The MADS-Box Transcription Factor BoAGL8 Is Involved in Regulating Flowering in Broccoli

Abstract

Broccoli (Brassica oleracea L. var. italica) is a biennial or annual herbaceous plant belonging to the species Brassica oleracea in the genus Brassica of the Cruciferae family. The green flower curd serves as the primary edible organ, with its development and preservation critically determining broccoli yield and quality. Given that these processes are regulated by flowering time, understanding the mechanisms underlying floral transition is essential for enhancing broccoli yield and quality. This study aimed to identify the MADS-box family in broccoli and to investigate the function of the BoAGL8 gene in floral induction. We identified a total of 176 MADS-box genes, of which 54 genes were up-regulated and 50 genes were down-regulated under low-temperature treatment. Notably, the expression of BoAGL8 was up-regulated by 6.70-fold under low-temperature induction, prompting us to select and clone this gene for further analysis. Tissue-specific expression profiling further revealed that BoAGL8 is expressed at relatively high level in both mature and young leaves. After 15 days of low-temperature treatment, BoAGL8 expression in shoot tip was significantly upregulated compared to untreated controls. Subcellular localization analysis showed that BoAGL8 protein was located to the nucleus. Ectopic over-expression of BoAGL8 in Arabidopsis exhibited accelerated bolting and flowering, reduced rosette leaf number, and increased seed yield per plant compared to wild-type plants. Furthermore, compared to wild-type controls, transgenic lines exhibited upregulated expression of AtFT, AtAP1 and AtSEP3, alongside downregulation of SVP expression. The above results indicate that BoAGL8 may play a key regulatory role in the process of floral organ development in broccoli, providing an important theoretical basis for future research on flowering time regulation and breeding in broccoli.

1. Introduction

MADS-box genes are a crucial class of transcription factors, prevalent across plants, animals, and fungi. They play key roles in essential biological processes including growth and development, secondary metabolic regulation, and responses to biotic and abiotic stresses [1]. The name “MADS” originates from four founding members: yeast’s MINI CHROMOSOME MAINTENANCE1 (MCM1), Arabidopsis thaliana’s AGAMOUS (AG), Antirrhinum majus’s DEFICIENS (DEF), and the human SERUM RESPONSE FACTOR (SRF). Each possesses a conserved 180 bp N-terminus MADS-box domain that encodes a DNA-binding domain and recognizing similar target DNA sequences [2]. Currently, research on MADS-box gene functions in plants is now well-established. Their primary role center on regulating floral organ development, plant morphology, and flowering time. These genes displayed distinct spatiotemporal expression patterns during the development of roots, stems, leaves, flowers, seeds, and other tissues, where they govern diverse regulatory functions [3]. Numerous MADS-box genes and their functions have been identified and validated in many plants such as Arabidopsis thaliana [4], Cucumis sativus [5], Oryza sativa [6], and Zea mays [7].

Flowering, a pivotal developmental phase in higher plants, represents the transition from vegetative to reproductive growth through floral bud differentiation, which determines flowering timing. Optimal flowering time is crucial for ensuring crop yield and reproductive success [8]. The flowering process in plants is coordinately regulated by endogenous factors and diverse environmental cues. MADS-box transcription factor genes play pivotal roles in flowering regulation by integrating environmental signals and developmental cues through multiple flowering pathways, including photoperiod vernalization, autonomous, thermosensory, gibberellin, and age-dependent [9,10,11]. The MADS-box transcription factor AGAMOUS-like 8 (AGL8), also known as AGL8 or FRUITFULL (FUL), has been extensively characterized for its role in flowering time regulation across crops. In Arabidopsis thaliana, FUL not only controls flowering time but also regulates meristem determinacy [12]. Ectopic expression of the Arabidopsis FUL gene in Brassica napus confers shatter-resistant siliques [13]. Heterologous expression of peach PpAGL8 [14] or soybean GmAGL8 [15] both lead to early flowering in Arabidopsis. Heterologous overexpression of BoFLC1 and BoFLC3 in cabbage (Brassica oleracea) or BcSVP in Chinese cabbage (Brassica rapa) delayed flowering [16,17]. In Chinese cabbage, BrSOC1b promotes flowering by interacting with other MADS-box proteins, such as AGL8, to integrate upstream signaling pathways [18]. Furthermore, the flowering promoter AGL19 in broccoli bypass the FT-dependent long-distance signaling pathway and indirectly regulate flowering through the floral integrator AGL24, which subsequently activates SOC1 [19]. Despite considerable progress in characterizing flowering genes in cruciferous vegetables, the role of broccoli BoAGL8 in regulating flowering time remains unclear.

