Metal Efflux Transporters Research Articles

Identification of metal tolerance proteins (MTP) and their gene expression under drought stress in potato (Solanum tuberosum L.)

Metal tolerance proteins (MTPs) are as metal efflux transporters, existing extensively at all plant sections and play significant roles in regulation of the metal levels in biological processes. In the current study, phylogenetic relationships, gene structures, conserved motifs, and StMTP domains were analyzed. Here, 12 MTP genes in S. tuberosum were detected and categorized in three major clusters namely Fe/Zn-MTP, Zn-MTP, and Mn-MTP and seven groups (1, 5, 6, 7, 8, 9, and 11) according to phylogenetic relationships. Based on in silico and qPCR analysis, all of StMTPs included a cation diffusion facilitator (CDF) domains and the putative Mn-MTP harbored the ZT-dimer. An evolutionary analysis indicated that StMTP genes had undergone gene duplication leading to gene loss and gene expansion events. Analysis of transcription factor binding sites (TFBS) and microRNA in promoter region and coding sequence of StMTP genes revealed the presence of 5312 putative TFBS and 13 StmiRNAs. The analysis of promoter regions of StMTP genes possess various frequencies of TFBS, illustrating various responses in different growth and developmental stages as well as under abiotic stress. Expression profile analysis revealed that the StMTP9 were up-regulated in leaves and stem, while, StMTP8 up-regulated in leaves. Both genes down-regulated in tubers, roots as well as under drought stress. These results will provide a better insight for functional characterization of StMTP genes and can be helpful to elucidate the biological structure of their genes in potato.

OsZIP1 functions as a metal efflux transporter limiting excess zinc, copper and cadmium accumulation in rice

BackgroundMetal homeostasis is critical for plant growth, development and adaptation to environmental stresses and largely governed by a variety of metal transporters. The plant ZIP (Zn-regulated transporter, Iron-regulated transporter-like Protein) family proteins belong to the integral membrane transporters responsible for uptake and allocation of essential and non-essential metals. However, whether the ZIP family members mediate metal efflux and its regulatory mechanism remains unknown.ResultsIn this report, we provided evidence that OsZIP1 is a metal-detoxified transporter through preventing excess Zn, Cu and Cd accumulation in rice. OsZIP1 is abundantly expressed in roots throughout the life span and sufficiently induced by excess Zn, Cu and Cd but not by Mn and Fe at transcriptional and translational levels. Expression of OsZIP-GFP fusion in rice protoplasts and tobacco leaves shows that OsZIP1 resides in the endoplasmic reticulum (ER) and plasma membrane (PM). The yeast (Saccharomyces cerevisiae) complementation test shows that expression of OsZIP1 reduced Zn accumulation. Transgenic rice overexpressing OsZIP1 grew better under excess metal stress but accumulated less of the metals in plants. In contrast, both oszip1 mutant and RNA interference (RNAi) lines accumulated more metal in roots and contributed to metal sensitive phenotypes. These results suggest OsZIP1 is able to function as a metal exporter in rice when Zn, Cu and Cd are excess in environment. We further identified the DNA methylation of histone H3K9me2 of OsZIP1 and found that OsZIP1 locus, whose transcribed regions imbed a 242 bp sequence, is demethylated, suggesting that epigenetic modification is likely associated with OsZIP1 function under Cd stress.ConclusionOsZIP1 is a transporter that is required for detoxification of excess Zn, Cu and Cd in rice.

Metal tolerance of arsenic-resistant bacteria and their ability to promote plant growth of Pteris vittata in Pb-contaminated soil

Soils contaminated with Pb and As are difficult to remediate. In this study, the utility of coupling As-hyperaccumulator Pteris vittata with metal-resistant rhizobacteria was explored. Siderophore-producing and P-solubilizing As-resistant bacteria from the P. vittata rhizosphere were screened for resistance to multiple metals. Results indicated Pseudomonas spp. strain PG-12 was most efficient in resisting multiple metals, i.e., up to 0.6 mM Cd and 10 mM Pb. Amplification of gene fragments encoding various metal efflux transporters (PbrA and CadA2) from genomic DNA of PG-12 suggested that metal efflux might play a role in its metal resistance and detoxification. In addition, PG-12 produced significant levels of plant growth hormones including 17.4 μg mL−1 indole acetic acid and 3.54 μg mL−1 gibberellin. P. vittata sporophytes inoculated with PG-12 were grown in Pb-contaminated medium and exhibited improved growth, increased P uptake, and reduced Pb uptake into plant tissue compared to the control. Results demonstrated that viable PG-12 cells were responsible for Pb immobilization and plant growth enhancement in P. vittata. The ability of PG-12 cells to solubilize P and display resistance to multiple metals combined with the production of plant hormones indole acetic acid and gibberellin make PG-12 a suitable candidate for plant growth promotion in metal-contaminated soil.

Characterization of two cation diffusion facilitators NpunF0707 and NpunF1794 in Nostoc punctiforme.

