SummaryThe 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 ef¯ux of malate from root apices. Malate forms a stable complex with Al that is harmless to plants and, therefore, this ef¯ux 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 F 2 and F 3 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 Alsensitive line. Heterologous expression of ALMT1 in Xenopus oocytes, rice and cultured tobacco cells conferred an Al-activated malate ef¯ux. Additionally, ALMT1 increased the tolerance of tobacco cells to Al treatment. These ®ndings demonstrate that ALMT1 encodes an Al-activated malate transporter that is capable of conferring Al tolerance to plant cells.

To study the chromosomal partitioning mechanism in cell division, we have isolated a novel type of Escherichia coli mutants which formed anucleate cells, by using newly developed techniques. One of them, named muk4M, is not lethal and produces normal-sized anucleate cells at a frequency of 0.5 to 3% of total cells in exponentially growing populations but does not produce filamentous cells. Results suggest that the mutant is defective in the chromosome positioning at regular intracellular positions and fails frequently to partition the replicated daughter chromosomes into both daughter cells, resulting in production of one anucleate daughter cell and one with two chromosomes. The mu)41 mutation causes pleiotropic effects: slow growth, hypersensitivity to sodium dodecyl sulfate, and tolerance to colicin El protein, in addition to anucleate cel formation.

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

Here, we report the aluminum (Al)-induced organ-specific expression of a WAK1 (cell wall-associated receptor kinase 1) gene and cell type-specific localization of WAK proteins in Arabidopsis. WAK1-specific reverse transcriptase-polymerase chain reaction analysis revealed an Al-induced WAK1 gene expression in roots. Short-and long-term analysis of gene expression in root fractions showed a typical "on" and "off" pattern with a first peak at 3 h of Al exposure followed by a sharp decline at 6 h and a complete disappearance after 9 h of Al exposure, suggesting the WAK1 is a further representative of Al-induced early genes. In shoots, upon root Al exposure, an increased but stable WAK1 expression was observed. Using confocal microscopy, we visualized Al-induced closure of leaf stomata, consistent with previous suggestions that the Al stress primarily experienced in roots associated with the transfer of root-shoot signals. Elevated levels of WAK protein in root cells were observed through western blots after 6 h of Al exposure, indicating a lag time between the Al-induced WAK transcription and translation. WAK proteins are localized abundantly to peripheries of cortex cells within the elongation zone of the root apex. In these root cells, disintegration of cortical microtubules was observed after Al treatment but not after the Al analog lanthanum treatments. Tip-growing control root hairs, stem stomata, and leaf stomatal pores are characterized with high amounts of WAKs, suggesting WAKs are accumulating at plasma membrane domains, which suffer from mechanical stress and lack dense arrays of supporting cortical microtubules. Further, transgenic plants overexpressing WAK1 showed an enhanced Al tolerance in terms of root growth when compared with the wild-type plants, making the WAK1 one of the important candidates for plant defense against Al toxicity.

The functions of two genes whose expression provides tolerance to aluminium (Al) stress were investigated using plants and Saccharomyces cerevisiae (yeast): the Arabidopsis thaliana blue copper binding gene (AtBCB) and Nicotiana tabacum guanosine diphosphate (GDP) dissociation inhibitor gene (NtGDI1). To determine the localization of these proteins, each gene was fused to the green fluorescent protein (GFP) gene and introduced into onion epidermal cells. AtBCB was localized to cell membrane region and NtGDI1 to cytoplasm. Transgenic lines over-expressing the AtBCB gene showed constitutive lignin production in whole roots. By contrast, wild-type Arabidopsis (Ler) produced a negligible level of lignin and enhanced lignin production in the root-tip region by Al stress. Compared with Ler, the AtBCB-expressing lines showed a lower deposition of malon dialdehyde after Al stress. Microscopic observation of the Al-treated roots indicated that the deposition of lipid peroxides was clearly low in the area where lignin accumulated. It was proposed that lipid peroxidation caused by Al stress was diminished by the formation of lignin. Expression of the NtGDI1 gene in yeast complemented the temperature-sensitive phenotype of a sec19 mutant at 37 degrees C. This gene also complemented an Al-sensitive phenotype shown by the sec19 mutant at the permissive temperature of 32 degrees C. These results suggested that the yeast Sec19 vesicle transport system has a function in providing basal Al resistance in yeast by the export of Al ions. It was also proposed that over-expression of the NtGDI1 protein activates an Al efflux system that protects Arabidopsis against Al toxicity.

