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Article

Natural Resistance-Associated Macrophage Protein (Nramp) Family in Foxtail Millet (Setaria italica): Characterization, Expression Analysis and Relationship with Metal Content under Cd Stress

by
Yang Yang
1,†,
Jie Zheng
2,†,
Yinpei Liang
1,
Xinyue Wang
1,
Kangping Li
1,
Liang Chen
2,
Amo Aduragbemi
3,
Yuanhuai Han
1,
Zhaoxia Sun
1,
Hongying Li
1 and
Siyu Hou
1,*
1
College of Agriculture, Houji Laboratory of Shanxi Province, Shanxi Agricultural University, Taiyuan 030031, China
2
State Key Laboratory of Crop Stress Biology for Arid Areas, College of Agronomy, Northwest A&F University, Yangling 712100, China
3
Institute of Plant Breeding, Genetics and Genomics, University of Georgia, Athens, GA 30602, USA
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(8), 2000; https://doi.org/10.3390/agronomy13082000
Submission received: 22 June 2023 / Revised: 20 July 2023 / Accepted: 24 July 2023 / Published: 28 July 2023
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Abstract

:
The excessive content of heavy metals and the deficiency of beneficial trace elements in cereals have threatened global food security and human health. As important metal transporters, Natural resistance-associated macrophage proteins (Nramps) are involved in the absorption and transport of various metal ions in plants, including beneficial elements and hazardous heavy metals, yet little is known about their roles in foxtail millet. In this study, 12 Nramps were identified in foxtail millet genome and divided into three clades. Expansion and functional differentiation of SiNramp gene family is evident in the high proportion of gene duplication as well as the diversity in protein structure and expression characteristics. The SiNramp genes exhibited different response patterns to Cd stress in different tissues. Based on the integration of ionome, RNA-seq and orthologous analysis, the association of SiNramp genes with the accumulation of different metal ions was investigated, and the possible functions of several SiNramp genes were predicted, such as SiNramp6 and SiNramp12. In general, this study provides a comprehensive theoretical framework for the study of Nramp genes in foxtail millet and other minor gramineous crops, which will lay a foundation for further research on the mechanism of metal transport and accumulation.

1. Introduction

Recently, the rapid increase in population, accelerated urbanization, and intensified human industrial and agricultural activities, including mining, metal smelting, excessive use of pesticides and fertilizers, and other anthropogenic activities, have released significant amounts of heavy-metal pollutants into the environment, resulting in heavy-metal pollution having become a serious environmental and public health problem worldwide [1]. Heavy metals in soil not only directly affect plant growth and development by influencing mineral element absorption, photosynthesis, and ROS systems [2], but also can be easily ingested by humans through the food chain, thus posing a serious threat to human health [3]. On the other hand, more than half of the global population suffers from severe deficiencies in essential trace elements such as Fe and Zn, and plants require multiple essential mineral and beneficial elements to maintain growth and development and resist biotic and abiotic stresses [4,5]. Due to the geochemical behavior similarities of heavy metal elements such as Cd, Pb, and Cr with essential elements like Fe, Zn, and Mn, and the poor specificity of plant metal transport proteins, there exists a certain degree of synergy/antagonism between these heavy-metal and trace-metal elements in their absorption and transfer in plants [6,7]. Therefore, identifying and characterizing important metal transport proteins in plants and their relationships with metal ion content (ionome) are crucial for understanding the molecular mechanisms of metal element absorption and transfer in plants, thereby improving food quality, and preserving human health.
Natural resistance-associated macrophage proteins (Nramps) are integral membrane transporters in plants responsible for the uptake, translocation, and detoxification of transition metals [8]. They have been confirmed to play critical roles in the absorption and transport of metal ions such as cadmium (Cd), iron (Fe), zinc (Zn) and manganese (Mn) [9,10,11,12,13,14]. The Nramp gene family in Arabidopsis has been investigated extensively, much like other major gene families. AtNramp1, located on the plasma membrane, acts as a high-affinity Mn transporter and plays an important role in Fe and Cd transport through collaboration with other proteins such as IRT1 [15,16]. AtNramp2, located on the trans-Golgi network (TGN), is involved in the transport of Mn from the TGN to the cytoplasm [17,18]. AtNramp3 and AtNramp4, both acting as vacuolar metal transporters, are responsible for maintaining the balance of Fe and Mn between the vacuole and cytoplasm under specific conditions and are involved in plant photosynthesis under Mn-deficient conditions [19,20]. AtNramp6 participates in the migration and distribution of Cd within cells, leading to cadmium toxicity [15]. In rice, different Nramp proteins specifically transport different metal ions. For instance, OsNramp1 transports Fe, As, Mn, and Cd, while OsNramp3 transports Mn to young leaves in low-manganese conditions [10,21,22]. Similar to AtNramp3 and AtNramp4, OsNramp2 also plays a role in the transportation of iron inside and outside the vacuole [23]. OsNramp4 has been shown to specifically transport aluminum (Al) but cannot transport divalent cations in yeast [24]. Additionally, OsNramp5 and OsNramp6 are responsible for transporting Fe, Mn, and Cd, as well as Fe and Mn, respectively [25,26]. In the heavy metal hyperaccumulator Sedum Alfredii, the expression of the SaNramp6 gene is strongly induced by Cd stress, and the overexpression of SaNramp6 significantly increases Cd accumulation in Arabidopsis [27]. In summary, different members of the Nramp gene family mediate the transport and distribution of different metal ions by encoding different Nramp proteins [28]. Therefore, investigating the effects of different Nramp proteins on metal ion transport and accumulation in plants is crucial for revealing the molecular mechanisms underlying plant metal ion absorption and transport.
Foxtail millet (Setaria italica), which originated from China, is considered one of the earliest domesticated cereals in human history, and was domesticated from Setaria viridis about 10,000 years ago [29]. Historically, foxtail millet has long been cultivated as a major food crop in arid and semi-arid regions, particularly in Asia and Africa, including China, India, and Nigeria. As a vital component of global food security and diversity of crops and food, the United Nations Food and Agriculture Organization (FAO) has designated 2023 as the International Year of Millets to address global food crises. Millet, the hulled product of foxtail millet, is rich in various nutrients such as essential amino acids, vitamins, and minerals, and has important dietary benefits in boosting immunity, promoting digestion, and preventing diseases [30]. Despite having a smaller cultivation area compared to major crops like rice and wheat, foxtail millet faces significant challenges in terms of food safety and quality due to the increasing global heavy-metal pollution. To minimize these adverse effects and improve the beneficial elements of millet while reducing the accumulation of toxic heavy metals, a better understanding of the interaction mechanisms of metal element absorption and transport in foxtail millet is crucial for breeding improvement and ensuring global food security.
To date, there have been no reports on the metal ion transporters in foxtail millet. Here, based on the latest foxtail millet reference genome, we systematically identified the Nramp gene family in foxtail millet at the whole-genome level and characterized them from chromosome localization, basic physicochemical properties, gene duplication patterns, selection pressure, phylogenetic relationships, gene structure, conserved domains, and three-dimensional structures. Furthermore, we analyzed the expression patterns of these Nramp genes in different tissues at different growth stages of foxtail millet. Finally, we predicted the functions of millet Nramp genes based on ionomics (10 metal ions) and transcriptomics analysis of their responses to different concentrations of Cd treatment and their relationship with metal ion accumulation. This study provides a fundamental theoretical framework for further functional studies of foxtail millet Nramp genes and provides a basis for a deeper understanding of the mechanism of metal ion transport and migration in foxtail millet.

