Seeds of B. rapa (inbred line Chiifu-401-42) were germinated in Petri dishes at 25°C, transplanted to pots with fertile substrate, and grown at 22°C under a 16 h/8 h light–dark cycle. Seedlings were subjected to salinity stress via irrigation with 100 mM or 200 mM NaCl. Leaf and root tissues from control and treated plants were collected 12 h after treatment, snap-frozen in liquid nitrogen, and stored at −80°C for RNA extraction. Three biological replicates were used for each condition (OMEGA, China). Wild-type Nicotiana benthamiana was grown in a 2:1 (v/v) peat:perlite mix at 25°C, approximately 70% RH, under a 16 h/8 h photoperiod, and fully expanded leaves were used for Agrobacterium-mediated GFP transient expression.

The full-length coding sequence (CDS) of the putative gene was retrieved from the B. rapa genome database (BRAD; http://brassicadb.org/brad/). The physicochemical properties of the identified proteins, including the theoretical isoelectric point (pI) and molecular weight (MW), were calculated using the ProtParam tool (Artimo et al., 2012). In addition, the BrPHD58 protein sequence was used as a BLASTP query to identify PHD58-like homologs in Arabidopsis thaliana and other Brassica species by searching online proteomes in BRAD (http://brassicadb.org/brad/). The resulting candidate sequences were then confirmed through the presence of the conserved PHD-finger domain, together with the associated Alfin-like domain, using the SMART and InterPro databases. The exon/intron structure of the BrPHD58 gene was determined using Gene Structure Display Server 2.0. Gene-specific primer pairs were designed using the Primer3Plus tool (https://www.primer3plus.com). High-quality total RNA was obtained from the young leaf tissue of B. rapa using an RNA purification kit (OMEGA, China), and 2 μg of high-quality RNA was subsequently used for first-strand cDNA synthesis using a cDNA Synthesis Kit (TransGen, China).

Evolutionarily conserved protein sequences from various Brassica species were identified through BLAST searches using the B. rapa genome database on the BRAD platform (http://brassicadb.org/brad/). Multiple sequence alignments were conducted using ClustalW, and ligand-binding residues were annotated with the use of BioEdit software (version 7.2.5). Phylogenetic relationships among the homologs were inferred using the neighbor-joining (NJ) method implemented in MEGA7 software (version 7.0.26), with statistical support evaluated through 1,000 bootstrap replicates.

To investigate subcellular localization, the BrPHD58 coding sequence, flanked by BamHI and XbaI restriction sites, was initially cloned into the pMD18-T cloning vector. The resulting recombinant plasmids were digested with the corresponding restriction enzymes and ligated into the 35S-GFP expression vector (35S-pBinGFP2) to generate the fusion construct 35S-BrPHD58-GFP. The integrity of the construct was confirmed by sequencing. The verified fusion plasmids were then transferred into A. tumefaciens strain GV3101 using standard transformation protocols. The fusion constructs and control vectors were introduced into the epidermal cells of N. benthamiana leaves via microinjection. The samples were then returned to the growth chamber for an additional 24 h, after which confocal microscopy was used to observe the transformed tobacco leaf cells (TCS sp8, Leica, Solms, Germany).

To amplify the target gene, PCR was performed using cDNA synthesized from total RNA as the template. The amplification fragments were separated using agarose gel electrophoresis and purified using a Gel Extraction Kit (OMEGA BioTek, USA). The purified DNA fragment obtained from gel extraction was subsequently ligated into the pMD-18T vector (TaKaRa, Dalian, China) for molecular cloning. The recombinant construct was digested using QuickCut restriction enzymes XbaI and BamHI (TaKaRa, Dalian, China). Following digestion, the products were separated on a 1% agarose gel, and DNA fragments of the expected size for the candidate gene were visualized and confirmed. The presence and accuracy of the target sequence were further verified using Sanger sequencing. The purified product was ligated into the expression vector pCAMBIA1301, driven by the CaMV35S promoter. The fusion plasmids were first validated by restriction enzyme digestion and subsequently confirmed by Sanger sequencing. After the successful insertion of the candidate gene into the 35S-pCAMBIA1301 vector, the recombinant construct was introduced into Agrobacterium tumefaciens strain GV3101 for expression studies.

