Systematic Analysis of the DOF Gene Family in Flowering Chinese Cabbage (Brassica campestris) and the Role of BcDOF56 in Abiotic Stress Adaptation

1. Introduction

The DNA-binding with One Finger (DOF) family is a plant-specific group within the zinc-finger superfamily. Following the isolation of ZmDOF1 from maize (Zea mays) in 1993 [1], DOF homologs have been characterized in organisms ranging from algae to higher plants [2,3,4,5]. DOF proteins, generally ranging from 200 to 400 amino acids in size, are structurally defined by a highly conserved N-terminal DOF domain of approximately 52 residues. This domain forms a single C₂-C₂ zinc finger with the consensus sequence CX₂CX₂₁CX₂C [3]. In contrast, the C-terminal segment harbors a highly divergent transcriptional regulatory domain, which serves as the primary structural basis for the functional diversification of DOF proteins [6].

DOF transcription factors are highly conserved across spermatophytes and early terrestrial plants [7], with an evolutionary origin predating the divergence of Chlorophyta and embryophytes [8]. The green alga Chlamydomonas reinhardtii retains only a single DOF homolog, indicating that the extant DOF family most likely originated from this ancestral gene copy [4]. Subsequent extensive gene duplications drove the expansion of the DOF family during angiosperm diversification, facilitating plant adaptation to dynamic terrestrial environments [4]. To date, DOF family members have been characterized in numerous plant species, with gene copy number varying considerably. In monocots, for instance, genome-wide surveys have reported 30, 96, and 27 DOF genes in rice (Oryza sativa) [9], wheat (Triticum aestivum) [10], and orchid (Cymbidium goeringii) [11], respectively. In dicots, 36 DOF members have been characterized in Arabidopsis thaliana [12], 33 in tomato (Solanum lycopersicum) [13], 76 in Chinese cabbage (Brassica rapa) [14], 117 in rapeseed (Brassica napus) [15], 44 in Populus trichocarpa [16], and 75 in tobacco (Nicotiana tabacum) [17]. This pronounced divergence in copy number highlights the contribution of DOF expansion in lineage-specific functional diversification, enabling plants to cope with multifaceted environmental stresses [4].

Functionally, DOF transcription factors participate in multiple physiological processes, such as seed germination [18], leaf senescence and floral morphogenesis [19,20], and fruit ripening [21]. In Arabidopsis, AtDOF6 represses seed germination by interacting with AtTCP14 [18], whereas AtDOF2.1 promotes leaf senescence by activating jasmonic acid biosynthesis [20]. Overexpression of rapeseed BnCDF1 (cycling DOF factor 1) in Arabidopsis delays flowering by regulating the photoperiod pathway [22], while AtDOF4.7 accelerates floral organ abscission by inhibiting cell wall hydrolase activity [19]. In banana (Musa acuminata), MaDOF23 negatively regulates ethylene biosynthesis to modulate fruit ripening [21]. Beyond these developmental roles, DOF proteins serve as critical integrators of stress signaling, mediating plant tolerance to environmental stress, including extreme temperatures, drought, salinity, and pathogen infection [23]. For instance, the transcription of AtDOF5.8 is significantly induced by drought and salt stresses in Arabidopsis [24]. In B. rapa, most BraDof genes exhibit distinct transcriptional changes under abiotic stress conditions: the transcription of BraDof003, BraDof045, and BraDof053 is significantly upregulated under extreme temperature stress, whereas BraDof072 is strongly repressed; furthermore, drought and salinity treatments induce the transcription of BraDof045, BraDof053, and BraDof074 [14]. In walnut (Juglans regia), JrDOF3 improves thermotolerance through targeted activation of heat shock proteins [25]. Both rapeseed BnCDF1 and rice OsDOF1 enhance plant cold tolerance [22,26]. Moreover, PsDOF genes in Populus simonii are consistently upregulated across multiple tissues under diverse stress conditions [27]. Notably, a recent study in tobacco revealed opposing functions among DOF family members under cold stress: NtDOF40 enhances cold tolerance, while NtDOF63 acts as a negative regulator [17]. Collectively, these studies establish DOF transcription factors as critical integrators of plant development and stress adaptation. However, DOF members exhibit pronounced functional divergence across plant species, executing distinct or even antagonistic biological roles.

Flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee), a variant of B. rapa, is an economically and nutritionally vital stalk vegetable widely cultivated across southern China [28,29]. Unlike most Brassicaceae crops that require low-temperature vernalization to initiate bolting, flowering Chinese cabbage exhibits vernalization-independent development [30,31]. However, biotic and abiotic stress conditions often disrupt normal stalk development, causing substantial yield losses and quality deterioration that severely constrain year-round production of flowering Chinese cabbage [29]. Global climate change further threatens crop productivity, underscoring the urgent need to explore stress-tolerant genetic resources for crop improvement [32]. Although DOF gene families have been extensively investigated in various plant species, systematic characterization and functional roles of DOF members remain lacking in flowering Chinese cabbage. The recently released reference genome of this crop provides a valuable genomic resource for systematic gene family studies [30].