Broccoli is a typical green-plant vernalization species, in which the transition to flowering is induced by exposure to low temperatures. Flower bud differentiation, a prerequisite for the formation of marketable curds, is promoted under relatively low temperatures [20]. Furthermore, curd development is optimized at 6 °C, as evidenced by the attainment of maximum curd weight at this temperature [21]. However, if plants encounter unsuitable low temperatures before the commercial curd formation, they may undergo premature transition from vegetative to reproductive growth. Under such conditions, the resulting curds are small, loose, and unmarketable, causing considerable yield reduction and economic damage [22]. In broccoli production, deviations in flowering time (either premature or delayed) can lead to either premature or delayed curd formation. Since the formation and maintenance of the curd are vital for yield and quality, controlling this process is essential. However, the molecular mechanisms that govern this critical flowering time are yet to be elucidated. Therefore, this study aims to elucidate the function of the BoAGL8, a MADS-box gene in broccoli, providing new insights into the molecular basis of flowering regulation and establishing a theoretical foundation for breeding new broccoli varieties with superior curd traits and broader adaptability.

2. Materials and Methods

2.1. Plant Materials

Plant materials included broccoli cultivar Zhongqing 10, Nicotiana benthamiana, and wild-type Arabidopsis thaliana (Columbia-0, Col-0). Zhongqing 10 seeds were obtained from Zhongshu Seeds Technology Co., Ltd. (Beijing, China) and cultivated and treated as described Chai et al. [23]. Broccoli cultivar Zhongqing 10 is an early-maturing F1 hybrid developed by the Institute of Vegetables and Flowers of the Chinese Academy of Agricultural Sciences. As one of the most widely cultivated broccoli varieties in China, it displays uniform growth and produces high-quality curds. Both N. benthamiana and Col-0 were maintained in the laboratory and grown in climate-controlled growth chambers at 25 ± 1 °C under 16 h light/8 h dark photoperiod.

2.2. Identification and Physicochemical Profiling of the MADS-Box Gene Family in Broccoli

The whole-genome and protein sequences of broccoli were retrieved from the Ensembl Plants database (http://plants.ensembl.org/, accessed on 8 May 2025), and those of Arabidopsis thaliana were sourced from The Arabidopsis Information Resource (TAIR, https://www.arabidopsis.org/, accessed on 8 May 2025). A BLASTP search was conducted against the broccoli proteome using each Arabidopsis MADS-box protein sequence as a query. Hits meeting the thresholds of E-value ≤ 1 × 10⁻²⁰ and sequence identity ≥ 30% were retained for subsequent analysis. Additionally, the MADS-box family HMM profiles SRF-TF (PF00319) and K-box (PF01486) were obtained from the Pfam database (http://pfam.xfam.org/, accessed on 8 May 2025) for domain identification. These profiles were employed to scan the broccoli proteome for conserved domains using the hmmsearch tool in HMMER 3.0, with a domain E-value cutoff 1 × 10⁻²⁰. The candidate sequences identified by both BLASTP and HMM searches were merged, and redundant or low-confidence entries were manually filtered out to produce a final non-redundant set of candidate genes. Finally, physicochemical properties of the identified proteins were analyzed using ExPASy-ProtParam tool (https://web.expasy.org/protparam/, accessed on 10 May 2025).

2.3. Phylogenetic Analysis and Classification of MADS-Box Gene Family in Broccoli

Protein sequence alignment of broccoli and Arabidopsis MADS-box proteins was performed using MUSCLE in MEGA7.0. Phylogenetic trees were reconstructed using the neighbor-joining (NJ) method with 1000 bootstrap replicates. Tree visualization and annotation were carried out using the Evolview online platform (https://www.evolgenius.info/evolview/, accessed on 10 May 2025).

2.4. Comprehensive Analysis of Gene Structure, Conserved Motifs, and Promoter Cis-Acting Elements in Broccoli MADS-Box Gene

Conserved motifs in broccoli MADS-box genes were analyzed using the MEME suite (https://web.mit.edu/meme/current/share/doc/overview.html, accessed on 12 May 2025) with a maximum motif count set to 10. Cis-acting elements in the promoter regions were predicted and analyzed with PlantCARE. All results were visualized using TBtools software (v2.331) (https://github.com/CJ-Chen/TBtools/, accessed on 12 May 2025).

