Genome-wide identification and analysis of the Delay Of Germination 1 (DOG1) gene family in Brassica napus and its potential role in Manganese (Mn) stress response

TL;DR

This study identifies and analyzes the DOG1 gene family in Brassica napus, revealing its role in responding to manganese stress and offering insights for developing stress-tolerant crops.

Contribution

The first genome-wide analysis of the DOG1 gene family in Brassica napus under Mn stress, revealing its regulatory roles and evolutionary patterns.

Findings

42 BnDOG1 genes were identified and distributed across 16 chromosome scaffolds.

Phylogenetic analysis grouped BnDOG1 genes into five subfamilies.

BnDOG1 genes were induced by Mn stress, suggesting a role in stress response.

Abstract

Manganese (Mn), a heavy metal, induces oxidative stress when present in excess, thereby inhibiting plant growth. The Delay of Germination 1 (DOG1) gene family plays a crucial role in seed dormancy and germination. However, the genome-wide organization and functional roles of the DOG1 gene family under Mn stress remain uncharacterized in Brassica napus.

In this study, we identified 42 BnDOG1 family members and elucidated their regulatory roles under Mn stress using whole-genome and differential transcriptomic analyses. The BnDOG1 genes were evenly distributed across 16 chromosome scaffolds, and all members contained the conserved DOG1 domain. Phylogenetic analysis classified the BnDOG1 genes into five subfamilies. Covariance analysis indicated that segmental duplication was the primary mechanism driving gene expansion. Gene Ontology (GO) enrichment analysis revealed the highest enrichment in biological process categories. Notably, BnDOG1 genes were induced by Mn stress.

In this study, we demonstrate that the BnDOG1 gene plays a critical role in mediating physiological responses to environmental stress, thereby establishing a foundation for the development of heavy metal–tolerant crops.

The online version contains supplementary material available at 10.1186/s12870-026-08296-1.

Figures

Phylogenetic tree of *DOG1* gene family in *Arabidopsis thaliana* and *Brassica napus*. Light gray squares represent *BnDOG1* members; light blue triangles represent *AtDOG1* members

Phylogenetic tree of *DOG1* gene family in *Arabidopsis thaliana* and *Brassica napus*. Light gray squares represent *BnDOG1* members; light blue triangles represent *AtDOG1* members

Phylogenetic tree of *BnDOG1* genes and analysis of conserved motifs (a), domains (b), and gene structures (c)

Chromosomal distribution of *DOG1* genes in *Brassica napus*

Segmental duplication events of *BnDOG1* genes

Synteny analysis of *DOG1* genes among *Brassica napus*, *Arabidopsis thaliana*, *Brassica oleracea*, and *Brassica rapa*. Gray lines represent syntenic blocks; red lines highlight orthologous *DOG1* pairs

Protein-protein interaction network of *BnDOG1* members

Protein-protein interaction network of *BnDOG1* members

Top 20 enriched GO terms for *BnDOG1* genes. Y-axis: GO terms; X-axis: gene ratio; dot size: number of genes

Full text

Background

Brassica napus is a globally important oilseed crop, and breeding high-quality varieties is essential. Identifying key genes and favorable genetic variations will accelerate varietal improvement [1]. Although abiotic stresses such as drought and lead induce cellular damage in B. napus [2, 3], the species exhibits strong tolerance to salinity and cadmium [4, 5]. Interestingly, while drought stress impairs maternal plants, it enhances the vigor of their offspring [3].