Here, we systematically surveyed and profiled the DOF gene family across the genome of flowering Chinese cabbage, elucidating their chromosomal localization, gene structures, phylogenetic relationships, syntenic patterns, and cis-regulatory elements. We further investigated the tissue-specific expression profiles of BcDOFs and explored their responses to heat and drought stresses. Collectively, these findings establish a comprehensive framework for deciphering BcDOF biological functions and provide promising candidate genes for breeding stress-resilient cultivars.

2. Materials and Methods

2.1. Identification and Chromosome Localization of DOF Gene Family in Brassica campestris

The genome sequences and annotations of B. campestris were retrieved from the China National Gene Bank (CNGB), based on data from Li et al. [30]. 36 Arabidopsis DOF proteins were obtained from TAIR (https://www.arabidopsis.org/; accessed on 10 June 2025), and served as queries for BLASTP searches against the B. campestris genome (E-value < 1× 10^-5) with the BLAST GUI Wrapper in TBtools-II v.2.482 [33]. In parallel, the Advanced HMMER Search module in TBtools-II was employed to screen candidate DOF members from this genome using the zinc-finger DOF (zf-DOF) domain model (Pfam: PF02701). Non-redundant candidates pooled from both searches were subsequently validated for intact, characteristic DOF motifs using the NCBI Batch CD-Search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 10 June 2025), thereby establishing the final catalog of BcDOF members.

Physicochemical properties of the predicted DOF proteins were analyzed with the ExPASY ProtParam tool (https://web.expasy.org/protparam/, accessed on 10 June 2025). Subcellular localization of DOF proteins was inferred using DeepLoc-2.0 (https://services.healthtech.dtu.dk/cgi-bin/webface2.cgi; accessed on 10 June 2025), and their chromosomal positions were visualized using TBtools-II [33].

2.2. Phylogenetic Analysis and Subfamily Classification of DOF proteins

To elucidate evolutionary relationships and subfamily classifications, DOF protein sequences from B. campestris and Arabidopsis were aligned using ClustalW under default parameter settings. MEGA 7.0 was employed to construct a neighbor-joining (NJ) tree supported by 1000 bootstrap replicates, followed by visualization in FigTree v1.4.4.

In parallel, to explore cross-species evolutionary dynamics, a multi-species phylogenetic tree covering ten representative plant species was constructed via OrthoVenn3 (https://orthovenn3.bioinfotoolkits.net/home, accessed on 12 July 2025) [34]. The species included C. reinhardtii, Z. mays, P. trichocarpa, O. sativa, Arabidopsis, S. lycopersicum, Cucumis sativus, B. rapa, B. napus, and B. campestris. Orthologous clusters were identified using the default OrthoMCL algorithm, and reconstructed the species phylogeny using the maximum likelihood (ML) method under the JTT+CAT evolutionary model. The DOF members previously identified in B. rapa [14] were manually refined; two putative sequences (Bra002057 and Bras007632) were excluded due to their high sequence homology to Arabidopsis Syntaxin of Plant 21 and Auxin Response Factor 18, leaving 74 authentic BraDOF members for downstream analysis. Subfamily demarcations were assigned according to established classifications and visualized using Adobe Illustrator CC 2019.

2.3. Orthology and Synteny Analyses of BcDOFs

Genome sequences and corresponding annotations of Arabidopsis, B. rapa, and B. napus were retrieved from TAIR (TAIR9), BRAD (Brara_Chiifu_v1.5), and GenoScope (Brassica_napus.annotation_v5), respectively. Orthologous gene clusters spanning these three species alongside B. campestris were identified and visualized using OrthoVenn3 server (https://orthovenn3.bioinfotoolkits.net/home, accessed on 4 July 2025) [34].

Within B. campestris, gene duplication events were identified via the One Step MCScanX-Super Fast plugin and mapped using the Advanced Circos module in TBtools-II [33]. Additionally, interspecific syntenies of DOFs among Arabidopsis, B. campestris, B. rapa, and B. napus were characterized and visualized using the MCScanX-Super Fast plugin and the Multiple Synteny Plot module, respectively. The nonsynonymous (Ka) and synonymous (Ks) substitution rates were determined for orthologous gene pairs via the Simple Ka/Ks Calculator module. The divergence time (T) of paired orthologs was estimated via the equation T = Ks/(2λ), where λ = 1.5 × 10⁻⁸ substitutions per site per year, a neutral mutation rate previously calibrated for B. rapa [35].

