The B. juncea cultivar “Purple Leaf Mustard” was grown in Cd-contaminated soils as described previously (Zhang et al., 2021).

The A. thaliana plants were grown in soil to generate the T3 homozygous transgenic lines. The wild type (Col-0) and T3 lines were cultured in 1/2MS medium containing 0, 10, 30, and 50 μM CdCl₂ for 10 days at 22°C under a 16-h light/8-h dark cycle.

Total RNA was extracted and cDNA synthesis was performed using a Thermo Fisher Scientific RevertAid First Strand cDNA Synthesis Kit. Full-length cDNA encoding BjNRAMP1 was amplified through PCR using gene-specific primers ( Table S1 ). The amplified products of the PCR were cloned into the pMD19-T vector (Takara) and sequenced via the Sanger method. The amino acid sequences of BjNRAMP1 and the other related species were employed to perform multi-sequence alignment using the ClustalW with default parameters in MEGA7. The phylogenetic tree was subsequently constructed using the neighbor-joining approach (no. of bootstrap replications = 1,000) implemented in MEGA7.

Confirmed BjNRAMP1 CDS in the pMD19-T vector were digested with restriction enzymes and inserted into the pYES2 expression vector. After sequencing verification, the empty and target vector was transferred into the Cd-sensitive yeast mutant Δyap1 and wild-type Y252, according to the yeast transformation and phenotype identification methods provided by Peng et al. (2017). In Δyap1, the vacuolar Cd sequestration was decreased already, resulting in hypersensitivity to Cd relative to wild-type cells (Wemmie et al., 1994). Meanwhile, the plasmids were also extracted and transformed into the zinc-sensitive yeast mutant Δzrc1 and its wild-type CM100, and copper-sensitive yeast mutant Δcup2 and its wild-type BY4741 using the lithium acetate method as we described before (Zhang et al., 2022).

The BjNRAMP1 gene fragment was ligated into pEGOEP35S-H, and transformed into A. thaliana wild-type Col-0 by the Agrobacterium tumefaciens GV3101 dip flowering method. The transformed lines were screened for hygromycin resistance and verified by sequencing. T3 generations of homozygous transgenic lines were used for phenotype identification.

A 1,917-bp promoter sequence located upstream of the BjNRAMP1 start codon was obtained by PCR amplification. The pBWA(V)BII-GUS vector was cleaved with enzymes BsaI and Eco31I and the target fragment was ligated to the linear vector using homologous recombination to construct the proBjNRAMP1:GUS fusion protein expression vector. The proBjNRAMP1:GUS vector was transformed into the A. thaliana wild type (Col-0).

To determine the localization of the BjNRAMP1 gene in plant cells, the full-length coding sequence without a stop codon of the BjNRAMP1 gene was amplified by RT-RCR and transformed into both A. thaliana protoplasts and tobacco (Nicotiana benthamiana) leaf epidermal cells using a plant expression binary vector pEGOEP35S-H with a GFP fluorescent tag (pEGOEP-H- 35S:BjNRAMP1-GFP). The preparation of protoplasts and their transformation into A. thaliana was performed following the polyethylene glycol method (Peng et al., 2017). Meanwhile, the Agrobacterium tumefaciens GV3101 solutions with the pEGOEP-H- 35S:BjNRAMP1-GFP vector were injected into the 3-week-old tobacco leaf epidermal cells. After incubation of A. thaliana protoplasts at 23℃ in the dark for 20 h and growth of tobacco for 2 days in dark, the GFP fluorescence was examined using a confocal microscope (FV1000; OLYMPUS). The localization marker of the cell plasma membrane is AtPIP2A-RFP.

Tissues from B. juncea and A. thaliana were collected and immediately frozen in liquid nitrogen with three biological replicates. First-strand cDNA was synthesized using a Tsingke Goldenstar RT6 cDNA Synthesis Kit ver.2 (Tsingke, Beijing, China). qRT-PCR was performed using SYBR Premix Ex TaqII on a Bio-Rad CFX 96 Real-Time Detection System as described previously (Zhang et al., 2020a).

For metal measurement in A. thaliana lines, the harvested plants were washed with distilled water, followed by drying at 75℃ until completely dehydrated. Plants were ground to fine powder, digested in 70% nitric acid at 100℃ for 2 h, and diluted with ultrapure water for metal determination.

For metal measurement in yeast, yeast cells that were transformed with empty vectors and BjNRAMP1 were cultured to OD = 0.8 with 20 μM CdCl₂ solution, treated, and incubated for 24 h. The cells were collected by centrifugation and washed with sterile water and 2 mmol/L EDTA. After washing well, the cells were dried at 70°C and subsequently nitrated with 70% concentrated nitric acid and diluted. The concentrations of Cd and other elementals in all samples were determined by inductively coupled plasma-mass spectrometry (ICP-MS).

Previously, we reported that the BjNRAMP1 was upregulated when B. juncea was exposed to high concentrations of Cd using RNA-Seq (Zhang et al., 2021). Herein, qRT-PCR result confirmed that the expression of BjNRAMP1 was silenced under either control or 10mg/kg Cd stress, but induced in both leaves and roots when plants were under 30 mg/kg Cd stress, suggesting that BjNRAMP1 was sensitive to higher concentration of Cd exposure ( Figure 1A ).