Various Arabidopsis transgenic lines were established via A. tumefaciens-mediated transformation using the floral dip method (Mara et al., 2010). Putative T1 transgenic lines were validated by PCR amplification of BrPHD58 from genomic DNA and RT-PCR analysis of RNA using specific primers (Supplementary Table S1). Total RNA was extracted and reverse-transcribed into cDNA according to the protocol provided by TransGen Bio-Tech, China. Genomic DNA was purified from the leaf tissue of transgenic Arabidopsis using the CTAB extraction technique. Seeds from confirmed T1 Arabidopsis lines were sown on MS medium containing 30 mg/L hygromycin to select for T2 and, subsequently T3 generations. Three homozygous T3 lines (2, 3, and 6) were selected for subsequent molecular and phenotypic characterization.

For the salt stress experiment, transgenic seeds were grown on half-strength MS medium. For the initial 5-day growth screen, seedlings were grown on half-strength MS medium with different NaCl concentrations (75 mM and 150 mM), and their phenotypic responses were recorded. For soil-based salinity experiments, 14-day seedlings were cultivated in soil and exposed to 200 mM NaCl solution to simulate salt stress conditions. Photographs were captured at various time points after exposure to salinity stress. To assess chlorophyll levels, 12-day seedlings of both transgenic and WT lines were treated with 200 mM NaCl for three days. Subsequently, 0.05 g of rosette leaves were harvested and homogenized in 3 mL of 80% acetone, and the extracts were centrifuged at 8,000 rpm for 5 min. After centrifugation, the clear supernatant was carefully decanted, and the absorbance was measured at 645 nm and 663 nm. The total chlorophyll content was quantified using the method described by Arnon (1949).

The qRT-PCR method was employed to quantify the expression of selected reference genes in BrPHD58 transgenic and WT (Col-0) plants under normal conditions and after exposure to salt stress using gene-specific primers (Supplementary Table S1). Twelve-day-old Arabidopsis seedlings cultivated on treated MS medium were subjected to salt stress via foliar application of 100 mM NaCl solution for 2 h. Total RNA was extracted from both transgenic lines and Col-0 plants using a Plant RNA Extraction Kit (OMEGA, China). First-strand cDNA synthesis was performed using the EasyScript cDNA Synthesis Kit (TransGen, China). qRT-PCR was conducted using the ABI 7500 Fast RT-PCR System (Applied Biosystems, USA) according to the manufacturer’s protocol. Each biological sample was analyzed in triplicate to obtain average Ct values, and relative gene expression levels were quantified using the 2⁻ΔΔCt method (Schmittgen and Livak, 2008). Relative expression levels of genes were normalized using Arabidopsis Actin-2 as the internal control gene.

Data are presented as mean ± standard error (SE) of three biological replicates. Statistical significance was evaluated using Student’s t-test (*P <0.05 and **P <0.01) in IBM SPSS Statistics v22.

The coding sequence of BrPHD58 contained 753 bp, encoding a peptide sequence of 251 amino acids, with a predicted molecular weight of 27.91 kDa and an isoelectric point (pI) of 5.12. The BrPHD58 gene is located on chromosome A06 and comprises five exons, interrupted by four introns. The SMART tool was used to analyze conserved domains and identified a canonical PHD finger domain of approximately 43 amino acids, typically implicated in chromatin recognition via histone methyl-lysine binding, and an Alfin-like region of approximately 128 amino acids, often associated with transcriptional regulation in plants (Figure 1A). BLASTP searches identified the closest homologs in A. thaliana and detected highly similar proteins across Brassica species (Figure 1A). A phylogenetic tree was established using the (NJ) method to evaluate the evolutionary association of Alfin-like PHD finger proteins among Brassica and Arabidopsis species. Phylogenetic analysis revealed that BrPHD58 is closely related to other Brassica species (Figure 1B). Furthermore, conserved domain analysis revealed that proteins from all Brassica species contained a highly conserved N-terminal Alfin/DUF3594 domain and a C-terminal PHD domain, confirming their classification within the Alfin-like PHD-finger gene family (Figure 1B).

Salt stress, drought, and temperature extremes are major abiotic factors that severely limit crop growth and lead to substantial yield loss (Gupta et al., 2022). In a previous study, we identified that BrPHD58 was highly expressed under salt and drought stress conditions (Alam et al., 2019). Expression analyses were further performed in B. rapa using qRT-PCR to verify induction of BrPHD58 by high salinity. The results clearly showed that BrPHD58 was highly upregulated (e.g., 5-fold increase) at 12 h after treatment with 100 mM and 200 mM NaCl compared to the control (Supplementary Figure S1). These results suggest that BrPHD58 may contribute to the regulation of plant responses to salt stress.

