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Identification and functional analysis of cation-efflux transporter 1 from Brassica juncea L.

BMC Plant Biology volume 22, Article number: 174 (2022) Cite this article

Abstract

Background

Brassica juncea behaves as a moderate-level accumulator of various heavy metal ions and is frequently used for remediation. To investigate the roles of metal ion transporters in B. juncea, a cation-efflux family gene, BjCET1, was cloned and functionally characterized.

Results

BjCET1 contains 382 amino acid residues, including a signature motif of the cation diffusion facilitator protein family, six classic trans-membrane-spanning structures and a cation-efflux domain. A phylogenetic analysis showed that BjCET1 has a high similarity level with metal tolerance proteins from other Brassica plants, indicating that this protein family is highly conserved in Brassica. BjCET1 expression significantly increased at very early stages during both cadmium and zinc treatments. Green fluorescence detection in transgenic tobacco leaves revealed that BjCET1 is a plasma membrane-localized protein. The heterologous expression of BjCET1 in a yeast mutant increased the heavy-metal tolerance and decreased the cadmium or zinc accumulations in yeast cells, suggesting that BjCET1 is a metal ion transporter. The constitutive expression of BjCET1 rescued the heavy-metal tolerance capability of transgenic tobacco plants.

Conclusions

The data suggest that BjCET1 is a membrane-localized efflux transporter that plays essential roles in heavy metal ion homeostasis and hyper-accumulation.

Peer Review reports

Background

Several heavy metal ions, such as zinc (Zn2+) and cobalt (Co2+), are essential trace elements involved in various vital biological processes [1]. However, large amounts of heavy metal ions, even essential ones, can inactivate functional proteins and block biological processes [2]. In addition, for the non-essential elements, such as cadmium (Cd2+), accumulation even at low concentrations can cause toxicity [3]. To cope with these challenges, plants have thus evolved a complex network for metal ion uptake, trafficking, storage and efflux [4, 5].

Many Brassicaceae plants have particular mechanisms for heavy metal ion detoxification and homeostasis [6,7,8]. Several cation transporter families are involved in metal transport and storage, such as cation diffusion facilitator (CDF) proteins, natural resistance-associated macrophage proteins, yellow-stripe 1-like proteins, zinc-regulated transporter and iron-related transporter proteins, and cation exchangers [9, 10].

In plants, CDF family members, also known as metal tolerance proteins (MTPs), are vacuole membrane-localized and heavy-metal tolerance-related proteins [11,12,13]. On the basis of phylogeny, the CDF family proteins are classified into three clusters: Zn-, iron (Fe)- and manganese (Mn)-CDFs [14]. The typical structure of a CDF protein consists of six transmembrane domains, one modified CDF signature and one C-terminal cation efflux domain [15]. The model plant Arabidopsis thaliana possesses 12 MTPs, and the functions of several AtMTPs have been well-studied. For example, AtMTP1 has a cytosolic histidine-rich loop and is involved in sensing cytosolic Zn2+ [16]. AtMTP3 maintains metal ion homeostasis by regulating the exclusion of Zn2+ from the shoots to the roots [17]. AtMTP5 forms a complex with AtMTP12 to transport Zn2+ from the cytoplasm to Golgi apparatus [18]. AtMTP11 is an Mn transporter that confers Mn tolerance [19].

Phytoremediation is an ecologically and economically sound strategy to eliminate heavy metal ions from contaminated soils [20]. Brassica juncea is frequently used for the remediation of soils contaminated with heavy metals, owing to its high ability to fix metal ions in aboveground plant parts [21, 22]. Several metal transporters have been identified in B. juncea. BjYSL7 encodes a plasma-localized transporter that is involved in the transport of Fe2+, Cd2+ and nickel (Ni2+) ions from roots to shoots [23]. BjHMA4R, a heavy metal efflux pump, specifically binds to Cd2+ in the cytosol at low concentrations [24]. The bZIP transcription factor BjCdR15 is a regulator of Cd2+ uptake and transport in shoots [25]. Thus, the identification and functional analyses of B. juncea CDF family proteins are important for designing and breeding metal-accumulating plants.

Previously, four cation-efflux transporter-encoding genes (BjCET1–4) were predicted in B. juncea, and functions for BjCET2, − 3 and − 4 in the regulation of ion homeostasis have been reported [25, 26]. However, the biological function of BjCET1 is largely unknown. Here, the contribution of BjCET1 to heavy-metal tolerance, as well as its potential role in the phytoremediation of heavy metal-contaminated soils, were investigated.

Results

Sequence analysis of the BjCET1 gene

According to the previously published sequence information (GenBank ID: AY187082), the full-length cDNA BjCET1 was cloned [27]. BjCET1 contains a putative open read frame of 1146 bp encoding 382 amino acid residues. The putative BjCET1 protein possesses a CDF family signature motif (SLAILTDAAHLLSD) at the N-terminus, six classic trans-membrane-spanning structures in the middle region, and a cation-efflux domain at the C-terminus (Fig. 1a).

