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Genome-wide identification, characterization, and expression analysis of BZR transcription factor family in Gerbera hybrida

BMC Plant Biology volume 25, Article number: 143 (2025) Cite this article

Abstract

Background

The BZR family genes encode plant-specific transcription factors as pivotal regulators of plant BR signaling pathways, critically influencing plant growth and development.

Results

In this study, we performed a genome-wide investigation of the BZR family gene in gerbera to identify the key components of the BR pathway that may function in petal growth. The identified BZR genes, named GhBEH1-7 (GhBEH1, GhBEH2, GhBEH3, GhBEH4, GhBEH5, GhBEH6, GhBEH7), are distributed across chromosomes 3, 5, 10, 11, 12, 14 and 15. These genes exhibit similar exon–intron structures and possess typical BZR family structures. Phylogenetic analysis clustered these genes into two distinct subgroups. Analysis of cis-acting elements revealed their involvement in hormone response, stress response, and growth regulation. Subcellular localization analysis indicated nuclear localization for GhBEH1 and GhBEH2, while the remaining five genes exhibited dual localization in the nucleus and cytoplasm. The transactivation assay indicated that GhBEH1 and GhBEH2 may function as transcriptional repressors, contrasting with the transcriptional activation observed for the other five genes. Notably, seven GhBEHs exhibit various expression patterns under different growth stages of ray florets and BR treatment conditions. Meanwhile GhBEH1 and GhBEH2 showed pronounced responsiveness to BR stimulation.

Conclusion

Our work explains genome-wide identification, characterization, and expression analysis of gerbera’s BZR transcription factor family. We hinted that these seven GhBEHs are involved in petal growth and development regulation. These findings provide a basis for further studies on the biological function of the BZR gene family in petal growth and a theoretical basis for future horticultural application in gerbera.

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Background

Brassinosteroids (BRs), a class of plant steroidal hormones [1], influence a wide range of cellular responses such as cell elongation, photomorphogenesis, flowering, stress tolerance, and pathogen resistance, thereby contributing significantly to both yield enhancement and flower quality improvement [2,3,4,5,6]. In plants, BRs are perceived by the cell membrane receptor brassinosteroids-insensitive 1 (BRI1) [7]. This perception leads to the activation of the BRI1 receptor kinase and triggers a phosphorylation cascade that inhibits the glycogen synthase kinase-3 (GSK3)-like kinase BIN2 [8, 9]. BIN2 undergoes phosphorylation and regulates other kinases as well as crucial transcription factors (TFs), including members of the brassinazole-resistant (BZR) gene family [8, 10]. BIN2 phosphorylates BZR1 family transcription factors, and after BIN2 inactivation, PP2A dephosphorylates BZR1 family transcription factors [11, 12]. Upon dephosphorylation, brassinazole-resistant 1(BZR1) and BRI-EMSSUPPRESSOR1 (BZR2/BES1) translocate into the nucleus, where they regulate the expression of target genes, thereby influencing cell growth [8, 13]. This intricate regulatory mechanism mediated by BRs plays a central role in gene expression and specific cellular developmental processes [14, 15].

As crucial phytohormones, BRs are essential for regulating plant growth and development, primarily mediating BZR transcription factors [2, 4, 5, 8, 10, 16,17,18]. In Arabidopsis, the BZR gene family is composed of three classes: brassinazole-resistant 1 (BZR1), BRI-EMSSUPPRESSOR1 (BZR2/BES1), and homologs 1–4 (BEH1-BEH4) of BZR1/2, sharing high sequence identity with BZR1/2 [19, 20]. BZR1 and BZR2/BES1, identified as transcriptional repressors and activators [19, 21, 22], are key transcription factors in the BR signaling pathway. These two proteins share 88% sequence homology and bind to DNA through a highly conserved N-terminal DNA-binding domain [19, 21]. This DNA-binding domain contains an atypical basic helix-loop-helix (bHLH) DNA-binding motif capable of specifically binding E-box (CANNTG) and BRRE (CGTGT/CG) elements [19, 21]. BZR genes are notable for modulating downstream gene expression throughout various plant growth and development stages [19, 21]. Although current research has identified numerous BR-responsive genes [23,24,25], only a subset are targets of BZR1 or BZR2/BES1 [26, 27]. BZR1 collaborates with the RLA1/SMOS1 transcription factors in rice to form a transcriptional complex that synergistically regulates downstream genes within the BR signaling pathway, thereby influencing growth and development [28]. Similarly, in maize, BZR1 directly regulates the expression of the GRACE and KRP6 genes involved in cell expansion and organ size determination [29]. In Chrysanthemum, CmBES1 binds directly to the promoter of CUC2, suppressing its expression and facilitating the transformation of tubular flowers into tongue-shaped flowers [30]. Furthermore, in litchi, BRs mitigate ethylene-induced fruit abscission by repressing the transcription of LcACS1/4 and LcACO2/3 through LcBZR1/2 [31]. Despite extensive research on BZR proteins in Arabidopsis, rice, Chrysanthemum, and litchi, the functions of BZR transcription factors in gerbera are still unclear. Therefore, the hypothesis has to be proved that BZR1 and BZR2/BES1 may regulate other transcription factors to modulate secondary BR-responsive genes and perform additional functions. Thus, identifying and characterizing new BZR genes from various plant species are promising avenues for gaining novel insights into this gene family.

