Organic acid secretion as a mechanism of aluminium resistance: a model incorporating the root cortex, epidermis, and the external unstirred layer

The resistance of some plants to Al (aluminium or aluminum) has been attributed to the secretion of Al(3+)-binding organic acid (OA) anions from the Al-sensitive root tips. Evidence for the 'OA secretion hypothesis' of resistance is substantial, but the mode of action remains unknown because the OA secretion appears to be too small to reduce adequately the activity of Al(3+) at the root surface. In this study a mechanism for the reduction of Al(3+) at the root surface and just beneath the epidermis by complexation with secreted OA(2-) is considered. According to our computations, Al(3+) activity is insufficiently reduced at the surface of the root tips to account for the Al resistance of Triticum aestivum L. cv. Atlas 66, a malate-secreting wheat. Experimental treatments to decrease the thickness of the unstirred layer (increased aeration and removal of root-tip mucilage) failed to enhance sensitivity to Al(3+). On the basis of additional modelling, the observed spatial distribution of Al in roots, and the anatomical responses to Al, it is proposed that the epidermis is an essential component of the diffusion pathway for both OA and Al. We suggest that Al(3+) in the cortex must be reduced to small concentrations in order substantially to alleviate the inhibition of root elongation and so that the outer surface of the epidermis can tolerate relatively large concentrations of Al(3+). If OA secretion is required for reducing Al(3+) mainly beneath the root surface, rather than in the rhizosphere, then the metabolic cost to plants will be greatly reduced.     

Toxicity of Al to Desulfovibrio desulfuricans

The toxicity of Al to Desulfovibrio desulfuricans G20 was assessed over a period of 8 weeks in a modified lactate C medium buffered at four initial pHs (5.0, 6.5, 7.2, and 8.3) and treated with five levels of added Al (0, 0.01, 0.1, 1.0, and 10 mM). At pH 5, cell population densities decreased significantly and any effect of Al was negligible compared to that of the pH. At pHs 6.5 and 7.2, the cell population densities increased by 30-fold during the first few days and then remained stable for soluble-Al concentrations of <5 x 10(-5) M. In treatments having total-Al concentrations of > or =1 mM, soluble-Al concentrations exceeded 5 x 10(-5) M and limited cell population growth substantially and proportionally. At pH 8.3, soluble-Al concentrations were below the 5 x 10(-5) M toxicity threshold and cell population density increases of 20- to 40-fold were observed. An apparent cell population response to added Al at pH 8.3 was attributed to the presence of large, spirilloidal bacteria (accounting for as much as 80% of the cells at the 10 mM added Al level). Calculations of soluble-Al speciation for the pH 6.5 and 7.2 treatments that showed Al toxicity suggested the possible presence of the Al(13)O(4)(OH)(24)(H(2)O)(12)(7+) "tridecamer" cation and an inverse correlation of the tridecamer concentration and the cell population density. Analysis by (27)Al nuclear magnetic resonance spectroscopy, however, yielded no evidence of this species in freshly prepared samples or those taken 800 days after inoculation. Exclusion of the tridecamer species from the aqueous speciation calculations at pHs 6.5 and 7.2 yielded inverse correlations of the neutral Al(OH)(3) and anionic Al(OH)(4)(-) monomeric species with cell population density, suggesting that one or both of these ions bear primary responsibility for the toxicity observed.     

