Use of comprehensive target analysis for determination of contaminants of emerging concern in a sediment core collected from Beppu Bay, Japan.

In recent years, concern about the release of anthropogenic organic micropollutants referred to as contaminants of emerging concern (CECs) has been growing. The objective of this study was to find potential CECs by means of an analytical screening method referred to as comprehensive target analysis with an automated identification and quantification system (CTA-AIQS), which uses gas and liquid chromatography combined with mass spectrometry (GC-MS and LC-QTOF-MS). We used CTA-AIQS to analyze samples from a sediment core collected in Beppu Bay, Japan. With this method, we detected 80 compounds in the samples and CTA-AIQA could work to useful tool to find CECs in environmental media. Among the detected chemicals, three PAHs (anthracene, chrysene, and fluoranthene) and tris(isopropylphenyl)phosphate (TIPPP) isomers were found to increase in concentration with decreasing sediment depth. We quantified TIPPP isomers in the samples by means of targeted analysis using LC-MS/MS for confirmation. The concentration profiles, combined with previous reports indicating persistent, bioaccumulative, and toxic properties, suggest that these chemicals can be categorized as potential CECs in marine environments.

Aluminium reduces sugar uptake in tobacco cell cultures: a potential cause of inhibited elongation but not of toxicity.

Aluminium is well known to inhibit plant elongation, but the role in this inhibition played by water relations remains unclear. To investigate this, tobacco (Nicotiana tabacum L.) suspension-cultured cells (line SL) was used, treating them with aluminium (50 microM) in a medium containing calcium, sucrose, and MES (pH 5.0). Over an 18 h treatment period, aluminium inhibited the increase in fresh weight almost completely and decreased cellular osmolality and internal soluble sugar content substantially; however, aluminium did not affect the concentrations of major inorganic ions. In aluminium-treated cultures, fresh weight, soluble sugar content, and osmolality decreased over the first 6 h and remained constant thereafter, contrasting with their continued increases in the untreated cultures. The rate of sucrose uptake, measured by radio-tracer, was reduced by approximately 60% within 3 h of treatment. Aluminium also inhibited glucose uptake. In an aluminium-tolerant cell line (ALT301) isogenic to SL, all of the above-mentioned changes in water relations occurred and tolerance emerged only after 6 h and appeared to involve the suppression of reactive oxygen species. Further separating the effects of aluminium on elongation and cell survival, sucrose starvation for 18 h inhibited elongation and caused similar changes in cellular osmolality but stimulated the production of neither reactive oxygen species nor callose and did not cause cell death. We propose that the inhibition of sucrose uptake is a mechanism whereby aluminium inhibits elongation, but does not account for the induction of cell death.

Mitochondrial alterations related to programmed cell death in tobacco cells under aluminium stress.

The present investigation was undertaken to verify whether mitochondria play a significant role in aluminium (Al) toxicity, using the mitochondria isolated from tobacco cells (Nicotiana tabacum, non-chlorophyllic cell line SL) under Al stress. An inhibition of respiration was observed in terms of state-III, state-IV, succinate-dependent, alternative oxidase (AOX)-pathway capacity and cytochrome (CYT)-pathway capacity, respectively, in the mitochondria isolated from tobacco cells subjected to Al stress for 18 h. In accordance with the respiratory inhibition, the mitochondrial ATP content showed a significant decrease under Al treatment. An enhancement of reactive oxygen species (ROS) production under state-III respiration was observed in the mitochondria isolated from Al-treated cells, which would create an oxidative stress situation. The opening of mitochondrial permeability transition pore (MPTP) was seen more extensively in mitochondria isolated from Al-treated cells than in those isolated from control cells. This was Ca(2+) dependent and well modulated by dithioerythritol (DTE) and Pi, but insensitive to cyclosporine A (CsA). The collapse of inner mitochondrial membrane potential (DeltaPsi(m)) was also observed with a release of cytochrome c from mitochondria. A great decrease in the ATP content was also seen under Al stress. Transmission electron microscopy analysis of Al-treated cells also corroborated our biochemical data with distortion in membrane architecture in mitochondria. TUNEL-positive nuclei in Al-treated cells strongly indicated the occurrence of nuclear fragmentation. From the above study, it was concluded that Al toxicity affects severely the mitochondrial respiratory functions and alters the redox status studied in vitro and also the internal structure, which seems to cause finally cell death in tobacco cells.

