BrMAF5 and Its Antisense lncRNA BrMAF5L Regulate Vernalization-Induced Bolting and Flowering in Chinese Cabbage

Abstract

Bolting and flowering time are critical agronomic traits affecting the commercial value and breeding efficiency of Chinese cabbage (Brassica rapa L. ssp. pekinensis). Although vernalization is a key environmental signal promoting flowering, its regulatory mechanisms remain poorly understood in this crop. Here, we identify the flowering repressor gene BrMAF5 and its antisense long non-coding RNA BrMAF5L as negative regulators of vernalization-induced flowering. During vernalization, transcript levels of both genes showed a decreasing trend as the vernalization period extended. Functional assays in Arabidopsis thaliana demonstrated that ectopic expression of BrMAF5 or BrMAF5L significantly delayed flowering, accompanied by increased expression of floral repressors (AtFLC, AtTEM1) and reduced expression of floral activators (AtFT, AtSOC1). Moreover, protein interaction analysis revealed that BrMAF5 associates with BrACP4 and BrRAB1A, linking it to fatty acid metabolism and membrane trafficking pathways. Collectively, our results reveal a novel regulatory module in vernalization-mediated flowering. These findings pave the way for developing bolting-resistant Brassicaceae crops by identifying promising molecular targets.

1. Introduction

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an important vegetable crop in China. As a typical biennial plant, it requires prolonged cold exposure (vernalization) to induce flowering. However, in commercial production, premature bolting significantly reduces market value and causes substantial economic losses for farmers [1], while accelerated bolting facilitates breeding cycle shortening. Therefore, elucidating the molecular mechanism of vernalization-mediated flowering regulation in Chinese cabbage is of particular importance. Flowering is a complex developmental process regulated by multiple internal and external factors [2]. In Arabidopsis, several flowering regulatory pathways have been identified, including the photoperiod, vernalization, autonomous, gibberellin, age, and ambient temperature pathways [3]. These pathways ultimately converge on floral integrators such as FT and SOC1, which activate floral meristem identity genes like LFY and AP1 to initiate flowering [4]. Among these, the vernalization pathway plays a critical role in overwintering crops. In Arabidopsis, vernalization epigenetically represses the flowering inhibitor FLC through PRC2-mediated trimethylation of histone H3K27me3 [5], with participation of auxiliary factors like (VIN3) and (VRN2) [6].

As a close relative of Arabidopsis, Chinese cabbage exhibits both conserved and unique flowering regulatory networks [7]. Due to whole-genome triplication events, the Chinese cabbage genome contains multiple FLC gene copies (BrFLC1–BrFLC5) with divergent expression patterns and functions [8]. For instance, natural variation in BrFLC2 and BrFLC3 is significantly associated with flowering time, and a transposon insertion in its first intron may lead to late-flowering phenotypes [9]. Beyond FLC, the MAF (MADS AFFECTING FLOWERING) subfamily of MADS-box genes also plays important roles in vernalization responses. In Arabidopsis, MAF genes (e.g., MAF2–5) act as paralogs of FLC, enhancing the robustness of flowering repression through functional redundancy [10]. Two DEK proteins (DEK3 and DEK4) regulate flowering time by controlling MAF5 expression [11], while root-specific activation of novel targets MAF4 and MAF5 can delay flowering through FRI expression [12]. Epigenetically, the plant-specific CK1 member MLK4 accelerates flowering by suppressing FLC/MAF transcription via H3T3 phosphorylation, providing a new paradigm for precise developmental regulation through epigenetic modifications [13]. However, the functions of MAF genes in Chinese cabbage remain unclear.

The suppression of the expression of the core flowering repressor, the FLC gene, is a critical step in vernalization-induced flowering [14]. Long non-coding RNAs (lncRNAs) have emerged as important epigenetic regulators in plant flowering control. In Arabidopsis, antisense lncRNAs COOLAIR and COLDAIR transcribed from the FLC locus recruit chromatin-modifying complexes to repress FLC expression [15,16]. Three COOLAIR-like lncRNAs (lncFLC1, lncFLC2a, and lncFLC2b) have been characterized in Chinese cabbage; their overexpression significantly downregulates FLC while activating flowering promoters FT and SOC1 [17]. Transcriptome sequencing further identified BrMAF5 and its antisense lncRNA BrMAF5L [18]. These findings demonstrate that lncRNAs play crucial roles in flowering regulation. However, the molecular functions of BrMAF5 and its associated lncRNA BrMAF5L remain to be experimentally validated.

