Genome-wide identification of NRAMP gene family in Brassica U’s triangle species and its response to Cadmium stress in B. napus

Abstract

Background

Soil cadmium contamination is a persistent environmental and agricultural problem worldwide, largely because it stays in the soil and can easily enter the food chain. In plants, the Natural Resistance-Associated Macrophage Protein (NRAMP) gene family plays key role in cadmium uptake, transport, and homeostasis. However, systematic investigation of NRAMP genes across all six Brassica U’s triangle species has been limited, even though these plants produce high biomass, have well-established genetic resources, and are considered promising candidates for phytoremediation.

Results

This study presents a genome-wide analysis of NRAMP genes across the six Brassica U’s triangle species, identifying 81 members in total (87 when including six Arabidopsis thaliana NRAMPs as an outgroup). Phylogenetic classification devided these genes into two distinct subfamilies: Subfamily I (NRAMP1/6) and Subfamily II (NRAMP2-5), which differ clearly in gene structure and conserved protein motifs. An evolutionary analysis revealed that the NRAMP family expanded mainly through whole-genome duplications followed by segmental duplications, with some gene loss later during diploidization. Nearly all duplicated and orthologous gene pairs had Ka/Ks ratios below 1, indicating functional conservation of these metal transporters. Expression profiling in Brassica napus under cadmium stress demonstrated clear tissue-specific functional specialization. Correlation analyses identified key genotype-phenotype relationships: BnNRAMP2c and BnNRAMP2d showed a strong positive correlation with root cadmium content, indicating potential roles in metal uptake or sequestration; BnNRAMP1c/1e/1f expression correlated with leaf cadmium levels, suggesting their involvement in shoot translocation; whereas BnNRAMP6a correlated negatively with root cadmium, hinting a possible exclusion mechanism.

Conclusions

This study provides basic understanding of the evolutionary history and functional diversification of NRAMP transporters in Brassica species. We reveal a division of labor among specific members. BnNRAMP2d was identified as a candidate gene that may play an important role in root cadmium accumulation. The BnNRAMP1s cluster may be involved in shoot distribution, and BnNRAMP6a may contribute to cadmium exclusion. The identified genes serve as potential targets for developing rapeseed cultivars with reduced cadmium content to enhance food safety, or alternatively, for optimizing cadmium-efficient phytoremediation to alleviate environmental contamination.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-09104-6.

Introduction

Cadmium contamination of soil is a serious environmental threat worldwide. As a highly toxic, mobile, and persistent heavy metal, cadmium harms ecosystem health by disrupting soil microbial communities and reducing fertility. Furthermore, it bioaccumulates in the food chain, posing serious risks to human health, including kidney damage, osteoporosis, and cancer [1, 2]. Phytoremediation, which uses plants to extract metals from soil, has emerged as a promising and sustainable way tackle this problem [3, 4]. However, this approach often depends on hyperaccumulator plants like Noccaea caerulescens and Sedum alfredii, which tend to grow slowly and produce little biomass. These limitations have pushed researchers to look for plant materials that combine high metal uptake with strong growth, and also to better understand the molecular mechanisms behind these traits [5–9].

The Brassicaceae family includes many metal-tolerant species and is therefore highly promising for phytoremediation [10]. Within this family, Brassica crops are particularly attractive. They combine high biomass, rapid growth, and established farming practices with a well-defined genetic structure known as “U’s triangle”. As described by Dr. Nagaharu U, this identifies three diploid species, B. oleracea, B. rapa, and B. nigra, as the progenitors of three allotetraploid species (B. napus, B. juncea, and B. carinata) [11]. The recent release of a high-quality genome for B. carinata has now provided a complete genetic resource for analyzing their evolutionary relationships [12, 13]. This offers an unmatched comparative system to investigate the genetic basis of heavy metal tolerance and accumulation, making it ideal for studying gene families central to these processes.

Significant genotypic variation in cadmium response exists among the species of U’s triangle, and this variation is likely driven by differences in key metal transporter families [14]. Among these proteins, the NRAMP family is critical for cadmium uptake, translocation and accumulation in plants [15]. NRAMP genes encode conserved transporters that maintain mineral balance and mediate heavy metal stress responses [16]. Structurally, these proteins usually contain 10–12 transmembrane domains (TMDs) that form a passage for ions, with a conserved motif between TMD8 and TMD9 that is essential for substrate binding [17–20]. Acting as H⁺-symporters, NRAMPs use the energy from the cell’ proton gradient to transport a broad range of divalent metal ions [21, 22]. This broad substrate specificity allows them to handle both essential nutrients (e.g., Fe²⁺, Mn²⁺, Zn²⁺, Ni²⁺) and toxic metals such as Al³⁺ and Cd²⁺, placing them at the center of plant nutrition and detoxification [23–28]. For example, in rice (Oryza sativa L.), OsNRAMP5 is a major route for manganese and cadmium uptake in roots, and disrupting its function significantly decreases the grain cadmium content [29, 30]. In Arabidopsis, AtNRAMP3 and AtNRAMP4 remobilize iron from vacuoles, which is vital for seed germination [31, 32]. Similar roles for NRAMP homologs have been confirmed in other major crop species, such as maize, wheat, and soybean [33–37].

