Genome-wide exploration of MTP gene family in mustard (Brassica juncea L.): evolution and expression patterns during heavy metal stress

Genome-wide exploration of MTP gene family in mustard (Brassica juncea L.): evolution and expression patterns during heavy metal stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Genome-wide exploration of MTP gene family in mustard (Brassica juncea L.): evolution and expression patterns during heavy metal stress Liang You, Jialin Sheng, Guoxiang Jiang, Hao Chen, Yuhui Yuan, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4296734/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Aug, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Members of the Metal Tolerance Protein (MTP) family are critical in mediating the transport and tolerance of divalent metal cations. Despite their significance, little is known about the MTP genes in mustard ( Brassica juncea ), particularly in relation to how they react to HM stress. In our study, we identified MTP gene sets in Brassica rapa (17 genes), Brassica nigra (18 genes), and B. juncea (33 genes) using the HMMER tool (Cation_efflux; PF01545) and BLAST analysis. Then, for the 33 BjMTPs , we carried out a detailed bioinformatics analysis covering the physicochemical properties, phylogenetic relationships, conserved motifs, protein structures, collinearity, spatiotemporal RNA-seq expression, GO enrichment, and expression profiling under six HM stresses (Mn 2+ , Fe 2+ , Zn 2+ , Cd 2+ , Sb 3+ , and Pb 2+ ). According to the findings of physicochemical characteristics and phylogenetic tree, the allopolyploid B. juncea ’s MTP genes were inherited from its progenitors, B. rapa and B. nigra , with minimal gene loss during polyploidization. The BjMTP gene family exhibited conserved motifs, promoter elements, and expression patterns that aligned with seven evolutionary branches (G1, G4-G9, and G12). Further, by co-expression analysis, the core and gene-specific expression modules of BjMTPs under six HM stresses were found. The HM treatments exhibited consistently upregulated of BjA04.MTP4 , BjA09.MTP10 , and BjB01.MTP5 genes, indicating their critical roles in enhancing HM tolerance in B. juncea . These discoveries may contribute to a genetic improvement in B. juncea 's HM tolerance, which would facilitate the remediation of HM-contaminated areas. Biological sciences/Molecular biology Biological sciences/Plant sciences/Plant stress responses Brassica juncea L. MTP gene Heavy metals Specific genes Expression profiles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Growing concern is being expressed over the rising levels of heavy metal (HM) contamination in the environment as a result of industrial operations such as mining, exhaust emissions, waste management, artificial fertilizer use in agriculture, and urban wastewater treatment. Effective mitigation measures are desperately required since this scenario presents a serious danger to agricultural production 1,2 . Trace levels of essential HMs such as manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) are present in plants to regulate numerous physiological processes 3 . However, high concentrations of these essential HMs or even low concentrations of non-essential HMs including cadmium (Cd), lead (Pb), mercury (Hg), and antimony (Sb), may trigger phytotoxic consequences. Such toxicity is characterized by growth restriction, chlorosis, root discoloration, and even plant death 2 ,4,5,6 . Plants have evolved many defense mechanisms against HMs, such as detoxification, sequestration, cell membrane transport processes, and raising the concentrations of compatible solutes 2,7 . By identifying metals and enabling their movement to particular places in plant tissues, some transport proteins are essential to these systems for preserving cellular homeostasis 2,8 . Studies show that families of transport proteins, such as the Zn-regulated iron-regulated transporter-like proteins (ZIPs), ATP-binding cassettes (ABCs), natural resistance-associated macrophage protein (NRAMPs), and cation diffusion facilitators (CDFs), are sequester HMs into vacuoles and chelate them to reduce their toxicity 7,9 . The metal tolerance proteins (MTPs), also known as the cation transporter family (CDF), are a class of proteins that were first discovered in bacteria. They have a role in the tolerance and transportation of divalent metal cations, including Mn 2+ , Fe 2+ , Zn 2+ , and Cd 2+ . MTPs are also necessary for plants to detoxify HMs 7,10,11 . Structurally, MTPs are characterized by transmembrane domains (TMDs), metal ion recognition motifs (HXXXD or DXXXD), and cation transport domains 12 . These proteins are categorized into three major subgroups based on substrate specificity: Zn-CDF, Zn/Fe-CDF, and Mn-CDF. They facilitate the transport of ions such as Zn 2+ , Fe 2+ , Mn 2+ , Co 2+ , Cd 2+ , and Ni 2+ 13 . In Arabidopsis thaliana , the 12 MTP proteins are further divided into seven branches: G1, G5, G6, G7, G8, G9, and G12, emphasizing the complexity and specificity of their roles 11 . Previous studies have shown the range of functions displayed by many MTP proteins. As a vacuolar zinc transporter, AtMTP3 is necessory to regulate intracellular Zn 2+ levels and maintaining homeostasis 14 . The AtMTP8 protein is essential for acting as a transporter for Mn 2+ , in addition to regulating Fe transport during seed germination and developmental processes 15 . Additionally, a protein combination AtMTP5 and AtMTP12 may create helps to enhance Zn 2+ transport into the Golgi apparatus 16 . Brassica juncea L ., known for its rapid vegetative growth and substantial aboveground biomass, has been recognized as an effective accumulator of various HMs, such as Pb, Cd, selenium (Se), Zn, chromium (Cr), Cu, and nickel (Ni). As a result, this plant is widely used in studies on soil decontamination and remediation 17 ,18,19 . To date, only a few cation-efflux family genes, namely BjCET1-4 , have been identified in B. juncea . Heterologous expression of these genes in yeast has demonstrated their ability to confer tolerance against Zn and Cd 20,21,22 . Understudied, however, are the thorough discovery and functional characterisation of other cation-efflux family members in B. juncea 's response to HMs. Here, we explored the MTP proteins in B. juncea using bioinformatics techniques, with an emphasis on the dynamics of the BjMTP genes expression under HM stress. This research lays a foundation for further investigation into the molecular mechanisms of HM homeostasis in B. juncea via BjMTP family members and offers valuable genetic resources for breeding programs aimed at soil remediation. Results 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 selectivity 23,24 . 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 categories 25 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 evolution 26 . 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 BjMTP s and the tissue-specific expression, potentially linked to the cis-regulatory elements in their promoters. Additionally, certain BjMTP s 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 facilitation 7,11 . 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 Mn 2+ , 250 mg/L Fe 2+ , and 10 mg/L Sb 3+ on plant morphologies were noticeable (Fig. 6). The leaves developed uneven white spots as a result of Mn 2+ exposure. Fe 2+ stress was connected to overt signs of dehydration. Additionally, a low Sb 3+ concentration resulted in root yellowing, which was subsequently connected to an unidentified bacterial strain (unpublished data). Thus, in B. juncea , both non-essential (Sb 3+ ) and necessary (Mn 2+ and Fe 2+ ) 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 Fe 2+ and Sb 3+ 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 Mn 2+ stress in roots after 48 h. All HM treatments activated MTP8, which is known to modulate Mn 2+ transport and tolerance 15,27 , in leaf tissues. Significantly, BjA04.MTP4 , BjA09.MTP10 , and B jB01.MTP5 genes showed the greatest levels of expression (997.1-, 370.5-, and 2137.3-fold greater than control, respectively) under Sb 3+ and Pb 2+ 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 essential 28 . 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 Zn 2+ 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. Discussion The allopolyploid B. juncea ’s MTP genes derive from diploid progenitors B. rapa and B. nigra MTPs are a broad family of transport proteins found in bacteria, fungi, plants, and animals. These proteins have a crucial role in regulating the cellular efflux of excess metal ions and maintaining ionic homeostasis 13 . MTP proteins have been identified in various plant species, such as A. thaliana (12 genes), Solanum Lycopersicum (11 genes), Medicago truncatula (12 genes), Glycine max (20 genes) 29 , Solanum tuberosum (21 genes) 28 , Arachis hypogaea L. (22 genes) 30 , and B. napus (33 genes) 31 . In this study, we identified 17, 18, and 33 MTPs in the genomes of the diploid parental species B. rapa , B. nigra , and the allopolyploid species B. juncea , respectively. Comparative studies of MTP genes indicated that BjMTPs are made up of the homologs BrMTP and BnMTP , without novel additions. Notably, the loss of BnB06.MTP4b and BnB01.MTP12 from B. nigra was observed during the B. juncea polyploidization event. This genomic complexity greatly raises the likelihood of various gene functions, but it also increases the difficulty of functional research. Furthermore, the B. juncea genome lacks homologs tor AtMTP2 and AtMTP3 , paralleling the absence of an AtMTP7 homolog in B. napus 31 . This might be the result of mismatches in gene classification caused by the high degree of homology among MTP genes within the subgroups. The seven-branch model may elucidate BjMTP functional diversity The functions of the BjMTP gene family were clarified by the use of integrated bioinformatic approaches such as phylogenetic tree, conserved motif, protein