Based on the established research background of vernalization in Brassicaceae species and our prior RNA-seq analysis of the vernalization pathway in Chinese cabbage, this study proposes the following central hypothesis: the flowering repressor BrMAF5 (BraA02g044940.3C) may function in concert with its genomically adjacent long non-coding RNA BrMAF5L (MSTRG.6639.1), with each regulating the vernalization response and the bolting–flowering process in Chinese cabbage. To test this hypothesis, we systematically characterized the molecular functions of BrMAF5 and BrMAF5L using the bolting-resistant doubled haploid (DH) line B24108C48 as experimental material, and elucidated their regulatory roles in the vernalization-mediated flowering pathway. Our findings provide novel insights into the flowering regulatory network of Chinese cabbage and identify potential molecular targets for the genetic improvement of bolting resistance.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

The plant material used in this study was the vernalization-requiring Chinese cabbage DH line ‘B24108C48’, maintained by our research group and provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, China. We conducted both germination and ovule vernalization treatments on ‘B24108C48’. Approximately 300 uniformly healthy seeds were carefully selected, washed with sterile water, and placed in Petri dishes lined with two layers of filter paper. The experiment was arranged in six vernalization treatments (0, 5, 10, 15, 20, and 25 days), with three biological replicates per treatment, each comprising an independent lot of approximately 50 seeds. The seeds were placed in a climate chamber at a constant temperature of 25 °C and 16 h light/8 h dark for two days to accelerate germination [18]. Vernalization was performed at 4 °C (under 2/22-h light/dark cycle and 150 μmol m⁻² s⁻¹ light intensity for 25 days), while non-vernalized seeds were maintained at 25 °C with the same light/dark cycle and light intensity for 25 days [19].

Two Arabidopsis lines were employed in this study: the Columbia wild-type (Col-0) and the MAF5 loss-of-function mutant maf5 (SALK_045623C). Both lines were cultivated under controlled conditions at 18–24 °C with a 16/8-h light/dark cycle, a light intensity of 150 µmol/m²/s, and a growth medium composed of a 1:1:1 (v/v/v) mixture of potting soil, vermiculite, and perlite. A total of 21 plants each of the Columbia wild-type and the maf5 mutant were grown in 54 × 28 cm trays. For DNA extraction, approximately 200 mg of young leaves were collected at the rosette stage, all plants were uniformly processed for genomic DNA extraction using the CTAB method [20]. For floral-dip transformation, the bacterial suspension was adjusted to an OD600 of 0.8–1.0 for infection. The maf5 mutants were screened using the triple-primer PCR method (protocol available at: http://signal.salk.edu/tdnaprimers.2.html, accessed on 17 March 2025).

2.2. RNA Isolation

Total RNA was extracted from materials subjected to different vernalization durations and samples collected from various plant tissues using the RNA extraction kit (TransGen Biotech, Beijing, China). The integrity of extracted RNA was verified by electrophoresis on 1.0% agarose gels, while RNA concentration and purity were determined using a micro-nucleic acid detection instrument. Qualified RNA samples were subsequently reverse transcribed into cDNA using the TransGen reverse transcription kit (TransGen Biotech, Beijing, China), with all procedures performed on ice. The extracted RNA was stored at −80 °C, and the resulting cDNA solutions were preserved at −20 °C for subsequent cloning of gene CDS regions and RT-qPCR (Real-Time Quantitative Polymerase Chain Reaction) analysis.