Apoplastic pH and Fe(3+) reduction in intact sunflower leaves

It has been hypothesized that under NO(3)(-) nutrition a high apoplastic pH in leaves depresses Fe(3+) reductase activity and thus the subsequent Fe(2+) transport across the plasmalemma, inducing Fe chlorosis. The apoplastic pH in young green leaves of sunflower (Helianthus annuus L.) was measured by fluorescence ratio after xylem sap infiltration. It was shown that NO(3)(-) nutrition significantly increased apoplastic pH at distinct interveinal sites (pH >/= 6.3) and was confined to about 10% of the whole interveinal leaf apoplast. These apoplastic pH increases presumably derive from NO(3)(-)/proton cotransport and are supposed to be related to growing cells of a young leaf; they were not found in the case of sole NH(4)(+) or NH(4)NO(3) nutrition. Complementary to pH measurements, the formation of Fe(2+)-ferrozine from Fe(3+)-citrate was monitored in the xylem apoplast of intact leaves in the presence of buffers at different xylem apoplastic pH by means of image analysis. This analysis revealed that Fe(3+) reduction increased with decreasing apoplastic pH, with the highest rates at around pH 5. 0. In analogy to the monitoring of Fe(3+) reduction in the leaf xylem, we suggest that under alkaline nutritional conditions at interveinal microsites of increased apoplastic pH, Fe(3+) reduction is depressed, inducing leaf chlorosis. The apoplastic pH in the xylem vessels remained low in the still-green veins of leaves with intercostal chlorosis.     

Interaction of Al(III) with the peptides AspAsp and AspAspAsp

The interactions of Al(III) with the dipeptide AspAsp and the tripeptide AspAspAsp in aqueous solutions were studied by pH-potentiometry and multinuclear 1H- and 13C- nuclear magnetic resonance (NMR) spectroscopy. Their numerous negatively charged COO(-) functions allow these ligands to bind Al(III) even in weakly acidic solutions. Various mononuclear 1:1 complexes are formed in different protonation states. 13C-NMR spectroscopy unambiguously proved participation of the COO(-) functions in a monodentate or chelating mode in Al(III) binding, however, the terminal-NH(2) group seems to be excluded from the coordination. Depending on the metal ion to ligand ratio precipitation occurs at pH approximately 5 to 6. This indicates that the COO(-) groups at the low level of preorganization in such small peptides are not sufficient to keep the Al(III) ion in solution and to prevent the precipitation of Al(OH)(3) at physiological pH. To achieve this, a more specific arrangement of the side-chain donors seems necessary.     

Aluminium accumulation in root cell walls coincides with inhibition of root growth but not with inhibition of magnesium uptake in Norway spruce

High levels of aluminium in the soil solution of forest soils cause stress to forest trees. Within the soil profile, pH and aluminium concentration in the soil solution vary considerably with soil depth. pH strongly influences the speciation of A1 in solution, and is a factor when considering toxicity of A1 to roots. Norway spruce ( Picea abies [L.] Karst.) seedlings were grown for 7 weeks in nutrient solutions at pH 3.2, 4.0 or 5.0 containing 0, 100 or 400 µ M A1. At the end of this period, seedling growth, the cation exchange capacity of the roots and the amount of exchangeable Ca and Mg in roots were determined. A1 concentrations in whole roots, root segments, and in needles were measured. Using X‐ray microanalysis, the concentrations of Al, Ca, Mg and P were determined in cortical cell walls. We wanted to test the hypotheses that (1) the amount of Al bound to cation exchange sites can be used as a marker for Al toxicity and (2) the Mg concentration of needles is controlled by the amount of Mg bound to cation exchange sites. Low pH reduced the inhibition of Al on root growth and shoot length. Both low pH and Al lowered the concentration of Ca and Mg in needles. Al concentrations in the roots decreased as the pH decreased. In the roots, Al displaced Mg and Ca from binding sites at the root cortical cell walls. A comparison of the effects of Al at the different pH values on root growth and Mg concentration in the needles, suggests that, at pH 5.0, an Al fraction in the apoplast inhibits root growth, but does not affect Mg uptake. This fraction of Al is not available for transport to the shoots. In contrast, Mg uptake is strongly affected by Al at pH 3.2, although only very low levels of Al were detected in the roots. Thus, Al accumulation in the apoplast is a positive marker for Al effects on root growth, but not Mg uptake. The Mg concentration of needles is not controlled by the amount of Mg bound to cation exchange sites.     