structure, promoter element, collinearity, RNA-seq expression pattern, and GO enrichment investigations. Phylogenetic analysis revealed that the BjMTP gene family, similar to those in A. thaliana 11 , B. nupus 31 , and G. max 29 , can be categorized into three subgroups (Zn-CDF, Mn-CDF, and Fe/Zn-CDF) and seven major branches (G1, G5, G6, G7, G8, G9, and G12). The BjMTPs were divided into three subgroups by earlier research based on the substrate-binding selectivity for Zn 2+ , Mn 2 + and Fe 2 + 13 . However, phylogenomic investigations of the genomes of plants and algae have revealed seven branches within the CDF family, which are believed to date back to the origin of land plants 11 . According to our analysis of the conserved motifs, promoter elements, and RNA-seq expression patterns, most BjMTP genes are probably more closely related to the seven-branch classification. Moreover, similar results were also observed for other crops 29,31,32 .Specifically, after several whole-genome duplication events, B. juncea grew from the 12 AtMTPs seen in the model plant A. thaliana to 33 BjMTPs . These genes consistently inherit all seven ancient branches, with gene counts per branch varying from one to six (Table S2). Through establishing a relationship between the conserved Cation_efflux domain's metal transport properties and the variations observed among the seven branches of the BjMTP family, one can infer that the functional variations in metal ion transport exhibited by BjMTPs are dependent on the particular branches. BjMTP genes exhibit a broad-spectrum response to heavy metal stress B. juncea is a globally important oilseed and vegetable crop, known for its high tolerance to various abiotic stresses, such as salt, drought, and heavy metals 18,33,34 . Prior research has clarified the function of MTP genes in the detoxification of divalent metals, such as Zn 2+ , Mn 2+ , Fe 2+ , Cu 2+ , Pb 2+ , Hg 2+ , and Se 2+ , both essential and non-essential 29,31 . In this study, six HM ions (Mn 2+ , Fe 2+ , Zn 2+ , Cd 2+ , Sb 3+ , and Pb 2+ ) were introduced to B. juncea seedlings in order to clarify the role of BjMTP genes in response to metal stress. Sb 3 + is a non-essential HM rarely included in studies; yet, it possesses phytotoxic threats and potential carcinogenic effects 6,35,36 . According to this investigation, B. juncea seedlings showed notable phenotypic alterations after 48 h treatments with 250 mg/L Mn 2+ , 250 mg/L Fe 2+ , and 10 mg/L Sb 3+ , indicating the highest concentration of these HMs that B. juncea can tolerate. The fact that the other three HMs had no effect on the plant phenotype suggests that B. juncea plants might be temporarily tolerant of this concentration levels. Within A. thaliana , the cation efflux transporter AtMTP1 is involved in Zn detoxification 37 . Numerous metals, including Zn, Fe, Co, and Ni, translocate due in part to OsMTP1 38,39 . In this investigation, Zn 2 + and Fe 2 + stresses caused BjA04.MTP1 to be elevated in B. juncea roots. But compared to BjA04.MTP4 (of the same G1 branch), which helps cucumbers maintain Zn 2 + homeostasis and sequester Cd 39 , its expression was less noticeable. Notably, in response to six HMs, root tissues showed a substantial upregulation of BjA04.MTP4 , BjB01.MTP5 , and BjA09.MTP10 , suggestingthe potential of comprehensive metal tolerance and transport across these genes. It has been shown that MTP8 proteins regulate Mn 2 + transport and provide Mn 2 + tolerance 15,27 . To improve resistance to Mn 2+ , OsMTP8 sequesters Mn in the vacuoles of rice panicle cells 27 . Surprisingly, our findings showed that BjA09.MTP8 overexpression was only stimulated by Fe 2 + in the roots; in contrast, BjA09.MTP8 reacted to almost all HMs in the leaves, perhaps explaining the speckled leaves that were seen after Mn 2 + exposure. Related to the Mn-CDF subfamily as well, BjB06.MTP9 showed opposing regulatory effects on HM tolerance and stress responses in the tissues of the roots and leaves. These findings highlight the MTP genes' interspecific functional variety and spatial expression in HM stress responses. Numerous transcriptome studies on HM stress have shown this behavior 40,41 . Co-expression networks are essential for studying gene regulation and play a critical role in identifying novel genes for plant growth and environmental adaptation 28,42 . A co-expression network study of the S. tuberosum OPT family has shown that StOPT1 / 3 is upregulated in the leaves under Cd and Cu stress and StYSL5 / 11 is upregulated in the roots in response to Cu and Zn stress 28 . These results emphasize the tissue-specific, metal-responsive, and synergistic regulation properties of HM-responsive genes. Our research, which used co-expression network analysis to examine the response patterns of BjMTPs to HMs, also revealed a similar trend. The results indicated that whereas modules G1 ( BjA04.MTP4 ), G5 ( BjB01.MTP5 ), G7 ( BjA06.MTP7 ), and G9 ( BjB06.MTP9 , BjA09.MTP10 , and BjA05.MTP11 ) participate in the HM stress response in the roots, modules G4 ( BjA04.MTP4 ), G5 ( BjB01.MTP5 ), and G8 ( BjA09.MTP8 ) co-respond to HM stress in the leaves. Furthermore, this is the first study that we are aware of that incorporates Sb 3 + into stress response tests on BjMTPs . According to our findings, most BjMTPs were triggered by low concentrations of Sb, indicating that MTP proteins may be involved in the uptake and transport of multivalent metals. The specific genes of B. juncea implicated in the response to HM stress were revealed by this study, laying the groundwork for future research into the characterisation of BjMTPs . Materials and methods Identification of MTPs The genome and protein sequences of B. rapa (Chiifu v3.5), B. nigra (NI100 v2), and B. juncea (Sichuan Yellow v1.2) were obtained from the Brassicaceae database ( http://brassicadb.cn/#/ ) and the Molecular Breeding of Oilseed website ( http://www.oilseedhunan.net/ ), respectively. In addition, 12 Arabidopsis MTP proteins were downloaded from the NCBI ( http://www.ncbi.nlm.nih.gov/ ), and the HMM file of the MTP conserved domain (Cation_efflux, PF01545) was acquired from the PFAM datebase ( http://pfam.xfam.org/ ). To identify MTP proteins in B. rapa , B. nigra , and B. juncea , the HMMER (v3.2.1) program under TBtools-Ⅱ(v2.028) 43 was used to screen the protein files for the cation efflux domain, setting the E-value cutoff at 1E-07. Subsequently, the identified MTP proteins were aligned with AtMTPs, retaining only those with an identity greater than 75% for further analysis. The ExPasy tool ( https://web.expasy.org/compute_pi/ ) was utilized to predict the isoelectric point (pI), molecular weight (MW), and physicochemical properties of BjMTP proteins. For subcellular localization and transmembrane domain (TMD) analysis, we used the Plant-mPLoc ( http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ ) and TMHMM-2.0 ( https://services.healthtech.dtu.dk/services/TMHMM-2.0/ ) programs, respectively, to perform our predictions. Phylogenetic, chromosomal location, conserved motif, and protein structure analyses The maximum likelihood (ML) method, implemented in MEGA7.0 44 , was utilized with 1000 bootstrap replicates to construct a phylogenetic tree of MTP protein families in A. thaliana , B. rapa , B. nigra , and B. juncea . For enhanced visualization, the phylogenetic tree was refined and displayed using EvolView ( https://www.evolgenius.info/evolview/ ). The chromosomal location of BjMTP genes was obtained from the "Sichuan Yellow" genome GFF3 file. Subsequently, the MEME Suite 5.5.3 ( https://meme-suite.org/meme/tools/meme ) was used to identify conserved motifs in the BjMTP proteins, setting the number of motifs to 15. The SMART database ( http://smart.embl.de/ ) was utilized to analyze the conserved domains of BjMTP proteins and acquire the corresponding annotation results. Finally, TBtools-Ⅱ 43 software was used to visualize the evolutionary tree, chromosomal location, conserved motifs, and structural domain results of the BjMTP proteins. To further understand the structure and function of the BjMTP proteins, a comprehensive 3D structural domain prediction was conducted using the AlphaFold Protein Structure Database ( https://alphafold.ebi.ac.uk/ ). Analysis of the promoter elements and collinearity of BjMTPs The 2-kb bp upstream sequences preceding the start codon of the BjMTPs were analyzed for promoter elements using PlantCare ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). The results were carefully evaluated, statistically analyzed, and categorized. TBtools-Ⅱ was then used to generate visual representations based on the processed data. To gain a deeper understanding of the evolutionary patterns and expansion within the BjMTP family members, MCScanX and Ka (non-synonymous)/Ka (synonymous) calculator programs (both integrated within TBtools-Ⅱ) were employed to analyze collinearity and calculate selection pressure among the MTPs in B. juncea . Expression pattern and GO enrichment (GO) analysis To elucidate the expression patterns of MTPs in various tissues of B. juncea , RNA-seq data for "Sichuan Yellow" were retrieved from the NCBI database. These datasets included RNA-seq data for various tissues such as roots (SRR11787772), stems (SRR11787777), leaves (SRR11787776), buds (SRR11787782), siliques at 7 (SRR11787779) and 15 (SRR11787783) days after flowering (DAF), pods at 20 DAF (SRR11787780), seeds (SRR11787781), and seed coats (SRR807368). The data analysis was conducted following the methodology described by Kang 45 , with fragments per kilobase of transcript per million fragments mapped (FPKM) values used to quantify gene expression levels. A heatmap was subsequently generated to visualize the expression patterns of the BjMTP genes by plotting the Log 2 (FPKM + 1) values. To gain insights into the biological functions of BjMTP genes, all genes in this family were selected as the target gene set for enrichment analysis