2.3. Cloning of BrMAF5 and Its Antisense lncRNA in Chinese Cabbage

The reference sequences of the BrMAF5 gene and its lncRNA were obtained from the Chinese cabbage database BRAD (http://brassicadb.cn/, accessed on 25 May 2024) and transcriptome data, and specific primers for DNA and CDS amplification were designed using Primer Premier 6 software. All designed primers were subsequently subjected to BLAST analysis in BRAD (http://www.brassicadb.cn, accessed on 17 June 2024) to verify their specificity. The amplification template consisted of cDNA samples from the vernalization-requiring Chinese cabbage cultivar B24108C48. Restriction enzyme digestion was performed according to the requirements of different vector constructions and specific restriction sites. The digested products and PCR-amplified fragments (using homologous arm primers) were gel-purified. Using the homologous recombination kit (TransGen Biotech, Beijing, China), the recombination products were transformed into DH5α competent E. coli cells. Positive clones were selected and verified by PCR amplification with universal vector primers, followed by sequencing confirmation. The successfully ligated constructs were identified, and all primer sequences used are listed in Supplementary Table S1.

2.4. Temporal and Spatial Expression Analysis of BrMAF5 and Its Antisense lncRNA

To validate the reliability of RNA-seq data, selected genes were verified by RT-qPCR analysis. First-strand cDNA was synthesized from total RNA using the TransGen Reverse Transcription Kit (TransGen Biotech, Beijing, China). Primer amplification efficiency was evaluated by constructing a standard curve via a five-fold serial dilution of pooled cDNA templates. Based on the analysis of amplification curves, primers meeting the criteria of 90–110% amplification efficiency and a linear correlation coefficient (R²) greater than 0.990 were selected for subsequent experiments. The expression stability of candidate reference genes (AtActin for Arabidopsis and BrActin for Chinese cabbage) under all experimental conditions was assessed using the geNorm algorithm, and the most stably expressed gene was used for normalization. Each sample was analyzed with three biological replicates and three technical replicates. Amplification was then carried out on a BIORAD CFX96 real-time PCR system. Relative gene expression levels were calculated using the 2^−ΔCT method [21], normalized against the reference genes. The corresponding primer sequences are listed in Supplementary Table S1.

2.5. Subcellular Localization Analysis

The coding sequences of BrMAF5 (lacking stop codons) were cloned into the pCAMBIA2300-eGFP vector and fused in-frame with the green fluorescent protein (GFP), successfully generating plasmids, including BrMAF5-eGFP. Using specifically designed homologous arm primers (sequences listed in Supplementary Table S1), the target fragments were amplified by PCR, gel-purified, and subsequently subjected to homologous recombination with the double-digested BrMAF5-eGFP vector. To validate subcellular localization, essential controls were implemented, including the 35S::eGFP empty vector. The recombinant BrMAF5-eGFP plasmid and the eGFP empty vector were transformed into Agrobacterium tumefaciens GV3101 competent cells. The resulting bacterial suspensions were infiltrated into the abaxial side of Nicotiana benthamiana leaves for transient expression assays. After 48 h of dark incubation, the samples were observed and imaged using a confocal laser scanning microscope (Zeiss LSM780, Oberkochen, Germany).

2.6. Fluorescence In Situ Hybridization (FISH) Analysis

Collected shoot apex and leaf tissues were fixed immediately with RNase-free formaldehyde fixative following paraffin sectioning. The fixed samples were then subjected to graded dehydration using a JJ-12J dehydration system (WHJJ, Wuhan, China), followed by paraffin embedding with a JB-P5 embedding system (WHJJ, Wuhan, China). Using a Leica RM2016 rotary microtome (Leica, Shanghai, China), the embedded blocks were serially sectioned at a thickness of 5 μm, and the resulting sections were transferred onto glass slides. The sections were thoroughly dewaxed with xylene, rehydrated through a graded ethanol series, and subsequently permeabilized with PBS containing 0.5% TritonX-100. The permeabilized sections were hybridized with specific probes, and non-specific binding was removed by stringent washes. Probe signals were detected using a confocal microscope (FV100, Olympus, Tokyo, Japan) for subcellular localization, while overall tissue expression profiles were captured via whole-slide scanning with a microscope (Leica DM2000 LED, Shanghai, China). Furthermore, fluorescence labeling was verified using a Nikon TS2 inverted fluorescence microscope, and entire slides were digitally archived using a fully automated slide scanner (3DHISTECH, Jinan, China).

