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. 2026 Jul 30;17:1905435. doi: 10.3389/fpls.2026.1905435

MdMYB41L mediates stamen development and anthocyanin biosynthesis in apple

Kuo Yang 1, Ruihong Sun 1, Yanfu Pan 1, Zhudong Xing 1, Yanyi Zhang 1, Hengjia Zhang 1,*, Chongyang Li 1,*
PMCID: PMC13468913  PMID: 42597613

Abstract

MYB proteins play crucial roles in plant growth and development. In this study, we characterized MdMYB41L, a MYB transcription factor from apple (Malus domestica). Expression analysis revealed that MdMYB41L was highly expressed in leaves and anthers, and the protein was exclusively localized to the nucleus. Overexpression of MdMYB41L in Arabidopsis caused shorter siliques, anther indehiscence, and a lower seed setting rate. Artificial pollination analysis demonstrated that the stamen developmental defect resulting from MdMYB41L overexpression led to decreased fertility. The seeds from transgenic lines showed a dramatic increase in length, width, area, and weight. Overexpression of MdMYB41L suppresses anthocyanin accumulation through downregulating the expression of anthocyanin biosynthesis‐related genes. Collectively, our findings highlight the important role of MdMYB41L in regulating stamen development and anthocyanin biosynthesis.

Keywords: anthocyanin, apple, MdMYB41L, seed development, stamen developmental

1. Introduction

In seed plants, upon deposition on the stigma, pollen grains undergo adhesion, hydration, and germination, followed by growth through the style prior to fertilization. Anthers are key components of stamens. Anther dehiscence and filament elongation represent the terminal phases of anther development (Zúñiga-Mayo et al., 2023). During the mature pollen stage, the septum degenerates, causing the anther to become bilocular, and the stomium subsequently ruptures to facilitate pollen release (Wilson et al., 2011). If any physiological process of stamen elongation, pollen maturation, or anther dehiscence is disrupted, it can lead to male sterility in plants (Dai et al., 2019; Åstrand et al., 2021; He et al., 2023). Numerous genes have been demonstrated to participate in the regulation of stamen development (Marciniak and Przedniczek, 2019; Su et al., 2023; Sui et al., 2023; Wiese et al., 2024; Li et al., 2025).

MYB transcription factors (TFs) constitute one of the largest transcription factor families in plants, with members distributed across diverse species. MYB proteins are characterized by a conserved MYB domain, among which the R2R3-MYB type represents the most extensively studied subfamily (Wu et al., 2024). MYB TFs are extensively involved in various biological processes, including plant growth, development, stress responses, and secondary metabolism (Dubos et al., 2010; Wu et al., 2025; Zhang et al., 2025). Notably, MYB proteins have been demonstrated to play critical roles in plant fertility. In Arabidopsis, myb108 myb24 double mutant exhibits reduced pollen tube elongation, accompanied by lower pollen viability and germination, leading to decreased fertility (Mandaokar and Browse, 2009). MYB transcription factors regulate anther development in plants. In pepper, silencing of CaMYB108 led to delayed anther dehiscence and decreased pollen viability (Sun et al., 2019). The myb21 myb24 double mutant of Arabidopsis displays abnormalities in pollen maturation, anther dehiscence, and filament elongation, which collectively cause male sterility (Song et al., 2011). MYB21 and MYB24 participate in jasmonate-mediated stamen development through the bHLH-MYB complexes (Chen et al., 2016; Huang et al., 2017). Accordingly, it is essential to identify MYB proteins implicated in stamen development. In addition to their functions in reproductive development, MYB proteins are also key regulators of plant secondary metabolism. Anthocyanin biosynthesis is a major branch of the phenylpropanoid pathway, which is catalyzed by enzymes encoded by structural genes such as CHS, CHI, F3H, DFR, ANS, and UFGT (Kuang et al., 2025). MYB proteins often assemble with bHLH and WD40 proteins into the MBW complex, which synergistically activates or represses the expression of structural genes, thereby fine-tuning anthocyanin accumulation (Liu et al., 2021; Muhammad et al., 2024; Sharma et al., 2024; Zhao et al., 2025; He et al., 2026). The MYB protein FaMYB5 positively regulates anthocyanin and proanthocyanidin biosynthesis by directly activating the transcription of F3’H and LAR in strawberry. FaMYB5 forms an MBW complex with FaEGL3 and FaLWD1/FaLWD1-like to modulate flavonoid metabolism (Jiang et al., 2023). Under low nitrate conditions, MsMYB62-like suppresses the expression of MsF3’H, thereby negatively regulating anthocyanin biosynthesis in Malus spectabilis (Meng et al., 2023). In apple, MdMYB73 negatively regulates anthocyanin biosynthesis through suppressing MdUFGT. Furthermore, MdMYB73 competes with MdMYB1, a positive regulator of anthocyanin biosynthesis, for binding to the MdUFGT promoter, thus exerting an inhibitory effect on anthocyanin accumulation (Zhang et al., 2026).

