Al-sensitive Line Research Articles

Repression of Mitochondrial Citrate Synthase Genes by Aluminum Stress in Roots of Secale cereale and Brachypodium distachyon.

Aluminum (Al) toxicity in acid soils influences plant development and yield. Almost 50% of arable land is acidic. Plants have evolved a variety of tolerance mechanisms for Al. In response to the presence of Al, various species exudate citrate from their roots. Rye (Secale cereale L.) secretes both citrate and malate, making it one of the most Al-tolerant cereal crops. However, no research has been done on the role of the mitochondrial citrate synthase (mCS) gene in Al-induced stress in the rye. We have isolated an mCS gene, encoding a mitochondrial CS isozyme, in two S. cereale cultivars (Al-tolerant cv. Ailés and Al-sensitive inbred rye line Riodeva; ScCS4 gene) and in two Brachypodium distachyon lines (Al-tolerant ABR8 line and Al-sensitive ABR1 line; BdCS4 gene). Both mCS4 genes have 19 exons and 18 introns. The ScCS4 gene was located on the 6RL rye chromosome arm. Phylogenetic studies using cDNA and protein sequences have shown that the ScCS4 gene and their ScCS protein are orthologous to mCS genes and CS proteins of different Poaceae plants. Expression studies of the ScCS4 and BdSC4 genes show that the amount of their corresponding mRNAs in the roots is higher than that in the leaves and that the amounts of mRNAs in plants treated and not treated with Al were higher in the Al-tolerant lines than that in the Al-sensitive lines of both species. In addition, the levels of ScCS4 and BdCS4 mRNAs were reduced in response to Al (repressive behavior) in the roots of the tolerant and sensitive lines of S. cereale and B. distachyon.

Uncovering the transcriptional response of popcorn (Zea mays L. var. everta) under long-term aluminum toxicity

To date, the investigation of genes involved in Al resistance has focused mainly on microarrays and short periods of Al exposure. We investigated genes involved in the global response under Al stress by tracking the expression profile of two inbred popcorn lines with different Al sensitivity during 72 h of Al stress. A total of 1003 differentially expressed genes were identified in the Al-sensitive line, and 1751 were identified in the Al-resistant line, of which 273 were shared in both lines. Genes in the category of “response to abiotic stress” were present in both lines, but there was a higher number in the Al-resistant line. Transcription factors, genes involved in fatty acid biosynthesis, and genes involved in cell wall modifications were also detected. In the Al-resistant line, GST6 was identified as one of the key hub genes by co-expression network analysis, and ABC6 may play a role in the downstream regulation of CASP-like 5. In addition, we suggest a class of SWEET transporters that might be involved in the regulation of vacuolar sugar storage and may serve as mechanisms for Al resistance. The results and conclusions expand our understanding of the complex mechanisms involved in Al toxicity and provide a platform for future functional analyses and genomic studies of Al stress in popcorn.

Rhizosphere pH difference regulated by plasma membrane H<sup>+</sup>-ATPase is related to differential Al tolerance of two wheat cultivars

Aluminum (Al)-tolerance of different cultivars shows considerable differences. Elevation of rhizosphere pH is an external Al-resistant mechanism of plants. To elucidate the correlation between Al tolerance and the capacity of plants to modify the rhizosphere pH at different Al-tolerant levels, a comparative study on the wheat (Triticum aestivum L.) cultivars ET8 (Al-tolerant) and ES8 (Al-sensitive) was performed. Rhizosphere pH of ET8 was much higher than that of ES8 under the same treatment, significant correlations were obtained among all the data of rhizosphere pH and relative root elongation (R<sup>2</sup> = 0.9209**), or Al content in root apex (R<sup>2 </sup>= 0.9321**), which indicated that Al tolerance may be related to pH changes in the rhizosphere. The elevation of rhizosphere pH was inhibited by H<sup>+</sup>-ATPase specific inhibitor DCCD (dicylcohexylcarbodiimide, 25 mmol). Relative PM (plasma membrane) H<sup>+</sup>-ATPase activity of ET8 was significantly higher than that of ES8 under the same treatment. Significant correlation between all the data of relative PM H<sup>+</sup>-ATPase activity and rhizosphere pH (R<sup>2</sup> = 0.8319**) were obtained. Taken together, these results suggest that PM H<sup>+</sup>-ATPase was involved in regulating rhizosphere pH. Under Al stress, the Al-tolerant line showed a stronger capacity of up-regulating rhizosphere pH by PM H+-ATPase than the Al-sensitive line, which may explain the observed differences in Al tolerance between the two wheat cultivars.

