Metal tolerance protein MTP6 affects mitochondrial iron and manganese homeostasis in cucumber.

Members of the cation diffusion facilitator (CDF) family have been identified in all kingdoms of life. They have been divided into three subgroups, namely Zn-CDF, Fe/Zn-CDF, and Mn-CDF, based on their putative specificity to transported metal ions. The plant metal tolerance protein 6 (MTP6) proteins fall into the Fe/Zn-CDF subgroup; however, their function in iron/zinc transport has not yet been confirmed. Here, we characterized the MTP6 protein from cucumber, Cucumis sativus. When expressed in yeast and in protoplasts isolated from Arabidopsis cells, CsMTP6 localized in mitochondria and contributed to the efflux of Fe and Mn from these organelles. Immunolocalization of CsMTP6 in cucumber membranes confirmed this association with mitochondria. Root expression and protein levels of CsMTP6 were significantly up-regulated in conditions of Fe deficiency and excess, but were not affected by Mn availability. These results indicate that MTP6 proteins contribute to the distribution of Fe and Mn between the cytosol and mitochondria of plant cells, and are regulated by Fe to maintain mitochondrial and cytosolic iron homeostasis under varying conditions of Fe availability.

Cucumber Golgi protein CsMTP5 forms a Zn-transporting heterodimer with high molecular mass protein CsMTP12.

Heterodimeric complexes formed by members of the cation facilitator (CDF) family catalyse the import of Zn into the secretory pathway of yeast and vertebrate cells. Orthologous proteins AtMTP5 and AtMTP12 from Arabidopsis have also been shown to form a heterodimeric complex at the Golgi compartment of plant cells that possibly transport Zn. In this study we show that cucumber proteins CsMTP5 and CsMTP12 form a functional heterodimer that is involved in the loading of Zn into the ER lumen under low Zn, and not in the detoxification of yeast from Zn excess through vesicle-mediated exocytosis. Using specific antibodies, we demonstrate that CsMTP5 is localized at the Golgi compartment of cucumber cells and is markedly up-regulated upon Zn deficiency. The level of CsMTP5 transcript in cucumber is also significantly elevated in Zn-limiting conditions, whereas the expression of CsMTP12 is independent of the availability of Zn. Therefore we propose that the cucumber heterodimeric complex CsMTP5-CsMTP12 functions to deliver Zn to Zn-dependent proteins of the Golgi compartment and is regulated by zinc at the level of CsMTP5 transcription.

Cucumber metal tolerance protein 7 (CsMTP7) is involved in the accumulation of Fe in mitochondria under Fe excess.

The plant metal tolerance protein family (MTP) includes 12 members that have been classified into three phylogenetically different subgroups - Zn-cation diffusion facilitator (CDF), Fe/Zn-CDF and Mn-CDF - based on their putative metal specificity. To date, only members belonging to the Zn-CDF or Mn-CDF group have been characterized functionally. The plant Fe/Zn-CDF subgroup includes two proteins, MTP6 and MTP7, but their function and metal specificity have not been confirmed. In this study we showed that cucumber CsMTP7 is a highly specific mitochondrial Fe importer that is able to confer yeast tolerance to Fe excess through increased accumulation of Fe in the mitochondria. We also demonstrated that CsMTP7 contributes to the increased accumulation of Fe in the mitochondria of Arabidopsis thaliana protoplasts. The transcripts and mitochondrial levels of CsMTP7 and ferritin - the iron-storing protein - are significantly increased in cucumber roots in response to Fe excess. This finding suggests that CsMTP7 and ferritin work in concert to accumulate Fe in plant mitochondria. As genes that encode orthologous proteins have been identified in phylogenetically distant organisms, including Archaea, cyanobacteria, humans and plants, but not in yeast, we concluded that the MTP7-mediated mitochondrial Fe accumulation may be conserved in the species, and express mitochondrial ferritin for mitochondrial Fe storage.