2. Materials and Methods

2.1. Genome-Wide Identification and Characterization of NRAMP Gene Family in Foxtail Millet

The latest reference genome data for foxtail millet (cv. xiaomi, a mutant of cultivar Jingu21 from EMS mutagenesis), including protein sequences, genome sequences, CDS sequences, and gene model annotation files (.gff3), were downloaded from the Multi-omics Database for Setaria italica (MDSi, http://foxtail-millet.biocloud.net/home). The latest reference genome information for seven species including Arabidopsis, soybean, potato, sorghum, maize, rice, and barley were obtained from the Ensemble Plants database (http://plants.ensembl.org/index.html). The identification numbers and names of the Nramp genes in Arabidopsis, soybean, potato, rice, and barley were obtained from published literature [31,32,33]. All these Nramp protein sequences were extracted as the seed sequences for the identification of the Nramp gene family in foxtail millet, sorghum, and maize. Then the seed sequences were used to perform multi-sequence alignments against three local protein databases constructed using the protein sequences in foxtail millet, sorghum, and maize with an e-value less than 1 × 10−5 and a similarity greater than 50% as the threshold. We utilized the HMMER tool (version 3.3.2) to search for the conserved domain (PF01566) of Nramp proteins, which was downloaded from the Interpro database. The BLASTP-identified candidate Nramp proteins were examined, and sequences containing the conserved domain were subsequently validated using the NCBI CDD database [34]. Finally, the Nramp gene family members in foxtail millet, sorghum, and maize were manually curated to remove sequences lacking the conserved domain, incomplete domain sequences, and those originating from the same gene but different transcripts.
The cDNA, protein, CDS sequence lengths and gene structure information of these Nramp genes were obtained from the gene model annotation file (.gff3). The theoretical isoelectric point (pI) and molecular weight (MW) of these Nramp proteins were calculated using the computer pI/MW tool in the ExPASy database (https://web.expasy.org/compute_pi/) [35]. Furthermore, the transmembrane (TM) regions of these Nramps were predicted using the TMHMM Server 2.0 (http://www.cbs.dtu.dk/services/TMHMM/), while their subcellular localization was determined using LocTree 3 (https://www.rostlab.org/services/loctree3/, accessed on 15 March 2023).

2.2. Chromosomal Localization, Duplication Events and Selection Pressures of NRAMP Genes in Foxtail Millet

Based on the gene structure annotation files of the foxtail millet genome, the distribution of foxtail millet Nramp genes on the chromosomes was displayed using the TBtools tool [36]. To investigate the expansion of the Nramp gene family in foxtail millet, collinearity analysis and duplication events of Nramp genes were performed using the Multiple Collonearity Scan toolkit (MCscanX) on foxtail millet and Arabidopsis genomes. Additionally, KaKs_Calculator 2.0 was employed to calculate the Ka/Ks ratio of non-synonymous mutations (Ka) to synonymous mutations (Ks) between these duplicated genes in foxtail millet [37].

2.3. Phylogenetic Analysis, Conserved Motifs, and Multiple Sequence Alignment of the Nramp Gene Family

To understand the evolutionary relationship between the Nramp gene family in foxtail millet and other species, a phylogenetic tree was constructed using Nramp protein sequences from eight species, including dicots of Arabidopsis, soybean and potato and monocots of sorghum, maize, rice, barley and foxtail millet. Multiple sequence alignments were performed using Clustal Omega and a maximum likelihood (ML) tree was constructed using MEGA 6.06 [38]. Furthermore, the conserved motifs of foxtail millet Nramp proteins were predicted using the Motif-based sequence analysis Server (MEME, http://meme-suite.org). For this analysis, motif widths ranging from 6 to 200 were employed, allowing any number of repetitions, and setting a maximum motif number of 10 as the threshold. The conservation of the TM region and conserved motifs in the foxtail millet Nramp sequence was also examined. Multiple sequence alignment of the foxtail millet Nramp protein sequence was conducted using Clustal W, and the results were visualized using Escript 3.0. Subsequently, the TM region and conserved motifs were manually annotated [39].

2.4. Three-Dimensional Structure and Molecular Docking of the Nramp Gene Family

To construct the three-dimensional models of Nramp proteins in foxtail millet, we employed the AlaphFold2 tool (version 2.3.2) (https://github.com/deepmind/alphafold, accessed on 22 April 2023) [40]. Subsequently, to validate the binding of Nramp proteins to metal ions, we created a molecular docking model of SiNramp5 with CdCO3. The 3D model file of the CdCO3 ligand was obtained from the Pubchem database (https://pubchem.ncbi.nlm.nih.gov/, accessed on 24 April 2023), and its format was converted using OpenBabel 3.1.1 software. The processing of the protein receptor and small molecule ligand, such as through hydrogenation, the merging of non-polar hydrogen, and charge calculation, was completed using Autodocktools 4.2.6 software, with the Grid Box set to global docking mode [41]. Following the processing of the protein receptor and small-molecule ligand model files, semi-flexible molecular docking was conducted using Autodock-vina 1.1.2 software. Upon completion of the docking process, the best docking conformation was visualized utilizing Pymol v2.4 software, while the visualization of the 2D docking results and subsequent analysis of molecular interactions were carried out employing LigPlot2 [42].

2.5. Cis-Acting Elements and Spatiotemporal Expression Patterns of Nramps in Foxtail Millet

The upstream 2000 bp region of the start codon of Nramp genes in foxtail millet was used to predict the cis-acting elements in the promoter regions. These sequences were extracted from the genome sequence using TBtools and uploaded to the PlantCare database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 28 April 2023) for online prediction [43]. Afterwards, the different types of cis-acting elements were manually classified and visualized [44].
To explore the spatiotemporal expression patterns of these Nramp genes, we performed a thorough scan of their expression characteristics on the foxtail millet multi-omics website (MDSi). The RNA-seq dataset encompasses the expression profiles of all genes in 29 important tissues throughout the entire growth period of foxtail millet. These tissues include germinated seeds (3 days), two-week-old plants, one-month-old leaves, flag leaves at the boot stage, leaves and panicles at the heading stage, panicles at the pollination stage, flag leaves, leaf sheaths, stems, neck-panicle internodes, roots, and top forth leaves at the filling stage. Additionally, the dataset covers primary and third panicle branches at the differentiation stage, and immature seeds (S1–S5), and spikelets (S2 and S4) at the filling stage. The expression levels of Nramps in foxtail millet were normalized by TPM (transcripts per million) values and visualized by TBtools.