Mechanisms of magnesium amelioration of aluminum toxicity in soybean at the gene expression level

Micromolar concentration of magnesium (Mg) in culture solution is known to ameliorate aluminum (Al) toxicity in soybean and other leguminous species. To advance the understanding of this phenomenon at the level of gene expression in soybean, we undertook a comparative transcriptome analysis using DNA microarrays and Al-tolerant and Al-sensitive genotypes treated with Al ions alone or Al plus Mg ions. We observed a more rapid alteration of transcription for Al-tolerant than Al-sensitive soybean after introduction of Mg into Al-containing medium, but at 72 h, far more genes were altered (both upregulated and downregulated) in the Al-sensitive line, reflecting the known greater saving effect of Mg for Al-sensitive than Al-tolerant lines. Mg appears to ameliorate Al toxicity in the sensitive genotype by the dual mechanisms of (i) specifically increasing the expression level of several genes that are upregulated in the Al-treated, Al-tolerant genotype in the absence of Mg and (ii) possibly saving energy by decreasing expression of most genes relative to expression under Al stress. Mg-mediated reduction in gene expression also appears to be an important mechanism of Mg protection of the Al-tolerant genotype.

Transcriptional profile of maize roots under acid soil growth.

BackgroundAluminum (Al) toxicity is one of the most important yield-limiting factors of many crops worldwide. The primary symptom of Al toxicity syndrome is the inhibition of root growth leading to poor water and nutrient absorption. Al tolerance has been extensively studied using hydroponic experiments. However, unlike soil conditions, this method does not address all of the components that are necessary for proper root growth and development. In the present study, we grew two maize genotypes with contrasting tolerance to Al in soil containing toxic levels of Al and then compared their transcriptomic responses.ResultsWhen grown in acid soil containing toxic levels of Al, the Al-sensitive genotype (S1587-17) showed greater root growth inhibition, more Al accumulation and more callose deposition in root tips than did the tolerant genotype (Cat100-6). Transcriptome profiling showed a higher number of genes differentially expressed in S1587-17 grown in acid soil, probably due to secondary effects of Al toxicity. Genes involved in the biosynthesis of organic acids, which are frequently associated with an Al tolerance response, were not differentially regulated in both genotypes after acid soil exposure. However, genes related to the biosynthesis of auxin, ethylene and lignin were up-regulated in the Al-sensitive genotype, indicating that these pathways might be associated with root growth inhibition. By comparing the two maize lines, we were able to discover genes up-regulated only in the Al-tolerant line that also presented higher absolute levels than those observed in the Al-sensitive line. These genes encoded a lipase hydrolase, a retinol dehydrogenase, a glycine-rich protein, a member of the WRKY transcriptional family and two unknown proteins.ConclusionsThis work provides the first characterization of the physiological and transcriptional responses of maize roots when grown in acid soil containing toxic levels of Al. The transcriptome profiles highlighted several pathways that are related to Al toxicity and tolerance during growth in acid soil. We found several genes that were not found in previous studies using hydroponic experiments, increasing our understanding of plant responses to acid soil. The use of two germplasms with markedly different Al tolerances allowed the identification of genes that are a valuable tool for assessing the mechanisms of Al tolerance in maize in acid soil.