2.7. Construction of Overexpression Vector and Arabidopsis Transformation

The overexpression vectors for BrMAF5 and BrMAF5L were constructed using the pCAMBIA1305 vector, with BamHI and HindIII serving as restriction sites for generating pCAMBIA1305-BrMAF5 and pCAMBIA1305-BrMAF5L constructs. The recombinant plasmids were subsequently transformed into Agrobacterium tumefaciens GV3101 competent cells. For Arabidopsis transformation, Agrobacterium infection solution was prepared following standard protocols [22]. One day prior to infection, Arabidopsis plants were watered thoroughly, with siliques and fully opened flowers removed. Floral dip transformation was performed by immersing inflorescences in the infection solution for 2 min with gentle agitation. Post-infection, plants were laid horizontally in trays and covered with plastic film to maintain humidity. After 24 h of dark treatment, plants were returned to normal growth conditions. Apex meristems were removed 10 days later, and the resulting seeds were collected as T₁ generation. After each generation of seeds was harvested and air-dried, positive lines were screened on 1/2 MS medium supplemented with hygromycin and timentin antibiotics. After 14 days of cultivation, Arabidopsis plants capable of rooting and growing on the antibiotic-containing medium were transplanted into soil and continued to be cultured under suitable temperature and humidity conditions to ensure healthy growth. When Arabidopsis developed five to six rosette leaves, DNA from transgenic Arabidopsis and control groups was extracted using the CTAB method [20] and subjected to PCR detection.

Commercially obtained mutants were screened and identified using the three-primer method. DNA was extracted from mutant plants, and homozygous mutants were identified by PCR in preparation for subsequent infection experiments.

Phenotypic observation included recording bolting time (defined as when the bolting stem reached 2 cm in height), flowering time (when the first flower was fully open), and the number of rosette leaves across different Arabidopsis lines. For molecular analysis, RNA was extracted from T₃ generation plants and reverse-transcribed into cDNA for quantitative real-time PCR (qRT-PCR) analysis.

2.8. Yeast Two-Hybrid Assay

The homologous arm primers pBT3-N-BrMAF5-F/R were designed using Primer Premier 6 software (Supplementary Table S1) to clone the BrMAF5 coding sequence into pBT3-N without altering its reading frame. Subsequently, the Coolaber NMY51 Yeast Two-Hybrid Interaction Verification Kit was employed to co-transform the following plasmid combinations into NMY51 yeast competent cells: pBT3-N-BrMAF5 with pPR3-N, pTSU2-APP with pNubG-Fe65 (positive control), and pTSU2-APP with pPR3-N (negative control). Autoactivation verification was performed on three types of media: SD/-Leu/-Trp, SD/-Leu/-Trp/-His, and SD/-Leu/-Trp/-His/X-α-gal. Colonies that grew normally on SD/-Leu/-Trp/-His/-Ade/X-α-gal medium were selected for PCR amplification, and the PCR products were sent for sequencing. Comparative analysis was conducted based on the Chinese cabbage reference genome sequence information to screen candidate interacting genes for subsequent one-to-one verification. The PCR amplification results and functional annotation information of the screened interacting protein genes are presented in Supplementary Table S2.

For one-to-one validation, BrACP4 (BraA01g015780.3C) and BrRAB1A (BraA06g041250.3C) were amplified using homologous-arm primers (pPR3-N-BrACP4-F/R and pPR3-N-BrRAB1A-F/R; Supplementary Table S1) and cloned into pPR3-N. The full-length BrMAF5 coding sequence was cloned into pBT3-N. Accordingly, pBT3-N-BrMAF5 was co-transformed with pPR3-N-BrACP4, pPR3-N-BrRAB1A, or empty pPR3-N (negative control) into NMY51 yeast cells. The known interacting pair pTSU2-APP + pNubG-Fe65 served as a positive control, whereas pTSU2-APP + pPR3-N served as a negative control. Transformants were selected on SD/−Leu/−Trp plates and incubated at 30 °C until colonies appeared. PCR-positive single colonies, together with positive and negative controls, were resuspended in 0.9% (w/v) NaCl, adjusted to OD600 = 0.2, and serially diluted (10⁰, 10⁻¹, 10⁻², and 10⁻³). Aliquots (4 μL) of each dilution were spotted onto SD/−Leu/−Trp/−His and SD/−Leu/−Trp/−His/−Ade plates. Plates were incubated inverted at 30 °C for 60 h to assess protein–protein interactions.

