Ca2+-dependent phosphorylation of NRAMP1 by CPK21 and CPK23 facilitates manganese uptake and homeostasis in Arabidopsis.

Homeostasis of the essential micronutrient manganese (Mn) is crucially determined through availability and uptake efficiency in all organisms. Mn deficiency of plants especially occurs in alkaline and calcareous soils, seriously restricting crop yield. However, the mechanisms underlying the sensing and signaling of Mn availability and conferring regulation of Mn uptake await elucidation. Here, we uncover that Mn depletion triggers spatiotemporally defined long-lasting Ca2+ oscillations in Arabidopsis roots. These Ca2+ signals initiate in individual cells, expand, and intensify intercellularly to transform into higher-order multicellular oscillations. Furthermore, through an interaction screen we identified the Ca2+-dependent protein kinases CPK21 and CPK23 as Ca2+ signal-decoding components that bring about translation of these signals into regulation of uptake activity of the high-affinity Mn transporter natural resistance associated macrophage proteins 1 (NRAMP1). Accordingly, a cpk21/23 double mutant displays impaired growth and root development under Mn-limiting conditions, while kinase overexpression confers enhanced tolerance to low Mn supply to plants. In addition, we define Thr498 phosphorylation within NRAMP1 as a pivot mechanistically determining NRAMP1 activity, as revealed by biochemical assays and complementation of yeast Mn uptake and Arabidopsis nramp1 mutants. Collectively, these findings delineate the Ca2+-CPK21/23-NRAMP1 axis as key for mounting plant Mn homeostasis.

Local distribution of manganese to leaf sheath is mediated by OsNramp5 in rice.

To play essential roles of manganese (Mn) in plant growth and development, it needs to be transported to different organs and tissues after uptake. However, the molecular mechanisms underlying Mn distribution between different tissues are poorly understood. We functionally characterized a member of rice natural resistance-associated macrophage protein (NRAMP) family, OsNramp5 in terms of its tissue specificity of gene expression, cell-specificity of protein localization, phenotypic analysis of leaf growth and response to Mn fluctuations. OsNramp5 is highly expressed in the leaf sheath. Immunostaining revealed that OsNramp5 is polarly localized at the proximal side of xylem parenchyma cells of the leaf sheath. Both the gene expression and protein abundance of OsNramp5 are unaffected by different Mn concentrations. Knockout of OsNramp5 decreased the distribution of Mn to the leaf sheath, but increased the distribution to the leaf blade at both low and high Mn supplies, resulting in reduced growth of leaf sheath. Furthermore, expression of OsNramp5 under the control of root-specific promoter in osnramp5 mutant complemented Mn uptake, but could not complement Mn distribution to the leaf sheath. These results indicate that OsNramp5 expressed in the leaf sheath plays an important role in unloading Mn from the xylem for the local distribution in rice.

NRAMP6c plays a key role in plant cadmium accumulation and resistance in tobacco (Nicotiana tabacum L.).

Tobacco plants (Nicotiana tabacum L.) exhibit considerable potential for phytoremediation of soil cadmium (Cd) pollutants, owing to their substantial biomass and efficient metal accumulation capabilities. The reduction of Cd accumulation in tobacco holds promise for minimizing Cd intake in individuals exposed to cigar smoking. NRAMP transporters are pivotal in the processes of Cd accumulation and resistance in plants; however, limited research has explored the functions of NRAMPs in tobacco plants. In this investigation, we focused on NtNRAMP6c, one of the three homologs of NRAMP6 in tobacco. We observed a robust induction of NtNRAMP6c expression in response to both Cd toxicity and iron (Fe) deficiency, with the highest expression levels detected in the roots. Subsequent subcellular localization and heterologous expression analyses disclosed that NtNRAMP6c functions as a plasma membrane-localized Cd transporter. Moreover, its overexpression significantly heightened the sensitivity of yeast cells to Cd toxicity. Through CRISPR-Cas9-mediated knockout of NtNRAMP6c, we achieved a reduction in Cd accumulation and an enhancement in Cd resistance in tobacco plants. Comparative transcriptomic analysis unveiled substantial alterations in the transcriptional profiles of genes associated with metal ion transport, photosynthesis, and macromolecule catabolism upon NtNRAMP6c knockout. Furthermore, our study employed plant metabolomics and rhizosphere metagenomics to demonstrate that NtNRAMP6c knockout led to changes in phytohormone homeostasis, as well as shifts in the composition and abundance of microbial communities. These findings bear significant biological implications for the utilization of tobacco in phytoremediation strategies targeting Cd pollutants in contaminated soils, and concurrently, in mitigating Cd accumulation in tobacco production destined for cigar consumption.