Gerbera hybrida, belonging to the Asteraceae family (Compositae), represents one of the largest branches within angiosperms. The inflorescence of gerbera consists of three types of florets: small central disc florets (dfs), medium intermediate trans florets (tfs), and large marginal ray florets (rfs). This species is extensively employed as a model organism for investigating the organogenesis and developmental processes within the Asteraceae family [32,33,34]. Although numerous TFs governing petal growth have been extensively studied in gerbera [35,36,37,38,39,40,41], research on the BZR TFs remains limited, particularly in elucidating the functions of BZR TFs in petal growth. Therefore, this study conducted a comprehensive and systematic analysis of the BZR family genes in gerbera, aiming to investigate this gene family within the context of gerbera thoroughly. The GhBEH (BZR/BES homologs) genes were initially identified through transcriptome analysis. Preliminary functional predictions were made through phylogenetic analysis, conserved domain sequence search, protein structure prediction, chromosome localization, gene structure, and promoter cis-element analysis. Additionally, we conducted subcellular localization experiments and transcriptional activity analysis of the GhBEH family. Furthermore, real-time quantitative reverse transcription (qRT) PCR analysis was employed to investigate the expression patterns of GhBEHs in various growth stages of ray florets. Concurrently, the response of GhBEHs to BR treatment was examined. These findings establish a foundation for further exploration into the function of GhBEH genes and for identifying and characterizing BZR genes in diverse species. Moreover, this study provides a theoretical basis for the in-depth investigation of gerbera’s molecular mechanisms governing petal growth.

Methods

Plant material and growth conditions

Gerbera hybrida (G.jamesonii Bolus ex Adlam × G.viridifolia Schultz-Bip.) cv “Terra Regina” and “Shenzhen No. 5” were used in this study, and these two cultivars are from the greenhouse of Prof. Paula Elomaa at the University of Helsinki and the School of Life Sciences greenhouse at South China Normal University, respectively. “Shenzhen No. 5” was used for the subcellular localization analysis, dual-luciferase reporter assay, and hormone treatments. In contrast “Terra Regina” was used for the expression pattern of GhBEHs in different petal growth stages.

Gerbera shoots were individually propagated in multiplication medium to induce bud clusters and subsequently maintained in a tissue-culture room at 24 ± 2 °C under a long-day photoperiod (16 h light/8 h dark). Upon root establishment, the plants were transferred to a greenhouse with a 22–28 °C temperature and relative humidity of 60–80%.

Identification of the GhBZR family gene

The BZR gene family sequences from Arabidopsis, sourced from the Arabidopsis Information Resource (https://www.arabidopsis.org/), were subjected to a BLASTP search against NCBI databases using an e-value of 10–10 to identify potential candidate genes. The domain structure of the BZR gene family was annotated based on prior investigations [42, 43], and redundant sequences were filtered out via sequence alignment conducted using DNAMAN software.

Phylogenetic analysis and multiple sequence alignment

The seven BZR proteins from gerbera were subjected to genome-wide searches on NCBI using BLASTp, followed by phylogenetic tree clustering analysis of the aligned homologous protein sequences. The phylogenetic tree of the BZR gene family was constructed using MEGA5.1 software based on the neighbor-joining (NJ) method. Bootstrapping was performed with 1000 replicates to assess branch support values. Multiple sequence alignment of these seven proteins, in addition to Arabidopsis BZR family members and four homologous proteins from Asteraceae, was performed using DNAMAN, and the typical BZR family structure was mapped.

Chromosome localization analysis and gene structure

The chromosomal location of GhBZR genes was obtained from the gerbera database using TBtools software. Subsequently, a genomic distribution map illustrating the spatial arrangement of BZR genes throughout the gerbera genome was constructed using MG2C software. The exon–intron structure of GhBZRs was analyzed based on the full-length genome sequences and the CDSs by TBtools. The gene structure was also visualized using the Gene Structure Display Server (GSDS 2.0) (http://gsds.cbi.pku.edu.cn/).

