SummaryPlant nutrition critically depends on the activity of membrane transporters that translocate minerals from the soil into the plant and are responsible for their intra-and intercellular distribution. Most plant membrane transporters are encoded by multigene families whose members often exhibit overlapping expression patterns and a high degree of sequence homology. Furthermore, many inorganic nutrients are transported by more than one transporter family. These considerations, coupled with a large number of so-far non-annotated putative transporter genes, hamper our progress in understanding how the activity of speci®c transporters is integrated into a response to¯uctuating conditions. We designed an oligonucleotide microarray representing 1096 Arabidopsis transporter genes and analysed the root transporter transcriptome over a 96-h period with respect to 80 mM NaCl, K starvation and Ca 2 starvation. Our data show that cation stress led to changes in transcript level of many genes across most transporter gene families. Analysis of transcriptionally modulated genes across all functional groups of transporters revealed families such as V-type ATPases and aquaporins that responded to all treatments, and families ± which included putative non-selective cation channels for the NaCl treatment and metal transporters for Ca 2 starvation conditions ± that responded to speci®c ionic environments. Several gene families including primary pumps, antiporters and aquaporins were analysed in detail with respect to the mRNA levels of different isoforms during ion stress. Cluster analysis allowed identi®cation of distinct expression pro®les, and several novel putative regulatory motifs were discovered within sets of co-expressed genes.
SummaryMechanisms are required by all organisms to maintain the concentration of essential heavy metals (e.g. Zn and Cu) within physiological limits and to minimise the detrimental effects of non-essential heavy metals (e.g. Cd). Heavy-metal P-type ATPases (HMAs) are a subgroup of the P-type ATPase superfamily that may contribute to metal homeostasis in plants. We cloned and characterised a member of this family, AtHMA4, from Arabidopsis thaliana that clusters with the Zn/Co/Cd/Pb subclass of HMAs on phylogenetic analysis. Sequencing of the AtHMA4 cDNA showed that it contained the conserved motifs found in all P-type ATPases and also motifs that are characteristic of heavy-metal ATPases. Escherichia coli mutants defective in the HMAs, CopA and ZntA, were used in functional complementation studies. AtHMA4 was able to restore growth at high [Zn] in the zntA mutant but not at high [Cu] in the copA mutant, suggesting a role in zinc transport. Heterologous expression of AtHMA4 in Saccharomyces cerevisiae made the yeast more resistant to Cd but did not affect sensitivity to other metals compared with vector-transformed controls. The organ speci®city of AtHMA4 was analysed in Arabidopsis and showed that AtHMA4 was expressed in a range of tissues with highest expression in roots. AtHMA4 was upregulated in roots exposed to elevated levels of Zn and Mn but downregulated by Cd. Possible physiological roles of this transporter in Arabidopsis are discussed.
The transition metal Zn is essential for many physiological processes in plants, yet at elevated concentrations this, and the related non-essential metal Cd, can be toxic. Arabidopsis thaliana HMA4, belonging to the Type P 1B subfamily of P-type ATPases, has recently been implicated in Zn nutrition, having a role in root to shoot Zn translocation. Using Arabidopsis insertional mutants, it is shown here that disruption of AtHMA4 function also results in increased sensitivity to elevated levels of Cd and Zn, suggesting that AtHMA4 serves an important role in metal detoxification at higher metal concentrations. AtHMA4 and a truncated form lacking the last 457 amino acids both confer Cd and Zn resistance to yeast but a mutant version of the full-length AtHMA4 (AtHMA4-C357G) does not; this demonstrates that the C-terminal region is not essential for this function. Evidence is presented that AtHMA4 functions as an efflux pump.
Molybdenum and tungsten have similar ionic radii and chemical properties. Tungsten is the heaviest atom and the only third-row transition element that exhibits biological activity in enzymes. Molybdenum is the only second-row transition metal that exhibits biological activity when it is present in a cofactor of a metalloenzyme. Both metals are present mainly in enzymes that catalyze oxygen atom transfer reactions. In these enzymes they are coordinated by the two dithiolene sulfur atoms of a pterin molecule, called a molybdopterin cofactor (25). In the case of tungsten, the metal is always coordinated by two pterin moieties, forming a so called bis-pterin cofactor (9, 13). Molybdenum is an essential trace metal for many forms of life whereas tungsten is found mostly in archaea and in some bacteria. The molybdenum cofactor-containing enzymes can be divided into three families depending on the coordination chemistry of the Mo ligand: the sulfite oxidases; the xanthine oxidoreductases, also including the aldehyde oxidases; and the dimethyl sulfoxide reductases, which are found only in prokaryotes (18, 35). The tungsten cofactor-containing enzymes consist only of the aldehyde oxidoreductases (AORs), formate dehydrogenases (FDHs), and an acetylene hydratase (13). Based on sequence comparison the FDHs and acetylene hydratase are part of the molybdenum-containing dimethyl sulfoxide reductase family.The transport of molybdate has been well characterized in particular for Escherichia coli, which expresses a high-affinity ABC transporter for molybdate encoded by the modABC genes (27). The periplasmic molybdate binding protein ModA binds specifically molybdate and tungstate and not sulfate or other anions (27). Crystal structures of the E. coli and the Azotobacter vinelandii ModA indicate that the specificity for molybdate and tungstate is mostly determined by the size of the binding pocket. The Cambridge Structural Database (2) gives 1.75 Ϯ 0.04 Å and 1.76 Ϯ 0.02 Å for molybdate and tungstate, respectively, and 1.47 Ϯ 0.02 Å for sulfate. The ModA proteins cannot discriminate between molybdate and tungstate. The first tungsten-specific ABC transporter was identified in Eubacterium acidaminophilum (17). The periplasmic tungsten uptake protein (TupA) was cloned and expressed in E. coli and was shown to bind only tungstate with a high affinity. A crystal structure of TupA is not yet available, and it is not clear what the structural basis is of the specificity for tungstate over molybdate. Recently, a high-affinity vanadate transporter, which was highly selective for vanadate compared to tungstate, was identified in the cyanobacterium Anabaena variabilis ATCC 29413 based on the sequence similarity with the TupA protein from E. acidaminophilum (58% sequence similarity) (24). A. variabilis ATCC 29413 expresses an alternative V-dependent nitrogenase for the fixation of nitrogen, and therefore it requires vanadate (24). The specificity of this transporter for vanadate indicates that high sequence similarities are not conclusive fo...
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