Identification and physicochemical characterization of MTP proteins
A total of 68 MTP proteins from three species, including B. rapa (17), B. nigra (18), and B. juncea (33), were identified based on validation results using Cation_efflux (PF01545) domain characteristic, PFAM, SMART, and BLAST analyses. Considering their similarity and phylogenetic relationship with AtMTPs, all MTPs were named BxMTP1-BxMTP12 (x = r, n, and j). Significantly, the analysis of gene number and physicochemical characteristics of MTPs reveals that most BjMTP proteins in the allopolyploid B. juncea (AABB, n = 38) were the result of combining MTP proteins from its diploid ancestors, B. rapa (AA, n = 20) and B. nigra (BB, n = 18). However, homologs for BnB06.MTP4b and BnB01.MTP12 were not detected in B. juncea. Furthermore, among the identified BjMTPs, orthologs of AtMTP2 and AtMTP3 were missing. Within the 33 BjMTPs, the number of orthologs ranged from 2 to 4. The distribution scores of these orthologs varied, with a minimum of 1 (BjMTP12) and a maximum of 6 (BjMTP8) (Table S1).
BjMTP proteins varied in length from 163 to 1106 amino acids, with molecular weights ranging from 18.34 kDa (BjB06.MTP4) to 122.28 kDa (BjA04.MTP1). Generally characterized as acidic, the BjMTPs had an average isoelectric point (pI) < 7, except for BjB06.MTP4, which had a pI of 10.46. Subcellular localization analysis indicated that most BjMTPs were present in the vacuoles, while some localized to the cell membrane. Further analysis of transmembrane domains revealed that most BjMTPs possessed 4 to 8 TMDs, with BjA04.MTP12 notably containing 14 TMDs. In contrast, BjA03.MTP6 and BjB08.MTP6 lacked discernible TMDs (Table S1).
Phylogenetic analysis and chromosomal localization of BjMTPs
Based on the phylogenetic tree and structural characteristics of 80 MTP proteins from A. thaliana, B. rapa, B. nigra, and B. juncea, the MTPs could be categorized into three main subgroups: Mn-CDF, Zn-CDF, and Fe/Zn-CDF. These subgroups could be further subdivided into seven branches: G1, G5, G6, G7, G8, G9, and G12 (Fig. 1a), where BjMTP proteins displayed a close phylogenetic relationship with their corresponding orthologs in BrMTPs and BnMTPs. In addition, the lowest and largest numbers of BjMTP genes (1 and 10, respectively) were found in branches G12 and G9. Chromosomal localization analysis revealed that the A and B subgenomes of B. juncea contain 17 and 16 BjMTP genes, respectively, dispersed unevenly throughout 11 chromosomes. Notably, chromosome B06 had the highest number of genes (8 genes), while chromosomes B03 and B04 each had only a single BjMTP gene. No BjMTP genes were found on chromosomes A01, A02, A08, A10, B02, B05, and B07 (Fig. 1b).
Structural analysis of BjMTP proteins
By analyzing 15 conserved motifs within the BjMTP proteins, subgroup-specific patterns were identified by means of motif conservation. This research revealed distribution patterns that were peculiar to each branch, with distinct motifs defining them all. Specifically, G1 was distinguished by motifs 6 and 7; G5 by motif 6; G6 by motif 14; G7 by motifs 4, 5, 8, 13, and 14; G8 by motifs 1, 2, 3, 4, 5, 8, 9, 11, and 12; and G9 by motifs 1, 2, 4, 8, and 11 (Fig. 2a-b). Using the SMART database, further protein domain analysis revealed that the BjMTP family members possessed a Cation_efflux domain. Members from branches G6, G8, and G9 also contained a Zinc Transporter dimerization (ZT_dimmer) domain (Fig. 2c), suggesting that motifs 2, 4, and 8, and motifs 1 and 11 correspond to the Cation_efflux and ZT_dimmer domains, respectively. Additionally, BjA04.MTP1, BjB01.MTP1, and BjB06.MTP1 contained a helix-loop-helix (HLH) domain. Investigation of 3D structures for 10 representative BjMTP proteins via the AlphaFold database indicated that the cation efflux domain typically comprised 4–6 transmembrane helices, arranged into a compact cluster structure (Fig. S1). In contrast, Zn-MTP and Fe/Zn-MTP subgroups displayed either 1–2 HXXXD or DXXXD residues (Fig. S1a-b), while the Mn-CDF subgroup consistently had two DXXXD residues (Fig. S1c). Notably, no clear pattern was seen in these residues across the seven branches that were detected. Prior studies have showed a strong correlation between these conserved residues and metal ion selectivity23,24. Therefore, Therefore, it was suggested by AlphaFold's predictions that the residues in BjMTPs are likewise intimately related to metal ion transport.