Effect of pH and nitrogen source on aluminium tolerance of rye (Secale cereale L.) and yellow lupin (Lupinus luteus L.)

Soluble aluminium (Al) is a major factor limiting plant growth in acid mineral soils. Aluminium concentrations in soil solutions are mainly determined by soil pH. However, pH also affects the ratio between activities of protons and cationic Al species and the equilibrium between mono-and polynuclear hydroxy-Al species. The phytotoxicity of these species is not yet clear. The objective of the present study was to clarify the role of minor changes of pH in the rhizosphere on Al phytotoxicity in two Al-tolerant plant species by direct control of the pH in the nutrient solution (4.1, 4.3, 4.5) and in addition by varying the pH in the root apoplast using either nitrate or ammonium as N source. The plants were grown in solution culture at constant external pH. Whereas the Al-sensitive plant species barley and horse bean were damaged at very low Al supplies (1.85 μM and 9.3 μM respectively), 222 μM had to be applied to rye and yellw lupin for a comparable inhibition of root elongation. Yellow lupin was initially severely inhibited in root growth by Al, but then gradually recovered from this ‘Al shock’ within 3 days. In contrast to lupin, rye was hardly affected by Al initially, and it took about 16 h until maximum inhibition of root elongation. In the presence of nitrate, raising the pH from 4.1 to 4.5 aggravated root-growth depression by Al in rye and lupin. Whereas rye roots were severely damaged by ammonium especially at low pH, lupin was rather indifferent to the N source. Aluminium toxicity was less severe in presence of ammonium compared to nitrate N. This effect was less clear with rye at lower pH, because of it's higher proton sensitivity compared to lupin. Less Al injury at lower pH and in presence of ammonium was related to lower Al concentrations in the 1 cm root tips. The results are compatible with data showing high phytotoxicity of mononuclear and polynuclear hydroxy-Al species. However, they could also be interpreted in the light of proton amelioration of Al toxicity owing to competition for Al-sensitive binding sites in the root apoplast.     

Hydroxyl radical-induced cell-wall loosening in vitro and in vivo: implications for the control of elongation growth

Hydroxyl radicals (OH) are capable of unspecifically cleaving cell-wall polysaccharides in a site-specific reaction. I investigated the hypothesis that cell-wall loosening underlying the elongation growth of plant organs is controlled by apoplastically produced OH attacking load-bearing cell-wall matrix polymers. Isolated cell walls (operationally, frozen/thawed, abraded segments from coleoptiles or hypocotyls, respectively) from maize, cucumber, soybean, sunflower or Scots pine seedlings were pre-loaded with catalytic Cu or Fe ions and then incubated in a mixture of ascorbate + H2O2 for generating OH in the walls. This treatment induced irreversible wall extension (creep) in walls stretched in an extensiometer. The reaction could be promoted by acid pH and inhibited by several OH scavengers. Generation of OH by the same reaction in living coleoptile or hypocotyl segments caused elongation growth. Auxin-induced elongation growth of maize coleoptiles could be inhibited by OH scavengers. Auxin promoted the production of superoxide radicals (O2(-)), an OH precursor, in the growth-controlling outer epidermis of maize coleoptiles. It is concluded that OH fulfils basic criteria for a wall-loosening factor acting in auxin-mediated elongation growth of plant species with widely differing cell-wall polysaccharide compositions.     