Genome-Wide Identification and Expression Analysis of SlNRAMP Genes in Tomato under Nutrient Deficiency and Cadmium Stress during Arbuscular Mycorrhizal Symbiosis.

Arbuscular mycorrhizal (AM) fungi are well known for enhancing phosphorus uptake in plants; however, their regulating roles in cation transporting gene family, such as natural resistance-associated macrophage protein (NRAMP), are still limited. Here, we performed bioinformatics analysis and quantitative expression assays of tomato SlNRAMP 1 to 5 genes under nutrient deficiency and cadmium (Cd) stress in response to AM symbiosis. These five SlNRAMP members are mainly located in the plasma or vacuolar membrane and can be divided into two subfamilies. Cis-element analysis revealed several motifs involved in phytohormonal and abiotic regulation in their promoters. SlNRAMP2 was downregulated by iron deficiency, while SlNRAMP1, SlNRAMP3, SlNRAMP4, and SlNRAMP5 responded positively to copper-, zinc-, and manganese-deficient conditions. AM colonization reduced Cd accumulation and expression of SlNRAMP3 but enhanced SlNRAMP1, SlNRAMP2, and SlNRMAP4 in plants under Cd stress. These findings provide valuable genetic information for improving tomato resilience to nutrient deficiency and heavy metal stress by developing AM symbiosis.

Iron in immune cell function and host defense.

The control over iron availability is crucial under homeostatic conditions and even more in the case of an infection. This results from diverse properties of iron: first, iron is an important trace element for the host as well as for the pathogen for various cellular and metabolic processes, second, free iron catalyzes Fenton reaction and is therefore producing reactive oxygen species as a part of the host defense machinery, third, iron exhibits important effects on immune cell function and differentiation and fourth almost every immune activation in turn impacts on iron metabolism and spatio-temporal iron distribution. The central importance of iron in the host and microbe interplay and thus for the course of infections led to diverse strategies to restrict iron for invading pathogens. In this review, we focus on how iron restriction to the pathogen is a powerful innate immune defense mechanism of the host called "nutritional immunity". Important proteins in the iron-host-pathogen interplay will be discussed as well as the influence of iron on the efficacy of innate and adaptive immunity. Recently described processes like ferritinophagy and ferroptosis are further covered in respect to their impact on inflammation and infection control and how they impact on our understanding of the interaction of host and pathogen.

NRAMP6 and NRAMP1 cooperatively regulate root growth and manganese translocation under manganese deficiency in Arabidopsis.

The essential micronutrient manganese (Mn) in plants regulates multiple biological processes including photosynthesis and oxidative stress. Some Natural Resistance-Associated Macrophage Proteins (NRAMPs) have been reported to play critical roles in Mn uptake and reutilization in low Mn conditions. NRAMP6 was demonstrated to regulate cadmium tolerance and iron utilization in Arabidopsis. Nevertheless, it is unclear whether NRAMP6 plays a role in Mn nutrition. Here, we report that NRAMP6 cooperates with NRAMP1 in Mn utilization. Mutation of NRAMP6 in nramp1 but not in a wild-type background reduces root growth and Mn translocation from the roots to shoots under Mn deficient conditions. Grafting experiments revealed that NRAMP6 expression in both the roots and shoots is required for root growth and Mn translocation under Mn deficiency. We also showed that NRAMP1 could replace NRAMP6 to sustain root growth under Mn deficiency, but not vice versa. Mn deficiency does not affect the transcript level of NRAMP6, but is able to increase and decrease the protein accumulation of NRAMP6 in roots and shoots, respectively. Furthermore, NRAMP6 can be localized to both the plasma membrane and endomembranes including the endoplasmic reticulum, and Mn deficiency enhances the localization of NRAMP6 to the plasma membrane in Arabidopsis plants. NRAMP6 could rescue the defective growth of the yeast mutant Δsmf2, which is deficient in endomembrane Mn transport. Our results reveal the important role of NRAMP6 in Mn nutrition and in the long-distance signaling between the roots and shoots under Mn deficient conditions.