Promoter sequence analysis of GhBEH genes

Approximately 2000 bp of sequence upstream from the start codon (ATG) in the GhBEH genes was identified as the putative regulatory promoter region retrieved from the gerbera database. Subsequently, these promoter sequences were analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Prediction and analysis of subcellular localization

The subcellular localizations of seven gerbera BZR proteins were predicted by predictprotein (https://open.predictprotein.org/). These proteins were expressed under the control of the CaMV 35S promoter in the YFP vector, while an empty YFP vector served as the negative control. Subsequently, the construct vectors were transformed into gerbera protoplasts, and fluorescence signals were visualized using a laser confocal scanning microscope (LSM 800, Zeiss, Germany). The nuclear localization marker NLS-mCherry was co-transfected into gerbera protoplasts to label the nucleus.

Dual-luciferase reporter assay

To evaluate the transcriptional activity of seven BZR proteins, their full-length sequences were inserted into the effector-GAL4 vector containing the GAL4 DNA-binding domain. The reporter vector contains the CaMV35S promoter driving the firefly luciferase gene, with five tandem copies of the GALRE element upstream. The internal control vector features the CaMV35S promoter driving the Renilla luciferase gene. Subsequently, the effector-GAL4 vector, internal control vector, and reporter vector carrying the luciferase gene were co-transfected into gerbera protoplasts. The luciferase assay was performed according to the manufacturer’s protocol (Dual-Luciferase® Reporter Assay, Promega, United States), with Firefly and Renilla luciferase activities quantified using an Enspire multimode microplate reader (PerkinElmer Inc.). For each experiment, three biological replicates were used.

Yeast two-hybrid assay

The full-length cDNA sequences of GhBEHs (GhBEH1, GhBEH2, GhBEH3, GhBEH4, GhBEH5, GhBEH6, GhBEH7) were cloned into the pGBKT7 vector to generate the BD construct. These constructs were co-transferred to yeast AH109 cells and pGADT7, respectively, to test the transcriptional activation ability of the GhBEHs. Positive transformants were selected on synthetic dropout media (SD/-Leu-Trp-His and SD/-Leu-Trp-His-Ade). X-α-gal was added to the synthetic dropout medium to increase selection stringency.

Quantitative RT-PCR (qRT-PCR)

RNA extraction was performed by reverse transcription using the Toyobo reverse transcription kit, following the protocols described in a previous study [44]. qRT-PCR was performed in a 20μL reaction. The reaction contains 10μL SYBR Master Mix, 0.4μL (10 μM) of each primer, 5μL cDNA template and 4.2μL Nuclease-free water. The PCR procedure is as follows: initial denaturation at 94 °C for 3 min, 30 s at 94 °C, 30 s at 58 °C, and 30 s at 72 °C, a total of 28–32 cycles, and final extension at 72 °C for 10 min. qRT-PCR was conducted using CFX96™ thermocycler (Real-Time PCR Detection Systems, Bio-Rad, USA). The expression levels of the GhBEHs (GhBEH1, GhBEH2, GhBEH3, GhBEH4, GhBEH5, GhBEH6, GhBEH7) were normalized to GhACTIN (GenBank AJ763915) gene expression, as described in gerbera studies [38, 45]. Relative expression levels were calculated by using 2−△△CT [46]. Each sample was analyzed with three biological replicates and three technical replicates.

Hormone treatments

Petals from the outermost whorl of ray florets were excised from the inflorescences at stage 3 for hormone treatment. The detached petals were placed on two layers of Whatman filter paper immersed in BL, BRZ, and PPZ, respectively, for durations of 0.5, 0.75, 1, 2, 4, 10, 12, and 24 h. Subsequently, morphological observations were conducted, and the resulting data were collected and analyzed. Each experiment was replicated at least three times.

Statistical analysis

A Student’s t-test was employed for statistical analysis to determine the significance of differences between the samples. The graphs were created using the GraphPad Prism software.

Results

Identification of BZR family genes in gerbera

Seven BZR family homologous genes were identified through extensive alignment and analysis using the transcriptome database of gerbera. The homologous gene (GenBank PQ538752) was designated GhBEH1 (BES1/BZR1 homolog 1), while the remaining six homologs were designated GhBEH2 (GenBank GACN01006390.1), GhBEH3 (GenBank PQ538753), GhBEH4 (GenBank GACN01030557.1), GhBEH5 (GenBank GACN01023011.1), GhBEH6 (GenBank PQ538754), and GhBEH7 (GenBank GACN01022598.1) (Additional file 1). The entire lengths of these seven proteins are as follows: GhBEH1, 294 amino acids; GhBEH2, 306 amino acids; GhBEH3, 312 amino acids; GhBEH4, 287 amino acids; GhBEH5, 315 amino acids; GhBEH6, 297 amino acids; GhBEH7, 322 amino acids. The cDNA sequence and protein sequence are included in Additional file 2.