Apple is rich in mineral elements, vitamins, and anthocyanins, which make it popular among consumers. Here, we isolated and characterized MdMYB41L, an MYB gene from apple. We found that MdMYB41L was highly expressed in anthers. MdMYB41L-overexpressing Arabidopsis exhibited significantly reduced fertility and produced larger seeds. Artificial pollination experiments confirmed that impaired pollination was the primary cause of the seed phenotype observed in these transgenic lines. Furthermore, overexpression of MdMYB41L in apple calli decreased anthocyanin accumulation and suppressed the expression of anthocyanin biosynthetic genes. Collectively, our findings demonstrate that MdMYB41L regulates stamen development and anthocyanin biosynthesis.

2. Materials and Methods

2.1. Plant materials and growth conditions

Arabidopsis thaliana plants were grown in the greenhouse under long day conditions (16 h light/8 h dark) at 22°C. The Columbia‐0 ecotype was used as the wild-type Arabidopsis. Arabidopsis seeds were surface-sterilized with 75% ethanol and 2.6% sodium hypochlorite, placed at 4 °C for 3 days. They were then sown on 1/2MS medium. The apple calli ‘Orin’ were cultured on MS medium supplemented with 1.5 mg/L 2,4-D and 0.4 mg/L 6-BA at 22 °C in the dark, and subcultured every two weeks (Yang et al., 2021a).

2.2. Vector construction and plant transformation

The full-length cDNA of MdMYB41L was inserted into the pRI-101 plasmid to generate the overexpression vector. The MdMYB41L overexpression vector was transformed into Agrobacterium tumefaciens strain EHA105, which was then introduced into apple calli (Yang et al., 2021b). In order to generate transgenic lines, we transferred overexpression vector into Agrobacterium tumefaciens strain GV3101 and used an Agrobacterium-mediated method to transform Arabidopsis. All phenotypic analyses of the transgenic Arabidopsis were conducted using homozygous T3 generation lines. The primers used in the study are listed in Supplementary Table 1.

2.3. Seed size measurement.

We observed and captured images of mature dry seeds under a stereomicroscope (SZX16, Olympus). The ImageJ 1.54g software was used to measure seed length, width, and area. Arabidopsis seeds were weighed by analytical microbalance.

2.4. Differential interference contrast (DIC) microscopy

Immature seeds extracted from siliques were fixed at 4°C in Hoyer’s solution (chloral hydrate : glycerol : water = 8 : 1 : 3) for DIC observations. A Nicon digital sight 10 cameras were used to capture images. ImageJ software was used to measure ovule size and embryo size from the DIC images.

2.5. Gene expression analysis

Total RNA was extracted using RNA plant extraction kits (Tiangen, Beijing, China). The cDNA was synthesized by reverse transcription kit (TaKaRa, Dalian, China). Quantitative real-time PCR (qRT-PCR) was performed on a real-time PCR system using SYBR Green reagents (Vazyme, Nanjing, China) with cDNA as the template. The MdActin was used as an internal control. Relative expression levels were calculated using the 2−ΔΔCt method (Wang et al., 2020; Sui et al., 2023).

