The seeds of Chinese cabbage (Cv. Guangdongzao) were pretreated by soaking them with 8% sodium hypochlorite for 3 min, followed by thorough washing with ddH₂O at least five times. These seeds were then germinated in half-strength MS media within a growth chamber set to day/night cycles of 16 h/8 h at 22°C/20°C. Seedlings with uniform size were transferred to a hydroponic culture. After 5 days, they were subjected to a 75-μM Cd treatment. This concentration was selected as it yielded the most pronounced tolerance phenotype in BrMYB116 transgenic yeast compared to the control, a finding supported by testing other concentrations (50 and 100 μM) as shown in Supplementary Figure 3 . The samples were collected at various time points: 0, 2, 4, 8, 12, and 24 h after Cd stress, and used for total RNA extraction and subsequent analysis. For the generation of transformed Chinese cabbage plants, the floral dip method and vernalization infiltration technique were employed (Chen et al., 2021).

The CDS was cloned into the pRS416-GFP vector to construct the overexpression vectors. NSR1 and VLD1 genes were cloned into the pRS423 vector in fusion with the RFP sequence. BrMYB116 was cloned from the cDNA of Chinese cabbage, while the coding sequences of ScFIT3, NSR1, and VLD1 were amplified using the cDNA of yeast (JRY472) with specific primers. Subsequently, the amplified sequences were inserted into pRS416-GFP and pRS423 in fusion with the RFP sequence. For expression constructs of BrMYB116 and ScFIT3, DNA was PCR amplified from genomic DNA of Chinese cabbage (Cv. Guangdongzao) and yeast (JRY472), respectively, with specific primers. The amplified sequences were subsequently inserted into the binary vector pCambia3300 to yield pBrMYB116::BrMYB116 and pScFIT3::ZmO2L1.

Total RNA was extracted from Chinese cabbage tissues using TRIzol. For yeast RNA extraction, the M5-EASTspin Yeast RNA Rapid Kit was employed, as previously described. The RNA was then used for cDNA synthesis. For qRT-PCR analysis, the SYBER premix-Ex-Taq kit was used. The experiment was performed using three biological replicates.

Yeast cells transformed with either the BrMYB116 and ScFIT3-expressing pRS416-GFP vectors, along with those transformed with the empty pRS416-GFP (WT) vector, were cultured overnight in an SC (URA⁻) fluid medium at 30°C. The cell solutions were then diluted to achieve an OD₆₀₀ of approximately 0.1 and allowed to grow until reaching an OD₆₀₀ of 0.3. After precisely adjusting the OD₆₀₀ to 0.3, the cell solutions were fivefold diluted and spotted onto plates with or without 75 μM of Cd. The cells were incubated at 30°C for 2 days. The experiments were repeated three times. The growth curve of BrMYB116 and WT was conducted after cultivation in a liquid SC (URA⁻) medium at 30°C. The solutions were diluted to an OD₆₀₀ of approximately 0.1 and then further incubated to reach an OD₆₀₀ of approximately 0.3. After precisely adjusting the OD₆₀₀ to 0.3, the solution was treated with 75 μM of Cd for 12 h. Subsequently, OD₆₀₀ was measured every 2 h to monitor the growth pattern.

The yeast cells co-transformed with pRS416-GFP-expressing vectors or BrMYB116 ScFIT3- and pRS423-expressing vectors NSR1 and VLD1 in fusion with RFP were cultured overnight in an SC (URA⁻ and HIS⁻) fluid medium at 30°C. The yeast cells transformed with an empty pRS416-GFP vector were also cultured overnight in an SC (URA⁻) fluid medium at 30°C. Then, the solutions were diluted to an OD₆₀₀ of approximately 0.1 and allowed to grow until reaching an OD₆₀₀ of approximately 0.3. After adjusting the OD₆₀₀ to exactly 0.3, the solutions were treated with or without 75 μM of Cd. After incubating for about 12 h at 30°C, the yeast cells were observed under a Zeiss 300 confocal microscope.

