Overexpression of MtDof32 in Medicago truncatula enhances leaf and flower organ size through modulation of cell expansion

As leaf organ size is a key determinant of plant morphology and development, elucidating the cellular and molecular mechanisms governing organ size regulation has become a central research focus in legume biology. DNA binding with one finger 32 (MtDof32), previously identified as a key regulatory factor in organ development, has been shown to influence leaf and flower organ size in Arabidopsis. Here, we demonstrated that MtDof32 overexpression in Medicago truncatula resulted in the enlargement of both leaf and flower organs. Cellular analysis revealed that MtDof32 primarily modulated organ size by controlling cell expansion. Yeast two-hybrid, co-immunoprecipitation (Co-IP), and bimolecular fluorescence complementation (BiFC) assays collectively established the nuclear-localized interaction between MtDof32 and MtEBP1. Additionally, we observed that MtDof32 overexpression led to delayed flowering and reduced branching in transgenic plants. Comparative analysis with wild-type plants indicated significant alterations in the expression levels of key flowering and branching regulatory genes. This study underscored the conserved role of MtDof32 in regulating organ size across leguminous species, offering valuable insights for the genetic modification and utilization of this gene in crop improvement strategies.

1 Introduction

Legume forage stands out as one of the most economically and nutritionally valuable forage resources, renowned for its strong adaptability, high biomass production, and elevated protein content. It plays a pivotal role in ensuring a stable forage supply (Hamacher et al., 2021). While enhancing forage yield remains a major goal in legume research, a deeper understanding of the genetic and regulatory mechanisms controlling plant architecture, such as leaf size, branching pattern, and resource allocation, is equally critical for targeted crop improvement. Elucidating the molecular pathways that govern organ development and phenotypic plasticity provides a fundamental basis for designing novel strategies to optimize plant form and function, thereby supporting the sustainable development of animal husbandry.

The morphogenesis of plant organs is regulated through multifactorial control systems, among which interspecific genomic divergence constitutes the primary evolutionary determinant governing organ size specification. Consequently, organ size within specific plant varieties tends to be relatively consistent, reflecting the precise regulation of organ growth and development. The ultimate size of plant organs is shaped by the long-term interplay between intrinsic genetic factors and external environmental conditions, involving intricate regulatory pathways (Powell and Lenhard, 2012; Zheng et al., 2023). This complex and finely tuned process engages a multitude of specific genes, including those related to hormones, ubiquitins, cytochromes, microRNAs, transcription factors, and other regulatory elements. To date, numerous growth-promoting and growth-inhibiting factors involved in plant organ development have been identified, providing a foundational understanding of the mechanisms governing organ size regulation. However, while substantial progress has been made in characterizing genes that modulate organ morphogenesis, the underlying regulatory mechanisms remain incompletely understood, and the regulatory network requires further refinement.

Studies at the cellular level have revealed that the plant organs size was governed by the regulation of cell division, differentiation, and growth during organ development, with both cell proliferation and cell expansion playing crucial roles in this process (Cadart et al., 2019). Cell growth serves as a fundamental determinant in the formation, organogenesis, and post-embryonic morphogenesis of plant tissues and organs. Key intermediates in certain cell growth regulatory pathways are essential for intercellular signal transduction, influencing the plant cell cycle, promoting cell differentiation, and indirectly modulating cell size and organ morphology. Furthermore, when cell proliferation is impaired, plants can compensate by increasing cell volume to achieve the mature organ size (Nomoto et al., 2022).

Dof (DNA binding with one finger) family transcription factors, which utilize a single zinc finger motif for DNA binding, are unique to plants and have been identified or predicted in numerous higher plant species (Lohani et al., 2021). These Dof proteins play crucial roles in plant growth and development (Alam et al., 2024; Song et al., 2024). It has been demonstrated that Dof participate in regulating diverse physiological and developmental processes in plants, such as carbon and nitrogen metabolism, flower and seed development, synthesis of secondary metabolites, and vascular and leaf development during plant growth. Notably, one of the key functions of Dof proteins was their involvement in regulating organ development through plant-specific biological processes, often linked to hormone signaling pathways. For instance, overexpression of the tomato gene SlCDF3 resulted in the enlargement of leaves, petals, and siliques (Corrales et al., 2014). Similarly, overexpression of OBP3, a member of the Dof family in Arabidopsis, led to delayed development of cotyledons and plants, whereas obp3 mutants exhibited enlarged cotyledons and plants (Ward et al., 2005). In contrast, plants overexpressing Dof6 displayed dwarf phenotype, compared to wild-type plants (Rueda-Romero et al., 2012). Interestingly, MtDof32 exhibited opposite functional characteristics to those of AtOBP3 and AtDof6, as its overexpression in Arabidopsis caused enlargement of leaf and flower organs (Guo et al., 2021). Additionally, certain Dof functions were closely associated with hormone signaling pathways. For example, Arabidopsis AtDof6 regulated abscisic acid (ABA) biosynthesis and metabolism, thereby influencing seed germination. AtDof6 overexpression disrupted normal plant development, leading to sterility. The high expression levels also conferred delayed progeny seed germination, ABA hypersensitivity, and upregulation of ABA biosynthesis genes. Molecular analyses revealed that AtDof6 physically interacts with TCP14 to form a transcriptional repressor complex that downregulates key genes involved in ABA biosynthesis and metabolism, ultimately inhibiting seed germination (Rueda-Romero et al., 2012). Despite significant progress in understanding the roles of Dof in organ development, the precise mechanisms by which they influence organ size through the regulation of plant cell size remain unclear. Furthermore, whether these processes depend on hormone signaling pathways need to be further investigated.

Our previous studies indicated that, among several predicted Dof transcription factors associated with development, MtDof32 exhibited the most significant variation in expression levels across various developmental stages of Medicago truncatula (M. truncatula), which prompted us to select it for further functional analysis. Arabidopsis plants overexpressing MtDof32 exhibited significant enlargement of leaves and flowers (Guo et al., 2021), along with altered expression levels of genes involved in auxin signaling transduction, such as AtEBP1, AtARL, and AtKLU. For functional characterization of MtDof32 in developmental processes, this study employed transgenic technology to generate MtDof32-overexpressing M. truncatula plants, building on prior findings. Additionally, using yeast two-hybrid screening, we identified and validated ERBB-3 BINDING PROTEIN 1 (MtEBP1) as an interacting protein of MtDof32. Notably, MtDof32 regulates cell size in M. truncatula through its interaction with MtEBP1, a mechanism not previously reported. This study provided a theoretical foundation and genetic resources for elucidating the regulatory mechanisms of organ development in M. truncatula, offered new insights for the advancement of high-yield alfalfa biotechnological breeding strategies.

