Genes encoding proteins of the cation diffusion facilitator family that confer manganese tolerance

The yeast Saccharomyces cerevisiae expressing a cDNA library prepared from Stylosanthes hamata was screened for enhanced Mn(2+) tolerance. From this screen, we identified four related cDNAs that encode membrane-bound proteins of the cation diffusion facilitator (CDF) family. One of these cDNAs (ShMTP1) was investigated in detail and found to confer Mn(2+) tolerance to yeast by internal sequestration rather than by efflux of Mn(2+). Expression of ShMTP1 in a range of yeast mutants suggested that it functions as a proton:Mn(2+) antiporter on the membrane of an internal organelle. Similarly, when expressed in Arabidopsis, ShMTP1 conferred Mn(2+) tolerance through internal sequestration. The ShMTP1 protein fused to green fluorescent protein was localized to the tonoplast of Arabidopsis cells but appeared to localize to the endoplasmic reticulum of yeast. We suggest that the ShMTP1 proteins are members of the CDF family involved in conferring Mn(2+) tolerance and that at least one of these proteins (ShMTP1) confers tolerance by sequestering Mn(2+) into internal organelles.     

Zinc transporter of Arabidopsis thaliana AtMTP1 is localized to vacuolar membranes and implicated in zinc homeostasis

Cation diffusion facilitator (CDF) proteins belong to a family of heavy metal efflux transporters that might play an essential role in homeostasis and tolerance to metal ions. We investigated the subcellular localization of Arabidopsis thaliana AtMTP1, a member of the CDF family, and its physiological role in the tolerance to Zn using MTP1-deficient mutant plants. AtMTP1 was immunochemically detected as a 43 kDa protein in the vacuolar membrane fractioned by sucrose density gradient centrifugation. The expression level of AtMTP1 in suspension-cultured cells was not affected by the Zn concentration in the medium. When AtMTP1 fused with green fluorescent protein was transiently expressed in protoplasts prepared from Arabidopsis suspension-cultured cells, green fluorescence was clearly observed in the vacuolar membrane. A T-DNA insertion mutant line for AtMTP1 displays enhanced sensitivity to high Zn concentrations ranging from 200 to 500 microM, but not to Zn-deficient conditions. Mesophyll cells of the mtp1-1 mutant plants grown in the presence of 500 microM Zn were degraded, suggesting that Zn at high concentrations causes serious damage to leaves and that AtMTP1 plays a crucial role in preventing this damage in plants. Thus we propose that AtMTP1 is localized in the vacuolar membrane and is involved in sequestration of excess Zn in the cytoplasm into vacuoles to maintain Zn homeostasis.     

A role for the AtMTP11 gene of Arabidopsis in manganese transport and tolerance

The Arabidopsis AtMTP family of genes encode proteins of the cation diffusion facilitator (CDF) family, with several members having roles in metal tolerances. Four of the 11 proteins in the family form a distinct cluster on a phylogenetic tree and are closely related to ShMTP8, a CDF identified in the tropical legume Stylosanthes hamata that is implicated in the transport of Mn(2+) into the vacuole as a tolerance mechanism. Of these four genes, AtMTP11 was the most highly expressed member of the Arabidopsis subgroup. When AtMTP11 was expressed in Saccharomyces cerevisiae, it conferred Mn(2+) tolerance and transported Mn(2+) by a proton-antiport mechanism. A mutant of Arabidopsis with a disrupted AtMTP11 gene (mtp11) was found to have increased sensitivity to Mn(2+) but not to Cu(2+) or Zn(2+). At a non-toxic but sufficient Mn(2+) supply (basal), the mutant accumulated more Mn(2+) than the wild type, but did not show any obvious deleterious effects on growth. When grown with Mn(2+) supplies that ranged from basal to toxic, the mutant accumulated Mn(2+) concentrations in shoots similar to those in wild-type plants, despite showing symptoms of Mn(2+) toxicity. AtMTP11 fused to green fluorescent protein co-localized with a reporter specific for pre-vacuolar compartments. These findings provide evidence for Mn(2+)-specific transport activity by AtMTP11, and implicate the pre-vacuolar compartments in both Mn(2+) tolerance and Mn(2+) homeostasis mechanisms of Arabidopsis.     

