Genome-Wide Characterization of the PbeDof Gene Family Reveals PbeDof9.1 as a Key Regulator of Salt Tolerance via Enhancing Antioxidant Capacity in Pyrus betulifolia

Abstract

Soil salinization severely restricts the sustainable development of the pear industry. Pyrus betulifolia, a vital native salt-tolerant rootstock in China, holds great significance for investigating stress resistance mechanisms. Plant-specific DNA-binding One Zinc Finger (Dof) transcription factors act as pivotal regulators in stress adaptation. However, their functions in P. betulifolia remain largely unexplored. In this study, we identified 43 PbeDof members within the P. betulifolia genome and classified them into eight subfamilies via phylogenetic analysis. Gene structure and conserved motif analyses revealed that PbeDof members within the same subfamily share similar exon-intron organizations and protein architecture, suggesting evolutionary conservation. Promoter analysis indicated that PbeDof genes are rich in cis-acting elements related to light, phytohormones (especially ABA and MeJA), and stress responses, implying their potential roles in diverse biological processes. Chromosomal localization and collinearity analyses revealed that segmental duplication was the primary driver of this family’s expansion. Combined transcriptomic profiling and qRT-PCR assays demonstrated that PbeDof9.1 is predominantly expressed in roots and is strongly induced by salt stress. Subcellular localization confirmed that PbeDof9.1 targets the nucleus. Functional characterization indicated that heterologous overexpression of PbeDof9.1 in Arabidopsis thaliana significantly enhances salt tolerance at germination and seedling stages. Notably, under 175 mM NaCl stress, the transgenic lines exhibited a superior root system architecture, with primary root length and lateral root numbers being approximately 1.5-fold higher than those of the wild type. Furthermore, homologous overexpression in pear calli confirmed that PbeDof9.1 mitigates oxidative damage by boosting the activities of peroxidase (POD) and catalase (CAT) to scavenge reactive oxygen species (ROS), thereby reducing malondialdehyde (MDA) accumulation. Collectively, this study characterizes the PbeDof family and establishes PbeDof9.1 as a key candidate gene for the genetic improvement of salt tolerance in pear rootstocks.

1. Introduction

Soil salinization is a global environmental issue that severely threatens the sustainable development of agricultural ecosystems [1]. Among these, salt stress serves as one of the most destructive abiotic stresses, significantly inhibiting plant growth and development primarily by inducing osmotic stress, disrupting ion homeostasis, and causing oxidative damage [2]. Among deciduous fruit trees, pear (Pyrus spp.) is particularly sensitive to salt stress, making soil salinization a key bottleneck restricting the sustainable development of the pear industry [3]. Given that grafting is standard practice in pear propagation, the salt tolerance of the rootstock is a determinant factor for the environmental adaptability of the scion [4]. This characteristic makes the breeding of salt-tolerant rootstocks an important breakthrough point for enhancing the stress resistance of the pear industry. In this context, analyzing the molecular mechanisms of plant salt tolerance and mining key salt-tolerance genes are the prerequisites and foundation for genetic improvement [5]. Particularly with the development of molecular biology technology, revealing the mechanism of rootstock salt tolerance at the gene level will provide important theoretical basis and technical support for pear stress resistance breeding [6].

The Dof (DNA binding with one finger) gene family consists of plant-specific transcription factors, first identified in maize (Zea mays) as regulators of photosynthesis-related genes [7]. Typically, the number of Dof members in plant genomes is relatively small and conserved across species [8]. The model plant Arabidopsis thaliana genome contains 36 Dof members, the monocot representative rice (Oryza sativa) has 30 Dof members [9], and the cultivated pear (Pyrus bretschneideri) has 45 Dof members [10]. Dof transcription factors belong to the single zinc finger protein superfamily, and their protein length is usually between 200 and 400 amino acids [8]. Members of this family have significant structural characteristics, the N-terminus contains a highly conserved Dof domain, the core of which is a C2/C2 zinc finger structure folded by 50 to 52 amino acid residues [11]. Unlike other zinc finger proteins, Dof transcription factors contain only one C2/C2 zinc finger [12]. This unique single-finger structure endows them with the ability to specifically recognize and bind to the core sequence 5′-(T/A) AAAG-3′ (commonly referred to as the AAAG motif) in the promoter region of target genes [13]. In terms of structural stability and function, four cysteine residues within the domain maintain conformational stability through coordination, while specific tryptophan residues located on the C-side of this domain play a key role in the DNA binding process. In addition, the C-terminal region of Dof proteins usually contains regulatory functional domains responsible for transcriptional activation or repression [11].