2.5. Chromosomal Localization and Collinearity Analysis of the MADS-Box Gene Family in Broccoli

The chromosomal location of broccoli MADS-box genes was mapped using MG2C (v2.1) (http://mg2c.iask.in/mg2c_v2.1/, accessed on 12 May 2025). Genomic collinearity within the broccoli genome was analyzed with MCScanX under default parameters, using BLASTP alignments and genome annotations as input to identify syntenic blocks. These blocks were visualized using Circos (v0.69-9). Furthermore, interspecies synteny analysis between broccoli and other species—Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, and Solanum lycopersicum—was conducted and visualized using the same methodology. All genomic and protein sequences for these species were obtained from the Ensembl Plants database (http://plants.ensembl.org/, accessed on 12 May 2025).

2.6. Expression Analysis of the MADS-Box Gene Family in Broccoli

RNA-seq analysis method in this study was the same as that of Chai et al. [23]. Based on transcriptome data from our laboratory, the Fragments Per Kilobase per Million mapped reads (FPKM) values of MADS-box genes in control (CK) and low-temperature-treatment (V15) groups were analyzed. Expression patterns were visualized through a heatmap generated with TBtools.

2.7. Cloning and Bioinformatics Analysis of the BoAGL8 Gene in Broccoli

At the five-leaf stage, nine healthy broccoli plants exhibiting uniform size and morphology were selected. Tissue samples were collected from five organs: roots, stems, shoot apical meristems (SAM), mature leaves, and young leaves. Tissues from three plants were pooled as one biological replicate, generating three biological replicates. All samples were immediately wrapped in aluminum foil, flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent analyses. Total RNA was extracted from broccoli shoot apical meristems using the High-Efficiency Plant Total RNA Extraction Kit (Jinsha Biotechnology Co., Ltd., Beijing, China). RNA integrity was verified by 1.0% agarose gel electrophoresis, while concentration and purity were assessed using a Nanodrop spectrophotometer. First-strand cDNA was synthesized from the total RNA using PrimeScript™ RT Master Mix (Takara, Dalian, China) according to the manufacturer’s instructions, and stored at −20 °C. Partial BoAGL8 cDNA sequences were obtained from our transcriptome database (PRJNA1022574). Gene-specific primers (Supplementary Table S1) were designed using SnapGene software (v6.0.2) based on the broccoli genome database (http://www.bogdb.com/genome/broccoli, http://plants.ensembl.org/, accessed on 8 May 2025). BoAGL8 was amplified from broccoli shoot apical meristem cDNA using PrimeSTAR Max DNA Polymerase (Takara, Dalian, China) with the following PCR protocol: initial denaturation at 94 °C for 1 min; 40 cycles of denaturation at 98 °C for 10 s, annealing at 57 °C for 15 s, and extension at 72 °C for 5 s; final extension at 72 °C for 5 min. The PCR product was cloned into pMD18-T vector (Takara, Dalian, China) and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China).

Physicochemical properties and hydrophobicity of deduced BoAGL8 protein were analyzed using ExPASY tools ProtParam and ProtScale (https://web.expasy.org/protparam/, accessed on 15 May 2025). The open reading frame (ORF) and conserved domains were identified with NCBI’s ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/, accessed on 15 May 2025) and CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/, accessed on 15 May 2025), respectively. Homologous sequences were retrieved via NCBI-BLAST, followed by multiple sequence alignment and phylogenetic tree construction using DNAMAN software (v9.0).

2.8. Tissue-Specific Expression Patterns of BoAGL8 in Broccoli

At the five-leaf stage, total RNA was extracted from roots, stems, shoot apical meristems, mature leaves, and young leaves of broccoli plants under control (CK) and 15-day low-temperature treatment (V15) conditions, using a High-Efficiency Plant Total RNA Extraction Kit (Jinsha Biotechnology, Beijing, China). First-strand cDNA was synthesized from total RNA using the PrimeScript™ RT Master Mix (Takara, Dalian, China). qRT-PCR primers (Supplementary Table S1) were designed with Premier 5 software. Expression levels of BoAGL8 in broccoli tissues under different treatments were quantified by qRT-PCR using 18S rRNA as the endogenous reference. qRT-PCR was performed on an Applied Biosystems^® 7500 Fast Real-Time PCR System (ABI, Foster, CA, USA) using TB Green^® Premix Ex Taq™ (Takara, Dalian, China) in 20 μL reactions containing: 10 μL 2×Green qPCR MasterMix, 0.5 μL each primer (10 μM), 2 μL cDNA template, and RNase-free water. The thermal profile included: 95 °C for 2 min (initial denaturation); 50 cycles of 95 °C for 15 s, 57 °C for 15 s, 72 °C for 30 s. Three biological replicates were analyzed per sample. Relative expression of target gene was calculated using the 2^−ΔΔCt method [24], with statistical significances (α = 0.05) determined by one-way Student’s t-test in SPSS v27.0.