The DELAY OF GERMINATION 1 (DOG1) gene plays a critical role in seed dormancy adaptation to diverse environments. DOG1 suppresses gibberellin (GA)-regulated gene expression and modulates endosperm properties [6, 7]. Moreover, the expression levels of DOG1 regulators correlate with altitude, temperature, and climate [8, 9]. As a central dormancy regulator, DOG1 likely interacts with plant hormones to control seed dormancy [10]. Other transcription factors also participate in dormancy regulation; for example, Basic LEUCINE ZIPPER TRANSCRIPTION FACTOR67 activates DOG1 to establish primary dormancy in Arabidopsis [11], and Arabidopsis thaliana SEED DORMANCY 4-LIKE regulates dormancy by mediating both DOG1 and GA pathways [12]. DOG1 forms a regulatory network by interacting with ABA HYPERSENSITIVE GERMINATION1 and heme [13]. The DOG1 protein can self-associate— a property not required for protein accumulation but essential for its function [14]. Natural variation in seed dormancy is primarily governed by two loci—REDUCED DORMANCY 5 and DOG1—which interact with the abscisic acid (ABA) signaling pathway to regulate germination [15]. Studies indicate that two type 2 C protein phosphatases, ABA-Hypersensitive Germination 1 (AHG1) and AHG3, play critical roles in releasing DOG1-mediated dormancy signals [16]. Additionally, Arabidopsis histone demethylases, LYSINE-SPECIFIC DEMETHYLASE LIKE 1 and 2, are crucial for establishing seed dormancy, although they repress the expression of dormancy-related genes such as DOG1 [17]. Dormant seeds are typically smaller than non-dormant ones [18], and environmental perception by the endosperm is vital [19]. Ethylene also regulates seed dormancy, and crosstalk between ethylene and DOG1 pathways has been reported [20]. Interestingly, nitric oxide release shows a negative correlation with dormancy [21]. Seed dormancy determines germination timing, and the amount of DOG1 protein in harvested seeds dictates the duration of dormancy release [22, 23]. Three effective methods to break seed dormancy include cold stratification, gibberellin application, and ultrasound pretreatment, all of which significantly enhance germination rate and speed [24].

Manganese (Mn) is both an essential micronutrient and a heavy metal, and Brassica napus plays a role in responding to heavy metal stress. Previous studies have shown that DOG1 genes are involved in various hormone signaling pathways. This study aims to investigate the role of DOG1 genes in Brassica napus under manganese (Mn) stress. Excessive Mn causes oxidative stress and cellular damage [25]. Mn toxicity has been studied in several crops: in peanut (Arachis hypogaea), it leads to leaf spotting, inhibited root growth, and increased proline content [26]; in rice (Oryza sativa), the OsZIP gene family may modulate Mn toxicity [27]; in tea (Camellia sinensis), overexpression of CsMetal Tolerance Protein 8 enhances stress tolerance under Mn stress [28]. Interestingly, selenium (Se) supplementation can alleviate Mn-induced stress in plants [25].

DOG1 is a key gene family involved in seed dormancy regulation. Previous stud-ies have shown that DOG1 expression can be induced by exogenous gibberellin and methyl jasmonate [29, 30]. However, the molecular mechanisms underlying DOG1 family functions under abiotic stress remain unclear. In this study, we conducted comprehensive analyses of the BnDOG1 gene family using bioinformatics and qRT-PCR, focusing on gene structure, phylogeny, physicochemical properties, selective pressure, homology, promoter cis-elements, protein interaction networks, GO enrichment, and transcriptomic expression patterns. To better understand the role of the DOG1 gene family in seed dormancy regulation and stress response, the specific scientific question addressed in this study is whether DOG1 genes participate in the response of Brassica napus to Mn stress.

Methods

Identification of BnDOG1 gene family members

Protein sequences and annotation files for Arabidopsis thaliana and Brassica napus were downloaded from Ensembl Plants (http://plants.ensembl.org/index.html)[31] and BRAD (http://brassicadb.cn/#/)[32]. The Brassica napus genome version used was Brana_ZS_HZAU_V1.0. Candidate BnDOG1 genes were identified using the Hidden Markov Model (HMM) profile of the DOG1 domain (PF14144) from PFAM (https://www.ebi.ac.uk/interpro/entry/pfam/#table)[33]. Additionally, known AtDOG1 protein sequences from UniProt ( https://www.uniprot.org/)[34] were used as queries in BLAST searches against the B. napus proteome with an E-value cutoff of 1e-5. The initial HMM screening yielded 73 candidates, while BLAST identified 44. The intersection of these two sets was taken, and redundant sequences were removed, resulting in 42 non-redundant BnDOG1 genes.

Multiple sequence alignment and phylogenetic analysis

To investigate evolutionary relationships, BnDOG1 and AtDOG1 protein sequences were combined. Multiple sequence alignment was performed using MUSCLE in MEGA11. A maximum likelihood (ML) [35] phylogenetic tree was constructed with 1000 bootstrap replicates and visualized in circular format using iTOL (https://itol.embl.de/)[36].

Gene structure and conserved motif prediction

Exon-intron structures were analyzed using TBtools based on genome annotations. Conserved motifs were identified using the MEME suite (https://meme-suite.org/meme/tools/meme) [37] with a setting of 10 motifs. Results for motifs, domains, gene structures, and phylogeny were integrated and visualized using TBtools.