2 Materials and methods

2.1 Plant materials and growth conditions

In this study, M. truncatula (ecotype R108) seeds were employed as the experimental material. The seeds were surface-sterilized with 75% ethanol for 12 minutes, followed by thorough rinsing with sterile distilled water. Subsequently, the sterilized seeds were placed on moistened sterile filter paper and subjected to cold stratification at 4°C for 5 days to break dormancy. After vernalization, the seeds were transferred to standard growth conditions with a photoperiod of 16 h light at 26°C and 8 h dark at 24°C, and maintained until cotyledon expansion. All seeds used in subsequent experiments underwent this standardized pretreatment protocol. Following germination, the seedlings were transferred to Hoagland nutrient solution and cultivated under the same temperature and light conditions (16 h light at 26°C/8 h dark at 24°C) for further growth and development.

2.2 Cloning of MtDof32 from M. truncatula

The gene and protein sequences of MtDof32 (Mtr7g010950) were retrieved from the National Center for Biotechnology Information (NCBI) database and subjected to sequence analysis using MEGA version 6.0 (Koichiro Tamura, Hachioji, Tokyo, Japan). Based on the obtained sequence, a pair of specific primers, Dof32-F/R (see Supplementary Table 1 for details), was designed to amplify the full-length coding sequence (CDS) from M. truncatula. The gene amplification was performed using PrimeSTAR^® Max DNA Polymerase (Takara Biotech, Beijing, China) following the manufacturer’s protocol. The resulting PCR products were subsequently cloned into the pEASY-T5 vector (TransGen Biotech, Beijing, China) for further molecular characterization and functional studies.

2.3 Quantitative analysis

The R108 M. truncatula plants were subjected to phytohormone treatments with 10 μmol/L Indole-3-acetic acid (IAA), 100 μmol/L ABA, 100 μmol/L Gibberellic acid (GA₃), and 5 mmol/L Salicylic acid (SA) for varying durations. For each phytohormone treatment, samples were collected at 0 h, 2 h, 4 h, 8 h, 12 h, and 24 h, with each treatment replicated three times. The collected samples were immediately flash-frozen in liquid nitrogen to preserve RNA integrity. Total RNA was extracted from the whole plant samples using the Total RNA Kit (Omega, Norcross, USA). Subsequently, cDNA was synthesized from the extracted RNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara Biotech, Beijing, China). The first-strand cDNA served as the template for real-time quantitative RT-PCR (qRT-PCR) analysis. The housekeeping gene Actin (Mtr3g095530) was used as an internal reference to normalize the expression levels, and the relative expression of MtDof32 was calculated using the 2^-ΔΔCT method. Three biological replicates were performed for each experiment. Primer sequences used in the study were provided in Supplementary Table 1. The data are shown as mean values ± SD. Independent t-tests demonstrated that there was significant difference (P<0.05).

2.4 Obtaining transgenic plants

The full-length CDS was cloned into the pCAMBIA3302 (p3302) vector, generating the recombinant construct p3302-35S::MtDof32. The construct was subsequently introduced into M. truncatula calli via Agrobacterium-mediated transformation. Following the selection of PCR-positive transgenic plants, two independent transgenic lines exhibiting high MtDof32 expression levels were selected for further analysis. To establish stable transgenic lines, T2 generation plants were obtained through self-pollination of primary transformants and used for subsequent functional characterization studies.

2.5 Subcellular localization analysis

The MtEBP1 CDS was cloned into the pSAT-GFP vector using gene-specific primers SAT-MtEBP1-F/R (Supplementary Table 1), generating the recombinant construct pSAT-35S::MtEBP1-GFP. The construct was subsequently introduced into M. truncatula leaf protoplasts via polyethylene glycol (PEG4000)-mediated transformation. Subcellular localization of the MtEBP1-GFP fusion protein was analyzed using laser scanning confocal microscopy 16–24 h post-transformation. Protoplast isolation and transformation were performed according to established protocols (Yoo et al., 2007).

2.6 Yeast two-hybrid assay

The MtDof32 CDS was amplified by PCR using pGBKT7-Dof32-F/R primers and subsequently fused into the pGBKT7 vector through the action of endonucleases and seamless ligases, generating the bait construct pGBKT7-Dof32. The recombinant plasmid was transformed into the Y187 yeast strain for library screening. Yeast two-hybrid screening was conducted using the Matchmaker GAL4 Two-Hybrid System (Clontech, USA), where the Y187 strain carrying the bait construct was mated with AH109 strain containing a M. truncatula cDNA library for protein interaction screening.

For prey vector construction, the MtEBP1 CDS was cloned into pGADT7 and transformed into yeast strain AH109 according to the manufacturer’s instructions (Clontech, USA). Protein-protein interactions were assessed by co-culturing the transformed Y187 and AH109 strains on selective media (SD/-Ade/-Trp and SD/-Ade/-His/-Leu/-Trp). Following incubation at 30°C for 48 h, positive colonies were subjected to β-galactosidase activity assays to confirm protein interactions.

2.7 Bimolecular fluorescence complementation assay

MtDof32 CDS was fused to YFP at its N-terminus in the pSYNE vector, generating the 35S promoter driven pSYNE-35S::MtDof32 construct. Similarly, the MtEBP1 CDS was inserted into the pSYCE vector with a C-terminal YFP tag, creating the pSYCE-35S::MtEBP1 fusion construct. Both recombinant plasmids were introduced into A. tumefaciens strain GV3101 and subsequently infiltrated into the abaxial epidermis of Nicotiana benthamiana leaves using agroinfiltration. YFP fluorescence was visualized 48 h post-infiltration using laser scanning confocal microscopy.

2.8 Co-immunoprecipitation assay

Tobacco leaves from one-month-old plants were infiltrated with Agrobacterium tumefaciens carrying 35S::MtDof32-FLAG and 35S::MtEBP1-GFP constructs. Two days post-infiltration, leaf samples were harvested, immediately frozen in liquid nitrogen, and stored at -80°C. The frozen tissue was ground to a fine powder in liquid nitrogen and homogenized in IP lysis buffer supplemented with a protease inhibitor cocktail. The homogenate was centrifuged at 15,000 × g for 10 min at 4°C, and the resulting supernatant was collected as the total protein extract. The total protein extract was incubated with pre-treated beads at 4°C to facilitate binding of the target protein. After incubation, the beads were washed with ice-cold PBS to remove non-specifically bound proteins. Subsequently, the beads were collected and resuspended in 45 μL of IP lysis buffer and 15 μL of 4× SDS loading buffer. The mixture was boiled at 100°C for 5 minutes to elute the proteins, followed by centrifugation at 15,000 × g for 5 minutes. The resulting supernatant, designated as the IP sample, was collected and stored at -80°C for subsequent analysis. An input sample was prepared by mixing 45 μL of the total protein extract with 15 μL of 4× SDS loading buffer, followed by denaturation at 100°C for 5 min. The sample was then stored at -80°C. For immunoblotting analysis, the input and IP samples were probed with anti-GFP and anti-FLAG antibodies to detect the respective proteins.