The evolutionary history of gene families is often accompanied by complex gene duplication and amplification events. Existing studies indicate that Whole Genome Duplication (WGD) and Segmental Duplication are the dominant forces driving the expansion of the Dof family, while the contribution of Tandem Duplication is relatively limited [8]. For example, Ma et al. found that the expansion of the Chinese cabbage (Brassica rapa) Dof family was primarily driven by segmental duplications, accompanied by limited tandem duplication events [14]. In radish (Raphanus sativus L.), 30 segmental duplications were detected, with only 2 clear tandem duplications [15]. Wen et al. also confirmed 6 segmental duplications and 2 tandem duplications in cucumber (Cucumis sativus) [16]. To trace the evolutionary history of the Dof family and analyze its functional differentiation, scholars have widely used comparative genomics for phylogenetic reconstruction. Taking the model plant A. thaliana as an example, its Dof proteins were initially divided into 7 subgroups [9]. Subsequently, Noguero et al. summarized the Dof gene family into 6 evolutionary groups based on sequence similarity, with primitive members mostly concentrated in groups I, III, and V [17]. Thereafter, Ma et al. proposed a classification framework of “four major branches, nine subfamilies” and pointed out that algae only have 1–2 Dof members [14], while most higher plants possess 40–50 copies; notably, the Camelina sativa Dof family expanded to 103 members, revealing the significant species-specific expansion characteristics of this family [18].

Since their discovery, Dof transcription factors have been confirmed as key regulators in plant growth, development, and stress adaptation networks [19,20]. Functional studies indicate that Dof proteins mediate salt stress responses through diverse pathways. For instance, A. thaliana AtDof5.8 and Tamarix hispida ThDof8 enhance salt tolerance by transcriptionally regulating the downstream genes ANAC069 and ThCrk10 [21,22], respectively. In contrast, the cotton transcription factor GhDof1.7 functions as a positive regulator through protein interaction, specifically by binding to GhCar4 [23]. Transcriptomic analyses indicate that Dof genes in fruit crops, such as sweet orange [24], apple [25], and pineapple [26], are significantly induced by abiotic stresses, suggesting conserved roles in stress adaptation. However, current knowledge of the Dof family in Pyrus is largely restricted to flowering time regulation in the cultivated white pear (P. bretschneideri) [27], with a complete absence of information regarding Dof-mediated salt stress responses. Consequently, the lack of systematic characterization of Dof genes in the elite salt-tolerant rootstock P. betulifolia hinders the molecular understanding of its superior adaptability. In light of this, we performed a genome-wide identification of the PbeDof gene family in P. betulifolia and focused on analyzing its expression patterns under salt stress. Furthermore, we screened one key salt-tolerant gene and verified its function, thereby providing a theoretical foundation and a valuable candidate gene for the molecular breeding of salt-tolerant rootstocks.

2. Results

2.1. Identification and Physicochemical Property Analysis of the PbeDof Gene Family

In this study, a total of 43 PbeDof transcription factor family members were identified through genome-wide analysis (Supplementary Table S1). Following the nomenclature system established for white pear (Pyrus bretschneideri) [10], these genes were systematically named PbeDof1.1 through PbeDof17.2 based on their chromosomal locations. Two members that could not be mapped to any chromosome were designated as PbeDof0.1 and PbeDof0.2. Analysis of physicochemical properties revealed significant diversity among the 43 PbeDof proteins. The protein lengths ranged from 149 amino acids (aa) (PbeDof5.2) to 527 aa (PbeDof13.1), with molecular weights (Mw) varying correspondingly from 16.80 kDa (PbeDof5.2) to 56.80 kDa (PbeDof13.1). The isoelectric point (pI) ranged from 4.81 to 9.56; PbeDof12.1 was the most basic (pI = 9.44), while PbeDof5.1 was the most acidic (pI = 4.89). In terms of gene structure, the vast majority of members (95.3%) contained 0–2 introns. Notably, PbeDof14.4 exhibited a unique structure with three introns. Subcellular localization predictions indicated that 39 members are localized in the nucleus, while PbeDof7.3 and PbeDof0.1 are predicted to target the mitochondria, and PbeDof3.1 and PbeDof11.2 are targeted to the chloroplast.

2.2. Chromosomal Localization Analysis of PbeDof Genes

Chromosomal localization analysis revealed that the PbeDof genes are unevenly distributed across all 17 chromosomes of P. betulifolia (Supplementary Figure S1). Chromosomes 2, 3, 4, 8, 9, 12, 13, and 16 each harbor a single PbeDof gene, whereas chromosomes 1, 5, and 10 contain the highest number, with five genes each. Additionally, two members (PbeDof0.1 and PbeDof0.2) were located on unanchored scaffolds and are therefore not displayed in the chromosomal map.