Phytoextraction of Zinc: Physiological and Molecular Mechanism

Zinc is an essential trace element, necessary for plants, animals, and microorganisms. Zn is required for many enzymes as a catalytic cofactor, for photosynthetic CO₂ fixation, and in maintaining the integrity of bio-membranes. However, Zn is potentially toxic when accumulated beyond cellular needs. Phytoextraction technique, which is a part of phytoremediation, has opened new avenues for remediation of Zn-contaminated places. Hyperaccumulators like Thlaspi caerulescens and Arabidopsis halleri have been identified, which can accumulate up to 40,000 mg kg^?1 Zn in the aerial parts of the plant body. Carboxylic acids, primarily malate, citrate, and oxalate, and amino acids are found to play an important role in Zn hyperaccumulation. Transmembrane metal transporters are assumed to play a key role in Zn metal uptake, xylem loading, and vacuolar sequestration. Members of CDF (cation diffusion facilitator) and ZIP (zinc-regulated transporter, iron-regulated transporter like protein) family have been implicated in Zn-metal-tolerance mechanisms. A potential metal-binding motif, containing multiple histidine residues, is found in the variable regions of almost all of the ZIP family, including ZIP1, ZIP2, ZIP4, ZRT1, and ZRT2. Overexpression of some Zn metal transporter genes like TcZNT1 (Thlaspi caerulescens Zn transporter1), TcHMA4 (Thlaspi caerulescens heavy metal ATPase) in Thlaspi caerulescens, AhMTP1;3 (Arabidopsis halleri metal transporter1;3) in Arabidopsis halleri, and PtdMTP1(Poplar metal transporter1) from a hybrid poplar confer Zn hypertolerance in Thlaspi, Arabidopsis, and Poplar plant species.     

A putative novel role for plant defensins: a defensin from the zinc hyper-accumulating plant, Arabidopsis halleri, confers zinc tolerance

The metal tolerance of metal hyper-accumulating plants is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the Zn hyper-accumulating plant Arabidopsis halleri ssp. halleri, we carried out a functional screening of an A. halleri cDNA library in the yeast Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. The screening revealed four A. halleri defensin genes (AhPDFs), which induced Zn but not cadmium (Cd) tolerance in yeast. The expression of AhPDF1.1 under the control of the 35S promoter in A. thaliana made the transgenic plants more tolerant to Zn than wild-type plants, but did not change the tolerance to Cd, copper (Cu), cobalt (Co), iron (Fe) or sodium (Na). Thus, AhPDF1.1 is able to confer Zn tolerance both to yeast and plants. In A. halleri, defensins are constitutively accumulated at a higher level in shoots than in A. thaliana. A. halleri defensin pools are Zn-responsive, both at the mRNA and protein levels. In A. thaliana, some but not all defensin genes are induced by ZnCl2 treatment, and these genes are not induced by NaCl treatment. Defensins, found in a very large number of organisms, are known to be involved in the innate immune system but have never been found to play any role in metal physiology. Our results support the proposition that defensins could be involved in Zn tolerance in A. halleri, and that a role for plant defensins in metal physiology should be considered.     

Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. Characterization of a novel heavy metal transporting ATPase

