Nramp Family Research Articles

FUNCTIONAL METATRANSCRIPTOMICS REVEALS A NOVEL MULTI METAL TOLERANT GENE OF NRAMP-FAMILY, ISOLATED FROM METAL-CONTAMINATED SOIL

The continuous release of heavy metal pollutants into soil through human activities posesthreat to both macro- and micro-organisms within the ecosystem. Functional metatranscriptomics is a robust method for discovering novel genes associated with a particular biological process. In this study, functional meta transcriptomic approach was employed to isolate a gene from size-fractionated cDNA libraries of micro-eukaryotes. The cDNA libraries were raised using high-quality RNA from heavy metal-contaminated soil. The identified gene aligned with Nramp (Natural resistance associated macrophage protein) family. The gene, JMC-10, was submitted to GenBank under Accession No. OR882126, and it encodes a protein with high sequence similarity to other Nramp family members, including Smf1p, Smf2p, and Smf3p in Saccharomyces cerevisiae. To study the function of JMC-10 gene, a series of growth assays were conducted in yeast cells that either overexpressed JMC-10 or carried a deletion of gene. The study revealed the putative role of JMC-10 in the transportation of divalent metal ions, including iron, manganese, zinc and cadmium. Notably, the overexpression of JMC-10 was induced by exposure to toxic levels of cadmium, suggesting it’s potential role in metal detoxification. The study provides new intuitions into the roles of Nramp family in metal homeostasis and highlights the potentiality of functional metatranscriptomics for identifying new genes having crucial roles in complex biological processes. Further, it may serve as a biomarker for identifying metal pollution.

Correction to: Genome‑wide identification of NRAMP family genes in Populus trichocarpa and their roles in transport of heavy metals

Correction to: Genome‑wide identification of NRAMP family genes in Populus trichocarpa and their roles in transport of heavy metals

Genome-wide identification of NRAMP family genes in Populus trichocarpa and their roles in transport of heavy metals

Genome-wide identification of NRAMP family genes in Populus trichocarpa and their roles in transport of heavy metals

Cloning and biological function analysis of Nramps in blueberry

The natural resistance-associated macrophage protein (Nramp) family comprises crucial membrane transport proteins that play vital roles in the transport and reuse of heavy metal ions in plants. Limited information is currently available on the Nramp family in blueberry (Vaccinium spp.; Vc). Elucidation of the mechanism underlying mineral-element deficiency and heavy metal toxicity in blueberry is essential for the sustainable development of the blueberry industry. In this study, three Nramp genes (VcNramp4, VcNramp6, and VcNramp8.2) were cloned and identified from blueberry, and their functions were analyzed in transgenic Arabidopsis thaliana and yeast. Sequence analysis showed that the VcNramp genes encoded 321–455 amino acids and contained 6–8 transmembrane structural domains. Expression analysis showed that VcNramp genes were significantly up-regulated under iron (Fe) deficiency. The expression of VcNramp genes was most significantly up-regulated in the roots, by 2.05-fold relative to the control group. VcNramp4 and VcNramp6 expression in yeast and A. thaliana alleviated Fe deficiency stress. Interestingly, yeast expressing VcNramp6 showed a 1.13- to 1.38-fold increase in Fe accumulation at different medium pH levels, which was significantly higher than that of yeast expressing the other VcNramp genes. All VcNramp genes respond to changes in Fe ion concentration. Among them, VcNramp6 can effectively ameliorate Fe deficiency symptoms in plants. The expression of VcNramp genes exhibited distinct tissue specificity. Notably, the expression levels of VcNramp6 and VcNramp8.2 in the stems was only 2 %–10 % of that in the roots. The results illustrated the Fe nutrient inefficiency of blueberry. Furthermore, all identified VcNramp genes responded to mild zinc or cadmium stress, but their expression levels decreased under severe zinc or cadmium stress.

High-resolution structures with bound Mn2+ and Cd2+ map the metal import pathway in an Nramp transporter.

Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+ and Mn2+ are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps.

Golgi in and out: multifaceted role and journey of manganese.

Manganese (Mn) is pivotal for plant growth and development but little is known about the processes that control its homeostasis in the cell. A spotlight on the pools of intracellular manganese and their cellular function has recently been gained through the characterization of new Mn transporters. In particular, transporters catalyzing the ins and outs of Mn at the various Golgi membranes have revealed the central role of the Golgi pool of Mn in the synthesis of the cellwall and as a reservoir for the numerous cellular Mn-dependent pathways whose calibration relies on a set of Golgi-resident transporters of the BICAT and NRAMP families.

