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Article

Unraveling the NRAMP Gene Family: Aegilops tauschii’s Prominent Barrier Against Metal Stress

by
Hongying Li
1,*,
Yibo Li
1,
Fuqiang Yang
1,
Xiaolin Liang
1,
Yifan Ding
1,
Ning Wang
1 and
Xiaojiao Han
2,*
1
College of Horticulture and Plant Protection, Henan University of Science and Technology, Luoyang 471023, China
2
Key Laboratory of Tree Breeding of Zhejiang Province, Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China
*
Authors to whom correspondence should be addressed.
Agronomy 2025, 15(8), 1919; https://doi.org/10.3390/agronomy15081919
Submission received: 28 June 2025 / Revised: 20 July 2025 / Accepted: 6 August 2025 / Published: 8 August 2025
(This article belongs to the Special Issue Physiological and Molecular Mechanisms of Plant Under the Abiotic Stress)
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Abstract

The natural resistance-associated macrophage proteins (NRAMPs) gene family represents a group of membrane transporter proteins with wide distribution in plants. This family of membrane transporters plays a pivotal role in mediating plant responses to metal stress by coordinating ion transport processes and maintaining cellular metal homeostasis, thereby effectively mitigating the detrimental effects of metal ion stress on plant growth and development. This study conducted a comprehensive genome-wide analysis of the NRAMP gene family in A. tauschii using integrated bioinformatics approaches, as well as the expression pattern when exposed to heavy metal-induced stress. By means of phylogenetic investigation, eleven AetNRAMP proteins were categorized into five distinct subgroups. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed that the majority of NRAMP genes exhibited marked differential expression patterns under specific stress treatments. Subsequently, yeast cells were employed to validate the functions of AetNRAMP1 and AetNRAMP3. It was confirmed that AetNRAMP1 functioned in copper transport, and AetNRAMP3 showed an increase in its expression level under manganese stress. These findings establish a molecular foundation for elucidating the functional specialization of NRAMP gene family members in A. tauschii’s heavy metal detoxification pathways, providing critical genetic evidence for their stress-responsive regulatory networks. Nevertheless, significant knowledge gaps persist regarding its functions in A. tauschii. Research on metal stress resistance in this wheat progenitor species may establish a theoretical foundation for enhancing wheat tolerance and developing improved cultivars.