The yeast cells expressing BrMYB116 were treated with 75 μM Cd for 14 h, and then the cells were harvested and sent to Noveogen for transcriptome analysis. Three independent biological replicates were performed. Briefly, RNA integrity was tested using the RNA Nano 6000 Assay Kit on a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as the input material for RNA sample preparation. mRNA was purified using poly-T oligo-attached magnetic beads. Fragmentation was performed using divalent cations under elevated temperatures in a first-strand synthesis reaction buffer. The first-strand cDNA was synthesized using a random hexamer primer and M-MuLV reverse transcriptase (RNase H). Subsequently, second-strand cDNA synthesis was carried out using DNA polymerase I and RNase H. Remaining overhangs were converted to blunt ends by exonuclease/polymerase. After the adenylation of the 3′ DNA fragments, adaptors with hairpin loop structures were prepared for hybridization. To select cDNA fragments within the preferred length range of 370–420 bp, the library fragments were purified with an AMPure XP system (Beckman Coulter, Beverly, USA). Subsequently, PCR was carried out with Phusion high-fidelity DNA polymerase, universal PCR primers, and the Index (X) Primer. The resulting PCR products were purified using the AMPure XP system, and library quality was evaluated using an Agilent Bioanalyzer 2100 system. The clustering of index-coded samples was carried out on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina). After cluster generation, the library was sequenced on an Illumina Novaseq platform, and 150-bp paired-end reads were generated.

The promoter activity assay was carried out in tobacco as previously described (He et al., 2016). Briefly, all reporter vectors were transformed into GV3101 strains. A single clone was inoculated into YEP medium with rifampicin and kanamycin and grown at 28°C overnight. The cells were collected and resuspended, and the OD₆₀₀ was adjusted to 1. The cells were incubated at room temperature for about 2 h, after which, they were infiltrated into 4-week-old tobacco leaves. Subsequently, these leaves were placed in a dark growth chamber overnight, followed by exposure to light for 2 days, as previously described. The Dual-Luciferase Reported Assay system (catalog No. E1910; Promega) was used for the quantification of luciferase values.

ChIP in yeast was performed according to an established method with minor modifications. Briefly, 100 ml of yeast cells (with an OD of 1.0) were cross-linked by 1% of formaldehyde for 20 min at 30°C. The sonicated protein–DNA complex was immunoprecipitated with or without (negative control) anti-flag antibody. The DNA from these complexes was then used for ChIP-qPCR assay. ChIP signals were calculated as previously described. All primers used for qPCR are listed in Supplementary Table S1 .

The content of Cd in Chinese cabbage seedlings was measured using an ICP-MC instrument as previously described (Gong et al., 2003).

In our previous report (He et al., 2022), a number of Cd response genes (CRGs) from the Chinese cabbage cultivar “Guangdongzao” have been identified via transcriptome profile analyses. As yeast is a common eukaryotic model for studying Cd-resistant mechanisms, it was employed in this study to investigate the CRGs of Chinese cabbage involved in Cd tolerance (He et al., 2022). The genes were fused with a glycerol phosphate dehydrogenase (GPD) promoter in the yeast expressing recombination vector pRS416 to yield pGPD::CRGs, which were then transformed into budding yeast (cv JRY472). Serial dilution assays were conducted to evaluate Cd tolerance. The results show that the strains expressing BrMYB116 demonstrated enhanced growth compared with the wild-type JRY472 with an empty vector ( Figure 1A ). Growth curves in yeast expressing BrMYB116, with or without Cd stress, revealed that overexpressing cells displayed a Cd tolerance phenotype, particularly between 8 and 16 h post-Cd treatment ( Figure 1B ).