2.6. Plant Material, Cd Treatment, Ionome and Transcriptome

To investigate the response of foxtail millet Nramp genes to Cd stress, we cultivated the foxtail millet variety ‘xiaomi’ in a modified Hoagland’s nutrient solution (0.72 mM K2SO4, 0.1 mM KCl, 2 mM Ca (NO3)2, 0.65 mM MgSO4, 0.25 mM KH2PO4, 1 μM MnSO4, 1 μM ZnSO4, 0.1 μM CuSO4, 5 × 10−3 μM Na2MoO4, 1 μM H3BO3, 0.2 μM FeNaEDTA) in a greenhouse at Shanxi Agricultural University, with a temperature of 22 °C and a 16 h light (12,000 lx)/an 8 h dark photoperiod. The seedlings at the four-leaf stage subjected to Cd stress for one week. The Cd stress was induced using CdCl2 solutions with concentrations of Cd1 (5 μM), Cd2 (10 μM), and Cd3 (30 μM), respectively. Samples of roots, stems, and leaves under different stress concentrations were quickly frozen and sent to PANQMIX Biomedical Tech Co., Ltd. (Suzhou, China) and PERSONAL GENE TECHNOLOGY Co., Ltd. (Nanjing, China) for ionome detection and transcriptome sequencing, with three biological replicates.
The ionome detection included 10 metal ions: Na, Mg, K, Ca, Mn, Fe, Cu, Zn, Mo, Cd. All measurements were conducted using a Thermo Fisher Scientific iCAP TQ ICP-MS/MS instrument, which was equipped with standard nickel cones. The measurements followed the standard protocol provided by PANQMIX Biomedical Tech Co., Ltd. The transcriptome sequencing data was carried out following the standard analysis workflow provided by PERSONAL GENE TECHNOLOGY Co., Ltd. (Nanjing, China). RNA-seq libraries were built and sequenced on a BGISEQ 500 sequencing platform with 150-PE. Trimmomatic, Hisat2, and Stringtie were used to filter raw reads, map the genome, and calculate gene expression, respectively [45,46]. In addition, the Spearman and Pearson correlation coefficients were used to calculate the correlation between the expression of foxtail millet Nramp genes and the metal content by SPSS 19.0 (SPSS, Inc., Chicago, IL, USA).

3. Results

3.1. Identification of Nramp Gene Family in Foxtail Millet

By conducting local BLAST multiple sequence alignment and conserved domain analysis, we identified 12, 11, and 12 high-confidence Nramp genes in foxtail millet, maize, and sorghum, respectively (Table 1 and Table S1). In comparison with dicot species like Arabidopsis (7) and potato (5), the number of Nramp genes in the five monocot species nearly doubled. For instance, foxtail millet (12), maize (11), sorghum (12), rice (11), and barley (11) all exhibited a substantial increase in the number of Nramp genes. Notably, this count even approached the number of Nramps found in tetraploid soybean (13).
All Nramp genes were renamed according to their chromosomal location. Table 1 presents detailed information regarding the gene structure and protein characteristics of foxtail millet Nramp genes (SiNramps). The mRNA lengths of these SiNramps ranged from 1344 bp for SiNramp11 to 5051 bp for SiNramp2, with intron numbers ranging from 3 to 16. The lengths of the encoded proteins range from 447 to 1272 aa. The molecular weights of these proteins range from 49,069.75 to 138,045.10 D with their pI ranging from 5.05 to 8.61. As typical integral membrane transport proteins, all SiNramp proteins contained varying numbers of TM regions, ranging from 7 to 12 (Table 1). For subcellular localization, 6 SiNramp proteins were located on the vacuolar membrane, 5 on the plasma membrane, and 1 on the endoplasmic reticulum membrane. Overall, although there were only 12 Nramp genes in foxtail millet, they exhibited rich diversity in gene structure, protein characteristics, TM regions, and subcellular localization.

3.2. Chromosomal Localization and Gene Duplication of SiNramp Genes

To explore the relationship between the expansion of the foxtail millet Nramp gene family and gene duplication events, we remapped SiNramps onto chromosomes and analyzed the duplication events and selection pressure among family members (Figure 1). The 12 SiNramp genes are unevenly mapped onto 6 chromosomes, with chromosome 9 and chromosome 2 having more genes, 4 and 3, respectively. With the exception of SiNramp7, other SiNramps were found to be distributed at the ends of the chromosomes. This pattern of distribution aligns with the overall gene distribution observed throughout the foxtail millet genome (Figure 1). Analysis of gene duplication events revealed that more than half of the 12 SiNramps (58.33%, 7 genes) were found to be associated with duplication events. Among these, five genes were involved in tandem duplications (TDs), forming two distinct tandem duplication groups (TD1 and TD2), while two genes formed a segmental duplication gene pair (SD1) (Figure 1). With the exception of TD1, the other two groups of duplicated gene pairs caused diversification in the TM regions or subcellular localization of SiNramps. For example, within the TD2 group, SiNramp8 and SiNramp9 exhibit 12 and 10 transmembrane regions, respectively. Additionally, SiNramp9 and SiNramp10 display variations in terms of subcellular localization. These duplicated genes are subject to varying degrees of selection pressure. The Ka/Ks values of all duplicated gene pairs were less than 1, indicating that SiNramps were generally subject to purifying selection (Figure 1). Comparatively, the Ka/Ks values of paralogous genes in the TD2 gene pair were relatively high, at 0.32 and 0.31, while the Ka/Ks values of genes in the TD1 and SD1 gene pairs were relatively small, not exceeding 0.2.

3.3. Phylogenetic Relationship, Gene Structure and Conserved Domains of SiNramps

The phylogenetic tree constructed by Nramp proteins from five monocots and three dicots showed that all Nramp proteins were accurately distinguished into three different evolutionary branches (Figure 2). Except for Clade III, which did not include the Nramp of soybean, the other two branches contained Nramp proteins from all species. Clade I and II respectively contained 32 Nramps. Within Clade I, a total of seventeen genes were identified, with three genes (25%) each originating from foxtail millet, maize, and sorghum representing 25%, 27%, and 25% respectively, and four genes (36%) each from rice and barley. The remaining 15 genes belonged to dicots, with four genes from Arabidopsis (57%), three genes from potato (60%), and eight genes from soybean (62%). Clade II consisted of 24 genes from monocots and eight genes from dicots. Among the monocots, there were five genes from foxtail millet (42%), five from maize (45%), five from sorghum (42%), five from rice (45%), four from barley (36%), two from Arabidopsis (29%), one from potato (20%), and five from soybean (38%). In Clade III, except for StNramp4 and AtNramp6 from dicots, the other 15 Nramp genes were from monocots. Overall, the majority of dicot Nramps clustered in Clade I, followed by Clade II, while monocot Nramps had a higher proportion in Clade II and III. Furthermore, based on the collinearity analysis of foxtail millet and Arabidopsis genomes, only SiNramp12 and AtNramp7 were identified as orthologs, which was similar to the clustering distribution of monocot and dicot Nramps in each clade in the phylogenetic tree (Figure 2 and Figure 3).
Based on the gene structure annotation information of foxtail millet and Arabidopsis reference genomes, we compared the differences in gene structure of these Nramp genes (Figure 3). Overall, Nramps from the same evolutionary branch showed similar exon/intron structures. For example, SiNramp5, SiNramp11, SiNramp12 and AtNramp5, AtNramp4, AtNramp2 in Clade I showed similar exon/intron patterns. However, some Nramps also showed a certain degree of diversity in gene structure, such as AtNramp7 and AtNramp2. Furthermore, homologous genes also exhibited differences in gene structure, such as the different number of exons of orthologous genes (AtNramp7 and SiNramp12) and paralogous genes (T D2, SiNramp9 and SiNramp10). Based on the MEME software (version 5.5.3), conserved motifs of all eight species Nramp proteins were predicted, and these conserved motifs were highly consistent with their phylogenetic relationships, with no significant differences between different species (Figure 4). Several motifs, such as Motif 1, Motif 7, Motif 10, Motif 4, Motif 5, and Motif 8, were conserved in almost all Nramp proteins from different species. Some motifs were specifically present in certain evolutionary branches, such as Motif 9 and Motif 6, which were only present in Clade I and II.
To further understand the relationship between conserved motifs and transmembrane functional regions, we annotated the conserved motifs and transmembrane regions on the multiple sequence alignment results (Figure 5). Most of the conserved motifs exhibited a close association with transmembrane regions. Notably, Motif 7, Motif 4, and Motif 6 encompassed the entire TM1, TM3, and TM4 regions, respectively. Additionally, Motif 1 and Motif 3 contained essential segments of TM2 and TM6 within the transmembrane region. In addition, some motifs were localized to intramembrane regions, such as Motif 8 in the intramembrane region between TM8 and TM9. These conserved motifs constituted the basic configuration of Nramp proteins to ensure their stable transport function.