2.9. Subcellular Localization of Broccoli BoAGL8

To investigate the subcellular localization of broccoli BoAGL8 protein, a pCAMBIA1300-GFP overexpression vector was constructed. The BoAGL8 coding sequence (excluding the stop codon) was PCR-amplified with primers containing XbaI/KpnI restriction sites (Supplementary Table S1). The purified fragment was cloned into the XbaI/KpnI-digested pCAMBIA1300-GFP vector using T4 DNA ligase, generating the recombinant plasmid pCAMBIA1300-GFP-BoAGL8. A empty pCAMBIA1300-GFP vector served as a positive control.

The recombinant plasmid pCAMBIA1300-GFP-BoAGL8 and the control plasmid pCAMBIA1300-GFP were separately transformed into Agrobacterium tumefaciens GV3101. The transformed cells were spread onto LB solid medium containing 20 mg/L rifampicin (Rif) and 50 mg/L kanamycin (Kana), followed by incubation at 28 °C for 2–3 days. Positive single colonies were selected and inoculated into 1 mL of LB liquid medium with the corresponding antibiotics, then cultured overnight at 28 °C with shaking at 180 rpm. A 100 μL aliquot of the culture was transferred into 100 mL of fresh LB liquid medium supplemented with antibiotic and grown under the same conditions until the OD₆₀₀ reached 0.6–0.8. The bacterial cells were collected by centrifugation at 5000 rpm for 5 min at room temperature. The pellet was resuspended in an infiltration buffer containing 10 mM MES, 10 mM MgCl₂, and 200 μM acetosyringone (AS), with the pH adjusted to 5.6 using KOH [25]. The bacterial suspension was adjusted to an OD₆₀₀ of 1.0 and incubated in the dark at room temperature for 2–3 h. Four-week-old Nicotiana benthamiana plants were used for agroinfiltration. The prepared bacterial suspension was gently infiltrated into the abaxial side of the leaves using a 1 mL sterile syringe (without a needle) until the mesophyll tissue was fully saturated, indicated by the appearance of water-soaked spots. Multiple sites were infiltrated per leaf, and each infiltrated zone was marked with a pen. After infiltration, the plants were initially maintained in the darkness for 24 h and then moved to normal light conditions for an additional 24–36 h. GFP fluorescence signals in the leaf epidermal cells were observed and imaged using a laser scanning confocal microscope (Leica TCS SP8, Berlin, Germany).

2.10. Heterologous Expression of BoAGL8 in Arabidopsis

To generate BoAGL8-overexpression lines, the coding sequence (CDS) of BoAGL8 was cloned into the pBI121 vector under the control of the CaMV 35S promoter, generating the recombinant construct pBI121-BoAGL8. The plasmid was then transformed into Arabidosis thaliana Col-0 ecotype via Agrobacterium-mediated floral dip transformation [26]. Transgenic plants were selected on 1/2 MS medium containing 50 mg·L⁻¹ kanamycin and confirmed by genomic PCR using primers listed in Supplementary Table S1. Homozygous T3 lines were then selected for subsequent experiments.

2.11. Phenotypic Analysis of BoAGL8-Overexpressing Arabidopsis

Homozygous T3 transgenic Arabidopsis plants were used as the test subjects, with wild-type (WT) plants as controls. Rosette leaf number was counted when bolting stems reached approximately 1 cm in length, and bolting time was recorded. Flowering initiation was determined upon the first flower opening, at which time plant height and inflorescence stem diameter (at the base) were also measured. Total seed weight per plant was assessed following complete seed harvest.

To analyze BoAGL8 expression in transgenic plants, total RNA was extracted from rosette leaves of 30-day-old transgenic and wild-type (WT) Arabidopsis plants. BoAGL8 expression levels in transgenic lines were quantified via qRT-PCR with AtACTIN11 as the internal reference gene. Reactions were performed on an Applied Biosystems^® 7500 Fast Real-Time PCR System (ABI, Foster, CA, USA) using TB Green^® Premix Ex Taq™ (Takara, Dalian, China), under identical conditions to those described in Section 2.4. Each sample included three biological replicates. The relative gene expression level was calculated using the 2^−ΔΔCT method [24]. The expression profiles of flowering-related genes (AtFT, AtAP1, AtSEP3, AtSVP) were analyzed similarly. All primer sequences are listed in Supplementary Table S1.

