E2F/DP (Eukaryotic Transcription Factor 2/Dimerization Partner) represents a class of protein complexes that play a pivotal role in the transcriptional regulation of genes in eukaryotes. It plays a crucial role for environmental stress responses (Mariconti et al., 2002). In eukaryotic cells, the RB/E2F signaling network orchestrates cell cycle progression, mediates mitotic division and DNA damage response, modulates cell size homeostasis, and ensures genomic integrity through dynamic phosphorylation by cyclin-dependent kinases (CDKs). Furthermore, this regulatory network mediates transcriptional reprogramming through sequence-specific interactions with cis-regulatory elements in target gene promoters. In planta, it plays a pivotal role in integrating photomorphogenic signaling with adaptive responses to abiotic/biotic stressors, thereby regulating spatiotemporal growth patterns, developmental transitions, and stress acclimation (Sozzani et al., 2006). In higher plants, E2F/DP transcription factors can be divided into three categories, namely E2F, DP, and DEL (DP - E2F - like), based on their conserved domains. The E2F gene group encompasses four functional domains: the retinoblastoma protein (RBR)-binding domain, the DNA-binding domain, the “mark box” domain, and the leucine-zipper dimerization domain. Typical E2Fs are able to act as either activators or repressors of gene expression. In contrast, non-typical E2Fs downregulate the expression of E2F-dependent genes by competing with typical E2Fs for DNA-binding sites. Notwithstanding recent advancements, current understanding of the E2Fs gene family evolutionary history, phylogenetic architectures, functional divergence among family members in plant species, and their roles within complex regulatory mechanism remains unclear. Currently, The E2F/DP transcription factor family in Arabidopsis thaliana represents the most comprehensively characterized member of this gene family among plant species. The Arabidopsis thaliana E2F/DP family consists of 8 members, including 3 types of canonical E2Fs (AtE2FA, AtE2FB, AtE2FC), 3 types of non-typical E2Fs (AtE2FD/DEL2, AtE2FE/DEL1, AtE2FF/DEL3), and 2 types of DP (Ramirez-Parra et al., 2004; Desvoyes et al., 2006). Functional studies have shown that E2Fs exhibit specific cooperative or antagonistic effects. The cross-regulation among various E2Fs has become part of the replication regulatory network in both animals and plants. In meristematic cells, AtE2F can activate cell-cycle genes to promote mitosis for cell proliferation. Cell proliferation in Arabidopsis thaliana depends on the transcriptional activation of E2F/DP. The deletion of the 3 typical E2F/DPs leads to plant sterility but does not affect vegetative growth (Magyar et al., 2005). The DcE2F1 factor in carrot is one of the earliest E2F factors to be studied. DcE2F1 is likely a homolog of AtE2FA in Arabidopsis thaliana. The phenotypes observed in Arabidopsis thaliana transformants with ectopic expression of DcE2F1 are highly similar to those of overexpressing AtE2FA. Conserved promotion of cell proliferation in embryonic and seedling tissues, coupled with ectopic hypocotyl formation, indicate its important role in the control of cell proliferation. In Arabidopsis thaliana, the cotyledon size of plants overexpressing AtE2FA or AtE2FB increases significantly, and the number of cells more than doubles. AtE2Fa acts downstream of the ERECTA kinase and is involved in regulating cell size and stomatal density. Studies on mammals also confirm that the E2F signaling pathway is crucial for cell growth and proliferation, demonstrating that E2F/DP plays a decisive role in the growth and development of organisms (Polager and Ginsberg, 2008). The E2F/DP gene family is extensively involved in plant growth, development, and responses to stress. Currently, E2F/DP genes have only been identified in a few plants such as Arabidopsis thaliana, moso bamboo, wheat, carrot, and tomato. There is no reported information on Brassica napus yet. For instance, salicylic acid is a key regulator in plant defense responses in Arabidopsis thaliana, the non-typical AtE2FE/DEL1 factor participates in the plant immune response and is found to inhibit the accumulation of salicylic acid to balance growth and defense. The promoters of PheE2F/DP transcription factors in moso bamboo harbor diverse cis-acting elements (Divya et al., 2024), which participate in leaf and root development, cell cycle regulation, and abiotic stresses responses. The expression of most PheE2F/DP genes is up-regulated under drought and salt stress (Li et al., 2021); The E2F/DP transcription factors in carrot regulate cell proliferation, size, and stomatal development, and are also involved in the growth and development processes of embryos and plants (Perrotta et al., 2021). Under drought stress, E2F/DP genes in wheat regulate the expression of downstream genes, maintain the balance of cell osmotic pressure, and enhance drought tolerance (Yu et al., 2022). Under drought stress, Sorbus pohuashanensis E2F/DP genes enhance the perception and response to water deficit by regulating the abscisic acid (ABA) signaling pathway, and regulate growth to adapt to a high-salt environment. Under drought stress, Medicago sativa E2F/DP genes maintain cell osmotic balance through multiple regulatory mechanisms, participate in hormone signal transduction pathways, and enhance stress tolerance, and the expression of four genes such as E2Fa is up-regulated by more than 2-fold under salt stress (Ma et al., 2014). In maize, the expression level of the ZmE2F6 gene in the stem is significantly up-regulated after 3 hours of drought treatment and down-regulated at 6, 12, and 24 hours of treatment, indicating that ZmE2F6 responds to drought stress.