Aluminum stress induces up-regulation of an efficient antioxidant system in the Al-tolerant maize line but not in the Al-sensitive line

The response of the antioxidant enzymes, superoxide dismutase (SOD; EC 1.15.1.1) and peroxidase (POD; EC 1.11.1.7), to Aluminum (Al) stress was studied in roots of two inbred lines of maize ( Zea mays L.) differing in their tolerance to Al. In addition, the production of malondialdehyde (MDA) was measured to evaluate the level of lipid peroxidation as well as the accumulation of proline (Pro) and carbohydrates under 72 h Al stress. Roots of Al (0, 120, 240, 360 and 480 μM, at pH 4.2) -treated plants were sampled at various times (12, 24, 48, 72 h) after commencement of stress. A major difference in the antioxidant enzymes between the two maize lines associated with Al tolerance was observed after 24 h of Al exposure. A gradual increase in the membrane lipid peroxidation in Al-stressed root of the susceptible maize line was accompanied by decreased activities of the antioxidant enzymes SOD and POD. In contrast, increased activities of the SOD and POD were found in Al-treated roots of the tolerant maize line, in which the level of membrane lipid peroxidation remained almost unchanged. After 72 h exposure to 480 μM Al the accumulation of Al in roots was almost from 90 times (tolerant) to 140 times (sensitive) than the control (without Al), while at the same time Al treatment resulted in 2.2 to 2.5-fold (at 240, 360 and 480 μM Al) increased Pro content in the roots of the tolerant line compared to 0 μM Al. Yet, 72 h exposure to 480 μM Al increased 1.7-fold the carbohydrate concentration in the roots of the Al tolerant maize line VA-22 while in the sensitive line A 4/67 remained almost unchanged. These data provide evidence of an internal mechanism of tolerance that increase the antioxidant system activity in order to limit cellular damages and possibly linked to the Al tolerance of the maize line VA-22. Analyses of the 12, 24, 48, and 72 h POD and SOD isoforms showed that in the Al-tolerant maize plants the anionic POD isoforms A 1, A 3 and A 4 and the SOD isoforms SOD 1 and SOD 2 were induced by increased Al-stress. It seemed that in the Al tolerant maize line, the anionic POD isoforms A 1, A 3 and A 4 and the SOD isoforms SOD 1 and SOD 2 were required for adaptation as the oxidant level increased by the increased Al stress. Our results suggest that Al toxicity may be mediated by oxidative stress and that the better protection of the Al tolerant maize roots from Al-induced oxidative damage results, at least partially, from the increased activity of their antioxidative system.

Effect of lime on root growth, morphology and the rhizosheath of cereal seedlings growing in an acid soil

The effect of soil acidity on root and rhizosheath development in wheat and barley seedlings was investigated in an acid Ferrosol soil to which various amounts of lime (CaCO3) were applied to modify soil Al concentrations (pH (CaCl2): 4.22 to 5.35 and Al (CaCl2 extract): 17.7 to 0.4 mg kg−1 soil; respectively), and Ferrosol soil from an adjacent location at the same site which had a higher Al concentration (pH 4.19; 29.2 mg kg−1 Al). The cereal lines were selected on the basis of differences in their rate of root growth, Al-resistance and root hair morphology. Root morphology was assessed after 7 days of growth. The length of fine (mainly lateral) roots of Al-sensitive genotypes was more sensitive to soil Al concentrations than that of the coarse (mainly primary) roots. The experiments demonstrated that even where root growth was protected by expression of the TaALMT1 gene for Al-resistance, root-soil contact was diminished by soil acidity because root hair length (in many lines), and root hair density and rhizosheath formation (all lines) were adversely affected by soil acidity. In the case of Al-sensitive lines, fine root growth and rhizosheath mass were reduced over much the same range of soil Al concentrations (i.e. >3–6 mg kg−1 Al). Although Al-resistant lines could maintain fine root length under these conditions, they were similarly unable to maintain rhizosheath mass. This finding may help to explain why Al-resistant wheats which yield relatively well in deep acid soils, may also benefit from application of lime to the surface layers of the soil.

Physiological and molecular characterization of aluminum resistance in Medicago truncatula.