[Characterization of the proteins involved in the transport and storage of iron in plants]. / Charakterystyka bialek zwiazanych z transportem i magazynowaniem zelaza u roslin.

Iron is a transient metal essential for the proper growth and development of plants because as a component of the enzymes with a wide redox potential, iron contributes to the key cellular processes. During evolution, plants have developed a wide range of molecular mechanisms for the efficient control of iron homeostasis within their cells, tissues and organs. These include membrane proteins involved in the uptake, long-distance transport and intracellular distribution of iron as well as the iron-storing and iron-chelating proteins, that are involved in the protection of the plant cells from iron excess and/or ensure the proper growth and development of plants under Fe deficiency. Since iron is crucial for the functioning of plants, the proteins involved in the transport, chelation and storage of iron within plant cells are currently thoroughly studied. This work presents the current state of the art in the knowledge of these proteins and their regulatory mechanisms.

Transmembrane topology of the arsenite permease Acr3 from Saccharomyces cerevisiae.

Acr3 is a plasma membrane transporter, a member of the bile/arsenite/riboflavin transporter (BART) superfamily, which confers high-level resistance to arsenicals in the yeast Saccharomyces cerevisiae. We have previously shown that the yeast Acr3 acts as a low affinity As(III)/H+ and Sb(III)/H+ antiporter. We have also identified several amino acid residues that are localized in putative transmembrane helices (TM) and appeared to be critical for the Acr3 activity. In the present study, the topology of Acr3 was investigated by insertion of glycosylation and factor Xa protease cleavage sites at predicted hydrophilic regions. The analysis of the glycosylation pattern and factor Xa cleavage products of resulting Acr3 fusion constructs provide evidence supporting a topological model of Acr3 with 10 TM segments and cytoplasmically oriented N- and C-terminal domains. Next, we investigated the role of the hydrophilic loop connecting TM8 and TM9, the large size of which is unique to members of the yeast Acr3 family of metalloid transporters. We found that a 28 amino acid deletion in this region does not affect Acr3 folding, trafficking substrate binding, or transport activity. Finally, we constructed a homology-based structural model of Acr3 using the crystal structure of the Yersinia frederiksenii homologue of the human bile acid sodium symporter ASBT.

Disentangling genetic and epigenetic determinants of ultrafast adaptation.

A major rationale for the advocacy of epigenetically mediated adaptive responses is that they facilitate faster adaptation to environmental challenges. This motivated us to develop a theoretical-experimental framework for disclosing the presence of such adaptation-speeding mechanisms in an experimental evolution setting circumventing the need for pursuing costly mutation-accumulation experiments. To this end, we exposed clonal populations of budding yeast to a whole range of stressors. By growth phenotyping, we found that almost complete adaptation to arsenic emerged after a few mitotic cell divisions without involving any phenotypic plasticity. Causative mutations were identified by deep sequencing of the arsenic-adapted populations and reconstructed for validation. Mutation effects on growth phenotypes, and the associated mutational target sizes were quantified and embedded in data-driven individual-based evolutionary population models. We found that the experimentally observed homogeneity of adaptation speed and heterogeneity of molecular solutions could only be accounted for if the mutation rate had been near estimates of the basal mutation rate. The ultrafast adaptation could be fully explained by extensive positive pleiotropy such that all beneficial mutations dramatically enhanced multiple fitness components in concert. As our approach can be exploited across a range of model organisms exposed to a variety of environmental challenges, it may be used for determining the importance of epigenetic adaptation-speeding mechanisms in general.

The mitogen-activated protein kinase Slt2 modulates arsenite transport through the aquaglyceroporin Fps1.