1. Introduction

In recent years, the continuous advancement of scientific and technological capabilities have significantly enhanced China’s industrial capacity. However, the environmental repercussions are also intensifying [1]. The uncontrolled mining of mineral resources, indiscriminate discharge of heavy metal wastewater, excessive application of pesticides and fertilizers, atmospheric deposition of dust, and accumulation of solid waste are primary contributors to heavy metal contamination in soil [2]. This has resulted in a significant increase in the concentration of toxic heavy metals in agricultural soil [3]. A series of resultant issues, including the persistent decline in soil productivity, contamination of agricultural products, and degradation of the ecological environment, have emerged as significant factors impeding agricultural development. Furthermore, the issue of elevated levels of heavy metal ions in various field crops, including rice (Oryza sativa), wheat (Triticum aestivum L.), and corn (Zea mays L.), has a more pronounced effect on grain production and food safety in China. Additionally, metal ions function as a double-edged sword in the plant growth process. Certain heavy metal ions serve as essential micronutrients for plant growth and metabolic processes. Representative elements including zinc (Zn), copper (Cu), and manganese (Mn) fulfill critical physiological functions through mediating enzymatic activation, maintaining redox homeostasis, and facilitating photosynthetic electron transport, respectively. A deficiency of these essential trace metal ions in plants can result in issues such as stunted growth and impaired development [4]. Conversely, if the concentration is excessively high, it becomes toxic to plants, inhibiting physiological activities such as cell growth and photosynthesis, thereby causing detrimental effects.
In recent years, the food-related issues resulting from heavy metal pollution have become increasingly severe. Consequently, there is an urgent need to investigate the molecular mechanisms underlying plants’ selective absorption of heavy metals from soil, the maintenance of ionic balance within tissues, and the detoxification of excessive concentrations. Natural resistance-associated macrophage proteins (NRAMPs) represent a phylogenetically ubiquitous gene family demonstrating evolutionarily conserved structural and functional characteristics across diverse organisms spanning from prokaryotes to mammals, with particular conservation in metal ion transport domains. NRAMPs have been characterized as a comprehensive family of membrane proteins and serve as crucial metal transporter proteins in plants [5,6,7]. This gene family serves as a multifunctional regulatory hub, coordinating cellular homeostasis maintenance, photosynthetic efficiency modulation, protein activity potentiation, and environmental stress signal transduction through integrated molecular networks. Currently, seven, thirteen, eleven, and eight NRAMP genes have been identified in Arabidopsis thaliana [8], Glycine max L. [9], Sedum alfredii Hance, and O. sativa L., respectively. Among these, the research on NRAMP genes in A. thaliana and O. sativa is the most comprehensive. Genes encoding NRAMP transporters play pivotal roles in mediating the cellular translocation of essential and toxic metal ions, including Mn [3], Fe [10], Zn [11], and cadmium (Cd) [12], through conserved transmembrane transport mechanisms [13]. In A. thaliana, AtNRAMP1 has been functionally characterized as a transporter mediating iron (Fe), Mn, and Cd uptake [13,14], whereas AtNRAMP2 localizes to the endomembrane system and orchestrates subcellular Mn partitioning through vesicular trafficking pathways. During seed germination in A. thaliana, AtNRAMP3 and AtNRAMP4 mediate Mn remobilization from vacuolar stores via tonoplast efflux transport, with their functional deficiency under Mn-deprived conditions leading to impaired photosystem II (PSII) assembly efficiency and subsequent biomass accumulation retardation [15]. AtNRAMP5 orchestrates rhizospheric Cd and Fe acquisition through root-specific metal influx channels, while AtNRAMP6 governs symplatic Cd trafficking via endomembrane-mediated subcellular compartmentalization, particularly in vascular bundle cells. These metalloproteins function as central regulators of ionic equilibrium by coordinating spatiotemporal metal allocation through xylem loading and phloem redistribution, while concurrently executing heavy metal detoxification via glutathione-mediated chelation and vacuolar sequestration in root epidermal cells. NRAMP proteins can maintain cellular metal ion homeostasis by sequestering toxic elements in vacuoles or exporting them out of sensitive cell compartments, thereby enhancing plants’ tolerance to heavy metal stress [16,17], or by regulating the transcription level of the NRAMP gene to translate a large amount of NRAMP protein in vivo, thereby preventing the massive accumulation of heavy metal ions to reduce cell damage [18]. NRAMP proteins can also reduce cellular stress by cooperating with other transporters to remove metal ions from the cytoplasm [19,20].
Aegilops tauschii Coss serves as the D genome donor species for common wheat (AABBDD). This genetic affinity endows the two species with a high degree of homology in gene sequences, regulatory networks, and metabolic pathways. Such a close genetic relationship means that genes associated with heavy metal stress tolerance identified in A. tauschii (e.g., the AetNRAMP family) can be directly introduced into the wheat genome via approaches like hybridization and gene editing. Their functions are more likely to be retained and exerted in wheat, which substantially lowers the barriers to cross-species gene transfer and serves as a “natural bridge” for wheat genetic improvement [21]. Hexaploid bread wheat (Triticum aestivum, AABBDD, 2n = 6x = 42) represents one of the principal food crops globally [22]. In light of the dual challenges posed by population growth and frequent extreme climatic events, it is essential to consistently enhance wheat yield to safeguard global food security. After long-term domestication and breeding, common wheat has a gradually narrowed genetic background, and many genes related to stress resistance have been lost due to artificial selection. As a wild relative of wheat, the genetic diversity of the wild population of A. tauschii is much higher than that of the D genome of common wheat. It contains abundant gene resources related to resistance, yield and quality, and is an important gene pool for the genetic improvement of the modern wheat D genome [23,24]. NRAMPs are proteins that facilitate the transport of metal ions in plants [2]. This gene family constitutes a significant component of metal transport mechanisms in plants, with numerous heavy metal transport processes relying on its involvement. For instance, metal ions including Cu [8], zinc (Zn) [11], Fe [25], Mn, and Cd [26,27,28] are significant. It sustains the dynamic balance of metal ions within plants by modulating their levels of transport [29]. The investigation identified the NRAMP gene family in A. tauschii through bioinformatics methodologies, and subsequently conducted a comprehensive analysis on the sequence attributes, gene architectures, evolutionary relationships, and distinct expression patterns of the family constituents. Concurrent with the previous analysis, the effects of the stress induced by four heavy metal ions, specifically Mn2+, Cd2+, Zn2+, and Cu2+, on the physiological and biochemical indices of A. tauschii seedlings and the expression of NRAMP genes were investigated. The findings of this study have established a basis for the effective exploration of the germplasm resources of A. tauschii and the introduction and utilization of superior genes into wheat, while also providing resources for the enhancement of the original germplasm in the genetic improvement of modern wheat.