BackgroundAluminum (Al) toxicity is an important factor limiting crop production on acid soils. However, little is known about the mechanisms by which legumes respond to and resist Al stress. To explore the mechanisms of Al toxicity and resistance in legumes, we compared the impact of Al stress in Al-resistant and Al-sensitive lines of the model legume, Medicago truncatula Gaertn.ResultsA screen for Al resistance in 54 M. truncatula accessions identified eight Al-resistant and eight Al-sensitive lines. Comparisons of hydroponic root growth and root tip hematoxylin staining in an Al-resistant line, T32, and an Al-sensitive line, S70, provided evidence that an inducible Al exclusion mechanism occurs in T32. Transcriptional events associated with the Al resistance response were analyzed in T32 and S70 after 12 and 48 h Al treatment using oligonucleotide microarrays. Fewer genes were differentially regulated in response to Al in T32 compared to S70. Expression patterns of oxidative stress-related genes, stress-response genes and microscopic examination of Al-treated root tips suggested a lower degree of Al-induced oxidative damage to T32 root tips compared to S70. Furthermore, genes associated with cell death, senescence, and cell wall degradation were induced in both lines after 12 h of Al treatment but preferentially in S70 after 48 h of Al treatment. A multidrug and toxin efflux (MATE) transporter, previously shown to exude citrate in Arabidopsis, showed differential expression patterns in T32 and S70.ConclusionOur results identified novel genes induced by Al in Al-resistant and sensitive M. truncatula lines. In T32, transcription levels of genes related to oxidative stress were consistent with reactive oxygen species production, which would be sufficient to initiate cell death of Al-accumulating cells thereby contributing to Al exclusion and root growth recovery. In contrast, transcriptional levels of oxidative stress-related genes were consistent with excessive reactive oxygen species accumulation in S70 potentially resulting in necrosis and irreversible root growth inhibition. In addition, a citrate-exuding MATE transporter could function in Al exclusion and/or internal detoxification in T32 based on Al-induced transcript localization studies. Together, our findings indicate that multiple responses likely contribute to Al resistance in M. truncatula.

Aluminum tolerance in maize is correlated with increased levels of mineral nutrients, carbohydrates and proline, and decreased levels of lipid peroxidation and Al accumulation

We investigated the uptake of aluminum (Al) and transport to shoots in two inbred maize lines (Zea mays L., VA-22 and A(4/67)) differing in Al tolerance. Seedlings were grown for 7 days in hydroponic culture with nutrient solution that contained 0, 240, 360, and 480microM Al at pH 4.2. After 7 days of exposure to Al, roots of sensitive maize line (A(4/67)) plants accumulated 2-2.5 times more Al than roots of tolerant line (VA-22) plants. Inductively coupled plasma atomic emission spectrometry (ICP-AES) showed that the tolerant line retained higher concentrations of Ca(2+), Mg(2+), and K(+) compared with the sensitive line. In response to Al treatment, proline (Pro) concentration increased three-fold in roots of tolerant plants, while a slight increase was observed in roots of sensitive-line plants. A substantial carbon surplus (two-fold increase) was observed in roots of the Al-tolerant maize line. Carbohydrate concentration remained almost unchanged in roots of Al-sensitive line plants. Al treatment triggered the enhancement of lipid peroxidation in the sensitive line, while no change in lipid peroxidation level was observed in the tolerant maize line. These data provide further support to the hypothesis that a mechanism exists that excludes Al from the roots of the tolerant maize line, as well as an internal mechanism of tolerance that minimizes accumulation of lipid peroxides through a higher Pro and carbohydrate content related to osmoregulation and membrane stabilization.

Aluminum Resistance in Maize Cannot Be Solely Explained by Root Organic Acid Exudation. A Comparative Physiological Study

Root apical aluminum (Al) exclusion via Al-activated root citrate exudation is widely accepted as the main Al-resistance mechanism operating in maize (Zea mays) roots. Nonetheless, the correlation between Al resistance and this Al-exclusion mechanism has not been tested beyond a very small number of Al-resistant and Al-sensitive maize lines. In this study, we conducted a comparative study of the physiology of Al resistance using six different maize genotypes that capture the range of maize Al resistance and differ significantly in their genetic background (three Brazilian and three North American genotypes). In these maize lines, we were able to establish a clear correlation between root tip Al exclusion (based on root Al content) and Al resistance. Both Al-resistant genotypes and three of the four Al-sensitive lines exhibited a significant Al-activated citrate exudation, with no evidence for Al activation of root malate or phosphate release. There was a lack of correlation between differential Al resistance and root citrate exudation for the six maize genotypes; in fact, one of the Al-sensitive lines, Mo17, had the largest Al-activated citrate exudation of all of the maize lines. Our results indicate that although root organic acid release may play a role in maize Al resistance, it is clearly not the only or the main resistance mechanism operating in these maize roots. A number of other potential Al-resistance mechanisms were investigated, including release of other Al-chelating ligands, Al-induced alkalinization of rhizosphere pH, changes in internal levels of Al-chelating compounds in the root, and Al translocation to the shoot. However, we were unsuccessful in identifying additional Al-resistance mechanisms in maize. It is likely that a purely physiological approach may not be sufficient to identify these novel Al-resistance mechanisms in maize and this will require an interdisciplinary approach integrating genetic, molecular, and physiological investigations.