3. Results

3.1. Identification and Characterization of the MADS-Box Gene Family in Broccoli

Genome-wide analysis identified 176 MADS-box genes in broccoli, designated BoMADS1-BoMADS176 (Supplementary Table S2). These genes were distributed unevenly across chromosomes, with chromosome C9 harboring the highest number. Theoretical isoelectric points (pI) ranged from 4.32 to 11.46, with the majority (138 genes) exhibiting a pI > 7 and 38 genes a pI < 7. Calculated molecular weights varied from 6884.04 to 91,018.37 Da, corresponding to proteins ranging from 61 to 825 amino acids in length (detailed in Supplementary Table S2). These findings reveal considerable diversity in the physicochemical properties of MADS-box proteins in broccoli.

3.2. Phylogenetic Analysis and Classification of MADS-Box Gene Family in Broccoli

Phylogenetic analysis of 176 broccoli (BoMADS) and 104 Arabidopsis (AtMADS) genes revealed that BoMADS proteins segregated into 16 distinct subfamilies: 77 M-type and 99 MIKC-type protein. Further classification of M-type proteins identified three conserved subgroups: Mα (n = 33, the most abundant), Mβ (n = 19, the least abundant), and Mγ (n = 25). Among MIKC-type protein, phylogenetic analysis resolved 12 MIKC* and 87 MIKC^C members. The MIKC^C group was further subdivided into 12 subclades, notably including the AP1/FUL subclade, which contained eight members, including BoAGL8 (Figure 1). Conserved orthologous relationships were observed between several BoMADS and AtMADS genes, within corresponding clades, suggesting functional conservation.

3.3. Gene Structure, Conserved Motifs, and Promoter Cis-Acting Elements of the MADS-Box Gene Family in Broccoli

To elucidate the structural diversity and evolutionary characteristics of BoMADS-box genes, a comprehensive analysis of gene structures, focusing on intron–exon organization was performed. All 176 genes contained complete coding sequences (CDSs), while untranslated regions (UTRs) were variably present. Most Type I genes (subfamilies Mα, Mβ, and Mγ) lacked introns, with a minority containing 1 to 2 introns. In marked contrast, Type II MIKC* genes exhibited complex structures with 5 to 9 introns and conserved C-terminal motifs, demonstrating significantly greater complexity than their Type I gene (Figure 2A).

Conserved motif analysis of BoMADS-box proteins using MEME identified 10 distinct motifs, designated Motif 1 to Motif 10 (Figure 2B). Genes within the same subfamily exhibited similar motif composition, suggesting potential functional similarity. Notably, Motif 1 and Motif 2 were ubiquitous, encoding the M-domain either individually or in combination. In contrast, Motif 4, Motif 6, and Motif 9, corresponding to the K domain, were exclusively identified in Type II genes. These findings indicate that Type II members exhibit greater evolutionary conservation than Type I members within this family.

To explore the potential functions of BoMADS-box genes, we analyzed cis-acting elements within their promoter regions (Figure 3). This analysis identified 45 distinct types of cis-acting elements in the promoters of BoMADS-box genes, which were functionally classified into four categories: stress-responsive (8 types), plant growth-related (9 types), hormone-responsive (9 types), and light-responsive elements (19 types). Notably, elements involved in basal transcription regulation, light response, abscisic acid (ABA) response and jasmonic acid (JA) response were highly abundant. Conversely, elements related to wound response, seed development regulation, and low-temperature response were less prevalent.

3.4. Chromosomal Localization and Collinearity Analysis of MADS-Box Gene Family in Broccoli

Chromosomal mapping of the 176 BoMADS-box genes revealed that 165 genes were precisely localized across the nine chromosomes of broccoli (Figure 4A), while 11 genes remained unmapped. Chromosome 9 exhibited the highest gene density, harboring 25 BoMADS-box genes, including a distinct cluster of 10 genes. In contrast, chromosome 5 contained the lowest number, with only 11 BoMADS-box genes.

Collinearity analysis was performed to elucidate the distribution patterns and evolutionary relationships of homologous BoMADS-box genes in broccoli. The analysis identified 112 segmentally duplicated gene pairs among the 176 MADS-box members, indicating extensive gene duplication events (Figure 4B). These duplications exhibited a non-random chromosomal distribution, with chromosome 1 showing the highest frequency and chromosome 7 the lowest. Comparative genomic analysis revealed distinct patterns of MADS-box gene collinearity between broccoli and related species: Arabidopsis thaliana exhibited the highest conservation with 101 syntenic gene pairs; Oryza sativa showed minimal synteny, with only 2 gene pairs localized to chromosomes 1 and 4; Solanum lycopersicum displayed intermediate conservation with 13 syntenic pairs distributed across chromosomes 2, 3, 4, 7 and 9; and Populus trichocarpa shared 12 syntenic gene pairs spanning chromosomes 1, 2, 3, 6, 7 and 8 (Figure 4C).