Assessing the Rhizotoxicity of the Aluminate Ion, Al(OH)(4)

Dissolved aluminum (III) in acidic soils or culture media is often rhizotoxic (inhibitory to root elongation). Alkaline solutions of Al are also sometimes rhizotoxic, and for that reason toxicity has been attributed to the aluminate ion, Al(OH)₄⁻. In the present study, seedlings of wheat (Triticum aestivum L. cv Tyler) and red clover (Trifolium pratense L. cv Kenland) were cultured in aerated aluminate solutions at pH 8.0 to 8.9. The bulk phases of these solutions were free of reactive polynuclear hydroxy-Al (including the extremely toxic species AlO₄Al₁₂[OH]₂₄[H₂O]⁷⁺₁₂ [Al₁₃]) according to the ferron (8-hydroxy-7-iodo-5-quinolinesulfonic acid) assay. At an aluminate concentration of 25 micromolar (23 micromolar activity) and a pH of 8, root elongation was less than 40% of Al-free controls, but at pH 8.9 elongation was 100% of controls. The hypothesis is offered that aluminate is nontoxic and that the inhibition at lower pH values is attributable to Al₁₃ postulated to have formed in the acidic free space of the roots where the ratio /{Al³⁺/}//{H⁺/}³ may rise above 10¹⁰. At this value hydroxy-Al in over-saturated, alkaline solutions begins to undergo rapid conversion to polynuclear species.     

Nitrate does not result in iron inactivation in the apoplast of sunflower leaves

It has been hypothesized that nitrate (NO(3)(-)) nutrition might induce iron (Fe) deficiency chlorosis by inactivation of Fe in the leaf apoplast (H.U. Kosegarten, B. Hoffmann, K. Mengel [1999] Plant Physiol 121: 1069-1079). To test this hypothesis, sunflower (Helianthus annuus L. cv Farnkasol) plants were grown in nutrient solutions supplied with various nitrogen (N) forms (NO(3)(-), NH(4)(+) and NH(4)NO(3)), with or without pH control by using pH buffers [2-(N-morpholino)ethanesulfonic acid or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. It was shown that high pH in the nutrient solution restricted uptake and shoot translocation of Fe independently of N form and, therefore, induced Fe deficiency chlorosis at low Fe supply [1 micro M ferric ethylenediaminedi(O-hydroxyphenylacetic acid)]. Root NO(3)(-) supply (up to 40 mM) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted (59)Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO(3)(-) (6 mM) showed no effect on (59)Fe uptake by the symplast of leaf cells. However, high light intensity strongly increased (59)Fe uptake, independently of apoplastic pH or of the presence of NO(3)(-) in the apoplastic solution. Finally, there are no indications in the present study that NO(3)(-) supply to roots results in the postulated inactivation of Fe in the leaf apoplast. It is concluded that NO(3)(-) nutrition results in Fe deficiency chlorosis exclusively by inhibited Fe acquisition by roots due to high pH at the root surface.     

Aluminium distribution in soybean root tip for a short time Al treatment

Using the lumogallion staining method which we developed (Kataoka et al. 1997a), Al movement in soybean (Glycine max. (L.)Merr. cv. Tsurunoko) root tips treated for a short time was studied. We have indicated that the majority of Al accumulated in the root was found between 0 and 2 mm from the root apex within 2 h (Kataoka et al. 1997a, b). In the study presented here two-day seedlings of the soybean were treated with 50 μmol/L AlCl₃ (pH 4.4), including 0.2 mmol/L CaCl₂, for 1 h, and Al accumulation in the root sections at both 1 and 2 mm apart from root apex was analyzed by a confocal laser microscopy. Although the early indicators, callose induction and the decrease of growth recovery, were not observed in the root when treated for 15 min, a trace amount of Al was already incorporated into the nucleus of cells and the middle tissue of the root. The non-toxic level of Al was more rapidly absorbed than previously thought. The initial increase of callose accumulation and the reduction of the growth recovery were found after 30 min. Therefore, the difference between Al accumulation profiles of 15 and 30 min was analyzed to find out what triggered a toxic Al effect. Increase of Al accumulation in whole root tissue was observed in the root sections, at both 1 and 2 mm from the root apex, and the greatest amount of Al was found in the cytoplasm of the outer cortex, 1 mm away from the root apex. These results are consistent with the fact that Al exclusion from root tip cells is an important mechanism of Al tolerance.     