A wheat gene encoding an aluminum-activated malate transporter.

The major constraint to plant growth in acid soils is the presence of toxic aluminum (Al) cations, which inhibit root elongation. The enhanced Al tolerance exhibited by some cultivars of wheat is associated with the Al-dependent efflux of malate from root apices. Malate forms a stable complex with Al that is harmless to plants and, therefore, this efflux of malate forms the basis of a hypothesis to explain Al tolerance in wheat. Here, we report on the cloning of a wheat gene, ALMT1 (aluminum-activated malate transporter), that co-segregates with Al tolerance in F2 and F3 populations derived from crosses between near-isogenic wheat lines that differ in Al tolerance. The ALMT1 gene encodes a membrane protein, which is constitutively expressed in the root apices of the Al-tolerant line at greater levels than in the near-isogenic but Al-sensitive line. Heterologous expression of ALMT1 in Xenopus oocytes, rice and cultured tobacco cells conferred an Al-activated malate efflux. Additionally, ALMT1 increased the tolerance of tobacco cells to Al treatment. These findings demonstrate that ALMT1 encodes an Al-activated malate transporter that is capable of conferring Al tolerance to plant cells.

An intracellular mechanism of aluminum tolerance associated with high antioxidant status in cultured tobacco cells

An aluminum (Al) tolerance mechanism, together with oxidative stress tolerance, was investigated in an Al tolerant cell line (ALT301) and the parental Al sensitive cell line (SL) of tobacco. During Al exposure in a simple calcium solution for 24 h, Al triggered the evolution of a reactive oxygen species (ROS) in SL much higher than ALT301 [Plant Physiol. 128 (2002) 63]. Under the conditions, Al enhanced comparable rates of citrate secretion from both cell lines to the same extent. Al enhanced the gene expression of manganese superoxide dismutase (MnSOD) in both cell lines, but at a significantly higher rate in SL than in ALT301, and also enhanced the enzyme activity of MnSOD in both cell lines to nearly the same level. These results suggest that the extracellular chelation of Al with organic acids and MnSOD is not involved in the mechanism of Al tolerance of ALT301. ALT301 contained ascorbate (ASA) and glutathione (GSH) levels that were higher than SL under normal growth conditions. During 24 h of post-Al treatment culture in growth medium, but not during 24-h Al exposure in a simple Ca 2+ solution, lipid peroxidation was enhanced in SL much higher than in ALT301, and the average SL amounts of ASA and GSH were exhausted compared to ALT301. Pre-loading of ASA prior to Al treatment improved the growth of SL during the post-Al treatment culture. ALT301 also exhibited cross-tolerance to H 2O 2, Fe 2+ and Cu 2+. Under these oxidant exposures, ALT301 contained lower levels of intracellular H 2O 2 or lipid peroxides, and maintained higher amounts of ASA and GSH than SL. Taken together, we conclude that the accumulation of Al in cells enhances the peroxidation of lipids exclusively under growing conditions, and that the higher content of ASA and GSH in ALT301 than in SL seems to be in part responsible for the tolerance mechanism of ALT301 to Al by protecting cells from either lipid peroxidation or H 2O 2 commonly enhanced by Al or other oxidants.