Physiological and genetic analyses of aluminium tolerance in rice, focusing on root growth during germination.

Aluminium (Al) ion limits root growth of plants in acidic soils, and rice exhibits the highest level of Al-tolerance among graminous crops. To elucidate Al-tolerance mechanisms in rice, response to Al was compared between rice (Oryza sativa L., cv. Nipponbare) and wheat (Triticum aestivum L., cv. ET8), focusing on seminal root growth at seedling stage and germination stage. At seedling stage, rice and wheat were similarly sensitive to Al in both dose- and time-dependent manner during a 24-h Al exposure. On the contrary, at germination stage, rice was more tolerant to Al than wheat, and wheat roots displayed the loss of plasma membrane integrity more extensively than rice. A rice mutant exhibiting Al hypersensitivity at germination stage was obtained by screening 42,840 R2 progeny derived from the regenerated R0 plants of Nipponbare and thereafter confirmation of the mutant phenotype in R3 progeny. At germination stage, root growth of the mutant was strongly inhibited in the presence of Al but not in the absence of Al. However, at seedling stage, root growth of the mutant and wild type was similarly tolerant to Al. Taken together, we conclude that rice possesses Al-tolerant function that is under genetic control and specifically operates for root growth at germination stage, making rice more tolerant to Al than wheat.

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

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

Differential effects of aluminium on osmotic potential and sugar accumulation in the root cells of Al-resistant and Al-sensitive wheat.

The changes in osmotic potential and the concentration of osmotic solutes in the cell sap of the root tips exposed to Al were examined in two cultivars of wheat (Triticum aestivum) differing in Al resistance. Root elongation was less influenced by an 8-h exposure to 20 microM or 50 microM Al in Al-resistant cv. Atlas 66 than in Al-sensitive cv. Scout 66. After Al treatment the osmotic potential of the root cells was decreased in Atlas 66 but increased in Scout 66 indicating that the Al treatment osmotically stimulated the driving force for water uptake in Atlas 66 but suppressed it in Scout 66. Al increased the concentration of soluble sugars, the major osmotic solute in the root cells in Atlas 66, but decreased it in Scout 66. Al at both low (5 microM) and high (50 microM) concentrations, also increased the concentration of soluble sugars in the Al-resistant genotype ET8 but a high Al concentration decreased it in Al-sensitive genotype ES8. Enzymatic analyses and thin-layer chromatography revealed that soluble sugars in the root cells of both Atlas 66 and Scout 66 mainly consisted of monosaccharides such as glucose, fructose and a small amount of sucrose. These results suggest that the accumulation of soluble sugars in Al-resistant wheat Atlas 66 keeps the osmotic potential in the root cells low and thus, enables the root cells to take up water and to elongate against the pressure produced by cell wall rigidification under Al stress.

Isolation of aluminum-tolerant cell lines of tobacco in a simple calcium medium and their responses to aluminum.

Aluminum (Al)-tolerant cell lines (ALT301 and ALT401) of tobacco were isolated in a simple calcium (Ca) solution from ethyl methane sulfonate (EMS)-treated suspension cultured tobacco cells (Nicotiana tabacum L. cv. Samsun, a cell line SL) at the logarithmic phase of growth. A highly tolerant cell line ALT301 exhibited the accumulation of Al and the deposition of callose to the same extent as the parental SL cells during the exposure to Al. However, the Al-treated ALT301 cells grew much better than the Al-treated SL cells after transfer to Al-free growth medium. Compared to SL cells, ALT301 cells were more tolerant to toxicity of copper and iron, but not to that of lanthanum. These results suggest that ALT301 cells have an internal tolerance mechanism, which makes cells grow normally in spite of Al accumulation and Al-induced lesion represented by the deposition of callose. This tolerance mechanism seems also to be effective against copper and iron toxicity. A slightly tolerant cell line ALT401 also accumulated Al to the same degree as SL cells, but deposited significantly less callose than did SL cells (43% of SL). The growth of ALT401 cells after Al treatment was only slightly better than that of SL cells. Thus, it seems that ALT401 cells have a mechanism to protect cells only from the Al-induced deposition of callose, which is not enough to overcome the Al-induced inhibition of growth. The different phenotypes exhibited by these Al-tolerant cell lines suggest that the deposition of callose is not directly related to the inhibition of growth in Al-treated cells.