The Cation_efflux domain and HXXXD or DXXXD residues, which were essential for metal ion binding, were present in all BjMTP proteins, as was clear from the analyses of conserved domains and the 3D structure of BjMTPs. Significant structural differences were found, meanwhile, across different CDF subfamilies or branches, underscoring the functional variety of BjMTP genes.
Analysis of Promoters in genes
Analysis of Promoters in BjMTP genes
Using PlantCARE software, the cis-regulatory components of the BjMTP promoters were examined in order to clarify the tissue-specific expression and stress response roles of these proteins. After then, these elements were categorized and subjected to statistics. Out of the 806 components found across the promoters of 33 BjMTP genes, apart from the commonly observed cis-regulatory elements, three distinct categories25 were recognized based on their association with development and growth (298), hormone responsiveness (335), and stress responsiveness (173) (Table S3). Development-related elements included those related to light response (ACE, ATC-motif, Box-4, and G-box), meristem development (CAT-box), flowering (CCAAT-box), and circadian rhythm (Circadian), with light response elements accounted for 64.8% of this category (Fig. 3a). The elements related to hormones were associated with auxin (AuxRR-core, TGA-element, TGA-box), MeJA (CGTCA-motif, TGACG-motif), gibberellin (GARE-motif, TATC-box, P-box), abscisic acid (ABRE), and salicylic acid (TCA-element). Of these, abscisic acid response elements were the most dominant, accounting for 33.4% (112/335), whereas salicylic acid response elements were the least abundant, with only 5.1% (Fig. 3b). Anaerobic induction (ARE), anoxic induction (GC-motif), cold (LTR), drought (MBS), and general stress (TC-rich repeats) were among the stress-related elements. ARE elements made up 54.3% of this category, suggesting that BjMTPs play a major role in adaptation to anaerobic stress (Fig. 3c). Except for BjA04.MTP4 and BjB01.MTP5, the number of ARE elements varied among BjMTP genes, ranging from one (BjA04.MTP8) to nine (BjA03.MTP1) (Fig. 3d).
Once again, distinct patterns among evolutionary branches and the distribution of each gene uniquely were identified by the examination of cis-regulatory elements in BjMTP gene promoters. In particular, genes in the G1 branch often shared highly conserved MeJA-responsive elements, despite variations in the number of promoter elements. The promoters of homologous genes in the G5, G6, G7, and G8 branches have comparable cis-regulatory elements. Interestingly, the G9 branch, comprising 10 genes, was characterized by the abundance of G-box, ABRE, and ARE elements, along with markedly conserved MeJA-responsive elements (Fig. 3d). As a result, a wide variety of cis-regulatory elements are present in BjMTP promoter, which aid in the full regulation of development, hormone responses, and stress mechanisms. Furthermore, conserved features in their promoters probably impact the functional variety of BjMTP genes throughout evolutionary branches.
Synteny analysis of BjMTP genes
Whole genome duplication, segmental duplication, and tandem duplication are crucial mechanisms driving gene family evolution26. Syntenic analysis was used to identify the BjMTP genes in order to investigate gene duplication occurrences in these genes. All 32 BjMTP genes, with the exception of BjA04.MTP12, showed evolutionary events involving 44 pairs, mostly related to segmental duplication (Fig. 4; Table S4). Furthermore, all gene pairs had their Ka/Ks ratios assessed; the results ranged from 0.05 to 0.30, which is much less than the neutral selection threshold of Ka/Ks = 1. This data strongly implies that purifying selection occurred in all 44 pairs of duplicated genes, proving that the BjMTP genes did not develop new functions. These results suggested that related BjMTP genes probably operate similarly, especially when subjected to HM stress.
Expression patterns and GO enrichment analysis
Utilizing RNA-seq data from the NCBI database, the expression patterns of BjMTP genes in the different tissues of B. juncea were clarified. 33 BjMTP genes were thoroughly analyzed to determine the expression levels of these genes in a variety of tissues, including roots, stems, leaves, buds, siliques at 7 and 15 DAF, pods at 20 DAF, seeds, and seed coats. The distribution of cis-regulatory elements inside the promoters of BjMTPs was shown to be correlated with their expression. Homologous BjMTP genes generally exhibited similar expression patterns across tissues, with few exceptions. In stems, leaves, seed coats, and seed, for instance, BjMTP4 was minimally expressed, while BjMTP11 showed greater expression levels. Moreover, unique expression features were shown by the G9 branch genes BjMTP9, BjMTP10, and BjMTP11, with higher expression in leaves and buds and reduced expression in seeds (Fig. 5a, Table S5). This implies a correlation between the evolutionary branches of BjMTPs and the tissue-specific expression, potentially linked to the cis-regulatory elements in their promoters. Additionally, certain BjMTPs showed patterns of tissue-specific or ubiquitous expression. For instance, BjA04.MTP1 had high expression levels in a variety of tissues, but BjB06.MTP9, BjA07.MTP9, and BjB01.MTP11 showed significant increases in expression in leaf tissues, indicating specialized functions in specific tissues.