Thlaspi caerulescens is a heavy metal hyperaccumulator plant species that is able to accumulate extremely high levels of zinc (Zn) and cadmium (Cd) in its shoots (30,000 microg g(-1) Zn and 10,000 microg g(-1) Cd), and has been the subject of intense research as a model plant to gain a better understanding of the mechanisms of heavy metal hyperaccumulation and tolerance and as a source of genes for developing plant species better suited for the phytoremediation of metal-contaminated soils. In this study, we report on the results of a yeast (Saccharomyces cerevisae) complementation screen aimed at identifying candidate heavy metal tolerance genes in T. caerulescens. A number of Thlaspi genes that conferred Cd tolerance to yeast were identified, including possible metal-binding ligands from the metallothionein gene family, and a P-type ATPase that is a member of the P1B subfamily of purported heavy metal-translocating ATPases. A detailed characterization of the Thlaspi heavy metal ATPase, TcHMA4, demonstrated that it mediates yeast metal tolerance via active efflux of a number of different heavy metals (Cd, Zn, lead [Pb], and copper [Cu]) out of the cell. However, in T. caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with its homolog in Arabidopsis (Arabidopsis thaliana), we suggest that it may not be involved in metal tolerance. Instead, we hypothesize that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T. caerulescens. Additionally, evidence is presented showing that the C terminus of the TcHMA4 protein, which contains numerous possible heavy metal-binding His and Cys repeats residues, participates in heavy metal binding. When partial peptides from this C-terminal domain were expressed in yeast, they conferred an extremely high level of Cd tolerance and Cd hyperaccumulation. The possibilities for enhancing the metal tolerance and phytoremediation potential of higher plants via expression of these metal-binding peptides are also discussed.     

Characterization of the ER-located zinc transporter ZnT1 and identification of a vesicular zinc storage compartment in Hebeloma cylindrosporum

Metal tolerance of filamentous fungi is a poorly understood mechanism. In order to unravel the molecular basis of zinc (Zn) tolerance in the ectomycorrhizal fungal model Hebeloma cylindrosporum, we carried out a functional screening of an H. cylindrosporum cDNA library in the zrc1Δ mutant strain of Saccharomyces cerevisiae to search for genes conferring Zn tolerance to yeast cells. This strategy allowed the isolation of HcZnT1, a gene belonging to the cation diffusion facilitator family, which induced tolerance to Zn, but not to other metals. HcZnT1 was constitutively expressed in Hebeloma cells, whatever the Zn status of the medium and the fungal cell type (mycelia, sporocarps, mycorrhizas). A HcZnT1:GFP fusion protein was expressed in yeast and the corresponding fluorescence was recorded on endoplasmic reticulum membranes. Taken together, these different findings suggest a dual role of HcZnT1 in Zn homeostasis of fungal cells, by supplying requested Zn ions for the functioning of the endoplasmic reticulum as well as by detoxifying the cytosol under Zn stress. Zn pools were also investigated by using the Zn-specific fluorophore zinquin in H. cylindrosporum cells. Zinquin labeling revealed compartmentalization in intracellular vesicles interspersed throughout the cytoplasm that do not correspond to vacuolar compartments. Altogether the present data represent the first steps into the understanding of Zn homeostasis and tolerance in Hebeloma.     

Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells

The proteins from the ZIP and the CDF families of zinc transporters contain a histidine-rich sequence in a loop domain located between transmembrane domains III and IV for the ZIP family and transmembrane domains IV and V for the CDF family. Topological predictions suggest that these loops are located in the cytoplasm. The loops contain a histidine-rich sequence with a variable number of histidine residues depending on the transporter. The histidine-rich sequence was postulated to serve as an extra-membrane metal binding site in these proteins. hZip1 is a human zinc transporter ubiquitously expressed. The histidine-rich motif located in the large loop of this transporter is composed of the following sequence, H(158)WHD(161). To determine if this motif is involved in the zinc transport activity of the protein, we performed site directed-mutagenesis to replace the loop histidines with alanines. Results suggest that both histidines are necessary for the zinc transport function and are not involved in the plasma membrane localization of the transporter as has been reported for the Zrt1 transporter in yeast. In addition, two histidine residues in transmembrane domains IV and V are also important in the zinc transport function. The results support an intermolecular exchange mechanism of zinc transport.     

Zeroing in on zinc uptake in yeast and plants.