In Brassica species, the NRAMP gene family in the AC genomes has been analyzed. However, that study primarily focused on three species (B. napus, B. rapa, and B. oleracea) representing the A and C genomes [38]. With the recent availability of high-quality genome assemblies for all six U’s triangle species, including the newly released genome of B. carinata, a more complete genome-wide analysis across the entire Brassica U’s triangle is now feasible [13]. To fill this knowledge gap, we performed a genome-wide analysis of the NRAMP gene family across the six species of Brassica U’s triangle to provide a more complete evolutionary and functional understanding.

In this study, we aimed to: (1) systematically identify and characterize the NRAMP gene family across all six Brassica U’s triangle species; (2) elucidate the evolutionary history of this family, including duplication events and selection pressures; and (3) investigate the expression patterns of BnNRAMP genes under cadmium stress and clarify their relationship with Cd accumulation. We identified and phylogenetically classified 81 NRAMP members from these six Brassica species, together with six Arabidopsis thaliana NRAMPs for comparison. Gene structures, conserved motifs, and evolutionary relationships of these gene were systematically characterized. Furthermore, taking advantage of the agronomic importance of Brassica napus, expression profiles of its NRAMP genes under cadmium stress in genotypes with different cadmium accumulation patterns were further analyzed. This work provides a key genomic resource and a theoretical basis for understanding the role of NRAMP transporters in cadmium tolerance and accumulation in rapeseed, with implications for the developing improved phytoremediation strategies and low-cadmium crop species.

Results

Genome-wide identification of the NRAMP family in Brassica U’s triangle species

Using six Arabidopsis thaliana NRAMP sequences as a reference, we scanned the genomes of the six Brassica U’s triangle species and identified 91 candidate proteins carrying the NRAMP domain. Subsequent phylogenetic and homology assessments revealed that ten of these candidates were more closely related to the ETHYLENE INSENSITIVE 2 (EIN2) protein. Although EIN2 shares some sequence similarity with NRAMP transporters, it has no metal transport activity, so we excluded it from the final set. The remaining 81 were taken as Brassica NRAMP members, distributed as follows: 11 in B. rapa, 9 in B. nigra, 10 in B. oleracea, 16 in B. juncea, 20 in B. napus, and 15 in B. carinata.

The basic characteristics of the 81 identified Brassica NRAMP proteins are summarized in Table S2. The analysis revealed substantial diversity in their physicochemical properties. Protein length ranged from 372 amino acids (BcaNRAMP6b) to 1,306 amino acids (BnaNRAMP3d), which correspond to molecular weights of 40.31 kDa and 142.37 kDa, respectively. The theoretical isoelectric point (pI) ranged from acidic (4.84 for BcaNRAMP3b) to basic (9.33 for BcaNRAMP6b). Based on the instability index, 73 proteins were classified as stable and 14 as unstable. The grand average of hydropathicity (GRAVY) values, which ranged from − 0.003 (BraNRAMP3a) to 0.755 (BolNRAMP1c), indicated that nearly all proteins are hydrophobic, a characteristic consistent with integral membrane transporters. This finding was confirmed by transmembrane domain prediction, which revealed that the Brassica NRAMP proteins contained 9 to 13 transmembrane helices, with the majority (80 proteins) possessing the typical 12 domains. These structural features strongly support their role as membrane-embedded transporters. Subcellular localization prediction using WoLF PSORT and Cell-PLoc 2.0 placed all 81 Brassica NRAMP proteins at the plasma membrane (Supplementary Table S2). However, it should be noted that this conclusion is based solely on computational prediction; experimental validation (e.g., via GFP fusion) is still needed.

Phylogenetic and evolutionary relationships of NRAMP proteins

To investigate the evolutionary history of the NRAMP family within the context of the Brassica U’s triangle, a phylogenetic tree from the protein sequences of the six Brassica species (81 sequences) together with A. thaliana (6 sequences) was constructed. The tree clearly segregated the 87 NRAMP proteins into two well-supported clades, which named as Subfamily I and Subfamily II (Fig. 1). Subfamily I contains NRAMP1 and NRAMP6 orthologs, whereas Subfamily II includes NRAMP2, NRAMP3, NRAMP4, and NRAMP5 orthologs. This classification confirms that all members within each subfamily share a close evolutionary origin. Based on their clustered with Arabidopsis orthologs, all Brassica NRAMP proteins were systematically renamed (Table S2).

Fig. 1.

Phylogenetic tree of NRAMP proteins from Arabidopsis and Brassica U’s triangle species

The NRAMP family is divided into two subfamilies, represented by green and blue outer rings, respectively. Species included in the phylogenetic tree are Brassica rapa (Bra, green circle), Brassica nigra (Bni, blue circle), Brassica oleracea (Bol, purple circle), Brassica juncea (Bju, blue star), Brassica napus (Bna, purple star), Brassica carinata (Bca, green star) and Arabidopsis thaliana (At, red triangle).