2. Materials and Methods

2.1. Identification of NRAMP Gene Family Members and Analysis of Their Physicochemical Properties

The protein, genome, and annotation files of A. tauschii (Version 4.0) and A. thaliana (Tair11) were obtained from the Ensembl Plants database (https://plants.ensembl.org, accessed on 25 September 2024) and TAIR database (https://www.arabidopsis.org, accessed on 25 September 2024), respectively. BlastP was performed using the NRAMP member sequences of Arabidopsis against the A. tauschii genome to preliminarily screen NRAMP family members. The NRAMP domain was acquired from the Pfam database (http://pfam.xfam.org, accessed on 25 September 2024) [30], and 11 AetNRAMP members were obtained by removing sequences lacking the NRAMP domain or containing incomplete regions.
The physicochemical properties of the identified A. tauschii NRAMP member sequences were analyzed using the ProtParam online tool (https://www.expasy.org/resources/protparam, accessed on 25 September 2024) in ExPASy [31]. The subcellular localization of AetNRAMPs was predicted using the Cell-Ploc 2.0 online tool.
The genome and gene annotation files of A. tauschii were utilized to extract the chromosome gene density data, providing the locations of NRAMP gene family members on the chromosomes. Subsequently, the Tbtools software (Version 2.154) was employed for visualization and mapping [32] to obtain the gene and protein sequences of NRAMP gene family members in S. alfredii Hance via the Ensembl Plants database (https://plants.ensembl.org, accessed on 25 September 2024), and those in rice, poplar, common wheat, maize, and soybean via the Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 25 September 2024). We utilized the maximum likelihood (ML) method in MEGA 11 for the construction of a multispecies phylogenetic tree comprising NRAMP gene families from A. tauschii, A. thaliana, O. sativa L., G. max, P. trichocarpa, and S. alfredii. Jones–Taylor–Thornton (TT) was selected as the first choice of model. Then, we configured 1000 bootstrap replicates and enhanced the phylogenetic tree using the online tool iTOL (https://itol.embl.de, accessed on 25 September 2024). The species and gene IDs involved in constructing the phylogenetic tree are detailed in Table S1.

2.2. Method for Collinearity Analysis of AetNRAMP

Utilizing the genome files and gene annotation files of rice and soybean, we employed Tbtools (Version 2.154) to construct the collinearity among A. tauschii, rice, and soybean as well as within each of these species themselves. Subsequently, the collinear relationships were determined through gene sequence alignment and analysis and conduct visualization and mapping.

2.3. The Cis-Acting Element Analysis of AetNRAMP

The gene sequence of 2000 bp upstream of the start codon (ATG) of the AetNRAMP genes were extracted. The online software PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 25 September 2024) was employed to predict the cis-acting elements of AetNRAMP, and then Tbtools (Version 2.154) were used for visualization and mapping.

2.4. Plant Materials and Treatment Methods

The A. tauschii seeds utilized in this study were sourced from the experimental field located at Xinxiang Academy of Agricultural Sciences in Henan Province (latitude 35°18′ north, longitude 113°52′ east). After being rinsed thrice with deionized water, the seeds were placed in sterilized vermiculite. Upon reaching a length of 2 cm during germination, they were watered with 1/2 Hoagland nutrient solution. They were subsequently moved to a constant temperature incubator set at 26 °C (with a photoperiod of 14 h light/10 h dark) and hydroponically cultivated using 1/2 Hoagland nutrient solution. When the seedlings were 21 days old, the plants displaying consistent growth and robust conditions were chosen for stress treatment using 1/2 Hoagland nutrient solution supplemented with 100 μmol/L CdSO4, 100 μmol/L CuSO4, 100 μmol/L MnSO4, and 100 μmol/L ZnSO4, individually. Six seedlings were used for each treatment group, with three biological replicates set up. Following stress treatments of 0 h, 6 h, 12 h, 24 h, 48 h, and 96 h, the roots and leaves of A. tauschii seedlings were sampled, rapidly frozen in liquid nitrogen, and subsequently stored at −80 °C to facilitate total RNA extraction.

2.5. RNA Extraction and qRT-PCR Analysis

Total RNA was extracted from the roots and leaves of A. tauschii using an RNA extraction kit (RNE03, NOBELAB, Beijing, China). The obtained RNA was then reverse transcribed into cDNA following the instructions of the reverse transcription kit (MF166plus, Mei5bio, Beijing, China). The resulting cDNA was used for quantitative real-time PCR (qRT-PCR), with EF1-α serving as the internal reference gene [33]. Primers were designed using the Primer 3 online tool, and detailed sequences are provided in Supplementary Table S2. The qRT-PCR was performed using the SYBR Green fluorescence method. The reaction mixture (10 μL total volume) consisted of 1 μL cDNA template, 0.2 μL each of forward and reverse primers, 5 μL of 2× M5 Hi SYBR Premix Es Taq master mix, and ddH2O to adjust the final volume. Each sample was analyzed in triplicate, and amplification was carried out on a rtPCR detection system (CFX96, Bio-Rad, Hercules, CA, USA). The thermal cycling conditions were as follows: initial denaturation at 95 °C for 30 s, followed by 39 cycles of denaturation at 95 °C for 5 s and annealing at 60 °C for 30 s. The relative gene expression levels were calculated using the 2−ΔΔCt method [34], and data visualization was performed using GraphPad Prism 9 software.
To construct the pYES2-AetNRAMP yeast expression vector, the cDNA of A. tauschii was utilized as a template for amplification with specific primers (Table S2) containing the pYES2 homologous arms (underlined) (Vazyme, Nanjing, China). In a specific procedure, the purified PCR products were ligated into the pYES2 vector through seamless cloning (Tsingke Biotechnology, Beijing, China). Subsequent sequencing was conducted to acquire the pYES2-AetNRAMP positive strains, and the plasmids were isolated. The plasmids pYES2-AetNRAMP1, pYES2-AetNRAMP3, and the empty pYES2 vector were introduced into INVSc1 yeast competent cells.
Yeast cells carrying the target gene and those with the empty vector were cultured in SC-Ura medium containing galactose. The medium was supplemented with manganese sulfate (MnSO4) at a final concentration of 200 μmol/L and copper sulfate (CuSO4) at 100 μmol/L, respectively. The experiment included three biological replicates and was conducted for four days. Subsequently, the samples were centrifuged and washed three times with sterile water to remove residual medium. After all samples were dried to a constant weight at 65 °C, they were digested with a 6:1 (v/v) mixture of 65% nitric acid (HNO3) and 30% hydrogen peroxide (H2O2). The concentrations of heavy metal elements were quantitatively analyzed using an inductively coupled plasma optical emission spectrometer [35] (ICP-OES, iCAP-7400, Thermo Fisher Scientific, Waltham, MA, USA). Bar charts were plotted using GraphPad Prism 9, and data were compared for differences by test (**: p < 0.05; ns: p > 0.05) [35].