Chromosomal localization and synteny analysis

Chromosomal positions of BnDOG1 genes were mapped using TBtools [38]. Within-species synteny analysis was conducted to detect gene duplication events. Cross-species synteny analyses were performed between B. napus and (A) thaliana, and among (B) oleracea, B. rapa, and B. napus to infer orthologous relationships. Protein and annotation data for B. oleracea and B. rapa were obtained from BRAD.

Physicochemical properties and subcellular localization

Protein characteristics—including amino acid count, molecular weight, and isoelectric point—were analyzed using ExPASy (https://web.expasy.org/protparam/)[39]. Subcellular localization was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/)[40].

Selective pressure analysis

Ka (non-synonymous substitution rate), Ks (synonymous substitution rate), and Ka/Ks ratios were calculated using TBtools, with AtDOG1 genes as references. Genes with fewer than one effective codon after alignment were excluded.

Promoter cis-regulatory element analysis

Upstream 2000 bp sequences from translation start sites were extracted using TBtools. Cis-elements were identified via PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/)[41] and visualized with TBtools.

Protein-protein interaction network prediction

Interacting partners of BnDOG1 proteins were predicted using STRING (https://cn.string-db.org/)[42] based on Arabidopsis orthologs. Networks were visualized and refined using Cytoscape.

GO enrichment analysis

Functional annotations were performed using EggNOG-mapper (http://eggnog-mapper.embl.de/)[43]. GO enrichment analyses were conducted and visualized using TBtools.

Tissue-specific expression analysis

Expression profiles of BnDOG1 genes across tissues and stress conditions were retrieved from BnIR (https://yanglab.hzau.edu.cn/BnIR)[44]. Heatmaps of Log2(FPKM + 1) values were generated using ChiPlot.

Plant materials and qRT-PCR analysis

Based on the expression patterns of BnDOG1 gene members, the Brassica napus cultivar “Zhongshuang 11” was selected for cultivation. Rapeseed plants were grown hydroponically, and manganese (Mn) stress was applied using a solution of 100 µg/L MnSO₄. The stress treatment was administered at the bolting stage via root immersion, and plant samples were collected 24 h after treatment. Lateral roots and leaf samples were, immediately ground in liquid nitrogen and stored at -80 °C or used for RNA extraction with TRIzol reagent. First-strand cDNA was synthesized using the BeyoRT™ II cDNA Synthesis Kit (Beyotime, Shanghai). Quantitative real-time PCR (qRT-PCR) was performed on a CFX96™ Real-Time System (Bio-Rad) with SYBR Green Master Mix (Sangon Biotech). Three independent biological replicates were established for the plant materials, and each was analyzed in triplicate during qRT-PCR assays. Relative gene expression levels were calculated using the 2^−ΔΔCt method [45]. Primers were designed using Primer5.0, and Actin served as the reference gene (Table S5).

Results

Identification and characterization of BnDOG1 members

We identified 42 BnDOG1 genes, each containing at least one DOG1 domain. The protein sequences of these members are provided in Table S6. Physicochemical analysis (Table 1) revealed coding sequences ranging from 230 (BnaC05G007530) to 1296 (BnaC08G032460) amino acids, molecular weights from 25.92kDa (BnaA09G059590) to 146.67 kDa (BnaC08G032460), and isoelectric points from 4.89 (BnaC01G012400) to 9.82 (BnaC05G006110). Half of the proteins were acidic and half were basic. Instability indices ranged from 38.66 (Basic. Instability indices ranged from 38.66 (BnaC02G066230)) to 67.33 (BnaC05G006110); only one protein (BnaC02G066230) was stable (index < 40), suggesting dynamic regulatory rather than structural functions. Grand Average of Hydropathy (GRAVY) scores ranged from − 0.75 to − 0.166, indicating a hydrophilic nature and potential roles in metabolism, defense, and signaling. Subcellular localization predictions indicated that 73.81% of BnDOG1 proteins localize to the nucleus, 14.29% to the cytosol, and 11.9% to chloroplasts.