2.4. Analysis of Conserved Motifs and Gene Structures of BcDOFs

Conserved motifs within BcDOFs were analyzed using the MEME Suite (https://meme-suite.org/meme/; accessed on 20 September 2025) with a maximum of 15 motifs. Their gene structural characteristics were visualized using TBtools-II.

2.5. Profiling of Cis-Acting Elements in BcDOF Promoters

The 2000 bp genomic regions upstream of all BcDOF genes were retrieved from the B. campestris genome to predict putative cis-acting elements via the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 25 September 2025). The identified elements were categorized based on their putative function and displayed with TBtools-II.

2.6. Expression Profiling of BcDOFs Across Tissues and under Abiotic Stress

To characterize the tissue-specific expression profilings of BcDOFs, publicly available RNA-seq datasets (SRP427920) derived from B. campestris cultivar ‘49 Caixin’ were analyzed. FPKM (fragments per kilobase of exon model per million mapped fragments) values were obtained across seven tissues: roots, stems, young leaves, mature leaves, senescent leaves, flowers, and siliques. Normalized FPKM values were used to construct a heatmap.

For abiotic stress treatments, B. campestris cultivar ‘Youlv 501’ served as experimental material. Seedlings were grown in a controlled environmental chamber (T: 25 ± 2 °C; relative humidity: 75%; photoperiod: 16 h light/8 h dark) for 14 days. Uniform seedlings were transplanted into half-strength Hoagland solution and cultured for 7 days. At the five-leaf stage, seedlings were transferred to distinct stress regimes: heat treatment was applied at 40 °C in a constant-temperature incubator, while osmotic drought was simulated by supplementing the nutrient solution with 10% (w/v) polyethylene glycol 6000 (PEG 6000). Root and leaf were harvested at 0, 3, 24, and 48 h after treatment. All assays were conducted with three biological replicates per condition.

2.7. Quantitative Real-time PCR (qRT-PCR) Analysis

Following total RNA extraction and first-strand cDNA synthesis, quantitative real-time PCR (qRT-PCR) was conducted on a CFX connect^TM system (Bio-Rad, Hercules, CA, USA) with SYBR^® Green Premix Pro Taq HS qPCR Kit (Accurate Biology, Changsha, China). All qRT-PCR primers are detailed in Table S1. Relative gene expression was determined via the 2^-ΔΔCt method [36].

2.8. Overexpression of BcDOF56 and Functional Analysis in Arabidopsis

The coding sequence (CDS) of BcDOF56 was inserted into overexpression vector pCAMBIA3301, and subsequently transformed into Arabidopsis ecotype Columbia-0 (Col-0) via Agrobacterium tumefaciens-mediated floral dip protocol [37]. Putative transformants were screened across multiple generations via phosphinothricin resistance selection and further validated by qRT-PCR. Homozygous T₃ progenies were used for subsequent function assays.

Both wild-type (WT) and transgenic seeds were germinated in a peat-vermiculite composite (3:1, v/v) supplied with quarter-strength Hoagland solution, and cultivated under standard growth conditions for four weeks. For heat stress, seedlings were incubated at 40 °C for 60 h. For drought stress, watering was completely withheld for 15 days. Rosette leaves from both WT and transgenic lines were sampled to determine malondialdehyde (MDA) concentration and superoxide dismutase (SOD) activity, according to the previously described protocol [17].

2.9. Subcellular Localization of BcDOF56

The CDS of BcDOF56 lacking the termination codon was inserted into the transient expression vector pBI121-GFP linearized with SmaI and XbaI. The recombinant pBI121-BcDOF56-GFP or pBI121-GFP was transformed into Agrobacterium tumefaciens GV3101. The NLS-mCherry served as a nuclear marker. Agrobacterium suspensions carrying NLS-mCherry and either pBI121-GFP or pBI121-BcDOF56-GFP were infiltrated into N. benthamiana leaves. After infiltration for 72 h, fluorescence signals were visualized using an Axio Imager D2 microscope (Zeiss, Jena, Germany).

2.10. Statistical Analysis

Data were presented as the mean ± standard deviation (SD). Pairwise comparisons between groups were evaluated via a two-tailed Student’s t-test in SPSS v21.0 (IBM, Armonk, NY, USA). Asterisks denote statistical significance: *P < 0.05, **P < 0.01, and ***P < 0.001.

3. Results

3.1. Identification and Characterization of the BcDOF Gene Family in B. campestris

To identify candidate DOF family members in the B. campestris genome, we performed BLASTP searches using 36 Arabidopsis DOF proteins retrieved from TAIR. Following structural validation of the signature zf-DOF domain (Pfam: PF02701), a final set of 67 BcDOF candidates was identified and sequentially designated as BcDOF01 to BcDOF67 according to their physical coordinates on chromosomes (Figure 1; Table 1).