2.9. Statistical Analysis

Data are presented as the mean ± standard deviation (SD) of at least three biological replicates. All statistical analyses were performed using GraphPad Prism 9.0 software. The normality of the data distribution was verified by the Shapiro–Wilk test, and the homogeneity of variances was confirmed by Levene’s test. For comparisons among multiple groups (e.g., different vernalization durations), one-way analysis of variance (ANOVA) was applied, followed by Tukey’s post hoc test for pairwise comparisons. A p-value of less than 0.05 was considered statistically significant.

3. Results

3.1. Spatiotemporal Expression Analysis of BrMAF5 and Its lncRNA

Based on previous RNA-seq data, we predicted and identified BrMAF5 and its antisense lncRNA [15], followed by spatiotemporal expression analysis of both molecules. Initially, we cloned BrMAF5 and its antisense lncRNA using cDNA from Chinese cabbage cultivar ‘B24108C48’ as template. The sequences of BrMAF5 and its antisense lncRNA were amplified, revealing a 600-bp coding region for BrMAF5. The lncRNA was designated as BrMAF5L, which possesses a 325-bp transcript (Figure 1a). The structural diagrams of BrMAF5 and its lncRNA are shown (Figure 1b), revealing that BrMAF5L is antisense-complementary to the last exon segment of BrMAF5. Sequence alignment between BrMAF5 and AtMAF5 showed that BrMAF5 has some nucleotide and fragment deletions compared to AtMAF5 (Figure 1c). A phylogenetic tree was constructed using the neighbor-joining method to elucidate the evolutionary relationships of the MAF5 gene family in Brassicaceae crops closely related to Chinese cabbage (Figure 1d).

The expression patterns of BrMAF5 and BrMAF5L genes were analyzed by real-time quantitative PCR across different vernalization durations and in various plant tissues. Results showed that BrMAF5 expression exhibited a fluctuating trend of initial increase followed by decrease from the non-vernalization (NV) stage to 25 days of vernalization (V25). Specifically, during vernalization, BrMAF5 expression demonstrated a dynamic pattern of “rise-decline-re-rise-final drop to the lowest point”, with expression at V25 being significantly lower than at NV (Figure 1e). In contrast, BrMAF5L expression at all vernalization time points was substantially lower than at NV, showing the most pronounced downregulation during the NV to V5 transition and maintaining consistently low expression levels throughout the remaining vernalization period (Figure 1g). Overall, both BrMAF5 and BrMAF5L showed progressive downregulation as vernalization advanced. Spatial expression analysis revealed that BrMAF5 displayed minimal expression in roots but abundant accumulation in stems, leaves, and shoot apices (Figure 1f). BrMAF5L exhibited distinctly different spatial expression characteristics from BrMAF5, showing low expression in leaves and shoot apices but elevated expression in roots (Figure 1f,h).

3.2. BrMAF5 Subcellular Localization Analysis

Using the pCAMBIA2300-eGFP empty vector as the control and the pCAMBIA2300-BrMAF5-eGFP construct as the experimental treatment, the subcellular localization of BrMAF5 was examined with the aid of nuclear and membrane markers. Nicotiana benthamiana leaves were infiltrated with Agrobacterium, kept in the dark for 48 h, and then observed using a laser scanning confocal microscope. Confocal imaging showed that BrMAF5 was mainly localized to the nucleus and the membrane.