2.6. Subcellular localization analysis

The full-length CDS of MdMYB41L was fused into the pPZP211-GFP vector to obtain 35S::MdMYB41L-GFP. The recombinant plasmid was introduced into A. tumefaciens strain GV3101. Then, they were injected into N. benthamiana leaves and incubated in the dark for 3 days. Fluorescence signals were captured using a confocal laser‐scanning microscope (FV1200, Olympus, Japan).

2.7. GUS staining

The 1592 bp promoter fragment of MdMYB41L was inserted into the pCAMBIA1391 vector to obtain the ProMdMYB41L::GUS. The recombinant plasmid was transformed into Arabidopsis. Leaves, stems, roots, and flowers were stained using a GUS staining kit (Solarbio, Beijing, China) according to the manufacturer’s instructions (Yang et al., 2021b).

2.8. Determination of anthocyanin contents

Total anthocyanins were extracted using the ethanol-HCl method. Apple calli were immersed in an extraction buffer (95% anhydrous ethanol mixed with 1.5 M HCl) at room temperature for 24 h. The absorbance of the extract was measured at 530, 620, and 650 nm using a spectrophotometer, and the anthocyanin content was calculated according to described methods (An et al., 2018).

3. Results

3.1. Bioinformatics characterization of the MdMYB41L in apple

MYB TFs are known to be extensively involved in plant development. Here, we isolated a MYB gene, designated MdMYB41L, from apple. MdMYB41L contains a 1077 bp open reading frame and encodes a protein of 358 amino acids. In addition, MdMYB41L has a theoretical pI of 6.37, a molecular weight of 39,675.76 Da, and a grand average of hydropathicity (GRAVY) of -0.488. Sequence alignment revealed that MdMYB41L protein shares 96.09% sequence identity with a MYB protein from Pyrus bretschneideri. We further compared the amino acid sequences of MdMYB41L with those from six other plant species. Consistent with other MYB proteins, MdMYB41L possesses two conserved MYB domains at its N-terminus (Figure 1).

Figure 1.

Multiple sequence alignment graphic of seven MYB41L proteins from different plant species, with R2 and R3 DNA-binding domains labeled at the top. Residues are color-coded by conservation level, and sequence positions are indicated at right.

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Amino acid sequence alignment of MYB proteins. The two conserved MYB domains (R2-domain and R3-domain) of MYB proteins are marked with black lines. MdMYB41L: MD06G1037300 Malus domestica; PbMYB41L: XP_009371224.2 Pyrus bretschneideri; RcMYB41L: XP_024173796.1 Rosa chinensis; CmMYB41L: KAF3964320.1 Castanea mollissima; VvMYB41L: CBI33861.3 Vitis vinifera; GhMYB41L: XP_016721289.1 Gossypium hirsutum; AtMYB41L: AT4G28110 Arabidopsis thaliana.

3.2. Expression pattern analysis and subcellular localization of MdMYB41L

MYB proteins are widely distributed across plant species (Li et al., 2019; Wang et al., 2021). We explored the tissue expression pattern of MdMYB41L through GUS staining. The promoter fragment of MdMYB41L was fused into the pCAMBIA1300::GUS vector and transformed into Arabidopsis to obtain ProMdMYB41L::GUS transgenic Arabidopsis. As shown in Figure 2A, the GUS staining displayed that MdMYB41L was expressed in roots, stems, leaves, and flowers, with the highest expression in leaves and anthers. To determine the subcellular localization of MdMYB41L, we generated 35S::MdMYB41L-GFP recombinant plasmid. The recombinant construct was introduced into A. tumefaciens strain GV3101, and then was injected into tobacco leaves. After 3 days, the GFP fluorescence of MdMYB41L was detected using a confocal laser‐scanning microscope. As shown in Figure 2B, MdMYB41L was specifically localized in the nucleus.

Figure 2.

Panel A presents four close-up images of Arabidopsis plant tissues showing blue GUS staining in seedling, stem with trichomes, flower, and root hair, each with scale bars. Panel B displays four microscopy images of cells stained with DAPI (blue), showing GFP fluorescence (green), bright field, and merged images to indicate colocalization, with each containing a single fluorescent nucleus.