Fig. 1
figure 1

Basic sequence analysis of BjCET1. a Sequence alignments of predicted amino acid sequences for BjCET1-4, AtMTP1, AtMTP3, AtMTPA1, and AtMTPB1 was performed by CLUSTALW. The classic protein features are highlighted: six transmembrane domains are underlined in dark yellow; the CDF signature is marked by red dashed box; the C-terminal LZ motifs are marked by green dashed boxes; and the C-terminal putative Zn binding site HD(E)XHXWXL(I)TX8H is marked by blue dashed box. b Phylogenetic analysis of the reported CDF/MTP proteins from different plant species. The Neighbor–Joining phylogenetic tree was constructed using MEGA6.1 after CLUSTALW alignment of the full-length amino acid sequences

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A phylogenetic analysis showed that BjCET1 has high similarity with other well-identified MTP proteins (Fig. 1b). Most of the selected proteins were classed into Groups I or II, and all the MTPs from the Brassica family, such as B. nigra, B. oleracea, B. rapa and B. napus, were placed into the same group (Group II). Our data indicated that MTP/CDF proteins are highly conserved in Brassica. BjCET1 and MTP1 from B. nigra were grouped into one gene pair in the evolutionary tree, suggesting similar biological functions.

Expression analysis of BjCET1 under different heavy-metal treatments

To investigate the basic biological function of BjCET1, a tissue-specific expression analysis was performed. The BjCET1 gene was expressed highest in the roots and lowest in the leaves (Additional file 1).

To reveal the BjCET1 expression pattern in response to different heavy-metal stresses, qRT-PCR was performed. Under CdCl2 or ZnCl2 treatment, the expression of BjCET1 was significantly up-regulated at early stages and peaked at 24 h (Fig. 2a and b). BjCET1 expression was induced by CdCl2 at all concentrations tested. The expression level of BjCET1 under at high CdCl2 concentrations (100 and 200 μM) was lower than at the low CdCl2 concentration (50 μM) (Fig. 2c). Compared with the control, the BjCET1 expression largely increased during ZnCl2 treatments, and no significant differences were observed between different ZnCl2 concentrations (Fig. 2d).

Fig. 2
figure 2

Expression analysis of BjCET1 under heavy metal treatments. a The relative mRNA levels of BjCET1 gene under 200 μM CdCl2 treatment. b The relative mRNA levels of BjCET1 gene under 400 μM ZnCl2 treatment. c The relative mRNA levels of BjCET1 under CdCl2 treatments at different concentrations. d The relative mRNA levels of BjCET1 under ZnCl2 treatments at different concentrations. Data are expressed as means±SD of three biological repeats. “*” indicates significant differences in relative mRNA levels of BjCET1 gene

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Subcellular localization of the BjCET1 protein

The resistant tobacco plants harboring pBIN-BjCET1-mGFP5-ER and pBIN-mGFP5-ER vectors were obtained through Agrobacterium tumefaciens-mediated leaf-disc transformation. The process from calli differentiation to seedling growth is shown in Fig. 3a-d. The genomic DNAs of WT and transgenic tobacco plants were extracted, and the positive transgenic plants were identified using PCR assay. The target PCR product (950 bp) was detected in the transgenic plants, indicating that the T-region of the expression vector was integrated into the tobacco genome. The BjCET1 protein was found to be localized at the plasma membrane, suggesting that BjCET1 might be a potential transporter (Fig. 3e-f).

Fig. 3
figure 3

Genetic transformation and subcellular localization of BjCET1 in tobaccos. a-e The growth process of tobacco after Agrobacterium mediated transformation. f Subcellular localization of BjCET1 protein in root cells of transgenic tobaccos. Fluorescence of GFP was observed by using confocal laser scanning microscopy under 485 nm and 364 nm

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Involvement of BjCET1 in broad-range metal stress tolerance

To determine its functional properties and substrate specificities, BjCET1 was heterologously expressed in the yeast cot1&zrc1 (YK44) deficient mutant. Yeast cells containing empty pYES2 vector served as controls. Yeast cells expressing the BjCET1 protein were exposed independently to CoCl2, NiCl2, ZnCl2 or CdCl2 at different concentrations. The over-expression of BjCET1 enhanced the viability of transgenic yeasts against Co2+, Ni2+, Zn2+ and Cd2+ treatments (Fig. 4a-d).

Fig. 4
figure 4

Involvement of BjCET1 in a broad range of metal stress tolerance. a The growth status of yeast strains expressing BjCET1 under CdCl2 treatment at different concentrations. b The growth status of yeast strains expressing BjCET1 under different concentrations of CoCl2 stresses. c The growth status of yeast strains expressing BjCET1 under different concentrations of NiCl2 stresses. d The growth status of yeast strains expressing BjCET1 under different concentrations of ZnCl2 stresses. e Growth of yeast YK44 cells expressing BjCET1 and the pYES2 empty-vector control cells in YPGAL plates for 48–72 h. The YPGAL plates contained 0.02 mM Cd, 0.3 mM Co, 1.0 mM Ni, or 0.1 mM Zn

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To confirm the role of BjCET1 in heavy-metal tolerances, the growth states of yeast cells expressing the BjCET1 protein was observed on solid medium. The growth rates of both control and transgenic yeast cells were slower under heavy metal stress than under the control conditions. However, the growth capabilities of transgenic yeast under Ni2+, Co2+, Zn2+ and Cd2+ stresses were significantly greater than under control conditions (Fig. 4e). The data suggest that BjCET1 improved the resistances of yeast cells to various heavy metal ions.

