Divalent Cation Transporter Research Articles

Thermogenic Adipocytes Promote M2 Macrophage Polarization through CNNM4-Mediated Mg Secretion.

M2 macrophages promote adipose tissue thermogenesis which dissipates energy in the form of heat to combat obesity. However, the regulation of M2 macrophages by thermogenic adipocytes is unclear. Here, it is identified magnesium (Mg) as a thermogenic adipocyte-secreted factor to promote M2 macrophage polarization. Mg transporter Cyclin and CBS domain divalent metal cation transport mediator 4 (CNNM4) induced by ADRB3-PKA-CREB signaling in thermogenic adipocytes during cold exposure mediates Mg efflux and Mg in turn binds to the DFG motif in mTOR to facilitate mTORC2 activation and M2 polarization in macrophages. In obesity, downregulation of CNNM4 expression inhibits Mg secretion from thermogenic adipocytes, which leads to decreased M2 macrophage polarization and thermogenesis. As a result, CNNM4 overexpression in adipocytes or Mg supplementation in adipose tissue ameliorates obesity by promoting thermogenesis. Importantly, an Mg wire implantation (AMI) approach is introduced to achieve adipose tissue-specific long-term Mg supplement. AMI promotes M2 macrophage polarization and thermogenesis and ameliorates obesity in mice. Taken together, a reciprocal regulation of thermogenic adipocytes and M2 macrophages important for thermogenesis is identified, and AMI is offered as a promising strategy against obesity.

Marked microcytosis and increased transferrin saturation: Think about variants in SLC11A2 (DMT1)

Congenital microcytic anemias are rare diseases associated with decreased hemoglobin synthesis and red blood cells of low corpuscular volume. DMT1/NRAMP2 is a highly conserved divalent cation transporter encoded by the SLC11A2 gene, expressed at the membrane of various cells. It ensures ferrous iron absorption from the apical membrane of enterocytes, iron recovery from urine by renal tubules, and acidified endosome uptake after transferrin internalization. Pathogenic DMT1 variants have been described in 10 individuals to date, associated with recessive hypochromic anemia and iron overload. Herein, we report a new variant of SLC11A2 (c.469A>G, p.Ile157Val) compound with known p.Arg416Cys associated with a frankly microcytic anemia and increased transferrin saturation. The clinical picture observed in the patient was exceptionally mild, extending the field of the DMT1 phenotypes to borderline anemias.

Heavy metal tolerance in crop plants: an in silico comprehensive analysis of Metal- tolerance gene family in barley

Heavy metal contamination in soil is a global concern due to its harmful effect to all living organisms. Phytoremediation is an emerging cost- effective technology, which utilizes different types of hyperaccumulator plants for the removal of heavy metal pollutants. Crop plants have been suggested as a good candidate for recultivation of agricultural soil in phytoremediation process, however the molecular mechanisms responsible for the crop tolerance to heavy metals is still unknown. Metal-tolerance proteins (MTPs) are divalent cation transporters that play critical roles in metal tolerance and ion homeostasis in plants. The current study identified 12 HvMTPs in the barley (Hordeum vulgare, Hv) genome; the majority of MTPs were hydrophobic proteins found in the vacuolar membrane. Gene expression profiling suggests that HvMTPs play an active role in maintaining barley nutrient homeostasis throughout its life cycle. The expression of barley HvMTP genes in the presence of heavy metals revealed that these MTPs were induced by at least one metal ion, implying their involvement in metal tolerance.

The dynamic roles of intracellular vacuoles in heavy metal detoxification by Rhodotorula mucilaginosa.

Rhodotorula mucilaginosa (Rho) can develop a range of strategies to resist the toxicity of heavy metals. This study aimed to investigate the physiological responses and transcriptomic regulation of the fungus under different heavy metal stresses. This study applied transmission electron microscopy and RNA-seq to investigate the fungal resistance to Pb, Cd, and Cu stresses. Under Pb stress, the activated autophagy-related genes, vesicle-fusing ATPase, and vacuolar ATP synthase improved vacuolar sequestration. This offsets the loss of lipids. However, the metal sequestration by vacuoles was not improved under Cd stress. Vacuolar fusion was also inhibited following the interference of intravacuolar Ca2+ due to their similar ionic radii. Cu2+ showed the maximum toxic effects due to its lowest cellular sorption (as low as 7%) with respect to Pb2+ and Cd2+, although the efflux pumps and divalent metal ion transporters partially contributed to the detoxification. Divalent cation transporters and vacuolar sequestration are the critical strategies for Rho to resist Pb stress. Superoxide dismutase (SOD) is the main strategy for Cd resistance in Rho. The intracellular Cu level was decreased by efflux pump and divalent metal ion transporters.