Chromosome locations and gene structures of GhBEHs

The chromosomal locations of 7 GhBEH genes were mapped using TBtools software. Analysis of chromosomal distribution reveals that these genes are distributed across chromosomes 3, 5, 10, 11, 12, 14, and 15 of gerbera, showing a uniform distribution pattern (Fig. 1A). Notably, most GhBEH genes are located at the proximal or distal ends of the chromosomes, with two members oriented in the forward direction and the remaining five in the reverse direction. We analyzed their exon–intron structure to elucidate the structural characteristics of GhBEH genes. The results indicated that all GhBEH genes have a length within 5 Kb, each containing a single intron, as illustrated in Fig. 1B.

Fig. 1
figure 1

Chromosomal distribution and gene structures of GhBEHs. A Chromosomal distribution of seven GhBEHs in the gerbera genome. The red line and black arrow indicate the position and the direction of the seven GhBEH genes, respectively. B The structure of GhBEHs is visualized by GSDS 2.0. The exons are yellow, and the lines between boxes represent the introns

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Phylogenetic analysis and multiple sequence alignment analysis of the GhBEHs

To elucidate the evolutionary relationships among these BEH proteins, we conducted genome-wide searches using NCBI BLASTp for the seven GhBEH proteins. Subsequently, the identified homologous protein sequences were subjected to phylogenetic tree clustering analysis (Fig. 2). The study revealed that these seven members could be broadly categorized into two groups. GhBEH1, GhBEH2, GhBEH3, and GhBEH4 are closely related, clustering near Arabidopsis BZR1 and BES1. Meanwhile, GhBEH5, GhBEH6, and GhBEH7 form a distinct cluster. Importantly, these GhBEH proteins show closer evolutionary relationships with those from the Asteraceae family, such as sunflowers, lettuce, and chamomile, suggesting evolutionary consistency within Asteraceae plants.

Fig. 2
figure 2

Phylogeny of the GhBEH proteins in different species. The phylogenetic tree was constructed with MEGA 5 using the neighbor-joining method. The bootstrap values indicated the robustness of each branch. Seven GhBEHs are marked with blue dots. The scale bar represented 0.1 substitutions per site

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To explore the diversity of BEH proteins, we conducted amino acid sequence alignment analysis on these seven proteins alongside homologous proteins from Arabidopsis and four Asteraceae plants (Fig. 3). The results demonstrate that GhBEH proteins possess characteristic BZR structures. Their N-termini are highly conserved, containing nuclear localization sequences (NLS) and DNA binding domains (DB). Additionally, GhBEH1, GhBEH2, GhBEH3, and GhBEH4 share putative phosphorylation regions like those found in homologous sequences from Asteraceae plants and contain conserved PEST motifs and proline residues. Proline residue plays a crucial role in the functionality of Arabidopsis BZR1 and BES1 [13].

Fig. 3
figure 3

Multiple sequence alignment analysis of the BZRs in gerbera and other species. Arabidopsis thaliana: AtBZR1, AtBES1, AtBEH1, AtBEH2, AtBEH3, AtBEH4. Gerbera hybrida: GhBEH1, GhBEH2, GhBEH3, GhBEH4, GhBEH5, GhBEH6, GhBEH7. Helianthus annuus: HaBEH1. Tanacetum cinerariifolium: TcBEH1. Mikania micrantha: McBEH1. Lactuca sativa: LsBEH1. PEST motifs, putative phosphorylation regions, nuclear localization sequences (NLS), and DNA binding domains (DB) are marked with underline

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Subcellular localization of GhBEH proteins

The nuclear localization signals of the seven GhBEH proteins were initially predicted using predictprotein (https://open.predictprotein.org/). To validate these predictions, we generated YFP vectors driven by the 35S promoter and transformed them into gerbera protoplasts. Localization of the proteins was conducted using confocal laser scanning microscopy. The results showed exclusive nuclear localization of YFP-GhBEH1 and YFP-GhBEH2, while the remaining five proteins exhibited fluorescence throughout the entire cell (Fig. 4A), suggesting potential localization in both the nucleus and cytoplasm.

Fig. 4
figure 4

Subcellular localization analysis of GhBEH proteins. A Subcellular localization of GhBEH proteins. Gerbera protoplasts transiently expressed GhBEH-YFP fusion proteins were observed through the laser scanning confocal microscope. B The schematic representation of proline mutation in GhBEH1, GhBEH2, GhBEH3 and GhBEH4 (GhBEH1P192L, GhBEH2P209L, GhBEH3P219L, GhBEH4P206L). C Subcellular localization of GhBEH1P192L, GhBEH2P209L, GhBEH3P219L and GhBEH4P206L. Scale bar = 10 μm

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To validate the nuclear localization signals of the seven GhBEH proteins, YFP vectors, and a nuclear localization signal marker NLS-mCherry were co-transformed into protoplasts to assess the subcellular localization. Our observations confirmed that YFP-GhBEH1 and YFP-GhBEH2 were exclusively localized within the cell nucleus, whereas the remaining five proteins also showed significant fluorescence in this compartment (Fig. 5A).