The metal ion transport activity of BjCET1

The metal ion transport activity of BjCET1 was determined using the ICP-MS method. Compared with the control, the yeast zrc1 mutant over-expressing BjCET1 exhibited a reduced Zn accumulation, from 1460 μg/g to 1234 μg/g upon the ZnCl2 treatment (Fig. 5a). Compared with the control, the yeast cot1 mutant over-expressing BjCET1 also exhibited a reduced Cd accumulation, from 489 μg/g to 398 μg/g upon the CdCl2 treatment (Fig. 5b). The data suggest that BjCET1 might be a metal ion transporter that exports Zn and Cd ions out of yeast cells.

Fig. 5
figure 5

Metal ion transport activity of BjCET1. a ICP-MS assay of Zn content in zrc1 yeast cells. The yeast cells were incubated in 125 μM ZnCl2 for 4 h before collection. b ICP-MS assay of Cd content in cot1 yeast cells. The yeast cells were incubated in 125 μM CdCl2 for 4 h before collection. A significant change in Zn or Cd accumulation was detected in BjCET1 expressing yeasts compared to control yeast (P < 0.01)

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Heavy-metal tolerance of BjCET1-transformed tobacco

To investigate the role of BjCET1 in plants, the BjCET1 gene was heterologously over-expressed in tobacco seedlings. The leaves from BjCET1 over-expressing tobacco seedlings and WT were placed in ddH2O, 200 μM NiCl2, 400 μM ZnCl2 or 200 μM CdCl2 solution. There were no obvious differences between the transgenic and WT tobacco leaves in the ddH2O solution. However, under various heavy-metal stresses, the WT leaves turned yellow and rotted, while the transgenic leaves remained green (Fig. 6). The damage on transgenic tobacco leaves was significantly lower than on WT leaves, suggesting that over-expressing BjCET1 greatly improved tobacco resistance to heavy-metal stresses.

Fig. 6
figure 6

Heavy metal tolerance of BjCET1 transgenic tobacco leaves. The healthy transgenic tobacco leaves were selected as control. The round blades were placed in ddH2O, 200 μM NiCl2, 400 μM ZnCl2, or 200 μM CdCl2. Ten independent pieces were placed in each Petri dish. The status of the leaves was observed after 5 days

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Heterologous expression of BjCET1 enhanced tobacco’s heavy metal tolerance

Tobacco seedlings independently over-expressing BjCET1 and the empty vector were selected to analyze the roles of BjCET1 in Cd tolerance. Four important physiological parameters, including relative conductivity, soluble sugar content, chlorophyll content and free proline content, were determined in the transgenic and WT tobacco leaves. The soluble sugar contents were largely up-regulated by the CdCl2 treatment at different time points. Compared with WT, the soluble sugar contents in the BjCET1 transgenic plants were significantly higher at time points 15 d and 20 d (Fig. 7a). The chlorophyll contents were obviously reduced by the CdCl2 treatment. At time points 15 d and 20 d, the chlorophyll contents in the BjCET1 transgenic tobacco were higher than in WT (Fig. 7b). The relative conductivities were also greatly increased during the CdCl2 treatment. Under the CdCl2 treatment, the relative conductivity levels in the bjCET1 transgenic tobacco were lower than that in WT (Fig. 7c). Compared with WT, the free proline contents in the BjCET1 transgenic plants were significantly higher than in WT at time points 15 d and 20 d (Fig. 7d).

Fig. 7
figure 7

Physiological and biochemical changes of tobacco during Cd treatment process. Several physiological parameters, including soluble sugar (a), chlorophyll content (b), relative conductivity (c), and free proline content (d), were analyzed under the CdCl2 treatment. A P value < 0.01 was considered to be statistically significant and indicated by “*”

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Discussion

Higher plants possess a complex system for heavy metal ion uptake, absorption, transport, and storage, allowing them to cope with various heavy-metal stresses [28]. B. juncea is a rapid-growth plant with an appreciable capacity to absorb various toxic heavy metals. Thus, B. juncea is frequently applied to the ‘green’ remediation of toxic metal-contaminated mining soils [29].

The CDF/MTP family plays significant roles in maintaining intracellular ion homeostasis and tolerance in plants [30]. To date, several CDF/MTP family genes of B. juncea have been cloned and functionally characterized, such as BjCET2, 3, and 4 [26, 31]. In the present study, the BjCET1 gene was cloned and functionally characterized, giving us an opportunity to understand the entire CDF/MTP family in B. juncea.

Most of the CDF/MTPs contain six transmembrane domains, a cytoplasmic N-terminus and a characteristic C-terminal cation efflux domain [14]. Furthermore, a Leu zipper motif at the C-terminus of CDF/MTP family members is highly conserved and critical for Zn detoxification [32]. Similar to the previously published characterization of BjCET proteins, BjCET1 possesses a classic Zn-CDF signature, indicating that BjCET1 is a heavy-metal transporter. BjCET2 and BjCET4 are mainly expressed in roots, and BjCET3 is constitutively expressed in all tested tissues [26, 31]. Similar expression patterns reflect the shared functions of BjCET1, − 2 and − 4. The root-specific expression of BjCET1 further suggested that it functions in the root system against heavy metal over-accumulation.