To characterize genes involved in maintaining homeostatic levels of zinc in the cyanobacterium Nostoc punctiforme. Metal efflux transporters play a central role in maintaining homeostatic levels of trace elements such as zinc. Sequence analyses of the N. punctiforme genome identified two potential cation diffusion facilitator (CDF) metal efflux transporters, Npun_F0707 (Cdf31) and Npun_F1794 (Cdf33). Deletion of either Cdf31or Cdf33 resulted in increased zinc retention over 3 h. Interestingly, Cdf31(-) and Cdf33(-) mutants showed no change in sensitivity to zinc exposure in comparison with the wild type, suggesting some compensatory capacity for the loss of each other. Using qRT-PCR, a possible interaction was observed between the two cdf's, where the Cdf31(-) mutant had a more profound effect on cdf33 expression than Cdf33(-) did on cdf31. Over-expression of Cdf31 and Cdf33 in ZntA(-) - and ZitB(-) -deficient Escherichia coli revealed function similarities between the ZntA and ZitB of E. coli and the cyanobacterial transporters. The data presented shed light on the function of two important transporters that regulate zinc homeostasis in N. punctiforme. This study shows for the first time the functional characterization of two cyanobacterial zinc efflux proteins belonging to the CDF family.

Functional properties of human ferroportin, a cellular iron exporter reactive also with cobalt and zinc

Iron homeostasis is achieved by regulating the intestinal absorption of the metal and its recycling by macrophages. Iron export from enterocytes or macrophages to blood plasma is thought to be mediated by ferroportin under the control of hepcidin. Although ferroportin was identified over a decade ago, little is understood about how it works. We expressed in Xenopus oocytes a human ferroportin-enhanced green fluorescent protein fusion protein and observed using confocal microscopy its exclusive plasma-membrane localization. As a first step in its characterization, we established an assay to detect functional expression of ferroportin by microinjecting oocytes with (55)Fe and measuring efflux. Ferroportin expression increased the first-order rate constants describing (55)Fe efflux up to 300-fold over control. Ferroportin-mediated (55)Fe efflux was saturable, temperature-dependent (activation energy, Ea ≈ 17 kcal/mol), maximal at extracellular pH ≈ 7.5, and inactivated at extracellular pH < 6.0. We estimated that ferroportin reacts with iron at its intracellular aspect with apparent affinity constant < 10(-7) M. Ferroportin expression also stimulated efflux of (65)Zn and (57)Co but not of (64)Cu, (109)Cd, or (54)Mn. Hepcidin treatment of oocytes inhibited efflux of (55)Fe, (65)Zn, and (57)Co. Whereas hepcidin administration in mice resulted in a marked hypoferremia within 4 h, we observed no effect on serum zinc levels in those same animals. We conclude that ferroportin is an iron-preferring cellular metal-efflux transporter with a narrow substrate profile that includes cobalt and zinc. Whereas hepcidin strongly regulated serum iron levels in the mouse, we found no evidence that ferroportin plays an important role in zinc homeostasis.

Roles of plant metal tolerance proteins (MTP) in metal storage and potential use in biofortification strategies

Zinc (Zn) is an essential micronutrient for plants, playing catalytic or structural roles in enzymes, transcription factors, ribosomes, and membranes. In humans, Zn deficiency is the second most common mineral nutritional disorder, affecting around 30% of the world's population. People living in poverty usually have diets based on milled cereals, which contain low Zn concentrations. Biofortification of crops is an attractive cost-effective solution for low mineral dietary intake. In order to increase the amounts of bioavailable Zn in crop edible portions, it is necessary to understand how plants take up, distribute, and store Zn within their tissues, as well as to characterize potential candidate genes for biotechnological manipulation. The metal tolerance proteins (MTP) were described as metal efflux transporters from the cytoplasm, transporting mainly Zn2+ but also Mn2+, Fe2+, Cd2+, Co2+, and Ni2+. Substrate specificity appears to be conserved in phylogenetically related proteins. MTPs characterized so far in plants have a role in general Zn homeostasis and tolerance to Zn excess; in tolerance to excess Mn and also in the response to iron (Fe) deficiency. More recently, the first MTPs in crop species have been functionally characterized. In Zn hyperaccumulator plants, the MTP1 protein is related to hypertolerance to elevated Zn concentrations. Here, we review the current knowledge on this protein family, as well as biochemical functions and physiological roles of MTP transporters in Zn hyperaccumulators and non-accumulators. The potential applications of MTP transporters in biofortification efforts are discussed.

