The beneficial effect of aluminium and the role of citrate in Al accumulation in Melastoma malabathricum

* Here we investigated the beneficial effect of aluminium (Al) on the development of the Al accumulating plant Melastoma malabathricum. * Seedlings of M. malabathricum were cultivated in a nutrient solution containing 0.5 mM Al and compared with barley (Hordeum vulgare). In addition, roots of M. malabathricum were divided into one part growing in a nutrient solution, and the other part growing in a calcium solution. Al (0.5 mM) was applied to either solution. * Al-induced improvements of the root activity contributed to a growth enhancement in M. malabathricum. Al exposure without nutrients did not increase root growth and Al accumulation in the leaves. The beneficial effect, however, was induced by the combination of Al and nutrients. * We suggest that without nutrients roots are not able to synthesize an adequate amount of citrate that is required for transporting Al to the leaves. High Al levels in the plant tissues and/or an interaction of Al with particular nutrient elements in the apoplast of root cells appear to be essential to exert the beneficial effect of Al.     

Rapid Uptake of Aluminum into Cells of Intact Soybean Root Tips (A Microanalytical Study Using Secondary Ion Mass Spectrometry)

A wide range of physiological disorders has been reported within the first few hours of exposing intact plant roots to moderate levels of Al3+. Past microanalytic studies, largely limited to electron probe x-ray microanalysis, have been unable to detect intracellular Al in this time frame. This has led to the suggestion that Al exerts its effect solely from extracellular or remote tissue sites. Here, freeze-dried cryosections (10 [mu]m thick) collected from the soybean (Glycine max) primary root tip (0.3-0.8 mm from the apex) were analyzed using secondary ion mass spectrometry (SIMS). The high sensitivity of SIMS for Al permitted the first direct evidence of early entry of Al into root cells. Al was found in cells of the root tip after a 30-min exposure of intact roots to 38 [mu]M Al3+. The accumulation of Al was greatest in the first 30 [mu]m, i.e. two to three cell layers, but elevated Al levels extended at least 150 [mu]m inward from the root edge. Intracellular Al concentrations at the root periphery were estimated to be about 70 nmol g-1 fresh weight. After 18 h of exposure, Al was evident throughout the root cross-section, although the rate of accumulation had slowed considerably from that during the initial 30 min. These results are consistent with the hypothesis that early effects of Al toxicity at the root apex, such as those on cell division, cell extension, or nutrient transport, involve the direct intervention of Al on cell function.     

Preliminary results from a microscopic examination on the effects of aluminium on the root tips of wheat

Root tips from aluminium (Al) tolerant (Waalt) and Al sensitive (Warigal) wheat (Triticum aestivum (L). Thell.) cultivars exposed to low concentrations of Al (10 M) for 10, 24 and 72 hours were examined under the light and electron microscope. After fixing and embedding, longitudinal and transverse thin and ultrathin sections were cut. There was no evidence of Al damage to the root tips of the Al tolerant cultivar under both the light and electron microscope. For the Al sensitive cultivar, Al had no observable effect on the root tips 10 hours after Al addition when examined under the light microscope. When examined under an electron microscope, electron dense globular deposits were observed between the cell wall and cell membrane of the epidermal cells. There was not obvious damage to the cell cytoplasm. Two or 3 days after Al addition, light microscopy showed that the cells in the root tips had become swollen and extensively vacuolated. The tissues appeared disorganised and degenerate, particularly in the epidermis and outer cortical cells. The electron microscope also revealed a thickening of the cell wall. The cell wall was broken down, particularly in the epidermis in the region 4–6 mm from the root tip. The tissue in the meristematic area was largely intact.     