3.5. Expression Analysis of the MADS-Box Gene Family in Broccoli

Previous studies have demonstrated that after 15 days of different temperature treatments, flower buds of the broccoli plants under the low-temperature condition initiated differentiation, whereas control plants maintained vegetative growth [23]. To further investigate the expression of MADS-box genes during the initiation of flower bud differentiation, transcriptome data were utilized to analyze the expression of 176 BoMADS-box genes in broccoli under normal temperature (CK) and low-temperature (V15) conditions. The result revealed that 54 (30.68%) genes were significantly up-regulated, whereas 50 genes (28.40%) were significantly down-regulated (Supplementary Figure S1). Through KEGG functional annotation, eight MADS-box transcription factors related to flowering pathway were identified, and the differentially expressed genes (DEGs) were visualized using TBtools. The results revealed that two MADS-box transcription factors were significantly downregulated under low-temperature treatment: FLC (Bo3g005470.1) and SVP (Bo8g101000.1). Specifically, FLC expression was downregulated by 2.29-fold. Conversely, six transcription factors were significantly upregulated: SOC1 (Bo4g024850.1), AGL8 (Bo7g098190.1), AGL19 (Bo7g108370.1), AGL21 (Bo1g003270.1), AGL24 (Bo7g109590.1) and AGL42 (Bo2g162920.1). Among these, AGL8 expression increased by 6.70-fold (Supplementary Table S3 and Figure 5).

3.6. Sequence Analysis and Physicochemical Properties of the BoAGL8 Gene

The BoAGL8 gene was PCR-amplified from broccoli shoot tip cDNA. Electrophoresis analysis revealed a specific band of approximately 750 bp in size (Figure 6A). Sequencing identified a complete 726 bp open reading frame (ORF), encoding 241 amino acids (Figure 6B). Physicochemical analysis of the BoAGL8 protein predicted a molecular weight of 27.508 kDa, a theoretical pI of 9.46, a fat coefficient of 88.22, and an instability index of 53.56.

ProtScale analysis predicted that most amino acid residues of BoAGL8 were located in hydrophilic regions, indicating its hydrophilic nature (Figure 6C). Conserved domain analysis identified both a MADS_MEF2_like (residue 2–79) and a K-box domains (residues 84–173) (Figure 7A). These findings confirm that BoAGL8 belongs to the MADS-box transcription factor family.

Multiple sequence alignment revealed significant homology between BoAGL8 and other AGL8 proteins from other species (Figure 7B), including: Brassica napus (XP_022561166.2, 100%), Brassica oleracea var. Oleracea (XP_013594261.1, 98.76%), Brassica oleracea var. Botrytis (CAD 47850.1, 97.93%), Brassica rapa (XP_009136412.2, 95.85%), Raphanus sativus (XP_018490011.1, 92.98%), Arabidopsis thaliana (XP_568929.1, 92.15%), Eutrema salsugineum (XP_ 024006932.1, 92.12%), Camelina sativa (XP_010456827.1, 90.91%), Capsella rubella (XP_006281003.1, 90.09%), and Tarenay hassleriana (XP_019058647.1, 71.49%). Phylogenetic analysis delineated the evolutionary relationships among these AGL8 proteins (Figure 7C), with BoAGL8 most closely clustering with Brassica napus AGL8 protein.

3.7. Expression Pattern of the BoAGL8 Gene in Broccoli

To examine the expression pattern of the BoAGL8 gene in different tissues under normal and low-temperature conditions, the expression levels of BoAGL8 gene in roots, stems, shoot apices, mature leaves, and young leaves were analyzed by qRT-PCR. BoAGL8 was expressed in all tissues examined (Figure 8). Under both conditions, BoAGL8 expression was significantly higher in mature and young leaves compared to other tissues, suggesting a potential role in leaf development. Conversely, expression levels were relatively low in roots and stems. After 15 days of low-temperature treatment, BoAGL8 expression in shoot apices was significantly upregulated compared to the control, indicating that BoAGL8 may play a crucial role in floral bud differentiation in broccoli.

3.8. Subcellular Localization of BoAGL8

To determine the subcellular localization of BoAGL8, the recombinant plasmid pCAMBIA1300-GFP-BoAGL8 and the control plasmid pCAMBIA1300-GFP were transiently expressed in Nicotiana benthamiana leaves and observed using laser scanning confocal microscope. In the control (pCAMBIA1300-GFP), GFP fluorescence was distributed throughout the cell, whereas the GFP-BoAGL8 fusion protein exhibited strong nuclear localization (Figure 9). These results demonstrated that BoAGL8 was a nuclear-localized protein.