using the Gene Ontology (GO) database ( https://geneontology.org/ ). Significant enrichment results were then visualized using the Omicsmart platform ( https://www.omicsmart.com/RNAseq/home.html ). Plant materials, growth conditions, and treatments The seeds of "Sichuan yellow" were disinfected with a 50% sodium hypochlorite (v/v) solution, follow by thorough rinsing with distilled water to eliminate residual disinfectant. Hoagland's solution-soaked germination beds were used to plant the seeds. Following a week germination, the seedlings were carefully transplanted into opaque black containers that were filled with Hoagland's solution and exposed to a 16 h light/8 h dark photoperiod. Each container lid was equipped with six 1 cm diameter holes, each accommodating one seedling. In the phytotron, environmental conditions suitable for plant growth were precisely regulated: a light/dark cycle of 16 h/8 h, relative humidity of 50–60%, and a temperature of 24 ± 2°C. After four weeks of growth, upon reaching the 4–5 leaf stage, the plants were subjected to various treatments with slight modifications to the parameters described by Wu 28 : fresh Hoagland solution for the control group (metal-free), 250 mg Mn 2+ (MnSO 4 ·H 2 O), 250 mg Fe 2+ (FeSO 4 ·7H 2 O), 200 mg Zn 2+ (ZnSO 4 ·7H 2 O), 100 mg Cd 2+ (CdCl 2 ·2.5H 2 O), 10 mg Sb 3+ (KSbC 4 H 4 O 7 ·0.5H 2 O), and 250 mg Pb 2+ (PbCl 2 ). Each treatment was replicated across three containers, with six seedlings per container. Subsequently, root and leaf samples were collected at 12, 24, and 48 h after HM stress for RNA isolation using the Trizol Up plus RNA kit (TransGen Biotech, Beijing, China). The isolated RNA was reverse transcribed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and analyzed by qRT-PCR to assess gene expression levels, employing the AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). β-actin 46 was used as an internal control. The relative expression levels of target genes were calculated using the 2 −ΔΔct method. Each experiment included three biological and three technical replicates per treatment. Relative expression values (n) > 1 and < 1 represented positive and negative regulation [denoted by −(1/n)], respectively. All primers were synthesized by TSINKE Biotech (Table S1 ). Furthermore, we conducted gene co-expression network analysis using Wekemo Bioincloud software 47 , based on relative expression levels and employing Spearman correlation coefficients with a significance threshold of p < 0.05. Conclusion The MTP family plays a crucial role in plant transport and tolerance of divalent metal cations. In this study, we identified 33 MTP members in B. juncea for the first time using a combination of the Cation_efflux domain (PF01545) and BLAST analyses. Comprehensive analysis of conserved motifs, promoter elements, and RNA-seq expression data revealed that the 33 BjMTP genes are derived from BrMTP and BnMTP , exhibiting diversification that aligns with the seven evolutionary branches of the MTP family. Spatiotemporal expression profilings under HM stress successfully identified specific genes and crucial expression modules associated with the response of BjMTPs to HM stress. These findings laid a solid foundation for further investigation into the functional aspects of BjMTP genes and provided valuable genetic resources for breeding HM-tolerance varieties to cultivate in HM-contaminated soils. Declarations Competing interests The authors declare no competing interests. Author Contribution YL, CY, and LXJ provided the research idea and wrote the manuscript. YL, JLS, JGX,CH, YYH, GS, YML, HJH, XGH, and DRY contributed reagents, materials, and analysis tools. YL, JLS, JGX and CH performed the experiments. YL, LXJ and DRY revised the manuscript and provided fund support. All authors participated in writing and reviewing the manuscript. Acknowledgement This research was supported by Research Foundation of Education Bureau of Hunan Province, China(23B0809, 22B0844), Hunan Provincial Natural Science Foundation of China(2023JJ50083) and the National Natural Science Foundation of China(32371589). Data Availability The RNA-seq data of "Sichuan Yellow" in this study were retrieved from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/). These datasets included RNA-seq data for various tissues such as roots (SRR11787772), stems (SRR11787777), leaves (SRR11787776), buds (SRR11787782), siliques at 7 (SRR11787779) and 15 (SRR11787783) days after flowering (DAF), pods at 20 DAF (SRR11787780), seeds (SRR11787781), and seed coats (SRR807368). References Tiwari, S. & Lata, C. Heavy metal stress, signaling, and tolerance due to plant-associated microbes: an overview. Front. Plant Sci. 9, 336111 (2018). Riyazuddin, R. et al . A comprehensive review on the heavy metal toxicity and sequestration in plants. Biomolecules 12(1), 43 (2021). Williams, L. E. & Mills, R. F. P(1B)-ATPases–an ancient family of transition metal pumps with diverse functions in plants. Trends Plant Sci. 10(10), 491–502 (2005). Clemens, S. 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CLV1 cause a multilocular trait in Brassica juncea . Sci. Rep. 8(1), 5339 (2018). Gao, Y., Zhang, G., Jiang, S. & Liu, Y. Wekemo Bioincloud: A user-friendly platform for meta-omics data analyses. iMeta 3(1), e175 (2024). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4296734","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":297333944,"identity":"b7bd4e8b-1f8c-4058-b3e2-b0e42ec10baf","order_by":0,"name":"Liang You","email":"","orcid":"","institution":"Hunan Unversity of Humanities, Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"You","suffix":""},{"id":297333948,"identity":"ea2ee1c5-1edf-4c8f-bd26-454964ef788d","order_by":1,"name":"Jialin Sheng","email":"","orcid":"","institution":"Hunan Agricultural 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09:09:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4296734/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4296734/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-68877-8","type":"published","date":"2024-08-01T15:57:47+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55706302,"identity":"25d0a158-d56a-475e-8121-28d2d93ed014","added_by":"auto","created_at":"2024-05-02 04:59:11","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2158277,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis and chromosomal localization. (a) Phylogenetic analysis of 80 MTP proteins in \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eB. rapa\u003c/em\u003e, \u003cem\u003eB. nigra\u003c/em\u003e, and \u003cem\u003eB. juncea\u003c/em\u003e. The blue star, green circle, orange aquare, and red triangle denote MTPs from \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eB. rapa\u003c/em\u003e, \u003cem\u003eB. nigra\u003c/em\u003e, and \u003cem\u003eB. juncea\u003c/em\u003e, respectively. (b) Chromosomal localization of \u003cem\u003eBjMTP \u003c/em\u003egenes. Fonts in varying colors indicate distinct branches of the \u003cem\u003eBjMTP \u003c/em\u003egenes.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4296734/v1/55885cce008948d28ec13681.jpeg"},{"id":55706303,"identity":"b4e81171-ddd0-4a0a-a112-376696a19860","added_by":"auto","created_at":"2024-05-02 04:59:11","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1057693,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationship, conserved motif, and protein domain analyses of BjMTP proteins. (a) Phylogenetic tree of BjMTP proteins. The phylogenetic tree was constructed with the ML method of MEGA 7(1000 bootstrap replicates). (b) The analysis of conserved motifs in BjMTP proteins. In total, 15 different motifs are represented by squares with different colors. (c) Analysis of protein domains in BjMTP proteins. Various symbols represent different protein structural domains.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4296734/v1/a25bebe6b61d5d258de3948a.jpeg"},{"id":55706756,"identity":"8ec1d08b-e807-4695-9075-695617db95a8","added_by":"auto","created_at":"2024-05-02 05:07:11","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2072051,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of cis-regulatory elements in \u003cem\u003eBjMTP \u003c/em\u003epromoters. (a)Pie chart of cis-regulatory elements associated with growth and development. (b) Pie chart of cis-regulatory elements associated with hormone response. (c) Pie chart of cis-regulatory elements associated with stress response. (d) Analysis of the distribution of cis-regulatory elements in the promoter regions of \u003cem\u003eBjMTP \u003c/em\u003egenes among various branches.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4296734/v1/64ab3c83b268e6b04e9916c0.jpeg"},{"id":55706305,"identity":"f3460020-777e-4faa-a66e-8f5811ed2e07","added_by":"auto","created_at":"2024-05-02 04:59:11","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1269332,"visible":true,"origin":"","legend":"\u003cp\u003eThe collinear relationship among \u003cem\u003eBjMTP \u003c/em\u003egenes. The 18 chromosomes are depicted with distinct colors. Gray lines represent all collinear blocks within \u003cem\u003eB. juncea \u003c/em\u003egenome, whereas red lines indicate duplicated \u003cem\u003eBjMTP \u003c/em\u003egene pairs.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4296734/v1/2059ac54b683f2e0b42b7910.jpeg"},{"id":55706306,"identity":"98e21aa3-2aec-48f1-9183-da4d6c5ad12a","added_by":"auto","created_at":"2024-05-02 04:59:11","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2079967,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression pattern and GO enrichment analysis. (a) Analysis of expression pattern of \u003cem\u003eBjMTP \u003c/em\u003egenes. (b) GO enrichment analysis of \u003cem\u003eBjMTP \u003c/em\u003egenes.