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Expression pattern and subcellular localization of MdMYB41L. (A) GUS staining of ProMdMYB41L::GUS transgenic Arabidopsis in roots, stems, leaves and flowers. (B) Subcellular localization of MdMYB41L protein. DAPI dye specific staining of cell nuclei. Bright represents a bright field. Scale bar: 10 μm.

3.3. Overexpression of MdMYB41L alters silique development

To investigate the function of MdMYB41L, we transformed MdMYB41L into Arabidopsis. As a result, three stable overexpression lines (OE1, OE2, and OE3) were generated. The MdMYB41L-OE lines were confirmed at the transcriptional and DNA levels (Figures 3A, B). Through phenotype analysis, we found no prominent difference in plant height between wild-type (WT) and MdMYB41L-OE lines under normal growth conditions. However, we found that siliques development in the MdMYB41L-OE lines was defective, with many aborted siliques and significantly shorter siliques than those of WT (Figures 3C–E).

Figure 3.

Panel A shows a bar chart comparing relative expression levels among WT, OE1, OE2, and OE3, with WT lowest and OE2, OE3 highest. Panel B showsgel electrophoresis bands for WT, OE1, OE2, OE3, and a positive control, indicating successful amplification in the OEs and control. Panel C displays four whole stems labeled WT, OE1, OE2, and OE3 from left to right, with WT appearing more robust. Panel D contains close-up images of seed pods from WT, OE1, OE2, and OE3, with WT pods visibly longer. Panel E presents a box plot of pod length in centimeters for each group,showing WT significantly longer than others, with statistical groupings indicated.

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MdMYB41L affects siliques development. (A) qRT-PCR detected the expression level of MdMYB41L in WT and MdMYB41L-OE. (B) The MdMYB41L-OE lines were identified through PCR amplification. (C) Main inflorescences of WT and MdMYB41L-OE lines. Bars: 1 cm. (D, E) Phenotype and length of siliques in WT and MdMYB41L-OE. Bars: 0.5 cm. For (E) error bars indicate SD (n=40). different lowercase letters represent significant differences by one-way ANOVA, followed by Tukey’s test (p < 0.05). All data were obtained from three independent biological replicates.

3.4. Overexpression of MdMYB41L reduces the fertility in Arabidopsis

We observed that, within the same inflorescence, most flowers either failed to set seeds or set only a few, whereas a small proportion developed normally into siliques. Furthermore, MdMYB41L-OE lines exhibited significant reduced fertility compared with WT (Figure 4A). The silique seed set rate in MdMYB41L-OE lines was notably lower than that of WT (Figure 4B). In addition, seed yield per plant was markedly decreased in the OE lines compared to WT (Figure 4C). These results indicate that MdMYB41L negatively regulates fertility in Arabidopsis.

Figure 4.

Panel A displays seed pods with the wild type (WT) showing more seeds compared to three overexpression lines (OE1, OE2, OE3). Panel B shows a bar graph with WT having a higher seed setting rate percentage than OE lines. Panel C presents a bar graph with WT having a greater seed yield per plant in milligrams compared to the OE lines.

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Overexpression of MdMYB41L reduces fertility in Arabidopsis. (A) Dissected silique phenotypes of Arabidopsis. Bar: 1 mm. (B) Seed setting rate in WT and MdMYB41L-OE lines (n=100). (C) Seed yield per plant of WT and MdMYB41L-OE lines (n=30). For B, C, different lowercase letters represent significant differences by one-way ANOVA, followed by Tukey’s test (p < 0.05). All data were obtained from three independent biological replicates.

To investigate the mechanism by which MdMYB41L affects fertility, we observed the anther morphology of WT and MdMYB41L-OE. As shown in Figures 5, MdMYB41L-OE flowers exhibited anther indehiscence, with only a minority of anthers showing normal dehiscence (Figures 5A, B). Subsequently, cross-pollination assays were conducted between WT and the MdMYB41L-OE1 line. When MdMYB41L-OE1 was used as the female parent and WT as the male parent, the silique seed set rate showed no notable difference compared with that of WT, indicating that female gametophyte function in MdMYB41L-OE1 remained normal (Figure 6). These results suggest that the reduced fertility of MdMYB41L-OE is largely attributable to defective anther dehiscence.