Integrated biochemical and transcriptomic analysis reveals the effects of Burkholderia sp. SRB-1 on cadmium accumulating in Chrysopogon zizanioides L. under Cd stress

Application of plant growth-promoting rhizobacteria plays a vital role in enhancing phytoremediation efficiency. In this study, multiple approaches were employed to investigate the underlying mechanisms of Burkholderia sp. SRB-1 (SRB-1) on elevating Cd uptake and accumulation. Inoculation experiment indicated that SRB-1 could facilitate plant growth and Cd tolerance, as evidenced by the enhanced plant biomass and antioxidative enzymes activities. Cd content in plant shoots and roots increased about 36.56%–39.66% and 25.97%–130.47% assisted with SRB-1 when compared with control. Transcriptomics analysis revealed that SRB-1 upregulated expression of amiE, AAO1-2 and GA2-ox related to auxin and gibberellin biosynthesis in roots. Auxin and gibberellin, as hormone signals, regulated plant Cd tolerance and growth through activating hormone signal transduction pathways, which might also contribute to 67.94% increase of dry weight. The higher expression levels of ATP-binding cassette transporter subfamilies (ABCB, ABCC, ABCD and ABCG) in Chrysopogon zizanioides roots contributed to higher Cd uptake in Cd15 B (323.83 mg kg−1) than Cd15 (136.28 mg kg−1). Further, SRB-1 facilitated Cd migration from roots to shoots via upregulating the expression of Nramp, ZIP and HMA families. Our integrative analysis provided a molecular-scale perspective on Burkholderia sp. SRB-1 contributing to C. zizanioides performance.

Structural basis of metal import by Nramp family transporters.

Transition metal ions like Mn2+ and Fe2+ play a crucial role in various metabolic processes in all living cells. Excess or deficiency of these essential nutrients leads to diseases including cancer, anemia etc. Cellular levels of these metals are thus tightly controlled and regulated. Nramps (natural resistance-associated macrophage proteins), a class of transition metal transporters present in all domains of life, regulate the levels of these divalent metal ions within cells and prevent disorders related to metal insufficiency or overload. In humans, there exists two Nramps: Nramp2 is indispensable for Fe2+ and Mn2+ homeostasis, while Nramp1 provides resistance against intracellular pathogens by competing with the pathogen's metal transporters (including Nramp homologs) for acquisition of essential metal ions. Nramp-mediated metal ion transport typically involves co-transported protons. A few structures of Nramps are reported to date which reveal their overall LeuT-fold and provide a preliminary understanding of metal binding and proton co-transport. To better understand the metal import mechanism, we resolved high-resolution structures of Deinococcus radiodurans Nramp (DraNramp) in three conformations in both Mn2+-bound and metal-free states, providing the first molecular map of the entire Mn2+ transport cycle. The structures reveal that global conformational changes during transport are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations and conserved networks of polar residues lining the inner and outer gates. A Cd2+-bound structure highlights differences in coordination geometry for Mn2+ and Cd2+. Binding studies using isothermal titration calorimetry along with transport and mutational analyses reveal that the thermodynamic landscape for binding and transporting physiological metals is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, our results lay a foundation for understanding how metal ion transporters like Nramps evolve their substrate selectivity to different ecological niches.

OsNRAMP2 facilitates Cd efflux from vacuoles and contributes to the difference in grain Cd accumulation between japonica and indica rice

Cadmium (Cd) accumulation in rice grain is of health concern. Identifying genes involved in grain Cd accumulation and performing molecular breeding may reduce it. In this study, knockout of OsNRAMP2, a member of the NRAMP family, reduced grain Cd concentrations by more than 38%, and overexpressing OsNRAMP2 increased grain Cd concentrations by more than 50%. Physiological experiments showed that OsNRAMP2 facilitated Cd translocation from root to shoot by positively regulating Cd efflux from the vacuoles. At filling stage, OsNRAMP2 was highly expressed in all tissues except for husk, suggesting its role in Cd remobilization. Changes in OsNRAMP2 expression affected the concentrations of Fe, Mn, Zn, and Cu in grain and also affected rice growth. Phylogenetic analysis showed that the distribution of OsNRAMP2 haplotypes between japonica and indica was different. Among the four haplotypes of OsNRAMP2, Hap 1, with a 6-bp nucleotide insertion in exon 1, had grain Cd concentration at least 45.3% lower than any of the other three haplotypes. Almost all (99.3%) japonica accessions but rare indica accessions (4.44%) from the 3K sequenced rice genomes carry Hap 1 of OsNRAMP2. Our study sheds light on the molecular mechanism of grain Cd accumulation and provides a promising target for low-Cd rice breeding.