3 Result

3.1 Identification and homology analysis of MtDof32 in M. truncatula

Structural analysis revealed that MtDof32 protein (Mtr7g010950) possessed a single characteristic Dof domain, confirming its classification within the Dof transcription factor family. We systematically classified 42 Dof family members in M. truncatula based on conserved motif organization and constructed a phylogenetic tree using full-length protein sequences (Figure 1A). Sequence homology analysis between MtDof32 and Dof proteins from other species was performed using MEGA6.0 software (Figure 1B). Comparative analysis demonstrated that MtDof32 shared the highest sequence identity (82.1%) with L195_g008848, a Dof protein identified in Trifolium pratense. Based on these findings, we hypothesize that both functional redundancy and divergence may exist between MtDof32 and other transcription factors in the Dof family.

Figure 1

Figure 1. Dof transcription factors phylogenetic tree analysis. (A) Phylogenetic tree analysis between Dof proteins from M. truncatula. Dof32 is circled by a red box. (B) Sequence homology analysis of Dof proteins across selected species was conducted based on protein sequences acquired from the National Center for Biotechnology Information (NCBI). Gene accession numbers: M. truncatula Dof32 (Mtr7g010950), Trifolium pretense (L195_g008848), Glycine max (Gm18G260500), Arabidopsis thaliana (At5g39660), Brassica napus (Bn04g09490D), Oryza sativa (Os01g15900), Zea mays (Zm2g162749), Sorghum bicolor (Sb1g045840).

3.2 Expression analysis of MtDof32 in M. truncatula

The regulation of plant growth, developmental processes, and stress responses is mainly mediated by plant hormones. To further investigate the response of MtDof32 to exogenous hormones, we analyzed its expression patterns under various treatments (Figure 2). Quantitative analysis demonstrated that under exogenous IAA treatment, the expression level of MtDof32 initially increased, peaking at 8 h, before subsequently declining. In contrast, treatments with exogenous ABA, GA3, and SA led to significant downregulation of MtDof32 expression within 24 h. These findings suggested that the transcriptional activity of MtDof32 was closely associated with IAA, ABA, GA3, and SA, implying its possible regulatory function in various hormone-mediated signaling cascades.

Figure 2

Figure 2. Analysis of expression patterns of MtDof32 under exogenous hormones treatment. Three-week-old M. truncatula seedlings were treated with (A) 10 μmol/L IAA, (B) 100 μmol/L ABA, (C) 100 μmol/L GA₃, and (D) 5 mmol/L SA via foliar application. Samples were collected at 0, 2, 4, 8, 12, and 24 h post-treatment. Data represent mean ± SD of three biological replicates. *denotes statistically significant differences at p < 0.05. **denotes statistically significant differences at p < 0.01.

3.3 Ectopic expression of MtDof32 led to enhanced organ size in M. truncatula

Ectopic expression of MtDof32 in M. truncatula promoted the enlargement of both flower and leaf organs (Figure 3). At 60 days after planting, the average width of the vexillum petal in the transgenic lines MtDof32-#2 and MtDof32-#7 was 6.29 mm and 6.37 mm, respectively, compared to 4.87 mm in wild-type plants. Additionally, the average length of the vexillum petal was 7.45 mm and 7.57 mm, respectively, significantly exceeding the 5.75 mm of the wild type. Similarly, in these two transgenic lines, the average width of the apical leaflets in trifoliate compound leaves was 2.47 cm and 2.53 cm, respectively, significantly larger than the 1.71 cm measured in the wild type. A similar trend in the enlargement of both leaf and floral organs was observed as early as 30 days after planting (Supplementary Figure 3). To further investigate the cellular basis of these phenotypic changes, scanning electron microscopy was employed to examine the size of leaf cells in MtDof32-overexpressing M. truncatula. Microscopic analysis demonstrated that transgenic plants exhibited substantially larger leaf cells than those of wild-type R108, suggesting that MtDof32 likely influences leaf size by promoting cell enlargement.

Figure 3

Figure 3. Comparative analysis of leaf and flower between MtDof32 transgenic plants and wild M. truncatula. (A) Comparative analysis of leaf and flower between MtDof32 transgenic plants and wild M. truncatula. Samples were collected at 60 days of growth. (B) Differences of the epidermal mesophyl cells between the wild-type and MtDof32 overexpressed transgenic M. truncatula in scanning electron microscopy. (C) Comparison of terminal leaflet and vexillum petal size between wild type and MtDof32 transgenic group M. truncatula (n=18). *denotes statistically significant differences at p < 0.05. **denotes statistically significant differences at p < 0.01.

3.4 Overexpression of MtDof32 delayed the flowering time of M. truncatula

MtDof32 transgenic M. truncatula exhibited a delayed flowering phenotype, with flowering time occurring 8–11 days later than wild-type controls (Figure 4). MtFTa1 (Flowering Locus T) and MtSOC1 (Suppressor of Overexpression of Constans 1) serve as pivotal integrating factors in regulating flowering time. Consequently, we examined the expression levels of these two crucial genes in both transgenic and wild-type plants. Analysis of gene expression revealed significant downregulation of MtFTa1 and MtSOC1 transcripts in MtDof32-overexpressing plants. These findings suggested that MtDof32 functioned as a key regulator of flowering time in M. truncatula.

Figure 4

Figure 4. Phenotypic comparison of flowering time in MtDof32-overexpressing transgenic lines versus wild-type M. truncatula. (A) Phenotypic comparison analysis of MtDof32 transgenic lines and WT at 45 days of growth (B) Flowering time phenotype comparison in MtDof32 transgenic lines versus WT (n=10). *denotes statistically significant differences at p < 0.05. (C) Quantitative expression analysis of MtFTa1 and MtSOC1 genes in MtDof32 transgenic lines and WT using qRT-PCR. Data represent mean values ± SD from three biological replicates. *denotes statistically significant differences at p < 0.05. **denotes statistically significant differences at p < 0.01.

3.5 Ectopic expression of MtDof32 reduced the number of primary branches in M. truncatula

After the cotyledons unfolded, the seedlings were transferred to soil, and the time of plant growth was initiated. The number of branches in MtDof32-overexpressing M. truncatula was recorded at 30, 45, and 90 days of growth (Figure 5). At 30 days, overexpressing MtDof32 transgenic M. truncatula exhibited an average of 0.7 fewer primary branches compared to wild-type M. truncatula. At 45 days, the WT had an average of 1.7-1.8 more primary branches than the MtDof32-overexpressing transgenic lines. At 90 days, the average difference in branch number was 1.2-1.4. These results demonstrated that transgenic M. truncatula plants exhibited a significant reduction in primary branch number compared to R108 wild-type plants, a phenotype consistent with that observed in transgenic Arabidopsis. qRT-PCR analysis showed that the relative expression levels of both MtCCD7 and MtBRC1 genes in MtDof32-overexpressing transgenic M. truncatula lines (#2 and #7) were significantly upregulated compared to the WT control. It is noteworthy that although MtDof32-overexpressing transgenic plants exhibited a significant increase in leaf size, their total aerial biomass was significantly lower than that of wild-type controls at maturity (Supplementary Figure 4). This reduction in biomass is attributable to the decreased branch number in the overexpression lines, highlighting a notable trade-off between leaf cell expansion and branching capacity under constitutive MtDof32 expression.