3.4. Three-Dimensional Structures and Molecular Docking of Foxtail Millet Nramp Proteins

The three-dimensional structures of all foxtail millet Nramp proteins were constructed by AlaphFold2 (version 2.3.2) (Figure 6). SiNramp proteins showed complex and conserved three-dimensional structures, including α-helices, β-turns, and other secondary structures. Proteins with higher similarity had more similar three-dimensional structures, such as SiNramp 9 and SiNramp 10. SiNramps with significant differences also exhibit certain differences in the three-dimensional structure of proteins. For instance, SiNramp7 lacks Motif 8, unlike SiNramp6, which leads to an incomplete α-helix in the three-dimensional structure of SiNramp7 (Figure 5 and Figure 6). In addition, to understand the binding mode of SiNramps to metal on molecular dynamics, we performed a semi-flexible molecular docking of SiNramp5 with the CdCO3 ligand (Figure 7). The results indicated that the docking score of the CdCO3 ligand within the active pocket of SiNramp5 was −3.7 kcal/mol. It is worth noting that a lower docking score indicates a more stable binding of the ligand to the protein. The theoretical binding mode showed that CdCO3 formed hydrogen bonds with Ser343, Glu345, Ala346, Gly347, Gly342, and Ala119, with bond lengths of 2.95, 2.88, 3.04, 2.95, 2.80, and 3.07, respectively, to maintain the stable complex formed between SiNramp5 and the Cd ligand.

3.5. The Cis-Regulatory Element and Spatiotemporal Expression Patterns of SiNramps

The cis-acting elements in promoter regions of 12 SiNramps were predicted and analyzed in the PlantCare database. Six categories of cis-acting elements were identified, including those involved in light response, hormone response, stress response, tissue-specificity, growth and development and MYB transcription factor binding (Figure 8). A large number of light response and Phytochrome related elements may be related to their participation in photosynthesis through the transport of metal ions. The presence of several plant hormone-related elements in the promoter region, such as gibberellin, auxin, MeJA, abscisic acid, and salicylic acid-related elements, indicates that SiNramps may be influenced by various growth regulators. Furthermore, stress-related elements associated with defense mechanisms, stress response, low-temperature response, and wound response, among others, likely play a critical role in the response of SiNramps to various stresses. Additionally, elements related to tissue-specific expression and growth development partially explained their involvement in various processes of plant growth and development.
SiNramps showed clear and abundant expression patterns in different tissues throughout the whole growth period (Figure 9). Overall, SiNramp1 and SiNramp11 showed relatively low expression levels, with TPM values of less than 1 in each tissue at all times. During the growth and development of foxtail millet, SiNramp12 and SiNramp10 were highly expressed specifically in germinating seeds and 2-week-old seedlings, respectively. SiNramp11 was specifically expressed in panicle branch during the critical period of panicle differentiation at anthesis. SiNramp7, SiNramp2 and SiNramp10 were highly expressed in the root, while SiNramp3 and SiNramp6 were highly expressed in the flag leaf and stem, respectively. Apart from their high expression levels in nutrient organs or tissues, SiNramp9 and SiNramp4, along with SiNramp8, exhibited specific high expression in the spikelet during the early and post-filling stages, respectively. Interestingly, two genes of the same duplicated gene pair exhibited distinct tissue expression patterns, such as SiNramp3 and SiNramp4 in SD1 and SiNramp5 and SiNramp11 in TD1. Overall, SiNramps showed abundant and clear tissue expression patterns at different stages without redundancy.

3.6. Response to Cd Stress and Correlation with Metal Ion Contents of SiNramps

Three different concentrations of CdCl2 were applied to foxtail millet seedlings for one week to explore the response of SiNramps to Cd stress. Different response patterns were observed for SiNramps in different tissues under different Cd concentrations (Figure 10). SiNramp5 and SiNramp8 were up-regulated in roots under all Cd concentrations, while SiNramp1, SiNramp7, and SiNramp12 were up-regulated in roots under high concentrations of Cd stress. The expression of SiNramp3 was inhibited under medium and low concentrations, but induced by high concentrations of Cd. In addition to being more sensitive to cadmium stress in roots, several SiNramp genes showed different expression changes in stems and leaves. The expression of SiNramp2 in stems and leaves was induced by Cd at all concentrations. SiNramp6 was up-regulated expressed in stems under medium concentration of Cd stress and SiNramp10 was up-regulated expressed under high and low Cd stress, respectively. Apart from these SiNramps, the expression levels of other SiNramps in stems were almost unaffected by Cd stress.
Based on iCAP (Inductively coupled plasma atomic emission spectrometry) technology, the contents of 10 metal ions in root, stem and leaf samples under different Cd concentrations were determined, and spearman and pearson correlation analysis was performed on metal contents and the expression level of SiNramps (Figure 11). The accumulation of all 10 metal ions in foxtail millet showed some correlation. Except for the extremely significant negative correlation between Cu and Ca, Na, Mg, Mn, Zn from 0.51 to 0.89, the content of other metal ions in foxtail millet showed extremely significant positive correlation. Among these metals, namely Na, Mg, Mn, and Zn, strong positive correlations were observed, with correlation coefficients ranging from 0.73 to 0.94. Notably, there was a highly significant positive correlation of 0.94 between Ca and Zn. Iron (Fe) displayed highly significant positive correlations with several elements. Specifically, Fe exhibited positive correlations with Ca, Cu, Zn, and Mo ranging from 0.42 to 0.67. Furthermore, there was a highly significant positive correlation of 0.81 between Fe and Cd. These complex correlations indicated that the accumulation of metals in foxtail millet showed a certain synergism and antagonism.
The expression correlations of SiNramps, SiNramp3, SiNramp5, SiNramp7, SiNramp8, and SiNramp12 showed highly significant positive correlations ranging from 0.72 to 0.97, while SiNramp9 showed highly significant negative correlations from 0.60 to 0.73 with these genes. Interestingly, there was almost no correlation between SiNramp6 and SiNramp10 with other SiNramps, confirming that the functions of SiNramps not only ensured certain specificity, but also had synergistic and competitive relationships. With the exception of SiNramp4, all other SiNramps exhibited noticeable correlations with metal content, and the relationship between gene expression and metal contents was quite apparent. SiNramp1, SiNramp2, SiNramp5, SiNramp7, SiNramp8, and SiNramp12 displayed highly significant positive correlations (ranging from 0.42 to 0.91) with Cd, Fe, and Cu, while exhibiting highly significant negative correlations (ranging from −0.45 to −0.84) with Na, Mg, Mn, and Zn. In contrast, SiNramp6 and SiNramp10 showed highly significant positive correlations (0.44–0.83) with Ca, Na, Mg, Mn, and Zn, while demonstrating relatively weak correlations with other metals. This observation aligns with their distinct expression patterns, which appear relatively independent compared to other SiNramps. Overall, foxtail millet exhibited some synergy and competition in the transport and enrichment of different metals, and different SiNramps exhibited specific transport for different metals.