SgNramp1, a plasma membrane-localized transporter, involves in manganese uptake in Stylosanthes guianensis.

Transporters belonging to the natural resistance-associated macrophage protein (Nramp) family play important roles in metal uptake and homeostasis. Although Nramp members have been functionally characterized in plants, the role of Nramp in the important tropical forage legume Stylosanthes guianensis (stylo) is largely unknown. This study aimed to determine the responses of Nramp genes to metal stresses and investigate its metal transport activity in stylo. Five SgNramp genes were identified from stylo. Expression analysis showed that SgNramp genes exhibited tissue preferential expressions and diverse responses to metal stresses, especially for manganese (Mn), suggesting the involvement of SgNramps in the response of stylo to metal stresses. Of the five SgNramps, SgNramp1 displayed the highest expression in stylo roots. A close correlation between SgNramp1 expression and root Mn concentration was observed among nine stylo cultivars under Mn limited condition. The higher expression of SgNramp1 was correlated with a high Mn uptake in stylo. Subsequent subcellular localization analysis showed that SgNramp1 was localized to the plasma membrane. Furthermore, heterologous expression of SgNramp1 complemented the phenotype of the Mn uptake-defective yeast (Saccharomyces cerevisiae) mutant Δsmf1. Mn concentration in the yeast cells expressing SgNramp1 was higher than that of the empty vector control, suggesting the transport activity of SgNramp1 for Mn in yeast. Taken together, this study reveals that SgNramp1 is a plasma membrane–localized transporter responsible for Mn uptake in stylo.

VrNramp5 is responsible for cadmium and manganese uptake in Vigna radiata roots

As a non-essential mineral, Cadmium inhibits the growth and development of plants. A cultivar (Sulu, SL as wild type) of mungbean is more sensitive to Cd toxicity than its mutant line (20#). However, there is little information about the underlying mechanism. In this study, mutant line (20#) of mungbean exhibited lower Cd accumulation than wild type SL based on the time-course and dose-dependent Cd content in mungbean roots. Meanwhile, compared to 20# roots, the root tips of SL possess a higher capacity for transient Cd influx during exposure to Cd alone or co-treatment with Cd and manganese (Mn). Cd exposure reduced Mn contents of roots in both SL and 20#. Likewise, the addition of Mn in solution or in Cd-polluted soils inhibited Cd uptake in roots. Such reciprocal inhibition shows that Cd competes with Mn for uptake in roots of mungbean. Comparative transcriptomic of roots in SL and 20# were analyzed by Illumina sequencing to elucidate the molecular mechanisms of competitive uptake between Cd and Mn for mungbean. The transcriptomic analysis demonstrated that 28 metal transporters genes showed the log 2 fold changes under Cd stress, including ABC, ZIP, CAX, CCX, MTP, and Nramp family members. Expression abundance of VrABCC15–1 and VrZIP8 genes were significant up-regulated, and VrNramp5 was down-regulated in roots of mungbean under Cd stress. Thus, the three genes were complementarily expressed in Cd sensitive yeast (Δycf1). Expression of VrNramp5 resulted in hypersensitivity to excess Cd owing to the increment of Cd uptake in yeasts during exposure to Cd, but not by VrABCC15–1 or VrZIP8. Expressing-VrNramp5-SL or -VrNramp5–20# yeasts also accumulate higher Mn than that of the expressing-empty vector yeasts. The findings of this study demonstrated that the differences of Cd accumulation between SL and 20# was due to Cd uptake differences, and VrNramp5 is involved in Mn and Cd uptake in mungbean roots.

Characterization of the NRAMP Gene Family in the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis.

Transporters of the NRAMP family are ubiquitous metal-transition transporters, playing a key role in metal homeostasis, especially in Mn and Fe homeostasis. In this work, we report the characterization of the NRAMP family members (RiSMF1, RiSMF2, RiSMF3.1 and RiSMF3.2) of the arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis. Phylogenetic analysis of the NRAMP sequences of different AM fungi showed that they are classified in two groups, which probably diverged early in their evolution. Functional analyses in yeast revealed that RiSMF3.2 encodes a protein mediating Mn and Fe transport from the environment. Gene-expression analyses by RT-qPCR showed that the RiSMF genes are differentially expressed in the extraradical (ERM) and intraradical (IRM) mycelium and differentially regulated by Mn and Fe availability. Mn starvation decreased RiSMF1 transcript levels in the ERM but increased RiSMF3.1 expression in the IRM. In the ERM, RiSMF1 expression was up-regulated by Fe deficiency, suggesting a role for its encoded protein in Fe-deficiency alleviation. Expression of RiSMF3.2 in the ERM was up-regulated at the early stages of Fe toxicity but down-regulated at later stages. These data suggest a role for RiSMF3.2 not only in Fe transport but also as a sensor of high external-Fe concentrations. Both Mn- and Fe-deficient conditions affected ERM development. While Mn deficiency increased hyphal length, Fe deficiency reduced sporulation.