Arsenite is widely present in nature; therefore, cells have evolved mechanisms to prevent arsenite influx and promote efflux. In yeast (Saccharomyces cerevisiae), the aquaglyceroporin Fps1 mediates arsenite influx and efflux. The mitogen-activated protein kinase (MAPK) Hog1 has previously been shown to restrict arsenite influx through Fps1. In this study, we show that another MAPK, Slt2, is transiently phosphorylated in response to arsenite influx. Our findings indicate that the protein kinase activity of Slt2 is required for its role in arsenite tolerance. While Hog1 prevents arsenite influx via phosphorylation of T231 at the N-terminal domain of Fps1, Slt2 promotes arsenite efflux through phosphorylation of S537 at the C terminus. Our data suggest that Slt2 physically interacts with Fps1 and that this interaction depends on phosphorylation of S537. We hypothesize that Hog1 and Slt2 may affect each other's binding to Fps1, thereby controlling the opening and closing of the channel.

Genes Encoding Cucumber Full-Size ABCG Proteins Show Different Responses to Plant Growth Regulators and Sclareolide.

Full-size members of the ABCG (ATP-binding cassette, subfamily G) subfamily of ABC transporters have been found only in plants and fungi. The plant genes encoding full-size ABCGs identified so far appeared to be differentially regulated under various environmental constraints, plant growth regulators, and microbial elicitors, indicating a broad functional role of these proteins in plant responses to abiotic and biotic stress. Nevertheless, the structure and physiological function of full-size ABCGs in many plant species are still unknown. We have recently identified 16 genes encoding full-size ABCG proteins in cucumber and found that the transcripts of two of them, CsABCG36 (CsPDR8) and CsABCG40 (CsPDR12), are most abundant in roots and are significantly affected by phytohormones and auxin herbicide. In this study, we analyzed the structure and phylogeny of all the full-size cucumber ABCG transporters and studied the organ expression profiles of the remaining 14 CsABCG genes. In addition, we investigated the effect of different plant growth regulators and the diterpene sclareolide on CsABCG expression in cucumber roots. Until now, the full-size plant ABCG transporters have been grouped into five different clusters. The new phylogenetic analysis of full-size ABCGs from model plants and cucumber clustered these proteins into six different subgroups. Interestingly, the expression profiles of cucumber ABCG genes assigned to the same clusters were not correlated, suggesting functional diversification or different regulatory mechanisms of the full-size cucumber ABCG proteins.

Copper-transporting ATPases: The evolutionarily conserved machineries for balancing copper in living systems.

Copper ATPases (Cu-ATPases) are ubiquitous transmembrane proteins using energy from ATP to transport copper across different biological membranes of prokaryotic and eukaryotic cells. As they belong to the P-ATPase family, Cu-ATPases contain a characteristic catalytic domain with an evolutionarily conserved aspartate residue phosphorylated by ATP to form a phosphoenzyme intermediate, as well as transmembrane helices containing a cation-binding cysteine-proline-cysteine/histidine/serine (CPx) motif for catalytic activation and cation translocation. In addition, most Cu-ATPases possess the N-terminal Cu-binding CxxC motif required for regulation of enzyme activity. In cells, the Cu-ATPases receive copper from soluble chaperones and maintain intracellular copper homeostasis by efflux of copper from the cell or transport of the metal into the intracellular compartments. In addition, copper pumps play an essential role in cuproprotein biosynthesis by the uptake of copper into the cell or delivery of the metal into the chloroplasts and thylakoid lumen or into the lumen of the secretory pathway, where the metal ion is incorporated into copper-dependent enzymes. In the recent years, significant progress has been made toward understanding the function and regulation of Cu-transporting ATPases in archaea, bacteria, yeast, humans, and plants, providing new insights into the specific physiological roles of these essential proteins in various organisms and revealing some conservative regulatory mechanisms of Cu-ATPase activity. In this review, the structural, biochemical, and functional properties of Cu-ATPases from phylogenetically different organisms are summarized and discussed, with particular attention given to the recent insights into the molecular biology of copper pumps in plants.

Cucumber metal tolerance protein CsMTP9 is a plasma membrane H⁺-coupled antiporter involved in the Mn²âº and Cd²âº efflux from root cells.