By using GO enrichment analysis, the molecular functions of the BjMTP genes were further clarified. Biological activities such as efflux, cation, and ion transmembrane transporter activity were covered by the top 20 GO terms (Fig. 5b). Notably, the predicted biological functions of BjMTP genes were associated with essential processes such as zinc ion homeostasis maintenance and vacuolar membrane transport facilitation7,11. These results align with earlier findings on protein structure and subcellular localization, highlighting the significant role of BjMTP genes in metal cation transport.
Plant phenotypes and expression profiles of BjMTPs under six HMs stresses
A crucial sign of how a plant will response to biotic or abiotic stressors is its phenotypic. In the early phases of HM treatment (12 and 24 hours), B. juncea plants in our research did not show any discernible phenotypic alterations. After 48 hours, however, the effects of the other metals were minimal, but the effects of 250 mg/L Mn2+, 250 mg/L Fe2+, and 10 mg/L Sb3+ on plant morphologies were noticeable (Fig. 6). The leaves developed uneven white spots as a result of Mn2+ exposure. Fe2+ stress was connected to overt signs of dehydration. Additionally, a low Sb3+ concentration resulted in root yellowing, which was subsequently connected to an unidentified bacterial strain (unpublished data). Thus, in B. juncea, both non-essential (Sb3+) and necessary (Mn2+ and Fe2+) metals may have a negative impact on plant health.
From the 33 BjMTP genes, 10 representative genes were chosen for qRT-PCR analysis under different HM conditions in order to thoroughly evalute the impacts of various HMs on BjMTP gene expression. The selected genes included BjA04.MTP1 and BjA04.MTP4 (G1); BjB01.MTP5 (G5); BjB08.MTP6 (G6); BjA06.MTP7 (G7); BjA09.MTP8 (G8); BjB06.MTP9, BjA09.MTP10, and BjA05.MTP11 (G9); and BjA04.MTP12 (G12). These genes displayed spatiotemporal and tissue-specific expression patterns across six HM conditions, with roots responding much more strongly than leaves (Fig. S2, Fig. 7a-b). BjA04.MTP4 and BjB01.MTP5 were markedly elevated in roots during the first 12 and 24 hours of Fe2+ and Sb3+ exposure. But after 48 h of HM treatments, the majority of genes showed significant changes in expression, which was consistent with the phenotypic alterations in B. juncea that were seen. Within the Mn-CDF subgroup, only BjA09.MTP10 responded to Mn2+ stress in roots after 48 h. All HM treatments activated MTP8, which is known to modulate Mn2+ transport and tolerance15,27, in leaf tissues. Significantly, BjA04.MTP4, BjA09.MTP10, and BjB01.MTP5 genes showed the greatest levels of expression (997.1-, 370.5-, and 2137.3-fold greater than control, respectively) under Sb3+ and Pb2+ exposures (Fig. S2). Furthermore, the consistently downregulated expression of BjB06.MTP9 under various HM stresses highlighted its potential negative regulatory role in HM stress responses.
Co-expression networks of BjMTPs
To elucidate the regulatory mechanisms underlying gene responses to HM stress, co-expression networks are essential28. In this study, expression data for BjMTP genes across various tissues, HM stresses, and treatment durations were used to construct co-expression networks (Fig. 7a-b; Table S6). Consistent gene expression patterns were found in the root tissues exposed to HMs, namely in the cases of G1 (BjA04.MTP4), G5 (BjB01.MTP5), G7 (BjA06.MTP7), and G9 (BjB06.MTP9, BjA09.MTP10, and BjA05.MTP11) (Fig. 7a, c). In response to each of the six tested HMs, these genes showed synergistic positive regulation. BjA04.MTP4 (G4), BjB01.MTP5 (G5), and BjA09.MTP8 (G8) were found to be involved in the possible co-regulation of Zn2+ transport in leaf tissues, with BjA09.MTP8 demonstrating a positive response to HM stressors, which was less evident in roots (Fig. 7b, d). Furthermore, there was no discernible alteration in the expression of the neutral gene BjA04.MTP12 in any of the tissues. 10 BjMTP genes were subjected to a thorough co-expression study, which partly supported the classification of the genes into seven phylogenetic branches. Specifically, genes from the G9 branch (BjB06.MTP9, BjA09.MTP10, and BjA05.MTP11) showed different expression patterns in leaf tissues but demonstrated synergistic responses to six different types of HM stresses in root tissues, indicating tissue-specific responses that correlate with the seven-branch classification.