Brassica napus, as an important crop with great economic value, is widely used in fields such as food production and oil processing. However, the growth of Brassica napus relatively sensitive to water deficit, and drought stress has become a key factor severely constraining the increase in its yield (Wei et al., 2022). Therefore, identifying and harnessing stress-resistant genes in rapeseed through molecular breeding techniques holds great significance for the development of the rapeseed industry. In this study, 29 BnE2F/DP genes were comprehensively identified in the Brassica napus genome. Using bioinformatics and other methods, we performed an in-depth analysis of the gene structures, physicochemical properties, chromosome locations, conserved motifs, cis-acting elements, intragenomic collinearity, and phylogenetic evolutionary characteristics of the BnE2F/DP gene family members in rapeseed. By integrating transcriptome sequencing and real-time fluorescence quantitative PCR (RT-qPCR) techniques, we analyzed the expression patterns of the BnE2F/DP gene family in different tissues of Brassica napus. The findings of this study not only enhance our knowledge of the biological functions of the BnE2F/DP gene family in Brassica napus, but also provide important gene resources and a theoretical basis for stress-resistant molecular breeding of rapeseed. Moreover, they are anticipated to expedite the process of molecular-assisted selection for stress-resistant in rapeseed breeding.

E2F/DP transcription factors are conserved regulators of plant cell cycle progression, photoperiodic responses, and seed/embryo development, while also mediating abiotic stresses (Mariconti et al., 2002; Lammens et al., 2009; Berckmans et al., 2011). In animal systems, they play critical roles in viral replication and host cell cycle manipulation during adenovirus infection. Investigating the functional roles of E2F/DP transcription factor genes is critical for deciphering plant development regulatory circutry. Recent high-throughput genomic approaches have enabled systematic characterization of E2F/DP gene families across diverse plant species, including Phyllostachys heterocycla (Pubescens), Glycine max, and Solanum lycopersicum (Bai et al., 2018). Nevertheless, the details of the BnE2F/DP gene family in Brassica napus remain poorly understood.

Genome-wide characterization of the BnE2F/DP gene family identified 29 members, representing a substantial expansion compared to other plant species: Arabidopsis thaliana (8 genes) (Xiaozhen et al., 2018), Solanum lycopersicum (8 genes) (Divya et al., 2024), Triticum aestivum (27 genes) (Yu et al., 2022), Zea mays (21 genes) (Lescot et al., 2002), Medicago sativa (Ma et al., 2014) (5 genes), Phyllostachys edulis (23 genes) (Li et al., 2021), and Daucus carota (10 genes) (Perrotta et al., 2021). The Phyllostachys edulis genome (2021 Mb) is slightly smaller than Zea mays (2300 Mb), significantly smaller than Triticum aestivum (17 Gb), and substantially larger than Brachypodium distachyon (300 Mb). The expanded PheE2F/DPs gene family in Phyllostachys edulis (23 members) suggests that gene family size correlates with genome duplication events rather than the genome size. This pattern aligns with the differential gene expansion observed across plant lineages. Notably, the Brassica napus genes arose from an additional whole-genome triplication(WGT) event subsequent to its divergence from Arabidopsis thaliana, further illustrating the role of polyploidization in shaping gene family evolution. Brassica napus is an allopolyploid formed by the interspecific hybridization of Brassica rapa (AA genome) and Brassica oleracea (CC genome). Given the paleo hexaploid nature of both progenitor species (each having undergone a whole-genome triplication event subsequent to their divergence from Arabidopsis thaliana) theoretical predictions suggest that B. napus should contain 48 E2F/DP orthologs (8 Arabidopsis genes × 3 copies in each diploid progenitor × 2 sub genomes). However, our genome-wide analysis identified only 29 E2F/DP genes, indicating significant gene loss during the evolutionary history of this allopolyploid crop. Moreover, collinearity analysis shows that the genes have undergone purifying selection during evolution, suggesting that due to functional divergence has driven the retention of critical family members in Brassica napus for environmental adaptation, while non-essential duplicates were lost through fractionation. Chromosomal localization analysis revealed that the BnE2F/DP genes are unevenly distributed across 15 of 19 chromosomes in Brassica napus.The discrepancies in genes number among different sub-families might be attributed to gene amplification and loss events that have occurred during evolution.