Figure 5.

Panel A shows close-up images of Arabidopsis flowers from wild type (WT) and three overexpression lines (OE1, OE2, OE3), highlighting differences in anther and petal morphology. Panel B displays enlarged images of single anthers from WT and the same overexpression lines, illustrating differences in pollen release and anther surface characteristics.

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Overexpression of MdMYB41L affects the stamens development. (A). Comparison of flowers. Bar: 1 mm. (B) Comparison of anther in WT and MdMYB41L-OE plants. WT showed obvious anther dehiscence. Bar: 200 μm.

Figure 6.

Bar chart comparing seed setting rate percentages for two groups: wild-type male crossed with wild-type female, and wild-type male crossed with MdMYB41L-OE female. Both groups show similar values near 100 percent with a non-significant difference indicated by “ns”.

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Crosses between the WT and the MdMYB41L-OE1 plants. The siliques are collected at 15 days after pollination. Statistical analysis of silique seed set. Student’s t test was performed (p < 0.05; n=20). ns, not significant.

3.5. Overexpression of MdMYB41L increases the seed size

MYB proteins function as established regulators of seed size in plants (Zhang et al., 2023; Wang et al., 2024). In this study, we found that overexpression of MdMYB41L not only led to decreased fertility, but also exhibited significantly enlarged seeds and mature embryos (Figures 7A, B). Statistical analysis showed that the length, width, area, and weight of seeds of MdMYB41L-OE lines were dramatically greater than those of WT (Figures 7D–G). Similarly, we observed that MdMYB41L-OE cotyledons were markedly larger than those of WT at 9 days post-germination (Figures 7C, H).

Figure 7.

Panel A shows a top view of seeds from wild type (WT) and three overexpression lines (OE1, OE2, OE3); Panel B displays close-up views of individual seeds for each line; Panel C presents cotyledons from seedlings of WT and overexpression lines; Panels D-H are bar graphs comparing seed length, seed width, seed area, seed weight, and relative cotyledon area among these genotypes, with significant increases observed in the overexpression lines compared to wild type as indicated by different letters.

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MdMYB41L affects seed size in Arabidopsis. (A) Seed phenotype of WT and MdMYB41L-OE lines. Bar=0.2 mm. (B) Mature embryo phenotype of WT and MdMYB41L-OE lines. Bar=0.2 mm. (C) The 9-d-old seedlings of WT and MdMYB41L-OE lines. Bar=0.2 mm. (D–F) the length (D), width (E), area (F) of WT and MdMYB41L-OE lines (n=200). (G) 500-seed weight of WT and MdMYB41L-OE lines (n=3). (H) Relative cotyledon area of WT and MdMYB41L-OE lines (n=20). The WT was set to 1. For (B–H), different lowercase letters represent significant differences by one-way ANOVA, followed by Tukey’s test (p < 0.05). All data were obtained from three independent biological replicates.

3.6. Overexpression of MdMYB41L affects the seed development

To elucidate the role of MdMYB41L in seed development, we performed cytological analyses on seeds collected at different days after pollination (DAP). Seeds at 3, 6, and 8 DAP were isolated from the siliques of WT and MdMYB41L-OE1, and were fixed in Hoyer’s solution. As shown in Figure 8, embryo development proceeded similarly in WT and MdMYB41L-OE1 from 3 to 8 DAP. After the globular stage (3 DAP), their differences in seed cavity became increasingly pronounced (Figure 8A). At both the torpedo and bent cotyledon stages, the embryo and ovule sizes in MdMYB41L-OE1 were significantly greater than those in WT (Figures 8B, C). Accordingly, these results suggest that the increased seed size of MdMYB41L-OE lines is caused by embryo enlargement.

Figure 8.

Panel A shows microscope images comparing ovule and embryo development at 3, 6, and 8 days after fertilization in WT and OE samples. Panel B is a bar graph displaying ovule size over time for WT and OE, with OE showing significantly larger ovules at 6 and 8 days. Panel C presents embryo size for WT and OE over time, with OE exhibiting significantly larger embryos at 6 and 8 days.