In plants, the expression levels of many CDF/MTP family genes are induced in different tissues during heavy-metal treatments. For example, the expression of OsMTP11 was substantially enhanced at 4 h after exposure to Cd, Zn, Ni and Mn treatments [33]. In Populus trichocarpa, Cd exposure significantly enhances the expression of MTP11.1, and Zn exposure significantly increases the expression of MTP10.3 [34]. The expression level of BjCET1 was significantly increased during both Cd and Zn treatments, indicating an important role of BjCET1 in responses to heavy-metal stresses. Interestingly, BjCET1 was significantly induced at 6 h, suggesting that BjCET1 is a rapid-response gene.

CDF/MTP family members are responsible for the transport of heavy metal ions, such as Zn2+, Co2+, Ni2+ and Cd2+, in plants [34, 35]. The heterologous expression of CDF/MTP genes in yeast deficiency mutants is a good method to reveal their roles in heavy-metal tolerance [11]. The heterologous expression of B. napus MTP3 in yeast mutant cells enhances tolerance to, and intracellular sequestration of, Zn2+ and Mn2+ [30]. The heterologous expression of Camellia sinensis MTP8.2 in yeast cells confers tolerance to Ni2+ and Mn2+ but not to Zn2+ [13]. The heterologous expression of rice MTP1 in tobacco relieves Cd stress-induced phototoxic effects [36], and the heterologous expression of OsMTP1 in the yeast zrc1cot1 complements the Zn2+ and Co2+ hypersensitivities of this mutant [37]. In our study, the growth rate of the control yeast mutant was lower than that of the transgenic yeast under various heavy-metal stresses, suggesting that the heterologous expression of BjCET1 created a broad-range metal-stress tolerance in YK44 yeast cells.

The ICP-MS data suggested a potential metal ion transport activity for BjCET1 in yeast cells. To confirm the biological function of BjCET1 in plants, it was heterologously expressed in tobacco plants. In BjCET1-GFP transgenic tobacco plant leaves, the fusion protein mainly was found to be localized at the cell membranes. The CDF family proteins may be efflux transporters that are involved in metal homeostasis by maintaining optimal metal concentrations in the cytoplasm [12, 38]. The constitutive expression of BjCET1 greatly rescued the heavy metal-tolerance capabilities of transgenic tobacco plants. The data suggested that BjCET1 is a membrane-localized efflux transporter that plays important roles in heavy metal ion homeostasis and hyper-accumulation [39].

Conclusion

In our study, a heavy metal transporter-encoding gene, BjCET1, was cloned and functionally characterized. A sequence analysis showed that BjCET1 contained a classic Zn-CDF signature and was highly similar to MTP1 from B. nigra. The expression of BjCET1 was rapidly up-regulated under various heavy metal ion treatments. Yeast experiments suggested that BjCET1 is involved in a broad-range metal stress tolerance. Furthermore, the heterologous expression of BjCET1 enhanced the heavy-metal tolerance of tobacco. BjCET1 has great potential in the phytoremediation of heavy metal-contaminated environments.

Methods

Plant materials

B. juncea materials were obtained from the North Central Regional Plant introduction station of the United States National Plant Germplasm System (order number: IP173874). Nicotiana tabacum Cv. W38 was used for heterologous expression assay. All plant materials were grown routinely at room temperature in a greenhouse at Hangzhou Normal University. Fourteen-day-old seedlings were selected and treated with heavy metal ions at various concentrations.

Isolation of the BjCET1 gene

Total RNAs of B. juncea seedlings were extracted using TRIzol reagent (Invitrogen, Shanghai, China) in accordance with the manufacturer’s protocol. RNAs were used as templates for the first- and second-strands cDNA synthesis using a cDNA preparation kit (Illumina, San Diego, CA, USA). Using cDNA as the template, the cDNA sequence of BjCET1 was cloned by PCR amplification. The primer sequences are listed in Additional file 2.

Bioinformatic analysis of BjCET1 protein

The BjCET1 full-length protein sequence and other CET family members from B. juncea and MTP proteins from Arabidopsis were used for multiple sequence alignments. The alignments were performed using ClustalW with default parameters and were visualized subsequently using GeneDoc software (http://www.nrbsc.org/gfx/genedoc/). An unrooted phylogenetic tree of CET family proteins was constructed using MEGA6.1 (http://www.megasoftware.net/) employing the neighbor-joining method.

Expression analysis of BjCET1

Up- to the 5-6 leaf stage, the seedlings were used for tissue-specific expression analysis. Three major tissues, including root, leaf and shoot, were used in tissue-specific expression analysis experiment. For stress treatments, seedlings were hydroponically planted in aerated liquid medium containing CdCl2 or ZnCl2. Several previous studies have focused on the responses of B. juncea to Cd treatment. In Shu’s study, 50 μM CdCl2 was used to treat B. juncea seedlings; In Bhardwaj’s study, 200, 400, and 600 μM CdCl2 were used to treat B. juncea seedlings; 50 μM of CdCl2 was used to treat B. juncea seedlings; In Misra’s study, 10-160 μM of CdCl2 were used to treat B. juncea seedlings [35, 40,41,42]. Thus, 0, 50, 100, 200 μM of CdCl2 were used in our study. For ZnCl2 treatment, the concentration of ZnCl2 was set according to Lang’ s study [26].