Conserved protein motifs and gene structure of NRAMP members

The conservation and divergence of the NRAMP family were further investigated through protein motif and gene structure analyses. Analysis of the ten most conserved motifs showed that Brassica NRAMP proteins contain between six and nine motifs (Fig. 2A, D). Except for seven proteins (BjuNRAMP2a, BniNRAMP5a, BcaNRAMP6b, BraNRAMP6a, BraNRAMP6b, BolNRAMP1b, and BcaNRAMP1d) had lost some motifs, all members retained motifs 1, 3, and 7, indicating strong evolutionary pressure on core functional elements. A clear structural distinction was observed between the two phylogenetic subfamilies. Subfamily I (NRAMP1/6) consistently had eight motifs, while subfamily II (NRAMP2/3/4/5) had nine. Notably, Subfamily I universally lacked motif 8, and the motifs lying between motifs 3 and 1 was different: motif 5 in Subfamily I versus motif 9 in Subfamily II. Furthermore, the entire motif arrangement of Subfamily I was shifted about 20–30 amino acids upstream compared with Subfamily II. These conserved, subfamily-specific motif patterns strongly suggest functional divergence during evolution.

Fig. 2.

The conservation and divergence of the NRAMP family. A Conserved motif analysis of NRAMP proteins. B Domain analysis. C Gene structure analysis. D Conserved motifs

Conserved domain analysis confirmed that all sequences contain the NRAMP domain (Fig. 2B). However, three proteins (BnaNRAMP3c, BnaNRAMP3d, and BraNRAMP3a) had extra domains of RING-Ubox and Arm superfamilies, and BraNRAMP6b carried a DFU212 domain, suggesting potential functional specialization. Examination of the exon-intron structure revealed that the number of exons across Brassica NRAMP genes varied from 3 to 17 (Fig. 2C). Genes in the same phylogenetic clade generally share similar structural patterns. A striking difference emerged between subfamilies: Subfamily I had an average 11.3 exons, much higher than the average of 4.0 exons in Subfamily II.

Cis-acting element analysis of NRAMP gene promoters

To investigate the potential regulatory mechanisms of NRAMP genes, we analyzed cis-acting elements in their 2,000 bp regions upstream of each gene. A total of 74 regulatory elements were identified across the Brassica U’s triangle species (excluding core promoter elements likeTATA-boxes and elements with unknown functions; Fig. 3, Supplementary Table S3). The number of elements per promoter varied substantially, with BnaNRAMP4b had the highest count of 69 and BjuNRAMP3c had the least of 22. Unlike the conserved gene structures, the abundance and variety of cis-elements diverged considerably, even among closely related genes on the same phylogenetic branch.

Fig. 3.

The cis-acting elements of NRAMP genes. The promoter regions in the 2000 bp upstream promoter in all selected members. A Total cis-element counts by functional category. B Distribution of individual regulatory elements. Dot sizes in the heatmap indicate element copy numbers. Color coding: Hormone Response (red), Stress Response (blue), Light and Circadian (green), Plant Development (purple), Others (orange)

Interestingly, all promoters contained elements linked to the hormone response, stress response, light response and circadian rhythm, suggesting that NRAMP expression is likely regulated by a wide range of internal and external signals (Fig. 3A). Hormone-responsive elements, such as binding sites for abscisic acid (ABRE), gibberellin (GARE-motif, P-box), methyl jasmonate (CGTCA-motif, TGACG-motif), salicylic acid (TCA-element), and auxin (AuxRR-core, TGA-element), were particularly abundant. Stress-responsive elements, including AREs (antioxidant response), LTRs (low-temperature response), and WUN motifs (wounding response), were also enriched. Furthermore, light-responsive elements such as Box4, the G-box, and the GATA motif were ubiquitous as well. On the other hand, 23 promoters completely lacked elements related to specific developmental processes (Fig. 3B, Table S2). This absence suggests that the NRAMP gene family may be primarily geared toward general stress and environmental responses rather than directly controlling developmental programs.

Chromosomal distribution and collinearity analysis of NRAMP genes

NRAMP genes were found on most chromosomes of the Brassica U’s triangle species, but their distribution was uneven (Fig. 4). In the diploid progenitors, the genes were located on several chromosomes: ten BraNRAMPs across chromosomes A02, A03, A06, A07, A09, and A10 (with 2 on unanchored scaffolds); nine BniNRAMPs on chromosomes B02, B04, B05, B06, and B07; and ten BolNRAMPs on chromosomes C02, C03, C05, C06, C07, and C08 (Fig. 4A-C). This pattern carried over to the allotetraploids and expanded. B. juncea harboured 16 BjuNRAMPs derived from its A (A01, A02, A07, A08, A09, A10) and B (B02, B05, B06, B08) subgenomes; B. napus had 20 BnaNRAMPs from its A (A02, A03, A06, A07, A08, A09, A10) and C (C02, C03, C05, C06, C07, C08) subgenomes; and B. carinata contained 14 BcaNRAMPs from its B (B02, B04, B05, B07) and C (C01, C03, C05, C07, C08) subgenomes, with one gene on a contig (Fig. 4, D-F). Most chromosomes carried only a single NRAMP gene. However, notable exceptions were chromosome B06 of B. nigra and chromosome B02 of B. juncea, each containing four genes (Fig. 4, B, D). To assess whether NRAMP genes are non-randomly clustered on specific chromosomes, we performed Poisson goodness-of-fit tests and permutation tests (10,000 shuffles, accounting for chromosome length). For all six Brassica species, the observed distributions did not deviate significantly from randomness after multiple testing correction (all adjusted permutation p > 0.05; Supplementary Table S4). The scattered arrangement of NRAMP genes across genomes suggests that no tandem duplication occurred and no gene clusters was formed over evolution.