Figure 5

Figure 5. Comparative analysis of primary branches number in MtDof32 transgenic lines and wild M. truncatula. (A) Comparative analysis of MtDof32 transgenic plants and wild M. truncatula at 30 days of growth. (B) Differences of the primary branches number between the wild-type R108 and MtDof32 overexpressed transgenic M. truncatula (n=18). *denotes statistically significant differences at p < 0.05. **denotes statistically significant differences at p < 0.01. (C) Quantitative expression profiling of branching-related genes in transgenic and WT plants using qRT-PCR. Data are presented as mean ± SD from three independent biological replicates. *denotes statistically significant differences at p < 0.05.

3.6 MtDof32 were involved in interactions with MtEBP1

Using pGBKT7-MtDof32 as the bait protein, we performed a yeast two-hybrid screen against a cDNA library derived from Medicago truncatula. After multiple rounds of stringent selection on quadruple-dropout medium (SD/–Trp/–Leu/–His/–Ade) supplemented with X-α-Gal, a number of candidates interacting proteins were identified. All candidate proteins identified in the yeast two-hybrid screen are listed in Supplementary Table 2. Among the candidate genes obtained from the screen, many encoded proteins of unknown function or were not directly relevant to the focus of this study. Therefore, we focused further validation and analysis on MtEBP1 (Mtr7g069390), a candidate with well-established biological roles and clear functional relevance to our investigation.

To investigate the subcellular localization of MtEBP1, 35S::MtEBP1-GFP construct was generated and transiently expressed in M. truncatula protoplasts to determine MtEBP1 localization. Confocal microscopy revealed nuclear localization of the fusion protein (Figure 6A), confirming MtEBP1 as a nuclear protein, consistent with bioinformatics predictions. Since the nuclear localization of MtDof32 has been previously demonstrated (Guo et al., 2021), we will not elaborate on it here.

Figure 6

Figure 6. Subcellular localization of the MtEBP1, and proteins interaction between MtDof32 and MtEBP1. (A) Subcellular localization of MtEBP1 in the protoplast cell of M. truncatula. Scale bars = 25 µm. (B) The protein-protein interaction between MtDof32 and MtEBP1 was examined using BiFC analysis in tobacco epidermal cells. SGT1-YFPn+SRC2-1-YFPc was used as a positive control for the BiFC system. Scale bars = 50 µm. (C) Yeast two-hybrid assays were conducted to examine the direct interaction between MtDof32 and MtEBP1 proteins.

The physical interaction between MtDof32 and MtEBP1 was subsequently confirmed through BiFC assays. Co-expression of pSYNE-MtDof32 and pSYCE-MtEBP1 in tobacco epidermal cells resulted in distinct yellow fluorescence signals specifically localized to the nucleus (Figure 6B), confirming their interaction within the nucleus.

The protein-protein interaction between MtDof32 and MtEBP1 was further validated using yeast two-hybrid assays. The yeast strains Y187 (containing pGBKT7-MtDof32) and AH109 (containing pGADT7-MtEBP1) exhibited normal growth on SD/-Ade -His-Leu-Trp selection medium, comparable to the positive control (pGBKT7-53 + pGADT7-RecT). Furthermore, these colonies demonstrated β-galactosidase activity, as evidenced by blue coloration on SD/-His-Leu-Trp-Ade medium supplemented with X-α-Gal (Figure 6C).

The CoIP assays, performed by co-expressing 35S::MtDof32-FLAG and 35S::MtEBP1-GFP in tobacco leaves, confirmed that FLAG-tagged MtDof32 specifically co-precipitates with GFP-tagged MtEBP1 in vivo (Supplementary Figure 5).

In MtDof32 transgenic plants, both MtEBP1 and MtCYCD3–1 exhibited markedly elevated transcript levels compared to wild-type controls (Figure 7A). Furthermore, evaluation of the organ-specific expression profile in wild-type plants demonstrated that MtEBP1 accumulates preferentially in leaf tissues (Figure 7B). Transgenic lines also showed substantial upregulation of key auxin signaling-related genes, including MtSAUR and MtARF (Figure 7C), along with increased expression of the cell expansion-related genes MtTOR and MtEXPA (Figure 7D). These consistent upregulation patterns across multiple functional categories suggest that MtDof32 may collaborate with MtEBP1 to coordinate the regulation of genes involved in cell proliferation, auxin response, and cell expansion, providing mechanistic insights into its role in promoting organ growth.

Figure 7

Figure 7. Molecular characterization of gene expression in MtDof32-overexpressing Medicago truncatula. (A) Expression levels of MtEBP1 and MtCYCD3–1 in wild-type (WT) and MtDof32 transgenic plants. (B) Organ-specific expression profile of MtEBP1 in wild-type M. truncatula. (C) Relative expression of auxin signaling-related genes MtSAUR and MtARF in leaves of wild-type and MtDof32 transgenic lines. (D) Expression analysis of cell expansion-associated genes MtTOR and MtEXPA in leaves of wild-type and transgenic plants. *denotes statistically significant differences at p < 0.05. **denotes statistically significant differences at p < 0.01.

4 Discussion

The Dof transcription factor family plays crucial regulatory roles in diverse plant-specific biological processes. Current research demonstrates their functional significance in multiple physiological pathways, including: photomorphogenesis and light signal transduction (Cai et al., 2024; Zheng et al., 2024), coordination of carbon-nitrogen metabolic networks (Gupta et al., 2015; Sun et al., 2025), embryogenesis and seed maturation (Da Silva et al., 2016), and abiotic/biotic stress responses (Jin et al., 2024; Wang et al., 2021). The model legume M. truncatula possesses 42 Dof transcription factors, yet the functional characterization of most members remains largely unexplored. In this study, we focused on MtDof32 (Mtr7g010950), which encodes a 518-amino acid protein. Phylogenetic tree analysis revealed that MtDof32 shares the highest sequence similarity (72% identity) with L195_g008848 from Trifolium pratense. Consistent with its predicted function as a transcription factor, our previous subcellular localization studies using a pSAT6::MtDof32-GFP fusion construct demonstrated nuclear localization of the fluorescent signal (Guo et al., 2021).

qRT-PCR analysis revealed that MtDof32 expression in M. truncatula responded dynamically to various exogenous hormone treatments, suggesting its potential involvement in hormone signal transduction cascades. Previous research results have shown that Dof could mediate responses to multiple hormones, including GA (Boccaccini et al., 2014), ABA (Zhai et al., 2022), SA (Kang et al., 2010), and MeJA (Hao et al., 2015). In buckwheat, the FtDof gene was downregulated under GA treatment. In Helianthus annuus, ABA treatment induced differential expression patterns among HaDof transcription factors, with distinct members showing either significant upregulation (e.g., HaDof12, HaDof23) or downregulation (e.g., HaDof05, HaDof17), suggesting functional diversification within this TF family during ABA signaling. This indicated that ABA had specificity in regulating DOF genes. In Arabidopsis, the expression of the OBP2 was significantly increased after MeJA treatment. Plant hormone response profiling revealed that FtDof in Fagopyrum tataricum was specifically induced by MeJA treatment (3.5-fold increase), while showing suppression under ABA (0.4-fold), GA (0.3-fold), IAA (0.5-fold), and SA (0.6-fold) treatments compared to untreated controls (Li et al., 2022). Gibberellins typically inhibit the expression of Dof, while ABA and MeJA induce their expression more. This regulatory pattern may be related to the adaptive responses of plants under different physiological and environmental conditions.