4. Discussion

The excessive accumulation of heavy metals in cereals and the “hidden hunger” caused by the deficiency of trace metal elements have seriously affected global food security and human health [3,47]. Numerous studies have confirmed the interaction between heavy metals (such as Cd) and trace metals (Fe, Mn and Zn) in absorption and migration in plants, and these metal transporters including Nramp, CAX (Ca2+/H+ exchanger antiporter), HMA (heavy metal transporting ATPase), etc. usually do not specifically distinguish between heavy and beneficial metals and transport them separately [1,6,7]. Therefore, accurately identifying important metal transporters and exploring their inherent relationship with metal absorption and migration in plants is crucial for understanding the physiological functions and accumulation patterns of these metal elements in plants. As an important integral membrane metal transporter family in plants, the Nramp gene family has been shown to be involved in the transport of various divalent metal ions such as Fe, Mn, Zn, Cu, Cd, and Pb in multiple species [48,49]. The Nramp gene family has been identified and characterized in multiple species, such as Arabidopsis [50], soybean [32], peanut [51], potato [31], rice [52], barley [33], etc. However, the detailed information of Nramp gene family in important monocot crops such as foxtail millet, maize, and sorghum are rarely reported. In this study, 12, 11, and 12 high-confidence Nramp genes were identified from the genomes of foxtail millet, maize and sorghum, respectively. The number of Nramps in these three crops is close to that in barley (11), but significantly higher than that in dicots such as Arabidopsis (7) and potato (5) [31,50]. The number of Nramps in these crops is at a comparable level to tetraploid peanut and soybean [32,51]. This indicated that the Nramp gene family has undergone significant expansion in monocots, which was consistent with the phylogenetic tree constructed from 8 species of Nramp proteins (Figure 2).
Although all Nramp proteins were clearly clustered into three evolutionary clades, and most of them contained Nramps from both monocots and dicots (except for Clade III). Nramps in monocots and dicots did not correspond to each other in each clade, but were unequally clustered. Nramps in dicots were mainly clustered in Clade I and Clade II, while those in monocots were mainly clustered in Clade II and Clade III. This suggested that although the Nramp gene family was formed before the differentiation of monocots and dicots, its massive expansion mainly occurred after the differentiation of monocots and dicots. Comparative genomics study has shown that Pooideae and Panicoideae separated from the common ancestor of the Poaceae about 48 million years ago (MYA), and then rice, foxtail millet, and sorghum separated from their respective subfamilies at about 34.1 MYA, 27 MYA, and 13 MYA, respectively [53]. Although the number of Nramps in foxtail millet, sorghum and maize was similar to that in rice and barley, there were more orthologs between foxtail millet, sorghum and maize. These findings suggest that the expansion of the Nramp gene family in Poaceae may have occurred through two distinct processes. The first process likely took place after the differentiation of monocots and dicots but before the separation of Pooideae and Panicoideae. The second process occurred following the separation of Pooideae and Panicoideae (Figure 2). The unequal expansion of Nramp gene families between monocots and dicots and between Pooideae and Panicoideae may further promote the differentiation of different species.
Duplication events that generate new genes serve as the raw materials for functional divergence of plant genes, and subsequent mutations of new genes greatly enhance the adaptability of plants to the environment [44,54,55]. In fact, we identified three duplication events involving seven SiNramps in the foxtail millet Nramp gene family, constituting two tandem duplications and one segmental duplication (Figure 1). Ka/Ks evaluation of these duplicate genes revealed that all duplicate gene pairs were subjected to purifying selection (Ka/Ks < 1), indicating that these genes may tend to maintain important biological functions, similar to other important transporter gene families in foxtail millet [56]. TD2 had a relatively higher Ka/Ks compared to TD1 and SD1, suggesting that gene duplication in TD2 may be more likely to generate new functions. In addition, there were differences in subcellular localization and TM domains between the duplicated gene pairs in SD1 and TD2. SiNramp5 consists of 11 TM regions, whereas its paralog SiNramp11 lacks a TM1 region due to the deletion of Motif 7. Furthermore, SiNramp9 is localized on the vacuolar membrane, while SiNramp10 is localized on the endoplasmic reticulum membrane (Table 1, Figure 5). Although we did not independently analyze the expression patterns of duplicated genes, obvious changes in tissue expression patterns of duplicated gene pairs were also observed. During the grain filling stage, the SiNramp9 of SD2 exhibited specific high expression in the spikelet, while its paralog SiNramp10 displayed specific high expression in the root. For SD1, SiNramp3 was mainly highly expressed in the flag leaf while SiNramp4 was mainly highly expressed in the spike (Figure 9). Overall, similar to other gene families, the high proportion of duplication events and new duplicated genes, as well as the abundant variations observed in gene sequences, protein structures, and expression characteristics, are the main reasons for the expansion and functional divergence of the foxtail millet Nramp gene family.
Clarifying the spatiotemporal expression patterns of genes is an important step in understanding important functional genes, which can provide important evidence for predicting gene function [57]. All SiNramps showed clear and abundant expression patterns in different tissues during the whole growth period in foxtail millet. There are many reports about the function of Nramp gene in rice and Arabidopsis. Considering that the far relationship of AtNramps and SiNramps, the cloned Nramp genes in rice were selected to help us predict the possible functions of SiNramps in foxtail millet. The information of Nramps in foxtail millet and their orthologs in rice on the expression characteristics and the relationship between metals are listed in Table 2. Several orthologous genes in rice and foxtail millet showed similar tissue expression characteristics. SiNramp12 was highly expressed in germinating seeds and correlation analysis showed that its expression was significantly positively correlated with Fe and Cu in foxtail millet (Figure 9 and Figure 11). The rice ortholog OsNramp3 (OsNRAMP2 in previous study, Os03g0208500) has been shown to affect rice plant growth and seed germination by transferring Fe from vacuoles to the cytoplasm [58]. Considering the orthologous relationship between SiNramp12 and OsNramp3, the identical subcellular localization, the highly significant correlation with Fe content and the highly similar expression patterns, we speculated that the function of SiNramp12 in foxtail millet may be similar to that of OsNramp3 in rice. Another typical functional conserved homologous gene pair was SiNramp6 and its ortholog OsNramp7 in rice (OsNRAMP3 in previous study, Os06g0676000). OsNramp7 was specifically expressed in the vascular bundle connection of rice leaves, stems, and spikes, and regulated Mn recycling in different organs of rice by mediating the redistribution of Mn ions [59]. Interestingly, the expression data of foxtail millet whole growth period we selected included stems from different parts, while SiNramp6 was specifically highly expressed in the stems connecting flag leaves and spikes. This confirmed SiNramp6 and OsNramp7 were highly conserved in tissue expression patterns, and the significant positive correlation between their expression levels and Mn content of up to 0.81 suggested that SiNramp6 may mediate Mn recycling in foxtail millet through a similar pathway (Figure 9). In addition to some conserved tissue-specific expression orthologs, we also found conserved constitutively expressed orthologs. Both SiNramp5 and its orthologous gene OsNramp11 (OsNRAMP7 in a previous study, Os12g0581600) showed constitutive expression patterns in roots, stems, leaves, and spikes, and were induced to up-regulate expression under non-biotic stress (Figure 9 and Figure 11) [60], indicating that these orthologs may have relatively conservative functions. Additionally, except SiNramp4, the expression levels of most SiNramp genes were significantly correlated with the contents of various metals. Different SiNramp genes showed different correlation patterns with different metal contents (including positive correlation and negative correlation), and the same SiNramp gene also showed some competition and cooperation with different metal contents (Figure 11, Table 2). For example, the expression of SiNramp7 was significantly positively correlated with Cd, Fe, and Cu content, while negatively correlated with Mn, Mg, etc. As an important metal transporter known for transporting Fe and Mn, it exhibited competitive (Fe-Mn) and cooperative (Fe-Cd-Cu) relationships in the transport process of Cd, Fe, Cu, Mn and Mg. Other SiNramp proteins also exhibited similar competitive patterns, such as the transport of Fe and Zn by SiNramp5 and the transport of Cd, Fe, and Mn by SiNramp12. In general, the correlations between these metal contents and the expression level of SiNramp genes were objective, but the molecular mechanism of cooperative or competitive transport and their evolutionary purpose need to be further studied.
In addition to these SiNramps, which have the same expression pattern as the orthologs in rice, some new SiNramps formed after the differentiation of rice and foxtail millet did not find orthologs in rice. These new duplicated genes show special expression patterns different from their paralogs in foxtail millet. For example, three new duplicated genes, SiNramp8, SiNramp9 and SiNramp11 were specifically highly expressed in spikelet during panicle differentiation or floret development (Figure 9). Considering the huge structural differences between spikes in foxtail millet and rice, the functions of these genes may be unique to the newly duplicated genes in the foxtail millet Nramp gene family. Furthermore, different SiNramps exhibited different response patterns under different concentrations of Cd stress (Figure 10). Similar to the study in potato, the roots of foxtail millet were more sensitive to the Cd stress, and SiNramps were highly responded to Cd stress in roots [31]. Two SiNramps located on the vacuolar membranes, SiNramp 5 and SiNramp 8, were continuously induced by Cd and were up-regulated under all Cd concentrations, which might be related to its more direct involvement in the metal transport across vacuolar membrane to regulate cell osmotic pressure.
Based on ionome technology, the combined analysis of the contents of various metal ions enabled the revelation of their synergistic and competitive absorption in plants [65]. Here, we found the mutual antagonism between Cu and most divalent cations, and the synergistic absorption of Cd and Fe, Ca, Mn, and Zn ions (Figure 11). In contrast to previous studies on Nramp transporters and minority heavy metals in other species, we measured more metals based on ionome technology and found extensive correlations between the gene expression of SiNramps and the contents of multiple heavy metals, especially between divalent cations, which is a huge advantage of ionome technology [7]. The significant positive and negative correlations between the expression levels of these SiNramps and different metal contents were the direct manifestations of different SiNramp transporters’ specificity and competition for metal transport. Fox example, SiNramp10 and SiNramp6 may have higher specificity for Mg, Mn, and Zn, while most other SiNramp proteins may be more prone to binding with Cd, Fe, Cu. Overall, we provided a basic research framework for the absorption and transport of metal ions in foxtail millet by comparing the expression of SiNramps and the contents of 10 metal ions.