Root-to-shoot iron partitioning in Arabidopsis requires IRON-REGULATED TRANSPORTER1 (IRT1) protein but not its iron(II) transport function.

IRON-REGULATED TRANSPORTER1 (IRT1) is the root high-affinity ferrous iron (Fe) uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous Fe supplementation. Here we provide evidence supporting a second role of IRT1 in root-to-shoot partitioning of Fe. We show that irt1 mutants overaccumulate Fe in roots, most prominently in the cortex of the differentiation zone in irt1-2, compared to the wild type. Shoots of irt1-2 are severely Fe-deficient according to Fe content and marker transcripts, as expected. We generated irt1-2 lines producing IRT1 mutant variants carrying single amino-acid substitutions of key residues in transmembrane helices IV and V, Ser206 and His232, which are required for transport activity in yeast. Root short-term 55 Fe uptake rates were uninformative concerning IRT1-mediated transport. Overall irt1-like concentrations of the secondary substrate Mn suggested that the transgenic Arabidopsis lines also remain incapable of IRT1-mediated root Fe uptake. Yet, IRT1S206A partially complements rosette dwarfing and leaf chlorosis of irt1-2, as well as root-to-shoot Fe partitioning and gene expression defects of irt1-2, all of which are fully complemented by wild-type IRT1. Taken together, these results suggest a regulatory function for IRT1 in root-to-shoot Fe partitioning that does not require Fe transport activity of IRT1. Among the genes of which transcript levels are partially dependent on IRT1, we identify MYB DOMAIN PROTEIN10, MYB DOMAIN PROTEIN72 and NICOTIANAMINE SYNTHASE4 as candidates for effecting IRT1-dependent Fe mobilization in roots. Understanding the biological functions of IRT1 will help to improve Fe nutrition and the nutritional quality of agricultural crops.

Manganese transport by Streptococcus sanguinis in acidic conditions and its impact on growth in vitro and in vivo.

Streptococcus sanguinis is an oral commensal and an etiological agent of infective endocarditis. Previous studies have identified the SsaACB manganese transporter as essential for endocarditis virulence; however, the significance of SsaACB in the oral environment has never been examined. Here we report that a ΔssaACB deletion mutant of strain SK36 exhibits reduced growth and manganese uptake under acidic conditions. Further studies revealed that these deficits resulted from the decreased activity of TmpA, shown in the accompanying paper to function as a ZIP-family manganese transporter. Transcriptomic analysis of fermentor-grown cultures of SK36 WT and ΔssaACB strains identified pH-dependent changes related to carbon catabolite repression in both strains, though their magnitude was generally greater in the mutant. In strain VMC66, which possesses a MntH transporter, loss of SsaACB did not significantly alter growth or cellular manganese levels under the same conditions. Interestingly, there were only modest differences between SK36 and its ΔssaACB mutant in competition with Streptococcus mutans in vitro and in a murine oral colonization model. Our results suggest that the heterogeneity of the oral environment may provide a rationale for the variety of manganese transporters found in S. sanguinis.

Plasma membrane-associated calcium signaling modulates cadmium transport.

Cadmium (Cd) is a toxic heavy element for plant growth and development, and plants have evolved many strategies to cope with Cd stress. However, the mechanisms how plants sense Cd stress and regulate the function of transporters remain very rudimentary. Here, we found that Cd stress induces obvious Ca2+ signals in Arabidopsis roots. Furthermore, we identified the calcium-dependent protein kinases CPK21 and CPK23 that interacted with the Cd transporter NRAMP6 through a variety of protein interaction techniques. Then, we confirmed that the cpk21 23 double mutants significantly enhanced the sensitive phenotype of cpk23 single mutant under Cd stress, while the overexpression and continuous activation of CPK21 and CPK23 enhanced plants tolerance to Cd stress. Multiple biochemical and physiological analyses in yeast and plants demonstrated that CPK21/23 phosphorylate NRAMP6 primarily at Ser489 and Thr505 to inhibit the Cd transport activity of NRAMP6, thereby improving the Cd tolerance of plants. Taken together, we found a plasma membrane-associated calcium signaling that modulates Cd tolerance. These results provide new insights into the molecular breeding of crop tolerance to Cd stress.