Previous research has demonstrated that Dof transcription factors could function as regulators of flowering in plants. Some Dof members can directly or indirectly affect the expression of key flowering regulators such as FT and CO (Zhang et al., 2019). In this study, MtDof32 transgenic M. truncatula displayed a pronounced late-flowering phenotype relative to wild-type plants. Further analysis revealed that MtDof32 overexpression downregulated MtFT and MtSOC1 expression, indicating its involvement in repressing flowering-related gene networks. Similar results have been found in our previously functional study of MtDof32 in Arabidopsis (Guo et al., 2021). Studies in Arabidopsis demonstrated that AtCDF1 controlled flowering time through specific binding to the promoter regions of CO and FT, ultimately regulating their transcriptional activity (Zhang et al., 2019). Meanwhile, previous studies have also shown that auxin affected the initiation of flower bud differentiation by regulating gene expression. It can work synergistically with other hormones such as GAs and cytokinins to promote flower bud formation (Chandler and Werr, 2015). Auxin regulates flowering time by affecting the expression of flowering integrator genes such as FT and SOC1. High concentrations of auxin may delay flowering, while low concentrations may promote it (Yamaguchi et al., 2014). These data indicated that the function of MtDof32 was similar to that of some Dof family members, playing an important role in regulating plant flowering, and this process might involve different pathways.

The overexpression of MtDof32 in M. truncatula led to a significant reduction in branch number, corroborating our earlier findings in Arabidopsis thaliana (Guo et al., 2021). Notably, transcript levels of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and BRANCHED1 (BRC1) were markedly elevated in MtDof32-overexpressing plants. CCD7 encodes a carotenoid cleavage dioxygenase that catalyzes a key step in strigolactone (SL) biosynthesis, a phytohormone that suppresses lateral branching. BRC1, a TCP-family transcription factor, acts downstream of SL signaling to directly repress axillary bud outgrowth. Auxin affects the synthesis of SL by regulating the expression of CCD7, then indirectly regulate the expression of BRC1, and finally inhibit plant branching. Studies have shown that exogenous NAA or IAA could significantly upregulate the expression of CCD7, promote the synthesis of SL, and thus inhibit branching (del Rosario Cárdenas-Aquino et al., 2022; Chabikwa et al., 2019). Auxin also inhibits axillary bud outgrowth through transcriptional regulation of BRC1 (Yan et al., 2020). These results collectively suggested that MtDof32 might regulate shoot branching through the auxin and SL pathway by modulating CCD7 and BRC1 expression.

The findings of this study suggested that overexpression of MtDof32 in M. truncatula promoted cell expansion and regulated organ size, resulting in larger flowers and leaves. These results aligned with our previous observations in Arabidopsis (Guo et al., 2021). Additionally, yeast two-hybrid screening identified MtEBP1 (ERBB-3 BINDING PROTEIN 1) as a potential interacting partner of MtDof32 in M. truncatula. The physical interaction between MtDof32 and MtEBP1 was subsequently confirmed through yeast two-hybrid analysis. EBP1, a member of the peptidase M24 family, functions as an RNA-binding protein that regulates cellular growth and differentiation processes (Squatrito et al., 2006).

Previous studies have demonstrated that Arabidopsis EBP1 was involved in ribosome assembly and the inhibition of eIF2 phosphorylation, thereby influencing cell proliferation and growth (Eguchi et al., 2006; Squatrito et al., 2006). Research on Solanum tuberosum has shown that EBP1 promoted both cell proliferation and expansion during early organogenesis (Horváth et al., 2006). Heterologous expression of Hevea brasiliensis HbEBP1 in Arabidopsis resulted in enlarged organs through prolonged vegetative growth (Cheng et al., 2016). Importantly, auxin has been found to regulate EBP1 through cascade signaling, thereby controlling organ size (Magyar et al., 2012; Perrot-Rechenmann, 2010). Based on these findings, we hypothesize that MtDof32-MtEBP1 module regulates plant organ size through modulation of auxin signaling cascades (Figure 8). However, the precise regulatory mechanism by which this module influences cell size requires further investigation. Elucidating the function of this complex can significantly advance our knowledge to the molecular mechanisms of plant organ size regulation.

Figure 8

Figure 8. Proposed role of MtDof32-MtEBP1 in cell size regulation.

Furthermore, MtSAUR (Small Auxin Up RNA) and MtARF (Auxin Response Factor) are key components in the auxin signaling pathway. MtARF acts as a transcription factor that directly activates a suite of auxin-responsive genes, while MtSAUR proteins inhibit PP2C.D phosphatase activity, thereby activating plasma membrane H⁺-ATPases to induce cell wall acidification and loosening, which promotes cell expansion (Bao et al., 2024; Cancé et al., 2022). Their significant upregulation suggests that MtDof32 may enhance the output of auxin signaling, thereby driving cell wall loosening and cell enlargement. Expansion A (EXPA) is a class of non-enzymatic cell wall-loosening proteins that disrupt hydrogen bonds between cellulose microfibrils and matrix polysaccharides (such as xyloglucan), inducing cell wall “creep” and irreversible extension (Sun et al., 2021). The observed upregulation of MtEXPA in transgenic plants is consistent with previously reported mechanisms by which EXPAs promote cell expansion (Chen et al., 2021), indicating that MtEXPA likely acts as a direct executor of cell wall loosening, leading to increased cell size and organ enlargement. TOR (Target of Rapamycin) has been shown to regulate EBP1 expression levels, and overexpression of both EBP1 and TOR can impact plant organ size (Jamsheer et al., 2022). Taken together, these results suggest that MtDof32, as a transcriptional regulator, may directly or indirectly activate the expression of MtSAUR, MtARF, MtEXPA and MtTOR, thereby integrating hormonal signals with cell wall remodeling at the transcriptional level to promote cell expansion and organ growth.