Responses of wheat and barley to liming on a sandy soil with subsoil acidity

A 3 years field trial examined the effect of newly and previously applied lime on the growth and yield of two near-isogenic wheat genotypes differing only in aluminium (Al) tolerance ( Triticum aestivum L. Al-sensitive line ES8 and Al-tolerant line ET8), and barley ( Hordeum vulgare cv. Mundak) on an acid soil (pH CaCl 2 4.6 in 0–10 cm and pH 4.1–4.3 in 10–40 cm) in the medium rainfall region of Western Australia. The trial consisted of four lime treatments: (i) no lime control; (ii) surface liming at 1.5 t ha −1 in 1999; (iii) surface liming at 2.5 t ha −1 in 1984; (iv) liming in 1984 and re-liming in 1999. Wheat crops were grown in 1999 and 2001, and barley was grown in 2000. Liming in 1984 increased the pH in both topsoil and subsoil and decreased toxic Al in the subsoil. Liming in 1999 largely increased soil pH in the 0–10 cm in previously unlimed and limed plots, but only slightly increased the pH in 10–20 cm 2 years after application. In 1999, there was an overall 14% grain yield increase by growing ET8, mostly due to much better performance (41%) of ET8 over ES8 in the treatment with surface liming in 1999. In 2001, ET8 had yield 24% higher in the no lime control and 14% higher in the treatment with liming in 1999 compared with ES8. While both genotypes had similar root length density in the topsoil, root length density in acid subsoil was 22–160% higher for ET8 than for ES8. Wheat genotypes produced 23–24% higher yield due to the liming in 1984 compared to the no lime control. In 2000 season, shoot biomass of barley increased by 45–70% in the limed treatments compared with the no lime control. Liming at 2.5 t ha −1 in 1984 or liming at 1.5 t ha −1 in 1999 increased yield by 25%. Liming in 1984 and re-liming in 1999 increased the yield by over 50%. The results suggest that surface liming can ameliorate subsoil acidity as measured 15–17 years after application, and that growing an Al-tolerant crop in combination with surface liming provides a good strategy to combat subsoil acidity. The genotypic variation in response to liming appears to result from the difference in the sensitivity of root proliferation to low pH and high Al.

Long-term persistence of aluminium-tolerant and sensitive Phalaris lines on acidic soils and associated changes in soil acidity

Persistence of an aluminium (Al)-tolerant phalaris F1 hybrid (Siro 1146 Phalaris aquatica × P.�arundinacea) was compared with that of 3 more Al-sensitive phalaris (P. aquatica L.) lines (cv. Australian and the progenitors of cvv. Sirosa and Sirolan) at 2 sites (Strathbogie and Baddaginnie) in north-eastern Victoria with strongly acidic soils 20 years after sowing. Soil pHCa and extractable aluminium (AlCa) were also measured to 1 m depth under Siro 1146 and annual grass pasture at each site. All grass treatments contained volunteer subclover. Siro 1146 persisted better than the other lines (P<0.05) at Strathbogie where the soil contained high AlCa concentrations down to 50 cm depth. Soils should be sampled and tested to at least this depth to determine their suitability for phalaris. At this site Australian phalaris persisted better than the progenitors of Sirosa and Sirolan (P<0.01), probably because Australian has more spreading ability and tolerance of set stocking than the winter active lines. At Baddaginnie, the soil contained lower concentrations of AlCa below 20 cm depth than at Strathbogie and persistence of the 3 Al sensitive phalaris lines was good despite the lower rainfall. The less drought-tolerant Siro�1146 persisted poorly at Baddaginnie, but had high ground cover due to high lateral spread of the survivors. At Strathbogie, the 10–20 cm layer of soil under Siro 1146 had a higher pHCa and lower AlCa than that under the other 4 phalaris and the annual grass treatments. Soil under Siro 1146 also had higher pHCa and lower AlCa down to 40�cm�depth compared with the annual grass treatment, the differences being significant in the 20–30 cm layer. These differences were reversed at 50–100 cm, but only the effect on Al was significant. Although soils were not sampled at the beginning of the experiment, adequate replication and randomisation of the grass treatments showed that it was statistically improbable for observed final differences to be due to initial soil differences. The final differences may be due to greater amounts of nitrate being taken up from the upper layers by Siro 1146 over its long growing season compared with the annual grasses, leading to greater nitrate leaching from the upper layers and greater nitrate uptake from lower layers under the annuals (i.e. spatial separation of acid generation and consumption processes within the profile). Another possible reason for the greater acidification below 50 cm by the perennial is that its roots may have taken up more cations in this zone. Because of rising concern in some sectors of the public about the off-site, environmental effects of agriculture, the future role of more acid-tolerant phalaris cultivars growing near native vegetation in the high rainfall zone is discussed.