CBL1/9-CIPK23-NRAMP1 axis regulates manganese toxicity.

Manganese (Mn) is an essential micronutrient in plants. However, excessive Mn absorption in acidic soils can cause Mn toxicity, which adversely affects plant growth and crop yields. At present, acidic soils cover c. 30% of the Earth's surface. However, the mechanism underpinning Mn uptake remains largely unknown. We identified cbl1/9 and cipk23 mutants exhibiting high-Mn-sensitive phenotype through the reverse genetics method. Furthermore, we identified the CIPK23 phosphorylated NRAMP1 through a variety of protein interaction techniques and protein kinase assays. Here, we demonstrated that two calcineurin B-like proteins, CBL1/9, and their interacting kinase CIPK23 positively regulated the tolerance of Mn toxicity in Arabidopsis. The cbl1 cbl9 double mutant and cipk23 mutants exhibited high-Mn-sensitive phenotypes, which manifested as decreased primary root length, biomass, and chlorophyll concentration, and higher accumulation of Mn. In addition, CIPK23 interacted with and phosphorylated the Mn transporter NRAMP1 primarily at Ser20/22 in vitro and in vivo, and thereby induced clathrin-mediated endocytosis of NRAMP1 to reduce its distribution on the plasma membrane and enhance plant tolerance to Mn toxicity. In summary, we found that the CBL1/9-CIPK23-NRAMP1 module regulates the tolerance to high-Mn toxicity and provide insight into a mechanism of the tolerance of plants to Mn toxicity.

Solute carrier family 11 member 1 genetic polymorphisms rs17235409 and rs3731865 associate with susceptibility to extremity post-traumatic osteomyelitis in a Chinese Han population.

Genetic variations in the solute carrier family 11 member 1 (SLC11A1) gene have been implicated in developing inflammatory disorders. However, it is still unclear whether such polymorphisms contribute to the pathogenesis of post-traumatic osteomyelitis (PTOM). Therefore, this study investigated the roles of genetic variations of the SLC11A1 gene (rs17235409 and rs3731865) in PTOM development in a Chinese Han cohort. The SNaPshot method was used for genotyping 704 participants (336 patients and 368 controls) for rs17235409 and rs3731865. Outcomes revealed that rs17235409 increased the risk of PTOM occurrence by dominant (p = .037, odds ratio [OR] = 1.44) and heterozygous models (p = .035, OR = 1.45), implying AG genotype as a risk factor for PTOM development. In addition, patients with AG genotype had relatively higher levels of inflammatory biomarkers than those with AA and GG genotypes, especially for the white blood cell count and C-reactive protein. Despite no statistically significant differences achieved, rs3731865 may reduce the PTOM susceptibility, suggested by the results of dominant (p = .051, OR = 0.67) and heterozygous (p = .068, OR = 0.69) models. In short, rs17235409 confers an elevated chance of developing PTOM, with AG genotype as a risk factor. Whether rs3731865 involves in the pathogenesis of PTOM requires further investigations.

Genome-wide identification of the NRAMP gene family in Populus trichocarpa and their function as heavy metal transporters.

The natural resistance-associated macrophage protein (NRAMP) gene family plays a key role in essential mineral nutrient homeostasis, as well as toxic metal accumulation, translocation, and detoxification. Although the NRAMP family genes have been widely identified in various species, they still require to be analyzed comprehensively in tree species. In this study, a total of 11 NRAMP members (PtNRAMP1-11) were identified in Populus trichocarpa, a woody model plant, and further subdivided into three groups based on phylogenetic analysis. Chromosomal location analysis indicated that the PtNRAMP genes were unevenly distributed on six of the 19 Populus chromosomes. Gene expression analysis indicated that the PtNRAMP genes were differentially responsive to metal stress, including iron (Fe) and manganese (Mn) deficiency, as well as Fe, Mn, zinc (Zn), and cadmium (Cd) toxicity. Furthermore, the PtNRAMP gene functions were characterized using a heterologous yeast expression system. The results showed that PtNRAMP1, PtNRAMP2, PtNRAMP4, PtNRAMP9, PtNRAMP10, and PtNRAMP11 displayed the ability to transport Cd into yeast cells. In addition, PtNRAMP1, PtNRAMP6, and PtNRAMP7 complemented the Mn uptake mutant, while PtNRAMP1, PtNRAMP6, PtNRAMP7, and PtNRAMP9 complemented the Fe uptake mutant. In conclusion, our findings revealed the respective functions of PtNRAMPs during metal transport as well as their potential role in micronutrient biofortification and phytoremediation.