Inhibition of growth and development of root border cells in wheat by Al.

The production and development of border cells vary with genotype, and they are released in wheat at an earlier stage of root development than other species studied so far. No significant difference was observed in the maximum number of border cells between Al-tolerant (Atlas 66) and Al-sensitive (Scout 66) cultivars in the absence of Al treatment. Al seriously inhibited the production and release of border cells, resulting in clumping of border cells in Scout 66, but less clustering in Atlas 66. The number of border cells released from roots treated with Al is significantly less than that from roots grown without Al treatment. Al treatment induced the death of detached border cells in vitro and they were killed by a 20-h treatment with 25 micro m Al. No significant difference in survival percentage of detached border cells was observed between Atlas 66 and Scout 66, regardless of the presence or absence of Al. The removal of border cells from root tips of both Atlas 66 and Scout 66 enhanced the Al-induced inhibition of root elongation concomitant with increased Al accumulation in the root. These results suggest that border cells adhered to the root tips play a potential role in the protection of root from Al injury in wheat.

Mechanisms of Al-induced iron chlorosis in wheat (Triticum aestivum). Al-inhibited biosynthesis and secretion of phytosiderophore.

Although Al-induced iron chlorosis has been observed in many plants, the mechanisms responsible for this phenomenon are yet to be understood. We investigated the effect of Al on iron acquisition in a Strategy II plant, wheat (Triticum aestivum L.) using both Al-tolerant (Atlas 66) and -sensitive (Scout 66) cultivars. When iron was supplied as insoluble iron, ferric hydroxide, in the culture solution, both cultivars without Al treatment grew normally, while those with 100 µM AlCl3 developed chlorosis of the young leaves after 3 days of the treatment. A 21-h treatment with 100 µM AlCl3 in 0.5 mM CaCl2 solution (pH 4.5) decreased the amount of 2'-deoxymugineic acid (DMA) secreted by Fe-deficient Atlas 66 and Scout 66 plants by 85 and 90%, respectively. The amount of DMA secreted decreased with increasing external Al concentrations. Al treatment during the biosynthesis process caused the inhibition of that of DMA within 3 h. The secretion process was also found to be inhibited by Al, resulting in the biosynthesized DMA remaining in the roots. These results demonstrate the inhibition by Al of both biosynthesis and secretion of DMA attributed to Al-induced iron chlorosis.

The role of phosphorus in aluminium-induced citrate and malate exudation from rape (Brassica napus).

Exudation of organic anions is believed to be a common tolerance mechanism for both aluminium toxicity and phosphorus deficiency. Nevertheless, which of these stresses that actually elicit the exudation of organic anions from rape (Brassica napus L) remains unknown, and the combined effects of Al toxicity and P deficiency on rape have not been reported before. Therefore, in the current study, Brassica napus var. Natane nourin plants grown with or without 0.25 mM P were exposed to 0 or 50 micro M AlCl(3) and several parameters related to the exudation of organic anions from the roots were investigated. Eight days of P deficiency resulted in a significant growth reduction, but P deficiency alone did not induce exudation of organic anions. In contrast, Al strongly induced organic acid exudation, while simultaneously inhibiting root growth. Increased in-vitro activity of citrate synthase (CS, EC 4.1.3.7), malate dehydrogenase (MDH, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), together with reduced root respiration, indicated that the Al-induced accumulation and subsequent exudation of citrate and malate were associated with both increased biosynthesis and reduced metabolism of citric and malic acid. Phosphorus-sufficient plants showed more pronounced aluminium-induced accumulation and exudation of organic anions than P-deficient plants. A divided root chamber experiment showed the necessity of direct contact between Al and roots to elicit exudation of organic anions. Prolonged exposure (10 days) to Al resulted in a decrease in the net exudation of citrate and malate, and the rate of decrease was much more rapid in P-deficient plants than in P-sufficient plants. It is concluded that P nutrition affects the level of Al-induced synthesis and exudation of organic anions. However, the mechanism needs further investigation.