Chromosomal mapping revealed that all 67 BcDOF genes were nonuniformly distributed across ten chromosomes of B. campestris. Chromosome A09 contained the largest number (13 members, BcDOF52–BcDOF64), followed by A03 (10, BcDOF12–BcDOF21), and A01 (6, BcDOF01–BcDOF06). Chromosomes A04, A07, and A08 each harbored seven genes; A02 and A06 each had five genes; and A05 and A10 carried four and three genes, respectively.

The detailed physicochemical properties of all predicted BcDOF proteins are summarized in Table 1. Polypeptide lengths spanned from 148 (BcDOF20) to 482 amino acids (BcDOF23), corresponding to molecular weights ranging from 17.03 to 52.17 kDa. Predicted theoretical isoelectric points (pI) spanned from 4.73 (BcDOF33) to 9.74 (BcDOF20). Most BcDOF proteins displayed instability indices over 40, indicating their relative instability. All BcDOF proteins exhibited negative grand average of hydropathy (GRAVY) values, varying from –0.323 to –1.092, confirming their hydrophilic characteristics. Furthermore, all BcDOF proteins were predicted to be localized to the nucleus, consistent with their function as transcription factors.

3.2. Phylogenetic Analysis of the BcDOF Gene Family

To elucidate the classification and evolutionary relationship of DOF transcription factors, a phylogenetic tree was constructed in MEGA 7.0, including 67 BcDOFs from B. campestris and 36 AtDOFs from Arabidopsis (Figure 2). Tree topology assigned all DOF members into four major clades (Groups A-D). Groups B, C, and D were each further split into specific subfamilies: B₁/B₂, C₁, C_2.1, C_2.2, C₃, and D₁/D₂. Among the major clades, Group C contained the highest proportion of BcDOF members (35.82%, 24 genes), followed by Group D (28.36%, 19 genes). Among the nine subgroups, Subgroup D₁ was the largest (19.40%), whereas Subgroup C_2.2 was the smallest, representing only 5.97% of total BcDOF members (Figure 2).

To elucidate the evolutionary characteristics of DOF proteins, a comparative phylogeny was reconstructed across ten representative plant species, covering the green alga (C. reinhardtii), two monocots (Z. mays and O. sativa), and seven dicots (Arabidopsis, S. lycopersicum, C. sativus, P. trichocarpa, B. campestris, B. rapa, and B. napus) (Figure 3). Intriguingly, C. reinhardtii possessed a solitary DOF gene, which clustered into Subgroup D₁. In terrestrial plants, moderate family sizes were observed in O. sativa (30), Z. mays (46), and A. thaliana (36). In comparison, 67, 74, and 117 DOF members were identified in B. campestris, B. rapa, and B. napus, respectively, indicating substantial expansion of the DOF family within Brassica species. Given its presence in the green alga C. reinhardtii and its broad conservation across land plants, Subgroup D1 may represent an ancient and highly conserved DOF lineage.

3.3. Orthologous Gene Clustering of DOF Families in Brassicaceae

Given the pronounced expansion of DOF genes observed in Brassica species, we performed an orthologous clustering analysis across four representative Brassicaceae species using the OrthoVenn3 platform, incorporating 294 DOF proteins from B. campestris (67), B. rapa (74), B. napus (117), and A. thaliana (36) (Figure 4). Among the 70 orthologous clusters, 29 were shared by all four species, whereas 30 were restricted to three Brassica species. The remaining clusters comprised species-specific or partially shared groups. Among the 70 orthologous clusters, 29 clusters comprising 134 DOF proteins was shared across all four species, whereas 30 clusters encompassing 91 DOFs were identified as Brassica-specific. Pairwise comparisons revealed three shared clusters (6 DOFs) between B. campestris and B. rapa, three (9 DOFs) between B. rapa and B. napus, and two (4 DOFs) between B. campestris and B. napus. Additionally, three-species overlaps were detected, with two clusters (7 DOFs) shared among Arabidopsis, B. rapa, and B. napus, and one cluster (3 DOFs) common to Arabidopsis, B. campestris, and B. rapa. In addition, 40 proteins were assigned as singletons, including one gene each from Arabidopsis (AT3G45610.1) and B. campestris (BcDOF14), alongside four from B. rapa, and 34 from B. napus. Detailed information regarding these orthologous clusters and singletons is listed in Table S2 and Table S3.

3.4. Conserved Motifs and Gene Structural Features of BcDOFs

To delineate the structural architectures of BcDOFs, conserved motifs and gene structures were systematically analyzed. In total, 15 distinct motifs were detected across 67 BcDOFs using MEME Suite (Figure 5A, B; Figure S1). Notably, Motif 1, corresponding to the signature zf-DOF domain, was present in all BcDOF proteins. In contrast, the remaining motifs exhibited clade-specific distribution patterns, primarily within the C-terminal regions. For instance, Subgroups C2.2 and C3 were characterized by Motif 10, whereas Subgroup C2.1 carried N-terminal Motifs 5/14 along with C-terminal Motifs 9/10/12. Subgroups A and C1 shared Motifs 4 and 15, while Subgroup D1 exhibited the greatest motif diversity, harboring Motifs 3–8 and 15 (Figure 5A).