Systematic Analysis of NRAMP Family Genes in Areca catechu and Its Response to Zn/Fe Deficiency Stress.

Areca catechu is a commercially important medicinal plant widely cultivated in tropical regions. The natural resistance-associated macrophage protein (NRAMP) is widespread in plants and plays critical roles in transporting metal ions, plant growth, and development. However, the information on NRAMPs in A. catechu is quite limited. In this study, we identified 12 NRAMPs genes in the areca genome, which were classified into five groups by phylogenetic analysis. Subcellular localization analysis reveals that, except for NRAMP2, NRAMP3, and NRAMP11, which are localized in chloroplasts, all other NRAMPs are localized on the plasma membrane. Genomic distribution analysis shows that 12 NRAMPs genes are unevenly spread on seven chromosomes. Sequence analysis shows that motif 1 and motif 6 are highly conserved motifs in 12 NRAMPs. Synteny analysis provided deep insight into the evolutionary characteristics of AcNRAMP genes. Among the A. catechu and the other three representative species, we identified a total of 19 syntenic gene pairs. Analysis of Ka/Ks values indicates that AcNRAMP genes are subjected to purifying selection in the evolutionary process. Analysis of cis-acting elements reveals that AcNRAMP genes promoter sequences contain light-responsive elements, defense- and stress-responsive elements, and plant growth/development-responsive elements. Expression profiling confirms distinct expression patterns of AcNRAMP genes in different organs and responses to Zn/Fe deficiency stress in leaves and roots. Taken together, our results lay a foundation for further exploration of the AcNRAMPs regulatory function in areca response to Fe and Zn deficiency.

Genome-Wide Identification and Expression Analysis Reveals Roles of the NRAMP Gene Family in Iron/Cadmium Interactions in Peanut.

The natural resistance-associated macrophage protein (NRAMP) family plays crucial roles in metal uptake and transport in plants. However, little is known about their functions in peanut. To understand the roles of AhNRAMP genes in iron/cadmium interactions in peanut, genome-wide identification and bioinformatics analysis was performed. A total of 15 AhNRAMP genes were identified from the peanut genome, including seven gene pairs derived from whole-genome duplication and a segmental duplicated gene. AhNRAMP proteins were divided into two distinct subfamilies. Subfamily I contains eight acid proteins with a specific conserved motif 7, which were predicted to localize in the vacuole membrane, while subfamily II includes seven basic proteins sharing specific conserved motif 10, which were localized to the plasma membrane. Subfamily I genes contained four exons, while subfamily II had 13 exons. AhNRAMP proteins are perfectly modeled on the 5m94.1.A template, suggesting a role in metal transport. Most AhNRAMP genes are preferentially expressed in roots, stamens, or developing seeds. In roots, the expression of most AhNRAMPs is induced by iron deficiency and positively correlated with cadmium accumulation, indicating crucial roles in iron/cadmium interactions. The findings provide essential information to understand the functions of AhNRAMPs in the iron/cadmium interactions in peanuts.

Slc11 Synapomorphy: A Conserved 3D Framework Articulating Carrier Conformation Switch.