3.9. Ectopic Overexpression of BoAGL8 in Arabidopsis Accelerates Flowering

Positive transgenic Arabidopsis lines were confirmed in the T3 generation by PCR analysis. The pBI121-BoAGL8 recombinant plasmid and wild-type Arabidopsis plant served as a positive and a negative controls, respectively. Specific amplification bands of the expected size were detected in all transgenic lines but not in negative control (Figure 10A), demonstrating successful integration of BoAGL8 into the Arabidopsis genome. qRT-PCR analysis revealed significantly higher BoAGL8 expression in transgenic lines compared to wild-type controls (Figure 10C). Under identical growth conditions, T3 transgenic and wild-type Arabidopsis plants were cultivated simultaneously. Transgenic plants produced fewer rosette leaves (8.7 vs. 10.8 in wild-type), with bolting time and initial flowering occurring 7.8–8.4 days and 4.9–7.4 days earlier, respectively. At initial flowering, transgenic plants were 4.5–5.1 cm taller than wild-type plant. Seed yield per transgenic plant (29.1–36.0 mg) also significantly exceeded that of wild-type plants (17.9 mg) (

Table 1. Phenotypic characterization of BoAGL8-overexpressing T3 transgenic Arabidopsis lines.

Plant	Bolting Time (d)	Rosette Leaf Number	Time to First Flowering (d)	Plant Height at First Flowering (cm)	Seed Weight per Plant (mg)
WT	29.6 ± 2.1 a	10.8 ± 1.1 a	36.7 ± 2.1 a	9.7 ± 1.2 b	17.9 ± 5.4 c
OE3	21.2 ± 0.7 b	8.7 ± 0.7 b	31.8 ± 1.0 b	14.8 ± 1.3 a	29.1 ± 5.4 b
OE4	21.8 ± 1.4 b	8.7 ± 0.5 b	29.3 ± 1.5 c	14.2 ± 1.4 a	36.0 ± 11.7 a

Note: Values represent mean ± standard deviation. Different letters within the same column indicate statistically significant differences at p < 0.05.

). These results demonstrate that BoAGL8 promotes flowering and accelerates reproductive development in Arabidopsis. Notably, heterologous overexpression of BoAGL8 in the vernalization-insensitive Arabidopsis Col-0 ecotype resulted in reduced rosette leaf number, significantly earlier bolting and flowering time, and an accelerated transition to the reproductive growth stage, ultimately leading to increased seed yield. These findings suggest that the flowering-promoting function of BoAGL8 may be independent of the classical vernalization pathway.

3.10. Expression Analysis of Flowering-Related Genes in Arabidopsis Overexpressing BoAGL8

We examined the expression of endogenous flowering regulators AtFT, AtAP1, AtSEP3, and AtSVP in transgenic Arabidopsis (Figure 11). qRT-PCR analysis revealed that the expression levels of AtFT, AtAP1, and AtSEP3 were upregulated, while AtSVP expression was markedly downregulated in BoAGL8-overexpressing lines (Figure 11). These findings demonstrate that BoAGL8 overexpression promotes flowering by enhancing the expression of AtFT, AtAP1, and AtSEP3, and simultaneously suppressing the floral repressor AtSVP.

4. Discussion

In commercial broccoli production, inconsistent curd initiation timing and suboptimal curd quality are frequently challenges. Curd formation and maintenance are critical determinants of both yield and quality. Although governed by flowering time regulation, the molecular mechanisms underlying these processes remain largely elusive. The MADS-box gene family plays a primary role in regulating flowering time, growth morphology, and organ development in plants [1]. In recent years, an increasing number of MADS-box genes have been identified and analyzed across diverse plant species, such as Brassica oleracea (36 MIKC^C, 6 MIKC*, 25 Mα, 11 Mβ, 11 Mγ) [27], Brassica rapa [28], and Raphanus sativus [29]. Functional analyses reveal conserved roles of MADS-box genes among these species, demonstrating the evolutionary preservation of MADS gene functions [30]. This study provides a preliminary understanding of the evolutionary relationships of MADS-box genes in broccoli. We identified 176 MADS-box family genes within the broccoli genome, comprising 87 MIKC^C, 12 MIKC*, 33 Mα, 19 Mβ, and 25 Mγ. Notably, MIKC*-type genes exhibit distinct structural complexity with conserved C-terminal motif, aligning with cabbage [27]. Furthermore, we successfully cloned the BoAGL8 gene. Bioinformatics analysis revealed that BoAGL8 contains a 726 bp ORF encoding a 241-amino acids protein, with conserved MADS and K-box domains confirming its classification as a MADS-box family member.