Elucidation of iron homeostasis in Acanthamoeba castellanii

Acanthamoeba castellanii is a ubiquitously distributed amoeba that can be found in soil, dust, natural and tap water, air conditioners, hospitals, contact lenses and other environments. It is an amphizoic organism that can cause granulomatous amoebic encephalitis, an infrequent fatal disease of the central nervous system, and amoebic keratitis, a severe corneal infection that can lead to blindness. These diseases are extremely hard to treat; therefore, a more comprehensive understanding of this pathogen’s metabolism is essential for revealing potential therapeutic targets. To propagate successfully in human tissues, the parasites must resist the iron depletion caused by nutritional immunity. The aim of our study is to elucidate the mechanisms underlying iron homeostasis in A. castellanii. Using a comparative whole-cell proteomic analysis of cells grown under different degrees of iron availability, we identified the primary proteins involved in Acanthamoeba iron acquisition. Our results suggest a two-step reductive mechanism of iron acquisition by a ferric reductase from the STEAP family and a divalent metal transporter from the NRAMP family. Both proteins are localized to the membranes of acidified digestive vacuoles where endocytosed medium and bacteria are trafficked. The expression levels of these proteins are significantly higher under iron-limited conditions, which allows Acanthamoeba to increase the efficiency of iron uptake despite the observed reduced pinocytosis rate. We propose that excessive iron gained while grown under iron-rich conditions is removed from the cytosol into the vacuoles by an iron transporter homologous to VIT/Ccc1 proteins. Additionally, we identified a novel protein that may participate in iron uptake regulation, the overexpression of which leads to increased iron acquisition.

Overexpression of ZNT1 and NRAMP4 from the Ni Hyperaccumulator Noccaea caerulescens Population Monte Prinzera in Arabidopsis thaliana Perturbs Fe, Mn, and Ni Accumulation.

Metalliferous soils are characterized by a high content of metal compounds that can hamper plant growth. The pseudometallophyte Noccaea caerulescens is able to grow on metalliferous substrates by implementing both tolerance and accumulation of usually toxic metal ions. Expression of particular transmembrane transporter proteins (e.g., members of the ZIP and NRAMP families) leads to metal tolerance and accumulation, and its comparison between hyperaccumulator N. caerulescens with non-accumulator relatives Arabidopsis thaliana and Thlaspi arvense has deepened our knowledge on mechanisms adopted by plants to survive in metalliferous soils. In this work, two transporters, ZNT1 and NRAMP4, expressed in a serpentinic population of N. caerulescens identified on the Monte Prinzera (Italy) are considered, and their expression has been induced in yeast and in A. thaliana. In the latter, single transgenic lines were crossed to test the effect of the combined over-expression of the two transporters. An enhanced iron and manganese translocation towards the shoot was induced by overexpression of NcZNT1. The combined overexpression of NcZNT1 and NcNRAMP4 did perturb the metal accumulation in plants.

Identification and characterization of Nramp transporter AoNramp1 in Aspergillus oryzae.

The online version contains supplementary material available at 10.1007/s13205-021-02998-z.

Involvement of several putative transporters of different families in β-cyclocitral-induced alleviation of cadmium toxicity in quinoa (Chenopodium quinoa) seedlings

Cadmium (Cd) has a serious negative impact on crop growth and human food security. This study investigated the alleviating effect of β-cyclocitral, a potential heavy metal barrier, on Cd stress in quinoa seedlings and the associated mechanisms. Our results showed that β-cyclocitral alleviated Cd stress-induced growth inhibition in quinoa seedlings and promoted quinoa seedling root development under Cd stress. Moreover, it maintained the antioxidant system of quinoa seedlings, including the enzymatic, i.e., superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and nonenzymatic, i.e., reduced glutathione (GSH) and ascorbic acid (ASA), antioxidants, which eliminate the damage from excessive reactive oxygen species (ROS). Our results showed that β-cyclocitral could reduce the amount of Cd absorbed by roots. Furthermore, we systematically identified five transporter families from the quinoa genome, and the RT-qPCR results showed that ZIP, Nramp and YSL gene families were downregulated by β-cyclocitral to reduce Cd uptake by roots. Thus, β-cyclocitral promoted the growth, photosynthetic capacity and antioxidant capacity of the aboveground parts of quinoa seedlings. Taken together, these results suggested that the β-cyclocitral-induced decrease in Cd uptake may be caused by the downregulation of several selected transporter genes.