Transmembrane carriers of the Slc11 family catalyze proton (H+)-dependent uptake of divalent metal ions (Me2+) such as manganese and iron-vital elements coveted during infection. The Slc11 mechanism of high-affinity Me2+ cell import is selective and conserved between prokaryotic (MntH) and eukaryotic (Nramp) homologs, though processes coupling the use of the proton motive force to Me2+ uptake evolved repeatedly. Adding bacterial piracy of Nramp genes spread in distinct environmental niches suggests selective gain of function that may benefit opportunistic pathogens. To better understand Slc11 evolution, Alphafold (AF2)/Colabfold (CF) 3D predictions for bacterial sequences from sister clades of eukaryotic descent (MCb and MCg) were compared using both native and mutant templates. AF2/CF model an array of native MCb intermediates spanning the transition from outwardly open (OO) to inwardly open (IO) carriers. In silico mutagenesis targeting (i) a set of (evolutionarily coupled) sites that may define Slc11 function (putative synapomorphy) and (ii) residues from networked communities evolving during MCb transition indicates that Slc11 synapomorphy primarily instructs a Me2+-selective conformation switch which unlocks carrier inner gate and contributes to Me2+ binding site occlusion and outer gate locking. Inner gate opening apparently proceeds from interaction between transmembrane helix (h) h5, h8 and h1a. MCg1 xenologs revealed marked differences in carrier shape and plasticity, owing partly to an altered intramolecular H+ network. Yet, targeting Slc11 synapomorphy also converted MCg1 IO models to an OO state, apparently mobilizing the same residues to control gates. But MCg1 response to mutagenesis differed, with extensive divergence within this clade correlating with MCb-like modeling properties. Notably, MCg1 divergent epistasis marks the emergence of the genus Bordetella-Achromobacter. Slc11 synapomorphy localizes to the 3D areas that deviate least among MCb and MCg1 models (either IO or OO) implying that it constitutes a 3D network of residues articulating a Me2+-selective carrier conformation switch which is maintained in fast-evolving clades at the cost of divergent epistatic interactions impacting carrier shape and dynamics.

Genetic system underlying responses of Cryptococcus neoformans to cadmium.

Cryptococcus neoformans, basidiomycetous pathogenic yeast, is basically an environmental fungus and, therefore, challenged by ever changing environments. In this study, we focused on how C. neoformans responds to stress caused by cadmium that is one of high-risk pollutants. By tracking phenotypes of the resistance or sensitivity to cadmium, we undertook forward and reverse genetic studies to identify genes involved in cadmium metabolism in C. neoformans. We found that the main route of Cd2+ influx is through Mn2+ ion transporter, Smf1, which is an ortholog of Nramp (natural resistance-associated macrophage protein 1) of mouse. We found that serotype A strains are generally more resistant to cadmium than serotype D strains and that cadmium resistance of H99, a representative of serotype A strains, was found to be due to a partial defect in SMF1. We found that calcium channel has a subsidiary role for cadmium uptake. We also showed that Pca1 (P-type-ATPase) functions as an extrusion pump for cadmium. We examined the effects of some metals on cadmium toxicity and suggested (i) that Ca2+ and Zn2+ could exert their protective function against Cd2+ via restoring cadmium-inhibited cellular processes and (ii) that Mg2+ and Mn2+ could have antagonistic roles in an unknown Smf1-independent Cd2+ uptake system. We proposed a model for Cd2+-response of C. neoformans, which will serve as a platform for understanding how this organism copes with the toxic metal.

Glutathione regulates transcriptional activation of iron transporters via S-nitrosylation of bHLH factors to modulate subcellular iron homoeostasis.

Glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants but its involvement in modulating subcellular Fe homoeostasis remains elusive. In this study, we report that the GSH-deficient mutants, cad2-1 and pad2-1 displayed increased sensitivity to Fe deficiency with significant downregulation of the vacuolar Fe exporters, AtNRAMP3 and AtNRAMP4, and the chloroplast Fe importer, AtPIC1. Moreover, the pad2-1 mutant accumulated higher Fe levels in vacuoles but lower Fe levels in chloroplasts compared to wild type (Columbia ecotype [Col-0]) under Fe limited conditions. Exogenous GSH treatment enhanced chloroplast Fe contents in Col-0 but failed to do so in the nramp3nramp4 double mutants demonstrating that GSH plays a role in modulating subcellular Fe homoeostasis. Pharmacological experiments, mutant analysis, and promoter assays revealed that this regulation involves the transcriptional activation of Fe transporter genes by a GSH-S-nitrosoglutathione (GSNO) module. The Fe responsive bHLH transcription factors (TFs), AtbHLH29, AtbHLH38, and AtbHLH101 were found to interact with the promoters of these genes, which were, in turn, activated via S-nitrosylation (SNO). Taken together, the present study highlights the role of the GSH-GSNO module in regulating subcellular Fe homoeostasis by transcriptional activation of the Fe transporters AtNRAMP3, AtNRAMP4, and AtPIC1 via SNO of bHLH TFs during Fe deficiency.

PcNRAMP1 Enhances Cadmium Uptake and Accumulation in Populus × canescens.