Table 1. Characterization of DOG1 proteinsGene nameNumber of Amino AcidMolecular Weight(KDa)Isoelectric pointInstability IndexGrand Average of HydropathicitySubcellular localizationBnaA09G05959023125.925.6549.09-0.166ChloroplastBnaC01G01240024527.494.8958.76-0.38NucleusBnaA10G02640036441.826.6142.59-0.569NucleusBnaA03G04627028531.505.5151.3-0.314CytosolBnaA09G02056028031.035.1862.2-0.499NucleusBnaC03G04157034137.898.6454.9-0.57NucleusBnaC02G06623037042.237.8138.66-0.534NucleusBnaA08G01250028732.805.3562.9-0.509NucleusBnaA03G03360033136.838.9954.67-0.606NucleusBnaC05G00753023026.326.2356.6-0.311ChloroplastBnaC02G00288032636.648.8761.73-0.735NucleusBnaC03G07850029633.765.4564.6-0.472CytosolBnaC01G05455033437.107.8952.3-0.535NucleusBnaA07G02661058767.386.2361.6-0.729NucleusBnaC07G05172028531.625.4247.47-0.368CytosolBnaC05G05350024428.166.2754.04-0.494NucleusBnaA06G00523082392.359.5761.38-0.567NucleusBnaA05G03325055061.289.4344.47-0.503NucleusBnaC02G00403037743.326.6841.5-0.515NucleusBnaC05G006110930104.689.8267.33-0.536NucleusBnaA01G00998023927.649.0343.5-0.193CytosolBnaC01G01238047855.359.144.78-0.194ChloroplastBnaA01G04087033437.127.8950.39-0.54NucleusBnaC08G05789023226.085.4345.32-0.166ChloroplastBnaC09G02712028031.305.1265.32-0.531NucleusBnaA05G03095024428.106.6154.59-0.488NucleusBnaC09G06925065173.099.7760.32-0.596NucleusBnaC03G05667036742.018.3341.06-0.555NucleusBnaA09G03933071681.515.8758.8-0.645NucleusBnaA09G00809075086.518.9347.39-0.644NucleusBnaA02G04458064473.288.5543.47-0.576NucleusBnaA06G02774036741.957.341.11-0.545NucleusBnaA02G00545036241.627.2241.96-0.538NucleusBnaA06G00650023026.266.3255.44-0.293ChloroplastBnaA01G00999027931.494.9959.3-0.518NucleusBnaC08G0324601296146.678.1752.52-0.53NucleusBnaC07G03111028031.155.557.29-0.428CytosolBnaA07G04076037643.15.8165.94-0.602NucleusBnaC09G00968030835.399.4640.13-0.614NucleusBnaA06G04081028031.055.6258.74-0.42CytosolBnaC08G05714048453.747.0459.07-0.498NucleusBnaC05G05659031134.99.4158.46-0.554Nucleus

Phylogenetic analysis of BnDOG1 gene family

A phylogenetic tree was constructed using BnDOG1 and AtDOG1 sequences (Fig. 1). The 58 total members clustered into five clades, with Clade III containing the most members [17] and Clade II the fewest [5]. BnDOG1 genes were interspersed within all five clades alongside AtDOG1 genes, indicating deep evolutionary conservation.

Fig. 1. Phylogenetic tree of DOG1 gene family in Arabidopsis thaliana and Brassica napus. Light gray squares represent BnDOG1 members; light blue triangles represent AtDOG1 members

Gene structure, conserved motifs, and domains

A phylogenetic tree of the 42 BnDOG1 genes was used to analyze gene structure, motifs, and domains (Fig. 2). MEME identified 10 conserved motifs. All proteins contained Motif 1 and Motif 8. Members of Clades IV and V shared Motifs 1, 4, 5, and 8. All proteins possessed the DOG1 domain (Fig. 2b). Clades I–III predominantly encoded proteins with multiple domains, including bZIP in addition to DOG1, whereas Clades IV and V primarily encoded single-domain proteins. Exon numbers correlated with clade membership: Clades I–III had 6–14 exons, while Clades IV–V had 1–3 exons. Eight genes lacked introns, suggesting evolutionary optimization for efficiency and functional conservation.

Fig. 2. Phylogenetic tree of BnDOG1 genes and analysis of conserved motifs (a), domains (b), and gene structures (c)

Chromosomal distribution, homology, and selection pressure analyses

BnDOG1 genes were evenly distributed across 16 chromosomes, with 1–4 genes per chromosome (Fig. 3). No genes were found on chromosomes A04, C04, or C06.

Fig. 3. Chromosomal distribution of DOG1 genes in Brassica napus

Synteny analysis revealed 146 paralogous gene pairs, all derived from segmental duplications (Fig. 4), indicating segmental duplication as the dominant expansion mechanism (Figure S1). Comparative synteny with (A) thaliana, (B) oleracea, and B. rapa identified 72, 130, and 149 orthologous pairs, respectively (Fig. 5), supporting functional conservation of BnDOG1 genes.