Molecular characterization and expression patterns of MTP genes under heavy metal stress in mustard (Brassica juncea L.)

Members of the Metal Tolerance Protein (MTP) family are critical in mediating the transport and tolerance of divalent metal cations. Despite their significance, the understanding of MTP genes in mustard (Brassica juncea) remains limited, especially regarding their response to heavy metal (HM) stress. In our study, we identified MTP gene sets in Brassica rapa (17 genes), Brassica nigra (18 genes), and B. juncea (33 genes) using the HMMER (Cation_efflux; PF01545) and BLAST analysis. For the 33 BjMTPs, a comprehensive bioinformatics analysis covering the physicochemical properties, phylogenetic relationships, conserved motifs, protein structures, collinearity, spatiotemporal RNA-seq expression, GO enrichment, and expression profiling under six HM stresses (Mn2+, Fe2+, Zn2+, Cd2+, Sb3+, and Pb2+) were carried out. According to the findings of physicochemical characteristics, phylogenetic tree, and collinearity, the allopolyploid B. juncea’s MTP genes were inherited from its progenitors, B. rapa and B. nigra, with minimal gene loss during polyploidization. Members of the BjMTP family exhibited conserved motifs, promoter elements, and expression patterns across subgroups, consistent with the seven evolutionary branches (G1, G4–G9, and G12) of the MTPs. Further, spatiotemporal expression profiling under HM stresses successfully identified specific genes and crucial cis-regulatory elements associated with the response of BjMTPs to HM stresses. These findings may contribute to the genetic improvement of B. juncea for enhanced HM tolerance, facilitating the remediation of HM-contaminated areas.

Molecular evolution and functional diversification of metal tolerance protein families in cereals plants and function of maize MTP protein

Plants employ metal tolerance proteins (MTPs) to confer tolerance by sequestering excess ions into vacuoles. MTPs belong to the cation diffusion facilitator (CDF) family, which facilitates the transport of divalent transition metal cations. In this study, we conducted a comprehensive analysis of the MTP gene families across 21 plant species, including maize (Zea mays). A total of 247 MTP genes were identified within these plant genomes and categorized into distinct subgroups, namely Zn-CDF, Mn-CDF, and Fe/Zn-CDF, based on phylogenetic analyses. This investigation encompassed the characterization of genomic distribution, gene structures, cis-regulatory elements, collinearity relationships, and gene ontology functions associated with MTPs. Transcriptomic analyses unveiled stress-specific expression patterns of MTP genes under various abiotic stresses. Moreover, quantitative RT-PCR assays were employed to assess maize MTP gene responses to diverse heavy metal stress conditions. Functional validation of metal tolerance roles was achieved through heterologous expression in yeast. This integrated evolutionary scrutiny of MTP families in cereals furnishes a valuable framework for the elucidation of MTP functions in subsequent studies. Notably, the prioritized MTP gene ZmMTP6 emerged as a positive regulator of plant Cd tolerance, thereby offering a pivotal genetic asset for the development of Cd-tolerant crops, particularly maize cultivars.

Genome-wide identification, expression and function analysis of the MTP gene family in tulip (Tulipa gesneriana).

Currently, soil heavy metal contamination is a severe issue, particularly with Cd pollution. The metal tolerance protein (MTP) proteins, as plant divalent cation transporters, play a crucial role in the transport and tolerance of heavy metals in plants. This study conducted comprehensive identification and characterization of the MTP gene family in the tulip. A total of 11 TgMTP genes were identified and phylogenetically classified into three subfamilies. Conserved motif and gene structure analyses unveiled commonalities and variations among subfamily members. Expression profiling demonstrated several TgMTPs were markedly upregulated under Cd exposure, including the TgMTP7.1. Heterologous expression in yeast validated that TgMTP7.1 could ameliorate Cd sensitivity and enhance its tolerance. These results provide primary insights into the MTP gene family in tulip. Phylogenetic relationships and functional analyses establish a framework for elucidating the transporters and molecular mechanisms governing Cd accumulation and distribution in tulip. Key TgMTPs identified, exemplified by TgMTP7.1, may illuminate molecular breeding efforts aimed at developing Cd-tolerant cultivars for the remediation of soil Cd contamination.

