation are modified. Treatment of mutant seedlings with an exogenous brassinosteroid partially rescued a wild-type size, suggesting that brassinosteroid biosynthesis might be affected in sax1 plants. Wild-type sensitivities to ABA, auxin and gibberellins were also restored in sax1 plants by exogenous application of brassinosteroid, illustrating the pivotal importance of the BR-related SAX1 gene.     

Overexpression of an Arabidopsis magnesium transport gene, AtMGT1, in Nicotiana benthamiana confers Al tolerance

Aluminium (Al) toxicity is the most important limiting factor for crop production in acid soil environments worldwide. In some plant species, application of magnesium (Mg(2+)) can alleviate Al toxicity. However, it remains unknown whether overexpression of magnesium transport proteins can improve Al tolerance. Here, the role of AtMGT1, a member of the Arabidopsis magnesium transport family involved in Mg(2+) transport, played in Al tolerance in higher plants was investigated. Expression of 35S::AtMGT1 led to various phenotypic alterations in Nicotiana benthamiana plants. Transgenic plants harbouring 35S::AtMGT1 exhibited tolerance to Mg(2+) deficiency. Element assay showed that the contents of Mg, Mn, and Fe in 35S::AtMGT1 plants increased compared with wild-type plants. Root growth experiment revealed that 100 microM AlCl(3) caused a reduction in root elongation by 47% in transgenic lines, whereas root growth in wild-type plants was inhibited completely. Upon Al treatment, representative transgenic lines also showed a much lower callose deposition, an indicator of increased Al tolerance, than wild-type plants. Taken together, the results have demonstrated that overexpression of ATMGT1 encoding a magnesium transport protein can improve tolerance to Al in higher plants.     

Genetic and Chemical Reductions in Protein Phosphatase Activity Alter Auxin Transport, Gravity Response, and Lateral Root Growth

Auxin transport is required for important growth and developmental processes in plants, including gravity response and lateral root growth. Several lines of evidence suggest that reversible protein phosphorylation regulates auxin transport. Arabidopsis rcn1 mutant seedlings exhibit reduced protein phosphatase 2A activity and defects in differential cell elongation. Here we report that reduced phosphatase activity alters auxin transport and dependent physiological processes in the seedling root. Root basipetal transport was increased in rcn1 or phosphatase inhibitor-treated seedlings but showed normal sensitivity to the auxin transport inhibitor naphthylphthalamic acid (NPA). Phosphatase inhibition reduced root gravity response and delayed the establishment of differential auxin-induced gene expression across a gravity-stimulated root tip. An NPA treatment that reduced basipetal transport in rcn1 and cantharidin-treated wild-type plants also restored a normal gravity response and asymmetric auxin-induced gene expression, indicating that increased basipetal auxin transport impedes gravitropism. Increased auxin transport in rcn1 or phosphatase inhibitor-treated seedlings did not require the AGR1/EIR1/PIN2/WAV6 or AUX1 gene products. In contrast to basipetal transport, root acropetal transport was normal in phosphatase-inhibited seedlings in the absence of NPA, although it showed reduced NPA sensitivity. Lateral root growth also exhibited reduced NPA sensitivity in rcn1 seedlings, consistent with acropetal transport controlling lateral root growth. These results support the role of protein phosphorylation in regulating auxin transport and suggest that the acropetal and basipetal auxin transport streams are differentially regulated.     

