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.

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.

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.

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.