3.3. Fluorescence In Situ Hybridization Analysis of BrMAF5 and Its lncRNAs

Understanding the dynamic localization of genes provides valuable insights into their potential regulatory functions in plants. To investigate the spatial relationship between BrMAF5 and BrMAF5L, we performed a dual-label fluorescence in situ hybridization (FISH) assay in non-vernalized (NV) Chinese cabbage shoot apices. Nuclei were visualized with DAPI (blue), BrMAF5 mRNA with a red fluorophore, and BrMAF5L mRNA with a green fluorophore. Both transcripts were abundantly detected and showed overlapping distribution patterns within the shoot tip. Importantly, high-magnification imaging of leaf primordia revealed a significant spatial co-localization of the red (BrMAF5) and green (BrMAF5L) signals (Figure 2b). This observed proximity provides preliminary positional evidence consistent with potential functional crosstalk, such as regulatory interactions.

3.4. Identification of Homozygous Mutants and Transgenic Plants

Screening and identification were performed using the three-primer method. PCR detection results showed that the plants marked in the red box are homozygous mutants (Supplementary Figure S1). DNA from T₁ generation Arabidopsis plants and control groups was extracted for PCR analysis. Only plants showing correct amplification bands of the expected size were retained to ensure successful T-DNA integration (Figure 3a). The corresponding positive transgenic plants were selected for seed preservation, and this screening process continued until the T₃ generation for subsequent phenotypic analysis.

3.5. Phenotypic Observation of Overexpressed Plants and Detection of Flowering Gene Expression

Phenotypic observations were conducted on eight distinct Arabidopsis overexpression lines, with recordings of bolting time, flowering time, and rosette leaf number. Compared to wild-type (WT), the flowering time was significantly delayed by 4–5 days with a concomitant increase of 4–5 rosette leaves in both OE-BrMAF5 and OE-BrMAF5L transgenic lines. Both OE-BrMAF5 and OE-BrMAF5L transgenic lines exhibited delayed bolting phenotypes, with bolting time being postponed by 3–5 days (Figure 3b–f). These results preliminarily demonstrate that BrMAF5 and BrMAF5L inhibit flowering in Arabidopsis.

To further confirm this inhibitory effect, we analyzed expression patterns of known flowering-related genes (AtFLC, AtTEM1, AtFT, AtSOC1) in Arabidopsis by qRT-PCR. The results showed: flowering repressor genes (AtFLC, AtTEM1) were significantly upregulated in OE-BrMAF5 and OE-BrMAF5L compared to WT and maf controls; whereas flowering promoter genes (AtFT, AtSOC1) were significantly downregulated (Figure 3g–l). This further confirms the inhibitory role of BrMAF5 and BrMAF5L in Arabidopsis flowering.

3.6. BrMAF5 Interacts with BrACP4 and BrRAB1A to Regulate Flowering in Plants

To further investigate the regulatory mechanism of BrMAF5 in plant flowering, we performed a yeast library screening assay. Through yeast library screening and sequence alignment, combined with functional annotation of reference genes, two candidate genes potentially involved in plant flowering process were identified: BrACP4 and BrRAB1A. Functional annotations of other screened genes are provided in Supplementary Table S2. To examine the potential physical interactions between BrMAF5 and BrACP4/BrRAB1A, we conducted yeast two-hybrid assays. BrACP4 (411 bp) and BrRAB1A (609 bp) genes were cloned (Figure 4a).

The results demonstrated that co-transformation of BrMAF5 with BrACP4, and BrMAF5 with BrRAB1A, both enabled yeast growth on SD/-Leu/-Trp/-His and SD/-Leu/-Trp/-His/X-α-gal media, confirming the existence of interaction relationships between BrMAF5-BrACP4 and BrMAF5-BrRAB1A (Figure 4b,c).

4. Discussion

Chinese cabbage, a cruciferous crop belonging to the Brassica genus, is one of China’s most important vegetable crops, with abundant genetic resources and numerous varieties. Bolting and flowering are critical agronomic traits in agricultural production. Premature bolting significantly reduces the commercial value of Chinese cabbage, causing substantial economic losses for farmers; however, for breeders, rapid bolting can shorten the breeding cycle and accelerate the breeding process. Therefore, investigating the flowering regulation mechanisms in Chinese cabbage not only provides theoretical foundations for addressing practical production issues like premature bolting but also offers support for accelerating vegetable breeding.