Analysis of exon-intron architecture revealed pronounced divergence among BcDOF subgroups (Figure 5C). Members of Subgroups A, C_2.2, C₃, and D₂ were universally intronless, whereas Subgroups C₁, C_2.1, and B₁, alongside the majority of B₂ and D₁ members, possessed a single intron. Such structural divergence implies that gene structural reorganization may play a pivotal role in functional specialization during BcDOF evolution.

3.5. Collinearity and Duplication Analysis of BcDOF Genes

To elucidate the evolutionary history of DOF members, both intraspecific collinearity analysis of B. campestris and interspecific synteny analysis across B. campestris, B. rapa, B. napus, and Arabidopsis were performed using TBtools-II v2.345 (Figure 6; Table S4). Among the identified BcDOF genes, two gene pairs were classified as tandem duplicates (BcDOF01/BcDOF02 and BcDOF02/BcDOF03), four genes (BcDOF24, BcDOF34, BcDOF62, and BcDOF65) were classified as products of dispersed duplication, while the majority arose from whole-genome triplication (WGT) or segmental duplication. Consequently, WGT or segmental duplication predominantly drove the expansion of BcDOF genes in B. campestris.

To assess the selective constraints and evolutionary rates acting on duplicated BcDOF genes, we calculated the nonsynonymous-to-synonymous substitution ratios (Ka/Ks) across 56 paralogous pairs (Table S5). The estimated divergence times ranged from 3.84 (BcDOF02 and BcDOF03) to 47.28 (BcDOF25 and BcDOF51) million years ago (MYA), averaging 12.07 MYA. Notably, one gene pair (BcDOF21 and BcDOF56) was excluded from subsequent analysis due to extremely high sequence divergence. Only one gene pair (BcDOF01/BcDOF02) exhibited neutral selection (Ka/Ks ≈ 1), whereas the remaining pairs had Ka/Ks ratios varying from 0.15 (BcDOF14/BcDOF30) to 0.82 (BcDOF02/BcDOF03). These findings suggest that purifying selection has dominated BcDOF evolution, thereby preserving the the structural integrity and functional conservation of the BcDOF family during long-term evolution.

Interspecific synteny analysis identified 104, 162, and 165 homologous gene pairs between B. campestris and A. thaliana, B. rapa, and B. napus, respectively (Figure 7; Table S6). The Ks values of these homologous pairs between B. campestris and Arabidopsis ranged from 0.25 to 2.88, with an average of 0.69, corresponding to an average divergence time of approximately 23.13 MYA (Table S6). For interspecific orthologous gene pairs within Brassica species, the Ks values varied from 0 to 1.43, corresponding to average divergence times of 3.86 to 6.81 MYA. These results indicate that, following its split from the common ancestor shared with Arabidopsis, the Brassica lineage experienced WGT, which reshaped the modern genomes and drove the expansion of the DOF family across Brassica species.

3.6. Analysis of cis-Acting Elements in BcDOF Promoters

To elucidate the transcriptional regulatory mechanisms governing BcDOFs, we analyzed the 2000 bp promoter regions using the PlantCARE database. In total, 60 distinct cis-acting elements were mapped and assigned into four functional modules: light response, phytohormone signaling, growth and development, and environmental stress response (Figure 8).

Among all functional classes, elements responsive to light predominated in both frequency and diversity, spanning 22 distinct motif types. The G-box, Box-4, GT1-motif, and TCT-motif were the most prevalent, occurring in 85.07%, 82.09%, 65.67%, and 59.70% of BcDOF promoters, respectively. Phytohormone-responsive elements constituted another major category, encompassing elements associated with auxin (AuxRR-core, TGA-box, and TGA-element,), gibberellin (TATC-box, P-box, and GARE-motif), abscisic acid (ABRE and AAGAA-motif), ethylene (ERE), methyl jasmonate (TGACG- and CGTCA-motif), and salicylic acid (SARE and TCA-element). Notably, ABRE elements were the most prevalent, occurring 137 times in 55 BcDOF promoters (82.09%). In terms of developmental regulation, several tissue-specific elements were recognized, including those modulating endosperm metabolism (GCN4-motif and AACA-motif), root-specific expression (motif I), meristem differentiation (CAT-box and CCGTCC-box), flower development (AP-1), and seed maturation (RY-element). Furthermore, 13 stress-responsive elements were detected, dominated by anaerobic-induction elements (ARE, present in 92.54% of promoters with 142 sites), followed by pathogen defense signaling (as-1, 76.12%), wounding (WUN-motif, 46.27%), and low temperature (LTR, 41.79%), alongside other defense elements (STRE, W-box, TC-rich repeats, and WRE3). Abundant MYB and MYC transcription factor binding sites were also detected across BcDOF promoters. Taken together, the extensive cis-acting element profiles suggest that BcDOFs act as pivotal regulatory nodes integrating phytohormonal signaling and environmental stimuli to coordinate development and stress adaptation in B. campestris.