Gene structure dictated functional diversity, with exons number variation potentially contributing to evolutionary divergence. During evolutionary, BnE2F/DP subfamilies such as BnE2FC and BnE2FA have retained a conserved exon number, suggesting functional constraint. In contrast, the BnDPA subfamily underwent functional divergence, accompanied by exon loss, which may have enabled the acquisition of novel biological roles distinct from other subfamilies. Conserved motifs analysis revealed that all 29 BnE2F/DP genes possess the E2F-TDP domain, with additional domains present in specific subfamilies. Motif 1 is universally conserved across all BnE2F/DP proteins, indicating its critical role in the evolutionary maintenance of the gene family in Brassica napus. The high degree of motif conservation among family members suggests functional constraint, with most subfamilies sharing common regulatory modules. The presence of the conserved TDP domain (PF00207) in all members confirms their classification within the E2F/DP transcription factor family, while additional lineage-specific domains reflect subfamily diversification. Motif 1 likely corresponds to the conserved TDP domain of the BnE2F/DP genes, consistent with previous reports that all SlE2F/DP proteins in Solanum lycopersicum harbor this domain (Divya et al., 2024). Even after the evolutionary divergence of monocots and dicots within the E2F/DP gene family, these conserved motifs remain similar (Divya et al., 2024). BnE2F/DP genes within the same subfamily exhibit high sequence similarity. For example, BnE2FC and BnE2FA subfamilies share four identical motifs.

In the course of evolution, gene duplication events are a major source of new genetic traits. Segmental duplication increases the number of genes in specific gene families, thereby enhancing plants’ adaptability to various environmental stresses. Synteny analysis identified 35 duplication events among 29 BnE2F/DP genes in Brassica napus, with 28 pairs classified as segmental duplications. Moreover, the BnE2F/DP genes participated in intragenomic replication in the form of whole-genome duplication. Two duplication pairs in the tomato SlE2F/DP gene family indicate that segmental gene duplication triggered the expansion of the tomato E2F/DP gene family during evolution (Divya et al., 2024). In the wheat E2F/DP gene family, there are multiple segmental duplication events, and chromosomal segmental duplication plays a crucial role in the expansion and evolution of the E2F family (Yu et al., 2022). Twelve pairs of duplicated genes have been observed in the E2F/DP gene family of Phyllostachys edulis (moso bamboo) (Li et al., 2021). It has been reported that the functional differentiation between segmentally duplicated genes is more significant. The occurrence of multiple duplication events indicates that the BnE2F/DP gene family has been continuously expanding during the evolutionary process of the Brassica napus genome. This is consistent with the research results of Zea mays (Lescot et al., 2002) and Phyllostachys edulis (Li et al., 2021), indicating conserved E2F/DP gene family expansion patterns across plant lineages. Ka/Ks ratio analysis of the duplicated BnE2F/DP gene revealed values<1 for all pairs, indicating that the BnE2F/DP genes mainly evolved under purifying selection pressure, which is consistent with the evolutionary pattern of most gene families. Interspecific synteny analysis identified 9 orthologous genes pairs between Arabidopsis thaliana AtE2F/DP and Brassica napus BnE2F/DP, suggesting shared ancestry and functional conservation.