Fig. 5
figure 5

The nuclear localization confirmation of GhBEH proteins. A The nuclear localization of GhBEH proteins. Gerbera protoplasts transiently expressed GhBEH-YFP fusion proteins, and a nuclear localization signal marker NLS-mCherry were observed through the laser scanning confocal microscope. B The nuclear localization of GhBEH1P192L, GhBEH2P209L, GhBEH3P219L and GhBEH4P206L. Scale bar = 10 μm

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It has been reported that the mutation of 234th proline to leucine in Arabidopsis significantly enhances the nuclear localization signal of BZR1, a pivotal regulator in its function [19, 47]. Therefore, we constructed YFP-GhBEH1P192L, YFP-GhBEH2P209L, YFP-GhBEH3P219L, and YFP-GhBEH4P206L vectors (designated as GhBEH1P192L, GhBEH2P209L, GhBEH3P219L, and GhBEH4P206L, respectively, upon introduction of the proline-to-leucine mutation) (Fig. 4B), and transformed them into protoplasts to assess their subcellular localization. Our observations revealed that YFP-GhBEH1P192L and YFP-GhBEH2P209L maintained their nuclear localization (Figs. 4C and 5B). In contrast, the nuclear localization of YFP-GhBEH3P219L and YFP-GhBEH4P206L was significantly enhanced, suggesting that the proline-to-leucine mutation promotes the nuclear localization of GhBEH3 and GhBEH4.

Transcriptional activity analysis of GhBEHs

According to bioinformatics, structural predictions, and subcellular localization, the seven members are classified within the BZR family of transcription factors. In addition to their nuclear localization, transcription factors typically exhibit either transcriptional activation or inhibition capabilities [38, 39]. Transactivation assays were initially performed in yeast to investigate the transcriptional activity of the BZR family member in gerbera. The seven GhBEH genes fused with the BD vector were co-transformed with the AD empty vector into AH109 yeast-competent cells. The results showed that GhBEH1 and GhBEH2 did not grow on SD/-Leu-Trp-His (SD/-LTH) and SD/-Leu-Trp-His-Ade (SD/-LTHA), similar to the negative control, thereby suggesting a lack of self-activation activity in these two members. Meanwhile, the remaining five members exhibited normal growth on SD/-LTH and SD/-LTHA, consistent with the positive control, indicating their self-activation capability. Further validation was conducted by adding X-gal to the SD/-LTH and SD/-LTHA plates. The results showed that GhBEH1 and GhBEH2 neither grew nor turned blue, whereas GhBEH3, GhBEH4, GhBEH5, GhBEH6, and GhBEH7 displayed growth and distinctive blue coloration (Fig. 6A). These experiments collectively demonstrate that GhBEH1 and GhBEH2 lack self-activation activity, consistent with Arabidopsis BZR1 and BES1 observations, thus suggesting functional homology. In contrast, GhBEH3, GhBEH4, GhBEH5, GhBEH6, and GhBEH7 all exhibit strong self-activation activity.

Fig. 6
figure 6

Transcriptional activity analysis of GhBEHs. A Transcriptional activity of GhBEHs using yeast two-hybrid assay. + , pGBKT7-p53 transformed with pGADT7-SV40 large T antigen; -, pGBKT7-p53 vector transformed with pGADT7-lam vector. B The vector construction of effector and reporters used in the dual-luciferase activity assay. C Transcriptional activity analysis of GhBEHs by dual-luciferase assay. Values were the means ± SD from three biological replicates, ∗ indicating a significant difference at p < 0.05 by a Student’s t-test

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Meanwhile, dual-luciferase assays were conducted to substantiate further the transcriptional activity of GhBEHs (Fig. 6B). The results showed that GhBEH1 and GhBEH2 had significantly lower ratios than the control group. Conversely, GhBEH3 through GhBEH7 demonstrated ratios significantly higher than the control (Fig. 6C). These findings strongly suggest that GhBEH1 and GhBEH2 may function as transcriptional repressors, while the remaining five members act as transcriptional activators.