Zinc is an essential micronutrient. Genes responsible for zinc uptake have now been identified from yeast and plants. These genes belong to an extended family of cation transporters called the ZIP gene family. Zinc efflux genes that belong to another transporter family, the CDF family, have also been identified in yeast and Arabidopsis. It is clear that studies in yeast can greatly aid our understanding of zinc metabolism in plants.     

Characterization of a vacuolar zinc transporter OZT1 in rice (Oryza sativa L.)

The CDF family is a ubiquitous family that has been identified in prokaryotes, eukaryotes, and archaea. Members of this family are important heavy metal transporters that transport metal ions out of the cytoplasm. In this research, a full length cDNA named Oryza sativa Zn Transporter 1 (OZT1) that closely related to rat ZnT-2 (Zn Transporter 2) gene was isolated from rice. The OZT1 encoding a CDF family protein shares 28.2 % ~ 84.3 % of identities and 49.3 % ~ 90.9 % of similarities with other zinc transporters such as RnZnT-2, HsZnT-8, RnZnT-8 and AtMTP1. OZT1 was constitutively expressed in various rice tissues. The OZT1 expression was significantly induced both in the seedlings of japonica rice Nipponbare and indica rice IR26 in response to Zn²⁺ and Cd²⁺ treatments. Besides, OZT1 expression was also increased when exposed to other excess metals, such as Cu²⁺, Fe²⁺ and Mg²⁺. Subcellular localization analysis indicated that OZT1 localized to vacuole. Heterologous expression of OZT1 in yeast increased tolerance to Zn²⁺ and Cd²⁺ stress but not the Mg²⁺ stress. Together, OZT1 is a CDF family vacuolar zinc transporter conferring tolerance to Zn²⁺ and Cd²⁺ stress, which is important to transporting and homeostasis of Zn, Cd or other heavy metals in plants.     

Histidine residues in the region between transmembrane domains III and IV of hZip1 are required for zinc transport across the plasma membrane in PC-3 cells

The proteins from the ZIP and the CDF families of zinc transporters contain a histidine-rich sequence in a loop domain located between transmembrane domains III and IV for the ZIP family and transmembrane domains IV and V for the CDF family. Topological predictions suggest that these loops are located in the cytoplasm. The loops contain a histidine-rich sequence with a variable number of histidine residues depending on the transporter. The histidine-rich sequence was postulated to serve as an extra-membrane metal binding site in these proteins. hZip1 is a human zinc transporter ubiquitously expressed. The histidine-rich motif located in the large loop of this transporter is composed of the following sequence, H₁₅₈WHD₁₆₁. To determine if this motif is involved in the zinc transport activity of the protein, we performed site directed-mutagenesis to replace the loop histidines with alanines. Results suggest that both histidines are necessary for the zinc transport function and are not involved in the plasma membrane localization of the transporter as has been reported for the Zrt1 transporter in yeast. In addition, two histidine residues in transmembrane domains IV and V are also important in the zinc transport function. The results support an intermolecular exchange mechanism of zinc transport.     

Molecular characterization of a rice metal tolerance protein, OsMTP1

Rice (Oryza sativa L. 'Nipponbare') cDNA subtractive suppression hybridization (SSH) libraries constructed using cadmium (Cd)-treated seedling roots were screened to isolate Cd-responsive genes. A cDNA clone, encoding the rice homolog of Metal Tolerance Protein (OsMTP1), was induced by Cd treatment. Plant MTPs belong to cation diffusion facilitator (CDF) protein family, which are widespread in bacteria, fungi, plants, and animals. OsMTP1 heterologous expression in yeast mutants showed that OsMTP1 was able to complement the mutant strains' hypersensitivity to Ni, Cd, and Zn, but not other metals including Co and Mn. OsMTP1 expression increased tolerance to Zn, Cd, and Ni in wild-type yeast BY4741 during the exponential growth phase. OsMTP1 fused to green fluorescent protein was localized in onion epidermal cell plasma membranes, consistent with an OsMTP1 function in heavy metal transporting. OsMTP1 dsRNAi mediated by transgenic assay in rice seedlings resulted in heavy metal sensitivity and changed the heavy metal accumulation in different organs of mature rice under low-concentration heavy metal stress. Taken together, our results show that OsMTP1 is a bivalent cation transporter localized in the cell membrane, which is necessary for efficient translocation of Zn, Cd and other heavy metals, and maintain ion homeostasis in plant.     