Fig. 4.

Chromosomal localization of the NRAMPs of Brassica U’s triangle species. A Chromosomal localization in B. rapa. B Chromosomal localization in B. nigra. C Chromosomal localization in B. oleracea. D Chromosomal localization in B. juncea. E Chromosomal localization in B. napus. F Chromosomal localization in B. carinata

Tandem and segmental duplications often drive the expansion of gene families and help create functional diversity in plants. To assess this phenomenon in the Brassica NRAMP family, we performed an intragenomic collinearity analysis. 11 duplicated NRAMP pairs in B. juncea, 38 in B. napus, and 23 in B. carinata were found (Fig. 5A–C). The evolutionary pressure acting on these duplicated pairs was determined by nonsynonymous (Ka) to synonymous (Ks) substitution ratio (Ka/Ks). All analysed pairs had Ka/Ks values below 1, falling between 0 and 0.5. A Kruskal-Wallis test confirmed that there was no significant difference in the distribution of Ka/Ks ratios across the three allotetraploid species (p = 0.13) (Fig. 5G). Together, these results strongly indicate that the NRAMP gene family in Brassica species has undergone strong purifying selection, which maintains conserved functions by removing deleterious mutations.

Fig. 5.

Collinearity relationships of the NRAMPs of Brassica U’s triangle species. A-C Collinearity relationships within Brassica U’s triangle allotetraploid species. The gray lines in the background indicate colinear blocks among Brassica species, while the red lines represent collinear NRAMP gene pairs. D-F Relationships of collinearity among the allotetraploid species and their diploid progenitors. G Inter-species and Intra-species Ka/Ks ratios

In terms of tracing the origin of NRAMP genes in allopolyploids, we performed an inter-genomic collinearity analysis with their diploid progenitors. This revealed extensive homology: 43 gene pairs were identified between B. rapa, B. nigra, and B. juncea; 91 pairs between B. rapa, B. oleracea, and B. napus; and 72 pairs between B. nigra, B. oleracea, and B. carinata (Fig. 5D-F). Our analysis confirmed that none of the NRAMP gene in U’s triangle species arose from tandem duplication. The vast majority originated from whole-genome duplication (WGD) or segmental duplication events. A few genes, namely, BraNRAMP2b, BolNRAMP4a, and BjuNRAMP1b/3c, were identified as dispersed duplicates, likely resulting from transposition events. The evolutionary pressure on these genes was further assessed by calculating Ka/Ks ratios between allopolyploid NRAMPs and their diploid orthologs. For nearly all homologous pairs (except BnaNRAMP2a and BolNRAMP2b), the Ka/Ks values were less than 1 (Fig. 5G). Some collinear pairs presented Ka/Ks values of 0, indicating extreme sequence conservation. These results demonstrate that NRAMP genes have been under strong purifying selection throughout the evolution of Brassica allopolyploids, which has kept them highly conserved.

Tissue-specific expression patterns of NRAMP genes in B. napus under cadmium stress

The bioinformatics analyses above established the evolutionary history, structural conservation, and promoter architecture of NRAMP family across the Brassica U’s triangle species. The presence of many stress- and hormone-related cis-elements in promoter regions suggested that these genes might respond to cadmium stress. To further test this hypothesis and explore potential functional diversification, we chose Brassica napus (rapeseed) for subsequent expression analysis. B. napus is the most widely grown Brassica species worldwide and a major oilseed crop with great agricultural and economic importance.

We analyzed expression patterns of B. napus NRAMP genes in two lines (H42 and H47) that differ in cadmium accumulation capacity, across different tissues and growth stages, under Cd stress (30 mg/kg CdCl₂) and control (0 mg/kg CdCl₂) conditions. The two lines showed distinct Cd distribution phenotypes. In the high-accumulating line H42, Cd was mainly concentrated in roots at the seedling stage. At bolting and flowering, Cd was redistributed to both roots and leaves, with very little in siliques. In contrast, in the low-accumulating line H47, Cd stayed largely in the roots throughout all stages, with limited movement to shoots and the lowest amounts found in siliques (Fig. 6A, B; Supplementary Table S6).

Fig. 6.

BnaNRAMPs expression in and their correlation with Cd content. A Cd accumulation and BnaNRAMP expression in Line H42. B Cd accumulation and BnaNRAMP expression in Line H47. C Correlation analysis between BnaNRAMPs relative expression level and Cd content. Expression values are shown as log₂-transformed fold change relative to the untreated control. For correlation analysis, data from both genotypes and developmental stages were pooled. Tissue abbreviations: R, Root; S, Stem; L, Leaf; F, Flower; G, Grain. Scale bars with color gradient from blue to red represents value changes: Cadmium content: Cd content in different tissues across growth stages; Gene relative expression level: log₂(fold change) relative to the control; red indicates upregulation and blue indicates downregulation; Person correlation coefficients: red indicates positive correlation and blue indicates negative correlation; * indicates p < 0.05, ** indicates p < 0.01