5. Conclusions

In this study, 12 Nramp genes were accurately identified in foxtail millet and clearly divided into three clades based on phylogenetic tree constructed by Nramps of eight species. We demonstrated that gene duplication was the main reason for the expansion of Nramp gene family in foxtail millet, and systematically characterized the Nramp gene family of foxtail millet on their chromosomal distribution, gene structure, conserved motifs and protein 3D structure. Then, we constructed the molecular docking of SiNramp5 with a Cd ligand to simulate the binding of Nramps with metal elements. Furthermore, the spatiotemporal expression patterns of SiNramps in various tissues during the whole growth period and response to different concentrations of Cd were evaluated. Finally, based on the correlation of metal content (ionome) and gene expression, and orthologous comparison of foxtail millet and rice, the function of some SiNramp genes was predicted. In general, this study provides a comprehensive theoretical framework for the study of Nramp genes in foxtail millet and other minor gramineous crops, which will lay a foundation for further research on the mechanism of metal transport and accumulation by Nramp and breeding improvement in foxtail millet with low toxicity and high beneficial elements.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13082000/s1, Table S1: The Nramp proteins from 8 species used for construction of phylogenetic tree; Table S2: Contents of 10 metals in different tissues of foxtail millet seedlings under Cd stress.

Author Contributions

Conceptualization, H.L. and S.H.; methodology, H.L. and S.H.; software, Y.Y. and J.Z.; validation, Y.Y., J.Z. and Y.L.; formal analysis, X.W., L.C. and K.L.; investigation, X.W. and K.L.; resources, Y.H., Z.S. and H.L.; data curation, Y.Y. and A.A.; writing—original draft preparation, Y.Y. and J.Z.; writing—review and editing, A.A., L.C. and S.H.; visualization, Y.Y. and Y.L.; supervision, H.L.; project administration, Z.S.; funding acquisition, Y.Y., Z.S. and H.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Fundamental Research Program of Shanxi Province (202103021223157), Local Science and Technology Development Fund Projects Guided by the Central Government, China (YDZJSX2022B007), Grand science and technology special project in Shanxi Province (202101140601027), the National Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding (in preparation) (202204010910001-02), the Scientific and Technological Innovation Programs of Shanxi Agricultural University (2021BQ80), the Scientific Research Project of Shanxi Province Outstanding Doctoral Work Award Fund (SXBYKY2022056).

Data Availability Statement

Not applicable.