Cloning and sequence analysis indicated that BjNRAMP1 was 1,566 bp in length, encoding a protein with 521 amino acid residues with 12 predicted transmembrane domains ( Figure S1 ). Multiple alignments indicated that the BjNRAMP1 exhibited high identities to that in B. rapa (99%), B. oleracea (98.84%), B. napus (99%), and A. thaliana (85.90%). The phylogenetic analysis showed that BjNRAMP1 was closely related to its homologous genes in Brassica, including B. rapa, B. oleracea, and B. napus, but evolutionarily far away from that in Poaceae, such as O. sativa, Z. mays, and H. vulgare ( Figure 1B ).

To investigate the role of BjNRAMP1 in metal accumulation, the gene was expressed in the yeast heterologous system. Ectopic expression in Cd-sensitive mutant Δyap1 showed that the growth of transformed yeasts with BjNRAMP1 was enhanced as compared with the control with empty vector in the presence of 15 and 30 μM Cd ( Figure 2A ). The concentration of Cd and Mn of yeasts transformed with BjNRAMP1 was significantly higher than that in control when yeasts were under 20 μM Cd treatment for 6 and 12 h ( Figures 2B–E ). After 6 h of Cd treatment, the Cd concentration in yeasts transformed with BjNRAMP1 (40.93 μg/g) was not only 1.5-fold higher than that in Δyap1 with empty vector (27.08 μg/g), but also significantly higher than that in wild type (31.18 μg/g). As to Mn, the BjNRAMP1 transformed yeasts (4.05 μg/g) exhibited a similar level of Mn concentration with wild type (4.09 μg/g), but significantly higher than that in Δyap1 with the empty vector (3.12 μg/g). Additionally, the results were also observed when yeasts were treated with 20 μM Cd for 12 h, suggesting that BjNRAMP1 could facilitate the uptake of Cd and Mn.

However, no significant increase in the concentration of Zn, Cu, and Fe was measured in BjNRAMP1 transformed yeasts as compared with the empty vector ( Table S2 ). Meanwhile, no significant phenotypic differences were observed when BjNRAMP1 is expressed in Zn- and Cu-sensitive mutants ( Figure S2 ), indicating that BjNRAMP1 might be one of the major transporters of Cd and Mn, but not that of Zn and Cu.

To determine the subcellular localization of BjNRAMP1, the gene was fused with GFP fluorescent tag and under the regulation of the CaMV 35S promoter (35S:BjNRAMP1-GFP vector), and then the transiently expressed vector was transformed into both tobacco leaf epidermal cells and A. thaliana protoplasts ( Figures 3A, B ). In the tobacco leaf epidermal cells, the green fluorescence of fusion proteins (BjNRAMP1-GFP) was observed at the plasma membrane and co-localized with the plasma membrane marker AtPIP2A-RFP ( Figure 3A ). Despite the fact that the fluorescence intensity of BjNRAMP1-GFP in A. thaliana was slightly weaker than that in tobacco, the co-localized BjNRAMP1-GFP and plasma membrane marker AtPIP2A-RFP was still found in its protoplasts, confirming that BjNRAMP1 is localized to the plasma membrane ( Figure 3B ). Histochemical staining of GUS activity driven by the promoter of BjNRAMP1 (proBjNRAMP1:GUS) showed that BjNRAMP1 was mainly expressed in the vascular system of roots and leaves at the seedling stage, as well as stigma at the flowering stage ( Figure 3C ).

To further verify the function of BjNRAMP1, its coding sequence was driven by the CaMV 35S promoter and transformed into A. thaliana Col-0 generating the overexpression lines ( Figure 4 ). After qRT-PCR confirmation, three lines, OE-1 (2.09- to 29.86-fold), OE-3 (9.96- to 59.30-fold), and OE-4 (1.59- to 6.73-fold), which exhibited significantly higher expression than the Col-0, were selected for further analysis ( Figure 4B ). In the presence of different concentrations (10, 30, and 50 μM) of Cd, the growth of BjNRAMP1 overexpressing lines was significantly inhibited, displaying smaller seedling size ( Figure 4A ), lower fresh weight ( Figure 4C ), and shorter root length ( Figure 2D ). For example, no significant differences in phenotype (p > 0.05) were observed between the non-transformed line Col-0 and the overexpressing line OE-3 when Cd was absent (0 μM). However, the fresh weight and root length of OE-3 were dramatically decreased approximately 29.7% and 32.7% (p < 0.05), respectively, as compared with Col-0 under 10 μM Cd treatment. The inhibition effect was increased with the increase in the concentration of Cd, resulting in 50.8% and 54.5% decrease in fresh weight, as well as 52.1% and 71.7% decrease in root length observed under 30 and 50 μM Cd treatment, respectively.

The Cd concentration in the shoots and roots of the overexpression line OE-3 was significantly higher than that of the non-transformed line Col-0 ( Figure 4E ). Although significantly more Cd was accumulated in the shoots of OE-4, the Cd concentration in the roots was decreased. Together, these results suggested that BjNRAMP1 could increase the Cd accumulation in plants. Meanwhile, the Mn concentration in the roots of overexpression lines was significantly lower under normal growth conditions (Cd0), but significantly higher than that in Col-0 when plants were under 50 μM Cd stress, indicating that high Cd stress could promote the Mn uptake in the roots of overexpression lines ( Figure 2G ). In accordance with the findings in yeast, no significant increase in concentration of Zn, Cu, and Fe was observed in the A. thaliana overexpression lines as compared to the wild type ( Table S3 ).