Localization and compartmentation of Al in the leaves and roots of tea plants

Under acid soil conditions, solubility of aluminum (Al) increases leading to toxicity for plants. Al accumulator species such as tea, however, accumulate high levels of Al in tissues without toxicity symptoms. In this work, Al localization and compartmentation were studied in tea [Camellia sinensis (L.) O. Kuntze] grown hydroponically at 0 or 100 µM Al for eight weeks. Plant dry matter production was significantly higher in the presence of Al and accumulated up to 1.21 and 6.18 mg Al/g DW in the leaves and roots, respectively. About 40-50% of Al was partitioned into cell wall (CW)-bound fraction without any difference among leaves of different age and roots. A significant increase of the soluble phenolics fraction by Al was observed in both leaves and roots. Conventional and confocal laser scanning microscopy images of morin-stained roots indicated a high fluorescence signal in the caps and adjacent mersitematic cells. Towards basal parts, however, Al tended to accumulate mainly in the root hairs, rhizodermal and endodermal cell layers and slightly in the cortex while it was clearly excluded from the central cylinder. A high Al-morin signal was detected from the CW compared with other parts of the cells. Relatively high green fluorescence signal was emitted from the epidermal cell layer, trichomes, vascular bundle region and stomatal cells of particularly young leaves. Our study provides evidences for involvement of both avoidance and tolerance mechanisms for Al in tea plants.     

Distribution and mobility of aluminium in an Al-accumulating plant, Fagopyrum esculentum Moench

Buckwheat (Fagopyrum esculentum Moench. cv. Jianxi) accumulateshigh concentrations of Al in the leaves without showing anytoxicity. To understand the accumulation mechanism of Al inbuckwheat, the distribution and mobility of Al in buckwheatwere investigated. Relatively long-term treatment (28 d) withAl led to a decrease in Al concentration from old to young leaves,while a short-term (1 d) exposure to Al resulted in a uniformdistribution of Al in the leaves. When the fourth leaf was wrappedinside a transparent plastic bag to suppress transpiration,the Al concentration of this leaf was only one-quarter of thatin the corresponding leaf without wrappng. Within a leaf, theAl concentration at the margins was much higher than that inthe centre. These results indicate that Al distribution in theleaves is controlled by both rate and duration of transpiration.The mobility of Al between old and new leaves was studied byfirst growing plants in a solution with Al, followed by culturein a solution without Al. The Al content in the two new leavesappeared after removal of external Al was very low, whereasthat in the old leaves did not decrease but continued to increase.The increased Al content was found to be translocated from Alremaining in the roots. It is concluded that Al is not mobileonce it is accumulated in the leaf. Key words: Accumulation, aluminium, buckwheat, distribution, mobility, transpiration.     

Localization of aluminium in tea leaves

More than 30,000 ppm of aluminium (Al) was found accumulatedin old tea leaves. The localization of Al in epidermal cellswas detected by light microscopic observation and electron microprobeX-ray analysis. A distinct thickening of epidermal cell wallwas seen in old leaves. The addition of AICI₃ to the culturalsolution promoted the growth of tea seedlings. (Received February 2, 1976; )     

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.     

The effect of aluminium and media on the growth of mycorrhizal and nonmycorrhizal highbush blueberry plantlets

A factorial experiment was conducted to determine the effect of aluminium (0 and 600M) and media (sand, and 1:1 sand:soil) on mycorrhizal (M) and non-mycorrhizal (NM) highbush blueberry plantlets. There were no differences in nutrient uptake and total plant dry weight between M and NM plantlets. However, more root growth, as determined by dry weight, was observed in M than NM plantlets. The plantlets growing in sand had more dry weight than did those in the soil medium. Although the root growth and shoot growth were reduced by the 600M Al treatment, the direct effect of Al on plantlet growth was not clear due to Al and P interactions. Plant nutrient uptake was reduced by high concentrations of Al, suggesting that high Al concentration limited the ability of roots to acquire most of the nutrients. Mycorrhizal cortical cell infection levels of 15–20% wene maintained in the roots in soil medium but decreased to about 5% over the 6 weeks of the experiment in the sand medium. Although M plantlets accumulated more Al in their roots, Al was readily transported to the leaf tissues of M and NM plantlets.     

The ability of the biological control agent Bacillus subtilis, strain BB, to colonise vegetable brassicas endophytically following seed inoculation

The ability of Bacillus subtilis, strain BB, to colonise cabbage seedlings endophytically was examined following seed inoculation. Strain BB was recovered from different plant parts including leaves (cotyledons), stem (hypocotyl) and roots. While high bacterial populations persisted in the roots and lower stem, they were lower in the upper stem and leaves through time. In addition to cabbage, strain BB colonised endophytically the roots of 5 other vegetable brassicas. Fatty acid methyl ester (FAME) and PCR fingerprinting analysis confirmed the reliability of the detection method. Studies conducted with transmission electron microscope (TEM) showed that BB mainly colonised intercellular spaces of cortical tissues including intercellular spaces close to the conducting elements of roots and stem of cabbage seedlings. Gold labelling was specifically associated with BB and the fibrillar material filling the intercellular spaces where bacterial cells were found.     