A novel major facilitator superfamily protein at the tonoplast influences zinc tolerance and accumulation in Arabidopsis

Zinc (Zn) is an essential micronutrient required by all cells but is toxic in excess. We have identified three allelic Zn-sensitive mutants of Arabidopsis (Arabidopsis thaliana). The gene, designated ZINC-INDUCED FACILITATOR1 (ZIF1), encodes a member of the major facilitator superfamily of membrane proteins, which are found in all organisms and transport a wide range of small, organic molecules. Shoots of zif1 mutants showed increased accumulation of Zn but not other metal ions. In combination with mutations affecting shoot-to-root Zn translocation, zif1 hma2 hma4 triple mutants accumulated less Zn than the wild type but remained Zn sensitive, suggesting that the zif1 Zn-sensitive phenotype is due to altered Zn distribution. zif1 mutants were also more sensitive to cadmium but less sensitive to nickel. ZIF1 promoter-beta-glucuronidase fusions were expressed throughout the plant, with strongest expression in young tissues, and predominantly in the vasculature in older tissues. ZIF1 expression was highly induced by Zn and, to a lesser extent, by manganese. A ZIF1-green fluorescent protein fusion protein localized to the tonoplast in transgenic plants. MTP1 has been identified as a tonoplast Zn transporter and a zif1-1 mtp1-1 double mutant was more sensitive to Zn than either of the single mutants, suggesting ZIF1 influences a distinct mechanism of Zn homeostasis. Overexpression of ZIF1 conferred increased Zn tolerance and interveinal leaf chlorosis in some transgenic lines in which ZIF1 expression was high. We propose that ZIF1 is involved in a novel mechanism of Zn sequestration, possibly by transport of a Zn ligand or a Zn ligand complex into vacuoles.     

Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance

Metallothioneins (MTs) are small cysteine-rich proteins found in various eukaryotes. Plant MTs are classified into four types based on the arrangement of cysteine residues. To determine whether all four types of plant MTs function as metal chelators, six Arabidopsis (Arabidopsis thaliana) MTs (MT1a, MT2a, MT2b, MT3, MT4a, and MT4b) were expressed in the copper (Cu)- and zinc (Zn)-sensitive yeast mutants, Deltacup1 and Deltazrc1 Deltacot1, respectively. All four types of Arabidopsis MTs provided similar levels of Cu tolerance and accumulation to the Deltacup1 mutant. The type-4 MTs (MT4a and MT4b) conferred greater Zn tolerance and higher accumulation of Zn than other MTs to the Deltazrc1 Deltacot1 mutant. To examine the functions of MTs in plants, we studied Arabidopsis plants that lack MT1a and MT2b, two MTs that are expressed in phloem. The lack of MT1a, but not MT2b, led to a 30% decrease in Cu accumulation in roots of plants exposed to 30 mum CuSO(4). Ectopic expression of MT1a RNA in the mt1a-2 mt2b-1 mutant restored Cu accumulation in roots. The mt1a-2 mt2b-1 mutant had normal metal tolerance. However, when MT deficiency was combined with phytochelatin deficiency, growth of the mt1a-2 mt2b-1 cad1-3 triple mutant was more sensitive to Cu and cadmium compared to the cad1-3 mutant. Together these results provide direct evidence for functional contributions of MTs to plant metal homeostasis. MT1a, in particular, plays a role in Cu homeostasis in the roots under elevated Cu. Moreover, MTs and phytochelatins function cooperatively to protect plants from Cu and cadmium toxicity.     