Defense Mechanisms of Pseudomonas aeruginosa PAO1 against Quantum Dots and Their Released Heavy Metals

The growing use of quantum dots (QDs) in numerous applications increases the possibility of their release to the environment. Bacteria provide critical ecosystem services, and understanding their response to QDs is important to assess the potential environmental impacts of such releases. Here, we analyze the microbial response to sublethal exposure to commercial QDs, and investigate potential defense and adaptation mechanisms in the model bacterium Pseudomonas aeruginosa PAO1. Both intact and weathered QDs, as well as dissolved metal constituents, up-regulated czcABC metal efflux transporters. Weathered QDs also induced superoxide dismutase gene sodM, which likely served as a defense against oxidative stress. Interestingly, QDs also induced antibiotic resistance (ABR) genes and increased antibiotic minimum inhibitory concentrations by 50 to 100%, which suggests up-regulation of global stress defense mechanisms. Extracellular synthesis of nanoparticles (NPs) was observed after exposure to dissolved Cd(NO(3))(2) and SeO(2). With extended X-ray absorption fine structure (EXAFS), we discerned biogenic NPs such as CdO, CdS, CdSe, and selenium sulfides. These results show that bacteria can mitigate QD toxicity by turning on energy-dependent heavy-metal ion efflux systems and by mediating the precipitation of dissolved metal ions as less toxic and less bioavailable insoluble NPs.

MrdH, a Novel Metal Resistance Determinant of Pseudomonas putida KT2440, Is Flanked by Metal-Inducible Mobile Genetic Elements

We report here the identification and characterization of mrdH, a novel chromosomal metal resistance determinant, located in the genomic island 55 of Pseudomonas putida KT2440. It encodes for MrdH, a predicted protein of approximately 40 kDa with a chimeric domain organization derived from the RcnA and RND (for resistance-nodulation-cell division) metal efflux proteins. The metal resistance function of mrdH was identified by the ability to confer nickel resistance upon its complementation into rcnA mutant (a nickel- and cobalt-sensitive mutant) of Escherichia coli. However, the disruption of mrdH in P. putida resulted in an increased sensitivity to cadmium and zinc apart from nickel. Expression studies using quantitative reverse transcription-PCR showed the induction of mrdH by cadmium, nickel, zinc, and cobalt. In association with mrdH, we also identified a conserved hypothetical gene mreA whose encoded protein showed significant homology to NreA and NreA-like proteins. Expression of the mreA gene in rcnA mutant of E. coli enhanced its cadmium and nickel resistance. Transcriptional studies showed that both mrdH and mreA underwent parallel changes in gene expression. The mobile genetic elements Tn4652 and IS1246, flanking mrdH and mreA were found to be induced by cadmium, nickel, and zinc, but not by cobalt. This study is the first report of a single-component metal efflux transporter, mrdH, showing chimeric domain organization, a broad substrate spectrum, and a location amid metal-inducible mobile genetic elements.

The cation-efflux transporter BjCET2 mediates zinc and cadmium accumulation in Brassica juncea L. leaves

Brassica juncea L. is a Zn/Cd accumulator. To determine the physiological basis of its metal accumulation phenotype, the functional properties and role of the metal efflux transporter BjCET2 were investigated using transgenic technology. Heterologous expression of BjCET2 in the double mutant yeast strain Deltazrc1Deltacot1 enhanced the metal tolerance of the yeast strain and led to decrease in Zn or Cd accumulation. Detection of green fluorescence from green fluorescent protein (GFP) in the root tip of transgenic tobacco further revealed that BjCET2::GFP is localized at the plasma membrane. Semi-quantitative RT-PCR analysis showed that BjCET2 was most abundant in the root and was weakly expressed in the stem and leaves. The expression of BjCET2 was up-regulated by heavy metals. However, exposure to low temperature, salt and drought did not affect the expression of BjCET2. Overexpression of BjCET2 in transgenic B. juncea plants conferred heavy metal tolerance and increased Cd/Zn accumulation in the leaves. BjCET2-deficient B. juncea mediated by antisense RNA resulted in hypersensitivity to heavy metals and decreased Zn/Cd accumulation in the plants. These results suggest that the heavy metal efflux of BjCET2 plays important roles in the metal tolerance of B. juncea and in Zn/Cd accumulation in B. juncea.

Rice P1B-Type Heavy-Metal ATPase, OsHMA9, Is a Metal Efflux Protein

P(1B)-type heavy-metal ATPases (HMAs) are transmembrane metal-transporting proteins that play a key role in metal homeostasis. Despite their importance, very little is known about their functions in monocot species. We report the characterization of rice (Oryza sativa) OsHMA9, a member of the P(1B)-type ATPase family. Semiquantitative reverse transcription-polymerase chain reaction analyses of seedlings showed that OsHMA9 expression was induced by a high concentration of copper (Cu), zinc (Zn), and cadmium. We also determined, through promoterbeta-glucuronidase analysis, that the main expression was in the vascular bundles and anthers. The OsHMA9:green fluorescence protein fusion was localized to the plasma membrane. Heterologous expression of OsHMA9 partially rescued the Cu sensitivity of the Escherichia coli copA mutant, which is defective in Cu-transporting ATPases. It did not rescue the Zn sensitivity of the zntA mutant, which is defective in Zn-transporting ATPase. To further elucidate the functional roles of OsHMA9, we isolated two independent null alleles, oshma9-1 and oshma9-2, from the T-DNA insertion population. Mutant plants exhibited the phenotype of increased sensitivity to elevated levels of Cu, Zn, and lead. These results support a role for OsHMA9 in Cu, Zn, and lead efflux from the cells. This article is the first report on the functional characterization of a P(1B)-type metal efflux transporter in monocots.

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.