Members of the plant metal tolerance protein (MTP) family have been classified into three major groups - Zn-CDF, Mn-CDF and Zn/Fe-CDF - however, the selectivity of most of the MTPs has not been confirmed yet. Cucumber gene CsMTP9 encoding a putative CDF transporter homologous to members of the Mn-CDF cluster is expressed exclusively in roots. The relative abundance of CsMTP9 transcript and protein in roots is significantly increased under Mn excess and Cd. Immunolocalization with specific antibodies revealed that CsMTP9 is a plasma membrane transporter that localizes to the inner PM domain of root endodermal cells. The plasma membrane localization of CsMTP9 was confirmed by the expression of the fusion proteins of GFP (green fluorescent protein) and CsMTP9 in yeast and protoplasts prepared from Arabidopsis cells. In yeast, CsMTP9 transports Mn(2+) and Cd(2+) via a proton-antiport mechanism with an apparent Km values of approximately 10 µm and 2.5 µm for Mn(2+) and Cd(2+) , respectively. In addition, CsMTP9 expression in yeast rescues the Mn- and Cd-hypersensitive phenotypes through the enhanced efflux of Mn(2+) and Cd(2+) from yeast cells. Similarly, the overexpression of CsMTP9 in A. thaliana confers increased resistance of plants to Mn excess and Cd but not to other heavy metals and leads to the enhanced translocation of manganese and cadmium from roots to shoots. These findings indicate that CsMTP9 is a plasma membrane H(+) -coupled Mn(2+) and Cd(2+) antiporter involved in the efflux of manganese and cadmium from cucumber root cells by the transport of both metals from endodermis into vascular cylinder.

Identification of critical residues for transport activity of Acr3p, the Saccharomyces cerevisiae†As(III)/H+ antiporter.

Acr3p is an As(III)/H(+) antiporter from Saccharomyces cerevisiae belonging to the bile/arsenite/riboflavin transporter superfamily. We have previously found that Cys151 located in the middle of the fourth transmembrane segment (TM4) is critical for antiport activity, suggesting that As(III) might interact with a thiol group during the translocation process. In order to identify functionally important residues involved in As(III)/H(+) exchange, we performed a systematic alanine-replacement analysis of charged/polar and aromatic residues that are conserved in the Acr3 family and located in putative transmembrane segments. Nine residues (Asn117, Trp130, Arg150, Trp158, Asn176, Arg230, Tyr290, Phe345, Asn351) were found to be critical for proper folding and trafficking of Acr3p to the plasma membrane. In addition, we found that replacement of highly conserved Phe266 (TM7), Phe352 (TM9), Glu353 (TM9) and Glu380 (TM10) with Ala abolished transport activity of Acr3p, while mutation of Ser349 (TM9) to Ala significantly reduced the As(III)/H(+) exchange, suggesting an important role of these residues in the transport mechanism. Detailed mutational analysis of Glu353 and Glu380 revealed that the negatively charged residues located in the middle of transmembrane segments TM9 and TM10 are crucial for antiport activity. We also discuss a hypothetical model of the Acr3p transport mechanism.

Functional and Biochemical Characterization of Cucumber Genes Encoding Two Copper ATPases CsHMA5.1 and CsHMA5.2.

Plant copper P1B-type ATPases appear to be crucial for maintaining copper homeostasis within plant cells, but until now they have been studied mostly in model plant systems. Here, we present the molecular and biochemical characterization of two cucumber copper ATPases, CsHMA5.1 and CsHMA5.2, indicating a different function for HMA5-like proteins in different plants. When expressed in yeast, CsHMA5.1 and CsHMA5.2 localize to the vacuolar membrane and are activated by monovalent copper or silver ions and cysteine, showing different affinities to Cu(+) (Km ∼1 or 0.5 µM, respectively) and similar affinity to Ag(+) (Km ∼2.5 µM). Both proteins restore the growth of yeast mutants sensitive to copper excess and silver through intracellular copper sequestration, indicating that they contribute to copper and silver detoxification. Immunoblotting with specific antibodies revealed the presence of CsHMA5.1 and CsHMA5.2 in the tonoplast of cucumber cells. Interestingly, the root-specific CsHMA5.1 was not affected by copper stress, whereas the widely expressed CsHMA5.2 was up-regulated or down-regulated in roots upon copper excess or deficiency, respectively. The copper-induced increase in tonoplast CsHMA5.2 is consistent with the increased activity of ATP-dependent copper transport into tonoplast vesicles isolated from roots of plants grown under copper excess. These data identify CsHMA5.1 and CsHMA5.2 as high affinity Cu(+) transporters and suggest that CsHMA5.2 is responsible for the increased sequestration of copper in vacuoles of cucumber root cells under copper excess.