A transporter in the endoplasmic reticulum of Schizosaccharomyces pombe cells mediates zinc storage and differentially affects transition metal tolerance

The cation diffusion facilitator (CDF) family represents a class of ubiquitous metal transporters. Inactivation of a CDF in Schizosaccharomyces pombe, Zhf, causes drastically different effects on the tolerance toward various metals. A deletion mutant is Zn(2+)/Co(2+)-hypersensitive yet displays significantly enhanced Cd(2+) and Ni(2+) tolerance. Accumulation of zinc, cobalt, and cadmium is reduced in mutant cells. Non-vacuolar zinc content, as measured by analytical electron microscopy, is lower in zhf(-) cells compared with wild-type cells in the presence of elevated Zn(2+) concentrations. The protective effect against cadmium toxicity is independent of the phytochelatin detoxification pathway. Phytochelatin synthase-deficient cells show extremely enhanced (about 200-fold) cadmium tolerance when zhf is disrupted. Immunogold labeling indicates endoplasmic reticulum (ER) localization of Zhf. Electron spectroscopic imaging shows that accumulation of zinc coincides with Zhf localization, demonstrating a major role of the ER for metal storage and the involvement of Zhf in cellular zinc homeostasis. Also, these observations indicate that Cd(2+) ions exert their toxic effects on cellular metabolism in the ER rather than in the cytosol.     

The Arabidopsis metal tolerance protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion from the shoot under Fe deficiency and Zn oversupply

Zinc ions are required to maintain the biological activity of numerous proteins. However, when mislocalized or accumulated in excess, Zn(2+) ions are toxic because of adventitious binding to proteins and displacement of other metal ions, among them Fe(2+), from their binding sites. Heterologous expression of a previously uncharacterized Arabidopsis thaliana metal tolerance protein, MTP3, in the zrc1 cot1 mutant of budding yeast restores tolerance to, and cellular accumulation of, zinc and cobalt. An MTP3-GFP fusion protein localizes to the vacuolar membrane when expressed in Arabidopsis. Ectopic over-expression of MTP3 increases Zn accumulation in both roots and rosette leaves of A. thaliana, and enhances Zn tolerance. Exposure of wild-type plants to high but non-toxic concentrations of Zn or Co, or Fe deficiency, strongly induce MTP3 expression specifically in epidermal and cortex cells of the root hair zone. Silencing of MTP3 by RNA interference causes Zn hypersensitivity and enhances Zn accumulation in above-ground organs of soil-grown plants and of seedlings exposed to excess Zn or to Fe deficiency. Our data indicate that, in wild-type A. thaliana, the AtMTP3 protein contributes to basic cellular Zn tolerance and controls Zn partitioning, particularly under conditions of high rates of Zn influx into the root symplasm.     

Metal selectivity determinants in a family of transition metal transporters

Metal tolerance proteins (MTPs) are plant members of the cation diffusion facilitator (CDF) transporter family involved in cellular metal homeostasis. Members of the CDF family are ubiquitously found in all living entities and show principal selectivity for Zn(2+), Mn(2+), and Fe(2+). Little is known regarding metal selectivity determinants of CDFs. We identified a novel cereal member of CDFs in barley, termed HvMTP1, that localizes to the vacuolar membrane. Unlike its close relative AtMTP1, which is highly selective for Zn(2+), HvMTP1 exhibits selectivity for both Zn(2+) and Co(2+) as assessed by its ability to suppress yeast mutant phenotypes for both metals. Expression of HvMTP1/AtMTP1 chimeras in yeast revealed a five-residue sequence within the AtMTP1 N-segment of the His-rich intracytoplasmic loop that confines specificity to Zn(2+). Furthermore, mutants of AtMTP1 generated through random mutagenesis revealed residues embedded within transmembrane domain 3 that additionally specify the high degree of Zn(2+) selectivity. We propose that the His-rich loop, which might play a role as a zinc chaperone, determines the identity of the metal ions that are transported. The residues within transmembrane domain 3 can also influence metal selectivity, possibly through conformational changes induced at the cation transport site located within the membrane or at the cytoplasmic C-terminal domain.