Acknowledgments

We would like to thank the editors and reviewers for their constructive scientific review of this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Distribution of Natural resistance-associated macrophage protein (Nramp) genes on chromosomes of foxtail millet. Tandem duplication, segmental duplication, and Ka/Ks values are displayed by purple shadows, red lines and black boxes, respectively.
Figure 1. Distribution of Natural resistance-associated macrophage protein (Nramp) genes on chromosomes of foxtail millet. Tandem duplication, segmental duplication, and Ka/Ks values are displayed by purple shadows, red lines and black boxes, respectively.
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Figure 2. Phylogenetic tree of Natural resistance-associated macrophage proteins in eight plants. Phylogenetic tree is constructed by NRAMP proteins of foxtail millet, sorghum, maize, rice, barley, Arabidopsis, potato and soybean with maximum likelihood (ML) method using MEGA 6.06. Different colored ellipses represent different evolutionary clades and three clades are labeled with I, II and III. NRAMP proteins in monocots, Dicots and foxtail millet are labeled in blue, orange and black, respectively. The Nramp proteins are renamed according to previous studies or physical locations.
Figure 2. Phylogenetic tree of Natural resistance-associated macrophage proteins in eight plants. Phylogenetic tree is constructed by NRAMP proteins of foxtail millet, sorghum, maize, rice, barley, Arabidopsis, potato and soybean with maximum likelihood (ML) method using MEGA 6.06. Different colored ellipses represent different evolutionary clades and three clades are labeled with I, II and III. NRAMP proteins in monocots, Dicots and foxtail millet are labeled in blue, orange and black, respectively. The Nramp proteins are renamed according to previous studies or physical locations.
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Figure 3. Collinearity and Gene Structure of Nramps in foxtail millet and Arabidopsis. The inner circle is composed of chromosomes from foxtail millet (Si) and Arabidopsis (At). The gray lines connect the orthologs of foxtail millet and Arabidopsis, while the black line connects the orthologous Nramp genes of foxtail millet and Arabidopsis. The radial rods outside the circle show the structure of Nramps extracted from the genome annotation information of foxtail millet and Arabidopsis. The green boxes, yellow boxes and black lines represent the exons, UTR regions and introns, respectively.
Figure 3. Collinearity and Gene Structure of Nramps in foxtail millet and Arabidopsis. The inner circle is composed of chromosomes from foxtail millet (Si) and Arabidopsis (At). The gray lines connect the orthologs of foxtail millet and Arabidopsis, while the black line connects the orthologous Nramp genes of foxtail millet and Arabidopsis. The radial rods outside the circle show the structure of Nramps extracted from the genome annotation information of foxtail millet and Arabidopsis. The green boxes, yellow boxes and black lines represent the exons, UTR regions and introns, respectively.
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Figure 4. Conserved motifs of Nramp proteins in eight plants. Different evolutionary branches are labeled with different colored backgrounds and labeled with I, II and III. MEME software (version 5.5.3) is used to predict the conserved motifs of all Nramps. The conserved sequences of different motifs are shown in the figure.
Figure 4. Conserved motifs of Nramp proteins in eight plants. Different evolutionary branches are labeled with different colored backgrounds and labeled with I, II and III. MEME software (version 5.5.3) is used to predict the conserved motifs of all Nramps. The conserved sequences of different motifs are shown in the figure.
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Figure 5. The multiple-sequence alignment of Nramp proteins in foxtail millet. Multiple sequence alignment and visualization are performed by Clustal Omega and Escript 3.0, respectively. The transmembrane (TM) regions and conserved motifs are labeled by line and box, respectively.
Figure 5. The multiple-sequence alignment of Nramp proteins in foxtail millet. Multiple sequence alignment and visualization are performed by Clustal Omega and Escript 3.0, respectively. The transmembrane (TM) regions and conserved motifs are labeled by line and box, respectively.
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Figure 6. The three-dimensional models of Nramp proteins in foxtail millet. All 3D models are constructed and visualized using the AlaphFold2 tool (version 2.3.2) and Pymol v2.4 software, respectively.
Figure 6. The three-dimensional models of Nramp proteins in foxtail millet. All 3D models are constructed and visualized using the AlaphFold2 tool (version 2.3.2) and Pymol v2.4 software, respectively.
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Figure 7. The molecular docking of the SiNramp5 protein and CdCO3 ligand. The dark orange portion of the 3D structure represents the conserved NRAMP domain. The green compound structure represents CdCO3. In the upper right corner is the 2D structure diagram of the molecular docking. Ligand-bound amino acid residues are shown, with dotted lines and numbers representing binding hydrogen bonds and bond energies. The 3D model of SiNramp5 is constructed by the AlaphFold2 tool, the model of CdCO3 is downloaded from the Pubchem database and semi-flexible molecular docking is performed using Autodock-vina 1.1.2 software.
Figure 7. The molecular docking of the SiNramp5 protein and CdCO3 ligand. The dark orange portion of the 3D structure represents the conserved NRAMP domain. The green compound structure represents CdCO3. In the upper right corner is the 2D structure diagram of the molecular docking. Ligand-bound amino acid residues are shown, with dotted lines and numbers representing binding hydrogen bonds and bond energies. The 3D model of SiNramp5 is constructed by the AlaphFold2 tool, the model of CdCO3 is downloaded from the Pubchem database and semi-flexible molecular docking is performed using Autodock-vina 1.1.2 software.
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Figure 8. The cis-acting elements in the promoter regions of 12 SiNramp genes. Different colored ellipses represent different types of cis-acting elements. All elements are predicted online by PlantCare and manually classified. Three clades are labeled with I, II and III, respectively.
Figure 8. The cis-acting elements in the promoter regions of 12 SiNramp genes. Different colored ellipses represent different types of cis-acting elements. All elements are predicted online by PlantCare and manually classified. Three clades are labeled with I, II and III, respectively.
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Figure 9. The spatiotemporal expression patterns of SiNramp genes in multiple tissues during whole growth period in foxtail millet. The expression matrices (TPM values) of 12 SiNramps in 29 important tissues of foxtail millet during the whole growth period are retrieved from foxtail millet multi-omics database (MDSi). These tissues include germinated seeds (3 days), two-week plants, one-month leaf, flag leaf at boot stage, leaf and panicle at heading stage, panicle at pollination stage, flag leaf, leaf sheath, stem, neck-panicle internode, root and top forth leaf at filling stage, primary and third panicle branch at differentiation stage, immature seed (S1–S5) and spikelet (S2 and S4) at filling stage. The visualization is achieved by TBtools with green to red representing the amount of expression from low to high. The orange and green boxes represent tandem duplicated gene pairs and segmental duplicated gene pairs, respectively.
Figure 9. The spatiotemporal expression patterns of SiNramp genes in multiple tissues during whole growth period in foxtail millet. The expression matrices (TPM values) of 12 SiNramps in 29 important tissues of foxtail millet during the whole growth period are retrieved from foxtail millet multi-omics database (MDSi). These tissues include germinated seeds (3 days), two-week plants, one-month leaf, flag leaf at boot stage, leaf and panicle at heading stage, panicle at pollination stage, flag leaf, leaf sheath, stem, neck-panicle internode, root and top forth leaf at filling stage, primary and third panicle branch at differentiation stage, immature seed (S1–S5) and spikelet (S2 and S4) at filling stage. The visualization is achieved by TBtools with green to red representing the amount of expression from low to high. The orange and green boxes represent tandem duplicated gene pairs and segmental duplicated gene pairs, respectively.