2.3. Sequence Analysis of PbeDof Protein Conserved Domains

To deeply explore the sequence characteristics of the P. betulifolia Dof family, this study used Clustal Omega software (v1.2.4) to perform multiple sequence alignment on the core domains of 43 PbeDof proteins (Supplementary Figure S2). The results showed that all PbeDof members contain a highly conserved Dof domain, and its amino acid sequence has maintained extremely high stability during evolution. A canonical C-X2-C-X21-C-X2-C motif is located at the N-terminus of the domain (Supplementary Figure S2, upper annotation). The fixed positions of these four cysteine residues (Cys) suggest they coordinate with zinc ions to maintain the conformational stability of the single zinc finger structure. The middle region of the domain (approximately positions 10–30) is rich in amino acid residues such as K-F-Y-N-L-Q-P-R-H-F (Supplementary Figure S2). This region is a key module for Dof proteins to specifically recognize and bind to the AAAG core cis-element in the promoter region of target genes. The high conservation of its sequence implies the homology of PbeDof family members in transcriptional regulation functions. The C-terminus of the domain (approximately positions 45–50) contains a G-R-K (Glycine-Arginine-Lysine) sequence rich in basic amino acids (Supplementary Figure S2). It is speculated that this motif may serve as a nuclear localization signal (NLS) to guide the protein into the cell nucleus, or assist in the tight binding of the protein to the DNA backbone through charge interactions.

2.4. Analysis of cis-Acting Elements in PbeDof Gene Promoters

To understand the transcriptional regulation mechanism of PbeDof family members, the PlantCARE database was used to predict and analyze the 2000 bp promoter sequences upstream of the start codon of 43 PbeDof genes. The results showed that the PbeDof promoter regions are rich in various cis-acting elements, mainly classified into four categories, light response, hormone response, abiotic stress response, and growth and development (Figure 1).

Light response elements (such as G-box, Box 4, etc.) had the highest abundance in most members, suggesting that this family is widely involved in photomorphogenesis or photosynthesis regulation. Among them, PbeDof9.1 contains as many as 21 light response elements, while PbeDof16.1 only contains 4, showing differentiation in regulation modes among family members. In terms of hormone and stress response, all PbeDof genes contain at least one hormone or stress response element. The promoters of PbeDof14.4 and PbeDof15.1 are rich in various hormone response elements such as salicylic acid (SA), auxin, abscisic acid (ABA), and methyl jasmonate (MeJA), showing complex hormone regulation potential. In addition, members such as PbeDof6.2 and PbeDof9.1 also significantly enriched low-temperature and MeJA response elements, foreshadowing their roles in complex environmental adaptation.

2.5. Phylogenetic Tree Analysis of PbeDof Genes

To analyze the evolutionary relationship of the P. betulifolia Dof family, this study constructed a phylogenetic tree using the protein sequences of 43 P. betulifolia (PbeDof) and 45 white pear (PbrDof) members (Figure 2). Cluster analysis results showed that all Dof members were clearly divided into 8 subfamilies: A, B1, B2, C1, C2.1, C2.2, D1, and D2. Notably, compared with A. thaliana (containing 9 subfamilies), Pyrus plants lack the C3 subfamily. In each subfamily, the distribution of gene numbers showed significant imbalance. The B2 subfamily is the largest group, containing 10 PbeDof members; the B1 subfamily is the smallest, containing only 2 members. The member numbers of other subfamilies are: D1 (9), C1 (6), C2.1 (5), C2.2 (4), D2 (4), and A (3). Although the subfamily classification patterns of P. betulifolia and white pear are highly consistent, there are still copy number differences in some subfamilies. This interspecific quantity differentiation reflects that the Dof family underwent asymmetric expansion or contraction events during evolution, which may be an important strategy to respond to specific environmental selections and achieve adaptive evolution.

2.6. Gene Structure Analysis of PbeDof

To explore the structural evolution characteristics of the PbeDof gene family, this study jointly analyzed the phylogenetic tree, gene structure, and conserved motifs of 43 PbeDof genes (Figure 3). In terms of gene structure, PbeDof family members have relatively simple structures and are highly conserved in evolution. Statistics show that the vast majority of members have 0~2 introns, a feature consistent with the structural characteristics of the Dof family in species like A. thaliana and O. sativa, implying that this family tends towards simple gene structures during evolution. Among them, PbeDof14.4 is the structurally most complex member in the family, containing 3 introns (4 exons), which may be related to its functional specialization. Members clustered in the same subfamily often display similar exon-intron organization patterns; this subfamily-internal structural conservation supports the reliability of the phylogenetic classification.