The apoplastic pH of the Zea mays root cortex as measured with pH-sensitive microelectrodes: aspects of regulation

In the root cortex of Zea mays the apoplastic pH and aspects of its regulation were investigated using pH-sensitive microelectrodes. To measure the pH directly in different cell layers of the apoplast sharp double-barrelled electrodes were applied, whereas blunt pH-electrodes were used simultaneously to measure the pH at the root surface. Recordings carried out 8-10 mm behind the root tip show that the apoplastic pH is maintained between 5.1 and 5.6, depending on the given experimental conditions, i.e. varying external [K⁺], [Ca²⁺], pH, weak buffering, as well as perfusion of the test medium. When the medium pH (bulk) differs considerably from the apoplastic pH, a small pH gradient is built up between the root surface (unstirred layer) and the outer cortex layers. In a standing medium these gradients equilibrate. The apoplastic pH responds to increases in external [K⁺] and [CA²⁺] with an acidification, which is attributed to ion-exchange properties of the cell wall constituents. Stimulation of proton pump activity with fusicoccin acidifies the apoplast from pH 5.6 to pH 4.8, while deactivation of the pump with cyanide/salicylhydroxamic acid increases the pH of the apoplast from 5.6 to 6.2, and further to pH 6.6 with CCCP. The Ca²⁺ channel antagonists nifedipine and La³⁺ also increase the apoplastic pH. It is suggested that not only the proton pump, but also the cation channels may contribute to the regulation of the apoplastic pH.Keywords: Apoplast, ion-selective microelectrodes, pH, unstirred layer, Zea mays, root.      

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.     

Entry and distribution of aluminum in Zea mays

Summary The electron microprobe X-ray analyzer (microprobe) has been used to determine the mode of entry of aluminum (Al) and its distribution and localization in the corn plant. Microprobe analysis is a non-destructive method allowing for multiple element analysis in the same tissues, cells or cell organelles.Al was found to be precipitated on the surface of the epidermal cells of the root with no penetration into the cortex as long as the root surface remained intact. The root cap was freely permeable and contained the highest concentration of Al. The epidermal layer behind the root cap prevented movement into the cortex and conductive tissue.The penetration of a lateral root through the endodermis, cortex and epidermis provided a channel of entry for Al into the cortex and conducting tissues of both the lateral and main root. Essentially no Al was found in the transition zone and only small quantities were present in the above-ground plant parts.The localization of phosphorus was exactly the same as that of Al. This suggested that there was a precipitation of P by Al. A similar analysis for calcium and phosphorus on control plants did not reveal such a precipitation.The method of sample preparation was critical in retaining and localizing the elements in question and is discussed in that light.Michigan Agricultural Experiment Station Journal Article No. 4269.     

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.     

Apoplastic pH and FeIII reduction in young sunflower (Helianthus annuus) roots

The relationship between the apoplastic pH in young sunflower roots ( Helianthus annuus L.) and the plasmalemma ferric chelate reductase (FC-R; EC 1.16.1.7) activity in roots was investigated. The hypothesis was tested that a high apoplastic pH depresses FC-R activity, thereby restricting the uptake of Fe²⁺ into the cytosol. Until recently, little has been known about this relationship, because pH and redox reaction measurements are difficult to perform within the confines of the root apoplast. We recorded the apoplastic pH by means of the fluorescence ratio in conjunction with video microscopy by covalently tagging fluorescein boronic acid to OH groups of the root cell wall. Fe^III reduction was measured using a similar approach by tagging ferrozine diboronic acid with OH groups of the cell wall. Ferrozine forms an Fe²⁺ complex, thus indicating the reduction of ferric iron. In roots bathing in buffered outer solutions of different pH, a high pH sensitivity of apoplastic Fe^III reduction was found, with the highest ferric iron reduction rates at an apoplastic pH of 4.9; above an apoplastic pH of 5.3, no reduction was observed. Nitrate in the bathing solution increased the apoplastic pH and hence depressed the Fe^III reduction; ammonium had the reverse effect. Nitrate together with HCO₃^–, a combination which is typical of calcareous soils, had the strongest depressing effect. From the results, it can be concluded that the main reason for the frequently occurring iron deficiency chlorosis of plants grown on calcareous soils is the inhibition of Fe^III reduction in the apoplast, and hence Fe²⁺ uptake into the cytosol.     