Analysis of cis-acting elements in the promoters of GhBEHs

To investigate the potential function of GhBEHs, we employed PlantCARE to predict cis-acting elements within the promoter regions of seven BEH genes. One hundred forty-seven cis-acting elements were identified within approximately 2000 bp upstream of the translation initiation site. These elements encompass categories associated with phytohormones, stress and light responses, tissue-specific expression, and cell division (Fig. 7). Predominantly represented are the elements associated with tissue-specific expression, followed by those involved in stress and phytohormone responses. These findings hint that GhBEH genes likely participate in plant hormone response and various stresses, potentially influencing plant growth and stress resistance. Notably, different genes contain different numbers of phytohormone response elements, such as GhBEH1 (4 elements) and GhBEH4 (3 elements), and elements responsive to various phytohormones. For instance, GhBEH7 contains elements responsive to abscisic acid, gibberellin, ethylene, and auxin, whereas GhBEH5 only exhibits gibberellin response elements. These results indicate that distinct GhBEH genes may play diverse regulatory roles by responding to specific phytohormone signals.

Fig. 7
figure 7

Cis-element analysis in the promoters of GhBEHs

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Expression analysis of GhBEHs under BR treatment

The BZR family genes are the key plant BR signaling pathway transcription factors. In their expression profile analysis, only one member of the BZR family, GhBEH1, was identified as differentially expressed following BL (brassinolide) treatment [48]. To investigate whether other GhBEHs also respond to BR, we examined the expression changes of seven genes at various time points following BL treatment. Samples were collected at 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, and 24 h following BL treatment, and RNA was extracted for expression analysis. The results showed that GhBEH1 and GhBEH2 exhibit a consistent pattern of initial upregulation followed by downregulation over time. GhBEH1 reaches peak expression at 2 h, while GhBEH2 peaks slightly earlier, at 1 h. GhBEH3 and GhBEH4 respond slower to BL treatment, showing increased expression trends after 8 h. In contrast, GhBEH5 shows downregulated expression, while GhBEH6 and GhBEH7 respond rapidly to BL, showing significant upregulation at 0.5 h post-treatment, gradually declining by 4 h (Fig. 8A). A corresponding heatmap visually represents these expression dynamics (Fig. 8B). The varied changes in GhBEH expression levels post-BR treatment suggest diverse response timings and intensities to BR.

Fig. 8
figure 8

Dynamic expression changes of GhBEHs with different times of BL treatment. A The expression level of GhBEHs under different times of BL treatment by qRT-PCR. The expression level of GhBEHs at 0 h was normalized to 1. B Heatmap of the expression level of GhBEHs under different times of BL treatment. The experiments were replicated at least three times, and the expression value = mean ± SE. The expression values mapped to a color gradient from low (green) to high expression (red) are shown at the right of the figure

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To assess the response of GhBEHs to endogenous BR, we treated stage 3 petals with two BR synthesis inhibitors, BRZ (brassinazole) and PPZ (propiconazole), and analyzed their expression levels. The results demonstrate that GhBEH1 and GhBEH2 exhibit a pattern of initial decrease followed by an increase, contrasting with their response to BR treatment. However, the expression patterns of the remaining five genes showed irregular fluctuations in up and down-regulation (Fig. 9). These findings suggest that GhBEH1 and GhBEH2 exhibit apparent responses to BR, indicating their strong capability to respond to endogenous and exogenous BR stimuli.

Fig. 9
figure 9

Dynamic expression changes of GhBEHs with different times of BRZ or PPZ treatment. A The expression heatmap of GhBEHs under different times of BRZ treatment by qRT-PCR. B The expression heatmap of GhBEHs under different times of PPZ treatment by qRT-PCR. The expression levels mapped to a color gradient from low (green) to high expression (red) are shown at the right of the figure

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Expression pattern of GhBEHs in different petal growth stages

The early study divided the petal growth and developmental stages into 11 phases in gerbera “Terra Regina” [49]. To gain deeper insights into the potential functions of GhBEHs in petal growth, quantitative real-time polymerase chain reaction (qRT-PCR) was employed to examine their expression patterns. The analysis of GhBEH expression profiles across these stages revealed distinct trends: GhBEH1 and GhBEH2 exhibited an initial increase from stage 1 to stage 6, followed by a decline from stage 7 to stage 11, peaking at stages 6–7 (Fig. 10A, B). In contrast, GhBEH3, GhBEH4, GhBEH5, and GhBEH6 consistently exhibited significant downregulation throughout stages 1–11. Meanwhile, GhBEH7 did not show significant changes in expression levels across these stages.

Fig. 10
figure 10

Expression patterns of GhBEHs during different ray floret growth stages in gerbera. A Expression level of GhBEHs during different floret growth stages by qRT-PCR. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11 represent different growth stages of ray florets respectively. The expression level of GhBEHs in Ray1 was normalized to 1. Ray: ray floret. B The expression heatmap of GhBEHs during different ray floret growth stages. The experiments were replicated at least three times, and values are means ± SE. The expression values mapped to a color gradient from low (green) to high expression (red) are shown at the right of the figure

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To further elucidate the expression dynamics of GhBEHs across different growth stages of ray floret, a relative expression level graph was generated using GhBEH1 expression at stage 1 as a reference baseline (Fig. 11). The analysis revealed that GhBEH1 displayed the highest relative expression level among ray petals, followed by GhBEH7, while the expression levels of other genes were comparatively lower. Notably, GhBEH2 and GhBEH6 exhibited expression levels of approximately 1/20th of GhBEH1. These data suggest different degrees of influence of these genes on the petal growth of ray floret.