The seedlings of 5-6 leaf stage were subjected to 200 μM CdCl2 or 400 μM ZnCl2 solutions for 6 h, 24 h, 48 h, or 144 h. After treatment, leaves were collected and washed with ddH2O and quickly kept in liquid nitrogen until used. The primer sequences for the qRT-PCR are listed in Additional file 2.

QRT-PCR was performed using a SYBR Premix Ex Taq Kit (TaKaRa, Dalian, China) on an ABI PRISM 7700 DNA Sequence Detection System (Applied Biosystems, Shanghai, China). The ACTIN gene of B. juncea was used as an internal reference (5#-AAGATCTGGCATCACACTTTC-3# and 5#-TAGTCAACAGCAACAAAGGAG-3#) for the relative quantification of transcript levels [26]. The relative fold differences in the BjCET1 gene were calculated by the values of comparative cycle threshold (2-ΔΔCt). In detail, 1 μL cDNA sample and 0.1 μM of each primer were added to 5 μL of 2 × SYBR Green solution [43]. Five technical repetitions and three biological replicates were performed in qRT-PCR experiments.

Yeast experiments for the metal-tolerance assay

The cDNA of BjCET1 was cloned into the pYES2 vector. The BjCET1-pYES2 construct and empty pYES2 vector were transformed independently into the YK44 Saccharomyces cerevisiae mutant strain (ura3-52, his3-200, zrc1, cot1, MATα). The transformed yeast mutant cells were selected on solid medium containing SMM-uracil, 6.7 g yeast nitrogen base without amino acids, 20 mg histidine, 2% glucose and 2% agar. The presence of the BjCET1 insert sequence in the selected yeast cells was confirmed by PCR amplification.

The metal-tolerance experiment was performed in accordance with a previously published protocol [26]. In detail, yeast cells were grown in 10 mL of SMM-uracil liquid medium until reaching OD600 = 1.5. Then, 1 mL of yeast culture solution was transferred to a 50 mL of SMM-uracil liquid medium. The mixed culture was poured into agar dishes and allowed to cool. Solutions containing Cd2+ or Zn2+ ions were spread onto the agar, and the plates were maintained in an incubator at 30 °C for 3 days.

Yeast experiments for the metal-ion accumulation assay

To investigate the efflux transport activity of the BjCET1 protein, the accumulation of Zn2+ (or Cd2+) in selected yeast cells treated with ZnCl2 (or CdCl2) was analyzed. With ZnCl2 (or CdCl2) supplements, zrc1 (or cot1) yeast mutant cells containing the BjCET1 were added to 200 mL SMM–uracil liquid medium and cultured to OD600 = 0.2. The yeast cell culture was allowed to grow to OD600 = 0.4. Yeast cells were harvested by 5000×g centrifugation and washed three times with a 20-mM EDTA solution. The clean cells were dried and weighed. The Zn (or Cd) contents were analyzed using inductively coupled plasma-atomic emission spectrophotometry [26].

Heterologous expression of the BjCET1:GFP fusion protein

The cDNA of BjCET1 was inserted into the XbaI and BamHI sites of the pBI121-GFP vector (Additional file 3). The pBI121-BjCET1-GFP and pBI121 empty vectors were introduced independently into Agrobacterium tumefaciens (EHA105 strain) in accordance with a previously published method [44]. All the yeast transformants were selected on MS medium supplemented with 200 mg·L− 1 kanamycin and 250 mg·L− 1 Cef. Successful transformations were confirmed by PCR amplification. The primer sequences for the PCR are listed in Additional file 2. Tissue samples from the positive transgenic lines were used for the further experiments.

Confocal microcopy analysis

The GFP fluorescence of the positive transgenic lines were visualized by confocal laser scanning microcopy (Zeiss, Jena, Germany). The parameters for the confocal microcopy were set as follows: 490 nm for excitation, 500 nm for cutoff, and 515 nm for emission.

Heavy metal accumulation assay

Approximately 1 cm2 regions from the wild type (WT) and transgenic tobaccos were excised and placed in MS medium or MS medium containing 200 μM CdCl2. Each treatment group, containing 10 independent explants, was cultured at 25 °C. After 30 d, the plant samples were harvested and washed twice in ddH2O. To determine Cd contents, approximately 100-mg plant samples were digested with 2 mL HNO3 by heating discontinuously in a microwave for 2 min. The cooled solution was transferred to a 50-mL flask and ddH2O was added up to a standard volume before being analyzed using the ICP-MS method [26].

Four physiological parameters, soluble sugar, chlorophyll content, relative conductivity and free proline content, were determined in accordance with previously published methods [45].

Availability of data and materials

All data generated or analysed during this study are included in this published article and its supplementary information files. The full-length membranes of qRT-PCR gel are showed in Additional file 4. No sequencing data was used in the present study.