Dimerization of the CNNM extracellular domain.

Cystathionine- -synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) are an evolutionarily conserved family of magnesium transporters. They mediate magnesium homeostasis directly by transport of Mg2+ ions and indirectly by regulation of the transient receptor potential ion channel subfamily M member 7 (TRPM7). Here, we report the crystal structure of the extracellular domain of tapeworm CNNM4. The domain forms a dimer of immunoglobulin-like (Ig-like) folds with electron density observed for three glycosylation sites. Analytical ultracentrifugation confirms that mutations in the extracellular domain of human CNNM4 prevent its dimerization. An analogous mutation in mouse CNNM2 impairs its activity in a cellular assay of Mg2+ transport.

Klebsiella pneumoniae DedA family proteins have redundant roles in divalent cation homeostasis and resistance to phagocytosis.

The DedA superfamily is a highly conserved family of membrane proteins. Deletion of Escherichia coli yqjA and yghB, encoding related DedA family proteins, results in sensitivity to elevated temperature, antibiotics, and alkaline pH. The human pathogen Klebsiella pneumoniae possesses genes encoding DedA family proteins with >90% amino acid identity to E. coli YqjA and YghB. We hypothesized that the deletion of K. pneumoniae yqjA and yghB will impact its physiology and may reduce its virulence. The K. pneumoniae ΔyqjA ΔyghB mutant (strain VT101) displayed a growth defect at 42°C and alkaline pH sensitivity, not unlike its E. coli counterpart. However, VT101 retained mostly wild-type resistance to antibiotics. We found VT101 was sensitive to the chelating agent EDTA, the anionic detergent SDS, and agents capable of alkalizing the bacterial cytoplasm such as bicarbonate or chloroquine. We could restore growth at alkaline pH and at elevated temperature by addition of 0.5-2 mM Ca2+ or Mg2+ to the culture media. VT101 displayed a slower uptake of calcium, which was dependent upon calcium channel activity. VT201, with similar deletions as VT101 but derived from a virulent K. pneumoniae strain, was highly susceptible to phagocytosis by alveolar macrophages and displayed a defect in the production of capsule. These findings suggest divalent cation homeostasis and virulence are interlinked by common functions of the DedA family.IMPORTANCEKlebsiella pneumoniae is a dangerous human pathogen. The DedA protein family is found in all bacteria and is a membrane transporter often required for virulence and antibiotic resistance. K. pneumoniae possesses homologs of E. coli YqjA and YghB, with 60% amino acid identity and redundant functions, which we have previously shown to be required for tolerance to biocides and alkaline pH. A K. pneumoniae strain lacking yqjA and yghB was found to be sensitive to alkaline pH, elevated temperature, and EDTA/SDS and displayed a defect in calcium uptake. Sensitivity to these conditions was reversed by addition of calcium or magnesium to the growth medium. Introduction of ΔyqjA and ΔyghB mutations into virulent K. pneumoniae resulted in the loss of capsule, increased phagocytosis by macrophages, and a partial loss of virulence. These results show that targeting the Klebsiella DedA family results in impaired divalent cation transport and, in turn, loss of virulence.

Comparative proteomic analysis of cold seep clam Archivesica marissinica and shallow water shellfish Ruditapes philippinarum provides insights into the adaptation mechanisms of deep-sea mollusks