3. Results

3.1. Analysis of the Physicochemical Properties of Members of the AetNRAMP Gene Family

Using both Blast alignment and Pfam analysis, 11 members of the NRAMP gene family were finally identified from the genome of A. tauschii. Based on the research in A. thaliana [9] and the clustering relationships, these members were classified into AetNRAMP1-AetNRAMP8, AetEIN2.1-AetEIN2.3. Through the analysis of the protein sequences of these 11 members, it was found that the number of amino acids in the proteins of most NRAMP members ranged from 517 to 637. The relative molecular masses of AetEIN2.1 and AetNRAMP8 are 1268 and 275 respectively. Among these 11 members, there were 3 basic amino acids (pI > 7) and 8 acidic amino acids (pI < 7). Among them, AetNRAMP1, AetNRAMP4, AetEIN2.1, and AetEIN2.2 were unstable proteins (Instability > 40), and the remaining 7 were stable proteins (Instability < 40). The average hydrophilicity ranged from −0.034 to 0.755. The average hydrophilicity of AetEIN2.1 was negative, making them hydrophilic proteins, while the remaining 10 members were all hydrophobic proteins. The results of subcellular localization prediction showed that among the 11 NRAMP proteins of A. tauschii, AetEIN2.1 proteins were solely located in the chloroplast, while the remaining NRAMP proteins were all located in the cell membrane (Table 1).

3.2. Phylogenetic Tree Analysis of AetNRAMP Genes in A. tauschii

Evolutionary analysis has unveiled the phylogenetic relationships among gene family members across different species. To delve deeper into the phylogenetic connections and evolutionary history of the NRAMP family members in A. tauschii, the protein sequences of NRAMP gene family members from A. tauschii, A. thaliana, rice, poplar, soybean, and S. alfredii were aligned to construct a maximum likelihood (ML) phylogenetic tree (Figure 1). According to Asima Rasheed’s study [36], the NRAMP family members were categorized into five subgroups. AetNRAMP1 was placed in subgroup A, while AetNRAMP2 and AetNRAMP7 were assigned to subgroup B. AetNRAMP3 was classified under subgroup C. Subgroup D included AetNRAMP1, AetNRAMP3, AetNRAMP4, AetNRAMP5, and AetNRAMP8, whereas subgroup E comprised AetEIN2.1, AetEIN2.2, and AetEIN2.3. Furthermore, their resemblances extend to similar physicochemical properties and chromosomal positions. This evidence prompts the speculation that these two genes may perform analogous roles in the transportation of heavy metal ions. Analysis of the phylogenetic tree reveals a smaller number of AetNRAMP members situated in the second subgroup compared to the other two subgroups. Contrasting with the phylogenetic assessments of NRAMP gene families in other plant species, it becomes apparent that distinct levels of conservation and divergence exist among NRAMP genes across different species. This observation serves as a valuable reference point for delving deeper into the functionalities of the AetNRAMP genes.

3.3. Chromosomal Location Analysis of the AetNRAMP Genes in A. tauschii

The Tbtools software (Version 2.154) was used to conduct chromosomal location analysis for the AetNRAMP genes (Figure 2). The results indicated that the 11 AetNRAMPs were irregularly dispersed on four chromosomes (3D, 4D, 5D, 6D, and 7D).

3.4. Collinearity Analysis of AetNRAMP

Collinearity analysis, a method applicable within and between species, is capable of identifying structural variations within or between species, elucidating species’ chromosomal evolution, and investigating phenomena like polyploidization within species. In the specific context of A. tauschii, collinearity analysis revealed the presence of a specific homologous gene pair (AetNRAMP6:AetNRAMP7). The interspecies collinearity analysis provided significant insights (Figure 3). Comparative genome analysis of A. tauschii, soybean, rice, and wheat revealed six pairs of NRAMP homologous gene pairs present in both soybean and rice. As a wild relative of wheat, A. tauschii exhibits 20 pairs of direct homologous gene pairs with wheat. Furthermore, the collinearity assessment among A. tauschii, soybean, wheat, and rice indicates a high level of homology among the NRAMP genes across these four species.