Modification of plasma membrane NADPH oxidase activity in cucumber seedling roots in response to cadmium stress.

The aim of this study was to investigate the effect of cadmium on plasma membrane (PM) NADPH oxidase activity in cucumber roots. Plants were treated with cadmium for 1, 3 or 6 days. Some of the plants after 3-day exposure to cadmium were transferred to a medium without the heavy metal for the next 3 days. Treatment of plants with cadmium for 6 days stimulated the activity of NADPH oxidase. The highest stimulation of O2(•-) production by NADPH oxidase was observed in post-stressed plants, which was correlated with the stimulation of activity of PM H(+)-ATPase in the same conditions. In order to examine the effects of cadmium stresses on the expression level of genes encoding NADPH oxidase, putative cucumber homologs encoding RBOH proteins were selected and a real-time PCR assay was performed. NADPH is a substrate for oxidase; thus alterations in the activity of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, NADP-isocitrate dehydrogenase and NADP-malic enzyme under cadmium stress were studied. The activity of NADPH dehydrogenases was increased under cadmium stress. The results indicate that PM NADPH oxidase could be involved in plants' response to cadmium stress by affecting the activity of PM H(+)-ATPase, and NADPH-generating enzymes could play important roles in this process.

Molecular and biochemical properties of two P1B2-ATPases, CsHMA3 and CsHMA4, from cucumber.

P1B-ATPases (heavy metal ATPases, HMAs) constitute a multigenic subfamily of P-ATPases involved in the transport of monovalent and divalent heavy metals in plant cells. Here, we present the organization of genes encoding the HMA family in the cucumber genome and report the function and biochemical properties of two cucumber proteins homologous to the HMA2-4-like plant HMAs. Eight genes encoding putative P1B -ATPases were identified in the cucumber genome. Among them, CsHMA3 was predominantly expressed in roots and up-regulated by Pb, Zn and Cd excess, whereas the CsHMA4 transcript was most abundant in roots and flowers of cucumber plants, and elevated under Pb and Zn excess. Expression of CsHMA3 in Saccharomyces cerevisiae enhanced yeast tolerance to Cd and Pb, whereas CsHMA4 conferred increased resistance of yeast cells to Cd and Zn. Immunostaining with specific antibodies raised against cucumber proteins revealed tonoplast localization of CsHMA3 and plasma membrane localization of CsHMA4 in cucumber root cells. Kinetic studies of CsHMA3 and CsHMA4 in yeast membranes indicated differing heavy metal cation affinities of these two proteins. Altogether, the results suggest an important role of CsHMA3 and CsHMA4 in Cd and Pb detoxification and Zn homeostasis in cucumber cells.

Two metal-tolerance proteins, MTP1 and MTP4, are involved in Zn homeostasis and Cd sequestration in cucumber cells.