Improved correlation between soil exchangeable K and plant K contents on a tissue water basis

In acid volcanic soils, plant roots are thought to be injured by acidity (low pH) and/or solubilized aluminium (Al) ions. An attempt was made to separate the effects of low pH from those of Al on the elongation and viability of alfalfa (Medicago sativa L.) radicles in water culture. Root elongation was irreversively curtailed by 20 hours treatment at pH 4.0 without Al or 20 mmol m^-3 Al at pH 5.0. Viability of surface cells of root tips was detected as a degrading activity of fluorescein diacetate (FDA) by cellular esterases and subsequent accumulation of derived fluorescein within cells. Large numbers of the surface cells lost their viability after four hours exposure at the low pH. In contrast, surface cells maintained both FDA degrading activity and ability to accumulate fluorescein 20 h after initial exposure to the Al solution (20 mmol Al m^-3, pH 5.0). These results suggest that there are some significant differences in the mechanisms of phytotoxicity to alfalfa root between the two stress factors.     

Aluminium Toxicity Modulates Nitrate to Ammonia Reduction

Two weeks-old maize (Zea mays cv. XL-72.3) plants were exposed to Al concentrations 0 (Al0), 9 (Al9), 27 (Al27) or 81 (Al81) g m-3 for 20 d in a growth medium with low ionic strength. Thereafter, the Al concentration-dependent interactions on root nitrate uptake, and its subsequent reduction to ammonia in the leaves were investigated. Al concentrations in the roots sharply increased with increasing Al concentrations while root elongation correspondingly decreased. Root fresh and dry masses, acidification capacity, and nitrate and nitrogen contents decreased from Al27 onwards, whereas leaf nitrogen, nitrate, nitrite, and ammonia concentrations decreased starting with Al9. Electrolytic conductance increased by 60 % in root tissues from Al0 to Al81 but it did not increase significantly in the leaves. In Al9, Al27, and Al81 plants a decrease in shoot fresh and dry masses was observed. Al concentrations between 0 and 27 g m-3 increased net photosynthetic rate, stomatal conductance, and the quantum yield of photosynthetic electron transport, whereas the intercellular CO2 concentration was minimum in Al27 plants. In the leaves, nitrate reductase (E.C. 1.6.6.1) activity increased until Al27, and nitrite reductase (E.C. 1.6.6.4) activity until Al81. Hence there may be an Al mediated extracellular and intracellular regulation of root net nitrate uptake. Nitrate accumulation in the roots affects the translocation rates and, therefore, the nitrate concentration in the leaves. The in vivo reducing power generated by the photosynthetic electron flow does not limit nitrate to ammonia reduction, and the increase of maximum nitrate and nitrite reductase activities parallels the decreasing nitrate, nitrite, and ammonia concentrations.     

The effect of aluminium on the distribution of calcium,magnesium and phosphorus in mycorrhizal and non-mycorrhizal seedlings of Eucalyptus rudis: a cryo-microanalytical study

The distribution of Al, Ca, Mg and P in the lateral roots and leaves of mycorrhizal and non-mycorrhizal seedlings of Eucalyptus rudis grown with and without Al was analysed using energy-dispersive X-ray microanalysis on a cryo-scanning electron microscope. Al accumulated in all tissues of nonmycorrhizal plants: the endodermis was not a barrier to the translocation of Al. In mycorrhizal roots, Al was concentrated within the sheath. The presence of Al reduced the levels of Ca and Mg in both mycorrhizal and non-mycorrhizal roots and shoots in comparison with control plants. The presence of mycorrhizas increased the levels of Ca and Mg in plants grown with Al in comparison with non-inoculated plants, although there was no evidence that mycorrhizas increased the levels of P in plants grown in Al-amended soils. P levels were higher in the mycorrhizal sheath of plants grown with Al than the controls.     