Poplars are proposed for the phytoremediation of heavy metal (HM) polluted soil. Characterization of genes involved in HM uptake and accumulation in poplars is crucial for improving the phytoremediation efficiency. Here, Natural Resistance-Associated Macrophage Protein 1 (NRAMP1) encoding a transporter involved in cadmium (Cd) uptake and transport was functionally characterized in Populus × canescens. Eight putative PcNRAMPs were identified in the poplar genome and most of them were primarily expressed in the roots. The expression of PcNRAMP1 was induced in Cd-exposed roots and it encoded a plasma membrane-localized protein. PcNRAMP1 showed transport activity for Cd2+ when expressed in yeast. The PcNRAMP1-overexpressed poplars enhanced net Cd2+ influxes by 39-52% in the roots and Cd accumulation by 25-29% in aerial parts compared to the wildtype (WT). However, Cd-induced biomass decreases were similar between the transgenics and WT. Further analysis displayed that the two amino acid residues of PcNRAMP1, i.e., M236 and P405, play pivotal roles in regulating its transport activity for Cd2+. These results suggest that PcNRAMP1 is a plasma membrane-localized transporter involved in Cd uptake and transporting Cd from the roots to aerial tissues, and that the conserved residues in PcNRAMP1 are essential for its Cd transport activity in poplars.

Transmembrane helix 6b links proton and metal release pathways and drives conformational change in an Nramp-family transition metal transporter.

The natural resistance-associated macrophage protein (Nramp) family encompasses transition metal and proton cotransporters that are present in many organisms from bacteria to humans. Recent structures of Deinococcus radiodurans Nramp (DraNramp) in multiple conformations revealed the intramolecular rearrangements required for alternating access of the metal-binding site to the external or cytosolic environment. Here, using recombinant proteins and metal transport and cysteine accessibility assays, we demonstrate that two parallel cytoplasm-accessible networks of conserved hydrophilic residues in DraNramp, one lining the wide intracellular vestibule for metal release and the other forming a narrow proton transport pathway, are essential for metal transport. We further show that mutagenic or posttranslational modifications of transmembrane helix (TM) 6b, which structurally links these two pathways, impede normal conformational cycling and metal transport. TM6b contains two highly conserved histidines, His232 and His237 We found that different mutagenic perturbations of His232, just below the metal-binding site along the proton exit route, differentially affect DraNramp's conformational state, suggesting that His232 serves as a pivot point for conformational changes. In contrast, any replacement of His237, lining the metal exit route, locked the transporter in a transport-inactive outward-closed state. We conclude that these two histidines, and TM6b more broadly, help trigger the bulk rearrangement of DraNramp to the inward-open state upon metal binding and facilitate return of the empty transporter to an outward-open state upon metal release.

Buckwheat FeNramp5 Mediates High Manganese Uptake in Roots.

Manganese (Mn) is an essential element for plant growth and development, but transporters required for Mn uptake have only been identified in a few plant species. Here, we functionally characterized a member of the natural resistance-associated macrophage proteins (Nramps) family, FeNramp5 in buckwheat (Fagopyrum esculentum Moench), which is known as a species well adapted to acidic soils. FeNramp5 was mainly expressed in the roots, and its expression was upregulated by the deficiency of Mn and Fe. Furthermore, spatial and tissue-specific expression analysis showed that FeNramp5 was expressed in all tissues of the basal root regions. FeNramp5-GFP protein was localized to the plasma membrane when transiently expressed in buckwheat leaf protoplast. FeNramp5 showed the transport activity for Mn2+ and Cd2+ but not for Fe2+ when expressed in yeast. Furthermore, the transport activity for Mn2+ was higher in yeast expressing FeNramp5 than in yeast expressing AtNramp1. FeNramp5 was also able to complement the phenotype of Arabidopsis atnramp1 mutant in terms of the growth and accumulation of Mn and Cd. The absolute expression level of AtNramp1 was comparable to that of FeNramp5 in the roots, but buckwheat accumulated higher Mn than Arabidopsis when grown under the same condition. Further analysis showed that at least motif B in FeNramp5 seems important for its high transport activity for Mn. These results indicate that FeNramp5 is a transporter for the uptake of Mn and Cd and its higher transport activity for Mn is probably associated with higher Mn accumulation in buckwheat.