In terms of conserved motifs, this study identified 10 conserved motifs (Motif 1–10). Analysis found that the distribution pattern of motifs is highly consistent with the topology of the phylogenetic tree, strongly supporting the reliability of the subfamily classification. Among them, Motif 1 widely exists in PbeDof members, presumed to correspond to the core Dof zinc finger domain. Different subfamilies show unique motif composition modes; for example, some members of the B2 subfamily specifically contain Motif 10, while D1 subfamily members specifically enrich Motif 5, suggesting that these subfamily-specific motifs may endow specific biological functions to the group. Notably, some members show high structural complexity. For example, PbeDof17.1 contains all 10 motifs, and PbeDof9.1, PbeDof6.3, PbeDof13.1, PbeDof14.3, and PbeDof16.1 all contain 9 motifs. This complex motif composition implies that these members may be in pivotal positions in the transcriptional regulatory network and possess stronger functional diversity.

2.7. Synteny Analysis of Dof Genes

Gene duplication events are the main driving force for gene family expansion and functional differentiation. To analyze the amplification mechanism and evolutionary history of the PbeDof gene family, this study conducted deep mining of whole-genome data combining intraspecific and interspecific synteny analysis (Figure 4). The intraspecific collinearity of P. betulifolia Dof genes is shown in Figure 4B. The PbeDof gene family underwent significant Segmental Duplication and Tandem Duplication events during evolution. Due to the effects of whole genome duplication (WGD) or large-scale segmental duplications, a total of 44 syntenic Dof gene pairs were identified within the P. betulifolia genome, suggesting that segmental duplication is the primary mechanism driving the expansion of the Dof gene family. In addition, some members showed unique duplication modes: PbeDof1.1 and PbeDof1.2 constituted a proximal duplication gene pair; Notably, tandem duplication events were identified on chromosomes 5 and 10. Specifically, PbeDof10.2–10.5 were closely arranged on chromosome 10, forming a typical tandem duplication gene cluster. Similarly, we also observed a gene cluster containing PbeDof5.2–5.4 on chromosome 5. These tandem duplications may provide a genetic basis for the rapid functional evolution of family members in specific environments.

To further explore the conservation and specificity of PbeDof genes in cross-species evolution, this study selected the dicot model plant A. thaliana and the congeneric cultivated species P. bretschneideri to construct an interspecific comparative genomic map (Figure 4A). Orthology analysis results showed that the degree of collinearity of the PbeDof gene family varies significantly between different species and is highly correlated with species affinity. Between P. betulifolia and A. thaliana, only 63 pairs of syntenic genes were identified, and the collinear lines connecting them were relatively sparse. This indicates that although the Dof family retained core members in dicots, most duplicated genes may have undergone subfunctionalization, neofunctionalization, or loss during the long evolutionary divergence process. P. betulifolia and white pear showed a high degree of synteny, with 109 pairs of orthologous genes identified. There are large dense collinear blocks between the two in the map, indicating that the Dof gene family maintained extremely high genetic conservation after the differentiation of Pyrus species. Furthermore, analysis based on the distribution of synonymous substitution rates (Ks) (Figure 4C) further confirmed the above results. The Ks distribution peaks of P. betulifolia and white pear highly overlap, showing similar evolutionary histories; while there is a clear difference from the distribution of A. thaliana. These results collectively indicate that Whole Genome Duplication (WGD) and subsequent purification selection are the main forces reshaping the evolutionary characteristics of the P. betulifolia Dof gene family.

2.8. Expression Pattern Analysis of PbeDof Genes Under Different Stresses

Leveraging our previously obtained transcriptomic dataset regarding salt stress in P. betulifolia, we analyzed the expression patterns of PbeDof genes based on RNA-seq FPKM values under control and salt treatment conditions (Figure 5). Under normal growth conditions, PbeDof family members exhibited significant tissue-specific expression patterns. Specifically, PbeDof9.1, PbeDof6.3, and PbeDof2.1 were highly expressed in roots, whereas PbeDof16.4 and PbeDof4.4 were predominantly expressed in leaves. In contrast, the stems harbored the largest number of highly expressed members, including PbeDof7.1, PbeDof10.2, PbeDof15.2, and PbeDof0.2. This differentiated spatial distribution suggests that PbeDof members of different subfamilies undertake specific biological functions in plant organ development and function maintenance.

Deep mining based on transcriptome data under salt stress revealed the differentiation of expression patterns of PbeDof family members in stress response, and there were obvious differences in the response of different tissues to salt stress. In roots, PbeDof9.1 showed significant induced expression, implying it might play a leading role in root stress resistance; stem response involved significant up-regulation of PbeDof10.5 and PbeDof15.3, which might be related to signal transmission in vascular tissues; in leaves, PbeDof6.1 was mainly up-regulated.