3.5. Analysis of the Cis-Acting Elements of AetNRAMP

Cis-acting elements play a crucial role in gene expression in the promoter region, particularly in response to abiotic stress. By analyzing the cis-acting elements of the NRAMP genes in A. tauschii using TBtools software (Figure 4), it was observed that all AetNRAMP members contained cis-elements associated with light response, totaling 130. Specifically, the promoter sequences of the AetNRAMP genes comprised 122 cis elements linked to various plant hormones, including abscisic acid (46), gibberellin (14), auxin (12), salicylic acid (8), and methyl jasmonate (MeJA) (39). Furthermore, six cis-acting elements related to defense and stress responses were identified, such as those responsive to low temperature (7), drought (16), anaerobic induction (10), hypoxi-specific induction (10), defense and stress induction (3), and wound response (10). These research results indicate that the NRAMP gene has a significant impact on the natural environment, development, and health-related biomarker quality. This evidence substantiates the diverse array of physiological regulatory roles played by the NRAMP gene family in plants.

3.6. Expression Analysis of AetNRAMP Genes Under the Stress of Different Heavy Metal Ions in Leaves and Root

To investigate the functional roles of AetNRAMP genes under heavy metal stress, we conducted transcriptional profiling via quantitative reverse transcription PCR (qRT-PCR). Seven representative candidates were systematically selected from the eleven identified NRAMP family members in A. tauschii and exposed to four distinct heavy metal treatments (Cd2+, Mn2+, Fe2+, Zn2+) to analyze tissue-specific expression modulation in root and leaf organs. The results derived from the qRT-PCR analysis, depicted in Figure 5, exhibit distinct expression profiles of various AetNRAMP genes when subjected to four different heavy metal ions. In response to zinc stress, the expression of AetNRAMP1 in leaves was significantly increased, up to 600-fold (Figure 5A). Moreover, AetNRAMP2, AetNRAMP3, AetNRAMP4, AetNRAMP5, AetNRAMP6, and AetEIN2.1 displayed varying degrees of upregulation in their expression levels (Figure 5B–G). Specifically, only AetNRAMP4 exhibited upregulation in roots at 12 h under zinc stress, while the other genes were either suppressed or not expressed at all.
Under copper stress, only AetNRAMP3 exhibited increased expression in leaves (Figure 5C), while the rest of the genes displayed varying degrees of decreased expression. Moreover, AetNRAMP4 and AetNRAMP5 were entirely unexpressed (Figure 5D,F). In the roots of A. tauschii, only AetEIN2.1 demonstrated higher expression levels compared to normal conditions (Figure 5G), whereas the other members of the AetNRAMP family exhibited varying degrees of reduced expression.
Amid cadmium stress, the expression levels of AetNRAMP1, AetNRAMP2, and AetNRAMP5 in leaves exhibited notable upregulation (Figure 5A,B,E). The expressions of the remaining genes displayed varying responses without significant distinctions among them. Furthermore, under cadmium stress, the expressions of other genes showed a diminishing tendency, with even AetNRAMP4 and AetEIN2.1 demonstrating nearly negligible expressions.
During manganese stress, the expression levels of NRAMP genes in the leaves of A. tauschii surpassed those in the roots. Most genes in the leaves exhibited relatively active expressions. Notably, although at 6 and 12 h the expression levels of AetNRAMP1/2/3/4/5/6 were below normal levels, with prolonged stress duration, their expression levels started to show an upward trajectory, exceeding the normal conditions (Figure 5A–F). In contrast, in the roots, the expression levels of AetNRAMP1 and AetNRAMP2 peaked at 96 h, while the expressions of the other genes were suppressed (Figure 5I).

3.7. Tolerance of AetNRAMP Heterologous Expression in Yeast to Metal Ions

The functions of the AetNRAMP3 and AetNRAMP1 genes in transporting various metal ions were further investigated through yeast heterologous expression assays (Figure 6A). The results indicated that under Cu2+ stress, AetNRAMP3 enhanced yeast tolerance to copper, while AetNRAMP1 reduced yeast tolerance to copper. Under manganese stress, AetNRAMP3 reduced yeast tolerance to manganese, while AetNRAMP1 enhanced yeast tolerance to manganese, indicating its intricate regulatory function in manganese transport and potential direct participation in the manganese ion transport process. Taken together, these results suggest that the members of the AetNRAMP gene family exhibit distinct functional divisions and regulatory mechanisms under heavy metal stress, collectively maintaining the homeostatic balance of metal ions in A. tauschii.
To investigate the transport functions of the AetNRAMP1 and AetNRAMP3 genes for various metal ions in transgenic yeast, this study assessed the Cu2+ and Mn2+ concentrations in yeast cells following heavy metal stress treatment (illustrated in the Figure 6B,C). The findings revealed that, under heavy metal stress conditions, the Mn2+ content in AetNRAMP1 transgenic yeast cells was significantly lower than that in non-transgenic counterparts. Similarly, AetNRAMP3 transgenic yeast cells exhibited a marked reduction in Cu content compared to the control group. These results suggest that the AetNRAMP1 and AetNRAMP3 genes may enhance the efflux or transport capabilities of transgenic yeast for heavy metal ions, thereby decreasing the intracellular accumulation of Mn2+ and Cu2+.