The relative expression profiles of BnNRAMP genes under Cd stress and control condition revealed both evolutionary conservation and clear tissue-specific distinction. Overall patterns were broadly similar between H42 and H47, but key differences appeared. A subset of genes, including BnNRAMP1c, 1d, 1e, 2c, 2d, 4a, and 4b, were consistently highly expressed across all tissues and developmental stages. In contrast, BnNRAMP1a, 1b, 5a, and 5b were generally expressed at low levels. Phylogenetic relatedness was a strong predictor: genes in the same clade tended to have similar expression profiles. Spatially and temporally, most BnNRAMPs expression peaked in roots at the seedling stage, in roots and leaves at the bolting stage, in leaves or flowers at the flowering stage, and in leaves or siliques at the pod development stage. Notably, BnNRAMP1c, 1d, 1e, 2a, 2b, 4a, and 4b maintained particularly high expression in leaves across all time points (Fig. 6A, B; Supplementary Table S6). These spatiotemporal expression patterns suggest a coordinated model of NRAMP function: some members are likely responsible for Cd uptake in roots, whereas others facilitate its translocation to the shoots and subsequent distribution to reproductive tissues, including seeds.

To directly link NRAMP gene expression and cadmium accumulation, we performed a correlation analysis between transcript levels and Cd content across different tissues. For each gene-tissue combination, expression and Cd content data from two genotypes across developmental stages were pooled, with three biological replicates per genotype per stage. Due to the limited number of data points available for flowers and siliques from a single sampling time, the analysis was conducted exclusively on root, stem, and leaf tissues. This revealed distinct tissue-specific correlation patterns for individual NRAMP genes (Fig. 6C; Supplementary Figure S2). In leaves, members of the NRAMP1 subfamily (BnNRAMP1c, 1e, and 1f) were significant positively correlated with Cd level (r = 0.732–0.867, p < 0.05). In roots, NRAMP2 subfamily members (BnNRAMP2c and 2d) showed even stronger positive correlations (r = 0.775–0.911, p < 0.05), with BnNRAMP2d showing the highest correlation overall (r = 0.911, p < 0.01). Significant positive correlations in roots were also observed for BnNRAMP3b (r = 0.75) and BnNRAMP4a (r = 0.71). In contrast, BnNRAMP6a displayed a significant negative correlation in roots (r = -0.75, p < 0.05). No significant correlations were detected for any of the tested genes in the stems. These results clear demonstrate functional divergence among NRAMP family members in a tissue-specific manner, with certain genes are strongly linked in Cd accumulation in leaves and roots, while others may be involved in exclusion or sequestration.

Materials and methods

Data acquisition and identification of NRAMP genes

The reference genome, protein sequences, and annotation files for A. thaliana were obtained from The Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/, TAIR_V10.1) [39]. The genomic data for the six Brassica species of U’s triangle were downloaded from the Brassica Database (BRAD, http://www.brassicadb.cn/) [40] and the Brassica napus Translational Genomics Resource (BnTIR, https://yanglab.hzau.edu.cn/BnTIR) [41]. The Brassica accessions and their genomic constitutions were as follows: Brassica rapa (cv. Chiifu_V1.5, AA, 2n = 20), Brassica nigra (cv. Ni100_V2, BB, 2n = 16), Brassica oleracea (cv. JZS_V2.0, CC, 2n = 18), Brassica juncea (cv. tum_V1.5, AABB, 2n = 36), Brassica napus (cv. ZS11 HZAU_V1.0, AACC, 2n = 38), and Brassica carinata (cv. zd-1.V0, BBCC, 2n = 34).

Identification of NRAMP gene family members

The hidden Markov model (HMM) profile for the NRAMP family (PF01566) was retrieved from the InterPro database (https://www.ebi.ac.uk/interpro/) [42]. This profile was used to to screen protein sequences from the six Brassica species. Concurrently, rigorous identification was performed using HMMER 3.0 with the same PF01566 domain from the Pfam database [43]. The genomes of U’s triangle species were aligned against with six known A. thaliana NRAMP (AtNRAMP) protein sequences using TBtools to identify homologous genes [44]. Candidate sequences from all approaches were combined and manually checked. Sequences that lacked the complete conserved domain, contained incomplete open reading frames, or represented redundant transcripts of the same gene were removed from the final set.

Phylogenetic analysis of the NRAMP gene family

The full-length amino acid sequences of the identified NRAMP proteins from the Brassica U’s triangle species and the six Arabidopsis thaliana NRAMPs were aligned using ClustalW in MEGA11 [45]. A neighbor-joining phylogenetic tree was constructed with 1,000 bootstrap replicates using the Poisson model, pairwise deletion, and uniform rates. The resulting tree was visualized and refined by Evolview (https://www.evolgenius.info/evolview-v2/) [46]. The Brassica NRAMP genes were systematically named on the basis of their phylogenetic relationship with Arabidopsis orthologs. Each gene name consists of a three-letter species code (e.g., Bna for B. napus), followed by the symbol of the closest Arabidopsis ortholog and a lowercase letter suffix (a, b, c, .), indicating the relative phylogenetic distance within the clade, with “a” denoting the closest ortholog.

Analysis of protein properties, gene structure, and conserved motifs

The physicochemical properties (molecular weight, theoretical isoelectric point) of the identified NRAMP proteins were predicted using the ExPASy ProtParam tool (http://web.expasy.org/protparam/). Transmembrane domains were predicted with TOPCONS (https://topcons.net), whereas subcellular localization was determined by WoLF PSORT (https://wolfpsort.hgc.jp/) and Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/plant/). Conserved motifs were identified with the MEME Suite, with the maximum number of motifs set to ten. Functional domains were confirmed by the NCBI Conserved Domain Database (CDD, https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). The exon-intron structures of the NRAMP genes were visualized via the Gene Structure View function in TBtools, based on the corresponding general feature format (GFF) annotation files.