2.9. Transcriptome Expression Verification

To investigate the potential roles of PbeDof genes in response to abiotic stress, the expression patterns of nine selected members were analyzed under 200 mM NaCl treatment using qRT-PCR (Figure 6). The results revealed divergent temporal expression profiles among the family members. Generally, the majority of the tested genes, such as PbeDof1.3, PbeDof14.2, PbeDof15.1, and PbeDof17.1, exhibited a downregulation trend, with their transcript levels significantly repressed at 6 h or 12 h compared to the control (0 h). Although genes like PbeDof6.1 and PbeDof6.4 showed a recovery in expression by 24 h, their initial response was inhibited by salt stress.

In contrast, PbeDof9.1 displayed a distinct and positive response pattern. Unlike the downregulated members, PbeDof9.1 was rapidly and significantly induced at 6 h (reaching the highest expression level). While its expression fluctuated at 12 h, it remained comparable to or higher than the basal level throughout the treatment period (0–24 h). Similarly, PbeDof5.4 and PbeDof7.3 also showed inducible expression patterns, peaking at 12 h and 24 h, respectively. These results suggest that PbeDof9.1, along with PbeDof5.4 and PbeDof7.3, may function as positive regulators in the salt stress response of P. betulifolia.

2.10. Subcellular Localization Assay

Based on transcriptomic analysis and expression profiling, PbeDof9.1, which is strongly induced by salt stress, was selected as a key candidate gene. To investigate its subcellular localization, a 35S::PbeDof9.1-GFP fusion construct was generated and transiently expressed in Nicotiana benthamiana leaves. Confocal microscopy revealed that the PbeDof9.1-GFP fusion protein was exclusively localized in the nucleus (Supplementary Figure S3), whereas fluorescence signals from the empty vector control (GFP) were observed in both the cell membrane and nucleus. Taken together with the significant up-regulation of PbeDof9.1 in roots, stems, and leaves under salt stress, these findings confirm that PbeDof9.1 possesses typical characteristics of a transcription factor, suggesting it functions as a core nucleus-localized regulator in the salt tolerance network of pear.

2.11. Overexpression of PbeDof9.1 Enhances Salt Tolerance at Germination and Seedling Stages in Transgenic A. thaliana

To evaluate the biological function of PbeDof9.1 in response to salt stress, we systematically compared the phenotypes of the wild type (WT) and three independent homozygous T₃ overexpression lines (OE1, OE2, and OE3). RT-PCR analysis confirmed the expression of PbeDof9.1 in these three transgenic lines, whereas no transcript was detected in the WT (Supplementary Figure S4). Subsequently, we assessed seed germination and root growth under various NaCl concentrations (0, 125, 150, 175, and 200 mM). Under control conditions (0 mM NaCl), no significant differences in germination rates were observed among the lines (Figure 7A,C). However, as NaCl concentrations increased, seed germination of the WT was significantly inhibited, whereas the OE lines exhibited superior tolerance. Specifically, under 175 mM NaCl treatment, the germination rate of the WT dropped to approximately 25%, while the OE lines maintained rates above 35% (Figure 7C), indicating that the overexpression of PbeDof9.1 effectively mitigated the inhibitory effect of high salt stress on seed germination. Vertical root growth assays further revealed that salt stress significantly compromised root development in A. thaliana (Figure 7B). Statistical analysis demonstrated that under 125–200 mM NaCl, the primary root lengths of the transgenic lines were significantly longer, and the number of lateral roots was significantly higher than those of the WT (Figure 7D,E). Notably, under 175 mM NaCl stress, both primary root length and lateral root number in the OE lines were approximately 1.5-fold higher than those of the WT.

Taken together, these results confirm that the heterologous expression of PbeDof9.1 significantly enhances salt tolerance during the germination and seedling stages in A. thaliana, suggesting that PbeDof9.1 functions as a key positive regulator in the salt stress regulatory network of pear.

2.12. Overexpression of PbeDof9.1 Enhances Salt Tolerance and Antioxidant Capacity in Pear Calli

To validate the function of PbeDof9.1 in a homologous system, we generated transgenic pear calli overexpressing PbeDof9.1 following the method of Teng et al. and treated them with 200 mM NaCl for 7 days [28]. Under control conditions (0 mM NaCl), OEPbeDof9.1 calli exhibited a growth phenotype similar to that of the wild type (WT). qRT-PCR analysis confirmed that the transcript level of PbeDof9.1 in the transgenic calli was significantly higher than that in the WT (Supplementary Figure S5). However, under 200 mM NaCl stress, WT calli displayed severe growth inhibition and extensive browning, whereas OEPbeDof9.1 calli maintained vigorous growth with significantly alleviated browning symptoms (Figure 8A). Physiological analysis revealed that salt stress induced excessive accumulation of reactive oxygen species (ROS) and membrane damage in WT calli. Specifically, the levels of H₂O₂ (Figure 8B) and malondialdehyde (MDA) (Figure 8E) were sharply increased in the WT under 200 mM NaCl treatment, whereas their accumulation was significantly repressed in OE-PbeDof9.1 calli. Furthermore, the activities of key antioxidant enzymes, including peroxidase (POD) (Figure 8C) and catalase (CAT) (Figure 8D), were significantly higher in OEPbeDof9.1 calli than in the WT under salt stress. These results demonstrate that PbeDof9.1 confers salt tolerance by boosting the antioxidant defense system to scavenge excess ROS and protect cell membrane integrity.