Abbreviations

CDF:

Cation diffusion facilitator

MTP:

Metal tolerance protein

PCR:

Polymerase chain reaction

QRT-PCR:

Quantitative reverse transcription PCR

WT:

Wild type

References

  1. Wang FH, Qiao K, Liang S, Tian SQ, Tian YB, Wang H, et al. Triticum urartu MTP1: its ability to maintain Zn(2+) and co(2+) homeostasis and metal selectivity determinants. Plant Cell Rep. 2018;37(12):1653–66.

    Article  CAS  Google Scholar 

  2. Goyer RA. Toxic and essential metal interactions. Annu Rev Nutr. 1997;17:37–50.

    Article  CAS  Google Scholar 

  3. Shaari NEM, Tajudin M, Khandaker MM, Majrashi A, Alenazi MM, Abdullahi UA, et al. Cadmium toxicity symptoms and uptake mechanism in plants: a review. Braz J Biol. 2022;84:e252143.

    Article  CAS  Google Scholar 

  4. Hossain Z, Komatsu S. Contribution of proteomic studies towards understanding plant heavy metal stress response. Front Plant Sci. 2012;3:310.

    PubMed  Google Scholar 

  5. Hall JL. Cellular mechanisms for heavy metal detoxification and tolerance. J Exp Bot. 2002;53(366):1–11.

    Article  CAS  Google Scholar 

  6. Benzarti S, Mohri S, Ono Y. Plant response to heavy metal toxicity: comparative study between the hyperaccumulator Thlaspi caerulescens (ecotype Ganges) and nonaccumulator plants: lettuce, radish, and alfalfa. Environ Toxicol. 2008;23(5):607–16.

    Article  CAS  Google Scholar 

  7. Malecka A, Konkolewska A, Hanc A, Ciszewska L, Staszak AM, Jarmuszkiewicz W, et al. Activation of antioxidative and detoxificative systems in Brassica juncea L. plants against the toxicity of heavy metals. Sci Rep. 2021;11(1):22345.

    Article  CAS  Google Scholar 

  8. Kania J, Krawczyk T, Gillner DM. Oilseed rape (Brassica napus): the importance of aminopeptidases in germination under normal and heavy metals stress conditions. J Sci Food Agric. 2021;101(15):6533–41.

    Article  CAS  Google Scholar 

  9. Tang RJ, Luan S. Regulation of calcium and magnesium homeostasis in plants: from transporters to signaling network. Curr Opin Plant Biol. 2017;39:97–105.

    Article  CAS  Google Scholar 

  10. Pittman JK, Hirschi KD. CAX-ing a wide net: Cation/H(+) transporters in metal remediation and abiotic stress signalling. Plant Biol. 2016;18(5):741–9.

    Article  CAS  Google Scholar 

  11. Wang FH, Qiao K, Shen YH, Wang H, Chai TY. Characterization of the gene family encoding metal tolerance proteins in Triticum urartu: Phylogenetic, transcriptional, and functional analyses. Metallomics. 2021;13(7):mfab038.

  12. Papierniak-Wygladala A, Kozak K, Barabasz A, Palusinska M, Calka M, Maslinska K, et al. Identification and characterization of a tobacco metal tolerance protein, NtMTP2. Metallomics. 2020;12(12):2049–64.

    Article  CAS  Google Scholar 

  13. Zhang X, Li Q, Xu W, Zhao H, Guo F, Wang P, et al. Identification of MTP gene family in tea plant (Camellia sinensis L.) and characterization of CsMTP8.2 in manganese toxicity. Ecotoxicol Environ Saf. 2020;202:110904.

    Article  CAS  Google Scholar 

  14. Montanini B, Blaudez D, Jeandroz S, Sanders D, Chalot M. Phylogenetic and functional analysis of the Cation diffusion facilitator (CDF) family: improved signature and prediction of substrate specificity. BMC Genomics. 2007;8:107.

    Article  Google Scholar 

  15. Gustin JL, Zanis MJ, Salt DE. Structure and evolution of the plant cation diffusion facilitator family of ion transporters. BMC Evol Biol. 2011;11:76.

    Article  CAS  Google Scholar 

  16. Tanaka N, Kawachi M, Fujiwara T, Maeshima M. Zinc-binding and structural properties of the histidine-rich loop of Arabidopsis thaliana vacuolar membrane zinc transporter MTP1. FEBS Open Bio. 2013;3:218–24.

    Article  CAS  Google Scholar 

  17. Arrivault S, Senger T, Kramer U. The Arabidopsis metal tolerance protein AtMTP3 maintains metal homeostasis by mediating Zn exclusion from the shoot under Fe deficiency and Zn oversupply. Plant J. 2006;46(5):861–79.

    Article  CAS  Google Scholar 

  18. Fujiwara T, Kawachi M, Sato Y, Mori H, Kutsuna N, Hasezawa S, et al. A high molecular mass zinc transporter MTP12 forms a functional heteromeric complex with MTP5 in the Golgi in Arabidopsis thaliana. FEBS J. 2015;282(10):1965–79.