Spatial aluminium sensitivity of root apices of two common bean (Phaseolus vulgaris L.) genotypes with contrasting aluminium resistance

The initial response of plants to aluminium (Al) is an inhibition of root elongation. In the present study, short and medium-term effects of Al treatment (20 muM) on root growth and Al accumulation of two common bean (Phaseolus vulgaris L.) genotypes, VAX-1 (Al-sensitive) and Quimbaya (Al-resistant), were studied. Root elongation of both genotypes was severely inhibited during the first 3-4 h of Al treatment. Thereafter, both genotypes showed gradual recovery. However, this recovery continued in genotype Quimbaya until the root elongation rate reached the level of the control (without Al) while the genotype VAX-1 was increasingly damaged by Al after 12 h of Al treatment. Short-term Al treatment (90 microM Al) to different zones of the root apex using agarose blocks corroborated the importance of the transition zone (TZ, 1-2 mm) as a main target of Al. However, Al applied to the elongation zone (EZ) also contributed to the overall inhibition of root elongation. Enhanced inhibition of root elongation during the initial 4 h of Al treatment was related to high Al accumulation in root apices in both genotypes (Quimbaya>VAX-1). Recovery from Al stress was reflected by decreasing Al contents especially in the TZ, but also in the EZ. After 24 h of Al treatment the high Al resistance of Quimbaya was reflected by much lower Al contents in the entire root apex. The results confirmed that genotypic differences in Al resistance in common bean are built up during medium-term exposure of the roots to Al. For this acquisition of Al resistance, the activation and maintenance of an Al exclusion mechanism, especially in the TZ but also in the EZ, appears to be decisive.     

The role of root exudates in aluminium resistance and silicon-induced amelioration of aluminium toxicity in three varieties of maize (Zea mays L.).

Aluminium (Al) toxicity is widely considered to be the most important growth-limiting factor for plants in strongly acid soils (pH<5.0). The inhibition of root elongation in three varieties of maize (Zea mays L. vars Clavito, HS701b and Sikuani) was followed over the first 48 h of Al treatment, and during the initial 10 h elongation was determined on an hourly basis. The silicon (Si)-induced amelioration of Al toxicity was investigated by pre-treating seedlings for 72 h in nutrient solutions with 1000 microM Si before transfer into solutions with 0, 20 or 50 microM Al (without Si). Plants were either grown in complete low ionic strength nutrient solutions (CNS) or in low salt solutions of 0.4 mM CaCl2 (LSS). In addition, the role of root exudation of organic compounds as a mechanism of Si-induced alleviation of Al toxicity was investigated. Aluminium-induced inhibition of root elongation in the maize var. HS701b was observed within 1 h of Al exposure. After a lag time of at least 8 h, Si-induced alleviation of Al toxicity was observed in this variety when grown in LSS. In the Al-resistant var. Sikuani, Al-resistance was only observed after exposure to 50 microM Al, and not after exposure to 20 microM Al, suggesting that there exists a threshold Al concentration before the mechanisms of Al resistance are activated. Aluminium stimulated root exudation of oxalic acid in all three varieties, but exudate concentrations did not increase with either Al resistance or with Si pretreatment. Aluminium and Si triggered release of catechol and of the flavonoid-type phenolics: catechin, and quercetin. In the Al-resistant variety, Sikuani, Al-exposed plants pretreated with Si exuded up to 15 times more phenolics than those plants not pretreated with Si. The flavonoid-type phenolics, to date unconsidered, appear to play a role in the mechanism(s) of Si-induced amelioration of Al toxicity.     

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.     

Genotypic differences in Al resistance and the role of cell-wall pectin in Al exclusion from the root apex in Fagopyrum tataricum

Background and Aims

Aluminium (Al) toxicity is one of the factors limiting crop production on acid soils. However, genotypic differences exist among plant species or cultivars in response to Al toxicity. This study aims to investigate genotypic differences among eight cultivars of tatary buckwheat (Fagopyrum tataricum) for Al resistance and explore the possible mechanisms of Al resistance.

Methods

Al resistance was evaluated based on relative root elongation (root elongation with Al/root elongation without Al). Root apex Al content, pectin content and exudation of root organic acids were determined and compared.