Promoter and cis-acting element analyses

Promoter sequences, defined as the 2,000 bp regions upstream of the translation start site (ATG), were extracted for all identified NRAMP genes via the GFF annotation files and the “Sequence Extractor” function in TBtools. Putative cis-acting regulatory elements within these promoter regions were identified by the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [47].

Chromosomal location, collinearity and gene duplication

The chromosomal locations of the identified NRAMP genes were mapped based on positional data from the GFF annotation files. Synteny analysis was performed to identify collinear gene pairs both within (intra-genomic) and between (inter-genomic) the Brassica U’s triangle species using the “One Step MCScanX” and “Amazing Super Circos” functions in TBtools. For duplicated NRAMP gene pairs identified in the synteny analysis, the nonsynonymous (Ka) and synonymous (Ks) substitution rates were calculated. The Ka/Ks ratio was used to infer the selection pressure acting on the genes: a ratio < 1 indicates purifying selection, > 1 indicates positive selection, and a ratio of 1 suggests neutral evolution.

Plant materials, cadmium treatment, and sampling

Two rapeseed (Brassica napus) varieties, H42 and H47, which exhibit contrasting cadmium (Cd) accumulation patterns, were selected for this study. Seeds were germinated and grown in plug trays., Seedlings were transplanted into soil for treatment at two-leaf stage. The soil was spiked with a solution of CdCl₂·2.5 H₂O to achieve a nominal cadmium concentration of 30 mg/kg (Cd treatment). Untreated plants grown in the same soil served as the control (0 mg/kg Cd, CK). Plant tissues were collected at several developmental stages: roots, stems, and leaves were collected at the seedling and bolting stages; roots, stems, leaves, and flowers were collected at the flowering stage; and roots, leaves, and siliques were collected at the fruiting stage. After collection, roots were rinsed thoroughly with double-distilled water to remove soil particles. All samples were immediately flash frozen in liquid nitrogen and stored at -80 °C until further analysis. The experiment was carried out with three independent biological replicates per treatment.

Determination of cadmium content

Tissues were collected as described above and oven-dried at 70 °C for 24 h. Approximately 150 mg dry biomass was digested using a mixed-acid procedure. Specifically, each sample was treated with 10 mL concentrated nitric acid, 5 mL hydrochloric acid, 5 mL hydrofluoric acid, and 1 mL perchloric acid, and predigest at room temperature for 2 h. The vessels were then opened and heated to evaporate the acids, continuing until the residue was almost dry. After cooling to room temperature, the digest was diluted with Milli-Q water to a final volume of 25 mL. Cadmium concentration was determined by Agilent 5110 inductively coupled plasma optical emission spectrometer (ICP‒OES).

Gene expression analysis by quantitative real-time PCR (qRT‒PCR)

Gene expression of each sample was examined by qRT-PCR. Total RNA was extracted following the protocol of M5 Quickspin Universal Plant RNA Rapid Extraction Kit (Mei5 Biotechnology Co., Ltd.). RNA purity was assessed by A260/280 (1.8–2.1) and A260/230 (> 1.8) ratios with a NanoDrop spectrophotometer (Supplementary Table S5). Extracted RNA was reverse-transcribed into cDNA with the ExonScript RT SuperMix with a dsDNase Kit (Exongen, Chengdu, China). qRT-PCR was performed on a QX Series Real-Time Fluorescence Quantitative PCR System (GLMgene/QX, Sichuan Jiale Mei Technology Co., Ltd.) using UltraStart SYBR Green qPCR Master Mix (Exongen, Chengdu, China). The total volume of each qRT‒PCR mixture was 10 µL, which consisting of 5 µL of 2× SYBR Green mixture, 2 µL of cDNA, 0.3 µL each of forward and reverse primers (Table S1), and 2.4 µL of double-distilled water (ddH₂O). The qRT-PCR program was set as follows: initial denaturation at 95 °C for 3 min, followed by 39 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s.

Primer specificity was validated by melting curve analysis (single peak). Primers amplification efficiency was calculated from five-point standard curves with template cDNA dilution by 5 times. Primer efficiency (%) was calculated as (10^{− 1/slope}−1)×100. Primers with R² > 0.99 were used for quantification (Supplementary Table S1 and Figure S1). The melting curve was acquired via the instrument’s default program. BnaActin (GenBank accession: BnaC02G0037200ZS) was used as the reference gene. Each qRT-PCR reaction was performed in three technical replicates per biological sample, and the mean Ct value of the three replicates was used for calculation. No-template controls (NTC) were included in each run to monitor potential contamination. Relative expression levels were calculated using the 2^−ΔΔCt method, with the untreated control (0 mg/kg Cd) as calibrator.

Statistical analysis

To identify NRAMP members potentially involved in Cd accumulation, correlation analysis was performed to assess the relationships between the transcript abundance of each BnNRAMP gene and the Cd content within the same tissue. For each tissue (root, stem, and leaf), relative gene expression and Cd content were obtained from each individual biological replicate. Data were pooled from genotypes (H42 and H47) across developmental stages (seedling, bolting, flowering, and fruiting), with three biological replicates per genotype per stage. This yielded a total of 24 or 18 (no stem at fruiting stage) independent paired observations per gene-tissue combination. Due to an insufficient number of biological replicates for flower and silique tissues, correlation analysis could not be reliably performed for these organs, and they were therefore excluded from the statistical evaluation.