3.7. Tissue-specific expression profiles of BcDOF genes

To investigate the spatial expression profiles of BcDOFs, publicly available RNA-seq data (SRP427920) of B. campestris cultivar ‘49 Caixin’ were analyzed across seven representative tissues, including roots, stems, flowers, siliques, young leaves, mature leaves, and senescent leaves (Figure 9).

BcDOF members exhibited highly divergent expression patterns across the surveyed organs. Five genes exhibited ubiquitous transcription, with BcDOF16 and BcDOF45 constitutively expressed at high levels, whereas BcDOF34, BcDOF49, and BcDOF62 demonstrated broad expression with no marked organ bias. By contrast, several genes showed pronounced spatial preference: BcDOF28, BcDOF29, BcDOF51, and BcDOF57 were predominantly expressed in roots; BcDOF17, BcDOF32, BcDOF38, and BcDOF48 were preferentially expressed in stems, flowers, and siliques; and a distinct cluster comprising BcDOF04, BcDOF08, BcDOF21, BcDOF24, and BcDOF66 shared elevated transcript abundance in roots, stems, and siliques. Additionally, 22 BcDOF members (e.g., BcDOF01, BcDOF02, BcDOF05, BcDOF61) displayed negligible or basal expression throughout all surveyed tissues, implying stage-specific or conditional activation.

3.8. Expression Analysis of BcDOFs Under Abiotic Stress

To comprehensively characterize the stress-responsive profiles of BcDOFs, five-leaf-stage B. campestris seedlings were subjected to heat (40 °C) and simulated drought (10% PEG 6000) treatments. The relative transcript levels of 12 selected BcDOFs in roots and leaves were quantified via qRT-PCR.

Under heat stress, the majority of BcDOF genes in leaves were significantly induced at 3 h after treatment. Transcripts of BcDOF13, BcDOF24, BcDOF33, BcDOF35, BcDOF48, and BcDOF61 peaked at 3 h and subsequently declined at 24 and 48 h, yet remained significantly elevated relative to the control. BcDOF07, BcDOF25, and BcDOF56 exhibited continuous increasing during the treatment period, reaching maximum expression at 48 h. In contrast, BcDOF04 was transiently induced at the early stage before being sharply repressed by 48 h (Figure 10A). In roots, most BcDOFs displayed slight changes at 3 h but were markedly upregulated at later stages. BcDOF04, BcDOF38, BcDOF48, and BcDOF51 exhibited strong induction at 24 h, whereas the remaining tested genes maintained elevated transcript abundance at both 24 and 48 h (Figure 10B).

Drought stress elicited divergent expression responses among BcDOFs. In leaves, most BcDOFs were significantly induced by drought stress. BcDOF33 and BcDOF61 responded rapidly, peaking at 3 h; BcDOF07, BcDOF25, BcDOF48, and BcDOF56 reached maximum transcript abundance at 24 h, whereas BcDOF38 and BcDOF51 peaked at 48 h (Figure 11A). Conversely, BcDOF04, BcDOF13, BcDOF24, and BcDOF35 were significantly downregulated throughout the treatment. In roots, the majority of BcDOFs were initially suppressed at the early stage of drought but recovered at subsequent time points. Notably, BcDOF04, BcDOF38, and BcDOF51 were markedly upregulated at 48 h relative to the control (Figure 11B). Collectively, these data indicate that BcDOFs display distinct transcriptional dynamics under heat and drought stresses, supporting their functional diversification in conferring abiotic stress resilience in B. campestris.

3.9. Subcellular localization of BcDOF56

To determine the subcellular distribution of BcDOF56, the fusion construct pBI121-BcDOF56-GFP was transiently expressed in N. benthamiana leaves. The GFP fluorescence strictly co-localized with the nuclear marker NLS-mCherry (Figure 12), verifying that BcDOF56 resides in the nucleus, in line with its functional annotation as a transcriptional regulator.

3.10. Overexpression of BcDOF56 Enhances Heat and Drought Tolerance in Arabidopsis

To evaluate the biological functions of BcDOFs under environmental stress, BcDOF56 was chosen for transgenic functional validation. Two independent T₃ homozygous Arabidopsis lines overexpressing BcDOF56 (Figure S2) were generated for stress tolerance assays. When four-week-old seedlings were exposed to 40 °C for 60 h, WT plants displayed severe leaf chlorosis and wilting; by contrast, the transgenic lines exhibited only mild leaf curling and slight wilting (Figure 13A). Consistent with these phenotypes, MDA contents in transgenic lines were reduced to 52.94% and 58.84% of WT levels, while SOD activity was significantly elevated (Figure 13B, C).