Fig. 11
figure 11

The relative expression level of GhBEHs during different ray floret growth stages. The expression of GhBEH1 in stage 1 was set as unit 1. Three independent experiments were replicated, and values are means ± SE

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Discussion

The BZR family comprises pivotal transcription factors in plants that play a crucial role in the BR signaling pathway, governing plant growth and development and participating in abiotic stress responses [50,51,52]. However, there have been no reports on the functions of BZR TFs in gerbera. In this study, we conducted a comprehensive and systematic analysis of the BZR genes in gerbera, including multiple sequence alignment analysis, phylogenetic analysis, subcellular localization, transcriptional activity analysis, expression pattern, and response to BR treatment. Identifying and characterizing the BZR family members in gerbera represents a crucial first step in exploring the functional roles of this gene family.

The subcellular localization of proteins is closely related to their cellular functions [53]. Typically, transcription factors are predominantly located within the cell nucleus, although some TFs exhibit dual localization in both the nucleus and the cytoplasm [54]. In our investigation, GhBEH1 and GhBEH2 were exclusively localized in the nucleus, while the remaining five proteins were observed in both the nucleus and cytoplasm. The localization of the BZR family genes in different species is inconsistent. For instance, in some species such as Zanthoxylum armatum DC [55], potato (Solanum tuberosum. L) [56], and celery (Apium graveolens) [57], the BZR family genes are localized in both the nucleus and cytoplasm, similar to the situation in Arabidopsis, and is consistent with the localization of BEH3-7 in gerbera (Figs. 4A and 5A). In contrast, Birch (Betula platyphylla) BZR1-6 [58], Garlic (Allium sativum L.) BZR11 [59], and kiwifruit (Actinidia deliciosa var. deliciosa) BZR1.1 and BZR1.4 [60] are exclusively localized in the nucleus, mirroring the nuclear localization of BEH1 and BEH2 in gerbera (Figs. 4A and 5A). These findings suggest that the subcellular localization of BZR family members is not universally conserved across species and may be influenced by various factors, including phosphorylation/dephosphorylation dynamics [56]. For instance, it has been documented that in Arabidopsis, BR induce the dephosphorylation of BES1 and BZR1, which in turn promotes their nuclear accumulation [11, 42, 43]. Additionally, the interactions with proteins such as RACK1 and 14–3-3 may also play a role in modulating the localization of BZR proteins [55, 61]. Studies on Arabidopsis BZR1 have demonstrated that substituting proline with leucine at position 234 enhances its nuclear localization signal, thereby influencing its functional activity [19, 47]. Our study observed that GhBEH1P192L and GhBEH2P209L maintained their nuclear localization patterns unchanged. In contrast, the nuclear localization of GhBEH3P219L and GhBEH4P206L was significantly enhanced, similar to the results of BZR1 in Arabidopsis. These findings suggest that GhBEH3 and GhBEH4 in gerbera may function similarly to Arabidopsis BZR1 in terms of their subcellular localization and potentially functional roles.

Cis-acting elements are non-coding DNA sequences within promoter regions, influencing gene expression and function [62]. This study’s comprehensive analysis identified 147 cis-acting elements in the promoters of 7 GhBEH genes linked to various aspects of plant physiology, such as hormonal responses, stress adaptation, light sensitivity, tissue-specific expression, and cell division. Analysis of these cis-acting elements indicated that all seven GhBEHs possess motifs related to stress responsiveness, suggesting their role in modulating gerbera’s adaptation to the environment. In addition, except for GhBEH6, promoters of the remaining 6 GhBEH genes contain elements responsive to hormones, implicating the BZR gene family in the intricate crosstalk between BR signaling and other hormone pathways. In Arabidopsis, the BZR1 protein interacts with the blue light receptor cryptochrome 1 (CRY1), which mediates blue light-induced inhibition of hypocotyl elongation. This interaction results in the formation of a novel CRY1-BIN2-BZR1 module through the phosphorylation of BIN2, thereby regulating the crosstalk between blue light and BR signaling pathways and coordinating plant growth [63]. Additionally, BES1/BEH4 forms a transcriptional module with the auxin response factor ARF5, known as the ARF5-BES1/BEH4 module, which promotes Arabidopsis seedling growth by modulating a series of growth-related genes [64]. To further elucidate the hormonal regulation of gerbera BZR genes, we assessed the expression profiles of each gene under BR treatment. The results revealed diverse expression patterns among the seven GhBEH genes in response to BL, with particular emphasis on GhBEH6 because of the most significant fluctuation, which may be attributed to the apparent lack of hormone-responsive elements in its promoter region.