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(2)O(2), Fe(2+) and Cu(2+). Under these oxidant exposures, ALT301 contained lower levels of intracellular H(2)O(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(2)O(2) commonly enhanced by Al or other oxidants.

Overexpression of alternative oxidase gene confers aluminum tolerance by altering the respiratory capacity and the response to oxidative stress in tobacco cells.

Aluminum (Al) stress represses mitochondrial respiration and produces reactive oxygen species (ROS) in plants. Mitochondrial alternative oxidase (AOX) uncouples respiration from mitochondrial ATP production and may improve plant performance under Al stress by preventing excess accumulation of ROS. We tested respiratory changes and ROS production in isolated mitochondria and whole cell of tobacco (SL, ALT 301) under Al stress. Higher capacities of AOX pathways relative to cytochrome pathways were observed in both isolated mitochondria and whole cells of ALT301 under Al stress. AOX1 when studied showed higher AOX1 expression in ALT 301 than SL cells under stress. In order to study the function of tobacco AOX gene under Al stress, we produced transformed tobacco cell lines by introducing NtAOX1 expressed under the control of the cauliflower mosaic virus (CaMV) 35 S promoter in sensitive (SL) Nicotiana tabacum L. cell lines. The enhancement of endogenous AOX1 expression and AOX protein with or without Al stress was in the order of transformed tobacco cell lines > ALT301 > wild type (SL). A decreased respiratory inhibition and reduced ROS production with a better growth capability were the significant features that characterized AOX1 transformed cell lines under Al stress. These results demonstrated that AOX plays a critical role in Al stress tolerance with an enhanced respiratory capacity, reducing mitochondrial oxidative stress burden and improving the growth capability in tobacco cells.

Development and evaluation of a novel in-clinic automated hematology analyzer, ProCyte Dx, for canine erythrocyte indices, leukogram, platelet counts and reticulocyte counts.

A novel hematology analyzer for small animal medicine, ProCyte Dx, was developed from combination of the fluorescence laser flow cytometry and laminar flow impedance technologies, and its accuracy was evaluated by comparing with the conventional impedance-based hematology analyzer, pocH-100iV Diff, or microscopic manual cell counting methods with staining blood smears in the canine blood. Blood samples of 59 dogs were hematologically analyzed and compared by Pearson's correlation coefficients. Analyses between the two analyzers showed excellent correlation in RBC (r=0.998), HGB (r=0.999), HCT (r=0.998), MCV (r=0.994), MCH (r=0.974), MCHC (r=0.906), WBC (r=0.998) and PLT (r=0.993). Analyses between ProCyte Dx and microscopic manual counting results showed excellent correlation in neutrophils (r=0.920), lymphocytes (r=0.913) and reticulocyte percentages (r=0.924), good correlation in eosinophils (r=0.815) and reticulocyte numbers (r=0.850) and fair correlation in monocytes (r=0.770). The present study indicates that ProCyte Dx is acceptably accurate and can be a powerful tool for canine clinical medicine.

Aluminum toxicity recovery processes in root apices. Possible association with oxidative stress.

Al inhibits root apex elongation with concomitant morphological injuries such as ruptures punctuated by the regions stained with Evans blue. The recovery can be investigated by transfer of Al-injured roots to a solution lacking Al. In the Al-injured root apex, superoxide anion, H(2)O(2), Al, and lignin accumulate. During the recovery process, the central cylinder elongates leaving the region stained with Evans blue without marked disappearance. The obvious function of the region is not clear but may trigger the elongation of central cylinder during the recovery process. Thus the function of the region stained with Evans blue might be derived from the programmed cell-like idea. Oxidative stress concerns events induced under Al toxicity and the recovery process. The superoxide anion is primarily formed by plasma membrane-associated NADPH oxidase and is dismuted to H(2)O(2) and O(2) by superoxide dismutase. H(2)O(2) provides the electrons for the polymerization of phenolics to lignin, which causes the stiffening of the cell wall. The distortion of the cell wall caused by lignin may induce the breaking and tearing of cells, which results in the formation of ruptures at the rhizodermis and outer cortex layers. The production of superoxide anion, H(2)O(2), and lignin was reduced during the recovery process and thereby the elongation of the central cylinder may be induced.