Microbeam analysis of Ca exchange and uptake in the fine roots of spruce: influence of pH and aluminum

Summary A novel stable isotope labelling procedure for microbeam analysis was developed to monitor exchange and uptake of nutrients, primarily Mg, K and Ca, by root tips at the cellular level. Initially root samples were analysed from 2-year-old spruce trees, originating both from a nursery and from a polluted forest site, (1) for the cortex cell wall accessibility and nutrient binding properties, (2) for the influence of low pH and elevated aluminum concentrations on Ca binding to cortex cell walls, and (3) for long-range transport into the secondary xylem, proximal to the labelled root tip. In nursery control plants, Ca is localized mainly in the apoplast of the cortex. Exchange of Mg, K, Ca in the cell wall of the cortex and the primary xylem with label in incubation solutions is almost completed to equilibration within 30 min. In the secondary xylem we could detect Mg, K, and Ca from labelling solutions in minute amounts after 30 min, and as a major fraction after 48 h. This indicates that stable isotope labelling can be used to study both ion-exchange properties of the apoplast and long-range transport. Slight acidification of the labelling incubation media to pH 4.5 reduced Ca binding to the cortex cell walls slightly, but acidification to the extreme value of pH 2.3 reduced binding 41%. A combination of pH 4.5 and increased free aluminum reduced the binding by 83%. In a preliminary attempt to analyse the nutrient binding capability of the root-tip apoplast from pollution affected trees, we exposed fine roots of 2-year-old spruce from an acidified and polluted site showing typical low levels of Ca and Mg in the cortical cell walls to Ca-enriched media. Under these conditions the Ca content of cortex cell walls doubled upon incubation at pH 4.7, reaching 40% of the total binding capacity of our nursey control plants.     

The use of root growth and modelling data to investigate amelioration of aluminium toxicity by silicon in Picea abies seedlings

Three-week-old Picea abies seedlings were grown for 7 days in 100 microM aluminium (Al), combined with 1000 or 2000 microM silicon (Si). Solution pH was adjusted to 4.00, 4.25, 4.50, 4.75, or 5.00. In the absence of Si, solution pH had no effect on the decrease in root growth caused by 100 microM Al. Silicon did not ameliorate toxic effects of Al on root growth at pH 4.00, 4.25 and 4.50, whereas significant, and apparently complete, amelioration was found at pH 4.75 and 5.00. An equilibrium speciation model (EQ3NR), with a current thermodynamic database, was used to predict the behaviour of Al and Si in growth solutions. When Si was not present in the 100 microM Al solutions, Al(3+) declined from 92.4% of total Al at pH 4.00 to 54.6% at pH 5.00, and there was a concomitant increase in hydroxyaluminium species as pH increased. The addition of 1000 microM Si to the 100 microM Al solutions caused a reduction in Al(3+) content over the whole pH range: at pH 4.00 Al(3+) fell from 92.4 to 83.3% in the presence of Si; and at pH 5.00 the fall was from 54.6 to 17.7%. These falls were attributed to the formation of hydroxyaluminosilicate (HAS) species. Similar, but somewhat greater, changes were observed in solutions containing 2000 microM Si. The match between root growth observations and the modelling data was not very good. Modelling predicted that change in Al(3+) content with pH in the presence of Si was gradual, but root growth was markedly increased between pH 4.50 and 4.75. Differences between root growth and modelling data may be due to the model not correctly predicting solution chemistry or to in planta effects which override the influence of solution chemistry.     