    Article  CAS  Google Scholar 

  19. Delhaize E, Gruber BD, Pittman JK, White RG, Leung H, Miao Y, et al. A role for the AtMTP11 gene of Arabidopsis in manganese transport and tolerance. Plant J. 2007;51(2):198–210.

    Article  CAS  Google Scholar 

  20. Cui X, Mao P, Sun S, Huang R, Fan Y, Li Y, et al. Phytoremediation of cadmium contaminated soils by Amaranthus Hypochondriacus L.: The effects of soil properties highlighting cation exchange capacity. Chemosphere. 2021;283:131067. https://doi.org/10.1016/j.chemosphere.2021.131067.

  21. Goswami S, Das S. A study on cadmium phytoremediation potential of Indian mustard, Brassica juncea. Int J Phytoremediation. 2015;17(1-6):583–8.

    Article  CAS  Google Scholar 

  22. Thakur AK, Parmar N, Singh KH, Nanjundan J. Current achievements and future prospects of genetic engineering in Indian mustard (Brassica juncea L. Czern & Coss.). Planta. 2020;252(4):56.

    Article  CAS  Google Scholar 

  23. Wang JW, Li Y, Zhang YX, Chai TY. Molecular cloning and characterization of a Brassica juncea yellow stripe-like gene, BjYSL7, whose overexpression increases heavy metal tolerance of tobacco. Plant Cell Rep. 2013;32(5):651–62.

    Article  CAS  Google Scholar 

  24. Wang J, Liang S, Xiang W, Dai H, Duan Y, Kang F, et al. A repeat region from the Brassica juncea HMA4 gene BjHMA4R is specifically involved in cd(2+) binding in the cytosol under low heavy metal concentrations. BMC Plant Biol. 2019;19(1):89.

    Article  Google Scholar 

  25. Farinati S, DalCorso G, Varotto S, Furini A. The Brassica juncea BjCdR15, an ortholog of Arabidopsis TGA3, is a regulator of cadmium uptake, transport and accumulation in shoots and confers cadmium tolerance in transgenic plants. New Phytol. 2010;185(4):964–78.

    Article  CAS  Google Scholar 

  26. Lang M, Hao M, Fan Q, Wang W, Mo S, Zhao W, et al. Functional characterization of BjCET3 and BjCET4, two new cation-efflux transporters from Brassica juncea L. J Exp Bot. 2011;62(13):4467–80.

    Article  CAS  Google Scholar 

  27. Lang M, Zhang Y, Chai T. Identification of genes up-regulated in response to cd exposure in Brassica juncea L. Gene. 2005;363:151–8.

    Article  CAS  Google Scholar 

  28. Rizwan M, Ali S, Adrees M, Rizvi H, Zia-Ur-Rehman M, Hannan F, et al. Cadmium stress in rice: toxic effects, tolerance mechanisms, and management: a critical review. Environ Sci Pollut Res Int. 2016;23(18):17859–79.

    Article  CAS  Google Scholar 

  29. Jeyasundar P, Ali A, Azeem M, Li Y, Guo D, Sikdar A, et al. Green remediation of toxic metals contaminated mining soil using bacterial consortium and Brassica juncea. Environ Pollut. 2021;277:116789.

    Article  CAS  Google Scholar 

  30. Gu D, Zhou X, Ma Y, Xu E, Yu Y, Liu Y, et al. Expression of a Brassica napus metal transport protein (BnMTP3) in Arabidopsis thaliana confers tolerance to Zn and Mn. Plant Sci. 2021;304:110754.

    Article  CAS  Google Scholar 

  31. Xu J, Chai T, Zhang Y, Lang M, Han L. The cation-efflux transporter BjCET2 mediates zinc and cadmium accumulation in Brassica juncea L. leaves. Plant Cell Rep. 2009;28(8):1235–42.

    Article  CAS  Google Scholar 

  32. Blaudez D, Kohler A, Martin F, Sanders D, Chalot M. Poplar metal tolerance protein 1 confers zinc tolerance and is an oligomeric vacuolar zinc transporter with an essential leucine zipper motif. Plant Cell. 2003;15(12):2911–28.

    Article  CAS  Google Scholar 

  33. Zhang M, Liu B. Identification of a rice metal tolerance protein OsMTP11 as a manganese transporter. PLoS One. 2017;12(4):e0174987.

    Article  Google Scholar 

  34. Gao Y, Yang F, Liu J, Xie W, Zhang L, Chen Z, et al. Genome-Wide Identification of Metal Tolerance Protein Genes in Populus trichocarpa and Their Roles in Response to Various Heavy Metal Stresses. Int J Mol Sci. 2020;21(5):1680.

  35. Persans MW, Nieman K, Salt DE. Functional activity and role of cation-efflux family members in Ni hyperaccumulation in Thlaspi goesingense. Proc Natl Acad Sci U S A. 2001;98(17):9995–10000.

    Article  CAS  Google Scholar 

  36. Das N, Bhattacharya S, Maiti MK. Enhanced cadmium accumulation and tolerance in transgenic tobacco overexpressing rice metal tolerance protein gene OsMTP1 is promising for phytoremediation. Plant Physiol Biochem. 2016;105:297–309.