3. Discussion

As a plant-specific zinc finger protein family, Dof transcription factors play pivotal roles in regulating plant growth, development, and environmental adaptation. In this study, we identified 43 PbeDof members in the P. betulifolia genome. The family size is comparable to that of A. thaliana (36) and P. bretschneideri (45), suggesting that the copy number of the Dof family has remained relatively stable during dicot evolution. Sequence analysis revealed that all members contain a highly conserved C2C2-type zinc finger domain, implying a high degree of homology in DNA-binding capabilities. However, significant divergence in physicochemical properties and motif composition provides a structural basis for the functional diversification of this family. Notably, while most members are localized to the nucleus, the non-canonical subcellular localization of certain members—such as PbeDof7.3/0.1 (mitochondria) and PbeDof3.1/11.2 (chloroplasts)—points towards potential non-classical functions in organellar retrograde signaling that warrant further investigation.

Gene duplication is a primary driving force for gene family expansion and the acquisition of new functions. Chromosomal localization and collinearity analyses indicated that segmental and tandem duplications synergistically drove the evolution of the PbeDof family. In particular, the identification of tandem duplication clusters on chromosomes 5 and 10 (containing PbeDof5.2–5.4 and PbeDof10.2–10.5, respectively) mirrors expansion patterns observed in Lycium barbarum and Glycine max [29,30], suggesting that these duplication events provided genetic material for subfunctionalization. Phylogenetic analysis revealed highly consistent classification patterns between the Dof families of P. betulifolia and P. bretschneideri, yet both species lack the C3 subfamily present in A. thaliana. Given that Rosaceae species shared a whole-genome triplication (γ) event [31], we speculate that the C3 subfamily may have undergone lineage-specific loss during the divergence of the Pyrus ancestor. Furthermore, synteny analysis identified 109 orthologous gene pairs between P. betulifolia and the cultivated P. bretschneideri, significantly more than those shared with A. thaliana. However, the orthologous relationships are not entirely one-to-one; this variation likely reflects genomic reshaping driven by differential selection pressures—natural selection for the wild species versus artificial domestication for the cultivar [32]. Specifically, as P. betulifolia is an elite rootstock resource, the specific retention or expansion of certain Dof members in its genome may constitute the genetic basis for its superior salt tolerance.

Cis-acting elements in promoter regions act as “switches” for transcriptional regulation. Although light-responsive elements are widespread, the distribution of hormone- and stress-responsive elements is more indicative for identifying stress-resistance genes [1]. Our analysis revealed a significant enrichment of ABA-responsive elements (ABREs) and stress-defense elements (TC-rich repeats) in PbeDof promoters. Notably, the promoter of the key candidate PbeDof9.1 features a high density of ABREs and MeJA-responsive elements (CGTCA-motifs). This combinatorial pattern suggests that PbeDof9.1 likely serves as a “crosstalk node” integrating ABA, MeJA, and salt stress signals, enabling a rapid transcriptional response to salinity via hormone-dependent pathways.

Tissue-specific expression analysis further highlighted the functional differentiation of PbeDof genes. The high abundance of PbeDof9.1 in roots aligns with its role in root-mediated stress tolerance. Transcriptomic and qRT-PCR data consistently demonstrated that PbeDof9.1 acts as an “early responder,” being rapidly induced within 6 h of salt stress. While heterologous expression in A. thaliana confirmed that PbeDof9.1 significantly enhances seed germination and root growth under salt stress, validation within a homologous background is critical for establishing its practical application value. Here, we further confirmed its conserved function by overexpressing PbeDof9.1 in pear calli. Under salt stress, transgenic pear calli exhibited more vigorous growth and significantly lower browning rates compared to the wild type. Physiological analysis showed that the activities of key antioxidant enzymes (POD and CAT) were significantly enhanced, while MDA accumulation was reduced in transgenic lines. Since salt stress often triggers reactive oxygen species (ROS) bursts leading to oxidative damage [20,33,34,35], these results strongly support a mechanism whereby PbeDof9.1 maintains membrane integrity and intracellular redox homeostasis by boosting the endogenous antioxidant system [6]. Based on these phenotypes, we hypothesize that PbeDof9.1 might confer salt tolerance via dual pathways: first, by remodeling root architecture (e.g., increasing lateral root density), possibly involving auxin-related genes to cope with osmotic stress; and second, by potentially regulating downstream ion transporters (e.g., SOS1) or antioxidant systems to maintain Na+/K+ balance and redox homeostasis. Future studies will utilize Agrobacterium rhizogene-mediated hairy root transformation combined with ChIP-seq to further elucidate its direct downstream targets.