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4296734/v1/ab4965a564121d6c3be17b97.jpeg"},{"id":55706308,"identity":"4cc489d2-1a4d-44a6-bbcd-6ae68afb0bb0","added_by":"auto","created_at":"2024-05-02 04:59:11","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2901508,"visible":true,"origin":"","legend":"\u003cp\u003eThe phenotypic characteristics of \u003cem\u003eB. juncea\u003c/em\u003e plants after 48 h of exposure to various HMs. A, B, and C. The leaf and root phenotypes of \u003cem\u003eB. juncea\u003c/em\u003e plants treated with 250 mg/L Mn\u003csup\u003e2+\u003c/sup\u003e (a), 250 mg/L Fe\u003csup\u003e2+\u003c/sup\u003e (b), and 10 mg/L Sb\u003csup\u003e3+\u003c/sup\u003e (c).\u003c/p\u003e","description":"","filename":"image6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4296734/v1/0720aa166ba5a152cc00a15f.jpeg"},{"id":55706309,"identity":"155fead9-7e9c-4d3c-8d1c-04b6d9e1ff1f","added_by":"auto","created_at":"2024-05-02 04:59:11","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1034350,"visible":true,"origin":"","legend":"\u003cp\u003eExpression analysis of \u003cem\u003eBjMTP \u003c/em\u003egenes. (a, b) Expression heatmap of \u003cem\u003eBjMTP \u003c/em\u003egenes in the root (a) and leaf (b) tissues under various HMs treatments. (c, d) Co-expression network diagram of \u003cem\u003eBjMTP \u003c/em\u003egenes in the root (c) and leaf (d) tissues in response to six HMs. The node sizes (circle sizes) represent the magnitude of betweenness centrality, whereas the line color indicates the level of correlation between \u003cem\u003eBjMTP \u003c/em\u003egenes. Different colors in the circles indicate different subgroups within the \u003cem\u003eBjMTP \u003c/em\u003egenes.\u003c/p\u003e","description":"","filename":"image7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4296734/v1/bce83ac1bf6579b1e151f345.jpeg"},{"id":61794355,"identity":"b3eda132-3c62-440f-9f97-e79822e52526","added_by":"auto","created_at":"2024-08-05 16:18:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13399041,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4296734/v1/1d4c78e8-6bbd-4ff5-90c1-f568fb1c8af8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide exploration of MTP gene family in mustard (Brassica juncea L.): evolution and expression patterns during heavy metal stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGrowing concern is being expressed over the rising levels of heavy metal (HM) contamination in the environment as a result of industrial operations such as mining, exhaust emissions, waste management, artificial fertilizer use in agriculture, and urban wastewater treatment. Effective mitigation measures are desperately required since this scenario presents a serious danger to agricultural production\u003csup\u003e1,2\u003c/sup\u003e. Trace levels of essential HMs such as manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) are present in plants to regulate numerous physiological processes\u003csup\u003e3\u003c/sup\u003e. However, high concentrations of these essential HMs or even low concentrations of non-essential HMs including cadmium (Cd), lead (Pb), mercury (Hg), and antimony (Sb), may trigger phytotoxic consequences.\u0026nbsp;Such toxicity is characterized by growth restriction, chlorosis, root discoloration, and even plant death\u003csup\u003e2\u003c/sup\u003e\u003csup\u003e,4,5,6\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePlants have evolved\u0026nbsp;many defense mechanisms against HMs, such as\u0026nbsp;detoxification, sequestration, cell membrane transport processes, and raising the concentrations of compatible solutes\u003csup\u003e2,7\u003c/sup\u003e. By identifying metals and enabling their movement to particular places in plant tissues, some transport proteins are essential to these systems for preserving cellular homeostasis\u003csup\u003e2,8\u003c/sup\u003e. Studies\u0026nbsp;show that\u0026nbsp;families of transport proteins, such as the Zn-regulated iron-regulated transporter-like proteins (ZIPs), ATP-binding cassettes (ABCs), natural resistance-associated macrophage protein (NRAMPs), and cation diffusion facilitators (CDFs), are sequester HMs into vacuoles and chelate them to\u0026nbsp;reduce their toxicity\u003csup\u003e7,9\u003c/sup\u003e. The metal tolerance proteins (MTPs), also known as the cation transporter family (CDF), are a class of proteins that were first discovered in bacteria. They have a role\u0026nbsp;in the tolerance and transportation of divalent metal cations, including Mn\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, and Cd\u003csup\u003e2+\u003c/sup\u003e. MTPs are also necessary for plants\u0026nbsp;to detoxify HMs\u003csup\u003e7,10,11\u003c/sup\u003e. Structurally,\u0026nbsp;MTPs are characterized\u0026nbsp;by transmembrane domains (TMDs), metal ion recognition motifs (HXXXD or DXXXD), and cation transport domains\u003csup\u003e12\u003c/sup\u003e. These proteins are categorized into three major subgroups based on substrate specificity: Zn-CDF, Zn/Fe-CDF, and Mn-CDF. They facilitate\u0026nbsp;the transport of ions such as Zn\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Co\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, and Ni\u003csup\u003e2+\u003c/sup\u003e\u003csup\u003e\u0026nbsp;13\u003c/sup\u003e. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the 12 MTP proteins are further divided into seven branches: G1, G5, G6, G7, G8, G9, and G12, emphasizing the complexity and specificity of their roles\u003csup\u003e11\u003c/sup\u003e. Previous studies have shown the range of functions displayed by many MTP proteins.\u0026nbsp;As a vacuolar zinc transporter, AtMTP3 is necessory to regulate intracellular Zn\u003csup\u003e2+\u003c/sup\u003e levels and maintaining homeostasis\u003csup\u003e14\u003c/sup\u003e. The AtMTP8 protein is essential for acting as a transporter for Mn\u003csup\u003e2+\u003c/sup\u003e, in addition to regulating Fe transport during seed germination and developmental processes\u003csup\u003e15\u003c/sup\u003e. Additionally, a protein combination AtMTP5 and AtMTP12 may create helps to enhance\u0026nbsp;Zn\u003csup\u003e2+\u003c/sup\u003e transport into the Golgi apparatus\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBrassica juncea\u0026nbsp;\u003c/em\u003eL\u003cem\u003e.,\u0026nbsp;\u003c/em\u003eknown for its rapid vegetative growth and substantial\u0026nbsp;aboveground biomass, has been recognized as an effective accumulator\u0026nbsp;of various HMs, such as Pb, Cd, selenium (Se), Zn, chromium (Cr), Cu, and nickel (Ni). As a result, this plant is widely\u0026nbsp;used in studies on soil decontamination and remediation\u003csup\u003e17\u003c/sup\u003e\u003csup\u003e,18,19\u003c/sup\u003e. To date, only a few cation-efflux family genes, namely \u003cem\u003eBjCET1-4\u003c/em\u003e, have been identified in \u003cem\u003eB. juncea\u003c/em\u003e. Heterologous expression of these genes in yeast has demonstrated their ability\u0026nbsp;to confer tolerance against Zn and Cd\u003csup\u003e20,21,22\u003c/sup\u003e.\u0026nbsp;Understudied, however, are the thorough discovery and functional characterisation of other cation-efflux family members in \u003cem\u003eB. juncea\u003c/em\u003e\u0026apos;s response to HMs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere, we explored the MTP proteins in \u003cem\u003eB. juncea\u003c/em\u003e using bioinformatics techniques, with an emphasis on the dynamics of the \u003cem\u003eBjMTP\u0026nbsp;\u003c/em\u003egenes expression under HM stress. This research lays a foundation for further investigation into the molecular mechanisms of HM homeostasis in \u003cem\u003eB. juncea\u003c/em\u003e via \u003cem\u003eBjMTP\u0026nbsp;\u003c/em\u003efamily members and offers valuable genetic resources for breeding programs aimed at soil remediation.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv\u003e\n\u003ch2\u003eIdentification and physicochemical characterization of MTP proteins\u003c/h2\u003e\n\u003cp\u003eA total of 68 MTP proteins from three species, including \u003cem\u003eB. rapa\u003c/em\u003e (17), \u003cem\u003eB. nigra\u003c/em\u003e (18), and \u003cem\u003eB. juncea\u003c/em\u003e (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\u0026thinsp;=\u0026thinsp;r, n, and j). Significantly, the analysis of gene number and physicochemical characteristics of MTPs reveals that most BjMTP proteins in the allopolyploid \u003cem\u003eB. juncea\u003c/em\u003e (AABB, n\u0026thinsp;=\u0026thinsp;38) were the result of combining MTP proteins from its diploid ancestors, \u003cem\u003eB. rapa\u003c/em\u003e (AA, n\u0026thinsp;=\u0026thinsp;20) and \u003cem\u003eB. nigra\u003c/em\u003e (BB, n\u0026thinsp;=\u0026thinsp;18). However, homologs for BnB06.MTP4b and BnB01.MTP12 were not detected in \u003cem\u003eB. juncea\u003c/em\u003e. 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 (\u003cem\u003eBjMTP12\u003c/em\u003e) and a maximum of 6 (\u003cem\u003eBjMTP8\u003c/em\u003e) (Table S1).\u003c/p\u003e\n\u003cp\u003eBjMTP 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)\u0026thinsp;\u0026lt;\u0026thinsp;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).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003ePhylogenetic analysis and chromosomal localization of BjMTPs\u003c/h3\u003e\n\u003cp\u003eBased on the phylogenetic tree and structural characteristics of 80 MTP proteins from \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eB. rapa\u003c/em\u003e, \u003cem\u003eB. nigra\u003c/em\u003e, and \u003cem\u003eB. juncea\u003c/em\u003e, 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.\u0026nbsp;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 \u003cem\u003eBjMTP\u003c/em\u003e genes (1 and 10, respectively) were found in branches G12 and G9. Chromosomal localization analysis revealed that the A and B subgenomes of \u003cem\u003eB. juncea\u003c/em\u003e contain 17 and 16 \u003cem\u003eBjMTP\u003c/em\u003e 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 \u003cem\u003eBjMTP\u003c/em\u003e gene. No \u003cem\u003eBjMTP\u003c/em\u003e genes were found on chromosomes A01, A02, A08, A10, B02, B05, and B07 (Fig.\u0026nbsp;1b).