IntroductionThe deep-sea environment is always characterized by high hydrostatic pressure, fluctuating temperatures, heavy metals, darkness, and others. The Vesicomyidae inhabit cold seep zones, hydrothermal vents, and other chemically reduced environments.MethodsTo enhance the understanding of the adaptation mechanisms of clams in extreme environments, a comprehensive proteomic study was conducted on the cold seep clam Archivesica marissinica and shallow water clam Ruditapes philippinarum.ResultsA total of 4,557 proteins were identified from the comparative groups. The Gene Ontology results indicated that the differentially expressed proteins (DEP) for the comparative group Rpgill vs. Amgill were enriched in the nitrogen compound metabolic process, and others. The comparative analysis for Amfoot vs. Amgill and Ammantle vs. Amgill revealed significant enrichment of the differential proteins that were involved in metal ion transport, divalent inorganic cation transport, and so on. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis for the comparative group Rpgill vs. Amgill was significantly enriched in the regulation of actin cytoskeleton, lysosome, and others. The proteins that exhibited differential expression in the cold seep clam’ different tissues were also enriched in important pathways, such as lysosome, fatty acid degradation, nitrogen metabolism.DiscussionThe further analysis identified crucial response proteins involved in various biological pathways. For example, the pattern recognition receptors, such as galectin and peptidoglycan recognition protein, participated in recognition of symbiotic microorganisms. The lysosome pathway members, such as cathepsin and saposins, were engaged in the degradation process of symbiont proteins during symbiont digestion. Profilin and gelsolin from actin cytoskeleton pathway might be pressure-related proteins. Furthermore, carbonic anhydrases from nitrogen metabolism KEGG pathway provide inorganic carbon for symbiotic bacteria. Additionally, Mn superoxide dismutase plays a role in the scavenging of superoxide anion radicals and antioxidant activity. Then, arginine kinases facilitate the low temperature adaptation of deep-sea shellfish with its cold adaptation characters. These findings offer novel perspectives on the proteins that are implicated in A. marisica’s response to cold seep environments, thereby contributing to the understanding of deep-sea biological adaptation and the preservation of deep-sea ecosystems.

Characterization of <i>Staphylococcus lugdunensis</i> biofilms through ethyl methanesulfonate mutagenesis

<p><italic>Staphylococcus lugdunensis</italic> is a coagulase-negative species responsible for a multitude of infections. These infections often resemble those caused by the more pathogenic staphylococcal species, <italic>Staphylococcus aureus</italic>, such as skin and soft tissue infections, prosthetic joint infections, and infective endocarditis. Despite a high mortality rate and infections that differ from other coagulase-negative species, little is known regarding <italic>S. lugdunensis</italic> pathogenesis. The objective of this study is to identify the essential factors for biofilm formation in <italic>S. lugdunensis</italic>. <italic>S. lugdunensis</italic> was mutagenized through ethyl methanesulfonate (EMS) exposure, and the individual cells were separated using a cell sorter and examined for biofilm formation at 8 hr and 24 hr timepoints. Mutations that resulted in either increased or decreased biofilm formation were sequenced to identify the genes responsible for the respective phenotypes. A mutation within the <italic>S. lugdunensis</italic> surface protein A (<italic>slsA</italic>) gene was common among all of the low biofilm formers, thus suggesting that high expression of this protein is important in biofilm formation. However, other mutations common among the mutants with decreased biofilm formation were in the putative divalent cation transport gene, <italic>mgtE</italic>. Conversely, a mutation in the gene that codes for the von Willebrand factor binding protein, <italic>vwbl</italic>, was common among the mutants with increased biofilm formation. Following proteinase K treatment, a significant dispersal of the <italic>S</italic>. <italic>lugdunensis</italic> biofilm matrix occurred, thus confirming the presence of primarily protein-mediated biofilms; this is in agreement with previous <italic>S. lugdunensis</italic> studies. Additionally, all low biofilm formers exhibited decreased protein levels (1.95–2.77 fold change) within the biofilm matrix, while no difference was observed with extracellular DNA (eDNA) or polysaccharides. This study presents a unique methodology to identify genes that affect biofilm formation and sheds light on <italic>S. lugdunensis</italic> pathogenesis.</p>

Phosphatase and Pseudo-Phosphatase Functions of Phosphatase of Regenerating Liver 3 (PRL-3) Are Insensitive to Divalent Metals In Vitro.

Phosphatase of regenerating liver 3 (PRL-3) is associated with cancer metastasis and has been shown to interact with the cyclin and CBS domain divalent metal cation transport mediator (CNNM) family of proteins to regulate the intracellular concentration of magnesium and other divalent metals. Despite PRL-3's importance in cancer, factors that regulate PRL-3's phosphatase activity and its interactions with CNNM proteins remain unknown. Here, we show that divalent metal ions, including magnesium, calcium, and manganese, have no impact on PRL-3's structure, stability, phosphatase activity, or CNNM binding capacity, indicating that PRL-3 does not act as a metal sensor, despite its interaction with CNNM metal transporters. In vitro approaches found that PRL-3 is a broad but not indiscriminate phosphatase, with activity toward di- and tri-nucleotides, phosphoinositols, and NADPH but not other common metabolites. Although calcium, magnesium, manganese, and zinc-binding sites were predicted near the PRL-3 active site, these divalent metals did not specifically alter PRL-3's phosphatase activity toward a generic substrate, its transition from an inactive phospho-cysteine intermediate state, or its direct binding with the CBS domain of CNNM. PRL-3's insensitivity to metal cations negates the possibility of its role as an intracellular metal content sensor for regulating CNNM activity. Further investigation is warranted to define the regulatory mechanisms governing PRL-3's phosphatase activity and CNNM interactions, as these findings could hold potential therapeutic implications in cancer treatment.