4. Discussion

Metal transporters, such as NRAMP, heavy metal ATPase (HMA), metal tolerance proteins (MTPs), and basic leucine zipper (bZIP), among others, can regulate the ion balance within plants, controlling the absorption, transportation, and sequestration of heavy metal ions [29]. This regulation helps maintain the stable state of metals inside cells, preventing toxicity from high concentrations of metal ions [30]. The presence of multiple gene families related to metal transport in plants indicates their diverse responses and regulatory effects on various metal ions [31].
In response to heavy metal stress, a series of physiological and biochemical changes in A. tauschii reveal intricate and complex molecular mechanisms [32]. Roots, being the primary organs for metal ion absorption and transport in plants, are usually more vulnerable to stress [34]. However, this study found that most NRAMP genes in A. tauschii were expressed at relatively low levels in the roots, with some genes showing no expression. In contrast, the AetNRAMP genes displayed higher activity in the leaves, indicating that these genes primarily regulate the transportation and control of metal ions in the leaves. This research systematically examined the essential regulatory roles of the NRAMP gene family in A. tauschii during its response to heavy metal ion stress. The variations in expression levels of NRAMP gene family members under different heavy metal ion stresses in A. tauschii signify their specific roles in metal ion absorption, transport, and distribution.
Manganese plays a pivotal role in various physiological processes such as photosynthesis, respiration, and regulation of enzyme activity [33]. The insufficiency of manganese ions can impede these physiological processes and hinder growth and development. In A. thaliana, NRAMP1 enhances the plant’s high affinity for manganese absorption [34]. In rice, the manganese transporter OsNRAMP5 was the first to be identified, facilitating the absorption and transportation of Mn2+ in roots. Gene knockout studies have demonstrated that OsNRAMP5 is responsible for Mn2+ uptake [35], elucidating its role in transporting Mn2+ from the soil to the xylem. Mutant lines with OsNRAMP5 knockout or RNA interference displayed reduced manganese accumulation in roots and shoots, resulting in growth retardation due to manganese deficiency [33]. AetNRAMP1, AetNRAMP4, and AetNRAMP5, being homologous genes, exhibit a high degree of sequence similarity. Quantitative results indicate that the expression level of AetNRAMP1 in leaves exhibits an upward trend and is significantly higher than that in the roots. When AetNRAMP1 was transferred into yeast cells, the growth condition of the yeast was found to be consistent with the expression of AetNRAMP1 in leaves, but markedly different from its expression in the roots. These findings suggest that the manganese transport process is associated with photosynthesis. Subsequently, by measuring the metal content in transgenic yeast under Mn stress, it was further demonstrated that AetNRAMP1 is implicated in the regulation and transport of Mn2+; however, the precise mechanism remains to be elucidated.
Zn, an essential trace element for plant growth, functions as a structural ion in transcription factors. Additionally, it plays a crucial role in numerous enzyme-catalyzed reactions within cells. The excessive accumulation of zinc can disrupt enzyme-dependent reactions [36]. Moreover, an excess of zinc may lead to the accumulation of other heavy metals such as copper and manganese in roots and shoots [37]. The NRAMP1 protein of S. alfredii shows restricted similarity to the NRAMP proteins identified in A. thaliana. Despite being localized in the plasma membrane, it still exhibits the typical characteristics of plant NRAMP genes. It has the capability to transport not only manganese and cadmium but also zinc [38]. AetNRAMP1 and SaNRAMP1 belong to the same subgroup in phylogenetic evolution. Furthermore, upon zinc induction, the expression level of AetNRAMP1 exceeds that of the control group by more than 600 times. This suggests that the regulatory mechanism of this gene for zinc transport, accumulation, and regulation is akin to that of SaNRAMP1. However, the specific regulatory manner still requires further research.
Cd is a nonessential metal element for plants; however, it can be readily absorbed by plants. Excessive cadmium, however, disrupts the ionic homeostasis along with many physiological and biochemical processes within plant cells [38]. Once Cd is absorbed by plant roots, it is rapidly transported to the shoots via the xylem. Similar to the cadmium uptake process, a significant number of NRAMP transporter proteins actively participate in transferring Cd from roots to buds or other plant tissues. The functionality of NRAMP genes has been demonstrated across various species, contributing to cadmium accumulation. In rice, NRAMP1 and NRAMP4 serve as primary transporter proteins for cadmium uptake [39]. In A. thaliana, transporter proteins AtNRAMP1, AtNRAMP3, and AtNRAMP4 are crucial for Cd translocation from vacuoles, significantly impacting the dynamic balance and distribution of cadmium within the plants [37]. In this experimental study, the expression levels of AetNRAMP4 and AetNRAMP5 in leaves were upregulated under Cd stress, suggesting that they may play crucial roles in Cd transportation. Using gene knockout technology, Sasaki et al. have already confirmed OsNRAMP4 and OsNRAMP5 are directly implicated in cadmium transport [35]. Being homologous to them, AetNRAMP4 and AetNRAMP5 exhibit highly similar sequences, indicating their potential involvement in cadmium transport. However, further verification is required in A. tauschii to investigate how AetNRAMP4 and AetNRAMP5 participate in the transport and accumulation of cadmium.
Cu plays multiple important roles in plants, but its excessive presence may pose a threat to plant survival. Studies have shown that copper is crucial for carbon assimilation, ATP synthesis, photosynthesis, and respiration [38]. However, the excessive accumulation of copper can trigger oxidative stress in plants, causing serious damage to membrane structures, macromolecules, and impacting multiple biochemical pathways and DNA [40]. It has been demonstrated in Lu Qin’s research that the gene expression level of GmNRAMP5a was significantly upregulated under copper stress [28]. GmNRAMP5a and AetNRAMP3 belong to the same clade in the phylogenetic evolutionary tree. Quantitative results showed that under copper treatment, the expression level of the AetNRAMP3 gene in leaves was significantly higher than that in the control group (0 h), indicating that AetNRAMP3 may have similar functions and plays a role in the transport and regulation of copper in A. tauschii to maintain ionic balance among cells. In the roots of A. tauschii, the expression of the remaining AetNRAMP genes was not significant and was even undetectable when compared with the control group. Furthermore, the results obtained from yeast metal-sensitivity assays were consistent with the quantitative results of AetNRAMP3 observed in leaves. By measuring the changes in metal content in transgenic and nontransgenic yeasts under Cu stress, it was confirmed that AetNRAMP3 plays a role in the transport of Cu2+ in leaves.
Yeast is a unicellular eukaryotic organism with a eukaryotic expression system similar to plants to some extent. Consequently, it serves as an ideal model for screening and validating stress resistance genes in plants due to its capacity for rapid in vitro validation of the functionality of stress resistance genes [41]. Future studies should employ advanced techniques such as CRISPR/Cas9, gene silencing, and gene overexpression to investigate the functions and roles of this gene family in the metal ion transport processes.