Aluminum-Induced Changes in Cell-Wall Glycoproteins in the Root Tips of Al-Tolerant and Al-Sensitive Wheat Lines

To investigate wheat (Triticum aestivumL.) responses to Al stress, KCl- and SDS-extracted glycoproteins (covalently bound proteins isolated by cell-wall digestion by cellulysine–pectolase mixture) and extensins (hydroxyproline-containing glycoproteins, HRGPs) were isolated from cell-wall preparations purified from the root apices of Al-sensitive and Al-tolerant near-isogenic lines ES8 and ET8. Under Al stress conditions, two lines differed mostly in their extensins. The untreated plants of two lines were low in covalently bound extensins, although the content of this protein fraction in ES8 was higher than in ET8. When the seedlings were treated with Al, the extensin content increased in both wheat lines and especially in the Al-tolerant ET8 plants. Using two-dimensional electrophoresis, the authors demonstrated the accumulation of polypeptides with mol wts of 22.2 kD (pI 5.5–6.5), 24.5 kD (pI 5.8–6.0), and 33.1 kD (pI 5.25) and polypeptides of 22.2 kD (pI 6.8–7.6) and 40.5 kD (pI 7.6) in the extensin fraction from the cell walls of the Al-sensitive plants. The regulation of cell responses to Al stress may involve extensin expression.

Direct Evaluation of the Ca2+-Displacement Hypothesis for Al Toxicity.

One explanation for Al toxicity in plants suggests that Al displaces Ca2+ from critical sites in the apoplasm. We evaluated the Ca2+-displacement hypothesis directly using near-isogenic lines of wheat (Triticum aestivum L.) that differ in Al tolerance at a single locus. We measured both the growth and total accumulation (apoplasmic plus symplasmic) of 45Ca and Al into roots that had been exposed to Al alone or to Al with other cations. Root growth in the Al-sensitive line was found to be severely inhibited by low activities of Al, even though Ca2+ accumulation was relatively unaffected. In solutions containing the same activity of the Al3+ and Ca2+ ions as above, but also including either 3.0 mM Mg2+, 3.0 mM Sr2+, or 30 mM Na+, growth improved, whereas 45Ca2+ accumulation was significantly decreased. Since most of the 45Ca2+ accumulated by roots during short-term treatments will reside in the apoplasm, these results indicate that displacement of Ca2+ from the apoplasm by Al cannot account for the Al-induced inhibition of root growth and, therefore, do not support the Ca2+-displacement hypothesis for Al toxicity. We also show that total accumulation of Al by root apices is greater in the Al-sensitive genotype than the Al-tolerant genotype and suggest that cation amelioration of Al toxicity is caused by the reduction in Al accumulation.

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

In this study, the role of root organic acid synthesis and exudation in the mechanism of aluminum tolerance was examined in Al-tolerant (South American 3) and Al-sensitive (Tuxpeno and South American 5) maize genotypes. In a growth solution containing 6 μM Al3+, Tuxpeno and South American 5 were found to be two- and threefold more sensitive to Al than South American 3. Root organic acid content and organic acid exudation from the entire root system into the bulk solution were investigated via high-performance liquid chromatographic analysis while exudates collected separately from the root apex or a mature root region (using a dividedroot-chamber technique) were analyzed with a more-sensitive ion chromatography system. In both the Al-tolerant and Al-sensitive lines, Al treatment significantly increased the total root content of organic acids, which was likely the result of Al stress and not the cause of the observed differential Al tolerance. In the absence of Al, small amounts of citrate were exuded into the solution bathing the roots. Aluminum exposure triggered a stimulation of citrate release in the Al-tolerant but not in the Al-sensitive genotypes; this response was localized to the root apex of the Al-tolerant genotype. Additionally, Al exposure triggered the release of phosphate from the root apex of the Al-tolerant genotype. The same solution Al3+ activity that elicited the maximum difference in Al sensitivity between Al-tolerant and Al-sensitive genotypes also triggered maximal citrate release from the root apex of the Al-tolerant line. The significance of citrate as a potential detoxifier for aluminum is discussed. It is concluded that organic acid release by the root apex could be an important aspect of Al tolerance in maize.