Structural Changes and Aluminium Distribution in Maize Root Tissues

Growth and structural responses of primary roots of Zea mays L. to aluminium chloride were studied. The treatment of seedlings with 50 μM AlCl₃ resulted in high accumulation of Al, partial inhibition of root growth, occurrence of surface lesions in peripheral tissues, root thickening caused by expansion of inner cortical cells, reduced root cap length, extensive vacuolation, cell distortion, and increased synthesis of callose within 24 h. This revised version was published online in July 2006 with corrections to the Cover Date.     

The role of amino acids and sugars in supporting of osmotic homeostasis in maize seedlings under salinization conditions and treatment with synthetic growth regulators

Stress state in plants caused by salinization conditions is characterized by the disturbance of ionic and osmotic homeostasis. The maintenance of the latter is reached by accumulation of osmolytes including free amino acids and soluble sugars in cells. The free amino acid level in the 8-day-old control seedling leaves was higher, than in the roots, whereas the contrary picture was observed in 17-day-old plant tissues. At the same time 8-day-old seedling roots contained more total sugars, than leaves, although the reduced sugar content was nearly a half of the total sugar content. A decrease of both total and reduced sugar levels was observed in 17-day-old seedling tissues. One-day exposure of 7-day-old seedlings to 0.1 M NaCl increased the free amino acid content especially in roots, than in leaves, and the total sugar content in maize leaves, whereas in roots this level remained without changes. The prolongation of salt exposure to 10 days leads to osmolyte content decrease. The seed treatment with Methyure and Ivine intensified accumulation of free amino acids and soluble sugars in the root and leaf tissues under salinization conditions.     

Endophytic colonization of rice by a diazotrophic strain of Serratia marcescens

Six closely related N2-fixing bacterial strains were isolated from surface-sterilized roots and stems of four different rice varieties. The strains were identified as Serratia marcescens by 16S rRNA gene analysis. One strain, IRBG500, chosen for further analysis showed acetylene reduction activity (ARA) only when inoculated into media containing low levels of fixed nitrogen (yeast extract). Diazotrophy of IRBG500 was confirmed by measurement of 15N2 incorporation and by sequence analysis of the PCR-amplified fragment of nifH. To examine its interaction with rice, strain IRBG500 was marked with gusA fused to a constitutive promoter, and the marked strain was inoculated onto rice seedlings under axenic conditions. At 3 days after inoculation, the roots showed blue staining, which was most intense at the points of lateral root emergence and at the root tip. At 6 days, the blue precipitate also appeared in the leaves and stems. More detailed studies using light and transmission electron microscopy combined with immunogold labeling confirmed that IRBG500 was endophytically established within roots, stems, and leaves. Large numbers of bacteria were observed within intercellular spaces, senescing root cortical cells, aerenchyma, and xylem vessels. They were not observed within intact host cells. Inoculation of IRBG500 resulted in a significant increase in root length and root dry weight but not in total N content of rice variety IR72. The inoculated plants showed ARA, but only when external carbon (e.g., malate, succinate, or sucrose) was added to the rooting medium.     

Inhibitory effect of aluminium on the axonal transport of HRP microinjected into dorsal root ganglion neurons in vitro

In the present study the function of axonal transport in individual neurons under aluminium intoxication was investigated experimentally in comparison with controls. We used the technique of microinjection of horseradish peroxidase (HRP) in dissociated dorsal root ganglia (DRG) neurons and neurons of explant cultures of DRG. Different exposure periods (1 and 6 hours as well as 6 and 10 days) to aluminium were analysed quantitatively. This analysis revealed an impaired anterograde transport of HRP already after a short aluminium intoxication period of only 1 hour in DRG cells in vitro, an effect that increased with a prolonged aluminium exposure for up to 10 days. Hence, functional alterations of the anterograde transport caused by aluminium could be detected even after short exposure periods. Furthermore, the effects of aluminium on anterograde transport mechanisms were reversible 8 days after removal of aluminium. To determine how aluminium affects the cytoskeleton, we performed immunohistochemistry and electron microscopy on cultured DRG neurons. Distinct morphological alterations of the cytoskeleton, especially the accumulation of phosphorylated neurofilaments, appeared after 6 days of aluminium exposure. Our results suggest that neurofilaments are indispensable to the functional integrity of the cytoskeleton and its ability to mediate microtubule-based axonal transport processes.     