Organ morphogenesis in plants requires the tight spatiotemporal coordination of cellular proliferation and expansion (Gonzalez et al., 2012). Existing research indicated that plant hormones played a pivotal role in regulating organ size. These hormones formed a complex signaling network that translated external or internal stimuli into developmental responses (Zhu et al., 2020). Among the diverse types of plant hormones, auxin, cytokinin, brassinosteroids, and gibberellins were particularly significant, each with distinct yet interconnected roles in modulating plant growth and development (Santner and Estelle, 2009). Auxin, in particular, was crucial for plant development, exerting multifaceted effects on organ size. Auxin regulated flowering time and branching patterns in plants by controlling the expression of flowering-related genes (such as the FT gene) and apical dominance (through polar transport and PIN protein-mediated signaling), respectively, while interacting with other hormones (such as GA, ethylene, and cytokinins) to coordinately regulate plant growth and development (Chandler and Werr, 2015; Cucinotta et al., 2021; Hu et al., 2022; Dubois et al., 2018). In this study, the altered flowering time (including key genes in the flowering pathway) and branching number observed in overexpressing MtDof32 plants might be attributed to the modulation of auxin signaling by the MtDof32-MtEBP1 module. This suggested that the MtDof32-MtEBP1 interaction could influence auxin-mediated signaling pathways, thereby impacting plant development.

The observed phenotype, characterized by significantly enlarged leaves yet reduced branch number and overall biomass in MtDof32-overexpressing plants, can be interpreted through the lens of source-sink balance and resource allocation. While MtDof32 promotes cell expansion, leading to larger individual leaves—potentially enhancing photosynthetic capacity (source strength)—it concurrently strongly upregulates key branching suppressors such as MtCCD7 and MtBRC1. This results in a severe reduction in sink number and capacity. We propose that the significantly diminished sink demand imposes a feedback inhibition on photosynthetic activity, ultimately constraining overall biomass accumulation despite the increase in leaf size. This illustrates a classic trade-off where the genetic enhancement of one organ comes at the cost of another, likely mediated through auxin signaling pathways involving MtEBP1, and potentially modulated by the TOR signaling network (Jamsheer et al., 2022).

From a practical perspective, MtDof32 may not serve as a universal “yield-increasing” gene for biomass crops but rather as a valuable “plant architecture shaping” tool. Its application potential lies in precision breeding strategies, such as improving leaf yield in vegetable crops (e.g., lettuce, tobacco) or developing compact, high-density planting ideotypes with reduced branching. Future efforts could explore tissue-specific promoters to spatially control its expression, thereby uncoupling its beneficial effect on leaf growth from its inhibitory effect on branching.

5 Conclusion

Based on the findings presented, we proposed that MtDof32 and MtEBP1 functioned as a protein complex to regulate cell size in M. truncatula, a process that likely involve plant hormone signaling pathways. However, as upstream regulatory factors, the specific downstream genes targeted by MtDof32 and MtEBP1, as well as the mechanisms underlying their regulation, remain to be elucidated. Further experimental studies are necessary to explore the roles of MtDof32 and MtEBP1 within the molecular networks that govern cell size and to uncover the precise regulatory patterns involved.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

Author contributions

TG: Methodology, Conceptualization, Writing – original draft. HW: Validation, Methodology, Writing – original draft. SW: Conceptualization, Validation, Writing – review & editing, Funding acquisition. LZ: Methodology, Writing – original draft, Validation. SS: Writing – review & editing, Resources.

Funding

The author(s) declare that financial support was received for the research and/or publication of this article. The program was supported by National Natural Science Foundation of China (32301479). Natural Science Foundation of Chongqing, China (CSTB2022NSCQ-BHX0744). The Scientific Research Project of Chongqing City Administration Bureau (No.CGK 2024-12).

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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The author(s) declare that no Generative AI was used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2025.1666846/full#supplementary-material

References

Alam, O., Khan, L. U., Khan, A., Salmen, S. H., Ansari, M. J., Mehwish, F., et al. (2024). Functional characterisation of Dof gene family and expression analysis under abiotic stresses and melatonin-mediated tolerance in pitaya (Selenicereus undatus). Funct. Plant Biol. 51, FP23269. doi: 10.1071/FP23269

PubMed Abstract | Crossref Full Text | Google Scholar

Bao, D. F., Chang, S. Q., Li, X. D., and Qi, Y. H. (2024). Advances in the study of auxin early response genes: Aux/IAA, GH3, and SAUR. Crop J. 12, 964–978. doi: 10.1016/j.cj.2024.06.011

Crossref Full Text | Google Scholar

Boccaccini, A., Santopolo, S., Capauto, D., Lorrai, R., Minutello, E., Belcram, K., et al. (2014). Independent and interactive effects of DOF affecting germination 1 (DAG1) and the Della proteins GA insensitive (GAI) and Repressor of ga1-3 (RGA) in embryo development and seed germination. BMC Plant Biol. 14, 200. doi: 10.1186/s12870-014-0200-z

PubMed Abstract | Crossref Full Text | Google Scholar

Cadart, C., Venkova, L., Recho, P., Lagomarsino, M. C., and Piel, M. (2019). The physics of cell-size regulation across timescales. Nat. Phys. 15, 993–1004. doi: 10.1038/s41567-019-0629-y

Crossref Full Text | Google Scholar

Cai, K. W., Xie, X. Y., Han, L., Chen, J. B., Zhang, J. W., Yuan, H. T., et al. (2024). Identification and functional analysis of the DOF gene family in Populus simonii: implications for development and stress response. Front. Plant Sci. 15. doi: 10.3389/fpls.2024.1412175

PubMed Abstract | Crossref Full Text | Google Scholar

Cancé, C., Martin-Arevalillo, R., Boubekeur, K., and Dumas, R. (2022). Auxin response factors are keys to the many auxin doors. New Phytol. 235, 402–419. doi: 10.1111/nph.18159

PubMed Abstract | Crossref Full Text | Google Scholar

Chabikwa, T. G., Brewer, P. B., and Beveridge, C. A. (2019). Initial bud outgrowth occurs independent of auxin flow from out of buds. Plant Physiol. 179, 55–65. doi: 10.1104/pp.18.00519

PubMed Abstract | Crossref Full Text | Google Scholar

Chandler, J. W. and Werr, W. (2015). Cytokinin-auxin crosstalk in cell type specification. Trends Plant Sci. 20, 291–300. doi: 10.1016/j.tplants.2015.02.003

PubMed Abstract | Crossref Full Text | Google Scholar

Chen, S. K., Ren, H. Y., Luo, Y. X., Feng, C. Z., and Li, H. F. (2021). Genome-wide identification of wheat (Triticum aestivum L.) expansin genes and functional characterization of TaEXPB1A. Environ. Exp. Bot. 182, 104307. doi: 10.1016/j.envexpbot.2020.104307

Crossref Full Text | Google Scholar

Cheng, H., Chen, X., Zhu, J. S., and Huang, H. S. (2016). Overexpression of a Hevea brasiliensis ErbB-3 Binding protein 1 gene increases drought tolerance and organ size in Arabidopsis. Front. Plant Sci. 7. doi: 10.3389/fpls.2016.01703