Fig. 4. Segmental duplication events of BnDOG1 genes

All Ka/Ks ratios of syntenic gene pairs were less than 1 (Table S1), indicating strong purifying selection acting on the BnDOG1 family.

Fig. 5. Synteny analysis of DOG1 genes among Brassica napus, Arabidopsis thaliana, Brassica oleracea, and Brassica rapa. Gray lines represent syntenic blocks; red lines highlight orthologous DOG1 pairs

Promoter cis-regulatory elements

Cis-elements in the 2000 bp upstream regions were classified into four categories: light-responsive, hormone-responsive, development-related, and abiotic stress-related (Fig. 6). Light-responsive elements were the most abundant, whereas development-related elements were the least common. Hormone-responsive elements included those responsive to ABA, auxin, methyl jasmonate, GA, and salicylic acid. Development-related elements were associated with endosperm expression, palisade mesophyll differentiation, circadian rhythm, and meristem expression. Abiotic stress-related elements included those involved in anaerobic induction, low-temperature response, and drought induction. The abundance of cis-elements suggests complex regulatory functions for BnDOG1 genes. Notably, promoter regions of multiple DOG1 family members contain three cis-acting elements directly responsive to abiotic stresses—anaerobiosis, cold, and drought—strongly implicating the DOG1 family in plant physiological responses to abiotic stress.

Fig. 6a Predicted cis-regulatory elements in BnDOG1 promoters. b Classification of cis-elements

Protein interaction network

Thirteen BnDOG1 proteins (Table S2) interacted with GRXC7, GRXC8, NIMIN-1, NIMIN-3, and SCL14 (Fig. 7). GRXC7 and GRXC8 regulate redox balance and stress responses. NIMIN-1 and NIMIN-3 fine-tune defense and development by suppressing NPR1 complex formation. SCL14 modulates detoxification gene networks under biotic and abiotic stress. This interaction network implies diverse regulatory roles for BnDOG1 in stress adaptation. Collectively, these findings strongly indicate that the DOG1 family plays a pivotal role in integrating environmental stress signals, maintaining redox homeostasis, and activating defensive metabolic pathways to cope with diverse abiotic stresses through multiple regulatory strategies.

Fig. 7. Protein-protein interaction network of BnDOG1 members

GO enrichment analysis

GO analysis categorized BnDOG1 genes into molecular function, cellular component, and biological process (Table S3). Biological processes exhibited the highest number of enriched terms. The top 20 enriched GO terms are shown in Fig. 8. Molecular functions included nucleic acid binding (GO:0003676), heterocyclic compound binding (GO:1901363), and organic cyclic compound binding (GO:0097159). Cellular components were enriched in the nucleus (GO:0005634), intracellular membrane-bounded organelle (GO:0043231), and membrane-bounded organelle (GO:0043227). Biological processes highlighted regulation of cellular process (GO:0050794), regulation of biological process (GO:0050789), and biological regulation (GO:0065007). These results suggest roles in metabolic and stress-responsive pathways.

Fig. 8. Top 20 enriched GO terms for BnDOG1 genes. Y-axis: GO terms; X-axis: gene ratio; dot size: number of genes

Tissue-specific expression patterns

Transcriptomic data revealed distinct expression patterns across tissues (bud, pollen, sepal, cotyledon, leaf, root, seed, silique, stem) and under stress conditions (salt, cold, heat) (Fig. 9, Table S4). BnDOG1 genes were predominantly expressed in roots and pollen. Under stress, expression remained high in roots but low in leaves. BnaA06G027740 and BnaC03G056670 exhibited high root expression, particularly under cold stress. BnaA07G040760 was highly pollen-specific, whereas BnaC07G031110 was seed-specific with minimal stress responsiveness. BnaC01G012400 showed the lowest overall expression. These patterns indicate tissue-specific functions during development and stress responses. Collectively, these results demonstrate that BnDOG1 exhibits distinct tissue-specific expression, performs specialized roles during Brassica napus growth and development, and contributes to abiotic stress responses.

Fig. 9a Tissue-specific expression of BnDOG1 genes. b Expression under stress: I/IV = salt-stressed leaf/root; II/V = cold-stressed leaf/root; III/VI = heat-stressed leaf/root

qRT-PCR validation of expression patterns

qRT-PCR confirmed expression changes under Mn stress (Fig. 10). BnaA06G027740, BnaC03G056670, BnaA06G005230, and BnaC05G006110 were upregulated in roots but downregulated in leaves. BnaC09G009680 was significantly downregulated in both tissues, while BnaA09G008090 was significantly upregulated, validating Mn-responsive regulation. Notably, BnDOG1 genes show higher expression levels in roots than in leaves and are inducible under Mn stress.