Floral development initiates with the transformation of SAM into the inflorescence meristem (IM). Subsequently, floral meristems (FM) form on the flanks of the IM and differentiate to produce floral organs, including perianth and reproductive structures [31]. As the primary site for temperature perception and floral bud differentiation in plants, the shoot apex is thus crucial for flowering transition functioning. Low temperature is a key environmental cue regulating broccoli’s transition from vegetative to reproductive growth [32]. Broccoli requires vernalization to initiate floral bud differentiation and subsequent curd formation, typically occurring in regions with mean temperatures below 18 °C. Under excessively high temperatures, curd development is significantly impaired, with plants remaining arrested in the vegetative growth phase [33]. Notably, seedlings cold-treated −5 °C, −3 °C, 1 °C, or 2 °C exhibit significantly advanced flowering compared to those receiving conventional low-temperature vernalization [34]. In this study, BoAGL8 expression was detected in all examined tissues of broccoli. Following 15 days of low-temperature treatment, BoAGL8 transcript levels in shoot apical meristem were significantly higher than in untreated controls. Previous studies indicated that broccoli plants initiate floral bud differentiation after 15 days under relatively low temperature (mean 17 °C day/9 °C night, V15). These findings suggest that BoAGL8 plays an critical in broccoli floral bud differentiation. In Arabidopsis, the AGL8/FUL gene plays a regulatory role in the flowering process; agl8/ful mutants exhibit delayed flowering, increased rosette leaf number, and prolonged bolting time. Conversely, overexpression of AGL8/FUL significantly reduces flowering time and accelerates the floral transition [35]. Consistent with this, heterologous overexpression of AGL8 orthologs from species such as Glycine max [15], Spinacia oleracea [36], and Solanum lycopersicum [37] in Arabidopsis also promotes flowering. In sweet cherry (Prunus avium), PavFUL interacts with PavLFY, PavSOC1, PavAP1, and PavSEP proteins to coordinately regulate flowering and multicarpel silique development [38]. CpFUL-overexpressing Arabidopsis lines, the expression of AtAP1, AtLFY, AtFUL, and AtSEP3 was upregulated prior to inflorescence primordium formation, while AtSVP expression was downregulated [39]. Similarly, in Arabidopsis lines overexpressing GhAGL8, the expression levels of AtFT, AtAP1, and AtLFY were markedly elevated, whereas those of AtCDF1 and AtSVP was significantly reduced [40]. In this study, heterologous overexpression of BoAGL8 in the Arabidopsis Col-0 ecotype resulted in an early flowering phenotype, accompanied by upregulation of flowering-promoting genes (AtFT, AtAP1, AtSEP3) and downregulation of the floral repressor AtSVP. Notably, the Col-0 ecotype is vernalization-insensitive ecotype because it possesses a non-functional FRI allele that is incapable of inducing FLC expression [41]. These results indicate that BoAGL8 promotes flowering in a heterologous system, and this function is likely independent of the classical vernalization pathway. Future studies utilizing vernalization-responsive ecotypes (e.g., Edi-0) [42] will be essential to fully elucidate the role of BoAGL8 in low temperature-mediated flowering regulation. However, whether BoAGL8 similarly promotes flowering in broccoli requires further experimental validation.

5. Conclusions

In this study, we identified 176 MADS-box family genes within the broccoli genome. We comprehensively characterized their physicochemical properties, conserved domains, conserved motifs, and cis-acting elements. Transcriptome analysis revealed that under low-temperature treatment, the expression of 54 BoMADS-box genes (30.68%) was significantly upregulated, while that of 50 genes (28.40%) was significantly downregulated; among these, the expression level of AGL8 increased by 6.70-fold. The BoAGL8 gene, containing 726 bp ORF, encodes a 241-amino-acid protein featuring conserved MADS-box and K-box domains, with subcellular localization being nuclear. BoAGL8 exhibits ubiquitous expression across various broccoli tissues. Notably, transcript levels in shoot apices were markedly elevated following a 15-day low-temperature treatment compared to normal conditions. Heterologous expression of BoAGL8 in Arabidopsis accelerated flowering, decreased rosette leaf number, and enhanced seed production. BoAGL8 likely accelerated flowering by upregulating AtFT, AtAP1 and AtSEP3, concomitant with downregulation of AtSVP. This study provides a foundation for understanding BoAGL8-regulated flowering mechanisms in broccoli. Functional validation was conducted via heterologous overexpression in Arabidopsis. Future studies could use broccoli transformation systems for directly characterize BoAGL8 function, accelerating broccoli breeding.