PubMed Abstract | Crossref Full Text | Google Scholar

Corrales, A., Nebauer, S. G., Carrillo, L., Fernández-Nohales, P., Marqués, J., Renau-Morata, B., et al. (2014). Characterization of tomato cycling Dof factors reveals conserved and new functions in the control of flowering time and abiotic stress responses. J. Exp. Bot. 65, 995–1012. doi: 10.1093/jxb/ert451

PubMed Abstract | Crossref Full Text | Google Scholar

Cucinotta, M., Cavalleri, A., Chandler, J. W., and Colombo, L. (2021). Auxin and flower development: a blossoming field. Cold Spring Harbor Perspect. Biol. 13, a039974. doi: 10.1101/cshperspect.a039974

PubMed Abstract | Crossref Full Text | Google Scholar

Da Silva, D. C., Da Silveira Falavigna, V., Fasoli, M., Buffon, V., Porto, D. D., Pappas, G. J., et al. (2016). Transcriptome analyses of the Dof-like gene family in grapevine reveal its involvement in berry, flower and seed development. Horticulture Res. 3, 16042. doi: 10.1038/hortres.2016.42

PubMed Abstract | Crossref Full Text | Google Scholar

Del Rosario Cárdenas-Aquino, M., Sarria-Guzmán, Y., and Martínez-Antonio, A. (2022). Isoprenoid and aromatic cytokinins in shoot branching. Plant Sci. 319, 111240. doi: 10.1016/j.plantsci.2022.111240

PubMed Abstract | Crossref Full Text | Google Scholar

Dubois, M., Van Den Broeck, L., and Inzé, D. (2018). The pivotal role of ethylene in plant growth. Trends Plant Sci. 23, 311–323. doi: 10.1016/j.tplants.2018.01.003

PubMed Abstract | Crossref Full Text | Google Scholar

Eguchi, S., Tokunaga, C., Hidayat, S., Oshiro, N., Yoshino, K. I., Kikkawa, U., et al. (2006). Different roles for the TOS and RAIP motifs of the translational regulator protein 4E-BP1 in the association with raptor and phosphorylation by mTOR in the regulation of cell size. Genes to Cells 7, 757–766. doi: 10.1111/j.1365-2443.2006.00977.x

PubMed Abstract | Crossref Full Text | Google Scholar

Gonzalez, N., Vanhaeren, H., and Inzé, D. (2012). Leaf size control: complex coordination of cell division and expansion. Trends Plant Sci. 17, 332–340. doi: 10.1016/j.tplants.2012.02.003

PubMed Abstract | Crossref Full Text | Google Scholar

Guo, T., Wang, S. M., Zhang, T. J., Xu, L. X., Li, Y. R. Z., Chao, Y. H., et al. (2021). Expression of the Medicago truncatula MtDof32 transcription factor regulates plant growth and enhances abiotic stress tolerances in transgenic Arabidopsis. Environ. Exp. Bot. 183, 104339. doi: 10.1016/j.envexpbot.2020.104339

Crossref Full Text | Google Scholar

Gupta, S., Malviya, N., Kushwaha, H., Nasim, J., Bisht, N. C., Singh, V. K., et al. (2015). Insights into structural and functional diversity of Dof (DNA binding with one finger) transcription factor. Planta 241, 549–562. doi: 10.1007/s00425-014-2239-3

PubMed Abstract | Crossref Full Text | Google Scholar

Hamacher, M., Malisch, C. S., Reinsch, T., Taube, F., and Loges, R. (2021). Evaluation of yield formation and nutritive value of forage legumes and herbs with potential for diverse grasslands due to their concentration in plant specialized metabolites. Eur. J. Agron. 128, 126307. doi: 10.1016/J.EJA.2021.126307

Crossref Full Text | Google Scholar

Hao, D. C., Chen, S. L., Osbourn, A., Kontogianni, V. G., Liu, L. W., and Jordán, M. J. (2015). Temporal transcriptome changes induced by methyl jasmonate in Salvia sclarea. Gene 558, 41–53. doi: 10.1016/j.gene.2014.12.043

PubMed Abstract | Crossref Full Text | Google Scholar

Horváth, B. M., Magyar, Z., Zhang, Y., Hamburger, A. W., Bakó, L., Visser, R. G., et al. (2006). EBP1 regulates organ size through cell growth and proliferation in plants. EMBO J. 25, 4909–4920. doi: 10.1038/sj.emboj.7601362

PubMed Abstract | Crossref Full Text | Google Scholar

Hu, J., Su, H. L., Cao, H., Wei, H. B., Fu, X. K., Jiang, X. M., et al. (2022). AUXIN RESPONSE FACTOR7 integrates gibberellin and auxin signaling via interactions between DELLA and AUX/IAA proteins to regulate cambial activity in poplar. Plant Cell 34, 2688–2707. doi: 10.1093/plcell/koac107

PubMed Abstract | Crossref Full Text | Google Scholar

Jamsheer, K. ,. M., Jindal, S., Sharma, M., Awasthi, P., S, S., Sharma, M., et al. (2022). A negative feedback loop of TOR signaling balances growth and stress-response trade-offs in plants. Cell Rep. (Cambridge) 39, 110631. doi: 10.1016/j.celrep.2022.110631

PubMed Abstract | Crossref Full Text | Google Scholar

Jin, X., Wang, Z. M., Ai, Q. Y., Li, X., Yang, J. W., Zhang, N., et al. (2024). DNA-binding with one finger (Dof) transcription factor gene family study reveals differential stress-responsive transcription factors in contrasting drought tolerance potato species. Int. J. Mol. Sci. 25, 3488. doi: 10.3390/ijms25063488

PubMed Abstract | Crossref Full Text | Google Scholar

Kang, H. G., Foley, R. C., Oñate Sánchez, L., Lin, C., and Singh, K. B. (2010). Target genes for OBP3, a Dof transcription factor, include novel basic helix-loop-helix domain proteins inducible by salicylic acid. Plant Journal: Cell Mol. Biol. 35, 362–372. doi: 10.1046/j.1365-313X.2003.01812.x

PubMed Abstract | Crossref Full Text | Google Scholar

Li, J., Zhang, Y. C., Xu, L., Wang, C. Y., Luo, Y., Feng, S., et al. (2022). Genome-wide identification of DNA binding with one finger (Dof) gene family in Tartary Buckwheat (Fagopyrum tataricum) and analysis of its expression pattern after exogenous hormone stimulation. Biol. (Basel Switzerland) 11, 173. doi: 10.3390/biology11020173

PubMed Abstract | Crossref Full Text | Google Scholar

Lohani, N., Babaei, S., Singh, M. B., and Bhalla, P. L. (2021). Genome-wide in silico identification and comparative analysis of dof gene family in Brassica napus. Plants (Basel) 10, 709. doi: 10.3390/plants10040709