In conclusion, this study systematically characterized the PbeDof gene family at the genome-wide level, elucidating gene duplication as the primary driver of its expansion. Through expression profiling and multi-dimensional functional verification, we identified PbeDof9.1 as a key candidate gene for salt stress response in P. betulifolia. This gene likely functions as an early root signal sensor, initiating downstream stress-resistance programs via ABA-dependent pathways. Our findings not only enrich the theoretical understanding of the Dof family in Rosaceae but also provide a promising genetic target for molecular breeding of pear rootstocks utilizing the superior genetic resources of wild P. betulifolia.

4. Materials and Methods

4.1. Identification and Characterization of PbeDof Family Members

The protein sequences of A. thaliana Dof family members were retrieved from the TAIR database (https://www.arabidopsis.org/ (accessed on 15 October 2025)). The whole-genome sequence of P. betulifolia was downloaded from the Pear Genome Database (PGDB, http://pyrusgdb.sdau.edu.cn/ (accessed on 15 October 2025)) [36]. Two strategies were employed to identify candidate PbeDof genes using TBtools II software v2.225 [37]. First, a BLASTP search was performed against the P. betulifolia proteome using A. thaliana Dof proteins as queries (E-value < 1 × 10⁻⁵). Second, the Hidden Markov Model (HMM) profile of the Dof domain (PF02701), obtained from the Pfam database (https://www.ebi.ac.uk/interpro/ (accessed on 15 October 2025)), was used to search for Dof-containing proteins. The results from both methods were merged, and redundant sequences were removed. The presence of the Dof domain was further confirmed using the NCBI Conserved Domain Database (CDD). Sequences lacking a complete Dof domain or exhibiting significant defects were manually discarded [38]. Finally, the physicochemical properties (e.g., molecular weight and isoelectric point) were calculated using the “Protein Parameter Calc” function in TBtools II, and chromosomal locations were visualized using the “Gene Location Visualize” module. Subcellular localization was predicted using the WoLF PSORT server (https://wolfpsort.hgc.jp/ (accessed on 22 October 2025)).

4.2. Phylogenetic, Gene Structure, and Conserved Motif Analysis of the PbeDof Gene Family

A phylogenetic tree was constructed using the “One Step Build a ML Tree” module in TBtools II with default parameters. The resulting tree was re-rooted and visualized using the Interactive Tree Of Life (iTOL, https://itol.embl.de/ (accessed on 28 October 2025)). The conserved motifs of PbeDof proteins were analyzed using the MEME suite (https://meme-suite.org/meme/ (accessed on 28 October 2025)), with the maximum number of motifs set to 10 and the motif width ranging from 6 to 100 residues; other parameters remained default. Gene structure diagrams (exon-intron organization) were generated based on the P. betulifolia genome annotation (GFF3) using TBtools II. Finally, the phylogenetic tree, conserved motifs, and gene structures were integrated and visualized using the “Gene Structure View” function in TBtools II.

4.3. Promoter cis-Acting Element Analysis

To analyze promoter regions, the 2000 bp genomic sequences upstream of the translation start site (ATG) of each PbeDof gene were extracted using the ‘Gff3 Sequences Extractor’ module in TBtools II. Subsequently, putative cis-acting elements within these sequences were predicted and classified using the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 5 November 2025)).

4.4. Chromosomal Localization and Synteny Analysis

Chromosomal positions of PbeDof genes were mapped based on the P. betulifolia genome annotation using the “Gene Location Visualize” function in TBtools II. Collinearity analyses, including intraspecific collinearity within P. betulifolia and interspecific collinearity against A. thaliana and white pear (P. bretschneideri), were performed using the “One Step MCScanX” module. The results were visualized using the “Advanced Circos” and “Dual Synteny Plot” functions, respectively. Additionally, the synonymous substitution rates (Ks) of collinear gene pairs were calculated using the “Simple Ka/Ks Calculator” in TBtools II to assess evolutionary selection pressure.

4.5. Salt Stress Treatment and Gene Expression Analysis in P. betulifolia

Transcriptome data of P. betulifolia under salt stress were previously generated by our research group (NCBI Accession: PRJNA812627) [27]. The dataset was derived from seedlings treated with 200 mM NaCl for 0 and 2 days. FPKM (Fragments Per Kilobase of transcript per Million mapped reads) values for Dof family genes were extracted and row-normalized to generate a heatmap for identifying differentially expressed members.