Structural insights into regulation of CNNM-TRPM7 divalent cation uptake by the small GTPase ARL15.

Cystathionine-β-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) are an evolutionarily conserved family of magnesium transporters. They promote efflux of Mg2+ ions on their own and influx of divalent cations when expressed with the transient receptor potential ion channel subfamily M member 7 (TRPM7). Recently, ADP-ribosylation factor-like GTPase 15 (ARL15) has been identified as CNNM-binding partner and an inhibitor of divalent cation influx by TRPM7. Here, we characterize ARL15 as a GTP and CNNM-binding protein and demonstrate that ARL15 also inhibits CNNM2 Mg2+ efflux. The crystal structure of a complex between ARL15 and CNNM2 CBS-pair domain reveals the molecular basis for binding and allowed the identification of mutations that specifically block binding. A binding deficient ARL15 mutant, R95A, failed to inhibit CNNM and TRPM7 transport of Mg2+ and Zn2+ ions. Structural analysis and binding experiments with phosphatase of regenerating liver 2 (PRL2 or PTP4A2) showed that ARL15 and PRLs compete for binding CNNM to coordinate regulation of ion transport by CNNM and TRPM7.

New insights into the structure and function of CNNM proteins.

Magnesium (Mg2+ ) is the most abundant divalent cation in cells and plays key roles in almost all biological processes. CBS-pair domain divalent metal cation transport mediators (CNNMs) are a newly characterized class of Mg2+ transporters present throughout biology. Originally discovered in bacteria, there are four CNNM proteins in humans, which are involved in divalent cation transport, genetic diseases, and cancer. Eukaryotic CNNMs are composed of four domains: an extracellular domain, a transmembrane domain, a cystathionine-β-synthase (CBS)-pair domain, and a cyclic nucleotide-binding homology domain. The transmembrane and CBS-pair core are the defining features of CNNM proteins with over 20 000 protein sequences known from over 8000 species. Here, we review the structural and functional studies of eukaryotic and prokaryotic CNNMs that underlie our understanding of their regulation and mechanism of ion transport. Recent structures of prokaryotic CNNMs confirm the transmembrane domain mediates ion transport with the CBS-pair domain likely playing a regulatory role through binding divalent cations. Studies of mammalian CNNMs have identified new binding partners. These advances are driving progress in understanding this deeply conserved and widespread family of ion transporters.

Genome-wide identification and expression profile analysis of metal tolerance protein gene family in Eucalyptus grandis under metal stresses

Metal tolerance proteins (MTPs) as Me2+/H+(K+) antiporters participate in the transport of divalent cations, leading to heavy metal stress resistance and mineral utilization in plants. In the present study, to obtain better knowledge of the biological functions of the MTPs family, 20 potential EgMTPs genes were identified in Eucalyptus grandis and classified into seven groups belonging to three cation diffusion facilitator groups (Mn-CDFs, Zn/Fe-CDFs, and Zn-CDFs) and seven groups. EgMTP-encoded amino acids ranged from 315 to 884, and most of them contained 4–6 recognized transmembrane domains and were clearly prognosticated to localize into the cell vacuole. Almost all EgMTP genes experienced gene duplication events, in which some might be uniformly distributed in the genome. The numbers of cation efflux and the zinc transporter dimerization domain were highest in EgMTP proteins. The promoter regions of EgMTP genes have different cis-regulatory elements, indicating that the transcription rate of EgMTP genes can be a controlled response to different stimuli in multiple pathways. Our findings provide accurate perception on the role of the predicted miRNAs and the presence of SSR marker in the Eucalyptus genome and clarify their functions in metal tolerance regulation and marker-assisted selection, respectively. Gene expression profiling based on previous RNA-seq data indicates a probable function for EgMTP genes during development and responses to biotic stress. Additionally, the upregulation of EgMTP6, EgMTP5, and EgMTP11.1 to excess Cd2+ and Cu2+ exposure might be responsible for metal translocation from roots to leaves.