PubMed Abstract | Crossref Full Text | Google Scholar

Magyar, Z., Horváth, B., Khan, S., Mohammed, B., Henriques, R., De Veylder, L., et al. (2012). Arabidopsis E2FA stimulates proliferation and endocycle separately through RBR-bound and RBR-free complexes. EMBO J. 31, 1480–1493. doi: 10.1038/emboj.2012.13

PubMed Abstract | Crossref Full Text | Google Scholar

Nomoto, Y., Takatsuka, H., Yamada, K., Suzuki, T., Suzuki, T., Huang, Y., et al. (2022). A hierarchical transcriptional network activates specific CDK inhibitors that regulate G2 to control cell size and number in Arabidopsis. Nat. Commun. 13, 1660. doi: 10.1038/s41467-022-29316-2

PubMed Abstract | Crossref Full Text | Google Scholar

Perrot-Rechenmann, C. (2010). Cellular responses to auxin: division versus expansion. Cold Spring Harbor Perspect. Biol. 2, a001446. doi: 10.1101/cshperspect.a001446

PubMed Abstract | Crossref Full Text | Google Scholar

Powell, A. E. and Lenhard, M. (2012). Control of organ size in plants. Curr. Biol. 22, R360–R367. doi: 10.1016/j.cub.2012.02.010

PubMed Abstract | Crossref Full Text | Google Scholar

Rueda-Romero, P., Barrero-Sicilia, C., Gómez-Cadenas, A., Carbonero, P., and Oñate-Sánchez, L. (2012). Arabidopsis thaliana DOF6 negatively affects germination in non-after-ripened seeds and interacts with TCP14. J. Exp. Bot. 63, 1937–1949. doi: 10.1093/jxb/err388

PubMed Abstract | Crossref Full Text | Google Scholar

Santner, A. and Estelle, M. (2009). Recent advances and emerging trends in plant hormone signalling. Nat. (London) 7250, 1071–1078. doi: 10.1038/nature08122

PubMed Abstract | Crossref Full Text | Google Scholar

Song, H. F., Ji, X. C., Wang, M. Y., Li, J., Wang, X., Meng, L. Y., et al. (2024). Genome-wide identification and expression analysis of the Dof gene family reveals their involvement in hormone response and abiotic stresses in sunflower (Helianthus annuus L.). Gene 910, 148336. doi: 10.1016/j.gene.2024.148336

PubMed Abstract | Crossref Full Text | Google Scholar

Squatrito, M., Mancino, M., Sala, L., and Draetta, G. F. (2006). Ebp1 is a dsRNA-binding protein associated with ribosomes that modulates eIF2α phosphorylation. Biochem. Biophys. Res. Commun. 3, 859–868. doi: 10.1016/j.bbrc.2006.03.205

PubMed Abstract | Crossref Full Text | Google Scholar

Sun, W. J., Yu, H. M., Liu, M. Y., Ma, Z. T., and Chen, H. (2021). Evolutionary research on the expansin protein family during the plant transition to land provides new insights into the development of Tartary buckwheat fruit. BMC Genomics 22, 252. doi: 10.1186/s12864-021-07562-w

PubMed Abstract | Crossref Full Text | Google Scholar

Sun, Y. Q., Zhang, Y. F., Jian, C. Y., Wang, T., Cao, G. L., Li, N. N., et al. (2025). Identification and functional analysis of the Dof transcription factor genes in sugar beet. J. Plant Res. 138, 105–117. doi: 10.1007/s10265-024-01588-3

PubMed Abstract | Crossref Full Text | Google Scholar

Wang, Z. M., Wang, Y., Tong, Q., Xu, G. Z., Xu, M. L., Li, H. Y., et al. (2021). Transcriptomic analysis of grapevine Dof transcription factor gene family in response to cold stress and functional analyses of the VaDof17d gene. Planta 253, 1–14. doi: 10.1007/s00425-021-03574-8

PubMed Abstract | Crossref Full Text | Google Scholar

Ward, J. M., Cufr, C. A., Denzel, M. A., and Neff, M. M. (2005). The Dof transcription factor OBP3 modulates phytochrome and cryptochrome signaling in Arabidopsis. Plant Cell 17, 475–485. doi: 10.1105/tpc.104.027722

PubMed Abstract | Crossref Full Text | Google Scholar

Yamaguchi, N., Wu, M., Winter, C. M., and Wagner, D. (2014). LEAFY and polar auxin transport coordinately regulate Arabidopsis flower development. Plants (Basel) 3, 251–265. doi: 10.3390/plants3020251

PubMed Abstract | Crossref Full Text | Google Scholar

Yan, Y. Y., Zhao, N., Tang, H. M., Gong, B., and Shi, Q. H. (2020). Shoot branching regulation and signaling. Plant Growth Regul. 92, 131–140. doi: 10.1007/s10725-020-00640-1

Crossref Full Text | Google Scholar

Yoo, S., Cho, Y., and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572. doi: 10.1038/nprot.2007.199

PubMed Abstract | Crossref Full Text | Google Scholar

Zhai, Z. F., Xiao, Y. Q., Wang, Y. Y., Sun, Y. T., Peng, X., Feng, C., et al. (2022). Abscisic acid-responsive transcription factors PavDof2/6/15 mediate fruit softening in sweet cherry. Plant Physiol. (Bethesda) 190, 2501–2518. doi: 10.1093/plphys/kiac440

PubMed Abstract | Crossref Full Text | Google Scholar

Zhang, L., Jiang, A., Thomson, G., Kerr-Phillips, M., Phan, C., Krueger, T., et al. (2019). Overexpression of Medicago MtCDFd1_1 causes delayed flowering in medicago via repression of MtFTa1 but not MtCO-Like genes. Front. Plant Sci. 10. doi: 10.3389/fpls.2019.01148

PubMed Abstract | Crossref Full Text | Google Scholar

Zheng, G. H., Hu, S. Q., Cheng, S. M., Wang, L. Y., Kan, L. J., Wang, Z. M., et al. (2023). Factor of DNA methylation 1 affects woodland strawberry plant stature and organ size via DNA methylation. Plant Physiol. (Bethesda) 191, 335–351. doi: 10.1093/plphys/kiac462

PubMed Abstract | Crossref Full Text | Google Scholar

Zheng, K. H., Lv, M. M., Qian, J. Y., Lian, Y. R., Liu, R. L., Huo, S. H., et al. (2024). Identification and characterization of the DOF gene family in phoebe bournei and its role in abiotic stress-drought, heat and light stress. Int. J. Mol. Sci. 25, 11147. doi: 10.3390/ijms252011147

PubMed Abstract | Crossref Full Text | Google Scholar

Zhu, M. Y., Chen, W. W., Mirabet, V., Hong, L. L., Bovio, S., Strauss, S., et al. (2020). Robust organ size requires robust timing of initiation orchestrated by focused auxin and cytokinin signalling. Nat. Plants 6, 686–698. doi: 10.1038/s41477-020-0666-7

PubMed Abstract | Crossref Full Text | Google Scholar