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.     

Effect of sub-lethal concentrations of aluminium on the filtration activity of the freshwater mussel Anodonta cygnea L. at neutral pH

Significant amounts of aluminium (Al) are commonly present in rivers and lakes, largely in particulate form in neutral waters. Freshwater bivalves, as filter feeders are therefore exposed to both particulate and dissolved metal and are potentially vulnerable to Al. The effect of Al on filtering behaviour of the freshwater mussel Anodonta cygnea L. was investigated during short (1 hour) and long-term (15 days) exposure to environmentally relevant concentrations (250 and 500 microg l(-1)) at neutral pH. Water flow through the outflow siphon was monitored as an indicator of pumping capacity. Short-term (1 hour) exposure to 500 microg l(-1) added Al produced an irreversible decrease in the duration of filtering periods, presumably as an avoidance response to the toxicant. One-hour exposure 250 microg l(-1) Al had no detectable effect. When mussels were exposed to 250 or 500 microg l(-1) added Al for 15 days, siphon activity measured in days 11-15 of exposure was inhibited by 50% and 65%, respectively, compared to pre-exposure levels. Recovery occurred following transfer of mussels to uncontaminated water. Interaction between Al and freshwater bivalves at neutral pH may affect both the performance of the mussels and the chemical speciation of the metal in the natural environment.     

Expression and Localization of Plant Protein Disulfide Isomerase

A cDNA clone encoding a putative protein disulfide isomerase (PDI, EC 5.3.4.1) from alfalfa (Medicago sativa L.) was expressed in Escherichia coli cells, and an antiserum was raised against the expressed PDI-active protein. The antiserum recognized a protein of approximately 60 kD in extracts from alfalfa, soybean, and tobacco roots and stems. Levels of this protein remained relatively constant on exposure of alfalfa cell suspension cultures to the protein glycosylation inhibitor tunicamycin, whereas a slightly lower molecular mass form, also detected by the antiserum, was induced by this treatment. A lower molecular mass form of PDI was also observed in roots of alfalfa seedlings during the first 5 weeks after germination. PDI levels increased in developing soybean seeds up to 17 d after fertilization and then declined. Tissue print immunoblots revealed highest levels of PDI protein in the cambial tissues of soybean stems and petioles and in epidermal, subepidermal, cortical, and pith tissues of stems of alfalfa and tobacco. Immunogold electron microscopy confirmed the localization of PDI to the endoplasmic reticulum in soybean root nodules.     

Influence of aluminium on phosphate and calcium uptake in beech (Fagus sylvatica) grown in nutrient solution and soil solution

The effects of AICI₃ on uptake of Ca²⁺ and phosphate in roots of intact beech ( Fagus sylvatica L. provenance Maramures) plants were studied in nutrient solution and soil solution. Aluminium reduced the concentrations of Ca, Mg and P in plants and increased that of K. In short term experiments, uptake of Ca²⁺(⁴⁵Ca) was reduced by exposure of the roots to Al. The effect of aluminium on Ca²⁺(⁴⁵Ca) uptake was immediate and primarily of a competitive nature, preventing Ca²⁺ from being adsorbed. Uptake of ³²P-phosphate increased with increasing Al concentration up to 0.1 m M and then decreased at higher Al concentrations. The effect of Al on ³²P-phosphate uptake was most pronounced during the first hours of exposure. Growth of plants for 15 days in soil solution, collected from the upper A horizon of a beech forest soil, had no effect on uptake of Ca²⁺(⁴⁵Ca) and ³²P-phosphate, probably because of a low concentration of labile bound monomeric Al and binding of Al to organic compounds. Soil solution from the deeper B horizon reduced Ca²⁺(⁴⁵Ca) uptake and increased ³²P-phosphate uptake in a manner similar to that with Altreatment in nutrient solution. It is concluded that in soil solution from the deeper regions of the soil, mineral uptake by roots was affected by Al.