A novel SmtB/ArsR family metalloregulator, denoted BxmR, has been identified and characterized from the cyanobacterium Oscillatoria brevis. Genetic and biochemical evidence reveals that BxmR represses the expression of both bxa1, encoding a CPx-ATPase metal transporter, as well as a divergently transcribed operon encoding bxmR and bmtA, a heavy metal sequestering metallothionein. Derepression of the expression of all three genes is mediated by both monovalent (Ag(I) and Cu(I)) and divalent (Zn(II) and Cd(II)) heavy metal ions, a novel property among SmtB/ArsR metal sensors. Electrophoretic gel mobility shift experiments reveal that apoBxmR forms multiple resolvable complexes with oligonucleotides containing a single 12-2-12 inverted repeat derived from one of the two operator/promoter regions with similar apparent affinities. Preincubation with either monovalent or divalent metal ions induces disassembly of both the BxmR-bxa1 and BxmR-bxmR/ bmtA operator/promoter complexes. Interestingly, the temporal regulation of expression of bxa1 and bmtA mRNAs is different in O. brevis with bxa1 induced first upon heavy metal treatment, followed by bmtA/bxmR. A dynamic interplay among Bxa1, BmtA, and BxmR is proposed that maintains metal homeostasis in O. brevis by balancing the relative rates of metal storage and efflux of multiple heavy metal ions.Transition metal ions such as zinc, copper, iron, and manganese are essential trace elements that play integral catalytic functions in myriad metalloenzymes and electron transfer in all organisms (1-3). However, they are required only in trace amounts and, when present in excess in the environment, even essential metals can be cytotoxic, like heavy metal pollutants (4 -6). All organisms have evolved a range of mechanisms that govern metal homeostasis, defined as maintaining the intracellular bioavailable concentrations of essential metal ions within a range compatible with cell viability (7-9). Multiple lines of evidence from the past decade reveal that heavy metal homeostasis is maintained in all organisms by a small number of critical processes that include metal sensing, chelation, and transport (10 -18).Two distinct mechanisms play prominent roles in governing metal ion homeostasis and resistance in many organisms. One involves the uptake or efflux of specific heavy metal ions across biomembranes, mediated by ATP-coupled high affinity metal ion transporters such as those derived from the CPx-ATPase family (19 -21). Another mechanism involves the specific chelation of metal ions by intracellular chelators, e.g. metallothioneins (MTs), 1 now known to be widely distributed in nature (13,22,23). To meet the diverse biological requirements of specific metal ions, various strategies have evolved to regulate the transcription of genes encoding these heavy metal homeostasis proteins. In prokaryotes, the expression of these genes is tightly controlled by specific metalloregulators or "metal-sensing" transcriptional regulators (12,24,25). One such family of homologous metal sensor pro...

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With the ultimate purpose of clarifying the mechanism for aluminium (Al) toxicity and for Al tolerance, we tried to isolate cDNAs whose expression is induced by Al treatment and phosphate (Pi) starvation. We performed Pi starvation and Al treatment (two‐step treatment) on suspension‐cultured cells of Nicotiana tabacum L. cv. Samsun and then constructed a cDNA library using poly(A)+‐RNA derived from the treated cells. Four independent cDNA clones (pAL 111, 139, 141 and 142) were isolated from the library by differential screening. Northern blot hybridization analysis indicated that the expression of these clones was induced by Pi starvation. Furthermore, we found that pAL 111 and pAL 142 are also induced by Al treatment. The complete cDNA sequencing of these 4 clones was determined. The results indicated that pAL111 is identical to the parA gene of N. tabacum, which is described as an auxin‐regulated gene and that pAL142 is highly homologous to the parB gene of N. tabacum whose product has glutathione S‐transferase (GST, EC 2.5.1.18) activity. Furthermore, we found a cysteine‐rich domain in the amino acid sequence of pAL139. No DNA and deduced amino acid sequences homologous to the pAL141 were found.