For drought treatment, water was withheld for 15 days, causing severe wilting and foliar chlorosis in WT plants, whereas transgenic lines maintained relatively healthy growth with only slight curling at the leaf margins (Figure 14A). Consistent with the observation under heat stress, transgenic lines accumulated lower MDA (50.24% and 51.11% of WT) and exhibited elevated SOD activity relative to WT (Figure 14B, C). Collectively, these phenotypic and physiological data support a positive role for BcDOF56 in enhancing heat and drought tolerance in Arabidopsis.

4. Discussion

Plant-specific DOF factors function as pivotal hubs mediating development and responses to environmental stresses [4,23,38]. Although DOF families have been characterized in multiple plant species [12,13,14,15,16,17,26], a systematic characterization of the DOF family remains lacking in B. campestris. Therefore, we carried out a genome-wide identification of this family in B. campestris, followed by analyses of its evolutionary relationships, structural characteristics, expression profiling, and potential roles in heat and drought responses.

4.1. Evolutionart Dynamics and Expansion of the BcDOF Gene Family

In B. campestris, a total of 67 BcDOF genes were systematically identified. A comparative cross-species survey revealed a progressive expansion of DOF members from algae to angiosperms. C. reinhardtii harbors only a single DOF gene, whereas both monocot and dicot genomes possess substantially more members. This observation supports the hypothesis that the DOF gene family may have originated from a shared ancestor gene retained as only one single copy in C. reinhardtii [15]. Throughout land plant evolution, the expansion of DOF genes was closely associated with the establishment of complex transcriptional networks that facilitate adaptation to fluctuating environments [4]. Within Brassicaceae, Arabidopsis harbors 36 DOF genes, whereas the three investigated Brassica species exhibit dramatic family expansion, especially B. napus, whose 117 members represent the largest DOF inventory documented to date [15]. The interspecific copy-number divergence is largely attributed to gene duplication events and modifications of genome size and structure [39].

Synteny analysis revealed that an average Ks value of 0.69 for orthologous DOF pairs between Arabidopsis and B. campestris, corresponding to a divergence time of approximately 23.13 MYA, consistent with the published 14–24 MYA divergence time for Arabidopsis and Brassica species [15,40]. Following divergence from Arabidopsis, Brassica species underwent whole-genome triplication (WGT), chromosomal rearrangements, and allopolyploidization, which collectively reshaped their modern genomic architecture and catalyzed substantial gene family expansion [41]. Selection pressure analysis demonstrated that nearly all BcDOF paralog pairs exhibited Ka/Ks ratios substantially below 1.0, with only one gene pair (BcDOF01/BcDOF02) displaying a ratio close to 1. These findings suggest that purifying selection acted as the primary evolutionary constraint preserving BcDOF functional stability, while WGT and segmental duplication served as the major drivers expanding the BcDOF family to adapt to fluctuating environments [15].

4.2. Phylogenetic Classification, Structural Conservation and cis-Regulatory Characteristics

According to the phylogenetic classification established in Arabidopsis [12], all 67 BcDOF members were classified into nine subfamilies. Subgroup D₁ comprised the largest proportion of members (19.40%), whereas Subgroup C_2.2 contained the fewest (5.97%). This distribution pattern is consistent with that of Arabidopsis [12] and B. rapa [14], but departs from that reported in B. napus [15], potentially reflecting distinct evolutionary histories and functional diversification among lineages. The conserved zf-DOF domain was detected in all BcDOF proteins. DOFs within the same clade shared similar motif architectures, with Subgroup D₁ exhibiting more motif types than other subgroups. This observation aligns with the findings of B. rapa [14], B. napus [15], and tobacco [17]. Furthermore, exon-intron architecture analysis revealed that BcDOFs possessed one or two exons, with the majority (55.22%) containing two exons, matching the structural features of the DOF family reported in Arabidopsis [12], B. napus [15], and tobacco [17].

Cis-acting element analysis revealed that BcDOF promoters harbor abundant elements responsive to abiotic stress (drought, low temperature, anaerobic induction) and phytohormones (ABA, SA, and MeJA). These features align closely with findings in B. napus [15], tobacco [17], and pea [42], further supporting the evolutionarily conserved role of DOF proteins in regulating abiotic stress tolerance [38,43]. Analogous promoter compositions have also been reported in kiwifruit AcDOFs and Dendrobium officinale DoDOFs [44,45]. The conservation of these cis-elements elements reinforces the notion that DOF transcription factors act as central regulatory hubs integrating phytohormone cascades with environmental stress adaptations [5,43,46].