The BrPHD58 gene (Bra026210), which encodes a PHD finger protein in B. rapa, was amplified using gene-specific primers (Supplementary Figure S2A). The PCR product was purified, ligated into the pMD18-T vector, and transformed into E. coli DH5α. Recombinant clones were confirmed by Sanger sequencing and double enzyme digestion, producing two expected bands of approximately 2,700 bp and 753 bp (Supplementary Figures S2B, D). For expression analysis, the BrPHD58 fragment was ligated into the 35S-GFP and 35S-pCAMBIA1301 vectors. Precise insertion into 35S-GFP was confirmed by sequencing and BamHI/XbaI digestion (Supplementary Figure S2C). Similarly, cloning into 35S-pCAMBIA1301 was validated by sequencing and XbaI/BamHI digestion, producing bands of approximately 12 kb and 753 bp, respectively (Supplementary Figure S2E). The confirmed constructs were introduced into A. tumefaciens GV3101 for downstream expression analysis. Six transgenic lines were further confirmed by genomic PCR and used for subsequent BrPHD58 expression analyses (Supplementary Figures S2F, G).

To investigate the subcellular localization of BrPHD58, a BrPHD58-GFP fusion construct under the 35S promoter was transiently expressed in N. benthamiana leaves via A. tumefaciens-mediated transformation. The 35S:GFP vector was used as a control. GFP fluorescence was visualized using confocal microscopy. The results confirmed that the BrPHD58 protein was specifically localized in the nucleus, as indicated by the distinct nuclear GFP fluorescence observed, whereas the control GFP signal was distributed across the cell (Figure 2). These findings suggest that BrPHD58 predominantly resides in the nucleus, supporting its potential role as a transcription factor (Figure 2).

To assess the physiological responses to salt stress, three independently derived homozygous BrPHD58 (Bra026210) overexpression lines were selected for a comprehensive analysis based on their high expression levels as determined by RT–qPCR (Supplementary Figure S3). Under standard one-half MS growth conditions, the growth performance of the three transgenic lines was largely comparable to that of WT (Col-0), with only minimal differences observed (Figures 3A, D). However, upon exposure to salt stress, the growth of all BrPHD58 transgenic lines was significantly affected compared to that of WT (Col-0) (Figures 3B, C). Moreover, the BrPHD58 transgenic lines exhibited a significant reduction in root length compared to the WT control under 75 mM and 150 mM NaCl treatment (Figures 3E, F). These findings suggest that ectopic expression of BrPHD58 adversely affects root development under salt stress and reduces the overall salt tolerance of A. thaliana seedlings, underscoring its potential role in the modulation of stress response mechanisms.

To investigate whether BrPHD58 transcriptional factors have a functional role in response to salt stress, we developed transgenic Arabidopsis lines expressing BrPHD58 driven by the constitutive CaMV 35S promoter. Three independent Arabidopsis transgenic lines (L2, L3, and L6) were cultivated in soil, and 14-day-old T3 seedlings were treated with 200 mM NaCl (Figure 4). Overexpression of BrPHD58 impaired salt stress tolerance in Arabidopsis, as evidenced by the phenotypic differences observed after 7 days of 200 mM NaCl treatment (Figure 4A). Transgenic lines displayed leaf chlorosis compared to the Col-0 controls (Figure 4B). After 14 days, some of the transgenic plants died, whereas the majority of Col-0 plants remained green and viable (Figure 4C). By 21 days, most of the transgenic lines had died (Figure 4E), and after 27 days almost all of the transgenic lines had wilted and died, whereas some of the Col-0 plants survived at 200 mM NaCl (Figure 4G). These results indicate that BrPHD58 overexpression reduces salt tolerance in Arabidopsis. Survival rates recorded across the three independent salt stress experiments revealed significant differences between the transgenic lines and WT (Col-0) plants (Figure 5A). Chlorophyll content was measured in 12-day-old seedlings of various transgenic and WT plants following a 3-day treatment with 200 mM NaCl, revealing significant differences between the two groups (Figure 5B). The results demonstrated a significant reduction in chlorophyll levels in the transgenic lines compared to the controls (Figure 5B). These findings imply that BrPHD58 overexpression impairs the plant’s capacity to cope with salt stress.

To explore the molecular mechanism of BrPHD58 in the salt stress response, we performed quantitative expression analysis of three stress-associated genes in both BrPHD58 transgenic lines and Col-0 plants. The results demonstrated that under normal conditions, the transcript levels of AtRD22, AtRD29A, and AtLEA14 were elevated in BrPHD58 plants compared to those in WT. However, following salt stress treatment, the expression levels of the stress-responsive genes AtRD22, AtRD29A, and AtLEA14 were significantly reduced in BrPHD58 transgenic plants compared to WT plants (Figure 6). These results suggest that BrPHD58 may function as a negative regulator of gene expression associated with salt stress response in plants.