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Figure 10. The expression patterns of 12 SiNramp genes in roots, stems, and leaves under different concentrations of Cd stress. The seedlings at four-leaf stage subjected to Cd stress for one week using hoagland nutrient solution with CdCl2 added of Cd1 (5 μM), Cd2 (10 μM), and Cd3 (30 μM), respectively. CK represent the control treatment with hoagland nutrient solution. The Y-axis represents the FPKM value based on RNA-seq. Bars represent the mean values of three replicates ± standard deviation (SD). R, S, and L represent root, stem and leaf, respectively. * and ** represent significance at p < 0.05, 0.01, respectively.
Figure 10. The expression patterns of 12 SiNramp genes in roots, stems, and leaves under different concentrations of Cd stress. The seedlings at four-leaf stage subjected to Cd stress for one week using hoagland nutrient solution with CdCl2 added of Cd1 (5 μM), Cd2 (10 μM), and Cd3 (30 μM), respectively. CK represent the control treatment with hoagland nutrient solution. The Y-axis represents the FPKM value based on RNA-seq. Bars represent the mean values of three replicates ± standard deviation (SD). R, S, and L represent root, stem and leaf, respectively. * and ** represent significance at p < 0.05, 0.01, respectively.
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Figure 11. The correlations between 10 metal contents and the expression of 12 SiNramp genes in foxtail millet. The contents of 10 metal elements in roots, stems, and leaves under Cd stress and normal control are determined by ICAP TQ ICP-MS/MS. Spearman and Pearson correlation coefficients were calculated by SPSS 19.0 and listed above and below the diagonal, respectively. The highly significant correlation values at p < 0.01 are highlighted in bold and by an enlarged size in the heatmap. Orange backgrounds represent positive correlations, while blue backgrounds represent negative correlations.
Figure 11. The correlations between 10 metal contents and the expression of 12 SiNramp genes in foxtail millet. The contents of 10 metal elements in roots, stems, and leaves under Cd stress and normal control are determined by ICAP TQ ICP-MS/MS. Spearman and Pearson correlation coefficients were calculated by SPSS 19.0 and listed above and below the diagonal, respectively. The highly significant correlation values at p < 0.01 are highlighted in bold and by an enlarged size in the heatmap. Orange backgrounds represent positive correlations, while blue backgrounds represent negative correlations.
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Table
Table 1. The detail information and sequence characterization of 12 putative Nramp genes in foxtail millet.
Table 1. The detail information and sequence characterization of 12 putative Nramp genes in foxtail millet.
No.Gene 1Locus 2LocationGene StructureProtein 5TM Region 6Subcellular
Localization		Gene
Duplication 7
Length 3Intron 4SizeMWpI
1SiNramp1Si1g102001:8,834,970–8,841,63130841654359,149.498.6110plasma membrane
2SiNramp2Si2g045302:3,509,238–3,516,309505171272138,045.105.8411vacuole membrane
3SiNramp3Si2g105602:9,563,819–9,568,86220461253258,143.647.1111plasma membraneTD1
4SiNramp4Si2g105802:9,633,094–9,642,73624041251655,801.047.9811plasma membraneTD1
5SiNramp5Si3g361303:46,979,376–46,983,7902468354659,165.995.0511vacuole membraneSD1
6SiNramp6Si4g251904:36,083,068–36,089,26336061257862,443.987.5512plasma membrane
7SiNramp7Si7g076007:15,422,253–15,429,38216621255359,475.038.5111plasma membrane
8SiNramp8Si9g126509:8,165,772–8,168,6701743558063,243.046.4912vacuole membraneTD2
9SiNramp9Si9g126609:8,170,373–8,176,5081404546750,740.206.7310vacuole membraneTD2
10SiNramp10Si9g126709:8,177,896–8,184,096368761228133,808.276.0210endoplasmic reticulum membraneTD2
11SiNramp11Si9g167309:11,844,953–11,848,0691344344749,069.757.057vacuole membraneSD1
12SiNramp12Si9g484209:53,333,216–53,337,9602739352256,814.566.179vacuole membrane
1 Systematic designation given to foxtail millet Nramps in this study. 2 Locus identity number of SiNramps in foxtail millet genome (cv ‘xiaomi’). 3 Gene full length of SiNramps. 4 No. of introns for SiNramps. 5 Protein characterization of SiNramp proteins. 6 Number of transmembrane regions of SiNramp proteins, predicted by the TMHMM Server v2.0. 7 Duplicated genes, TD and SD represent tandem duplication and segmental duplication, respectively. The following numbers represent different pairs of duplicated genes.
Table
Table 2. The expression patterns of orthologous Nramp genes in foxtail millet and rice and association with metals.
Table 2. The expression patterns of orthologous Nramp genes in foxtail millet and rice and association with metals.
Nramps in Foxtail MilletRelated MetalsExpression OrganizationOsNramp IDs in This StudyOsNramp IDs in Previous StudyReported Functions of Nramps
SiNramp1Cd(+), Fe(+), Cu(+), K(−), Ca(−), Na(−), Mg(−), Mn(−), Zn(−)2-week shoot (H), Root (H), Stem (H), SpikeletOsNramp2
(Os02g0131800)		NRAT1; OsNRAMP4Expressed in root, Specific transport of Al [7]
SiNramp2Cd(+), Fe(+), Cu(+), K(−), Mg(−)Germinated seed, 2-week shoot, Root (H), Stem, leaf, SpikeOsNramp8
(Os07g0155600)		OsEIN2Highly expressed in root and coleoptile, Positive component in ethylene signaling [61], Induced expression by Fe and Mn (SpEIN2) [62]
SiNramp3
(TD1)		Fe(+), Cu(+), K(−), Ca(−), Na(−), Mg(−), Zn(−)Leaf sheath (H), Flag leaf (H), Stem, RootOsNramp10
(Os07g0258400)		OsNRAMP1Similar as OsNramp5, Responsible for the absorption of Cd, Mn, Fe, Ni, Pb and other divalent cations in the root and transport to the shoot [63]
SiNramp4
(TD1)		NoneSpike (H), S1 Immature seed (H), SpikeletOsNramp9
(Os07g0257200)		OsNRAMP5Responsible for the absorption of Cd, Mn, Fe and other divalent cations in the root and transport to the shoot [64]
SiNramp5
(SD1)		Cd(+), Fe(+), Cu(+), K(−), Na(−), Mg(−), Zn(−)Flag leaf (H), Stem (H), Root (H), Leaf sheath (H), SpikeOsNramp11
(Os12g0581600)		OsNRAMP7Expressed in root, stem, leaf and spike, and transport Fe and Zn [60]
SiNramp6Ca(+), Na(+), Mg(+), Mn(+), Zn(+), Cu(−)Germinated seed, 2-week shoot, Root, Stem (H), Flag leaf, SpikeOsNramp7
(Os06g0676000)		OsNRAMP3Specifically expressed at the binding point of vascular bundles connecting leaves, stems, and spikes, Specific distribution of Mn in plants [59]
SiNramp7Cd(+), Fe(+), Cu(+), K(−), Ca(−), Na(−), Mg(−), Mn(−)Root (H), Stem, Leaf sheathOsNramp1
(Os01g0503400)		OsNRAMP6Transport Fe and Mn [26]
SiNramp8
(TD2)		Cd(+), Fe(+), Cu(+), K(−), Ca(−), Na(−), Mg(−), Mn(−)S4 spikelet (H), S4 immature seedNANANA
SiNramp9
(TD2)		K(+), Na(+), Mg(+), Cu(−), Fe(−)S4 spikelet (H)OsNramp6
(Os03g0700800)		unknownunknown
SiNramp10
(TD2)		Ca(+), Na(+), Mg(+), Mn(+), Zn(+), Cu(−)Germinated seed, 2-week shoot (H), Root (H), Spike, S4 spikeletNANANA
SiNramp11
(SD1)		Fe(+), K(−)Germinated seed, pancile branch at spike differentation stage (H)NANANA
SiNramp12Cd(+), Fe(+), Cu(+), K(−), Na(−), Mg(−), Mn(−)Germinated seed (H), Leaf, Stem, Root, SpikeOsNramp3
(Os03g0208500)		OsNRAMP2; qCd3-2Transport Fe, Cd, Mn and play a key role in the process of seed germination [58]
‘+’ and ‘−’ in brackets represent the significant positive and negative correlations between SiNramp and a metal, respectively. The bold type represents the similar tissue or metal relationship of SiNramp with its rice homologous gene.
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Yang, Y.; Zheng, J.; Liang, Y.; Wang, X.; Li, K.; Chen, L.; Aduragbemi, A.; Han, Y.; Sun, Z.; Li, H.; et al. Natural Resistance-Associated Macrophage Protein (Nramp) Family in Foxtail Millet (Setaria italica): Characterization, Expression Analysis and Relationship with Metal Content under Cd Stress. Agronomy 2023, 13, 2000. https://doi.org/10.3390/agronomy13082000

AMA Style

Yang Y, Zheng J, Liang Y, Wang X, Li K, Chen L, Aduragbemi A, Han Y, Sun Z, Li H, et al. Natural Resistance-Associated Macrophage Protein (Nramp) Family in Foxtail Millet (Setaria italica): Characterization, Expression Analysis and Relationship with Metal Content under Cd Stress. Agronomy. 2023; 13(8):2000. https://doi.org/10.3390/agronomy13082000

Chicago/Turabian Style

Yang, Yang, Jie Zheng, Yinpei Liang, Xinyue Wang, Kangping Li, Liang Chen, Amo Aduragbemi, Yuanhuai Han, Zhaoxia Sun, Hongying Li, and et al. 2023. "Natural Resistance-Associated Macrophage Protein (Nramp) Family in Foxtail Millet (Setaria italica): Characterization, Expression Analysis and Relationship with Metal Content under Cd Stress" Agronomy 13, no. 8: 2000. https://doi.org/10.3390/agronomy13082000

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