This study elucidates the expression patterns of BrMAF5 and BrMAF5L, finding that their expression levels decreased significantly with prolonged vernalization, a trend similar to the vernalization response pattern of the Arabidopsis FLC gene [12]. AtMAF5 has been functionally verified to be a flowering repressor [23], Meanwhile, we also noted the opposite spatial expression patterns of BrMAF5 and BrMAF5L in roots, leaves, and shoots. This phenomenon could be attributed to the fact that Chinese cabbage is a vegetable that has undergone whole-genome triplication [24,25]. This event has led to structural and expressional changes in numerous genes during evolution. Our experimental validation confirmed that the expression levels of both BrMAF5 and BrMAF5L have indeed been altered.

FISH analysis revealed that BrMAF5L is adjacent to BrMAF5 in shoot apex tissues, implying potential spatial regulatory relationships between them. Subcellular localization showed that BrMAF5 protein localizes to both the nucleus and cell membrane, consistent with the typical nuclear function of MADS-box transcription factors in regulating target gene expression [26]. Fluorescence in situ hybridization further confirmed that BrMAF5L might regulate BrMAF5 expression through a cis-acting mechanism, analogous to the COOLAIR-mediated repression of FLC in Arabidopsis [6]. Heterologous overexpression of both BrMAF5 and BrMAF5L in Arabidopsis delayed flowering and increased rosette leaf number, supporting the hypothesis that BrMAF5 acts as a flowering repressor. Further analysis revealed significant upregulation of flowering repressor genes (AtFLC and AtTEM1) and downregulation of flowering promoters (AtFT and AtSOC1) in overexpression lines. Notably, AtTEM1 is a transcriptional repressor that negatively regulates the juvenile-to-adult transition and flowering transition by participating in multiple flowering pathways [27]; SOC1 encodes a MADS-box transcription factor that integrates diverse flowering signals from photoperiod, temperature, hormone, and age-related pathways [28]. These findings suggest that BrMAF5 likely delays flowering by suppressing key flowering integrators such as FT and SOC1. This aligns with reports that FRI expression in roots delays flowering by activating other MADS-box targets including MAF4 and MAF5 [29]. Additionally, BrMAF5L overexpression produced phenotypes similar to BrMAF5, indicating it may regulate BrMAF5 stability or transcriptional activity.

We verified the interactions between BrMAF5 and BrACP4 as well as BrRAB1A using yeast two-hybrid assays. Notably, the Arabidopsis homolog ACP4 is involved in chloroplast lipid synthesis and responds to photoperiod regulation [23,30], while RAB1A plays a central role in vesicle trafficking and pollen development [31]. Therefore, BrMAF5 may connect photoperiod and metabolic pathways through BrACP4, and influence membrane trafficking and reproductive development through BrRAB1A, thereby jointly regulating flowering time. Future research should focus on the dynamic changes in these interactions under different photoperiods and reproductive stages to reveal how BrMAF5 integrates environmental and intracellular signals for precise flowering regulation.

In Arabidopsis, studies have revealed that the flowering repressor MAF5 is precisely regulated through multiple pathways: the deubiquitinase OTU5 activates the expression of FLC, MAF4, and MAF5 through histone modifications to delay flowering [32]; simultaneously, the T-hook/PPC domain protein TEK has been shown to directly negatively regulate flowering-related genes, including MAF4 and MAF5 [33]. These findings provide critical clues for deciphering the regulatory mechanisms of BrMAF5 in Chinese cabbage, particularly its potential integration of epigenetic and transcriptional inputs. The dosage accumulation of FLC, FLM, and MAF genes (including MAF5) determines whether plants exhibit annual or perennial habits [34], suggesting that introducing MAF5 may convert annual plants into perennial ones. This can be considered an extension of MAF5’s flowering regulatory function in Arabidopsis and other plants. Thus, studying the BrMAF5 gene holds significant importance for Chinese cabbage and other Brassica crops.