It has been reported that members of the E2F/DP family in some plant species actively respond to abiotic stresses. Analysis of Brassica napus BnE2F/DP promoters revealed significant variation in the number and types of cis-regulatory elements across the 29 family members. However, all members contain 3 to 13 light-responsive elements, suggesting their involvement in light signaling pathways. This finding aligns with the previous reports that most PheE2FDPs in Phyllostachys edulis are regulated by the diurnal cycle (Li et al., 2021), implying that light plays a broad and fundamental role in modulating BnE2F/DP expression. Cis-regulatory elements analysis related that 28 BnE2F/DP promoters contain abiotic stress-responsive motifs, second only to light-responsive elements in abundance. This suggested that the BnE2F/DP family plays a broad role in Brassica napus stress responses, potentially mediating mechanisms of environmental sensing, signal transduction, and adaptive responses thereby helping the plants to adapt to external stresses and maintain normal physiological functions. Twenty-five BnE2F/DP genes contain cis-acting elements related to hormone responses, mainly involving abscisic acid, methyl jasmonate, gibberellin, salicylic acid, and auxin response elements. Notably, these genes exhibited significant enrichment in ABRE (ABA-responsive element) and MeJA-responsive motifs, with additional detection of abscisic acid, auxin, salicylic acid, and gibberellin-responsive elements in specific members. This suggests that their potential involvement in integrating multiple hormonal signaling pathways under environmental stresses. QRT-PCR analysis following abiotic stress treatments revealed that most BnE2F/DP genes exhibited high expression levels under both salt and drought stress, consistent with the expression patterns observed in the transcriptome data suggesting their potential roles in abiotic stress toleranceBnDPA-4 and BnE2FC-2 showed significant up-regulation under salt stress. Previous studies have found that SlE2F/DP2, SlE2F/DP7, and SlE2F/DP8 members in tomato (Solanum lycopersicum) exhibit high expression under salt stress (Divya et al., 2024); E2Fa, E2Fb, E2Fc, and E2FDPa in alfalfa (Medicago sativa) show more than two-fold up-regulation in expression under salt stress, meanwhile after 6 hours of salt stress, the expression of E2Fc increases in leaves, while in roots, its expression first rises and then declines (Ma et al., 2014). In Arabidopsis thaliana, studies report changes in expression at 6 h and 24 h under salt stress, indicating that salt stress can trigger co-expression of E2F and DP genes (Ramirez-Parra et al., 2004; Desvoyes et al., 2006; Sozzani et al., 2006). The TaDP2III-3 and TaDEL2II-27 genes in wheat (Triticum aestivum) were upregulated under salt stress, while TaE2F1I-19 and TaDP3III-15 were up-regulated under drought stress (Yu et al., 2022). These results are consistent with the findings in Phyllostachys edulis (moso bamboo), where PheE2F/DP genes exhibited upregulation at various time points in response to both drought and salt stresses (Li et al., 2021).Notably, BnE2FC-2 exhibited over two-fold up-regulation at different time points across all three stresses, while BnE2FC-1 and BnDPA-2 were repressed by nearly two-fold at various time points under the three stresses. These results suggest that the BnE2FC subfamily actively responds to diverse abiotic stresses, which is consistent with the predictions from the preceding cis-acting element analysis. The expression of BnDPB-3 and BnE2FC-2 was up-regulated by more than two-fold under ABA treatment. This observation aligns with the previous studies reporting significant enrichment in ABRE and MeJA response elements with Phyllostachys edulis PheE2F/DP genes and tomatoes SlE2F/DP genes (Li et al., 2021; Divya et al., 2024). Notably, plant genes expression is modulated not only by cis-regulatory elements but also by additional factors such as transcription factors and epigenetic modifications. Functional annotation of the BnDPB-4 promoter revealed an absence of development-related cis-regulatory elements but enrichment in hormone-responsive elements. This suggests that BnDPB-4 primarily mediates hormone signaling pathways in Brassica napus rather than participating in development process (Lescot et al., 2002).