\u003c/p\u003e\n\u003ch3\u003eStructural analysis of BjMTP proteins\u003c/h3\u003e\n\u003cp\u003eBy 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.\u0026nbsp;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.\u0026nbsp;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\u0026ndash;6 transmembrane helices, arranged into a compact cluster structure (Fig. S1). In contrast, Zn-MTP and Fe/Zn-MTP subgroups displayed either 1\u0026ndash;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 selectivity\u003csup\u003e23,24\u003c/sup\u003e. Therefore, Therefore, it was suggested by AlphaFold's predictions that the residues in BjMTPs are likewise intimately related to metal ion transport.\u003c/p\u003e\n\u003cp\u003eThe 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.\u003c/p\u003e\n\u003ch3\u003eAnalysis of Promoters in genes\u003c/h3\u003e\n\u003cdiv\u003eAnalysis of Promoters in \u003cem\u003eBjMTP\u003c/em\u003e genes\u003c/div\u003e\n\u003cp\u003eUsing PlantCARE software, the cis-regulatory components of the \u003cem\u003eBjMTP\u003c/em\u003e 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 \u003cem\u003eBjMTP\u003c/em\u003e genes, apart from the commonly observed cis-regulatory elements, three distinct categories\u003csup\u003e25\u003c/sup\u003e 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.\u0026nbsp;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.\u0026nbsp;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 \u003cem\u003eBjMTPs\u003c/em\u003e play a major role in adaptation to anaerobic stress (Fig.\u0026nbsp;3c). Except for \u003cem\u003eBjA04.MTP4\u003c/em\u003e and \u003cem\u003eBjB01.MTP5\u003c/em\u003e, the number of ARE elements varied among \u003cem\u003eBjMTP\u003c/em\u003e genes, ranging from one (\u003cem\u003eBjA04.MTP8\u003c/em\u003e) to nine (\u003cem\u003eBjA03.MTP1\u003c/em\u003e) (Fig.\u0026nbsp;3d).\u003c/p\u003e\n\u003cp\u003eOnce 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.\u0026nbsp;3d). As a result, a wide variety of cis-regulatory elements are present in \u003cem\u003eBjMTP\u003c/em\u003e 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 \u003cem\u003eBjMTP\u003c/em\u003e genes throughout evolutionary branches.\u003c/p\u003e\n\u003ch3\u003eSynteny analysis of BjMTP genes\u003c/h3\u003e\n\u003cp\u003eWhole genome duplication, segmental duplication, and tandem duplication are crucial mechanisms driving gene family evolution\u003csup\u003e26\u003c/sup\u003e. Syntenic analysis was used to identify the \u003cem\u003eBjMTP\u003c/em\u003e genes in order to investigate gene duplication occurrences in these genes. All 32 \u003cem\u003eBjMTP\u003c/em\u003e genes, with the exception of \u003cem\u003eBjA04.MTP12\u003c/em\u003e, showed evolutionary events involving 44 pairs, mostly related to segmental duplication (Fig.\u0026nbsp;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\u0026thinsp;=\u0026thinsp;1. This data strongly implies that purifying selection occurred in all 44 pairs of duplicated genes, proving that the \u003cem\u003eBjMTP\u003c/em\u003e genes did not develop new functions. These results suggested that related \u003cem\u003eBjMTP\u003c/em\u003e genes probably operate similarly, especially when subjected to HM stress.\u003c/p\u003e\n\u003cdiv\u003e\n\u003ch2\u003eExpression patterns and GO enrichment analysis\u003c/h2\u003e\n\u003cp\u003eUtilizing RNA-seq data from the NCBI database, the expression patterns of \u003cem\u003eBjMTP\u003c/em\u003e genes in the different tissues of \u003cem\u003eB. juncea\u003c/em\u003e were clarified. 33 \u003cem\u003eBjMTP\u003c/em\u003e 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 \u003cem\u003eBjMTPs\u003c/em\u003e was shown to be correlated with their expression. Homologous \u003cem\u003eBjMTP\u003c/em\u003e genes generally exhibited similar expression patterns across tissues, with few exceptions. In stems, leaves, seed coats, and seed, for instance, \u003cem\u003eBjMTP4\u003c/em\u003e was minimally expressed, while \u003cem\u003eBjMTP11\u003c/em\u003e showed greater expression levels. Moreover, unique expression features were shown by the G9 branch genes \u003cem\u003eBjMTP9\u003c/em\u003e, \u003cem\u003eBjMTP10\u003c/em\u003e, and \u003cem\u003eBjMTP11\u003c/em\u003e, with higher expression in leaves and buds and reduced expression in seeds (Fig.\u0026nbsp;5a, Table S5). This implies a correlation between the evolutionary branches of \u003cem\u003eBjMTP\u003c/em\u003es and the tissue-specific expression, potentially linked to the cis-regulatory elements in their promoters. Additionally, certain \u003cem\u003eBjMTP\u003c/em\u003es showed patterns of tissue-specific or ubiquitous expression. For instance, \u003cem\u003eBjA04.MTP1\u003c/em\u003e had high expression levels in a variety of tissues, but \u003cem\u003eBjB06.MTP9\u003c/em\u003e, \u003cem\u003eBjA07.MTP9\u003c/em\u003e, and \u003cem\u003eBjB01.MTP11\u003c/em\u003e showed significant increases in expression in leaf tissues, indicating specialized functions in specific tissues.\u003c/p\u003e\n\u003cp\u003eBy using GO enrichment analysis, the molecular functions of the \u003cem\u003eBjMTP\u003c/em\u003e genes were further clarified. Biological activities such as efflux, cation, and ion transmembrane transporter activity were covered by the top 20 GO terms (Fig.\u0026nbsp;5b). Notably, the predicted biological functions of \u003cem\u003eBjMTP\u003c/em\u003e genes were associated with essential processes such as zinc ion homeostasis maintenance and vacuolar membrane transport facilitation\u003csup\u003e7,11\u003c/sup\u003e. These results align with earlier findings on protein structure and subcellular localization, highlighting the significant role of \u003cem\u003eBjMTP\u003c/em\u003e genes in metal cation transport.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003ePlant phenotypes and expression profiles of \u003cem\u003eBjMTPs\u003c/em\u003e under six HMs stresses\u003c/h2\u003e\n\u003cp\u003eA 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 Mn\u003csup\u003e2+\u003c/sup\u003e, 250 mg/L Fe\u003csup\u003e2+\u003c/sup\u003e, and 10 mg/L Sb\u003csup\u003e3+\u003c/sup\u003e on plant morphologies were noticeable (Fig.\u0026nbsp;6). The leaves developed uneven white spots as a result of Mn\u003csup\u003e2+\u003c/sup\u003e exposure. Fe\u003csup\u003e2+\u003c/sup\u003e stress was connected to overt signs of dehydration. Additionally, a low Sb\u003csup\u003e3+\u003c/sup\u003e concentration resulted in root yellowing, which was subsequently connected to an unidentified bacterial strain (unpublished data). Thus, in \u003cem\u003eB. juncea\u003c/em\u003e, both non-essential (Sb\u003csup\u003e3+\u003c/sup\u003e) and necessary (Mn\u003csup\u003e2+\u003c/sup\u003e and Fe\u003csup\u003e2+\u003c/sup\u003e) metals may have a negative impact on plant health.\u003c/p\u003e\n\u003cp\u003eFrom the 33 \u003cem\u003eBjMTP\u003c/em\u003e 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 \u003cem\u003eBjMTP\u003c/em\u003e gene expression. The selected genes included \u003cem\u003eBjA04.MTP1\u003c/em\u003e and \u003cem\u003eBjA04.MTP4\u003c/em\u003e (G1); \u003cem\u003eBjB01.MTP5\u003c/em\u003e (G5); \u003cem\u003eBjB08.MTP6\u003c/em\u003e (G6); \u003cem\u003eBjA06.MTP7\u003c/em\u003e (G7); \u003cem\u003eBjA09.MTP8\u003c/em\u003e (G8); \u003cem\u003eBjB06.MTP9\u003c/em\u003e, \u003cem\u003eBjA09.MTP10\u003c/em\u003e, and \u003cem\u003eBjA05.MTP11\u003c/em\u003e (G9); and \u003cem\u003eBjA04.MTP12\u003c/em\u003e (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.\u0026nbsp;7a-b). \u003cem\u003eBjA04.MTP4\u003c/em\u003e and \u003cem\u003eBjB01.MTP5\u003c/em\u003e were markedly elevated in roots during the first 12 and 24 hours of Fe\u003csup\u003e2+\u003c/sup\u003e and Sb\u003csup\u003e3+\u003c/sup\u003e 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 \u003cem\u003eB. juncea\u003c/em\u003e that were seen. Within the Mn-CDF subgroup, only \u003cem\u003eBjA09.MTP10\u003c/em\u003e responded to Mn\u003csup\u003e2+\u003c/sup\u003e stress in roots after 48 h. All HM treatments activated MTP8, which is known to modulate Mn\u003csup\u003e2+\u003c/sup\u003e transport and tolerance\u003csup\u003e15,27\u003c/sup\u003e, in leaf tissues. Significantly, \u003cem\u003eBjA04.MTP4\u003c/em\u003e, \u003cem\u003eBjA09.MTP10\u003c/em\u003e, and B\u003cem\u003ejB01.MTP5\u003c/em\u003e genes showed the greatest levels of expression (997.1-, 370.5-, and 2137.3-fold greater than control, respectively) under Sb\u003csup\u003e3+\u003c/sup\u003e and Pb\u003csup\u003e2+\u003c/sup\u003e exposures (Fig. S2). Furthermore, the consistently downregulated expression of \u003cem\u003eBjB06.MTP9\u003c/em\u003e under various HM stresses highlighted its potential negative regulatory role in HM stress responses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003eCo-expression networks of \u003cem\u003eBjMTPs\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eTo elucidate the regulatory mechanisms underlying gene responses to HM stress, co-expression networks are essential\u003csup\u003e28\u003c/sup\u003e. In this study, expression data for \u003cem\u003eBjMTP\u003c/em\u003e genes across various tissues, HM stresses, and treatment durations were used to construct co-expression networks (Fig.\u0026nbsp;7a-b; Table S6). Consistent gene expression patterns were found in the root tissues exposed to HMs, namely in the cases of G1 (\u003cem\u003eBjA04.MTP4\u003c/em\u003e), G5 (\u003cem\u003eBjB01.MTP5\u003c/em\u003e), G7 (\u003cem\u003eBjA06.MTP7\u003c/em\u003e), and G9 (\u003cem\u003eBjB06.MTP9\u003c/em\u003e, \u003cem\u003eBjA09.MTP10\u003c/em\u003e, and \u003cem\u003eBjA05.MTP11\u003c/em\u003e) (Fig.\u0026nbsp;7a, c). In response to each of the six tested HMs, these genes showed synergistic positive regulation. \u003cem\u003eBjA04.MTP4\u003c/em\u003e (G4), \u003cem\u003eBjB01.MTP5\u003c/em\u003e (G5), and \u003cem\u003eBjA09.MTP8\u003c/em\u003e (G8) were found to be involved in the possible co-regulation of Zn\u003csup\u003e2+\u003c/sup\u003e transport in leaf tissues, with \u003cem\u003eBjA09.MTP8\u003c/em\u003e demonstrating a positive response to HM stressors, which was less evident in roots (Fig.