Prior to correlation analysis, the normality of Cd content and gene expression levels was checked with Shapiro-Wilk test for each gene-tissue combination. For variables with p > 0.05 in both groups, Pearson’s correlation was used. In our data, most gene-tissue combinations satisfied the normality assumption (see Supplementary Table S7 for details). Therefore, Pearson’s correlation coefficient was used as the primary measure, and results are reported as r with two-tailed p-values. The resulting correlation coefficients were visualized as a heatmap, with hierarchical clustering applied to group genes and tissues with similar correlation patterns. Correlation coefficients are shown inside each heatmap cell, and statistical significance is marked with asterisks (* indicates p < 0.05, ** indicates p < 0.01). All analyses were carried out using R Studio software (version 4.4.3).

Discussion

The NRAMP gene family has been extensively characterized in model plants and major crop species such as Arabidopsis, rice, and wheat, where its members are known to play key roles in heavy metal transport and homeostasis [30–32, 37, 48]. Nevertheless, a comprehensive view of this family in economically important Brassica species and its involvement in the cadmium stress response has remained missing. Our genome-wide analysis across the Brassica U’s triangle species offering new insights into the evolutionary history and possible functional mechanisms of NRAMP proteins in rapeseed under Cd stress.

The evolutionary history of the Brassica lineage, marked by a whole-genome triplication (WGT) and subsequent allopolyploidization, which provides an essential backdrop for understanding NRAMP family dynamics [49]. Our identification of 81 Brassica NRAMP genes across U’s triangle reveals a pattern consistent with post-polyploidization restructuring. The gene counts in allotetraploids (16 in B. juncea (AABB), 20 in B. napus (AACC), and 15 in B. carinata (BBCC)) are not simply the sum of their diploid parents, indicating significant gene loss during the diploidization (Fig. 4). This idea is reinforced by the scattered chromosomal distribution and complete lack of tandem duplicates, confirming that the expansion of this family was driven solely by WGD and segmental duplication [50, 51]. The evolutionary forces acting on the family have been remarkably consistent. Widespread purifying selection (Ka/Ks < 1) across both recently duplicated and orthologous gene pairs highlights the strong functional constraints needed to maintain metal homeostasis. The fact that some homologous pairs have Ka/Ks = 0 indicates extreme sequence conservation, suggesting those genes might encode core transporters that are essential for basic cellular ion balance (Fig. 5). Moreover, the uneven distribution of genes, such as the concentration on chromosome B06 of B. nigra and B02 of B. juncea, provides tangible support for subgenome dominance, where one parental genome retains more genes due to differential selection pressure [52]. Taken together, our analysis of the NRAMP family illustrates a classic evolutionary path in allopolyploids: initial expansion through WGD, then fractionation and diploidization, all under tight purifying selection to preserve indispensable physiological functions.

The NRAMP proteins identified in the Brassica U’s triangle species are structurally highly similar to their orthologs in other plants. The majority possess a canonical length of approximately 500 amino acids and contain the definitive NRAMP domain (Fig. 2, Table S2). The predicted transmembrane topology is consistent with known structures, generally featuring 10–12 transmembrane helices (TMs). The number of TMs in Brassica NRAMPs ranges from 9 to 13, with the conserved consensus transport motif (CTM) lying between TM8 and TM9, a feature reported in rice, peanut, and soybean [19, 35, 53, 54]. Subcellular localization prediction placed all 81 Brassica NRAMP proteins at the plasma membrane This uniform prediction strongly suggests that the primary, ancestral function of this family in Brassica is linked to plasma membrane activities, such as metal ion influx or efflux. This is in contrast to model species, where NRAMPs can be found on the plasma membrane, tonoplast and Golgi apparatus [25, 26, 32, 34, 55–63]. However, this conclusion is based on in silico predictions and still need experimental validation.

Phylogenetic analysis robustly classified the Brassica NRAMP family into two primary subfamilies, subfamily I (NRAMP1/6) and subfamily II (NRAMP2-5), which is consistent with the classification established in Arabidopsis (Fig. 1). Although recent studies have proposed alternative grouping systems based on exon number or a third clade found in monocots, our data from the Brassica U’s triangle strongly support the classical bipartite division [16, 64]. AA deep structural difference underlies this phylogenetic split. Subfamily I genes are intron-poor, with an average of 4.0 exons, whereas subfamily II genes are intron-rich, with an average of 11.3 exons (Fig. 2C). This difference is mirrored at the protein level, where Subfamily I universally lacks Motif 8 and has a different motif arrangement between Motif 3 and Motif 1 compared to Subfamily II (Fig. 2A). Such stark structural differentiation strongly suggests subfamily-specific functional or regulatory specialization. We propose that the lineage-specific WGT event provided the raw genetic material for this divergence. The resulting abundance of redundant gene copies created an evolutionary playground where exon gain, loss, and rearrangement could happen rapidly. This process of “divergent resolution” led to the formation of two structurally and likely functionally distinct subfamilies we observe today, a pattern of post-polyploidization evolution commonly observed in other Brassica gene families [65–69].