Genome-wide identification and expression analysis of metal tolerance protein (MTP) gene family in soybean (Glycine max) under heavy metal stress.

Plant metal tolerance proteins (MTPs) are plant membrane divalent cation transporters that specifically contribute to heavy metal stress resistance and mineral uptake. However, little is known about this family's molecular behaviors and biological activities in soybean. A total of 20 potential MTP candidate genes were identified and studied in the soybean genome for phylogenetic relationships, chromosomal distributions, gene structures, gene ontology, cis-elements, and previous gene expression. Furthermore, the expression of MTPs has been investigated under different heavy metals treatments. All identified soybean MTPs (GmaMTPs) contain a cation efflux domain or a ZT dimer and are further divided into three primary cation diffusion facilitator (CDF) groups: Mn-CDFs, Zn-CDFs, and Fe/Zn-CDFs. The developmental analysis reveals that segmental duplication contributes to the GmaMTP family's expansion. Tissue-specific expression profiling revealed comparative expression profiling in similar groups, although gene expression differed between groups. GmaMTP genes displayed biased responses in either plant leaves or roots when treated with heavy metal. In the leaves and roots, nine and ten GmaMTPs responded to at least one metal ion treatment. Furthermore, in most heavy metal treatments, GmaMTP1.1, GmaMTP1.2, GmaMTP3.1, GmaMTP3.2, GmaMTP4.1, and GmaMTP4.3 exhibited significant expression responses. Our findings provided insight into the evolution of MTPs in soybean. Overall, our findings shed light on the evolution of the MTP gene family in soybean and pave the path for further functional characterization of this gene family.

Metal tolerance gene family in barley: an in silico comprehensive analysis

Metal-tolerance proteins (MTPs) are divalent cation transporters that play critical roles in metal tolerance and ion homeostasis in plants. However, a comprehensive study of MTPs is still lacking in crop plants. The current study aimed to comprehensively identify and characterize the MTP gene family in barley (Hordeum vulgare, Hv), an important crop. In total, 12 HvMTPs were identified in the barley genome in this study. They were divided into three phylogenetic groups (Zn-cation diffusion facilitator proteins [CDFs], Fe/Zn-CDFs, and Mn-CDFs) and further subdivided into seven groups (G1, G5, G6, G7, G8, G9, and G12). The majority of MTPs were hydrophobic proteins found in the vacuolar membrane. Gene duplication analysis of HvMTPs revealed one pair of segmental-like duplications in the barley genome. Evolutionary analysis suggested that barley MTPs underwent purifying natural selection. Additionally, the HvMTPs were analyzed in the pan-genome sequences of barley (20 accessions), which suggests that HvMTPs are highly conserved in barley evolution. Cis-acting regulatory elements, microRNA target sites, and protein–protein interaction analysis indicated the role of HvMTPs in a variety of biological processes. Expression profiling suggests that HvMTPs play an active role in maintaining barley nutrient homeostasis throughout its life cycle, and their expression levels were not significantly altered by abiotic stresses like cold, drought, or heat. The expression of barley HvMTP genes in the presence of heavy metals such as Zn2+, Cu2+, As3+, and Cd2+ revealed that these MTPs were induced by at least one metal ion, implying their involvement in metal tolerance or transportation. The identification and comprehensive investigation of MTP gene family members will provide important gene resources for the genetic improvement of crops for metal tolerance, bioremediation, or biofortification of staple crops.

The OsNramp4 aluminum transporter is involved in cadmium accumulation in rice grains

Cadmium (Cd) toxicity affects many crops. Members of the OsNramp (Natural resistance-associated macrophage protein) gene family play important roles in the transport of divalent or trivalent cations in rice (Oryza sativa L.). This study explored a possible involvement of OsNramp4 in Cd2+ transport in rice. We employed the CRISPR/cas9 technique to obtain rice OsNramp4 knockout lines. Using NMT (Non-invasive Micro-test Technology) system for real-time measurement, it was found that the net Cd2+ flux was significantly lower than that of wild-type. The yeast strain expressing OsNramp4 grew poorly under Cd2+ stress, and accumulated more Cd2+ than the control strain, which enhanced the sensitivity of yeast to Cd2+. OsNramp4 was membrane-localized and mainly expressed in roots, but after tillering, its expression shifted to the nodes and glumes. Furthermore, the loss-of-function OsNramp4 mutation lowered the root cell sap Cd2+ content, resulting in a significant decrease in Cd2+ content in shoot and Cd2+ accumulation in grains. We characterized the OsNramp4, an Al3+ transporter with the ability to alter the cellular distribution of Cd2+ in rice and to reduce Cd2+ content in the grain. Our study highlights the complexity of ion uptake and transport in plants.