    Article  CAS  Google Scholar 

  37. Menguer PK, Farthing E, Peaston KA, Ricachenevsky FK, Fett JP, Williams LE. Functional analysis of the rice vacuolar zinc transporter OsMTP1. J Exp Bot. 2013;64(10):2871–83.

    Article  CAS  Google Scholar 

  38. Li J, Zheng L, Fan Y, Wang Y, Ma Y, Gu D, et al. Pear metal transport protein PbMTP8.1 confers manganese tolerance when expressed in yeast and Arabidopsis thaliana. Ecotoxicol Environ Saf. 2021;208:111687.

    Article  CAS  Google Scholar 

  39. Kim D, Gustin JL, Lahner B, Persans MW, Baek D, Yun DJ, et al. The plant CDF family member TgMTP1 from the Ni/Zn hyperaccumulator Thlaspi goesingense acts to enhance efflux of Zn at the plasma membrane when expressed in Saccharomyces cerevisiae. Plant J. 2004;39(2):237–51.

    Article  CAS  Google Scholar 

  40. Zhang ZW, Deng ZL, Tao Q, Peng HQ, Wu F, Fu YF, et al. Salicylate and glutamate mediate different Cd accumulation and tolerance between Brassica napus and B. juncea. Chemosphere. 2022;292:133466. https://doi.org/10.1016/j.chemosphere.2021.133466.

  41. Kapoor D, Kaur S, Bhardwaj R. Physiological and biochemical changes in Brassica juncea plants under cd-induced stress. Biomed Res Int. 2014;2014:726070.

    Article  Google Scholar 

  42. Seth CS, Kumar Chaturvedi P, Misra V. The role of phytochelatins and antioxidants in tolerance to cd accumulation in Brassica juncea L. Ecotoxicol Environ Saf. 2008;71(1):76–85.

    Article  CAS  Google Scholar 

  43. Yu C, Guo H, Zhang Y, Song Y, Pi E, Yu C, et al. Identification of potential genes that contributed to the variation in the taxoid contents between two Taxus species (Taxus media and Taxus mairei). Tree Physiol. 2017;37(12):1659–71.

    Article  CAS  Google Scholar 

  44. Walkerpeach CR, Velten J. Agrobacterium-mediated gene transfer to plant cells: cointegrate and binary vector systems. Plant Mol Biol Manual. 1994;261(2):226-35.

  45. Blunden CA, Wilson MF. A specific method for the determination of soluble sugars in plant extracts using enzymatic analysis and its application to the sugar content of developing pear fruit buds. Anal Biochem. 1985;151(2):403–8.

    Article  CAS  Google Scholar 

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Acknowledgements

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Funding

This study was supported by research grants from the Zhejiang Provincial Natural Science Foundation of China (LBY21H090005, LQ18C090006, LY19C16000). There is no role of the funding body in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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Authors and Affiliations

  1. College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, 310036, China

    Lu Han, Xiaohua Wu, Xinyu Zhang, Kailin Hou, Hongshan Zhang & Chenjia Shen

  2. Zhejiang Key Laboratory of Organ Development and Regeneration, College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, 310036, Zhejiang, China

    Lu Han, Xiaohua Wu & Xinyu Zhang

  3. Zhejiang Provincial Key Laboratory for Genetic Improvement and Quality Control of Medicinal Plants, Hangzhou Normal University, Hangzhou, 310036, China

    Kailin Hou, Hongshan Zhang & Chenjia Shen

Authors
  1. Lu Han
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Contributions

L.H. X.W. and C.S. made substantial contributions to conception, was involved in drafting the manuscript, and given final approval of the version to be published. L.H. X.Z. K.H. and C.S. made substantial contributions to acquisition of data analysis, was involved in revising the manuscript, and given final approval of the version to be published. L.H., X.W. and H.Z. made substantial contributions to analysis and interpretation of data. L.H. and C.S. was involved in revising the manuscript, and given final approval of the version to be published. All authors reviewed the manuscript.

Corresponding author

Correspondence to Chenjia Shen.

Ethics declarations

Ethics approval and consent to participate

This project uses plant materials. B. juncea materials were obtained from the North Central Regional Plant introduction station of the United States National Plant Germplasm System (order number: IP173874). Dr. Shangguo Feng undertook the formal identification of the plant material used in our study. A voucher specimen of B. juncea has been deposited in the herbarium of Hangzhou Normal University.

All the study procedures were carried out in accordance with relevant guidelines.

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Not applicable.

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The authors declare that they have no competing interests.

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Supplementary Information

Additional file 1: Figure S1

. Tissue-specific expression analysis of BjCET1 gene.

Additional file 2: Table S1

. The detail information of the primer sequences.

Additional file 3: Figure S2

. The vector map of pBI121-BjCET1-GFP.

Additional file 4. Figure S3

. The full-length membranes of qRT-PCR gel. 

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Han, L., Wu, X., Zhang, X. et al. Identification and functional analysis of cation-efflux transporter 1 from Brassica juncea L.. BMC Plant Biol 22, 174 (2022). https://doi.org/10.1186/s12870-022-03569-x

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Keywords

BMC Plant Biology

ISSN: 1471-2229

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