Genome-wide analysis of the NRAMP gene family in potato (Solanum tuberosum): Identification, expression analysis and response to five heavy metals stress

NRAMP family genes participate in the absorption and transport of heavy metals such as cadmium (Cd), zinc (Zn), copper (Cu), lead (Pb), iron (Fe) and manganese (Mn) and play an important role in the response to heavy metal stress. There is an abundance of research on these genes in bacteria, plants and fungi, although not in S. tuberosum. A total of 48 members(potato(5), Arabidopsis(7), Tomato(9), pepper(9), rice(12) and tobacco(6)) were identified from 6 species (potato (Solanum tuberosum), Arabidopsis thaliana, Tomato (Solanum lycopersicum), pepper (Capsicum annuum), rice (Oryza sativa) and tobacco (Nicotiana attenuate)) and were classified into four subgroups. Across NRAMP gene family members, there are 15 highly conserved motifs that have similar genetic structures and characteristics. In addition, a total of 16 pairs of colinear genes were found in eight species. Analysis of cis-elements indicated that, in response to abiotic stress, NRAMPs are mainly regulated by phytohormones and transcription factors. In addition, analysis of expression profiles indicated that StNRAMP4 is mainly expressed in the roots. According to a qRT-PCR-based analysis of the StNRAMP family, with the exception of Pb2+ stress, StNRAMPs positively responded to stress from Cu2+, Cd2+, Zn2+ and Ni2+ and The expression patterns is similar of StNRAMP2, under Pb2+, and Cu2+ treatment, the relative expression peaked at 24 h. the relative expression peaked at 12 h and was upregulated 428-fold in the roots under Ni2+ stress. Under Cd2+ stress, StNRAMP3 was upregulated 28-fold in the leaves. StNRAMP1, StNRAMP4 and StNRAMP5 showed significant upregulation under Cu2+, Cd2+ and Zn2+ stress, respectively. Expression of StNRAMPs could be specifically induced by heavy metals, implying their possible role in the transport and absorption of heavy metals. This research explains the colinear characteristics of NRAMPs in several food crop species, which is useful for providing important genetic resources for cultivating food crop that accumulate low amounts of heavy metals and for explaining the biological functions of NRAMPs in plants.

Electrophysiology Measurements of Metal Transport by MntH2 from Enterococcus faecalis.

Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.

Identification and functional characterization of natural resistance-associated macrophage protein 2 from sea cucumber Apostichopus japonicus

As a member of natural resistance-associated macrophage protein (Nramp) family, Nramp2 conservatively exists in the cell membrane across species and is essential for normal iron homeostasis in an H+-dependent manner. Withholding available iron represents an important host defense strategy. However, the function of Nramp2 in response to invading pathogens is largely unknown in invertebrates. In this study, a unique echinoderm Nramp2 was identified from sea cucumber Apostichopus japonicus (designated as AjNramp2). The cDNA sequence of AjNramp2 was 2360 bp, with a putative open reading frame of 1713 bp, encoding a typical Nramp domain containing protein with 570 amino acid residues. Structural analysis revealed that AjNramp2 consisted of highly conserved helix regions similar with the human Nramp2. Spatial expression analysis revealed that AjNramp2 was ubiquitously expressed in all examined tissues, with the highest level found in the intestine. Immunohistochemistry assay showed that AjNramp2 was mainly located in the cellular membrane in coelomocytes. Vibrio splendidus challenge and lipopolysaccharide (LPS) stimulation could significantly promote the expression of AjNramp2, which was consistent with the cellular iron level in coelomocytes. Moreover, when the expression of AjNramp2 was knocked down by siRNA-AjNramp2, the cellular iron level was coordinately decreased in coelomocytes under LPS stimulation. Taken together, results indicated that AjNramp2 serves as an iron transport receptor to withhold available iron and may contribute to the nutritional immunity defense system of sea cucumber.

Insights into the molecular mechanism of metal transport by NRAMP family transporters

Insights into the molecular mechanism of metal transport by NRAMP family transporters