\u0026nbsp;7b, d). Furthermore, there was no discernible alteration in the expression of the neutral gene \u003cem\u003eBjA04.MTP12\u003c/em\u003e in any of the tissues. 10 \u003cem\u003eBjMTP\u003c/em\u003e 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 (\u003cem\u003eBjB06.MTP9\u003c/em\u003e, \u003cem\u003eBjA09.MTP10\u003c/em\u003e, and \u003cem\u003eBjA05.MTP11\u003c/em\u003e) 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.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eThe allopolyploid\u003c/strong\u003e\u003cstrong\u003eB. juncea\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;s\u003c/strong\u003e\u003cstrong\u003eMTP\u003c/strong\u003e\u003cstrong\u003egenes derive from diploid progenitors\u003c/strong\u003e\u003cstrong\u003eB. rapa\u003c/strong\u003e\u003cstrong\u003eand\u003c/strong\u003e\u003cstrong\u003eB. nigra\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMTPs are a broad family of transport proteins found in bacteria, fungi, plants, and animals. These proteins have a crucial role in regulating the cellular efflux of excess metal ions and maintaining ionic homeostasis\u003csup\u003e13\u003c/sup\u003e. MTP proteins have been identified in various plant species, such as \u003cem\u003eA. thaliana\u003c/em\u003e (12 genes), \u003cem\u003eSolanum Lycopersicum\u003c/em\u003e (11 genes), \u003cem\u003eMedicago truncatula\u003c/em\u003e (12 genes), \u003cem\u003eGlycine max\u003c/em\u003e (20 genes)\u003csup\u003e29\u003c/sup\u003e, \u003cem\u003eSolanum tuberosum\u003c/em\u003e (21 genes)\u003csup\u003e28\u003c/sup\u003e, \u003cem\u003eArachis hypogaea\u003c/em\u003e L. (22 genes)\u003csup\u003e30\u003c/sup\u003e, and \u003cem\u003eB. napus\u003c/em\u003e (33 genes)\u003csup\u003e31\u003c/sup\u003e. In this study, we identified 17, 18, and 33 MTPs in the genomes of the diploid parental species \u003cem\u003eB. rapa\u003c/em\u003e, \u003cem\u003eB. nigra\u003c/em\u003e, and the allopolyploid species \u003cem\u003eB. juncea\u003c/em\u003e, respectively. Comparative studies of \u003cem\u003eMTP\u003c/em\u003e genes indicated that \u003cem\u003eBjMTPs\u003c/em\u003e are made up of the homologs \u003cem\u003eBrMTP\u003c/em\u003e and \u003cem\u003eBnMTP\u003c/em\u003e, without novel additions. Notably, the loss of \u003cem\u003eBnB06.MTP4b\u003c/em\u003e and \u003cem\u003eBnB01.MTP12\u003c/em\u003e from \u003cem\u003eB. nigra\u003c/em\u003e was observed during the \u003cem\u003eB. juncea\u003c/em\u003e polyploidization event. This genomic complexity greatly raises the likelihood of various gene functions, but it also increases the difficulty of functional research. Furthermore, the \u003cem\u003eB. juncea\u003c/em\u003e genome lacks homologs tor \u003cem\u003eAtMTP2\u003c/em\u003e and \u003cem\u003eAtMTP3\u003c/em\u003e, paralleling the absence of an \u003cem\u003eAtMTP7\u003c/em\u003e homolog in \u003cem\u003eB. napus\u003c/em\u003e\u003csup\u003e31\u003c/sup\u003e. This might be the result of mismatches in gene classification caused by the high degree of homology among MTP genes within the subgroups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe seven-branch model may elucidate\u003c/strong\u003e\u003cstrong\u003eBjMTP\u003c/strong\u003e\u003cstrong\u003efunctional diversity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe functions of the BjMTP gene family were clarified by the use of integrated bioinformatic approaches such as phylogenetic tree, conserved motif, protein structure, promoter element, collinearity, RNA-seq expression pattern, and GO enrichment investigations. Phylogenetic analysis revealed that the BjMTP gene family, similar to those in \u003cem\u003eA. thaliana\u003c/em\u003e\u003csup\u003e11\u003c/sup\u003e, \u003cem\u003eB. nupus\u003c/em\u003e\u003csup\u003e31\u003c/sup\u003e, and \u003cem\u003eG. max\u003c/em\u003e\u003csup\u003e29\u003c/sup\u003e, can be categorized into three subgroups (Zn-CDF, Mn-CDF, and Fe/Zn-CDF) and seven major branches (G1, G5, G6, G7, G8, G9, and G12). The \u003cem\u003eBjMTPs\u003c/em\u003e were divided into three subgroups by earlier research based on the substrate-binding selectivity for Zn\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;and Fe\u003csup\u003e2\u0026thinsp;+\u0026thinsp;13\u003c/sup\u003e. However, phylogenomic investigations of the genomes of plants and algae have revealed seven branches within the CDF family, which are believed to date back to the origin of land plants\u003csup\u003e11\u003c/sup\u003e. According to our analysis of the conserved motifs, promoter elements, and RNA-seq expression patterns, most \u003cem\u003eBjMTP\u003c/em\u003e genes are probably more closely related to the seven-branch classification. Moreover, similar results were also observed for other crops\u003csup\u003e29,31,32\u003c/sup\u003e.Specifically, after several whole-genome duplication events, \u003cem\u003eB. juncea\u003c/em\u003e grew from the 12 \u003cem\u003eAtMTPs\u003c/em\u003e seen in the model plant \u003cem\u003eA. thaliana\u003c/em\u003e to 33 \u003cem\u003eBjMTPs\u003c/em\u003e. These genes consistently inherit all seven ancient branches, with gene counts per branch varying from one to six (Table S2). Through establishing a relationship between the conserved Cation_efflux domain's metal transport properties and the variations observed among the seven branches of the BjMTP family, one can infer that the functional variations in metal ion transport exhibited by BjMTPs are dependent on the particular branches.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBjMTP\u003c/strong\u003e\u003cstrong\u003egenes exhibit a broad-spectrum response to heavy metal stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eB. juncea\u003c/em\u003e is a globally important oilseed and vegetable crop, known for its high tolerance to various abiotic stresses, such as salt, drought, and heavy metals\u003csup\u003e18,33,34\u003c/sup\u003e. Prior research has clarified the function of MTP genes in the detoxification of divalent metals, such as Zn\u003csup\u003e2+\u003c/sup\u003e, Mn\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Cu\u003csup\u003e2+\u003c/sup\u003e, Pb\u003csup\u003e2+\u003c/sup\u003e, Hg\u003csup\u003e2+\u003c/sup\u003e, and Se\u003csup\u003e2+\u003c/sup\u003e, both essential and non-essential\u003csup\u003e29,31\u003c/sup\u003e. In this study, six HM ions (Mn\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Sb\u003csup\u003e3+\u003c/sup\u003e, and Pb\u003csup\u003e2+\u003c/sup\u003e) were introduced to \u003cem\u003eB. juncea\u003c/em\u003e seedlings in order to clarify the role of \u003cem\u003eBjMTP\u003c/em\u003e genes in response to metal stress. Sb\u003csup\u003e3\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;is a non-essential HM rarely included in studies; yet, it possesses phytotoxic threats and potential carcinogenic effects\u003csup\u003e6,35,36\u003c/sup\u003e. According to this investigation, \u003cem\u003eB. juncea\u003c/em\u003e seedlings showed notable phenotypic alterations after 48 h treatments with 250 mg/L Mn\u003csup\u003e2+\u003c/sup\u003e, 250 mg/L Fe\u003csup\u003e2+\u003c/sup\u003e, and 10 mg/L Sb\u003csup\u003e3+\u003c/sup\u003e, indicating the highest concentration of these HMs that \u003cem\u003eB. juncea\u003c/em\u003e can tolerate. The fact that the other three HMs had no effect on the plant phenotype suggests that \u003cem\u003eB. juncea\u003c/em\u003e plants might be temporarily tolerant of this concentration levels.\u003c/p\u003e\n\u003cp\u003eWithin \u003cem\u003eA. thaliana\u003c/em\u003e, the cation efflux transporter \u003cem\u003eAtMTP1\u003c/em\u003e is involved in Zn detoxification\u003csup\u003e37\u003c/sup\u003e. Numerous metals, including Zn, Fe, Co, and Ni, translocate due in part to \u003cem\u003eOsMTP1\u003c/em\u003e\u003csup\u003e38,39\u003c/sup\u003e. In this investigation, Zn\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;and Fe\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;stresses caused \u003cem\u003eBjA04.MTP1\u003c/em\u003e to be elevated in \u003cem\u003eB. juncea\u003c/em\u003e roots. But compared to \u003cem\u003eBjA04.MTP4\u003c/em\u003e (of the same G1 branch), which helps cucumbers maintain Zn\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;homeostasis and sequester Cd\u003csup\u003e39\u003c/sup\u003e, its expression was less noticeable. Notably, in response to six HMs, root tissues showed a substantial upregulation of \u003cem\u003eBjA04.MTP4\u003c/em\u003e, \u003cem\u003eBjB01.MTP5\u003c/em\u003e, and \u003cem\u003eBjA09.MTP10\u003c/em\u003e, suggestingthe potential of comprehensive metal tolerance and transport across these genes. It has been shown that MTP8 proteins regulate Mn\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;transport and provide Mn\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;tolerance\u003csup\u003e15,27\u003c/sup\u003e. To improve resistance to Mn\u003csup\u003e2+\u003c/sup\u003e, OsMTP8 sequesters Mn in the vacuoles of rice panicle cells\u003csup\u003e27\u003c/sup\u003e. Surprisingly, our findings showed that \u003cem\u003eBjA09.MTP8\u003c/em\u003e overexpression was only stimulated by Fe\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;in the roots; in contrast, \u003cem\u003eBjA09.MTP8\u003c/em\u003e reacted to almost all HMs in the leaves, perhaps explaining the speckled leaves that were seen after Mn\u003csup\u003e2\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;exposure. Related to the Mn-CDF subfamily as well, \u003cem\u003eBjB06.MTP9\u003c/em\u003e showed opposing regulatory effects on HM tolerance and stress responses in the tissues of the roots and leaves. These findings highlight the \u003cem\u003eMTP\u003c/em\u003e genes' interspecific functional variety and spatial expression