To explore the specific role of BrMYB116 in Cd stress tolerance, the CDS was fused with GFP and then expressed in yeast cells. The pRS423 vector, expressing the nuclear-localized yeast gene NSR1 in fusion with RFP, was used as a positive control (Yan and Melese, 1993), while EV (empty vector; pRS416-EGFP) was used as a negative control. In the absence of Cd, BrMYB116 was predominately localized in the cytoplasm. However, when exposed to 75 µM of CdCl₂, BrMYB116 was translocated into the nucleus, while the GFP signal of empty vectors remained unaffected by Cd stress ( Figure 2 ), indicating the specific nuclear localization of BrMYB116 under Cd stress. In contrast, when BrMYB116 fused with GFP was expressed in Arabidopsis protoplasts, no noticeable difference in cellular localization was observed regardless of Cd presence or absence ( Supplementary Figure 1 ). While BrMYB116 is consistently localized in the nucleus of Arabidopsis protoplasts irrespective of external Cd levels ( Figure 1 ), its specific nuclear localization in yeast suggests a potential regulatory role in Cd stress tolerance at the molecular level. These results also imply that BrMYB116 may interact with the cis-acting elements of its target genes in the nucleus, which are essential for Cd stress response in yeast.

The response of BrMYB116 to Cd stress may be due to changes in transcript abundance of genes regulated by BrMYB116. An RNA-seq analysis was carried out on Cd-treated yeast cells transformed with BrMYB116-expressing vectors to examine whether the gene expression pattern has changed. A total of 331 DEGs with statistically significant changes [log2 (FoldChange) ≤1 or ≥1 and adjusted p-value ≤0.05] were selected ( Supplementary Table S2 ; Figure 3A ). GO enrichment analysis showed that numerous DEGs are involved in transporter and transmembrane transporter activity ( Supplementary Table S3 ) indicating that BrMYB116 might enhance Cd tolerance by regulating Cd ion transport. Given that BrMYB116 could regulate more than one target gene to confer Cd tolerance, we focused on a smaller group of DEGs with more pronounced expression changes [log2(FoldChange) ≤−1.5 or ≥3). These DEGs, labeled as DEG1 to DEG18 in descending order of expression in the clustering analysis map, include gene-CENPK1137D_4855, gene-CENPK1137D_521, gene-CENPK1137D_574, gene-CENPK1137D_5283, gene-CENPK1137D_2397, gene-CENPK1137D_417, gene-CENPK1137D_2396, gene-CENPK1137D_5368, gene-CENPK1137D_699, gene-CENPK1137D_138, gene-CENPK1137D_3368, gene-CENPK1137D_3854, gene-CENPK1137D_4708, gene-CENPK1137D_3277, gene-CENPK1137D_4991, gene-CENPK1137D_2539, gene-CENPK1137D_4598, and gene-CENPK1137D_3634 ( Figure 3B ).

To validate transcriptome analysis, 18 DEGs were selected and used for quantitative real-time PCR as presented in Figure 3C . The qRT-PCR results were consistent with the RNA-seq data for most genes ( Figure 3C ). To investigate their functions, these 18 DEGs were expressed in yeast cells under the control of a GPD promoter. Under Cd stress (75 µM), only yeast cells overexpressing DEG5 (CENPK1137D_2397), which encodes a putative member of the facilitator of iron transport 3 (FIT3), exhibited more substantial growth than the wild-type control ( Figure 4A ; Supplementary Figure 2 ).

This finding indicates that the overexpression of DEG5 alone is sufficient to confer Cd tolerance, underscoring its vital role in managing Cd stress. Furthermore, assessments of Cd content in yeast cells revealed reductions in Cd accumulation for both BrMYB116 and ScFIT3 compared to JRY472 (EV(WT)) ( Figure 4B ) indicating that the Cd tolerance phenotype mediated by BrMYB116 and ScFIT3 is mainly attributed to reductions in Cd accumulation. Notably, JRY472 (ScFIT3) showed greater reductions in Cd levels than JRY472 (BrMYB116) ( Figure 4B ). Given that diminished Cd accumulation is a key indicator of Cd tolerance, these findings suggest ScFIT3 as the potential primary target gene regulated by the transcription factor BrMYB116 in the Cd tolerance pathway.