Fig. 10a Expression of BnDOG1 genes in roots under Mn stress. b Expression in leaves under Mn stress

Discussion

The DOG1 gene family primarily regulates seed dormancy and germination. This study reveals an additional role for DOG1 in mitigating Mn stress in B. napus. The dual functionality may stem from the presence of both DOG1 and bZIP domains in many BnDOG1 proteins (Fig. 2b). The bZIP transcription factor family is well known for its roles in abiotic stress response, development, and secondary metabolism [46–49]. Previous studies suggest a close relationship between the DOG1 and bZIP gene families [50]. In the Brassica napus bZIP family, subgroup I is associated with heavy metal stress [51], implying that DOG1 genes may not only regulate seed dormancy but also contribute to abiotic stress responses. Our qRT-PCR results provide partial support for this hypothesis, showing significant expression changes under Mn stress. Notably, DOG1 is not absolutely required for dormancy [52], and its functions extend beyond primary dormancy to other aspects of seed maturation [53].

DOG1 controls primary and secondary dormancy in Arabidopsis, regulated by chromatin retention and mRNA stability [54]. The chromatin remodeler PICKLE and the evening complex of the circadian clock directly regulate DOG1 expression [55]. DOG1 homologs have been identified in other species: 24 in moso bamboo (Phyllostachys edulis) [50] and 53 in wheat (Triticum aestivum) [56]. In moso bamboo, the DOG1 gene is highly expressed in roots and leaves and can be induced by GA.

In wheat, DOG1 gene expression is associated with ABA and methyl jasmonate (MeJA) signaling pathways. In B. napus, we identified 42 members, equally split between acidic and basic proteins, possibly reflecting charge balance requirements. Most contain bZIP domains, similar to bamboo, though proportionally more so in bamboo [50]. Nuclear localization of most BnDOG1 proteins aligns with their regulatory roles.

Phylogenetically, BnDOG1 genes cluster into five clades, intermixed with AtDOG1 homologs, unlike the three-clade classification observed in bamboo and wheat. Motifs 1 and 8 are universally conserved, suggesting core functional significance. Even chromosomal distribution and absence on A04, C04, and C06 may reflect genomic constraints. Segmental duplication dominates gene expansion. High synteny with B. oleracea and B. rapa (130 and 149 pairs, respectively) versus (A) thaliana (72 pairs) supports the hybrid origin of (B) napus. Ka/Ks < 1 indicates strong purifying selection. Promoter cis-elements related to development (e.g., endosperm, circadian control) align with known dormancy mechanisms [6, 55], while stress-related elements (e.g., anaerobic, cold) may underlie Mn responsiveness. Interaction with stress-linked TFs (GRX, NIMIN, SCL14) further supports multifaceted stress regulation. GO enrichment in biological processes underscores the dynamic regulatory roles of BnDOG1 genes.

Transcriptome data analysis revealed substantial differences in the expression patterns of BnDOG1 family members across various tissues and under stress conditions. Roots are the primary site of Mn uptake. qRT-PCR results showed that BnDOG1 genes were predominantly upregulated in roots but downregulated in leaves. Additionally, the promoters of BnDOG1 genes contain regulatory elements associated with palisade mesophyll cell differentiation and meristematic expression, suggesting a potential role in regulating Mn transporter gene expression. The downregulation in leaves may serve to prevent excessive stress responses that could damage the photosynthetic system. These findings indicate significant differential expression of BnDOG1 genes, suggesting that DOG1 genes play a functional role in response to Mn stress.

Conclusions

This study performed a comprehensive bioinformatics analysis of the DOG1 gene fam-ily in Brassica napus and specifically examined its response to Mn heavy metal stress. Bioinformatics analyses indicated that BnDOG1 genes are functionally involved in abiotic stress responses. From cis-element identification to protein–protein interaction predictions, our work extended insights into stress regulation to the level of functional molecular interactions. GO enrichment and transcriptome analyses further corroborated the role of this gene family in abiotic stress adaptation. Finally, qRT-PCR validation confirmed that BnDOG1 genes are inducible under Mn stress. Future work will focus on transgenic functional validation of key BnDOG1 genes to assess their potential for breeding heavy metal–tolerant crops.

Supplementary Information

Supplementary Material 1.