NAD + Kinase Activity in Root Tips of Nearly Isogenic Linesof Wheat ( Triticum aestivum L.) that Differ in their Tolerance to Aluminium

Aluminium tolerance and NAD + kinase activity were measured in apical segments of roots from wheatcultivars Chinese Spring (C.S.) and BH 1146, as well as in lines C.S.ditelo2Ds and C.S.allo2Ds. Plants were grown for 4 days in solutions containing aluminium, calcium or magnesium salts. In the presence of 30 μM AICl 3 root growth was greatly reduced in the ditelosomic line; root growth of cultivar C.S. and its allosomic line was less affected, while root growth of the Al tolerant cultivar, BH 1146, was not affected at all. Transfer of the long arm of chromosome 2D from BH 1146 to C.S. markedly increased Al tolerance in the developed C.S.allo2Ds line compared with the C.S.ditelo2Ds line. In the presence of Al the total NAD+ kinase activity increased 2-5 times in root tips of tested genotypes with a maximum activity observed after 6 h of exposure to aluminium. In the Al tolerant allosomic line changes in total and calmodulin-dependent activity of NAD + kinase under conditions of Al stress did not follow the same pattern as changes in activity in Al tolerant BH 1146, but resembled changes in activity found in the Al sensitive ditelosomic line. These unexpected observations could be explained if genes located on the 2Dl chromosome segment were not involved in the operation of an Al tolerance mechanism based on NAD + kinase activity, but were involved in another mechanism(s} of Al tolerance, for example exclusion of AI. Both calcium and magnesium ameliorated the toxic effects of aluminium on root growth and NAD + kinase activity.

Aluminum-induced deposition of (1,3)-?-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)-β-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 β-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.

Effects of growth period, plant age and changes in solution aluminium concentrations on aluminium toxicity in wheat

The effects of growth period (time between transplanting and harvesting), plant age at which aluminium (Al) was added to solution, changes in Al concentration, and solution culture techniques (monitoring and adjusting solution Al concentrations thrice weekly or weekly replacement of the solutions) were investigated using a low ionic strength (2.7×10−3M) solution culture technique. The wheat (Triticum aestivum L.) cultivars Waalt (Al-tolerant) and Warigal (Al-sensitive), or the near isogenic lines bred from these cultivars (RR for the Al-tolerant line and SS for the Al-sensitive line) were grown. In all experiments and treatments, Al additions were required to maintain the nominal concentration. The decline in solution Al concentrations was partially attributed to formation of an Al-hydroxy-phosphate precipitate with an Al:P molar ratio of 2.8 to 4.0. Increasing the growth period from 14 to 28 days increased Al sensitivity in Warigal but not in Waalt. When plants were exposed to Al for the same time, increasing the age of the plants that Al was added to solution decreased sensitivity to Al. Differential Al tolerance between the two lines was evident when solutions were monitored thrice weekly or replaced weekly. However, the Al concentration required to reduce relative yield by a given amount when the solutions were replaced weekly was about twice that when the solutions were monitored. With a constant growth period of 28 days, increasing solution Al concentrations for 3 or more days resulted in decreased yields at harvest. The exact effect depended on the cultivar, plant part (tops or roots), when solution Al concentrations were increased and the duration of the increase. For example, increasing Al concentrations from 5 μM to 20 μM for 10 days reduced yield in the RR line by approximately 50% in the tops and 30% in the roots beyond the effect of 5 M but had no effect in the SS line due to yields already being low at 5 μM. Adding 10 μM Al to solution for 6 days at the beginning of the experiment reduced yield by 25% in the RR line and 50% in the SS line. In contrast, adding 10 μM Al for 6 days in the middle of the growth cycle had no effect on the RR line but reduced yield by approximately 25% in the SS line. These results show that growth period, the age of the plants at which Al is added and the technique used (monitored or weekly replacement) all need to be considered when comparing results from different experiments. These results also show that the Al concentrations in solution need to be regularly monitored in long term experiments.