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Seed development of WT and MdMYB41L-OE. (A) DIC microscopy of WT and MdMYB41L-OE lines from 3 DAP, 6 DAP, and 8 DAP. Scale bars: 50 µm. (B, C) The ovule size (B) and embryo size (C) of WT and MdMYB41L-OE. Asterisks represent significant differences (p< 0.05, Student’s t test).

3.7. MdMYB41L negatively regulates anthocyanin accumulation

MYB proteins play critical roles in regulating anthocyanin biosynthesis across various plant species (Yang et al., 2022). To investigate the function of MdMYB41L in anthocyanin biosynthesis, we generated two MdMYB41L-overexpressing apple calli lines (OE1 and OE2) (Figure 9B). Compared with WT, both MdMYB41L-OE transgenic calli lines exhibited lower anthocyanin content (Figures 9A, C). Consistent with anthocyanin phenotype, qRT-PCR analysis revealed that the transcript levels of anthocyanin biosynthesis-related genes MdDFR, MdANS, and MdF3H were significantly downregulated in MdMYB41L-OE calli (Figure 9D). Collectively, these results demonstrate that MdMYB41L functions as a negative regulator of anthocyanin accumulation in apple.

Figure 9.

Panel A shows three tissue samples labeled WT, OE1, and OE2, with WT exhibiting a deeper red color than OE1 and OE2. Panel B presents a bar graph of MdMYB41L expression levels, with WT lowest, OE1 intermediate, and OE2 highest. Panel C displays a bar graph of anthocyanin content, showing WT highest, OE1 intermediate, and OE2 lowest. Panel D is a grouped bar graph comparing MdDFR, MdANS, and MdF3H gene expression in WT, OE1, and OE2, with WT consistently higher than the two overexpression lines.

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MdMYB41L inhibits anthocyanin accumulation. (A) Phenotype of anthocyanin accumulation. The 15-day-old WT and MdMYB41L-OE apple calli were maintained in a plant greenhouse at 22 °C under light conditions (photon flux density of approximately 70 μmol·m−2·s−1) for 10 days. (B) The expression of MdMYB41L in apple calli. (C) The anthocyanin content. (D) The expression of anthocyanin biosynthesis‐related genes (MdDFR, MdANS, and MdF3H). MdActin acted as the reference. For B-D, different lowercase letters represent significant differences by one-way ANOVA, followed by Tukey’s test (p < 0.05). All data were obtained from three independent biological replicates.

4. Discussion

The normal dehiscence of anthers and the timely release of pollen are closely related to plants fertility (Åstrand et al., 2021). MYB proteins are involved in plant reproductive processes, such as anther development, anther dehiscence, and tapetum development (Zhang et al., 2025). Here, we identified an R2R3 MYB gene, MdMYB41L, from apple. We demonstrated that overexpression of MdMYB41L led to anther indehiscence in Arabidopsis (Figure 5), thereby reducing the seed setting rate. Sorbitol participates in the processes of stamen development, pollen germination, and pollen tube growth through the apple R2R3 MYB transcription factor MdMYB39L (Meng et al., 2018; Li et al., 2020). Given the close phylogenetic relationship between MdMYB41L and MdMYB39L, MdMYB41L may exert similar functions in reproductive development.

Overexpression of MdMYB41L in Arabidopsis resulted in a similar number of ovules as in WT; however, not all ovules developed into seeds, leading to a significant reduction in fertility in the MdMYB41L transgenic Arabidopsis. Artificial crossing experiments showed that female gametophyte function was normal in transgenic Arabidopsis (Figure 6). Taken together with the observed anther indehiscence phenotype, these findings demonstrate that the reduced fertility of MdMYB41L-OE lines is primarily attributable to defect in stamen development.

The Arabidopsis MYB26 regulates secondary thickening by altering the expression of NST1 and NST2, and its overexpression causes ectopic secondary thickening and anther dehiscence failure (Yang et al., 2007). Similarly, ectopic expression of the Brassica campestris R2R3-MYB gene BcMF28 in Arabidopsis leads to shortened filaments, indehiscent anthers, and aborted pollen, due to the non-degeneration of the septum and stomium during late anther development (Shen et al., 2019). In eggplant, overexpression of SmMYB108 promotes early anther dehiscence. The anther development-related genes (SmMYB21, SmARF6, and SmARF8) directly activate SmMYB108 transcription (Hu et al., 2023). The specific pathway through which MdMYB41L regulates anther development requires further investigation.