Genome-wide analyses of metal tolerance protein genes in apple (Malus domestica): Identification, characterization, expression and response to various metal ion stresses

The growth and production of fruit trees are adversely affected by heavy metal pollution in orchard lands. Plant metal homeostasis and resistance are regulated by metal tolerance proteins (MTP), which function as divalent cation transporters. To understand the possible role of MTPs in fruit trees, we identified 20 MdMTPs in the apple genome. Phylogenetic and collinear relationship analyses revealed that they belonged to Zn-MTP, Fe/Zn-MTP and Mn-MTP subfamilies, and have gone through two tandem and six segmental duplication events under purifying selection. All MdMTPs were predicted to form homodimer, with each monocase containing a conserved cation efflux domain (CED), the typical structural feature of MTP family, and one or two HXXXD or DXXXD residues. Further spatiotemporal expression analyses indicated that all MdMTP genes were ubiquitously expressed, and most of them were induced by at least three heavy metal ions, in roots, stems or leaves. Yeast and Arabidopsis mutant complementation experiments verified the function of MdMTP11.1 in response to heavy metal stress. Our findings provide an important genetic foundation for the functional elucidation of MdMTP genes, which can be used as the potential gene candidates for the breeding of new fruit tree cultivars with improved tolerance to heavy metal stress. • A total number of 20 metal tolerance protein (MTP) genes were identified in apple. • All MdMTP proteins contained a cation efflux domain to work for metal ion efflux. • Tandem and segmental duplication events occurred in MdMTPs under purifying selection. • Most of MdMTPs were induced by at least three heavy metal ions in various organs.

SLC41A1 knockout mice display normal magnesium homeostasis.

Transcellular Mg2+ reabsorption in the distal convoluted tubule (DCT) of the kidneys plays an important role in maintaining systemic Mg2+ homeostasis. SLC41A1 is a Na+/Mg2+ exchanger that mediates Mg2+ efflux from cells and is hypothesized to facilitate basolateral extrusion of Mg2+ in the DCT. In this study, we generated a SLC41A1 knockout mouse model to examine the role of SLC41A1 in Mg2+ homeostasis. Slc41a1-/- mice exhibited similar serum and urine Mg2+ levels as their wild-type littermates. Dietary restriction of Mg2+ resulted in reduced serum Mg2+ concentration and urinary Mg2+ excretion, which was similar in the wild-type and knockout groups. Expression of genes encoding Mg2+ channels and transporters such as transient receptor potential melastatin 6 (Trpm6), transient receptor potential melastatin 7 (Trpm7), cyclin and CBS domain divalent metal cation transport mediator 2 (Cnnm2), and Slc41a3 were unchanged based on genotype. We investigated the potential redundancy of SLC41A1 and its homolog SLC41A3 by generating a double knockout mouse. Although Slc41a3-/- knockout mice showed significantly reduced serum Mg2+ compared with wild-type and Slc41a1-/- knockout groups, double knockout mice displayed similar serum Mg2+ levels as Slc41a3-/- knockout mice. In conclusion, our data show that SLC41A1 is not involved in the regulation of systemic Mg2+ homeostasis in mice. Our data also demonstrate that SLC41A1 does not compensate for the loss of SLC41A3, suggesting different functions of these SLC41 proteins in vivo.NEW & NOTEWORTHY SLC41A1 has been hypothesized to mediate Mg2+ extrusion in the distal convoluted tubule and thus regulate Mg2+ homeostasis. This study investigated the role of SLC41A1 in Mg2+ homeostasis in vivo using a transgenic mouse model. Our results demonstrate that SLC41A1 is not required to maintain normal Mg2+ balance in mice. We also show that SLC41A3 is more important than SLC41A1 in regulating systemic Mg2+ levels.