in HM stress responses. Numerous transcriptome studies on HM stress have shown this behavior\u003csup\u003e40,41\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCo-expression networks are essential for studying gene regulation and play a critical role in identifying novel genes for plant growth and environmental adaptation\u003csup\u003e28,42\u003c/sup\u003e. A co-expression network study of the \u003cem\u003eS. tuberosum\u003c/em\u003e OPT family has shown that \u003cem\u003eStOPT1\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e is upregulated in the leaves under Cd and Cu stress and \u003cem\u003eStYSL5\u003c/em\u003e/\u003cem\u003e11\u003c/em\u003e is upregulated in the roots in response to Cu and Zn stress\u003csup\u003e28\u003c/sup\u003e. These results emphasize the tissue-specific, metal-responsive, and synergistic regulation properties of HM-responsive genes. Our research, which used co-expression network analysis to examine the response patterns of BjMTPs to HMs, also revealed a similar trend. The results indicated that whereas modules G1 (\u003cem\u003eBjA04.MTP4\u003c/em\u003e), G5 (\u003cem\u003eBjB01.MTP5\u003c/em\u003e), G7 (\u003cem\u003eBjA06.MTP7\u003c/em\u003e), and G9 (\u003cem\u003eBjB06.MTP9\u003c/em\u003e, \u003cem\u003eBjA09.MTP10\u003c/em\u003e, and \u003cem\u003eBjA05.MTP11\u003c/em\u003e) participate in the HM stress response in the roots, modules G4 (\u003cem\u003eBjA04.MTP4\u003c/em\u003e), G5 (\u003cem\u003eBjB01.MTP5\u003c/em\u003e), and G8 (\u003cem\u003eBjA09.MTP8\u003c/em\u003e) co-respond to HM stress in the leaves. Furthermore, this is the first study that we are aware of that incorporates Sb\u003csup\u003e3\u0026thinsp;+\u003c/sup\u003e\u0026thinsp;into stress response tests on \u003cem\u003eBjMTPs\u003c/em\u003e. According to our findings, most \u003cem\u003eBjMTPs\u003c/em\u003e were triggered by low concentrations of Sb, indicating that MTP proteins may be involved in the uptake and transport of multivalent metals. The specific genes of \u003cem\u003eB. juncea\u003c/em\u003e implicated in the response to HM stress were revealed by this study, laying the groundwork for future research into the characterisation of \u003cem\u003eBjMTPs\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003eIdentification of MTPs\u003c/h2\u003e\n\u003cp\u003eThe genome and protein sequences of \u003cem\u003eB. rapa\u003c/em\u003e (Chiifu v3.5), \u003cem\u003eB. nigra\u003c/em\u003e (NI100 v2), and \u003cem\u003eB. juncea\u003c/em\u003e (Sichuan Yellow v1.2) were obtained from the Brassicaceae database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://brassicadb.cn/#/\u003c/span\u003e\u003c/span\u003e) and the Molecular Breeding of Oilseed website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.oilseedhunan.net/\u003c/span\u003e\u003c/span\u003e), respectively. In addition, 12 \u003cem\u003eArabidopsis\u003c/em\u003e MTP proteins were downloaded from the NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003c/span\u003e), and the HMM file of the MTP conserved domain (Cation_efflux, PF01545) was acquired from the PFAM datebase (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003c/span\u003e). To identify MTP proteins in \u003cem\u003eB. rapa\u003c/em\u003e, \u003cem\u003eB. nigra\u003c/em\u003e, and \u003cem\u003eB. juncea\u003c/em\u003e, the HMMER (v3.2.1) program under TBtools-Ⅱ(v2.028)\u003csup\u003e43\u003c/sup\u003e was used to screen the protein files for the cation efflux domain, setting the E-value cutoff at 1E-07. Subsequently, the identified MTP proteins were aligned with AtMTPs, retaining only those with an identity greater than 75% for further analysis. The ExPasy tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/compute_pi/\u003c/span\u003e\u003c/span\u003e) was utilized to predict the isoelectric point (pI), molecular weight (MW), and physicochemical properties of BjMTP proteins. For subcellular localization and transmembrane domain (TMD) analysis, we used the Plant-mPLoc (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/bioinf/plant-multi/\u003c/span\u003e\u003c/span\u003e) and TMHMM-2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/TMHMM-2.0/\u003c/span\u003e\u003c/span\u003e) programs, respectively, to perform our predictions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003ePhylogenetic, chromosomal location, conserved motif, and protein structure analyses\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe maximum likelihood (ML) method, implemented in MEGA7.0\u003csup\u003e44\u003c/sup\u003e, was utilized with 1000 bootstrap replicates to construct a phylogenetic tree of MTP protein families in \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eB. rapa\u003c/em\u003e, \u003cem\u003eB. nigra\u003c/em\u003e, and \u003cem\u003eB. juncea\u003c/em\u003e. For enhanced visualization, the phylogenetic tree was refined and displayed using EvolView (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.evolgenius.info/evolview/\u003c/span\u003e\u003c/span\u003e). The chromosomal location of \u003cem\u003eBjMTP\u003c/em\u003e genes was obtained from the \"Sichuan Yellow\" genome GFF3 file. Subsequently, the MEME Suite 5.5.3 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003c/span\u003e) was used to identify conserved motifs in the BjMTP proteins, setting the number of motifs to 15. The SMART database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl.de/\u003c/span\u003e\u003c/span\u003e) was utilized to analyze the conserved domains of BjMTP proteins and acquire the corresponding annotation results. Finally, TBtools-Ⅱ\u003csup\u003e43\u003c/sup\u003e software was used to visualize the evolutionary tree, chromosomal location, conserved motifs, and structural domain results of the BjMTP proteins. To further understand the structure and function of the BjMTP proteins, a comprehensive 3D structural domain prediction was conducted using the AlphaFold Protein Structure Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://alphafold.ebi.ac.uk/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003eAnalysis of the promoter elements and collinearity of \u003cem\u003eBjMTPs\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe 2-kb bp upstream sequences preceding the start codon of the \u003cem\u003eBjMTPs\u003c/em\u003e were analyzed for promoter elements using PlantCare (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003c/span\u003e). The results were carefully evaluated, statistically analyzed, and categorized. TBtools-Ⅱ was then used to generate visual representations based on the processed data. To gain a deeper understanding of the evolutionary patterns and expansion within the BjMTP family members, MCScanX and Ka (non-synonymous)/Ka (synonymous) calculator programs (both integrated within TBtools-Ⅱ) were employed to analyze collinearity and calculate selection pressure among the MTPs in \u003cem\u003eB. juncea\u003c/em\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eExpression pattern and GO enrichment (GO) analysis\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo elucidate the expression patterns of \u003cem\u003eMTPs\u003c/em\u003e in various tissues of \u003cem\u003eB. juncea\u003c/em\u003e, RNA-seq data for \"Sichuan Yellow\" were retrieved from the NCBI database. These datasets included RNA-seq data for various tissues such as roots (SRR11787772), stems (SRR11787777), leaves (SRR11787776), buds (SRR11787782), siliques at 7 (SRR11787779) and 15 (SRR11787783) days after flowering (DAF), pods at 20 DAF (SRR11787780), seeds (SRR11787781), and seed coats (SRR807368). The data analysis was conducted following the methodology described by Kang\u003csup\u003e45\u003c/sup\u003e, with fragments per kilobase of transcript per million fragments mapped (FPKM) values used to quantify gene expression levels. A heatmap was subsequently generated to visualize the expression patterns of the \u003cem\u003eBjMTP\u003c/em\u003e genes by plotting the Log\u003csub\u003e2\u003c/sub\u003e (FPKM\u0026thinsp;+\u0026thinsp;1) values.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo gain insights into the biological functions of \u003cem\u003eBjMTP\u003c/em\u003e genes, all genes in this family were selected as the target gene set for enrichment analysis using the Gene Ontology (GO) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://geneontology.org/\u003c/span\u003e\u003c/span\u003e). Significant enrichment results were then visualized using the Omicsmart platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.omicsmart.com/RNAseq/home.html\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003ePlant materials, growth conditions, and treatments\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe seeds of \"Sichuan yellow\" were disinfected with a 50% sodium hypochlorite (v/v) solution, follow by thorough rinsing with distilled water to eliminate residual disinfectant. Hoagland's solution-soaked germination beds were used to plant the seeds. Following a week germination, the seedlings were carefully transplanted into opaque black containers that were filled with Hoagland's solution and exposed to a 16 h light/8 h dark photoperiod. Each container lid was equipped with six 1 cm diameter holes, each accommodating one seedling. In the phytotron, environmental conditions suitable for plant growth were precisely regulated: a light/dark cycle of 16 h/8 h, relative humidity of 50\u0026ndash;60%, and a temperature of 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. After four weeks of growth, upon reaching the 4\u0026ndash;5 leaf stage, the plants were subjected to various treatments with slight modifications to the parameters described by Wu\u003csup\u003e28\u003c/sup\u003e: fresh Hoagland solution for the control group (metal-free), 250 mg Mn\u003csup\u003e2+\u003c/sup\u003e (MnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;H\u003csub\u003e2\u003c/sub\u003eO), 250 mg Fe\u003csup\u003e2+\u003c/sup\u003e (FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO), 200 mg Zn\u003csup\u003e2+\u003c/sup\u003e (ZnSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO), 100 mg Cd\u003csup\u003e2+\u003c/sup\u003e (CdCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2.5H\u003csub\u003e2\u003c/sub\u003eO), 10 mg Sb\u003csup\u003e3+\u003c/sup\u003e (KSbC\u003csub\u003e4\u003c/sub\u003eH\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e\u0026middot;0.5H\u003csub\u003e2\u003c/sub\u003eO), and 250 mg Pb\u003csup\u003e2+\u003c/sup\u003e (PbCl\u003csub\u003e2\u003c/sub\u003e). Each treatment was replicated across three containers, with six seedlings per container. Subsequently, root and leaf samples were collected at 12, 24, and 48 h after HM stress for RNA isolation using the Trizol Up plus RNA kit (TransGen Biotech, Beijing, China). The isolated RNA was reverse transcribed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China) and analyzed by qRT-PCR to assess gene expression levels, employing the AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). \u0026beta;-actin\u003csup\u003e46\u003c/sup\u003e was used as an internal control. The relative expression levels of target genes were calculated using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;ct\u003c/sup\u003e method. Each experiment included three biological and three technical replicates per treatment. Relative expression values (n)\u0026thinsp;\u0026gt;\u0026thinsp;1 and \u0026lt;\u0026thinsp;1 represented positive and negative regulation [denoted by \u0026minus;(1/n)], respectively. All primers were synthesized by TSINKE Biotech (Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Furthermore, we conducted gene co-expression network analysis using Wekemo Bioincloud software\u003csup\u003e47\u003c/sup\u003e, based on relative expression levels and employing Spearman correlation coefficients with a significance threshold of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe MTP family plays a crucial role in plant transport and tolerance of divalent metal cations. In this study, we identified 33 MTP members in \u003cem\u003eB. juncea\u003c/em\u003e for the first time using a combination of the Cation_efflux domain (PF01545) and BLAST analyses. Comprehensive analysis of conserved motifs, promoter elements, and RNA-seq expression data revealed that the\u0026nbsp;33 \u003cem\u003eBjMTP\u003c/em\u003egenes are derived from \u003cem\u003eBrMTP\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBnMTP\u003c/em\u003e, exhibiting diversification that aligns with the seven evolutionary branches of the MTP family. Spatiotemporal expression profilings under HM stress successfully identified specific genes and crucial expression modules associated with the response of \u003cem\u003eBjMTPs\u003c/em\u003e to HM stress. These findings laid a solid foundation for further investigation into the functional aspects of \u003cem\u003eBjMTP\u0026nbsp;\u003c/em\u003egenes and provided valuable genetic resources for breeding HM-tolerance varieties to cultivate in HM-contaminated soils.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eYL, CY, and LXJ provided the research idea and wrote the manuscript. YL, JLS, JGX,CH, YYH, GS, YML, HJH, XGH, and DRY contributed reagents, materials, and analysis tools. YL, JLS, JGX and CH performed the experiments. YL, LXJ and DRY revised the manuscript and provided fund support. All authors participated in writing and reviewing the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was supported by Research Foundation of Education Bureau of Hunan Province, China(23B0809, 22B0844), Hunan Provincial Natural Science Foundation of China(2023JJ50083) and the National Natural Science Foundation of China(32371589).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe RNA-seq data of \"Sichuan Yellow\" in this study were retrieved from the National Center for Biotechnology Information database (https://www.ncbi.nlm.nih.gov/). These datasets included RNA-seq data for various tissues such as roots (SRR11787772), stems (SRR11787777), leaves (SRR11787776), buds (SRR11787782), siliques at 7 (SRR11787779) and 15 (SRR11787783) days after flowering (DAF), pods at 20 DAF (SRR11787780), seeds (SRR11787781), and seed coats (SRR807368).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTiwari, S. \u0026amp; Lata, C. Heavy metal stress, signaling, and tolerance due to plant-associated microbes: an overview. Front. Plant Sci. 9, 336111 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiyazuddin, R. \u003cem\u003eet al\u003c/em\u003e. A comprehensive review on the heavy metal toxicity and sequestration in plants. Biomolecules 12(1), 43 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWilliams, L. E. \u0026amp; Mills, R. F. P(1B)-ATPases\u0026ndash;an ancient family of transition metal pumps with diverse functions in plants. 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Genet. 53(9), 1392\u0026ndash;1402 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao, L. \u003cem\u003eet al\u003c/em\u003e. Mutations in the CDS and promoter of \u003cem\u003eBjuA07. CLV1\u003c/em\u003e cause a multilocular trait in \u003cem\u003eBrassica juncea\u003c/em\u003e. Sci. Rep. 8(1), 5339 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao, Y., Zhang, G., Jiang, S. \u0026amp; Liu, Y. Wekemo Bioincloud: A user-friendly platform for meta-omics data analyses. iMeta 3(1), e175 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Brassica juncea L., MTP gene, Heavy metals, Specific genes, Expression profiles","lastPublishedDoi":"10.21203/rs.3.rs-4296734/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4296734/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMembers of the Metal Tolerance Protein (MTP) family are critical in mediating the transport and tolerance of divalent metal cations. Despite their significance, little is known about the MTP genes in mustard (\u003cem\u003eBrassica juncea\u003c/em\u003e), particularly in relation to how they react to HM stress. In our study, we identified \u003cem\u003eMTP \u003c/em\u003egene sets in \u003cem\u003eBrassica rapa\u003c/em\u003e (17 genes), \u003cem\u003eBrassica nigra\u003c/em\u003e (18 genes), and \u003cem\u003eB. juncea\u003c/em\u003e (33 genes) using the HMMER tool (Cation_efflux; PF01545) and BLAST analysis. Then, for the 33 \u003cem\u003eBjMTPs\u003c/em\u003e, we carried out a detailed\u0026nbsp;bioinformatics analysis covering the physicochemical properties, phylogenetic relationships, conserved motifs, protein structures, collinearity, spatiotemporal RNA-seq expression, GO enrichment, and expression profiling under six HM stresses (Mn\u003csup\u003e2+\u003c/sup\u003e, Fe\u003csup\u003e2+\u003c/sup\u003e, Zn\u003csup\u003e2+\u003c/sup\u003e, Cd\u003csup\u003e2+\u003c/sup\u003e, Sb\u003csup\u003e3+\u003c/sup\u003e, and Pb\u003csup\u003e2+\u003c/sup\u003e). According to the findings of physicochemical characteristics and phylogenetic tree,\u0026nbsp;the allopolyploid \u003cem\u003eB. juncea\u003c/em\u003e’s MTP genes\u003cem\u003e \u003c/em\u003ewere inherited\u0026nbsp;from its progenitors, \u003cem\u003eB. rapa\u003c/em\u003e and \u003cem\u003eB. nigra\u003c/em\u003e, with minimal gene loss during polyploidization. The \u003cem\u003eBjMTP \u003c/em\u003egene family exhibited conserved motifs, promoter elements, and expression patterns that aligned\u0026nbsp;with seven evolutionary branches (G1, G4-G9, and G12). Further, by co-expression analysis, the core and gene-specific expression modules of \u003cem\u003eBjMTPs\u003c/em\u003e under six HM stresses were found. The HM treatments exhibited consistently\u0026nbsp;upregulated of \u003cem\u003eBjA04.MTP4\u003c/em\u003e, \u003cem\u003eBjA09.MTP10\u003c/em\u003e, and \u003cem\u003eBjB01.MTP5 \u003c/em\u003egenes, indicating\u0026nbsp;their critical\u0026nbsp;roles in enhancing HM tolerance in \u003cem\u003eB. juncea\u003c/em\u003e. These discoveries may contribute to a genetic improvement in \u003cem\u003eB. juncea\u003c/em\u003e's HM tolerance, which would facilitate the remediation of HM-contaminated areas.\u003c/p\u003e","manuscriptTitle":"Genome-wide exploration of MTP gene family in mustard (Brassica juncea L.): evolution and expression patterns during heavy metal stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 04:59:06","doi":"10.21203/rs.3.rs-4296734/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-30T05:10:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-27T13:41:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-27T09:54:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"82383396178003031420445820942250037855","date":"2024-05-15T11:08:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"119941254525509422545779045937704273129","date":"2024-05-14T16:50:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-13T03:52:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-13T03:44:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-27T16:48:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-26T06:47:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-04-20T09:02:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4081a091-485c-48bb-a719-a29afc210e2b","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":31368269,"name":"Biological sciences/Molecular biology"},{"id":31368270,"name":"Biological sciences/Plant sciences/Plant stress responses"}],"tags":[],"updatedAt":"2024-08-05T16:14:22+00:00","versionOfRecord":{"articleIdentity":"rs-4296734","link":"https://doi.org/10.1038/s41598-024-68877-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-08-01 15:57:47","publishedOnDateReadable":"August 1st, 2024"},"versionCreatedAt":"2024-05-02 04:59:06","video":"","vorDoi":"10.1038/s41598-024-68877-8","vorDoiUrl":"https://doi.org/10.1038/s41598-024-68877-8","workflowStages":[]},"version":"v1","identity":"rs-4296734","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4296734","identity":"rs-4296734","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}