Distribution and chemical speciation of aluminum in the Al accumulator plant,Melastoma malabathricum L.

The Al accumulation mechanisms in an Al accumulator plant, Melastoma malabathricum L. (Melastoma), was investigated. Al was located in the upper epidermal cells and also distributed in mesophyll cells in leaf sections. In root sections, Al was found in all the root tissues, particularly in the epidermis and endodermis. Al concentrations in young leaves, mature leaves, old leaves, and roots were 8.0, 9.2, 14.4, and 10.1 mg g¹, respectively. Approximately 45% of total Al in oldest leaves, and approximately 60% of total Al in leaves of other positions and roots were extracted in Tris-HCl buffer (pH 7.0). Since Al in the residual parts was mostly dissolved in hot 0.5 M H₂SO₄ containing 2% cetyl trimethylammonium bromide, residual Al seemed to consist mainly of monomeric Al and Al bound to pectic substances and hemicellulose. Al in the Tris-HCl extract consisted of non-monomeric Al (complexed form). Oxalate concentration in the Tris-HCl extract in leaves was significantly higher in the +Al treatment than in the –Al treatment and there was a positive correlation between the Al concentration and oxalate concentration. ²⁷Al NMR spectrum of fresh leaves indicated the presence in the order of monomeric Al, Al-oxalate, Al-(oxalate)₂, and Al-(oxalate)₃ in intact leaves.     

Probing the role of calmodulin in Al toxicity in maize

The role of calmodulin on Al toxicity was studied in two maize (Zea mays L.) inbred lines, Cat 100-6 (Al-tolerant) and S 1587-17 (Al-sensitive). Increasing levels of Al induced the release of malate at similar rate by roots of both genotypes, while the exudation of citrate, a stronger Al-binding compound, was 3.5 times higher in Cat 100-6 seedlings exposed to 16.2x10(-6) Al(3+) activity. The calmodulin inhibitor trifluoperazine significantly reduced the root growth in both genotypes, mimicking the main effect of Al. However, when Cat 100-6 and S 1587-17 seedlings were challenged with Al in conjunction with trifluoperazine, no further reduction in root growth or any other effect of Al toxicity was observed. The rate of Al-induced citrate exudation by both genotypes was not affected by treatment with trifluoperazine or calmidazolium, another calmodulin inhibitor. The Al(3+) interaction with cytoplasmic CaM was estimated using models for the binding of Al(3+) and Mg(2+) with CaM and physiological concentrations of citrate, CaM, InsP(3), ATP, ADP, Al(3+) and Mg(2+). In this simulation, Al(3+) associated with citrate and InsP(3), but not with CaM. We conclude that calmodulin is not relevant to the physiological processes leading to the Al tolerance in maize, nor is it a primary target for Al toxicity.     

High pH Causes Disintegration of the Root Surface in Lupinus angustifolius L.

The effect of high pH on the morphology and anatomy of the rootsof lupin (Lupinus angustifolius L. cv. Yandee) and pea (Pisumsativum L. cv. Dundale) was examined in buffered solution. Themorphology and anatomy of lupin roots were markedly altered,and root growth was reduced by increasing solution pH from 5·2to 7·5, whereas pea roots were unaffected. In lupin roots,pH 7·5 caused disintegration of the root surface andimpaired root hair formation. Lupin roots grown at pH 7·5also had decreased cell lengths but increased cell diameterin both the epidermis and the cortex in comparison to rootsgrown at pH 5·2. High pH reduced cell volume greatlyin the epidermis, to a lesser extent in the outer cortex andnot at all in the inner cortex. It appears that in lupins, theprimary detrimental effects of growth at pH 7·5 is reducedlongitudinal growth of cells near the root surface with a consequentreduction in elongation of the cells in inner cortex.Copyright1993, 1999 Academic Press Lupinus angustifolius L., Pisum sativum L., high pH, root morphology, root anatomy