Metal-tolerance proteins (MTPs) are divalent cation transporters that have been shown to be essential for metal homeostasis and tolerance in model plants and hyperaccumulators. Due to the lack of genomic resources, studies on MTPs in cultivated crops are lacking. Here, we present the first functional characterization of genes encoding cucumber proteins homologous to MTP1 and MTP4 transporters. CsMTP1 expression was ubiquitous in cucumber plants, whereas CsMTP4 mRNA was less abundant and was not detected in the generative parts of the flowers. When expressed in yeast, CsMTP1 and CsMTP4 were able to complement the hypersensitivity of mutant strains to Zn and Cd through the increased sequestration of metals within vacuoles using the transmembrane electrochemical gradient. Both proteins formed oligomers at the vacuolar membranes of yeast and cucumber cells and localized in Arabidopsis protoplasts, consistent with their function in vacuolar Zn and Cd sequestration. Changes in the abundance of CsMTP1 and CsMTP4 transcripts and proteins in response to elevated Zn and Cd, or to Zn deprivation, suggested metal-induced transcriptional, translational, and post-translational modifications of protein activities. The differences in the organ expression and affinity of both proteins to Zn and Cd suggested that CsMTP1 and CsMTP4 may not be functionally redundant in cucumber cells.

Elucidating the response of Kluyveromyces lactis to arsenite and peroxide stress and the role of the transcription factor KlYap8.

All organisms need to sense and respond to a range of stress conditions. In this study, we used transcriptional profiling to identify genes and cellular processes that are responsive during arsenite and tert-butyl hydroperoxide exposure in Kluyveromyces lactis. Many arsenite-responsive genes encode proteins involved in redox processes, protein folding and stabilization, and transmembrane transport. The majority of peroxide-responsive genes encode functions related to transcription, translation, redox processes, metabolism and transport. A substantial number of these stress-regulated genes contain binding motifs for the AP-1 like transcription factors KlYap1 and KlYap8. We demonstrate that KlYap8 binds to and regulates gene expression through a 13 base-pair promoter motif, and that KlYap8 provides protection against arsenite, antimonite, cadmium and peroxide toxicity. Direct transport assays show that Klyap8Δ cells accumulate more arsenic and cadmium than wild type cells and that the Klyap8Δ mutant is defective in arsenic and cadmium export. KlYap8 regulates gene expression in response to both arsenite and peroxide, and might cooperate with KlYap1 in regulation of specific gene targets. Comparison of KlYap8 with its Saccharomyces cerevisiae orthologue ScYap8 indicates that KlYap8 senses and responds to multiple stress signals whereas ScYap8 is only involved in the response to arsenite and antimonite. Thus, our data suggest that functional specialization of ScYap8 has occurred after the whole genome duplication event. This is the first genome-wide stress response analysis in K. lactis and the first demonstration of KlYap8 function.

Cucumber metal transport protein MTP8 confers increased tolerance to manganese when expressed in yeast and Arabidopsis thaliana.

Cation diffusion facilitator (CDF) proteins are ubiquitous divalent cation transporters that have been proved to be essential for metal homeostasis and tolerance in Archaebacteria, Bacteria, and Eukaryota. In plants, CDFs are designated as metal tolerance proteins (MTPs). Due to the lack of genomic resources, studies on MTPs in other plants, including cultivated crops, are lacking. Here, the identification and organization of genes encoding members of the MTP family in cucumber are described. The first functional characterization of a cucumber gene encoding a member of the Mn-CDF subgroup of CDF proteins, designated as CsMTP8 based on the highest homology to plant MTP8, is also presented. The expression of CsMTP8 in Saccharomyces cerevisiae led to increased Mn accumulation in yeast cells and fully restored the growth of mutants hypersensitive to Mn in Mn excess. Similarly, the overexpression of CsMTP8 in Arabidopsis thaliana enhanced plant tolerance to high Mn in nutrition media as well as the accumulation of Mn in plant tissues. When fused to green fluorescent protein (GFP), CsMTP8 localized to the vacuolar membranes in yeast cells and to Arabidopsis protoplasts. In cucumber, CsMTP8 was expressed almost exclusively in roots, and the level of gene transcript was markedly up-regulated or reduced under elevated Mn or Mn deficiency, respectively. Taken together, the results suggest that CsMTP8 is an Mn transporter localized in the vacuolar membrane, which participates in the maintenance of Mn homeostasis in cucumber root cells.