ScFIT3, a member of the facilitator of iron transporter family, is known to transfer Cd ions in addition to iron ions. We investigated the subcellular localization of ScFIT3 by fusing its CDS with GFP and expressing it in yeast cells. The pRS423-vector-expressing vacuole-localized gene VLD1 was fused with RFP as the positive control, and pRS416-GFP was used as the negative control (Huh et al., 2003). Under Cd stress, ScFIT3 was localized in the vacuole. However, ScFIT3 was distributed throughout the cell upon Cd treatment ( Figure 5 ). The absence of VLD1 fused with RFP in yeast cells under Cd stress suggests that Cd might have disrupted its expression. ScFIT3 changed its subcellular localization when treated with Cd stress. Under normal conditions without Cd stress, ScFIT3 may play a role in facilitating the transport of iron ions in the vacuole. However, in the presence of Cd, ScFIT3 appears to facilitate the transport of the Cd ions from the vacuole and throughout the whole cell. Cd content measurements ( Figure 4B ) showed that the altered localization of ScFIT3 might contribute to the exclusion of Cd ions from the cell membrane.

To explore the interaction between BrMYB116 and ScFIT3, a promoter–luciferase fusion was created by combining the 2-kb promoter of the firefly luciferase gene, including the 5′ UTR fragment from ScFIT3, into a promoter–luc fusion construct. Both p35S::BrMYB116 and empty vector (control) constructs were transiently introduced into tobacco leaves. In transactivation assay, enhanced expression of pScFIT3-luc via p35S::BrMYB was observed under Cd treatment compared to that without Cd ( Figure 6A ). These results indicated that BrMYB116 specifically activated pScFIT3 under Cd stress. A ChIP-qPCR experiment was carried out to confirm the interaction of BrMYB116 with the promoter fragments in transgenic yeast cells producing GFP-tagged BrMYB116. Following the immunoprecipitation with an anti-GFP antibody, four primer pairs (F1, F2, F3, and F3) were used for targeting different sections (500 bp) of the corresponding promoter ( Figure 6B ). The findings indicated a positive interaction for BrMYB116 at F1 ( Figure 6B ), but not with other fragments, including ACT1 (XM_009117825, NCBI) used as a negative control. This suggests that BrMYB116 binds specifically to a certain region of the ScFIT3 promoter to regulate its expression.

To further explore the role of BrMYB116 in Cd stress tolerance in Chinese cabbage, the expression of BrMYB116 was tested in response to Cd stress. Chinese cabbage seedlings were grown in MS nutrient solution with or without 200 µM of CdCl₂, and then the samples were collected at different time intervals. The qRT-PCR results revealed a steady increase in BrMYB116 mRNA levels, with the highest expression observed at the eighth hour post-Cd treatment ( Figure 7A ). These findings indicate that the expression of BrMYB116 was activated by Cd stress suggesting its involvement in Cd responses within the Chinese cabbage. Subsequently, we transferred BrMYB116 into the “Guangdongzao” cultivar and generated transgenic lines. After confirming the successful overexpression of BrMYB116 via RT-qPCR, two independent lines were selected for further analyses of the Cd tolerance phenotype. However, these lines did not exhibit enhanced Cd tolerance in comparison to wild-type plants, as depicted in Figures 7B, C . As a transcription factor activated by Cd stress, the downstream target gene in the Cd tolerance pathway might be missing in Chinese cabbage.

To explore the function of FIT3 in preventing Cd accumulation in plants, we transferred ScFIT3 to the “Guangdongzao” cultivar of Chinese cabbage. After confirming the successful integration of ScFIT3 via RT-PCR ( Supplementary Figure 4 ), two independent lines were selected to explore its involvement in Cd stress tolerance and uptake. Under Cd stress, transgenic seedlings exhibited improved growth and longer root lengths compared to the wild-type control ( Figures 7B, C ). Moreover, the Cd content was significantly reduced in these two transgenic lines compared to the wild-type control ( Figure 8A ) indicating that the improved growth in the transgenic lines was attributed to reduced Cd accumulation and toxicity. Recognizing the importance of Chinese cabbage as a dietary vegetable, we also analyzed the content of several essential nutritional ions in transgenic seedlings. As shown in Figures 8B–D , the contents of K, Cd, and P ions were indistinguishable between the transgenic seedlings and the wild-type control suggesting that the expression of FIT3 did not affect the uptake of the primary nutritional ions.