The growth of petals in gerbera involves complex cell proliferation and expansion mechanisms. Changes in cell number characterize the early stages of petal growth, while the middle and late stages emphasize cell size dynamics [65,66,67]. BR is pivotal in promoting cell elongation and expansion, significantly influencing overall petal growth [8, 68, 69]. We examined the expression profiles of GhBEHs in ray floret at different growth stages, revealing distinct patterns among the seven members. GhBEH3, GhBEH4, GhBEH5, and GhBEH6 exhibited high expression levels during the early stages of petal growth, whereas GhBEH7 maintained consistent expression throughout the entire growth period. In contrast, GhBEH1 and GhBEH2 displayed predominant expression in the middle and late stages, with GhBEH2 showing a pronounced increase during the middle stage and maintaining high expression levels later. These observations suggest that GhBEH3, GhBEH4, GhBEH5, and GhBEH6 likely contribute to early petal growth through involvement in petal cell proliferation. Conversely, GhBEH1 and GhBEH2 may play roles in petal development’s middle and late stages, potentially in cell elongation and expansion processes. Nevertheless, further experimental evidence is necessary to confirm these hypotheses.

In recent years, the BZR family genes have been studied in several species. The researches on tomato, legumes, and cucumber have primarily concentrated on the expression of BZR genes across different tissues and in response to abiotic stresses [70,71,72]. In wheat, studies have emphasized BZR responses to a range of biotic and abiotic stresses [73]. While the research on beet BZR genes has focused on taproot development and sugar accumulation [74]. In our research, we mainly aim at the function of BZR genes in the growth of ray floret petals (Figs. 10 and 11), in order to provide a basis for further studies on the biological function of the BZR gene family in petal growth and development of gerbera.

Conclusions

In this study, the whole genome of the gerbera BZR family gene was investigated, and seven BZR genes named GhBEH1-7 were identified. Research showed that GhBEH1 and GhBEH2 localized in the nucleus and may function as transcriptional repressors, while the remaining five genes serve as transcriptional activators. Expression analysis hinted that GhBEH1 and GhBEH2 may play roles in petal growth’s middle and late stages. Notably, all seven GhBEHs respond to BR, especially GhBEH1 and GhBEH2, which may play a role in BR-regulated plant growth and development. This is the first report to identify the BZR gene in gerbera and provides a basis for further research on the biological function of the BZR gene family in petal growth.

Data availability

All data presented in this research are available in the article and supplementary materials except for the unpublished genomic data of gerbera.

Abbreviations

BR:

Brassinosteroid

BRZ:

Brassinazole

Dfs:

Disc florets

PPZ:

Propiconazole

qRT-PCR:

Real-time quantitative reverse transcription PCR

Rfs:

Ray florets

Tfs:

Trans florets

TF:

Transcription factor

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Acknowledgements

We thank Prof. Paula Elomaa and Dr. Teng Zhang for instructing on some experiments and advising on the manuscript. We thank Dr. Fan Li and Prof. Liangsheng Zhang for providing the genome of gerbera.

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Funding

This work was supported by the Natural Science Foundation of Guangdong Province (2023A1515012864), Guangdong Key Laboratory of Plant Adaptation and Molecular Design (2022B1212010013-7), Laboratory of Lingnan Modern Agriculture Project (NZ2021009) and National Key R&D Program of China (2018YFD1000404).

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  1. Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Sciences, South China Normal University, Guangzhou, Guangdong Province, 510631, China

    Qishan Luo, Gan Huang, Xiaohui Lin, Xiaojing Wang & Yaqin Wang

  2. College of Landscape Architecture and Art, Henan Agricultural University, Zhengzhou, Henan Province, 450002, China

    Gan Huang

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  1. Qishan Luo
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  2. Gan Huang
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Contributions

Qishan Luo wrote the original draft and visualization. Gan Huang did conceptualization, data curation and formal analysis. Xiaohui Lin did data curation and formal analysis. Xiaojing Wang did supervision, writing-review and editing. Yaqin Wang did resources, supervision, funding acquisition, writing-review and editing. All authors read and approved the final manuscript.

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Correspondence to Yaqin Wang.

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Luo, Q., Huang, G., Lin, X. et al. Genome-wide identification, characterization, and expression analysis of BZR transcription factor family in Gerbera hybrida. BMC Plant Biol 25, 143 (2025). https://doi.org/10.1186/s12870-025-06177-7

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BMC Plant Biology

ISSN: 1471-2229

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