Genome-Wide Identification of Natural Resistance-Associated Macrophage Protein (NRAMP) and Expression Analysis Under Heavy Metal Stress in Sorghum bicolor L.

The natural resistance-associated macrophage protein gene is associated with Crohn's disease

The NRAMP (natural resistance-associated macrophage protein) family of genes has been widely characterized in organisms ranging from bacteria to yeast, plants, mice, and humans. This gene family plays vital roles in divalent metal ion transport across cellular membranes. As yet, comprehensive analysis of NRAMP family genes has not been reported for soybean. In this study, bioinformatics analysis was conducted to identify 13 soybean NRAMP genes, along with their gene structures, phylogenetic relationships, and transmembrane domains. Expression analysis suggests that GmNRAMP genes function in numerous tissues and development stages. Moreover, soybean NRAMP genes were differentially regulated by deficiencies of N, P, K, Fe, and S, along with toxicities of Fe, Cu, Cd, and Mn. These results indicate that GmNRAMP genes function in many nutrient stress pathways, and might be involved in crosstalk among nutrient stress pathways. Subcellular localization analysis in Arabidopsis protoplasts confirmed the tonoplast or plasma membrane localization of selected soybean NRMAP proteins. Protein-protein interaction analysis found that the networks of three GmNRAMP proteins which putatively interact with nodulin-like proteins, almost distinct from the network that is common to the other 10 soybean NRAMP proteins. Subsequent qRT-PCR results confirmed that these three GmNRMAP genes exhibited enhanced expression in soybean nodules, suggesting potential functions in the transport of Fe or other metal ions in soybean nodules. Overall, the systematic analysis of the GmNRAMP gene family reported herein provides valuable information for further studies on the biological roles of GmNRAMPs in divalent metal ion transport in various soybean tissues under numerous nutrient stresses and soybean-rhizobia symbiosis.

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.

Studies using natural resistance-associated macrophage protein (NRAMP) identification indicated that cattle could be selected for immunity. Several studies performed on intracellular organisms such as Mycobacterium, Salmonella, Brucella and Leishmania in human and mouse revealed that resistance against these bacteria was dependent on high activity of NRAMP in macrophages. However, hardly any researches have been done on Staphylococcus aureus in bovine mastitis, which is an intracellular organism and the main cause of bovine mastitis. The objectives of this study were to establish reverse transcriptase polymerase chain reaction (RT-PCR) methods, through which NRAMP mRNA expression could be compared and analyzed between mastitis-resistant and -susceptible cows. NRAMP gene and its expression were investigated using 20 cows (Holstein Friesian) in Korea. Cows were evenly split into two groups, with and without histories of clinical mastitis. Equivalent numbers of cows were randomly selected from each group. Monocytes were isolated from the bovine peripheral blood of each selected cows and activated with lipopolysaccharide (LPS). mRNA was separated from the monocytes and cDNA of NRAMP was synthesized and amplified using RT-PCR with amplification of β-actin as a control. The difference in NRAMP expressions of mastitis-resistant (n=10) and -susceptible (n=10) Holstein cows was analyzed. Results demonstrate that resistant cows produced more NRAMP mRNA than the susceptible ones, and ratios of NRAMP1:β-actin expression were higher in resistant cows with or without LPS activation. Therefore, this study could be applied to select bovine mastitis resistant cows before infection based on the expression of NRAMP1.

Transition metals are essential for many metabolic processes and their homeostasis is crucial for life. Aberrations in the cellular metal ion concentrations may lead to cell death and severe diseases. Metal ion transporters play a major role in maintaining the correct concentrations of the various metal ions in the different cellular compartments. Recent studies of yeast mutants revealed key elements in metal ion homeostasis, including novel transport systems. Several of the proteins discovered in yeast are highly conserved, and defects in some of the yeast mutants could be complemented by their human homologs. The studies of yeast metal ion transporters helped to unravel the molecular mechanism of macrophage defense against bacterial infection and hereditary diseases.

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

Analysis of NRAMP genes in the Triticeae reveals that TaNRAMP5 positively regulates cadmium (Cd) tolerance in wheat (Triticum aestivum)

Two novel transporters NtNRAMP6a and NtNRAMP6b are involved in cadmium transport in tobacco (Nicotiana tabacum L.)

Heavy metal stress is a critical challenge to agricultural productivity, necessitating deeper insights into the molecular mechanisms of metal transport in plants. In this study, we conducted a comprehensive genome-wide characterization of the Natural Resistance-Associated Macrophage Protein (NRAMP) gene family in Arabidopsis thaliana and identified six AtNRAMP genes. Phylogenetic and synteny analyses revealed their distribution into two distinct clades and evolutionary conservation with legumes such as Glycine max and Arachis hypogaea, indicating functional divergence and gene duplication events maintained under purifying selection. Conserved protein motifs and domains, particularly the NRAMP transmembrane domain, highlighted their conserved role in divalent metal ion transport, while cis-regulatory element analysis demonstrated enrichment of stress- and hormone-responsive elements, pointing to tight transcriptional regulation under environmental challenges. Structural modeling further supported the functional conservation of AtNRAMP proteins. Expression profiling showed clear tissue-specific expression under normal conditions and strong, differential regulation in response to cadmium and other heavy metals, as well as to the phytohormone abscisic acid (ABA). Collectively, these results provide foundational insights into the evolutionary relationships, regulatory mechanisms, and stress-responsive expression of the AtNRAMP gene family, offering a framework for future functional studies and potential applications in developing crops with enhanced heavy metal tolerance and improved growth under stress conditions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-025-07396-8.