For RNA extraction and qRT-PCR analysis, the plant total RNA extraction kit “VAMNE Magnetic Universal Total RNA Kit (Vazyme)” was used to extract sample RNA. First-strand cDNA was synthesized using “HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme)”. A 20 µL real-time fluorescence quantitative PCR system was established using “ChamQ Universal SYBR qPCR Master Mix (2×) (Vazyme)”. The amplification program was: 95 °C pre-denaturation for 30 s; followed by 40 cycles (95 °C 10 s, 60 °C 30 s); finally, the melting curve was collected. Using PbeEF1α (GWHPAAYT007384) as the internal reference gene [39], the relative expression levels were calculated using the 2^−ΔCt method (for stress expression profiling) or the 2^−ΔΔCt method (for overexpression analysis). For the identification of transgenic A. thaliana lines, RT-PCR was performed to verify the transcript expression of PbeDof9.1, and the PCR products were visualized by 1.2% agarose gel electrophoresis.

4.6. Subcellular Localization Assay

To generate the 35S::PbeDof9.1-GFP fusion construct, the full-length coding sequence of PbeDof9.1 (without the stop codon) was amplified and cloned into the pCAMBIA1300 vector. The recombinant plasmid was verified by sequencing and subsequently transformed into Agrobacterium tumefaciens strain GV3101. Transient expression assays were performed by infiltrating healthy leaves of Nicotiana benthamiana [40]. Fluorescence signals were monitored 48 h post-infiltration using a laser scanning confocal microscope. The empty 35S::GFP vector was used as a negative control.

4.7. Functional Verification of Salt Resistance in Transgenic A. thaliana Lines

To validate the function of PbeDof9.1 in conferring salt tolerance, we generated stable transgenic A. thaliana lines expressing the 35S::PbeDof9.1-GFP fusion protein. Three independent homozygous T3 lines (OE1, OE2, and OE3) were selected for comparison with the wild type (WT). To evaluate salt tolerance during germination, seeds were sown on 1/2 MS medium containing 0, 125, 150, 175, or 200 mM NaCl. After stratification at 4 °C for 3 days and subsequent growth for 7 days under long-day conditions (16 h light/8 h dark), germination rates were recorded. Additionally, to assess root system architecture under salt stress, seedlings were grown vertically on 1/2 MS plates with the corresponding NaCl concentrations. After 15 days, primary root length and lateral root numbers were quantified using ImageJ (v1.54g) and manual counting, respectively (n > 30 seedlings per replicate).

4.8. Genetic Transformation and Salt Stress Treatment of Pear Calli

To generate the transgenic pear calli, the full-length coding sequence of PbeDof9.1 was cloned into the pCAMBIA1300 vector to form the 35S::PbeDof9.1-GFP fusion construct. The empty vector 35S::GFP was used as a negative control. The constructs were separately introduced into Agrobacterium tumefaciens strain EHA105, followed by transformation into pear calli using the A. tumefaciens-mediated method as described previously [41]. The transgenic calli were cultured under continuous dark conditions on MS-based solid medium at 25 °C. The medium was supplemented with 10 mg/L hygromycin for selection and 200 mg/L timentin to inhibit bacterial growth. After confirming the transgene expression by PCR, the transgenic lines were subcultured onto fresh medium every 15–20 days.

For salt stress treatments, healthy and consistent transgenic calli (WT, OE-PbeDof9.1) were transferred to MS medium supplemented with 200 mM NaCl. The phenotypes were documented, and samples were collected for physiological and gene expression analyses after 7 days of treatment.

4.9. Determination of Antioxidant Enzyme Activities and MDA Content

The activities of peroxidase (POD) and catalase (CAT), as well as the contents of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂), were determined using the corresponding analytical kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) following the manufacturer’s instructions. The enzyme activities and the contents of MDA and H₂O₂ were calculated based on the fresh weight (FW) of the callus samples.

4.10. Statistical Analyses

All experiments were performed with three independent biological replicates, and each biological replicate included three technical replicates. The results are presented as the mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism 8 software (v8.0.2). Differences among treatments were evaluated using one-way analysis of variance (ANOVA), followed by Duncan’s multiple range test. Significance levels were set at p < 0.05 (*).

5. Conclusions

In summary, this study provides the first comprehensive genomic characterization of the PbeDof gene family in P. betulifolia, elucidating that segmental duplication acted as the dominant force driving its evolutionary expansion. Integrative functional analyses identified PbeDof9.1 as a pivotal salt-responsive regulator. Characterized by its predominant root expression and rapid induction, PbeDof9.1 orchestrates salt tolerance by mobilizing the antioxidant enzymatic system (POD, and CAT) to efficiently scavenge reactive oxygen species (ROS). These findings not only deepen our understanding of Dof-mediated stress adaptation but also highlight PbeDof9.1 as a highly promising target for the molecular breeding of robust, salt-tolerant pear rootstocks.