5. Conclusions

In this study, a comprehensive analysis was undertaken on the NRAMP gene family of A. tauschii, followed by the application of four types of metal ion stress treatments to seven of these genes. The members of these gene families exhibited heightened activity in leaves compared to roots under heavy metal stress, specifically Cu2+, Zn2+, Mn2+, and Cd2+. Through qRT-PCR experiments and yeast heterologous expression, the study discovered that AetNRAMP1 could enhance manganese transport capacity in the leaves of A. tauschii while reducing copper tolerance; AetNRAMP3 could elevate its expression level in leaves during copper stress and lower its transport capacity for manganese ions. In future studies, we can employ techniques such as CRISPR/Cas9, gene silencing, and gene overexpression to explore the functions and roles of this gene family in the process of metal ion transport. These findings will provide valuable genetic resources for cultivating A. tauschii with low heavy metal accumulation, thus laying a foundation for the development of new wheat varieties with high yield, quality, and stress resistance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15081919/s1. Table S1: Species, gene IDs, and gene names involved in constructing the phylogenetic tree; Table S2: The primers used for qRT-PCR of AetNRAMP genes and constructing yeast vectors with AetNRAMP1 and AetNRAMP3. Table S3: Analysis of the ten conserved motifs of AetNRAMP proteins in Aegilops tauschii.

Author Contributions

H.L. and X.H. designed and conducted the experiments. Y.L., F.Y., X.L. and Y.D. performed the experiments and analyzed the data. Y.L. and H.L. conducted the data and wrote the manuscript. X.H. and N.W. contributed to discussion in writing process. H.L. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The research was supported by the Public Welfare Industry Special Research Projects of Luoyang (2202022A) to N.W. and the National Natural Science Foundation of China (No. 31872168) to X.H.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Conflicts of Interest

All the authors declare that they have no conflict of interest or financial conflicts to disclose.