The Natural Resistance-Associated Macrophage Protein (NRAMP) family consists of integral membrane transporters essential for divalent metal ion transport in plants. This study aimed to identify and characterize NRAMP genes in tomato (Solanum lycopersicum), performing genome-wide classification, and cis-elements analysis, revealing four SlNRAMP genes in the tomato genome. Phylogenetic analysis classified the four SlNRAMP proteins into two distinct groups, group A and group B. The encoded proteins ranged in length from 509 amino acids in SlNRAMP3 to 773 amino acids in SlNRAMP4. The number of predicted transmembrane domains in SlNRAMPs ranged from 12 to 14. Expression analysis in 20 samples from three key developmental stages were collected for metabolic profiling and RNA-seq, revealing that group A genes, including SlNRAMP1 and SlNRAMP2, were predominantly expressed in flowers and mature roots, while group B genes, including SlNRAMP3 and SlNRAMP4, exhibited relatively high expression in leaves and roots, respectively. The SlEIN2 gene, previously misclassified as SlNRAMP5, has now been reassigned to a separate category. Quantitative RT‒PCR analysis demonstrated differential regulation of SlNRAMP expression in roots under cadmium and salt stress. The results highlighted that SlNRAMP4 is critical for cadmium sensitivity, whereas SlNRAMP2 plays a role in the early detection and signaling of NaCl stress. This study provides a detailed characterization of the SlNRAMP family in tomato, advancing our understanding of their roles in metal ion homeostasis and stress responses, while serving as a valuable resource for future research.

The NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN1 (NRAMP1) transporter guarantees plant survival of manganese (Mn) deficiency by mediating Mn entry into root cells. Unlike other high-affinity metal transporters, NRAMP1 is only slightly regulated at the transcriptional level. We show here that adequate Mn content in tissues is safeguarded through a tight control of the quantity of NRAMP1 present at the surface of root cells. Depending on Mn availability, an NRAMP1-GFP fusion protein cycles dynamically between the plasma membrane (PM) and endosomal compartments. This involves a clathrin-mediated endocytosis pathway, as disrupting this pathway in auxilin-overexpressor lines prevents NRAMP1 internalization. Mutation of the phosphorylated serine residues 20, 22 and 24 in the cytosol-exposed N terminus of NRAMP1 alters its membrane distribution. Indeed, a phospho-dead mutation stabilizes NRAMP1 at the PM, regardless of the Mn regime, and dramatically reduces plant tolerance to Mn toxicity. Conversely a phosphomimetic mutant is constitutively internalized into endosomes. Together, these data establish that phosphorylation of NRAMP1 is the trigger for its Mn-induced endocytosis and represents the main level of regulation of this transporter. Furthermore, the extent of Mn toxicity observed when interrupting NRAMP1 membrane cycling undermines the dogma that Mn is only marginally toxic to plants.

Heavy metals such nickel (Ni) can cause toxicity by 1) displacing essential components in the biomolecules, 2) blocking the functional group of molecules, or 3) modifying enzymes, proteins, the plasma membrane, and membrane transporters. The main objective of the present study was to investigate the effect of nickel (Ni) on gene expression of nitrate on gene expression with a focus on the genes coding for the high affinity Ni transporter family protein AT2G16800, and natural resistance-associated macrophage protein (NRAMP). Ni toxicity was assessed by treating seedlings with an aqueous solution of nickel nitrate salt [Ni(NO3)2] at the concentrations of 150 mg, 800 mg, and 1600 mg of nickel per 1 kg of dry soil. RT-qPCR was used to measure the expression of AT2G16800, and NRAMP genes in samples treated with nickel nitrates and controls. The results revealed that P. glauca is resistant to Ni based on lack of plant damage at all nickel concentrations. Ni has no effect on the expression of the AT2G16800 gene in needles or roots. However, it induced an upregulation of the NRAMP genes in roots at all the doses tested (150 mg/kg, 800 mg/kg, and 1600 mg/kg). On the other hand, Ni has no effect on the expression of the NRAMP gene in needle but the lowest dose of potassium (150 mg/kg) upregulated this gene in needle tissues.

A novel plasma membrane-based NRAMP transporter contributes to Cd and Zn hyperaccumulation in Sedum alfredii Hance

Transporter proteins play an essential role in the uptake, trafficking and storage of metals in plant tissues. The Natural Resistance-Associated Macrophage Protein (NRAMP) family plays an essential role in divalent metal transport. We conducted bioinformatics approaches to identify seven NRAMP genes in the Phaseolus vulgaris genome, investigated their phylogenetic relation, and performed transmembrane domain and gene/protein structure analyses. We found that the NRAMP gene family forms two distinct groups. One group included the PvNRAMP1, -6, and -7 genes that share a fragmented structure with a numerous exon/intron organization and encode proteins with mitochondrial or plastidial localization. The other group is characterized by few exons that encode cytoplasmic proteins. In addition, our data indicated that PvNRAMP6 and -7 may be involved in mineral uptake and mobilization in nodule tissues, while the genes PvNRAMP1, -2, -3, -4 and -5 are potentially recruited during plant development. This data provided a more comprehensive understanding of the role of NRAMP transporters in metal homeostasis in P. vulgaris.

Silencing of PpNRAMP5 improves manganese toxicity tolerance in peach (Prunus persica) seedlings