Key Results

Genotypic differences among the eight cultivars were correlated with exclusion of Al from the root apex. However, there was a lack of correlation between Al exclusion and Al-induced oxalate secretion. Interestingly, cell-wall pectin content of the root apex was generally lower in Al-resistant cultivars than in Al-sensitive cultivars. Although we were unable to establish a significant correlation between Al exclusion and pectin content among the eight cultivars, a strong correlation could be established among six cultivars, in which the pectin content in the most Al-resistant cultivar ‘Chuan’ was significantly lower than that in the most Al-sensitive cultivar ‘Liuku2’. Furthermore, root apex cell-wall pectin methylesterase activity (PME) was similar in ‘Chuan’ and ‘Liuku2’ in the absence of Al, but Al treatment resulted in increased PME activity in ‘Liuku2’ compared with ‘Chuan’. Immunolocalization of pectins also showed that the two cultivars had similar amounts of either low-methyl-ester pectins or high-methyl-ester pectins in the absence of Al, but Al treatment resulted in a more significant increase of low-methyl-ester pectins and decrease of high-methyl-ester pectins in ‘Liuku2’.

Conclusions

Cell-wall pectin content may contribute, at least in part, to differential Al resistance among tatary buckwheat cultivars.     

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

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

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.     

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.     

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.     

Simultaneous Overexpression of Citrate Synthase and Phosphoenolpyruvate Carboxylase in Leaves Augments Citrate Exclusion and Al Resistance in Transgenic Tobacco

Phosphoenolpyruvate carboxylase (PEPC) and citrate synthase (CS) are two key enzymes in organic acid synthesis metabolism. In the present study, a cytoplasmic form of CS from tobacco and a mutant (with reduced sensitivity to organic acid inhibition) PEPC from Synechococcus vulcanus were overexpressed simultaneously using a light-inducible promoter in tobacco leaves. The analysis for enzyme activity showed that CS and PEPC enzyme activities were increased by 235% to 257% and 218% to 236% in the selected cs and pepc (double-gene) overexpression lines, respectively, compared with those in the wild-type plants (WT). The measurement for the relative root elongation rate of the tobacco plants exposed to 30???M aluminum (Al) indicated that Al tolerance in the double-gene overexpression lines was stronger than that of the transgenic cs or pepc lines and WT plants. The ¹³C-NMR analysis with NaH¹³CO₃ showed that overexpression of CS and PEPC in the transgenic tobacco successfully constructed a new citrate synthesis pathway. Under the conditions with Al stress, the amount of citrate secreted from the double-transgenic tobacco roots was the largest among the tested plants. When grown on sandy soil supplied with a nutritional solution containing 500???M Al, the growth of the double-transgenic tobacco was better than that of the transgenic cs or pepc tobacco and WT, and their root biomass was the highest among the tested plants. These results demonstrated that construction of a new citrate synthesis pathway by simultaneous overexpression of CS and PEPC in the cytoplasm of transgenic plant leaves could enhance Al resistance in plants.     

Intracellular distribution and binding state of aluminum in root apices of two common bean (Phaseolus vulgaris) genotypes in relation to Al toxicity

The role of the intracellular distribution and binding state of aluminum (Al) in Al toxicity, using Al exchange and Al fractionation methodologies, were studied in two common bean ( Phaseolus vulgaris L.) genotypes differing in Al resistance. These two genotypes are characterized by a similar initial period (4 h) of Al sensitivity followed by a contrasting recovery period (8–24 h). A higher initial Al accumulation in Quimbaya (Al resistant) in the 5-mm root apex compared with VAX-1 (Al sensitive) could be related to its higher content of unmethylated pectin and thus higher negative charge of the cell walls (CWs). The binding state and cellular distribution of Al in the root apices revealed that the root elongation rate was significantly negatively correlated with the free apoplastic and the stable-bound, not citrate-exchangeable CW Al representing the most important Al fraction in the root apex (80%), but not with the symplastic and the labile-bound, citrate-exchangeable CW Al. It is postulated that the induced and sustained recovery from the initial Al stress in the Al-resistant genotype Quimbaya requires reducing the stable-bound Al in the apoplast thus allowing cell elongation and division to resume.