DNA display selection of peptide ligands for a full-length human G protein-coupled receptor on CHO-K1 cells.

The G protein-coupled receptors (GPCRs), which form the largest group of transmembrane proteins involved in signal transduction, are major targets of currently available drugs. Thus, the search for cognate and surrogate peptide ligands for GPCRs is of both basic and therapeutic interest. Here we describe the application of an in vitro DNA display technology to screening libraries of peptide ligands for full-length GPCRs expressed on whole cells. We used human angiotensin II (Ang II) type-1 receptor (hAT1R) as a model GPCR. Under improved selection conditions using hAT1R-expressing Chinese hamster ovary (CHO)-K1 cells as bait, we confirmed that Ang II gene could be enriched more than 10,000-fold after four rounds of selection. Further, we successfully selected diverse Ang II-like peptides from randomized peptide libraries. The results provide more precise information on the sequence-function relationships of hAT1R ligands than can be obtained by conventional alanine-scanning mutagenesis. Completely in vitro DNA display can overcome the limitations of current display technologies and is expected to prove widely useful for screening diverse libraries of mutant peptide and protein ligands for receptors that can be expressed functionally on the surface of CHO-K1 cells.

Changes in rupture formation and zonary region stained with Evans blue during the recovery process from aluminum toxicity in the pea root apex.

We investigated how the pea (Pisum sativum cv. Harunoka) root, upon return to an Al-free condition, recovers from injury caused by exposure to Al. Elongation and re-elongation of the root during the recovery process from Al injury occurred only in the apical 5-mm region of the pea root. With the model system of the pea root for recovery from Al injury, images of the root characterized by zonal staining with Evans blue showed the existence of two regions in the root apex consisting of rupture and zonary stained regions. Ruptures enlarged by increase in their depth but without widening of the intervals among zonary stained regions in the roots treated with Al continuously. On the other hand, intervals of the zonary stained regions were widened due to re-elongation of the root and were narrow in the rupture region in the recovery root.

Aluminum stress signaling in plants.

Aluminum (Al) toxicity is a major constraint for crop production in acidic soil worldwide. When the soil pH is lower than 5, Al(3+) is released to the soil and enters into root tip cell ceases root development of plant. In acid soil with high mineral content, Al is the major cause of phytotoxicity. The target of Al toxicity is the root tip, in which Al exposure causes inhibition of cell elongation and cell division, leading to root stunting accompanied by reduced water and nutrient uptake. A variety of genes have been identified that are induced or repressed upon Al exposure. At tissue level, the distal part of the transition zone is the most sensitive to Al. At cellular and molecular level, many cell components are implicated in the Al toxicity including DNA in nucleus, numerous cytoplastic compounds, mitochondria, the plasma membrane and the cell wall. Although it is difficult to distinguish the primary targets from the secondary effects so far, understanding of the target sites of the Al toxicity is helpful for elucidating the mechanisms by which Al exerts its deleterious effects on root growth. To develop high tolerance against Al stress is the major goal of plant sciences. This review examines our current understanding of the Al signaling with the physiological, genetic and molecular approaches to improve the crop performance under the Al toxicity. New discoveries will open up new avenues of molecular/physiological inquiry that should greatly advance our understanding of Al tolerance mechanisms. Additionally, these breakthroughs will provide new molecular resources for improving the crop Al tolerance via molecular-assisted breeding and biotechnology.

Aluminum toxicity and Ca depletion may enhance cell death of tobacco cells via similar syndrome.