4.3. Divergent Transcriptional Dynamics of BcDOFs under Heat and Drought Stresses

Previous studies have illustrated that the vital functions of DOF proteins in plant responses and adaptation to environmental stress [38]. In Arabidopsis, AtDOF5.8 is significantly upregulated by drought and salt stress and enhances seedling stress resilience via interaction with ANAC069 [24]. In B. napus, most BnaDOF genes respond strongly to extreme temperatures, BnaDOF54 (BnCDF1) is induced by cold [15], and its overexpression markedly improves cold tolerance [22]. Stress-responsive DOF homologs have also been implicated in stress responses in non-model species: PeDOF-11 is strongly activated by heat stress in Passiflora edulis [47]; DoDOF7 and DoDOF26 are significantly upregulated by heat and drought in Dendrobium officinale [45]; and PsDOFs show broad stress-responsive expression across various tissues in Populus simonii [27].

In the present study, qRT-PCR revealed distinct stress-dependent expression profiles of BcDOFs. Most BcDOFs were significantly induced in both foliar and root tissues under heat stress (Figure 10). Under drought stress, BcDOFs exhibited distinct tissue-specific expression patterns: several genes (BcDOF07, BcDOF25, BcDOF33, BcDOF38, BcDOF51, and BcDOF56) were markedly induced in leaves, whereas BcDOF04 and BcDOF13 were significantly repressed; furthermore, the majority of BcDOFs were significantly downregulated in roots (Figure 11). Similar heterogeneous and tissue-specific DOF expression profiles have been reported in B. rapa [14] and D. officinale [45]. In tobacco, NtDOF40 and NtDOF63 even exert opposing regulatory effects on cold tolerance [17]. DOF genes exhibit divergent transcriptional expression and biological functions under diverse stresses, with these differences shaped by plant species, genotype, and even tissue type [15]. Collectively, these findings suggest that DOF paralogs may have undergone functional diversification. Different members may regulate distinct downstream stress-responsive genes, leading to variation in stress-tolerance phenotypes across species and tissues.

4.4. BcDOF56 Positively Regulates Stress Tolerance Through ROS Homeostasis

Given its marked transcriptional induction under both heat and drought stresses, BcDOF56 was selected as a promising candidate for heterologous overexpression in Arabidopsis. Phenotypic and physiological analyses revealed that BcDOF56-overexpressing lines survived better than WT under both heat and drought treatments, with substantially lowered MDA content and significantly higher SOD activity (Figure 13 and Figure 14). MDA serves as a reliable marker for cellular lipid peroxidation, whereas SOD acts as a core antioxidant enzyme that catalyzes the dismutation of superoxide radicals, thereby alleviating reactive oxygen species (ROS) damage [48]. Consequently, these physiological results suggest that BcDOF56 may enhance heat and drought tolerance, potentially by promoting antioxidant defenses to alleviate ROS-induced oxidative damage.

Similar ROS-modulating circuits mediated by DOF have been reported in diverse species. For instance, in tobacco, NtDOF40 and NtDOF63 antagonistically regulate cold tolerance by differentially modulating antioxidant enzyme activities and ROS homeostasis [17]. Likewise, sweetpotato IbDOF2.1 interacts with ABA-binding factor 2 (IbABF2) to trigger stomatal closure and ROS scavenging under drought conditions [49]. These findings collectively suggest that DOF transcription factors participate in a multifaceted defense regulatory network by mediating protein-protein interactions, integrating hormonal signals, and activating the transcription of downstream antioxidant and protective genes [50]. In conclusion, this work elucidates the core functional characteristics of the DOF family B. campestris and identifies BcDOF56 as a promising candidate gene for breeding of climate-resilient varieties. However, the molecular mechanisms by which BcDOF56 regulates heat and drought tolerance remain to be elucidated.

5. Conclusions

In the present study, we identified 67 BcDOF genes in flowering Chinese cabbage (Brassica campestris) and assigned them to nine subfamilies. All BcDOFs harbor a conserved zf-DOF domain, display distinct gene structures, and contain numerous cis-acting elements related to hormone signaling and stress responses. Phylogenomic analysis indicated that the DOF family has an ancient origin and underwent substantial expansion in angiosperms. In B. campestris, whole-genome triplication and segmental duplication were the primary drivers of the BcDOF family expansion. Expression profiling demonstrated that BcDOFs exhibit diverse responses to heat and drought stresses. Moreover, heterologous overexpression of BcDOF56 in Arabidopsis enhanced tolerance to heat and drought stresses, possibly through the modulation of ROS homeostasis. Collectively, this work offers a framework for understanding the evolution and stress-responsive roles of the BcDOF family and identify BcDOF56 as a potential target for molecular breeding to improve abiotic stress tolerance.