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Agronomy 15 01919 g001
Figure 1. A phylogenetic tree was constructed using the maximum likelihood method for A. tauschii, A. thaliana, G. max, O. sativa, P. trichocarpa, S. alfredii, T. aestivum L., and Z. mays L. The tree is divided into five groups, each represented by a different color. It is divided into 5 distinct groups (A–E) based on the tree topology.
Figure 1. A phylogenetic tree was constructed using the maximum likelihood method for A. tauschii, A. thaliana, G. max, O. sativa, P. trichocarpa, S. alfredii, T. aestivum L., and Z. mays L. The tree is divided into five groups, each represented by a different color. It is divided into 5 distinct groups (A–E) based on the tree topology.
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Figure 2. Chromosomal localization of NRAMP gene family members in A. tauschii.
Figure 2. Chromosomal localization of NRAMP gene family members in A. tauschii.
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Figure 3. Analysis of intraspecific (A) and interspecific (B–D) collinearity within the natural resistance-associated macrophage protein gene family in A. tauschii.
Figure 3. Analysis of intraspecific (A) and interspecific (B–D) collinearity within the natural resistance-associated macrophage protein gene family in A. tauschii.
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Figure 4. Distribution map of cis-acting elements of NRAMP gene family members in A. tauschii (A) and quantity heatmap (B).
Figure 4. Distribution map of cis-acting elements of NRAMP gene family members in A. tauschii (A) and quantity heatmap (B).
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Figure 5. Expression levels of 7 AetNRAMP genes in the leaf (A–G) and root (H–N) of A. tauschii exposed to excess metals for 0 h, 6 h, 12 h, 24 h, 48 h, 96 h.
Figure 5. Expression levels of 7 AetNRAMP genes in the leaf (A–G) and root (H–N) of A. tauschii exposed to excess metals for 0 h, 6 h, 12 h, 24 h, 48 h, 96 h.
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Figure 6. A phenotypic characterization of the metal ion resistance exhibited by AetNRAMP1 and AetNRAMP3 in the yeast model (A) evaluating the concentrations of heavy metals copper (B) and manganese (C) in transgenic yeast (p < 0.05). The two-tailed t-test showed significant differences at the 0.05 significance level, with “**” indicating p < 0.05 and “ns” indicating p > 0.05.
Figure 6. A phenotypic characterization of the metal ion resistance exhibited by AetNRAMP1 and AetNRAMP3 in the yeast model (A) evaluating the concentrations of heavy metals copper (B) and manganese (C) in transgenic yeast (p < 0.05). The two-tailed t-test showed significant differences at the 0.05 significance level, with “**” indicating p < 0.05 and “ns” indicating p > 0.05.
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Table 1. Physicochemical properties of NRAMP protein in A. tauschii a molecular weight; b isoelectric point; c grand average of hydropathicity.
Table 1. Physicochemical properties of NRAMP protein in A. tauschii a molecular weight; b isoelectric point; c grand average of hydropathicity.
Gene NameGene IDNo. of aaMW apI bInstability IndexAliphatic IndexGRAVY cSubcellular Localization
AetNRAMP1AET7Gv2081350051756,063.078.5740.97122.40.614Cell membrane
AetNRAMP2AET4Gv2063220051956,699.386.1735.6115.740.516
AetNRAMP3AET7Gv2113640054859,511.968.0130.26117.990.552
AetNRAMP4AET3Gv2049770063769,455.157.8448.67111.950.509
AetNRAMP5AET4Gv2072500053858,277.66.2832.75117.810.55
AetNRAMP6AET5Gv2020800054759,061.85.233.39113.860.464
AetNRAMP7AET4Gv2016190054659,793.725.2137.44108.830.447
AetNRAMP8AET6Gv2022850027529,315.856.1331.0591.440.195
AetEIN2.1AET5Gv204064001268138,419.916.5347.7592.48−0.03Chloroplast
AetEIN2.2AET4Gv2086010055860,808.146.4546.66119.140.647Cell membrane
AetEIN2.3AET4Gv2085960052757,280.195.9237.79122.090.775
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Li, H.; Li, Y.; Yang, F.; Liang, X.; Ding, Y.; Wang, N.; Han, X. Unraveling the NRAMP Gene Family: Aegilops tauschii’s Prominent Barrier Against Metal Stress. Agronomy 2025, 15, 1919. https://doi.org/10.3390/agronomy15081919

AMA Style

Li H, Li Y, Yang F, Liang X, Ding Y, Wang N, Han X. Unraveling the NRAMP Gene Family: Aegilops tauschii’s Prominent Barrier Against Metal Stress. Agronomy. 2025; 15(8):1919. https://doi.org/10.3390/agronomy15081919

Chicago/Turabian Style

Li, Hongying, Yibo Li, Fuqiang Yang, Xiaolin Liang, Yifan Ding, Ning Wang, and Xiaojiao Han. 2025. "Unraveling the NRAMP Gene Family: Aegilops tauschii’s Prominent Barrier Against Metal Stress" Agronomy 15, no. 8: 1919. https://doi.org/10.3390/agronomy15081919

APA Style

Li, H., Li, Y., Yang, F., Liang, X., Ding, Y., Wang, N., & Han, X. (2025). Unraveling the NRAMP Gene Family: Aegilops tauschii’s Prominent Barrier Against Metal Stress. Agronomy, 15(8), 1919. https://doi.org/10.3390/agronomy15081919

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