The main objective of this work is to find out whether aluminum (Al) toxicity and Ca depletion cause cell death of tobacco cells via similar sequence of events. Tobacco cell suspension culture exhibited maximum fresh weight in the presence of a wide range of Ca concentrations between 0.1-1.0 mM whereas higher concentrations (>1.0-5.0 mM) gradually lowered cell fresh weight. However, this decrease in fresh weight does not imply a negative impact on cell viability since cell growth recommenced in fresh MS medium with rates mostly higher than those of low Ca. In addition, high Ca seems to be crucial for survival of Al-treated cells. On the other side, tobacco cells exhibited extreme sensitivity to complete deprivation of Ca. Without Ca, cells could not survive for 18 h and substantially lost their growth capability. Evans blue uptake proved membrane damage of Ca-depleted same as Al-treated cells; relative to maintained membrane intactness of calcium-supplemented (control) ones. Percentage of membrane damage and the growth capability (survival) of tobacco cells exhibited a clear negative correlation.Alterations in growth (fresh weight per aliquot) could not be ascribed neither to cell number nor to decreased dry matter allocation (dry weight/fresh weight percentage) but was mainly due to decreased cellular water content. In this context, Ca-depleted cells lost about half their original water content while 100 microM Al-treated ones retained most of it (ca 87%). This represented the single difference between the two treatments (discussed in the text). Nevertheless, such high water content of the Al-treated cells seems physiologically useless since it did not result in improved viability. Similarities, however, included negligible levels of growth capability, maximum levels of membrane damage, and comparable amounts of NO(3) (-) efflux. As well, both types of treatments led to a sharp decline in osmotic potential that is, in turn, needed for water influx. The above-mentioned sequence of events, induced by Al application looks, to a great extent, similar to Ca depletion syndrome leading finally to cell death of tobacco cells.

The BnALMT1 Protein That is an Aluminum-Activated Malate Transporter is Localized in the Plasma Membrane.

We have previously reported that Al-induces citrate and malate efflux from P-sufficient and P-deficient plants of rape (Brassica napus L.) and that P-deficiency alone could not induce this response. Further investigation showed that the transcript of two genes designated BnALMT1 and BnALMT2 is accumulated in roots by Al-treatment. Transgenic tobacco cells (Nicotiana tabacum) and Xenopus laevis oocytes expressing the BnALMT1 and BnALMT2 proteins released more malate than control cells in the presence of Al, indicating that the BnALMT genes encode an Al-activated malate transporter. The transgenic tobacco cells exposed to toxic level of Al grew better than control cells indicating that the genes can enhance Al-resistance of plant cells. In this study we showed the subcellular localization of BnALMT1 fused to the green fluoresce protein (GFP). The BnALMT1:: GFP construct was transiently expressed in protoplasts prepared from Arabidopsis leaves using the polyethylene glycol (PEG) method. The result showed that the BnALMT1 protein is localized in the plasma membrane. This provides further evidence that the BnALMT proteins facilitate the transport of malate across the plasma membrane (PM).

Overexpression of an auxilin-like gene (F9E10.5) can suppress Al uptake in roots of Arabidopsis.

Plants resistant to aluminium (Al) stress were isolated from Arabidopsis thaliana enhancer-tagged mutant lines. Compared with the parental Col-7 control line, one of the resistant candidates, #355-2, showed a higher expression of the F9E10.5 gene (At1g75100) on chromosome 1, a lower Al content in whole roots, and a shorter root hair length (approximately 30%). Both Al influx and associated oxidative stress occurred in root hairs, as well as in root tips of Col-7; however, they were seen only in root tips of #355-2. Transgenic plants overexpressing the F9E10.5 gene showed a slightly higher Al resistance than their parental control line (Ler). The F9E10.5 gene encodes an auxilin-like protein related to the clathrin-uncoating process in endocytosis. Microscopic observation indicated that both Al ion influx and endocytosis activity were lower in root hair cells of the #355-2 line than in those of Col-7. These results suggested that overexpression of this auxilin-like protein inhibits endocytosis in root hair cells by a disturbance of the transport system as in animal cells shown previously. It was also suggested that a part of the Al influx occurred via endocytosis in root hair cells in Arabidopsis. The Al resistance in the #355-2 line may therefore be due to a lower Al uptake via endocytosis in the root hair region.