Interestingly, MdMYB41L-OE Arabidopsis seeds were larger than those of the WT (Figure 7). Seed size is determined by the coordinated development of the seed coat, endosperm, and embryo, as well as on genetic, environmental, and physiological factors (Doughty et al., 2014; Gnan et al., 2014; Orozco-Arroyo et al., 2015; Zhang et al., 2023). Given that artificial cross-pollination of MdMYB41L-OE plants restored the WT seed size phenotype, the increased seed size in MdMYB41L-OE transgenic lines is likely due to reduced seed set. In MdMYB41L-OE plants, nutrients allocated to flowers are distributed among fewer developing seeds, which may consequently accumulate more storage compounds (Herridge et al., 2011; Rolletschek et al., 2021; Wang et al., 2023; Li et al., 2026). Similar observations have been reported in other genes and species (Vergès et al., 2021). Nevertheless, an alternative possibility cannot be excluded. Numerous studies have demonstrated that MYB transcription factors can directly regulate seed size (Zhang et al., 2025). Therefore, it is plausible that MdMYB41L may directly influence seed development, although this hypothesis requires further validation.

MYB TFs play indispensable roles in plant secondary metabolism (Zhang et al., 2025). Overexpression of MdMYB41L reduced anthocyanin accumulation in apple calli. Consistently, qRT-PCR analysis revealed that the transcript levels of MdDFR, MdF3H, and MdANS were downregulated in MdMYB41L-OE apple calli (Figure 9), suggesting that MdMYB41L may suppress anthocyanin biosynthesis by suppressing the expression of anthocyanin synthesis related genes. MdMYB1 and its alleles act as positive regulators of anthocyanin biosynthesis by activating MdDFR and MdUF3GT to promote anthocyanin biosynthesis (Takos et al., 2006; An et al., 2020). MYB proteins that negatively regulate anthocyanin biosynthesis have also been identified in various plant species (Li et al., 2022). MdMYB6 inhibited anthocyanin accumulation and downregulated the expression of anthocyanin biosynthesis-related genes (Wang et al., 2018). Overexpression of MdMYBL2 decreased anthocyanin accumulation in apple calli and inhibited the transcript levels of MdDFR, MdUFGT, MdMYB10, and MdbHLH3 (Gao et al., 2011).

5. Conclusion

In this study, we identified and characterized MdMYB41L, an R2R3 MYB TF from apple. Overexpression of MdMYB41L leads to anther indehiscence, thereby reducing plant fertility. In addition, MdMYB41L negatively regulates anthocyanin accumulation in apple calli. Our findings offer novel insights into the regulatory mechanisms underlying MYB transcription factor-mediated stamen development and anthocyanin biosynthesis in plants and will contribute to future studies aimed at deciphering and utilizing this regulatory network.

Funding Statement

The author(s) declared that financial support was received for this work and/or its publication. This research was funded by National Natural Science Foundation of China (32402530) and Natural Science Foundation of Shandong Province, China (ZR2023QC035 and ZR2023QC110).

Footnotes

Edited by: Daqiu Zhao, Yangzhou University, Yangzhou, China

Reviewed by: Jianling Zhang, Chongqing University, Chongqing, China

Dan-Dan Liu, Yunnan University, Kunming, China

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/s.

Author contributions

KY: Conceptualization, Data curation, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. RS: Investigation, Methodology, Writing – original draft, Writing –review & editing. YP: Investigation, Methodology, Writing – original draft, Writing – review & editing. ZX: Resources,Writing – review & editing. YZ: Resources, Writing – review & editing. HZ: Funding acquisition, Resources, Supervision, Writing – review & editing. CL: Conceptualization, Resources, Supervision, Project administration, Writing – riginal draft, Writing – review & editing.

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.

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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.2026.1905435/full#supplementary-material

Table1.pdf (97.6KB, pdf)

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