Quantitative real-time PCR (qRT-PCR) validation exhibited high consistency with developmental transcriptome data. Throughout the developmental stages of Brassica napus, BnE2FA and BnE2FE subfamilies genes exhibited consistently low expression levels. This observation suggests limited or no significant roles for these genes during the growth and development. In contrast, BnE2FC and BnDPB subfamily members displayed constitutive high expressions across tissues (roots, leaves, flowers, and seeds) and developmental stages ( Figure 6 ). This is consistent with the results that AtE2F in Arabidopsis thaliana (Xiaozhen et al., 2018) and DcE2F1 in Daucus carota (Perrotta et al., 2021) activate cell cycle genes in meristematic cells to promote cell mitotic proliferation. In tomato (Solanum lycopersicum), SlE2F/DP3, SlE2F/DP6, and SlE2F/DP8 are mainly expressed in vegetative tissues (roots), while SlE2F/DP1 and SlE2F/DP7 displayed reproductive tissue-specific expression (e.g., flowers, immature fruits). The remaining genes showed constitutive expression across both vegetative and reproductive tissues. SlE2F/DP genes play a regulatory role in the development, enlargement, and ripening processes of tomato fruits. This indicates that these genes may be involved in cell division and maturation processes (Li et al., 2021). A similar trend can be observed in the expression of E2F/DP genes in Medicago sativa (Ma et al., 2014). AtE2FC is generally regarded as a major repressor of E2F-regulated genes. Its overexpression reduces cell division and leads to an increase in endoreplication. It is hypothesized that BnE2FC may guide cells from division to differentiation in the late stage of seed maturation, thereby promoting the enlargement of seed cells and the accumulation of contents (Xiaozhen et al., 2018). In Daucus carota, DcE2F4, a homolog of AtE2FC, is reported to be well-expressed in young leaves, calli and other differentiated tissues, and is involved in regulating cell proliferation and the activities of mature cells (Perrotta et al., 2021). In summary, we speculate that the BnE2FC subfamily exhibits specific and high-level expression in seeds and embryos of rapeseed varieties, and shows a highly active response to abiotic stresses. Functional verification indicates that they play a key role in the development of seeds and embryos, and actively respond when plants are subjected to salt stress, ABA stress, and drought stress. This contrasts with previous findings in Arabidopsis thaliana (Xiaozhen et al., 2018), where overexpression of AtE2FA or AtE2FB significantly increased cotyledon size and doubled cells number. These observations suggest that during the evolution, homologous recombination events during gene duplication may have resulted in functional divergence of the BnE2FC subfamily in Brassica napus. These results suggest conserved roles for orthologous BnE2F/DP genes in regulating tissues and organ development across Brassica napus growth stages.

In this study, 29 members of the BnE2F/DP gene family were identified in Brassica napus and classified into 8 subfamilies based on phylogenetic analysis. Functional annotation revealed abundant cis-regulatory elements within BnE2F/DP genes, which are involved in processes including growth and development, abiotic stress responses, hormone signaling, and light perception. Notably, all 29 gene members harbored light-responsive elements, suggesting their critical roles in mediating light signaling pathways in Brassica napus. Additionally, 28 genes contained abiotic stress-responsive cis-regulatory elements, indicating their significant involvement in mediating stress tolerance mechanisms under adverse environmental conditions.

Collinearity and duplication pattern analyses revealed that genome expansion of the BnE2F/DP gene family in Brassica napus occurred primarily occurred through whole-genome duplication events, with 35 collinear gene pairs identified. This finding reflects strong evolutionary conservation among family members. Ka/Ks ratio analysis further indicated that BnE2F/DP genes have undergone purifying selection during evolution. Transcriptomic analysis and qRT-PCR validation demonstrated that BnE2F/DP genes play pivotal regulatory functions across Brassica napus developmental stages, with particularly high expression during seed and embryo development. This highlights their active involvement in coordinating reproductive growth processes, including seed maturation and embryo patterning. Transcriptome prediction under stress and fluorescence quantitative PCR analysis revealed that members of the BnE2F/DP gene family exhibited upregulated or even highly expressed patterns after salt stress and drought stress treatments at different time points. BnE2FC-2 showed more than two-fold high expression under different stresses and time points, indicating that the BnE2FC subfamily actively participates in stress responses, which is consistent with the previous cis-element prediction results. We speculate that BnE2FC-2 is a candidate gene for salt and drought tolerance.

In summary, this study performed genome-wide identification and systematic bioinformatics analysis of the BnE2F/DP family in rapeseed (Brassica napus), with a focus on analyzing their expression patterns during growth and development stages and under abiotic stresses. The results provide insights for further functional studies of the BnE2F/DP gene family and identify potential candidate genes for genetic improvement of rapeseed in regulating growth and development as well as enhancing abiotic stress resistance. This study also offers new perspectives for a better understanding of the BnE2F/DP family in crops.