Our correlation analysis between gene expression and cadmium content provides compelling evidence for the sophisticated, tissue-specific functional diversification of the NRAMP family in B. napus (Fig. 6). The strong positive correlation of NRAMP2 subfamily members (BnNRAMP2c and 2d) with root Cd content, particularly BnNRAMP2d (r = 0.91^**), suggests that they may serve as important candidates for cadmium uptake or root sequestration, a role that is consistent with their orthologs in foxtail millet and potato [70, 71]. At the same time, the significant positive correlations of BnNRAMP3b and BnNRAMP4a in roots suggest that these genes might operate synergistically in a coordinated network at the soil-root interface. In contrast, the significant positive correlations of NRAMP1 subfamily members (BnNRAMP1c, 1e, and 1f) with leaf cadmium levels point to a specialized role in cadmium translocation, distribution, or sequestration within aerial tissues. This spatial specialization between root and shoot fits a division of labour where different NRAMP members handle cadmium in different parts of the plant. A particularly intriguing finding was the significant negative correlation of BnNRAMP6a in roots, suggesting a potential role in cadmium exclusion or cytoplasmic detoxification, perhaps through efflux or vacuolar sequestration. The absence of significant correlations in stems for all members suggests that stems may act primarily as passive conduits, with less active NRAMP-mediated transport. This functional divergence parallels what has been reported in model species. In Arabidopsis, different NRAMPs exhibit tissue-specific expression and distinct roles under Cd stress, whereas in rice, a network of NRAMPs (OsNRAMP1, 2, 4, 5) regulates Cd uptake, root-to-shoot translocation, and distribution [56, 61, 64, 72].

While our study provides an evolutionary and expression landscape of the NRAMP family in Brassica species, several limitations should be acknowledged regarding the functional claims. First, the proposed roles of specific BnNRAMP genes (e.g., BnNRAMP2d in root cadmium accumulation, BnNRAMP1c/1e/1f in leaf distribution, and BnNRAMP6a in potential exclusion) are based solely on correlative evidence between gene expression and cadmium content. Correlation does not imply causation, and these hypotheses require further experimental validation through gene knockout, overexpression, or complementation assays in stable transgenic lines or transient systems. Second, our subcellular localization predictions are computational and await experimental confirmation (e.g., via GFP fusion). Third, the cadmium treatment used a single concentration (30 mg/kg), and the expression profiles may differ under other doses or chronic exposure. Therefore, the functional interpretations presented here should be considered as testable hypotheses rather than established mechanisms.

Conclusion

In summary, our study provides a systematic genomic and functional analysis of the NRAMP gene family across the Brassica U’s triangle species. The evolutionary analyses reveal a typical post-polyploidization pattern of duplication, purifying selection, and gene loss, along with a conserved intron-based subfamily dichotomy. The correlations between gene expression and cadmium content in B. napus generate testable hypotheses of BnNRAMP2d as a root accumulation candidate, BnNRAMP1s for leaf distribution, and BnNRAMP6a for potential exclusion. These hypotheses await functional validation via gene editing. Although the correlations did not reach genome-wide significance after multiple correction, they are strong enough to provide a clear set of priority targets. Together, our findings establish a genomic foundation and offer experimentally tractable hypotheses for engineering cadmium accumulation traits in rapeseed, either for phytoremediation or food safety, pending functional confirmation.

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Abbreviations

Cd

Cadmium

A. thaliana

Arabidopsis thaliana

B. carinata

Brassica carinata

B. juncea

Brassica juncea

B. napus

Brassica napus

B. nigra

Brassica nigra

B. oleracea

Brassica oleracea

B. rapa

Brassica rapa

CTM

Consensus transport motif

GRAVY

Grand average of hydropathicity

MW

Molecular weight

NRAMP

The Natural Resistance-Associated Macrophage Protein

pI

Isoelectric points

TMDs

Transmembrane domains

WGD

Whole-genome duplications

WGT

Whole-genome triplication

Authors' contributions

JL conceived and designed the experiments. FZ performed most of the experiments. XL, HT, YD and YK helped with data analysis. FZ and KG draft the manuscript. SG and JL supervised and complemented the writing. All authors have reviewed the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (32301762); Sichuan Provincial Science and Technology Support Program, 2025ZNSFSC0177.

Data availability

The reference genome, protein sequences, and annotation files for *A. thaliana* were obtained from The Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/, TAIR\_V10.1). The genomic data for the six *Brassica* species of U’s Triangle were sourced from the *Brassica* Database (BRAD, http://www.brassicadb.cn/) and the *Brassica napus* Translational Genomics Resource (BnTIR, https://yanglab.hzau.edu.cn/BnTIR). The *Brassica* accessions and their genomic constitutions were as follows: *Brassica rapa* (cv. Chiifu\_V1.5, AA, 2n=20), *Brassica nigra* (cv. Ni100\_V2, BB, 2n=16), *Brassica oleracea* (cv. JZS\_V2.0, CC, 2n=18), *Brassica juncea* (cv. tum\_V1.5, AABB, 2n=36), *Brassica napus* (cv. ZS11 HZAU\_V1.0, AACC, 2n=38), and *Brassica carinata* (cv. zd-1. V0, BBCC, 2n=34).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.